NZ764037B2 - Compositions and methods for modulating growth hormone receptor expression - Google Patents

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

COMPOSITIONS AND METHODS FOR MODULATING GROWTH HORMONE RECEPTOR EXPRESSION Seguence Listing The present application is being filed along with a Sequence g in electronic format. The Sequence Listing is provided as a file entitled BIOL0253WOSEQ_ST25 .txt created April 27, 2015, which is 1.29 MB in size. The information in the electronic format of the sequence g is incorporated herein by reference in its entirety.

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

Background Growth e 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 grth factor-1 (IGF- 1). IGF-l is produced mainly in the liver, but also in adipose tissue and the kidney, and secreted into the tream. Several disorders, such as galy and gigantism, are associated with elevated grth hormone levels and/or elevated IGF-I levels in plasma and/or tissues. ive production of growth hormone can lead to es such as acromegaly or gigantism.

Acromegaly and gigantism are associated with excess grth 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 grth related outcomes are mediated by elevated levels of serum IGF-l.

My Embodiments provided herein relate to methods, compounds, and compositions for ng, preventing, or ameliorating a disease associated with excess growth hormone. Several ments provided herein are drawn to antisense compounds or oligonucleotides targeted to grth 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 ing 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 speci?cally stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. rmore, the use of the term "including" as well as other forms, such as "includes" and ded", is not ng. 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 nts, 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 c definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic try, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for al synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in "Carbohydrate Modifications in nse Research" Edited by Sangvi and Cook, American Chemical Society Pharmaceutical , Washington DC, 1994; "Remington's es," Mack Publishing Co., Easton, Pa., 21St edition, 2005; and ense Drug Technology, Principles, Strategies, and Applications" Edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida; and Sambrook et al., "Molecular Cloning, A laboratory Manual," 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby orated by reference for any purpose. Where ted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the following meanings: "2’-F nucleoside" refers to a side sing a sugar comprising ?uorine at the 2’ position. Unless otherwise indicated, the ?uorine in a 2’-F nucleoside is in the ribo position (replacing the OH of a natural ribose). "2’-O-methoxyethyl" (also 2’-MOE and 2’-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2’ position of a furanose ring. A 2’-O-methoxyethyl modified sugar is a modified sugar. "2’-MOE nucleoside" (also 2’-O-methoxyethyl nucleoside) means a nucleoside comprising a 2’- MOE ed sugar moiety. "2’-substituted nucleoside" means a nucleoside comprising a substituent at the ition other than H or OH. Unless otherwise indicated, a stituted nucleoside is not a bicyclic nucleoside. "2’-substituted sugar moiety" means a furanosyl comprising a substituent at the 2’-position other than H or OH. Unless otherwise indicated, a 2’-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2 ’-substituent of a 2’-substituted sugar moiety does not form a bridge to another atom of the ?Jranosyl ring. "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 tide of a particular nse compound. hylcytosine" means a cytosine modified with a methyl group attached to the 5 position. A 5- methylcytosine is a modified nucleobase. " 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 ed herein to a subject to perform its ed 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 ion or infusion.

"Alkyl," as used herein, means a saturated straight or branched arbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12alkyl) with from 1 to about 6 carbon atoms being more preferred.

As used herein, "alkenyl," means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one -carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, yl, butenyl, 1-methylbutenyl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. l groups as used herein may optionally include one or more r substituent groups.

As used herein, "alkynyl," means a straight or ed hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more lly from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.

As used herein, "acyl," means a l formed by removal of a hydroxyl group from an organic acid and has the general Formula -C(O)-X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic yls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, tic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include ?thher tuent groups.

As used herein, clic" means a cyclic ring system wherein the ring is aliphatic. The ring system can se one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further sub stituent groups.

As used herein, atic" means a straight or ed hydrocarbon radical ning up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond.

An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be upted with one or more heteroatoms that include nitrogen, oxygen, sul?Jr and phosphorus. Such aliphatic groups upted by heteroatoms include without limitation, koxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further sub stituent groups.

As used herein, "alkoxy" means a radical formed n an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without tion, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, toxy, lerl-butoxy, n- pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include ?thher substituent groups.

As used herein, "aminoalkyl" means an amino substituted C1-C12 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a ?thher substituent group at the alkyl and/or amino portions.

As used herein, "aralkyl" and "arylalky " mean an ic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include t limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.

As used herein, "aryl" and "aromatic" mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include ?thher substituent groups.

"Amelioration" refers to a ing of at least one indicator, sign, or m 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 ion 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 man animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and man primates, including, but not limited to, monkeys and chimpanzees. ense ty" means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, nse 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 e of oing hybridization to a target nucleic acid through hydrogen g. Examples of nse compounds include single-stranded and double-stranded compounds, such as, antisense ucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

"Antisense inhibition" means reduction of target nucleic acid levels in the presence of an antisense nd complementary to a target nucleic acid ed 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 ce 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 bases 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 ?Jranosyl. In certain such embodiments, the bridge ts the 2 ’-carbon and the 4’-carbon of the furanosyl.

"Bicyclic nucleic acid" or " BNA" or "BNA nucleosides" means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby g a bicyclic ring system.

In certain embodiments, the bridge connects the 4’-carbon and the 2 ’-carbon of the sugar ring.

"Cap structure" or "terminal cap moiety" means al modi?cations, which have been incorporated at either terminus of an antisense compound.

"Carbohydrate" means a naturally occurring ydrate, a ed carbohydrate, or a carbohydrate derivative.

"Carbohydrate r" means a compound having one or more carbohydrate residues attached to a scaffold or linker group. (see, e.g., Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting," Bioconjugate Chemistry, 2003, (14): 18-29, which is incorporated herein by reference in its entirety, or Rensen et al., "Design and Synthesis of Novel N- Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor," J. Med. Chem. 2004, (47): 5798-5808, for examples of carbohydrate ate clusters).

"Carbohydrate derivative" means any compound which may be sized using a carbohydrate as a starting material or intermediate. "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 a: 4’-CH(CH3)-O-2’.

"Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4’-CH(CH3)-O-2’ bridge.

"Chemically distinct region" refers to a region of an antisense compound that is in some way chemically ent than another region of the same antisense compound. For example, a region having 2’- O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2’-O- methoxyethyl cations.

"Chemical modification" means a chemical difference in a nd when compared to a naturally occurring counterpart. Chemical modifications of ucleotides include nucleoside modi?cations (including sugar moiety modifications and nucleobase modifications) and intemucleoside linkage modifications. In nce to an oligonucleotide, chemical modification does not include differences only in base sequence.

"Chimeric antisense compounds" means nse compounds that have at least 2 chemically distinct s, each position having a plurality of subunits.

"Cleavable bond" means any chemical bond capable of being split. In certain ments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.

"Cleavable moiety" means a bond or group that is capable of being split under physiological conditions. In certain embodiments, a cleavable moiety is cleaved inside a cell or sub-cellular compartments, such as a lysosome. In certain embodiments, a cleavable moiety is cleaved by endogenous enzymes, such as nucleases. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. ministration" means administration of two or more pharmaceutical agents to an individual.

The two or more pharmaceutical agents may be in a single ceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

"Complementarity" means the capacity for pairing between nucleobases of a ?rst c acid and a second nucleic acid. ise, comprises" and ising" 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.

"Conjugate" or "conjugate group" means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular bution, cellular uptake, charge, and/or clearance properties.

"Conjugate linker" or "linker" in the context of a conjugate group means a portion of a conjugate group comprising any atom or group of atoms and which covalently link (1) an oligonucleotide to another portion of the ate group or (2) two or more portions of the conjugate group.

Conjugate groups are shown herein as radicals, providing a bond for forming covalent attachment to an oligomeric compound such as an antisense oligonucleotide. In certain embodiments, the point of attachment on the oligomeric compound is the 3'-oxygen atom of the roxyl group of the 3 ’ terminal nucleoside of the eric compound. In certain embodiments the point of attachment on the eric compound is the 5'-oxygen atom of the 5'-hydroxyl group of the 5’ terminal nucleoside of the oligomeric compound. In certain embodiments, the bond for forming attachment to the oligomeric compound is a cleavable bond. In certain such embodiments, such cleavable bond constitutes all or part of a cleavable moiety.

In certain ments, ate groups comprise a ble moiety (e.g., a cleavable bond or cleavable nucleoside) and a carbohydrate cluster portion, such as a GalNAc cluster portion. Such carbohydrate cluster portion comprises: a targeting moiety and, optionally, a conjugate linker. In certain embodiments, the carbohydrate cluster portion is fied by the number and identity of the ligand. For example, in certain embodiments, the carbohydrate cluster portion ses 3 GalNAc groups and is designated c3". In certain embodiments, the carbohydrate cluster n comprises 4 GalNAc groups and is designated "GalNAc4". Specific carbohydrate cluster portions g specific tether, branching and ate linker groups) are described herein and designated by Roman numeral followed by subscript "a". Accordingly "GalNAc3-1a" refers to a specific carbohydrate cluster portion of a conjugate group having 3 GalNAc groups and specifically identified tether, branching and linking groups. Such carbohydrate cluster fragment is attached to an oligomeric compound via a cleavable moiety, such as a cleavable bond or cleavable nucleoside.

"Conjugate compound" means any atoms, group of atoms, or group of linked atoms suitable for use as a conjugate group. In certain ments, conjugate compounds may possess or impart one or more properties, including, but not limited to pharmacodynamic, pharmacokinetic, g, absorption, cellular distribution, cellular uptake, charge and/or clearance properties. guous nucleobases" means nucleobases immediately adjacent to each other.

"Constrained ethyl nucleoside" or "cEt" means a nucleoside comprising a ic sugar moiety comprising a 4’-CH(CH3)-O-2’bridge.

"Deoxynucleoside" means a nucleoside comprising 2’-H ?Jranosyl sugar , as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2’-deoxynucleoside may comprise a modi?ed nucleobase or may comprise an RNA nucleobase (e.g., uracil).

"Designing" or "Designed to" refer to the process of designing an oligomeric compound that speci?cally hybridizes with a selected nucleic acid molecule.

"Differently ed" mean chemical cations or chemical substituents that are ent from one r, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are rently ed," even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are "differently ed," even though both are naturally-occurring unmodified nucleosides.

Nucleosides that are the same but for comprising ent bases are not differently modified. For example, a nucleoside comprising a 2’-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2’-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.

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

"Dose" means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain ments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.

"Double-stranded" refers to two separate oligomeric compounds that are hybridized to one another.

Such double stranded compounds may have one or more or non-hybridizing nucleosides at one or both ends of one or both strands (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) provided there is sufficient complementarity to maintain hybridization under physiologically nt conditions.

"Downstream" refers to the relative ion towards the 3 ’ end or C-terminal end of a nucleic acid.

"Effective amount" means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological e 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 duals to be treated, the formulation of the composition, assessment of the individual’s medical ion, and other relevant factors.

"Effective amount" in the context of modulating an activity or of treating or ting a condition means the administration of that amount of ceutical agent to a subject in need of such modulation, treatment, or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the ition, assessment of the individual’s medical condition, and other relevant factors.

"Ef?cacy" means the ability to produce a desired effect.

"Essentially unchanged" means little or no change in a particular ter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is ially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target c acid does, but the change need not be zero.

"Expression" means the s by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5’-cap), and translation.

"Fully complementary" or "100% complementary" means each nucleobase of a first nucleic acid has a complementary nucleobase in a second c acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

"Furanosyl" means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.

"Gapmer" means a chimeric antisense nd in which an internal region having a ity 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 sing the external regions. The internal region may be referred to as the "gap" and the al regions may be referred to as the "wings." "Growth Hormone Receptor (GHR)" means any nucleic acid or protein of GHR. "GHR c 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 c DNA comprising introns and exons), including a non-protein encoding (Le. non-coding) RNA sequence, and an mRNA sequence encoding GHR. "GHR mRNA" means an mRNA ng 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 ic tors include nucleic acids (including antisense compounds), peptides, dies, small les, and other agents capable of inhibiting the expression of GHR RNA and/or GHR n.

"Halo" and "halogen," mean an atom selected from ?uorine, chlorine, bromine and iodine. oaryl," and "heteroaromatic," mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or ?Jsed ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sul?Jr, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, lyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like.

Heteroaryl radicals can be ed to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include ?thher substituent groups.

"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 e, but are not limited to, an antisense oligonucleotide and a c 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 rd clinical tests or assessments.

"Immediately nt" means there are no ening elements between the ately adjacent elements.

"Individual" means a human or non-human animal selected for treatment or y.

"Inhibiting the expression or ty" refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

"Intemucleoside linkage" refers to the al bond between nucleosides.

"Intemucleoside l linking group" means a neutral linking group that directly links two nucleosides.

"Internucleoside phosphorus linking group" means a phosphorus linking group that directly links two sides.

"Lengthened" antisense oligonucleotides are those that have one or more additional sides relative to an antisense oligonucleotide disclosed herein. ge motif’ means a pattern of linkage cations in an oligonucleotide or region thereof.

The nucleosides of such an oligonucleotide may be modified or unmodified. Unless ise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

"Linked ucleoside" means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a tide.

"Linked nucleosides" means adjacent nucleosides linked together by an internucleoside linkage.

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

E O BX E E O BX BX O "‘11:of!) BX -0 95:1", "'1",- (A) (B) (C) (D) (E) 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 ed from -[C(R1)(R2)]n-, -C(R1)=C(R2)-, -C(R1)=N- and -N(R1)-; wherein: x is 0, , -C(=NR1)-, -C(=O)—, -C(=S)-, -O-, -Si(R1)2-, -S(=O)x- 1, or 2; n is 1, 2, 3, or 4; each R1 and R2 is, ndently, H, a ting group, hydroxyl, C1-C12 alkyl, tuted C1- C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, tuted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ 1, NJ1J2, SJ 1, N3, COOJ 1, acyl (C(=O)—H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a tuted cycle radical, C 1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a ting group.

Examples of 4’- 2’ bridging groups encompassed within the definition ofLNA e, but are not limited to one of ae: -[C(R1)(R2)]n-, -[C(R1)(R2)]n-O-, -C(R1R2)-N(R1)-O- or —C(R1R2)-O-N(R1)-.

Furthermore, other bridging groups encompassed with the definition ofLNA are 4'-CH2-2', 4'-(CH2)2-2', 4'- (CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-O-N(R1)-2' and 4'-CH2-N(R1)-O-2'- bridges, wherein each R1 and R2 is, independently, H, a protecting group or C1-C12 alkyl.

Also included within the definition ofLNA according to the ion 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 eneoxy (4’-CH2-O-2’) bridge to form the bicyclic sugar . The bridge can also be a methylene (-CH2-) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4’-CH2-O-2’) LNA is used. Furthermore; in the case of the bicylic sugar moiety haVing an ethylene bridging group in this position, the term ethyleneoxy (4’-CH2CH2-O-2’) LNA is used. 0t -L- methyleneoxy (4’-CH2-O-2’), an isomer of eneoxy (4’-CH2-O-2’) LNA is also encompassed within the definition of LNA, as used herein.

"Metabolic disorder" means a disease or condition principally characterized by dysregulation of metabolism — the complex set of chemical reactions associated with breakdown of food to produce energy.

"Mismatch" or omplementary 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 ydrate" means any carbohydrate haVing one or more chemical modifications relative to naturally ing carbohydrates.

"Modi?ed intemucleoside linkage" refers to a substitution or any change from a naturally occurring intemucleoside bond (i.e. a phosphodiester intemucleoside bond).

"Modified nucleobase" means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An "unmodi?ed 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.

"Modi?ed nucleotide" means a nucleotide haVing, independently, a modified sugar moiety, modified intemucleoside linkage, or modified nucleobase. ied ucleotide" means an oligonucleotide comprising at least one modified intemucleoside e, a modified sugar, and/or a modified nucleobase.

"Modified sugar" means substitution and/or any change from a natural sugar moiety. "Modified sugar moiety" means a substituted sugar moiety or a sugar surrogate.

"Modulating" refers to changing or adjusting a feature in a cell, tissue, organ or organism. For e, 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 e, a GHR antisense compound can be a tor that decreases the amount of GHR mRNA and/or GHR protein in a cell, tissue, organ or organism.

"MOE" means -OCH2CHZOCH3.

"Monomer" refers to a single unit of an oligomer. rs e, but are not limited to, nucleosides and nucleotides, whether naturally occuring or modified.

"Mono or clic ring system" is meant to include all ring systems selected from single or polycyclic radical ring s wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from tic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, heteroaromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of tion or each, independently, have varying degrees of tion including ?Jlly saturated, lly saturated or ?Jlly unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to heterocyclic rings as well as rings comprising only C ring atoms which can be present in a mixed motif such as for example benzimidazole wherein one ring has only carbon ring atoms and the ?Jsed ring has two nitrogen atoms. The mono or clic ring system can be further substituted with substituent groups such as for example phthalimide which has two =0 groups attached to one of the rings. Mono or polycyclic ring systems can be attached to parent molecules using various strategies such as directly through a ring atom, fused through le ring atoms, through a substituent group or through a bifunctional linking moiety.

"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 ). "Naturally occurring sugar moiety" means a ribo?Jranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.

"Naturally ing intemucleoside linkage" means a 3' to 5' phosphodiester linkage.

"Neutral linking group" means a linking group that is not charged. Neutral linking groups include without limitation otriesters, methylphosphonates, MMI (-CH2-N(CH3)-O-), amide-3 (-CH2-C(=O)- N(H)-), amide-4 (-CH2-N(H)-C(=O)-), formacetal (-O-CH2-O-), and thioformacetal (-S-CH2-O-). Further neutral g groups include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and PD. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)).

Further neutral linking groups include nonionic linkages comprising mixed N, O, S and CH2 component parts.

WO 68618 2015/028887 "Non-complementary nucleobase" refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

"Non-internucleoside neutral linking group" means a neutral linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside neutral linking group links a nucleoside to a group other than a nucleoside. In certain embodiments, a non-internucleoside neutral g group links two groups, neither of which is a nucleoside.

"Non-internucleoside orus linking group" means a phosphorus linking group that does not directly link two nucleosides. In certain embodiments, a non-internucleoside phosphorus linking group links a nucleoside to a group other than a nucleoside. In certain ments, a non-internucleoside phosphorus linking group links two groups, neither of which is a nucleoside. ic acid" refers to molecules composed of monomeric tides. A nucleic acid es, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded c acids.

"Nucleobase" means a heterocyclic moiety capable of g with a base of another c acid.

"Nucleobase complementarity" or "complementarity" when in reference to nucleobases means 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 mentary to uracil (U). In certain embodiments, complementary base means a nucleobase of an nse 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 en bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising n modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.

"Nucleobase modification motif" means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.

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

"Nucleoside" means a compound comprising a nucleobase moiety and a sugar moiety. sides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. sides may be linked to a ate moiety.

"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 nd such as for example peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=O)—O- or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (?Jranose 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 cleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-intemucleoside e combination, and the base is maintained for hybridization to a selected target.

"Nucleoside motif’ means a pattern of nucleoside modi?cations in an oligonucleotide or a region thereof. The es of such an ucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein bing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.

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

"Off-target effect" refers to an ed or deleterious biological effect ated with modulation ofRNA or protein expression of a gene other than the intended target nucleic acid.

"Oligomeric compound" means a polymeric structure comprising two or more sub-structures. In certain ments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric nd ses one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. Oligomeric compounds also include naturally occurring nucleic acids. In certain embodiments, an oligomeric compound comprises a ne of one or more linked monomeric subunits where each linked monomeric subunit is directly or indirectly ed to a heterocyclic base moiety. In n embodiments, eric compounds may also include monomeric subunits that are not linked to a heterocyclic base moiety, thereby providing abasic sites. In certain embodiments, the linkages joining the monomeric subunits, the sugar es or surrogates and the heterocyclic base moieties can be independently modified. In certain embodiments, the e-sugar unit, which may or may not include a heterocyclic base, may be substituted with a mimetic such as the rs in peptide nucleic acids.

"Oligonucleoside" means an oligonucleotide in which the intemucleoside linkages do not contain a phosphorus atom.

"Oligonucleotide" means a polymer of linked nucleosides each of which can be ed 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 stration, or intracranial administration, e.g. hecal or intracerebroventricular administration.

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

"Pharmaceutical agent" means a substance that provides a therapeutic bene?t when administered to an indiVidual. For e, in certain embodiments, a conjugated antisense oligonucleotide targeted to GHR is a pharmaceutical agent. aceutical composition" means a mixture of substances suitable for administering to an indiVidual. For example, a pharmaceutical composition may se 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.

"Phosphorus linking group" means a linking group comprising a phosphorus atom. Phosphorus linking groups include t limitation groups haVing the formula: Rb=l|3-RC Ra and Rd are each, independently, O, S, CH2, NH, or NJ1 wherein J1 is C1-C6 alkyl or substituted C1- C6 alkyl; Rb is O or S; Rc is OH, SH, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, tuted C1-C6 alkoxy, amino or substituted amino; and J1 is Rb is O or S.

Phosphorus linking groups include t limitation, phosphodiester, phosphorothioate, phosphorodithioate, onate, phosphoramidate, orothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.

"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 intemucleoside linkage.

"Portion" means a defined number of uous (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 n 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 inde?nitely. Prevent also means reducing the risk of developing a disease, disorder, or ion.

"Prodrug" means an inactive or less active form of a compound which, when administered to a subject, is metabolized to form the active, or more active, nd (e.g., drug).

"Prophylactically effective amount" refers to an amount of a pharmaceutical agent that es a prophylactic or preventative benefit to an animal.

"Protecting group" means any nd or protecting group known to those having skill in the art.

Non-limiting examples of protecting groups may be found in "Protective Groups in Organic Chemistry", T.

W. Greene, P. G. M. Wuts, ISBN 062301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

"Region" is de?ned as a portion of the target nucleic acid having at least one identifiable structure, on, or characteristic.

"Ribonucleotide" means a nucleotide having a hydroxy at the 2’ position of the sugar portion of the nucleotide. cleotides may be modified with any of a variety of substituents.

"RISC based antisense compound" means an antisense compound wherein at least some of the antisense activity of the antisense nd is attributable to the RNA Induced Silencing Complex (RISC).

"RNase H based antisense compound" means an antisense compound wherein at least some of the antisense activity of the antisense compound is attributable to hybridization of the antisense compound to a target nucleic acid and subsequent ge of the target nucleic acid by RNase H.

"Salts" mean a physiologically and pharmaceutically acceptable salt of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological s thereto.

"Segments" are d as smaller or sub-portions of regions within a target nucleic acid.

"Separate regions" means portions of an oligonucleotide wherein the al modifications or the motif of chemical modifications of any neighboring portions include at least one difference to allow the separate regions to be distinguished from one another.

"Sequence motif’ means a pattern of bases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.

"Side s" means physiological disease and/or ions attributable to a ent other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function alities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may te liver toxicity or liver ?Jnction abnormality. For example, increased bilirubin may te liver toxicity or liver on abnormality.

"Single-stranded" means an oligomeric compound that is not hybridized to its complement and which lacks sufficient self-complementarity to form a stable self-duplex.

"Sites," as used herein, are de?ned as unique nucleobase ons within a target c 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 c acid to induce a desired effect, while exhibiting minimal or no effects on rget nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in viva assays and therapeutic treatments.

"Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric nd 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.

"Substituent" and "substituent group," means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2’-substuent is any atom or group at the ition of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, nds of the present disclosure have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.

Likewise, as used herein, "substituent" in reference to a chemical ?mctional group means an atom or group of atoms that differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than en which replaces one of the hydrogen atoms of an unsubstituted methyl . Unless otherwise ted, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (-C(O)Raa), yl (-C(O)O-Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O-Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (-N(Rbb)(Rcc)), =NRbb), amido (-C(O)N(Rbb)(Rcc) 0r -N(Rbb)C(O)Raa), azido (-N3), nitro (-N02), cyano (-CN), carbamido ('OC(O)N(Rbb)(Rcc) 0r )C(O)ORaa)o ureido ('N(Rbb)C(O)N(Rbb)(Rcc))9 thioureido b)C(S)N(Rbb)' (Rea): guanidinyl ('N(Rbb)C(ZNRbb)N(Rbb)(Rcc))o amidinyl ('C(=NRbb)N(Rbb)(Rcc) or 'N(Rbb)c(:NRbb)(Raa))o thiol (-SRbb), sul?nyl (-S(O)Rbb), sulfonyl (-S(O)2Rbb) and sulfonamidyl (-S(O)2N(Rbb)(Rcc) or -N(Rbb)S- (0)2Rbb). Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including t limitation, alkyl, alkenyl, alkynyl, tic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are t to a recursive .

"Substituted sugar moiety" means a syl that is not a naturally occurring sugar .

Substituted sugar moieties include, but are not limited to ?Jranosyls comprising tuents at the 2’- position, the ition, the 5’-position and/or the 4’-position. Certain substituted sugar moieties are bicyclic sugar moieties.

"Sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.

"Sugar motif ’ means a pattern of sugar modi?cations in an oligonucleotide or a region thereof.

"Sugar surrogate" means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside sub-units are capable of linking together and/or linking to other nucleosides to form an eric compound which is capable of hybridizing to a complementary oligomeric compound. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sul?Jr, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar es (e.g., ered carbocyclic bicyclic sugar ates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide c acid). Sugar surrogates include without limitation morpholinos, exenyls and cyclohexitols.

"Target" refers to a protein, the modulation of which is desired. t gene" refers to a gene encoding a target.

"Targeting" or "targeted" means the process of design and selection of an nse compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

"Target nucleic acid," "target RN 99 a; , target RNA transcript" and "nucleic acid target" all mean a nucleic acid capable of being targeted by antisense compounds. "Target nucleic acid" means a nucleic acid molecule to which an antisense compound is intended to hybridize to result in a desired antisense actiVity.

Antisense oligonucleotides have sufficient complementarity to their target nucleic acids to allow hybridization under physiological conditions.

"Target region" means a portion of a target c acid to which one or more antisense compounds is targeted.

"Target segment" means the ce 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.

"Terminal group" means one or more atom attached to either, or both, the 3’ end or the 5’ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.

"Terminal intemucleoside linkage" means the linkage between the last two sides of an oligonucleotide or de?ned region f.

"Therapeutically effective amount" means an amount of a pharmaceutical agent that es a therapeutic benefit to an individual.

"The same type of modifications" refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA sides have "the same type of modification," even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.

"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 ceutical itions can be administered to the animal.

"Type of modification" in reference to a nucleoside or a nucleoside of a "type" means the chemical cation of a nucleoside and includes modified and unmodified sides. Accordingly, unless otherwise ted, a "nucleoside having a modification of a first type" may be an unmodified nucleoside.

"Unmodified" nucleobases or "naturally occurring nucleobase" means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

"Unmodified nucleotide" means a nucleotide composed of naturally ng nucleobases, sugar moieties, and intemucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. B-D-ribonucleosides) or a DNA nucleotide (i.e. B-D-deoxyribonucleoside).

"Upstream" refers to the relative direction towards the 5 ’ end or N-terminal end of a nucleic acid.

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

Certain Embodiments Certain embodiments provide methods, compounds and itions for inhibiting grth hormone receptor (GHR) expression.

Certain ments 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. 163.4 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 00 (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 porated herein as SEQ ID NO: 5), GENBANK Accession No. AF230800.1 (incorporated herein as SEQ ID NO: 6), the complement of GENBANK ion 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), K Accession No. NM_001242400.2 (incorporated herein as SEQ ID NO: 10), GENBANK Accession No. NM_001242401.3 (incorporated herein as SEQ ID NO: 11), GENBANK Accession No. NM_001242402.2 (incorporated herein as SEQ ID NO: 12), GENBANK Accession No. 242403.2 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No.

NM_001242404.2 (incorporated herein as SEQ ID NO: 14), GENBANK Accession No. NM_001242405.2 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No. NM_001242406.2 (incorporated herein as SEQ ID NO: 16), GENBANK Accession No. NM_001242460.1 (incorporated herein as SEQ ID NO: 17), GENBANK Accession NM_001242461.1 (incorporated herein as SEQ ID NO: 18), or GENBANK Accession No. NM_001242462.1 (incorporated herein as SEQ ID NO: 19).

Certain ments provide a compound comprising a modi?ed oligonucleotide and a ate group, wherein the modi?ed ucleotide consists of 10 to 30 linked nucleosides and has a nucleobase ce comprising at least 8 contiguous nucleobases of any of the nucleobase ces of SEQ ID NOs: Certain embodiments provide a compound comprising a modi?ed oligonucleotide and a conjugate group, wherein the modi?ed oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 9 uous nucleobases of any of the nucleobase sequences of SEQ ID NOs: -2295.

Certain embodiments provide a compound comprising a modi?ed oligonucleotide and a conjugate group, wherein the modi?ed oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 10 contiguous nucleobases of the nucleobase sequences of any of SEQ ID NOs: -2295.

Certain embodiments provide a compound comprising a modi?ed oligonucleotide and a ate group, wherein the modi?ed oligonucleotide ts of 10 to 30 linked nucleosides and has a nucleobase sequence comprising at least 11 contiguous nucleobases of the nucleobase sequences of any of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a d oligonucleotide and a conjugate group, wherein the modi?ed oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase ce sing at least 12 contiguous nucleobases of the nucleobase sequences of any of SEQ ID NOs: -2295.

Certain embodiments provide a compound sing a modi?ed ucleotide and a conjugate group, wherein the modi?ed oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequences of any of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modi?ed oligonucleotide and a conjugate group, n the modi?ed oligonucleotide consists of the nucleobase sequences of any one of SEQ ID NOs: 20-2295.

Certain embodiments provide a compound comprising a modi?ed oligonucleotide and a conjugate group, wherein the modi?ed oligonucleotide consists of 10 to 30 linked nucleosides complementary within tides 30-51, 63-82, 103-118, 143-159, 164-197, 206-259, 8, 554-585, 0, 736-776, 862- 887, 923-973, 978-996, 1127-1142, 1170-1195, 1317-1347, 1360-1383, 1418-1449, 1492-1507, 1524-1548, 1597-1634, 660, 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, 410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 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, 17596, 17943-17958, 18353-18368, 18636-18651, 19256-19271, 19814-19829, 20365-20380, 20994, 21566-21581, 22150-22165, 22803-22818, 29049-29064, 29554-29569, 30245- 30260, 30550-30565, 30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34407-34422, 34846-34861, 35684, 36312-36327, 36812-36827, 37504-37519, 38841-38856, 40250- 40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43306, 43500-43515, 43947-43962, 44448-44463, 45162-45177, 46025, 46476-46491, 47447-47462, 47752- 47767, 48001-48016, 48423-48438, 50195-50210, 50470-50485, 51119, 51756-51771, 52015-52030, 52245, 52588-52603, 53532-53547, or 54645-54660 of SEQ ID NO: 1, wherein said modi?ed oligonucleotide is at least 90% complementary to SEQ ID NO: 1.

Certain embodiments provide a nd comprising a modi?ed oligonucleotide and a conjugate group, wherein the modi?ed oligonucleotide consists of 10 to 30 linked sides having a nucleobase ce comprising a portion of at least 8 contiguous nucleobases 100% complementary to an equal length portion ofnucleobases 30-51, 63-82, 103-118, 143-159, 7, 206-259, 8, 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, 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, 30930, 31468-31483, 32366-32381, 32897-32912, 33187-33202, 33780-33795, 34422, 34846-34861, 35669-35684, 36312-36327, 36812-36827, 37504-37519, 38841- 38856, 40250-40265, 40706-40721, 40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43500-43515, 43947-43962, 44448-44463, 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 modi?ed oligonucleotide and a conjugate group, wherein the modi?ed oligonucleotide ts 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, 410, 9153-9168, 9554-9569, 9931-9946, 10549-10564, 10660-10679, 11020- 11793-12229, 12469-12920,13351-13415, 13717-13732, 14149-14164, 14361-14555,14965-15279, 15849-16001, 16253-16272, 16447-16545, 17130-17149, 17377-17669, 17958, 18353-18368, 18636- 18773, 19661-19918, 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, 33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37276-37295, 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, 68742, 69203- 69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 73061-73076, 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, 87262, 88063-88082, 88293-88308, 88967, 89160-89175, 89940-90255, 90528, 91073-91088, 91273-91292, 91647- 91662, 91930-92126, 92371, 93190-93443, 94111, 94374-94389, 94581-94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 97013, 97539-97554, 97800-97889, 98132-98151, 98624- 98672, 98810-99115, 99258-99273, 99503, 99791-99858, 100281-100300, 100406-100421, 100742- 100828, 101080-101103, -101320, 101788-101906, 102549-102568, 103566-103625, 104067- 104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, -107735, 108514- 108788, 109336-109505, 109849-109864, 110403-110442, 110701-110974, -111322, 112030- VV()2015/168618 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, -122013, 122180-122199, 122588-122770, 123031-123050, 123152-123167, 123671- 124055, -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, -133877, 134086- , 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, -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, -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, -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, - 165059, 165419-165575, 165856-165875, 166241-166450, 166837-166852, 167107-167122, 168004- 168019, -168823, 169062-169092, 169134-169153, -169711, 170081-170291, 170407- 170426, 170703-170814, 171021-171036, 171207-171226, 171431-171568, -171945, 172447- 172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, 178895-180539, 181514- 187644, -189904, 190109-194159, 194425-195723, 196536-196873, 197326-197961, 198145- , 198307-198381, 198715-199007, 199506-199563, 199816-199838, 200249-200635, 201258- , 202079-202094, 202382-202717, 203098-203934, 204181-204740, 205549-205915, 206412- 206764, 207510-207532, -210014, 210189-210296, 210502-210583, 210920-211418, 211836- 212223, 212606-212816, 213025-213044, -213440, -213933, 214479-214498, 214622- 214647, 214884-214951, -215508, 215932-215951, 216192-217595, 218132-218248, - 218541, -21219037, 219342-219633, 219886-220705, 221044-221059, 221483-221607, 221947- 221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, 224409-224430, 224717- , 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, -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- WO 68618 2015/028887 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, -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, -268051, 268366-269447, 270038- 271850, 271950-271969, 272631-274145, 274205-275747, 275808-276636, 276932-277064, 277391- 278380, 278932-279063, -281001, 281587-281610, 282229-283668, 290035-290474, 290924- 292550, 292860-294408, -297012, 297587-298115, 298161-298418, 298489-298738, 299082- 299187, -299669, 299723-299749, 299788-300504, or 300835-301295 of SEQ ID NO: 2, wherein said modi?ed oligonucleotide is at least 90% complementary to SEQ ID NO: 2.

Certain embodiments provide a compound comprising a modi?ed oligonucleotide and a conjugate group, wherein the modi?ed oligonucleotide consists 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, 695, 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, 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, 32382, 32827-33202, 33635-33795, 34138-34157, 34407-34422, 34845-34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36721-36827, 37032-37130, 37295, 37504-37675, 38094- 38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41342-41439, 42141-42164, 42700-42760, 43173-43537, 43765-46025, 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, 66183, 66566-66581, 66978-67080, 67251-67270, 67662-67929, 68727- 68742, 69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 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, 83416, 83884-84063, 84381-85964, 86220-86392, 86554-86655, 86901-86920, 87181-87262, 88063-88082, 88293-88308, 88605-88967, 89160-89175, 90255, 90473-90528, 91073-91088, 91273- 91292, 91662, 91930-92126, 92356-92371, 93190-93443, 93762-94111, 94389, 94581-94653, 94858, 95292-95583, 95829-95844, 96137-96503, 97013, 97554, 97800-97889, 98132- 98151, 98624-98672, 98810-99115, 99258-99273, 99478-99503, 99791-99858, 100281-100300, 100406- 100421, -100828, 101080-101103, 101242-101320, 101788-101906, -102568, 103566- 103625, 104067-104086, 104277-104858, 105255-105274, 106147-106364, 106632-106647, 106964- 107735, -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, -122199, -122770, 123031-123050, 123152- 123167, 123671-124055, 124413-124608, 125178-125197, -125616, 126357-126434, 126736- 126751, 126998-127236, 127454-127682, 128467-128482, 128813-129111, 129976-130013, 130308- 130323, 131036-131056, -131305, 131676-131691, 132171-132517, -133241, 133522- 133877, 134086-134101, 134240-134259, 134441-134617, -135030, 135431-135519, 135818- 135874, 136111-136130, 136282-136595, 136996-137152, 137372-137387, -137765, 138048- 138067, 138782-139840, 140343-140358, 140593-140701, 141116-141131, 141591-141719, 142113- , 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, -149877, 150236-150251, 150588- 151139, 151373-151659, 152201-152388, 152549-152771, 153001-153026, 153349-153364, 153831- 154112, -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, - 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, -171036, -171226, -171568, 171926- 171945, 172447-172462, 172733-172956, 173045-173756, 174122-174885, 175014-177830, - 180539, 181514-187644, -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, -203934, 204181-204740, 205549- 205915, 206412-206764, 207510-207532, 209999-210014, 210189-210296, 210502-210583, 210920- 211418, -212223, 212606-212816, -213044, 213425-213440, 213825-213933, 214479- 214498, 214622-214647, 214884-214951, 215446-215508, 215932-215951, 216192-217595, - 218248, 218526-218541, 218734-21219037, 219342-219633, -220705, 221044-221059, 221483- 221607, 221947-221962, 222569-222584, 222914-222998, 223436-223451, 223948-224122, - 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, -232848, 232884- 232899, 233210-233225, 233623-233646, 234447-234466, 234876-234918, 235258-235328, 235770- 235785, 236071-236213, -237196, 237585-237698, -237557, 244873-244897, 245319- , 245701-245780, 246152-246523, 246936-247031, 247203-247240, 247431-247450, 247644- 247659, 248223-248363, 248694-248762, 249494-249509, 250001-250020, 250693-250708, 251214- , 251601-251637, 251950-252060, -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, -266518, 266861-267131, 267375-268051, 268366- 269447, 270038-271850, -271969, -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, - 298738, 299082-299187, -299669, 299723-299749, 299788-300504, or 300835-301295 of SEQ ID NO: 2, wherein the base sequence of the modi?ed oligonucleotide is complementary to SEQ ID NO: 2.

In certain aspects, the compound comprises a modi?ed oligonucleotide consisting of 10 to 30 linked nucleosides complementary within nucleotides 155594-155613, 72107-72126, 153921-153940, 159252- , 213425-213440,153004-153019,155597-155612, 248233-248248 of SEQ ID NO: 2. n embodiments provide a compound comprising a modi?ed oligonucleotide and a ate group, wherein the modified oligonucleotide consists 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 modi?ed oligonucleotide and a ate group, wherein the modified ucleotide consists of the nucleobase ce of any one of SEQ ID NOs: -2295.

In n embodiments, a compound comprising an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a grth hormone receptor nucleic acid and 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, 698, 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, 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, a compound comprising an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or ucleotide is targeted to a growth hormone receptor nucleic acid and targets the following nucleotide regions of SEQ ID NO: 1: 30-51, 63-82, 8, 143-159, 164-197, 206-259, 361-388, 554-585, 625-700, 6, 862-887, 3, 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, 092, 2095-2110, 2306-2321, 2665-2683, 2685-2719, 2739-2770, 2859-2880, 960, 2963-2978, 3037-3052, 3205-3252, 3306-3332, 3371-3386, 3518-3542, 3975-3990, 4041-4087, 4418-4446, 45284546, 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, 33795, 34407-34422, 34846-34861, 35669- 35684, 36312-36327, 36812-36827, 37504-37519, 38856, 40265, 40706-40721, 40922-40937, 41424-41439, 41999-42014, 42481-42496, 42700-42715, 43291-43306, 43515, 43947-43962, 44448- 44463, 45162-45177, 46010-46025, 46476-46491, 47462, 47752-47767, 48001 -48016, 4843 8, 50210, 50485, 51119, 51756-51771, 52015-52030, 52230-52245, 52588-52603, 53532- 53547, or 54645-54660.

In certain embodiments, a compound comprises an nse compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to a region of a grth 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 uous bases 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, 087, 446, 4528-4546, 246, 7570-7585, 8395-8410, 9153- 9168, 9554-9569, 9931-9946,10549-10564, 11035, 11793-11808,12214-12229, 12474-12489, 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, 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, a compound comprising an antisense nd or oligonucleotide and a ate group, n the antisense compound or oligonucleotide is targeted to a growth hormone receptor nucleic acid is complementary within the following nucleotide regions of SEQ ID NO: 2: 2571- 2586, 059, 3097-3116, 3341-3695, 4024-4039, 4446-4894, 5392-5817, 6128-6265, 890, 7231- 7246, 8395-8410, 9153-9168, 9554-9569, 946,10549-10564,10660-10679, 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, 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, 34422, 34864, 35466-35485, 35669-35684, 36023-36042, 36266-36327, 36827, 37032-37130, 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, 54660, 54886-54901, 64662, 64882-65099, 65378, 65600-65615, 65988-66183, 66566-66581, 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, 94653, 94839-94858, 95292-95583, 95829-95844, 96137-96503, 96793-97013, 97539-97554, 97800-97889, 98132-98151, 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, -207532,209999-210014,210189-210296,210502-210583,210920-211418,211836-212223, 212606-212816,213025-213044,213425-213440,213825-213933,214479-214498,214622-214647, -214951,215446-215508,215932-215951,216192-217595,218132-218248,218526-218541, 218734-21219037,219342-219633,219886-220705,221044-221059,221483-221607,221947-221962, -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, -254083, 254246-254345, -254660, 254905 -254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317-257332, 257818-259305, 259500-259515, 261294-261656, -262036, 262453-262779, 263338-266518, 266861 -267131, 267375-268051, 268366-269447, -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, -298738, -299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, a nd comprising an antisense compound or oligonucleotide and a conjugate group, wherein the antisense nd or oligonucleotide is targeted to a growth hormone receptor nucleic acid targets the ing nucleotide regions of SEQ ID NO: 2: : 2571 -2586, 2867-3059, 3097-3116, 695, 40244039, 4446-4894, 5392-5817, 6128-6265, 6499-6890, 7231-7246, 410, 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, 36327, 36721- 36827, 37032-37130, 37276-37295, 37504-37675, 38094-38118, 38841-38856, 39716-40538, 40706-40937, 41164-41183, 41439, 42141-42164, 42700-42760, 43537, 43765-46025, 46476-46532, 48423- 48438, 50072-50210, 50470-50485, 50719-51234, 51797, 52015-52143, 52230-52245, 52652, 53466-54660, 54886-54901, 63751-64662, 64882-65099, 65363-65378, 65600-65615, 65988-66183, 66566- 66581, 66978-67080, 67251-67270, 67662-67929, 68742, 69203-69242, 69565-69620, 69889-70145, 70352-70584, 70925-71071, 71314-71329, 71617-71769, 72107-72241, 72584-72670, 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, 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, 99503, 99791-99858, 100281-100300, -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- ,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- ,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, -245334, 245701-245780, 246152-246523, 246936- 247031, 247203-247240, 247431-247450, 247644-247659, 248223-248363, 248694-248762, 249494- 249509, 250001-250020, -250708, -251233, 251601-251637, -252060, 252665- 252680, 252838-252863, -253166, 253594-253819, 254036-254083, 254246-254345, - 254660, -254920, 255397-255422, 255618-255633, 255992-256704, 257018-257092, 257317- 257332, 257818-259305, 259500-259515, 261294-261656, 262021-262036, 262453-262779, - 266518, 266861-267131, -268051, 268366-269447, -271850, 271950-271969, 272631- 274145, 274205-275747, 275808-276636, -277064, 277391-278380, 278932-279063, 279303- 281001, 281587-281610, -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, a compound comprises an nse compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is ed to a region of a grth 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 bases portion complementary to an equal length portion of a region recited herein. In certain embodiments, such compounds or ucleotide target the following nucleotide regions of SEQ ID NO: 2: : 2571-2586, 2867-3059, 3097-3116, 3341-3695, 039, 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, 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, 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, 79505, 80277-80292, 80575-80939, 81222, 81524-81543, 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- ,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- ,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, -220705, 221044-221059, 221483-221607, 221947- 221962, 222569-222584, 222914-222998, -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, -237196, -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, - 251637, 251950-252060, 252665-252680, 252838-252863, -253166, 253594-253819, 254036- 254083, 254246-254345, -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, -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, - 299187, 299276-299669, 299723-299749, 299788-300504, or 300835-301295.

In certain embodiments, a compound comprises an antisense nd or oligonucleotide and a conjugate group, wherein the antisense compound or ucleotide is targeted to 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 e receptor nucleic acid haVing the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 ted from nucleotides 42411001 to 42714000).

In certain ments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 2 of a grth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 145047-208139 (intron 2) of a grth hormone receptor nucleic acid haVing the nucleobase sequence of SEQ ID NO: 2 (GENBANK ion No. NT_006576.16 truncated from nucleotides 42411001 to 00).

In certain embodiments, a nd comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense nd or oligonucleotide is targeted to intron 3 of a grth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 208206-267991 (intron 3) of a grth 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 ments, a compound comprises an nse compound or oligonucleotide and a ate group, wherein the antisense compound or oligonucleotide is targeted to intron 4 of a growth hormone or nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 268122-274018 n 4) of a grth 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 n embodiments, a compound comprises an nse compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 5 of a grth hormone receptor nucleic acid. In n aspects, antisense nds or oligonucleotides target within nucleotides 274192-278925 (intron 5) of a grth hormone receptor nucleic acid haVing the nucleobase sequence of SEQ ID NO: 2 (GENBANK ion No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 6 of a grth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 279105-290308 (intron 6) of a grth hormone receptor nucleic acid haVing the nucleobase sequence of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 01 to 42714000).

In certain embodiments, a nd comprises an antisense compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 7 of a grth hormone receptor nucleic acid. In certain aspects, nse compounds or oligonucleotides target within nucleotides 290475-292530 (intron 7) of a grth hormone receptor c acid haVing the nucleobase sequence of SEQ ID NO: 2 (GENBANK ion No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an antisense compound or oligonucleotide and a conjugate group, wherein the nse compound or oligonucleotide is targeted to intron 8 of a grth hormone or nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within nucleotides 292622-297153 (intron 8) of a grth hormone receptor nucleic acid haVing the nucleobase ce of SEQ ID NO: 2 (GENBANK Accession No. NT_006576.16 truncated from nucleotides 42411001 to 42714000).

In certain embodiments, a compound comprises an nse compound or oligonucleotide and a conjugate group, wherein the antisense compound or oligonucleotide is targeted to intron 9 of a growth hormone receptor nucleic acid. In certain aspects, antisense compounds or oligonucleotides target within tides 297224-297554 n 9) of a grth 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 modi?ed internucleoside e, at least one ed sugar, and/or at least one modi?ed nucleobase.

In certain ments, any of the ing compounds or oligonucleotides comprises at least one d sugar. In certain aspects, at least one modi?ed sugar comprises a 2’-O-methoxyethyl group. In certain aspects, at least one modi?ed sugar is a bicyclic sugar, such as a 4’-CH(CH3)-O-2’ group, a - O-2’ group, or a 4’-(CH2)2-O-2’group.

In certain aspects, the modi?ed oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside e.

In certain embodiments, any of the ing nds or oligonucleotides comprises at least one modi?ed nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises: a gap segment consisting of linked deoxynucleosides; ’ wing segment consisting of linked nucleosides; and ’ 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. n embodiments provide a compound comprising a modi?ed oligonucleotide consisting of 10 to 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 ed oligonucleotide has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 918, 479 or 703, wherein the modified ucleotide comprises a gap segment consisting of ten linked deoxynucleosides; ’ wing segment consisting of five linked nucleosides; and ’ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, wherein each nucleoside of each wing segment comprises a 2’-O-methoxyethyl sugar; n each internucleoside linkage is a orothioate linkage and wherein each cytosine is a 5-methylcytosine.

In certain s, the modified oligonucleotide has a nucleobase ce comprising the sequence recited in SEQ ID NOs: 1800, 1904, 2122, 2127, or 2194, wherein the modi?ed oligonucleotide comprises of nucleosides that have either a MOE sugar modi?cation, an (S)-cEt sugar modi?cation, or a deoxy modi?cation; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In n embodiments, a compound ses a -stranded modi?ed oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides and has a nucleobase sequence comprising the sequence recited in SEQ ID NOs: 918, 479 or 703, wherein the modified ucleotide comprises a gap segment consisting of ten linked deoxynucleosides; ’ wing segment consisting of five linked nucleosides; and ’ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, n each nucleoside of each wing segment comprises a 2’-O-methoxyethyl sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises a single-stranded d oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 16 linked nucleosides and has a nucleobase sequence sing the sequence recited in SEQ ID NOs: 1800, 1904, 2122, 2127, or 2194, wherein the modi?ed oligonucleotide comprises of nucleosides that have either a MOE sugar modification, an (S)-cEt sugar modi?cation, or a deoxy modi?cation; wherein each ucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR and a conjugate group. For instance, in certain embodiments, a compound comprises ISIS 532401and a conjugate group.

In any of the ing embodiments, the compound or oligonucleotide can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a nucleic acid encoding grth hormone receptor.

In any of the foregoing embodiments, the nucleic acid encoding growth hormone or 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.

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

In certain embodiments, at least one internucleoside e of the modified ucleotide is a modified internucleoside linkage.

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

In certain embodiments, the modified oligonucleotide comprises at least 1, 2, 3, 4, 5, 6, or 7 phosphodiester internucleoside linkages.

In certain embodiments, each internucleoside linkage of the modified oligonucleotide is selected from a phosphodiester internucleoside linkage and a phosphorothioate internucleoside linkage.

In n embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate linkage.

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

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

In certain embodiments, the modified oligonucleotide comprises at least one ed sugar.

In certain embodiments, the modified sugar is a 2’ modified sugar, a BNA, or a THP.

In certain embodiments, the modified sugar is any of a 2 ’-O-methoxyethyl, 2’-O-methyl, a constrained ethyl, a LNA, or a 3 ’-?uoro-HNA.

In certain embodiments, the nd comprises at least one 2’-O-methoxyethyl nucleoside, 2’-O- methyl nucleoside, constrained ethyl nucleoside, LNA nucleoside, or 3’-?uoro-HNA nucleoside.

In n embodiments, the modified oligonucleotide comprises: a gap segment ting of 10 linked deoxynucleosides; a 5’ wing segment consisting of 5 linked nucleosides; and a 3’ wing segment consisting of 5 linked nucleosides; wherein the gap segment is positioned between the 5 ’ wing segment and the 3 ’ wing segment and n each nucleoside of each wing segment comprises a modified sugar.

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

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

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

Certain embodiments provide compounds ting of a ate group and a modified oligonucleotide according to the following formula: mCes mCes Aes mCes mCes Tds Tds Tds Gds Gds Gds Tds Gds Ads Ads Tes Aes Ges mCes Ae; wherein, A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine nucleobase, T = a thymine nucleobase, e = a 2’-O-methoxyethyl modified sugar moiety, d = a 2’-deoxy sugar moiety, and s = a phosphorothioate internucleoside linkage.

In certain ments, a nd comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc on the 5’ end. For instance, in certain embodiments, a compound comprises ISIS 532401 conjugated to GalNAc on the 5’ end. . In further embodiments, the compound has the following chemical ure comprising or consisting of ISIS 532401 with 5’-X, wherein X is a conjugate group comprising GalNAc as described herein: wherein X is a conjugate group comprising GalNAc.

In certain embodiments, a compound comprises an ISIS oligonucleotide ing GHR conjugated to GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate e. In further embodiments, a compound haVing the following chemical structure comprises or consists of ISIS 719223 with a 5’-X, wherein X is a conjugate group sing GalNAc as described herein: In certain embodiments, a compound comprises an ISIS oligonucleotide ing GHR conjugated to GalNAc, and wherein each internucleoside e of the oligonucleotide is a phosphorothioate linkage or a phosphodiester linkage. In ?thher embodiments, a compound haVing the following chemical structure comprises or ts of ISIS 719224 with a 5’-X, wherein X is a conjugate group comprising GalNAc as described herein: In certain ments, a compound comprises an ISIS oligonucleotide ing GHR conjugated to , and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage or a phosphodiester linkage. In ?thher embodiments, a compound haVing the following chemical structure comprises or consists of ISIS 766720 with a 5’-X, wherein X is a conjugate group comprising GalNAc as described herein: In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc. In further such embodiments, the compound comprises the sequence of ISIS 532401 conjugated to GalNAc, and is represented by the following al structure: wherein either R1 is —OCH2CH20CH3 (MOE)and R2 is H; or R1 and R2 together form a bridge, wherein R1 is —O- and R2 is —CH2-, -CH(CH3)-, or -CH2CH2-, and R1 and R2 are directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O-CH2CH2-; and for each pair of R3 and R4 on the same ring, independently for each ring: either R3 is selected from H and -OCH2CH20CH3 and R4 is H; or R3 and R4 together form a bridge, wherein R3 is —O-, and R4 is —CH2-, -CH(CH3)-, or -CH2CH2-and R3 and R4 are directly ted such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O- CHZCH2-; and R5 is ed from H and —CH3; and Z is selected from S' and O'.

In certain embodiments, a compound comprises an antisense oligonucleotide haVing a nucleobase sequence of any of SEQ ID NOs disclosed in nucleobase sequences of all of the aforementioned referenced SEQ ID NOs are incorporated by reference herein. For example, a compound comprises an oligonucleotide disclosed in GalNAc, and wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate linkage and has the following chemical structure: For example, a compound comprises an oligonucleotide disclosed in WO 78922 conjugated to GalNAc, and wherein each internucleoside e of the ucleotide compound is a phosphorothioate linkage or a phosphodiester linkage, and has the following chemical structure: Certain embodiments provide a ition 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 n 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 ments provide a method of treating a e 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 entioned embodiments, thereby treating the disease associated with excess growth e. In n aspects, the disease associated with excess growth hormone is acromegaly. In certain aspects, the ent reduces IGF-l levels.

Certain ments provide a method of preventing a disease associated with excess grth 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 grth hormone. In certain embodiments, the disease ated with excess grth hormone is acromegaly.

Certain embodiments provide a method of reducing grth 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 ated with excess growth hormone. In certain aspects, the disease associated with excess grth hormone is acromegaly.

In n aspects, the ing methods comprise co-administering the compound or composition and a second agent. In certain aspects, the compound or composition and the second agent are stered 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 ization to a target nucleic acid h hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the ’ 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 ucleotide 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 . In certain embodiments, an antisense compound is 12 to 22 subunits in . In certain embodiments, an antisense compound is 14 to 30 subunits in length. In n 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 n 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 . In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense nd is 18 to 20 subunits in length. In n embodiments, an antisense compound is 20 to 30 subunits in . In other words, such antisense nds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 ts, 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, 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 nse compound is 18 subunits in length. In certain ments, an antisense nd is 19 subunits in length.

In certain embodiments, an antisense compound is 20 ts 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 ts.

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 ned 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 ’ end and ’ an antisense compound having one nucleoside deleted from the 5 one nucleoside deleted from the 3 end.

When a single additional subunit is present in a ened 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 t, the added subunits may be nt to each other, for example, in an antisense compound having two ts added to the 5’ end (5’ addition), or atively to the 3’ end (3’ addition), of the nse 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’ It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or uce 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 speci?c cleavage of the target mRNA, albeit to a lesser extent than the nse ucleotides that contained no mismatches. rly, target c cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al. (J. Natl. Cancer Inst. -471, March 2001) demonstrated the ability of an oligonucleotide having 100% mentarity 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 nse 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 ments, antisense nds have chemically modi?ed subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding ty for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric nse nds typically contain at least one region modi?ed 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 d 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 ism 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 c acid is reduced. In certain embodiments, ization of the nse 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 ments, the presence of the nse compound ized with the target nucleic acid ancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of al 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 NA mechanisms; and occupancy based mechanisms. Certain antisense nds may act through more than one such mechanism and/or through additional mechanisms.

RNase H—Medialed 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 ke 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 ed nucleosides. In n 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 n such embodiments, the gap of the gapmer comprises DNA nucleosides. In n such ments, the gap of the gapmer comprises DNA-like sides. 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 ts RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the al 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 sing each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include B-D-ribonucleosides, B-D-deoxyribonucleosides, 2'- modified nucleosides (such 2’-modified nucleosides may include 2’-MOE and 2’-O-CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar ed nucleosides may include those having a constrained . 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 ments, 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 ntly 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 xynucleosides."Y" may comprise 2’-deoxynucleosides. As used herein, a gapmer bed as "X-Y-Z" has a configuration such that the gap is positioned immediately adjacent to each of the ’-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 ent. 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 sides.

In certain embodiments, the antisense compound targeted to a GHR nucleic acid has a gapmer motif in which the gap consists of6, 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 f0110WSi (J)m'(B)n'(J)p'(B)r'(A)t'(D)g'(A)V'(B)W'(J)X'(B)y'(J)Z each A is ndently a 2 ’-substituted nucleoside; each B is independently a bicyclic side; 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 ofW 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 nds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and - 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 ments, nse compounds comprise modifications that make them ularly suited for such mechanisms. 1'. 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 ed ate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5’-phosphate). In certain embodiments, such minal nucleosides stabilize the 5’-phosphorous moiety. Certain modified 5’- terminal nucleosides may be found in the art, for example in WO/2011/139702.

In certain embodiments, the 5’-nucleoside of an ssRNA compound has Formula IIc: T1_A M3 BXl J4 J5 J6 J7 (I) G T2 wherein: T1 is an optionally protected phosphorus moiety; T2 is an internucleoside linking group linking the compound of Formula IIc to the eric compound; A has one of the formulas: Q1_Q2 " Q3 Q1Q2 §_§ Q1Q2Q3 2 "a Q2: mews era; its: Q1 and Q2 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, tuted C2-C6 alkynyl 0r N(R3)(R4); Q3 is O, S, N(R5) or C(R6)(R7); each R3, R4 R5, R6 and R7 is, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl or C1-C6 alkoxy; M3 is O, S, NR14, C(R15)(R16), C(R15)(R16)C(R17)(R1g), C(R15)=C(R17), OC(R15)(R16) 01‘ OC(R15)(BX2); R14 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 , C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; R15, R16, R17 and R18 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; Bxl is a heterocyclic base moiety; or ifsz is present then sz is a heterocyclic base moiety and Bxl is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2- C6 alkynyl or tuted C2-C6 alkynyl; J4, J5, J6 and J7 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, tuted 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, NR19, C(R20)(R21), C(R20)=C(R21), C[=C(R20)(R21)] and C(=O) and the other two of J5, J6 and J7 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 l, substituted C2-C6 alkenyl, C2-C6 l or substituted C2-C6 alkynyl; each R19, R20 and R21 is, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 , C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; G is H, OH, halogen or O-[C(Rg)(R9)]n-[(C=O)m-X1]j-Z; each R8 and R9 is, independently, H, halogen, C1-C6 alkyl or substituted C1-C6 alkyl; X1 is O, S or N(E1); Z is H, n, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 l, substituted C2-C6 alkynyl or N(E2)(E3); E1, E2 and E3 are each, independently, H, C1-C6 alkyl or substituted C1-C6 alkyl; n is from 1 to about 6; m is 0 or 1; j is 0 or 1; each tuted group comprises one or more optionally protected substituent groups independently selected from halogen, 0J1, N(J1)(J2), =NJ1, SJ1, N3, CN, OC(=X2)J1, OC(=X2)N(J1)(J2) and C(=X2)N(J1)(J2); X2 is O, S or NJ3; each J1, J2 and J3 is, independently, H or C1-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, OCHZ or OC(H)(Bx2). In certain embodiments, M3 is O.

In certain embodiments, J4, J5, J6 and J7 are each H. In n embodiments, J4 forms a bridge With one 0st or J7.

In certain embodiments, A has one of the formulas: Q1_Q2 Q1_"% a?yora Q2 wherein: Q1 and Q2 are each, independently, H, n, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy or substituted C1-C6 alkoxy. In certain embodiments, Q1 and Q2 are each H. In certain embodiments, Q1 and Q2 are each, independently, H or halogen. In certain embodiments, Q1 and Q2 is H and the other of Q1 and Q2 is F, CH3 or OCH3.

In certain ments, T1 has the formula: wherein: R2, and R0 are each, independently, protected hydroxyl, protected thiol, C 1-C6 alkyl, tuted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, protected amino or substituted amino; and Rb is O or S. In certain embodiments, Rb is O and R2, and R0 are each, independently, OCH3, OCH2CH3 01‘ CH(CH3)2.

In certain embodiments, G is n, OCH3, OCHZF, OCHFZ, OCF3, 3, O(CH2)2F, OCHZCHFZ, OCH2CF3, OCHZ-CH=CH2, O(CH2)2-OCH3, 2-SCH3, O(CH2)2-OCF3, O(CH2)3- N(R10)(R11), O(CH2)2-ON(R10)(R11), O(CH2)2-O(CH2)2-N(R10)(R11), OCH2C(=O)-N(R10)(R11), OCH2C(=O)- N(R12)-(CH2)2-N(R10)(R11) or O(CH2)2-N(R12)-C(=NR13)[N(R10)(R11)] wherein R10, R11, R12 and R13 are each, independently, H or C1-C6 alkyl. In certain embodiments, G is halogen, OCH3, OCF3, 3, OCH2CF3, OCHz-CH=CH2, O(CH2)2-OCH3, O(CH2)2—O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)- (CH2)2-N(CH3)2 or OCHZ-N(H)-C(=NH)NH2. In certain embodiments, G is F, OCH3 or O(CH2)2-OCH3. In certain embodiments, G is O(CH2)2-OCH3.

In certain embodiments, the 5'—terminal nucleoside has Formula IIe: O\\ ,OH HO — O BXl In n embodiments, antisense compounds, including those particularly le for ssRNA comprise one or more type ?ed sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modi?cation motif. Such motifs may e any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain ments, the ucleotides 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 m region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region tutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar cations, wherein the sides ate between nucleotides haVing a sugar modification of a first type and nucleotides haVing a sugar modi?cation of a second type. In n 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; K 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 side of a type. For example, oligonucleotides may e one or more regions of any of the following nucleoside motifs: AABBAA; ABBABB; AABAAB; ABB; ABABAA; AABABAB; ABBAABBABABAA; BABBAABBABABAA; or ABABBAABBABABAA; wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2’-F, 2’-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides haVing such an alternating motif also comprise a modified 5 ’ terminal nucleoside, such as those of formula IIc or He.

In certain embodiments, ucleotides comprise a region having a 23 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 sides that are differently modified than A, however, B and C may have the same or different cations 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: ’- (Q)- (AB)XAy'(D)Z wherein: Q is a side comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside haVing Formula IIc or IIe; A is a first type of modifed nucleoside; B is a second type of modified nucleoside; D is a ed nucleoside comprising a modification different from the nucleoside adjacent to it.

Thus, if y is 0, then D must be ently modified than B and if y is 1, then D must be differently modified than A. In n embodiments, D differs from both A and B.

X is 5-15; Y is 0 or 1; Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif: 5’- (Q)- (A)x'(D)Z wherein: Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside haVing Formula IIc or IIe; A is a first type of modifed nucleoside; D is a modi?ed nucleoside comprising a ation 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 n embodiments, such terminal sides are not designed to hybridize to the target nucleic acid h 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 on of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

In certain embodiments, antisense compounds, ing those particularly suited for use as ssRNA comprise modified intemucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified intemucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating intemucleoside 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 ments, the oligonucleotide is uniformly linked by phosphorothioate cleoside linkages.

In certain embodiments, each intemucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each intemucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphoro- thioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate intemucleoside linkages. In n embodiments, the oligonucleotide comprises at least 8 phosphorothioate cleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate intemucleoside 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 cleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 utive phosphorothioate intemucleoside es. In certain embodiments, the oligonucleotide ses at least one block of at least one 12 consecutive phosphorothioate intemucleoside es. 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 sides 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: PS Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS ii. siRNA nds In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such ments, one or both strands may se any modi?cation motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodi?ed RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified intemucleoside linkages. l 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 ized 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 l 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 izing to a nucleic acid target resulting in loss of its normal ?mction. In some ments, the target nucleic acid is GHR. In certain embodiment, the degradation of the targeted GHR is facilitated by an activated RISC x that is formed with compositions of the invention.

Several embodiments are directed to double-stranded compositions wherein one of the strands is use?Jl in, for example, in?uencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are use?Jl for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention ize to a n 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 ating motif.

Each strand of the compositions of the present invention can be modified to ?Jlfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modi?cations in each strand permits ing the nse strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modi?ed such that it is ed 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 mentary to tide 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 ucleotides, 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 -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 tide sequence that is complementary to tide 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 tides 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 ucleotide, where the self- mentary 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 ce or a portion thereof. The -stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem sing 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 n embodiments, the double-stranded ucleotide 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 tide 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, -stranded oligonucleotides need not be limited to those molecules containing only RNA, but ?thher encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack roxy (2'-OH) containing nucleotides. In certain embodiments short ering 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 ated 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 be nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short ering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA ), 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 . In on, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post riptional gene silencing, ational tion, 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 tin ure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 6; 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 ism, including, e.g., "hairpin" or stem- loop double-stranded RNA effector molecules in which a single RNA strand with omplementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules sing 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 sed, for example, by WO 00/63364, ?led Apr. 19, 2000, or US. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single le 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 ts ly 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 t 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 ments, 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 es 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 es 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 n any single stranded regions, such as single stranded ends, or the dsRNA is a n. In other embodiments, the dsRNA has one or more single ed s 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 bed herein or those described in WO 00/63364, filed Apr. 19, 2000, or US. 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 ar 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 US.

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 es one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as ?uorine 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 en 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 oramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically d nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped s, as sed, for example, by WO 00/63364, ?led Apr. 19, 2000, or U.S. Ser. No. 60/130,377, ?led Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 64, 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 , published on April 29, 2004 as WO 2004/035765, 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 ge 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 ncy, 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 mly modi?ed or may comprise a mix of modi?cations and/or modi?ed and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide ces Nucleotide sequences that encode growth hormone receptor (GHR) targetable with the compounds provided herein include, without limitation, the following: GENBANK ion 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. 95.1 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. DB052048.1 (incorporated herein as SEQ ID NO: 5), K 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. 96.1 porated herein as SEQ ID NO: 8), GENBANK Accession No. NM_001242399.2 (incorporated herein as SEQ ID NO: 9), GENBANK Accession No. NM_001242400.2 (incorporated herein as SEQ ID NO: 10), GENBANK ion No. NM_001242401.3 (incorporated herein as SEQ ID NO: 11), GENBANK Accession No. NM_001242402.2 (incorporated herein as SEQ ID NO: 12), GENBANK Accession No. NM_001242403.2 (incorporated herein as SEQ ID NO: 13), GENBANK Accession No.

NM_001242404.2 (incorporated herein as SEQ ID NO: 14), GENBANK Accession No. NM_001242405.2 (incorporated herein as SEQ ID NO: 15), GENBANK Accession No. NM_001242406.2 (incorporated herein as SEQ ID NO: 16), GENBANK Accession No. NM_001242460.1 (incorporated herein as SEQ ID NO: 17), GENBANK Accession NM_001242461.1 (incorporated herein as SEQ ID NO: 18), GENBANK Accession No. 242462.1 (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: 2332). ization In some embodiments, hybridization occurs between an antisense compound sed herein and a GHR nucleic acid. The most common mechanism of hybridization es hydrogen bonding (e.g., Watson- Crick, Hoogsteen or reversed Hoogsteen en 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 speci?cally izable 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 bases 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 c acid.

Moreover, an antisense compound may hybridize over one or more segments of a GHR c acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop ure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds ed herein, or a specified n 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 , target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target c acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are mentary to a target region, and would therefore speci?cally hybridize, would represent 90 percent complementarity. In this e, 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 ?anked 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 er al., J. M01. 3101., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, ce identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, cs 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 n embodiments, the antisense compounds ed 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 ?Jlly complementary to a GHR nucleic acid, or a target region, or a target segment or target sequence f. As used herein, "?Jlly complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the ponding nucleobases of a target nucleic acid. For e, a 20 nucleobase nse compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 base portion ofthe target nucleic acid that is ?Jlly 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 "?Jlly 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 nd. At the same time, the entire 30 base antisense compound may or may not be ?Jlly complementary to the target ce, depending on whether the remaining 10 nucleobases of the antisense nd 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 nse compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. ) or ntiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense nds 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, , 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length se 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 c acid, or speci?ed portion thereof.

The antisense nds 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) bases 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 ments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain ments, the antisense compounds are mentary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the nse compounds are complementary to at least an 11 nucleobase portion of a target t. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In n embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In n 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 ofa 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 n thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same base pairing ability. For example, a RNA which contains uracil in place of ine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and ine pair with adenine. Shortened and lengthened versions of the nse 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 ns thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion ofthe 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 ed to an equal length n ofthe 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.

Modi?cations A nucleoside is a base-sugar combination. The nucleobase (also known as base) n of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a ate 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 h the covalent linkage of nt nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly ed to as forming the internucleoside linkages of the oligonucleotide.

Modi?cations to antisense compounds ass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. d antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced af?nity for nucleic acid target, sed stability in the presence of nucleases, or increased tory activity.

Chemically modi?ed nucleosides may also be employed to increase the g y 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.

Modi?ed Iniemucleoside Linkages The naturally occuring ucleoside e 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 es because of desirable properties such as, for example, enhanced cellular uptake, enhanced af?nity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified intemucleoside linkages include intemucleoside linkages that retain a phosphorus atom as well as intemucleoside linkages that do not have a phosphorus atom.

Representative phosphorus containing intemucleoside linkages e, but are not limited to, phosphodiesters, phosphotriesters, phosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and osphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a GHR c acid comprise one or more modi?ed intemucleoside linkages. In certain ments, the modified intemucleoside linkages are phosphorothioate linkages. In certain embodiments, each intemucleoside linkage of an antisense compound is a phosphorothioate intemucleoside linkage.

In certain embodiments, oligonucleotides comprise modi?ed cleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified intemucleoside linkage motif. In n ments, intemucleoside linkages are arranged in a gapped motif In such ments, the intemucleoside linkages in each of two wing regions are different from the cleoside linkages in the gap region. In certain embodiments the intemucleoside linkages in the wings are phosphodiester and the cleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped intemucleoside linkage motif may or may not have a gapped nucleoside motif and if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.

In n embodiments, oligonucleotides comprise a region having an alternating intemucleoside e motif. In certain ments, oligonucleotides of the present invention se a region of uniformly modified intemucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by orothioate intemucleoside linkages. In certain embodiments, the ucleotide is uniformly linked by phosphorothioate. In certain embodiments, each intemucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each intemucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one intemucleoside linkage is phosphorothioate.

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

In certain embodiments, oligonucleotides comprise one or more methylphosponate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosponate linkages. In certain embodiments, one methylphosponate linkage is in the central gap of an oligonucleotide haVing a gapmer nucleoside motif.

In n embodiments, it is desirable to e the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to e the number and position of orothioate internucleoside linkages and the number and position of phosphodiester internucleoside es to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside es may be decreased and the number of odiester internucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate ucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments it is desirable to decrease the number of phosphorothioate internucleoside linkages while retaining nuclease resistance. In certain embodiments it is desirable to increase the number of phosphodiester internucleoside linkages while retaining nuclease resistance. d Sugar Moieties Antisense nds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, sed binding ty, or some other beneficial biological property to the antisense nds. In certain ments, nucleosides comprise chemically ed ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R, R1 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 disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with ?thher substitution at the ition (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application Published on 11/22/07 wherein LNA is substituted with for example a 5'-methyl or a 5'-Vinyl group).

Examples of sides haVing modified sugar moieties include without limitation nucleosides comprising yl, 5'-methyl (R or S), 4'—S, 2'-F, 2'-OCH3, 2’-OCH2CH3, 2’-OCH2CH2F and 2'- O(CH2)20CH3 substituent groups. The substituent at the 2’ position can also be selected from allyl, amino, azido, thio, O-allyl, O-Cl-Clo alkyl, OCF3, OCHzF, 2SCH3, O(CH2)2-O-N(Rm)(Rn), O-CHz-C(=O)- N(Rm)(Rn), and O-CHZ-C(=O)-N(R1)-(CH2)2-N(Rm)(Rn), where each R1, Rm and RH is, independently, H or substituted or unsubstituted C1-C10 alkyl.

As used herein, "bicyclic nucleosides" refer to modi?ed sides comprising a bicyclic sugar moiety. es of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, antisense nds provided herein include one or more bicyclic nucleosides comprising a 4’ to 2’ bridge. Examples of such 4’ to 2’ bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as constrained ethyl or cEt) and 4'-CH(CH20CH3)- O-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-O-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'- CHZ-N(OCH3)-2' (and analogs thereof see published International Application WO/2008/150729, published December 11, 2008); 4'-CH2-O-N(CH3)-2' (see hed U.S. Patent ation US2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-O-2', n R is H, C1-C12 alkyl, or a ting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Zhou et al., J. Org. Chem, 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008).

Further reports related to bicyclic sides can also be found in published literature (see for example: Singh et al., Chem. Commun, 1998, 4, 455-456; Koshkin et al., edron, 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, ) 8362-8379; i et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; and Drum et al., Curr. Opinion Moi. Ther., 2001, 3, 239-243; U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Serial Nos. 61/026,995 and 61/097,787; Published PCT International applications 2010/036698; nucleosides can be prepared haVing one or more chemical sugar configurations including for example 0t-L-ribofuranose and B-D-ribo?iranose (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 pento?iranosyl sugar moiety wherein such bridges ndently comprises 1 or from 2 to 4 linked groups independently ed from - [C(R,)(Rb)]n—, -C(Ra)=C(Rb)-, =N-, -C(=0)-, -C(=NRa)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)x-, and —N(R,)—; wherein: x is 0, 1, or 2; nis 1,2, 3,or4; each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, aryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic l, halogen, OJ 1, NJ1J2, SJ 1, N3, COOJ 1, acyl (C(=O)- H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)—J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)- H), substituted acyl, a heterocycle radical, a substituted cycle 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-O- or —C(RaRb)-O-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'- , -C(RaRb)-N(R)-O- (CH2)3-2', 4'—CH2-O-2', 4'—(CH2)2-O-2', 4'—CH2-O-N(R)-2' and 4'-CH2-N(R)-O-2'- wherein each R is, independently, H, a protecting group or C1-C12 alkyl.

In certain embodiments, bicyclic sides are ?thher defined by ic configuration. For example, a side comprising a 4’-2’ ene-oxy bridge, may be in the Ot-L configuration or in the B- D configuration. Previously, a-L-methyleneoxy (4’-CH2-O-2’) BNA‘s have been incorporated into antisense oligonucleotides that showed antisense actiVity (Frieden et al., Nucleic Acids Research, 2003, 21 , 6365- 6372).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L-methyleneoxy (4’-CH2-O-2’) BNA BNA BNA , (B) B-D-methyleneoxy (4’-CH2-O-2’) , (C) ethyleneoxy (4’-(CH2)2-O-2’) , (D) aminooxy (4’-CH2-O-N(R)-2’) BNA, (E) oxyamino (4’-CH2-N(R)-O-2’) BNA, and (F) methyl(methyleneoxy) (4’-CH(CH3)-O-2’) BNA, (G) methylene-thio (4’-CH2-S-2’) BNA, (H) ene- amino (4’-CH2-N(R)-2’) BNA, (I) methyl yclic (4’-CH2-CH(CH3)-2’) BNA, (J) ene carbocyclic (4’-(CH2)3-2’) BNA and (K) Vinyl BNA as depicted below: MEXO E 0 BX E 0 BX E 0 BX % 31\ w "ago "a ‘o (A) R (B) (C) (D) Ego—Z/BX E O BX Ewa E O BX EQo BX gQ/BX E 0 BX "R a (1) CH3 (J) (K) CH2 wherein Bx is the base moiety and R is independently H, a protecting group, C1-C12 alkyl or C1-C12 alkoxy.

In certain ments, bicyclic nucleosides are provided having Formula I: a BX Qa\ \Q/QC Tb I wherein: Bx is a heterocyclic base moiety; -Qa-Qb-Qc- is (Rc)-CH2-, -C(=O)-N(Rc)-CH2-, -CH2-O-N(Rc)-, -CH2—N(Rc)-O- or -N(Rc)-O- Rc 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 n embodiments, bicyclic nucleosides are ed having Formula II: 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; Z2, is C1-C6 alkyl, C2-C6 l, C2-C6 alkynyl, substituted C1-C6 alkyl, tuted 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 n, oxo, hydroxyl, OJC, NJCJd, SIC, N3, OC(=X)JC, and NJeC(=X)NJch, wherein each Jo, Id and I6 is, independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJc.

In certain embodiments, bicyclic nucleosides are provided having Formula III: O BX O O I III wherein: Bx is a heterocyclic base moiety; Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive orus group, a phosphorus moiety or a covalent attachment to a support medium; Z, is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 l or substituted acyl (C(=O)-).

In n embodiments, bicyclic nucleosides are provided having Formula IV: qa qb Ta_0 BX 1?] IV 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 nt attachment to a support medium; Rd is C1-C6 alkyl, tuted C1-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, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1- C6 alkoxyl, acyl, tuted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl; In n embodiments, bicyclic nucleosides are provided having Formula V: Ta—O BX Bx is a heterocyclic base moiety; Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive orus group, a phosphorus moiety or a covalent attachment to a support medium; qa, qb, qe and qf are each, independently, hydrogen, n, C1-C12 alkyl, substituted C1-C12 alkyl, C2- C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 , substituted C1-C12 alkoxy, OJj, SJj, SOJj, SOZJJ', NJJ-Jk, N3, CN, C(=O)OJJ-, C(=O)NJij, C(=O)Jj, O-C(=O)NJJ-Jk, N(H)C(=NH)NJJ-Jk, N(H)C(=O)NJJ-J1< or N(H)C(=S)NJJ-Jk; or qe and qf together are =C(qg)(qh); qg and qh are each, independently, H, halogen, C 1-C12 alkyl or substituted C1-C12 alkyl.

The synthesis and ation of the methyleneoxy (4’-CH2-O-2 ’) BNA monomers adenine, cytosine, e, 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 W0 99/14226.

Analogs of methyleneoxy 2-O-2’) BNA and 2'-thio-BNAs, have also been prepared (Kumar er al., . Med. Chem. Lett, 1998, 8, 2219-2222). ation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel er 61]., WO 99/14226 ). Furthermore, synthesis of 2'—amino-BNA, a novel comformationally restricted high-af?nity 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 ed 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: Taio BX wherein: Bx is a heterocyclic base ; Ta and Tb are each, ndently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; each qi, qj, qk and q1 is, ndently, H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 l, substituted C1- C12 l, OJj, SJj, SOJj, SOZJJ', NJJ-Jk, N3, CN, JJ-, C(=O)NJJ-Jk, C(=O)Jj, O-C(=O)NJJ-Jk, N(H)C(=NH)NJJ-Jk, N(H)C(=O)NJJ-J1< or N(H)C(=S)NJJ-Jk; and qi and qj or q1 and qk together are =C(qg)(qh), wherein qg and qh are each, independently, H, halogen, C1-C12 alkyl 01’ substituted C1-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 er al., c Acids Research, 1997, 25(22), 4429-4443 and Albaek er al., J. Org. Chem, 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along With their oligomerization and biochemical s have also been described (Srivastava er 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 ?Jranose ring comprising a bridge ting 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 side 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 ments, such modifications include substituents selected from: a halide, including, but not limited to substituted and tituted alkoxy, substituted and tituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2’ modifications are selected from substituents including, but not limited to: O[(CH2)nO]mCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nF, O(CH2)nONH2, OCH2C(=O)N(H)CH3, and O(CH2)nON[(CH2)nCH3]2, Where n and m are from 1 to about 10. Other 2'- substituent groups can also be selected from: C1-C12 alkyl, tuted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, ryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, F, CF3, OCF3, SOCH3, SOZCH3, ONOZ, N02, N3, NHZ, 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 sides comprise a 2’-MOE side chain (Baker et al., J.

Biol. Chem, 1997, 272, 11944-12000). Such 2'—MOE tution have been described as having improved binding af?nity compared to unmodi?ed nucleosides and to other modi?ed nucleosides, such as 2’- 0- methyl, 0-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 n, Helv.

Chim. Acla, 1995, 78, 486-504; Altmann er al., Chimia, 1996, 50, 168-176; Altmann er al., Biochem. Soc.

Trans., 1996, 24, 630-637; and Altmann er al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, a "modi?ed tetrahydropyran side" or "modi?ed THP nucleoside" means a nucleoside having a mbered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modi?ed 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 n, Bioorg. Med. Chem, 2002, 10, 841-854) or ?uoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below: Ho? Ho? HOQ‘. BX "‘10; ; HO§ BX Ho‘s. BX F 5CH3 In certain embodiments, sugar surrogates are selected having Formula VII: Cll q2 Ta_O (13 C17 C14 C16 BX Tb/ R1 R2 C15 wherein ndently for each of said at least one ydropyran 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 ucleoside 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; q1, (12, q3, q4, q5, q6 and q7 are each independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 l, C2-C6 alkynyl or substituted C2-C6 l; and each of R1 and R2 is ed from hydrogen, hydroxyl, halogen, subsitituted or unsubstituted alkoxy, NJ1J2, SJ 1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, wherein X is O, S or NJ1 and each J1, J2 and J3 is, independently, H or C1-C6 alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R1 and R2 is fluoro. In certain ments, R1 is ?uoro and R2 is H; R1 is methoxy and R2 is H, and R1 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 lino sugar moieties and their use in oligomeric nds has been reported (see for example: Braasch er al., mistry, 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 ate having the following formula: ‘01:1Bx In n embodiments, morpholinos may be ed, for example by adding or ng various substituent groups from the above morpholino ure. Such sugar surrogates are referred to herein as "modifed morpholinos." Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl tuted nucleosides (see PCT International Application 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 Intemational Application the 5' on 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, eg, Srivastava er al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or more modi?ed cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in lly occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application published on April 10, 2010, Robeyns et al., J. Am. Chem. Soc, 2008, 130(6), 984; HorVath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 12980), 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; s et al., Acta Crystallographica, Section F: ural Biology and llization 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 842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modi?ed cyclohexenyl sides have Formula X.

C11 (12 ,0 C15 C17 C16 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 g the cyclohexenyl nucleoside analog to an antisense compound or one of T3 and T4 is an ucleoside 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 q1, (12, q3, q4, q5, q6, q7, qg and (19 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2- C6 alkenyl, tuted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or other sugar substituent group.

As used herein, "2’-modified" or bstituted" refers to a nucleoside comprising a sugar comprising a substituent at the 2’ position other than H or OH. ified 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-Cl-Clo alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)ZSCH3, O-(CH2)2-O- N(Rm)(Rn), or O-CHZ-C(=O)-N(Rm)(Rn), where each Rm and RH is, independently, H or tuted or unsubstituted C1-C10 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 ?uoro group at the 2’ position of the sugar ring.

As used herein, "2’-OMe" or "2’-OCH3" or "2’-O-methyl" each refers to a nucleoside comprising a sugar comprising an -OCH3 group at the 2’ position of the sugar ring.

As used herein, "MOE" or "2’-MOE" or H2CHZOCH3" or "2’-O-methoxyethyl" each refers to a side comprising a sugar comprising a -OCH2CHZOCH3 group at the2’ 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, 4). 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 US. 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; 811; 427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 873; 5,646,265; 5,670,633; 920; 5,792,847 and 6,600,032 and International ation , filed June 2, 2005 and published as WC 21371 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 al, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more sides haVing modified sugar moieties. In certain embodiments, the modified sugar moiety is 2’-MOE. In certain embodiments, the 2’-MOE modified sides are arranged in a gapmer motif In n ments, the modified sugar moiety is a bicyclic nucleoside having a (CH3)-O-2’) bridging group. In certain embodiments, the (4’- CH(CH3)-O-2’) modi?ed nucleosides are arranged throughout the Wings of a gapmer motif.

Modi?ed Nucleobases Nucleobase (or base) modi?cations or substitutions are structurally distinguishable from, yet functionally interchangeable With, naturally occurring or synthetic unmodi?ed nucleobases. Both natural and modi?ed nucleobases are capable of participating in hydrogen bonding. Such base modi?cations can impart nuclease stability, binding af?nity or some other bene?cial biological property to nse compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5- methylcytosine (5-me-C). Certain nucleobase substitutions, including ylcytosine substitutions, are particularly use?Jl for increasing the binding af?nity of an antisense compound for a target nucleic acid. For e, 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 modi?ed nucleobases include 5-hydroxymethyl cytosine, ne, hypoxanthine, 2- denine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of e and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, -propynyl (-CEC-CH3) uracil and ne and other alkynyl derivatives of pyrimidine bases, 6-azo , cytosine and thymine, il (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted es and guanines, 5-halo particularly 5-bromo, 5-tri?uoromethyl and other S- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, o-adenine, 8- nine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base es 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 S- substituted dines, 6-azapyrimidines and N—2, N—6 and 0-6 substituted purines, including 2 ropyladenine, 5-propynyluracil and 5-propynylcytosine.

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

Conjugated Antisense compounds In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure es conjugated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In n embodiments, the present sure provides methods comprising contacting a cell with a conjugated antisense compound comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure provides methods comprising ting a cell with a conjugated antisense compound comprising an antisense oligonucleotide and reducing the amount or activity of a nucleic acid transcript in a cell.

The asialoglycoprotein receptor (ASGP-R) has been described previously. See e.g., Park et al., PNAS vol. 102, No. 47, pp 17125-17129 (2005). Such ors are expressed on liver cells, ularly hepatocytes. Further, it has been shown that compounds comprising clusters of three N- acetylgalactosamine (GalNAc) ligands are capable of binding to the ASGP-R, resulting in uptake of the compound into the cell. See e.g., Khorev et al., Bioorganic and Medicinal Chemistry, 16, 9, pp 5216-5231 (May 2008). ingly, conjugates comprising such GalNAc rs have been used to facilitate uptake of certain compounds into liver cells, speci?cally hepatocytes. For example it has been shown that certain GalNAc-containing conjugates increase activity of duplex siRNA compounds in liver cells in vivo. In such instances, the GalNAc-containing conjugate is typically attached to the sense strand of the siRNA duplex.

Since the sense strand is discarded before the antisense strand ultimately hybridizes with the target nucleic acid, there is little n that the conjugate will interfere with activity. Typically, the conjugate is attached to the 3’ end of the sense strand of the siRNA. See e.g., U.S. Patent 8,106,022. Certain ate groups bed herein are more active and/or easier to size than conjugate groups previously described.

In certain embodiments of the t invention, conjugates are attached to single-stranded antisense compounds, including, but not limited to RNase H based antisense nds and antisense compounds that alter splicing of a pre-mRNA target nucleic acid. In such embodiments, the conjugate should remain attached to the antisense compound long enough to provide benefit ved uptake into cells) but then should either be cleaved, or otherwise not interfere with the subsequent steps necessary for activity, such as hybridization to a target nucleic acid and interaction with RNase H or enzymes associated with splicing or splice modulation. This balance of properties is more important in the setting of single-stranded antisense compounds than in siRNA compounds, where the conjugate may simply be attached to the sense strand.

Disclosed herein are conjugated single-stranded antisense compounds having improved potency in liver cells in vivo compared with the same antisense compound lacking the ate. Given the required e of properties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a ble moiety. As noted, without wishing to be bound by ism, it is logical that the ate should remain on the compound long enough to provide ement in uptake, but after that, it is desirable for some portion or, ideally, all of the conjugate to be cleaved, releasing the parent compound (e.g., antisense compound) in its most active form. In certain embodiments, the cleavable moiety is a cleavable nucleoside. Such embodiments take advantage of endogenous nucleases in the cell by attaching the rest of the conjugate (the cluster) to the antisense oligonucleotide through a nucleoside via one or more cleavable bonds, such as those of a phosphodiester linkage. In certain embodiments, the cluster is bound to the cleavable nucleoside through a phosphodiester linkage. In certain embodiments, the cleavable nucleoside is attached to the antisense oligonucleotide (antisense compound) by a phosphodiester linkage. In certain embodiments, the conjugate group may comprise two or three cleavable nucleosides. In such embodiments, such cleavable nucleosides are linked to one r, to the antisense compound and/or to the cluster via cleavable bonds (such as those of a phosphodiester linkage). Certain conjugates herein do not se a cleavable nucleoside and instead comprise a ble bond. It is shown that that sufficient ge of the conjugate from the oligonucleotide is provided by at least one bond that is able to cleavage in the cell (a cleavable bond).

In certain embodiments, conjugated antisense compounds are prodrugs. Such prodrugs are administered to an animal and are ultimately metabolized to a more active form. For example, conjugated antisense compounds are cleaved to remove all or part of the conjugate resulting in the active (or more active) form of the antisense compound lacking all or some of the conjugate.

In certain embodiments, conjugates are ed at the 5’ end of an oligonucleotide. Certain such 5’- conjugates are cleaved more efficiently than counterparts having a similar conjugate group attached at the 3 ’ end. In certain embodiments, improved activity may correlate with improved cleavage. In certain embodiments, oligonucleotides comprising a conjugate at the 5’ end have greater efficacy than oligonucleotides comprising a conjugate at the 3’ end (see, for e, Examples 56, 81, 83, and 84).

Further, 5’-attachment allows simpler oligonucleotide sis. Typically, oligonucleotides are sized ’ direction. To make on a solid support in the 3’ to 5 a 3 ’-conjugated oligonucleotide, typically one attaches a pre-conjugated 3’ nucleoside to the solid support and then builds the oligonucleotide as usual. However, attaching that conjugated nucleoside to the solid support adds complication to the synthesis. Further, using that ch, the conjugate is then present throughout the synthesis of the oligonucleotide and can become degraded during subsequent steps or may limit the sorts of reactions and reagents that can be used. Using the structures and techniques described herein for 5’-conjugated oligonucleotides, one can synthesize the oligonucleotide using rd automated techniques and introduce the conjugate with the final (5’-most) nucleoside or after the oligonucleotide has been d from the solid support.

In view of the art and the present disclosure, one of ordinary skill can easily make any of the conjugates and conjugated oligonucleotides herein. er, synthesis of certain such conjugates and conjugated oligonucleotides disclosed herein is easier and/or requires few steps, and is therefore less expensive than that of conjugates previously disclosed, ing advantages in manufacturing. For example, the synthesis of certain ate groups consists of fewer tic steps, resulting in sed yield, relative to conjugate groups previously described. Conjugate groups such as GalNAc3-10 in Example 46 and 3-7 in Example 48 are much simpler than previously described ates such as those described in U.S. 8,106,022 or U.S. 7,262,177 that require assembly of more chemical intermediates . Accordingly, these and other conjugates described herein have advantages over previously described compounds for use with any oligonucleotide, including single-stranded ucleotides and either strand of -stranded oligonucleotides (e.g., siRNA).

Similarly, disclosed herein are conjugate groups having only one or two GalNAc ligands. As shown, such conjugates groups improve activity of antisense compounds. Such compounds are much easier to prepare than conjugates comprising three GalNAc ligands. Conjugate groups comprising one or two GalNAc ligands may be attached to any antisense nds, including single-stranded oligonucleotides and either strand of double-stranded oligonucleotides (e.g., siRNA).

In certain embodiments, the conjugates herein do not substantially alter certain measures of tolerability. For example, it is shown herein that conjugated antisense compounds are not more immunogenic than unconjugated parent compounds. Since potency is improved, embodiments in which bility remains the same (or indeed even if tolerability worsens only slightly ed to the gains in potency) have improved properties for therapy.

In certain embodiments, conjugation allows one to alter antisense compounds in ways that have less attractive uences in the absence of conjugation. For example, in certain embodiments, replacing one or more phosphorothioate linkages of a fully phosphorothioate antisense compound with phosphodiester linkages results in improvement in some measures of tolerability. For example, in certain instances, such antisense compounds having one or more phosphodiester are less immunogenic than the same compound in which each linkage is a phosphorothioate. However, in certain instances, as shown in Example 26, that same ement of one or more phosphorothioate linkages with phosphodiester linkages also results in reduced cellular uptake and/or loss in potency. In certain ments, conjugated antisense compounds described herein tolerate such change in linkages with little or no loss in uptake and potency when compared to the conjugated full-phosphorothioate counterpart. In fact, in certain embodiments, for example, in Examples 44, 57, 59, and 86, oligonucleotides comprising a conjugate and at least one phosphodiester ucleoside linkage actually exhibit increased potency in vivo even relative to a full phosphorothioate counterpart also comprising the same conjugate. Moreover, since ation results in substantial increases in uptake/potency a small loss in that substantial gain may be acceptable to achieve improved tolerability.

Accordingly, in certain embodiments, conjugated antisense compounds se at least one odiester linkage.

In n embodiments, conjugation of antisense compounds herein results in sed delivery, uptake and activity in hepatocytes. Thus, more compound is delivered to liver . However, in certain embodiments, that increased delivery alone does not explain the entire increase in activity. In certain such embodiments, more compound enters hepatocytes. In certain embodiments, even that increased hepatocyte uptake does not explain the entire se in activity. In such ments, tive uptake of the conjugated compound is sed. For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates increase enrichment of antisense oligonucleotides in hepatocytes versus nonparenchymal cells. This enrichment is bene?cial for oligonucleotides that target genes that are expressed in hepatocytes.

In certain ments, conjugated antisense compounds herein result in reduced kidney exposure.

For example, as shown in Example 20, the concentrations of antisense oligonucleotides comprising certain embodiments of GalNAc-containing conjugates are lower in the kidney than that of antisense oligonucleotides lacking a GalNAc-containing conjugate. This has several bene?cial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding bene?t. Moreover, high concentration in kidney lly results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney s, kidney accumulation is undesired.

In certain embodiments, the present disclosure es conjugated antisense compounds represented by the formula: A—B—C—D—éE—F) q wherein A is the antisense ucleotide; B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether; each F is a ; and q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the ing group "D" branches as many times as is necessary to accommodate the number of (E-F) groups as indicated by "q". Thus, where q = 1, the a is: A—B—c—D—E—F where q = 2, the formula is: where q = 3, the formula is: A—B—C—D— E—F Where q = 4, the formula is: Where q = 5, the formula is: A—B—C—D In certain embodiments, ated antisense compounds are provided having the structure: Targeting moiety HO OH ‘ ,OH NH 0 H H 04" 0 NWN 0 Ho W (N K7,»: l N/J HO OH 0 H H 9‘ NHAC o Cleavahle moiety O OWN Branching group In certain embodiments, conjugated antisense compounds are provided having the structure: Cell targeting moiety 0/|\O eava y HO OH 0 o O (/?N HO UPC N 0 (I) \ ACHN OH O Q T th6 er Ligand 'o-P=o o \ HOOH "\ A80 o 0W0? 0 NHAC Branching group In certain embodiments, conjugated antisense compounds are provided having the structure: ASO Cleavable moiety H0—1|’=0 (/IiZ/S 2 / N ‘91" J Cell targeting moiety l'—_O%l HO O\/\/\/\ 0 (5-01 / \(IF? ACHN §?OH HO OH 0 (<2; Conjugate 0 (up? O linker HO o’éfo o I— .ACHN o OH Tether '—' Ligand HO OH o 110 NHAC Branching group In certain embodiments, conjugated antisense compounds are ed having the structure: Tether Cleavable moiety HO_1|3=O ACHN 0 HOW WYO H H l O N 3 ACHN 0 Conjugate Ho&w up linker O H O N ACHN 0 ing group Cell targeting moiety The present disclosure provides the following non-limiting numbered embodiments: Embodiment 1. The conjugated antisense compound of any of embodiments 1179 to 1 182, wherein the tether has a structure selected from among: 0 O WW1 WW1 E or H , ; wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or Embodiment 2. The conjugated antisense compound of any of embodiments 1179 to 1 182, wherein the tether has the structure: WO 68618 2015/028887 Embodiment 3. The conjugated antisense compound of any of embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has a structure selected from among: EWWAM—Ic;P-0——§OH and fWMAt/fa Embodiment 4. The conjugated antisense compound of any of embodiments 1179 to 1 182 or 1688 to 1689, wherein the linker has a structure selected from among: 35W,"AM:-EH0——E and EWM/WE wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

Embodiment 5. The conjugated antisense compound of any of embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has the structure: 0 O In ments having more than one of a particular variable (e.g., more than one ma; 99 a;n") unless otherwise ted, each such particular variable is selected independently. Thus, for a structure having more than one n, each n is selected independently, so they may or may not be the same as one another. i. Certain Cleavable Moieties In certain ments, a cleavable moiety is a cleavable bond. In n embodiments, a cleavable moiety comprises a cleavable bond. In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety attaches to the antisense oligonucleotide. In certain such embodiments, the cleavable moiety attaches directly to the cell-targeting moiety. In certain such embodiments, the cleavable moiety attaches to the conjugate linker. In certain embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a cleavable nucleoside or nucleoside analog. In certain ments, the side or nucleoside analog comprises an optionally protected heterocyclic base selected from a purine, substituted purine, dine or substituted pyrimidine. In certain ments, the cleavable moiety is a nucleoside comprising an optionally protected heterocyclic base selected from uracil, e, ne, 4-N— benzoylcytosine, 5-methylcytosine, 4-N-benzoylmethylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certain embodiments, the cleavable moiety is 2'—deoxy nucleoside that is attached to the 3' position ofthe antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2'- deoxy adenosine that is ed to the 3' position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester or phosphorothioate linkage. In certain embodiments, the cleavable moiety is 2'—deoxy adenosine that is attached to the 3' position of the antisense oligonucleotide by a phosphodiester linkage and is attached to the linker by a phosphodiester linkage.

In certain embodiments, the cleavable moiety is attached to the 3' position of the antisense ucleotide. In certain embodiments, the cleavable moiety is attached to the 5' position of the antisense oligonucleotide. In certain embodiments, the cleavable moiety is attached to a 2' position of the antisense oligonucleotide. In certain ments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester linkage. In certain embodiments, the cleavable moiety is ed to the linker by either a phosphodiester or a phosphorothioate linkage. In certain embodiments, the cleavable moiety is attached to the linker by a phosphodiester linkage. In certain embodiments, the conjugate group does not include a cleavable moiety.

In certain ments, the ble moiety is cleaved after the complex has been administered to an animal only after being alized by a targeted cell. Inside the cell the cleavable moiety is cleaved y releasing the active nse oligonucleotide. While not wanting to be bound by theory it is ed that the cleavable moiety is cleaved by one or more nucleases within the cell. In certain embodiments, the one or more nucleases cleave the phosphodiester linkage between the cleavable moiety and the linker. In certain embodiments, the cleavable moiety has a structure selected from among the following: O=F|’-OH O BX1 l 9‘3 OH O=F|’-OH O O l o a O=5) OH— I I O=F|’-OH O=F|’-OH O O O BX O BX2 O BX3 ; ;and CF 93 C? O: -OH O=P-OH O=P-OH n each of Bx, Bxl, Em, and Bx3 is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following: O=P-OH NH2 O N 0: -OH ii. Certain Linkers In certain embodiments, the conjugate groups comprise a linker. In certain such embodiments, the linker is covalently bound to the cleavable moiety. In certain such embodiments, the linker is covalently bound to the antisense oligonucleotide. In certain embodiments, the linker is covalently bound to a cell- ing . In certain embodiments, the linker further comprises a nt attachment to a solid support. In certain embodiments, the linker ?thher comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker ?thher comprises a covalent ment to a solid support and further comprises a covalent attachment to a protein binding moiety. In certain ments, the linker includes multiple positions for attachment of tethered ligands. In certain embodiments, the linker includes multiple positions for ment of tethered ligands and is not attached to a branching group. In certain embodiments, the linker r comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a linker.

In certain embodiments, the linker includes at least a linear group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether (-S-) and hydroxylamino (-O-N(H)-) groups. In certain embodiments, the linear group comprises groups selected from alkyl, amide and ether groups. In certain embodiments, the linear group comprises groups selected from alkyl and ether . In certain embodiments, the linear group comprises at least one phosphorus linking group. In certain embodiments, the linear group comprises at least one phosphodiester group. In certain embodiments, the linear group es at least one neutral linking group. In certain embodiments, the linear group is covalently attached to the cell- targeting moiety and the cleavable moiety. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety and the nse oligonucleotide. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety and a solid support. In certain embodiments, the linear group is covalently attached to the cell-targeting moiety, the cleavable moiety, a solid support and a protein binding moiety. In certain ments, the linear group includes one or more cleavable bond.

In certain embodiments, the linker includes the linear group covalently attached to a scaffold group.

In certain embodiments, the ld includes a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene , ether, thioether and ylamino groups. In certain ments, the scaffold es a branched aliphatic group comprising groups selected from alkyl, amide and ether groups. In certain embodiments, the scaffold includes at least one mono or polycyclic ring system.

In n embodiments, the scaffold includes at least two mono or polycyclic ring systems. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety and the linker. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable moiety, the linker and a solid support. In n embodiments, the linear group is covalently ed to the scaffold group and the ld group is covalently attached to the cleavable moiety, the linker and a protein binding moiety. In certain embodiments, the linear group is covalently attached to the scaffold group and the scaffold group is covalently attached to the cleavable , the linker, a protein binding moiety and a solid support. In certain embodiments, the scaffold group includes one or more cleavable bond.

In certain embodiments, the linker includes a protein binding moiety. In certain embodiments, the protein binding moiety is a lipid such as for example including but not limited to cholesterol, cholic acid, adamantane acetic acid, ne c acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)g1ycerol, geranyloxyhexyl group, cylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, ic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, n E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic ent, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, edelanol derivatized lithocholic acid), or a ic lipid. In certain embodiments, the protein g moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, Vitamin E, adamantane or 1-penta?uoropropyl.

In certain embodiments, a linker has a structure selected from among: H H w E—NH N N | 31/ VHW O,’ ('3' o R0251 [k N o o—F|>—OH N \5 "'W O HVNK - H \ ’ N H n O 3/ "3&0 ; ( n 0—; ( )n ,5 Wlw O ‘S\O I N I X 0,, O—P-OH , [3V 31A 5 OH - ; p [l] "I" MIN 0 m o, 5mm (DE-[Ni 0 EMS/s??g ; rri Eff/"Sykes; WW Héo S n O 1" N / H"Mn" W .00 H " \f ; a/Nws/SH#O ; wherein each n is, independently, from 1 to 20; and p is from 1 to 6.

In certain embodiments, a linker has a structure selected from among: wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure ed from among: 0 O O O EWnHMu/?f ; a?h?HWu/E .EAWEME ; n n O 2 wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among: QWEMN 0 o ; aiMHWu/‘t .EAWEM"? - ’ o o n n ’ wherein each L is, independently, a phosphorus linking group or a neutral linking group; and each n is, independently, from 1 to 20.

In certain embodiments, a linker has a ure selected from among: 5mm .1 S H N :5\HW HggoN "T" 00. 0’ UV0 E/%H4M3\/\Ig N N ~\;, UV O \f N ‘3 "\s/Swggo. H , /0 a: 95% 0\ .... o\,,o §4<_\ W00}:\"' 3—8 0 jOH 5mm 9&0N O \..

In certain embodiments, a linker has a ure selected from among: 0 O O O k"MRI/YL97. H L19. )1 )K/H . EkNWM {19‘ W;; O O ’ In certain embodiments, a linker has a structure ed from among: 0 O O O )k/HMRI/YHE ii)J\/N\n/\/\MH 1%. )1 )k/H . ;‘ZLL We;; O HO’ 0 In certain embodiments, a linker has a structure selected from among: " )1 o Q\}D}l O ZEVO 1W0 and EWO wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among: EKG/\Ajs; fe\O/\/\O/\/\Jr‘ ;and ;\O/\/\O/\/\O/\/\§' In certain embodiments, a linker has a structure selected from among: 0" OH g—o—g—o?ovL/oWo—g—o—é and OH 3 3 OH O?0v&0mo\;_OH 3 In n embodiments, a linker has a structure selected from among: O O 3‘ o—IFi—o—E 3 HANG (5H 13 H 6 o and In certain embodiments, the conjugate linker has the structure: In certain embodiments, the conjugate linker has the structure: O O In certain embodiments, a linker has a structure selected from among: {WWAM—Ic;P-0——§OH and fWM/xw? In certain embodiments, a linker has a ure selected from among: EN,"AVISP0—‘E 9’5me"3 OH and 0 wherein each n is independently, 0, l, 2, 3, 4, 5, 6, or 7. iii. Certain Cell-Targeting Moieties In certain embodiments, conjugate groups comprise cell-targeting moieties. Certain such cell-targeting moieties increase cellular uptake of antisense compounds. In certain embodiments, cell- targeting moieties se a branching group, one or more tether, and one or more ligand. In n embodiments, cell-targeting moieties comprise a branching group, one or more tether, one or more ligand and one or more cleavable bond. 1. Certain Branching Groups In certain embodiments, the conjugate groups se a targeting moiety comprising a branching group and at least two tethered s. In certain embodiments, the branching group attaches the conjugate linker. In certain ments, the branching group attaches the cleavable moiety. In certain embodiments, the branching group attaches the antisense oligonucleotide. In certain embodiments, the branching group is ntly attached to the linker and each of the tethered ligands. In certain embodiments, the branching group ses a ed aliphatic group comprising groups selected from alkyl, amide, disulfide, hylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises groups ed from alkyl, amide and ether groups. In certain embodiments, the branching group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system. In certain embodiments, the branching group comprises one or more cleavable bond. In certain embodiments, the conjugate group does not include a branching group.

In certain embodiments, a branching group has a structure selected from among: wherein each n is, independently, from 1 to 20; j is from 1 t0 3; and m is from 2 t0 6.

In certain embodiments, a branching group has a structure selected from among: wherein each n is, independently, from 1 to 20; and m is from 2 to 6.

In certain embodiments, a branching group has a ure selected from among: "at 0 it E 0 00 x" ha [\IIH o ’ NH #1 0 J ‘55 ; 9:: 3L /NH 9" _ a u , m 0 o o ‘5;\ H - ; éi g" u 5V3? u o 1:: 0 :3" HN\£5 tit/NH EWKNH ?‘zWkNH O O H EWLN NEEH ‘9?-W1 ; and M "1?? E/NH J‘MNH In certain embodiments, a branching group has a structure selected from among: A1 A1 "Lima—A171", ( g—A1WAFE n A nA / and |1 W W wherein each A1 is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure ed from among: wherein each A1 is independently, O, S, C=O or NH; and each n is, independently, from 1 to 20.

In n embodiments, a branching group has a structure selected from among: )n EL )n Ila "2,11— n n and "11L n n 344 5‘4 wherein A1 is O, S, C=O or NH; and each n is, independently, from 1 to 20.

In certain embodiments, a branching group has a structure selected from among: "m, Mongia In certain ments, a branching group has a structure selected from among: O "m W5, .

In certain embodiments, a ing group has a structure selected from among: In certain embodiments, conjugate groups comprise one or more tethers ntly attached to the branching group. In certain embodiments, conjugate groups comprise one or more tethers covalently ed to the linking group. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amide and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group sing one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amide, phosphodiester and polyethylene glycol groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether and amide groups in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, substituted alkyl, phosphodiester, ether and amide groups in any combination. In n embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester in any combination.

In n embodiments, each tether comprises at least one phosphorus linking group or l linking group.

In certain embodiments, the tether includes one or more cleavable bond. In certain embodiments, the tether is attached to the branching group h either an amide or an ether group. In certain embodiments, the tether is attached to the branching group through a phosphodiester group. In certain embodiments, the tether is attached to the branching group through a phosphorus linking group or neutral linking group. In certain embodiments, the tether is attached to the branching group through an ether group.

In certain ments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is ed to the ligand through an ether group. In certain ments, the tether is attached to the ligand through either an amide or an ether group. In certain embodiments, the tether is attached to the ligand through an ether group.

In certain embodiments, each tether comprises from about 8 to about 20 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises from about 10 to about 18 atoms in chain length between the ligand and the branching group. In certain embodiments, each tether group comprises about 13 atoms in chain length.

In certain embodiments, a tether has a ure selected from among: O H '31 EMHWOVbOAW? ; Emo?r; ; ii/NMEVE ; 3W ; H H H o H —N E H H . E o o N §_ N E, n HW W, n O {on m w 2 p H o o O O EMMA??kf‘ .

E E N E , m ,and ?g M/n n each n is, independently, from 1 to 20; and each p is from 1 to about 6.

In certain embodiments, a tether has a structure selected from among: E/QLMNOwOA/I‘LLL H ' N\/\/\/\ J44" ’ 511/ ‘95 1 WE, "HMO/fr" ; meA/OV‘LL ; wit/N\/\/\/\Tg;and gV\0/\€e _ In n embodiments, a tether has a structure selected from among: NH "71 wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among: 0 21 MW} g??uwt?‘: wherein L is either a phosphorus linking group or a neutral linking group; 21 is C(=O)O-R2; Z2 is H, C1-C6 alkyl or substituted C1-C6 alky; R2 is H, C1-C6 alkyl or substituted C1-C6 alky; and each m1 is, independently, from 0 to 20 wherein at least one m1 is greater than 0 for each .

In certain embodiments, a tether has a structure selected from among: In n embodiments, a tether has a structure selected from among: COOH OH ‘71,, me—(ili—om OH O—(IIDP——O—(—%/‘1:L"‘1 H wherein Z2 is H or CH3; and each m1 is, independently, from 0 to 20 wherein at least one m1 is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among: 0 O Wm?f or warm; , ; wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

In certain embodiments, a tether comprises a phosphorus linking group. In certain embodiments, a tether does not comprise any amide bonds. In n embodiments, a tether comprises a orus linking group and does not comprise any amide bonds. 3. Certain Ligands In certain ments, the present disclosure provides s n each ligand is covalently attached to a tether. In certain embodiments, each ligand is selected to have an affinity for at least one type of receptor on a target cell. In certain embodiments, ligands are selected that have an af?nity for at least one type of receptor on the surface of a mammalian liver cell. In certain ments, ligands are selected that have an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In n embodiments, each ligand is, independently selected from galactose, N—acetyl galactoseamine, mannose, glucose, glucosamone and fucose. In n embodiments, each ligand is N—acetyl galactoseamine (GalNAc). In n embodiments, the targeting moiety comprises 2 to 6 ligands. In certain embodiments, the targeting moiety comprises 3 ligands. In certain embodiments, the targeting moiety comprises 3 N—acetyl oseamine ligands.

In certain embodiments, the ligand is a carbohydrate, carbohydrate derivative, modified carbohydrate, multivalent carbohydrate cluster, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain embodiments, the ligand is an amino sugar or a thio sugar. For example, amino sugars may be selected from any number of compounds known in the art, for example glucosamine, sialic acid, a-D- galactosamine, N—Acetylgalactosamine, amidodeoxy-D-galactopyranose (GalNAc), 2-Amino0- [(R)carboxyethyl]deoxy-B-D-glucopyranose (B-muramic acid), y—2-methylamino-L- glucopyranose, 4,6-Dideoxy—4-formamido-2,3-dimethyl-D-mannopyranose, 2-Deoxy-2 -sulfoamino-D- glucopyranose and N—sulfo-D-glucosamine, and N-Glycoloyl—a-neuraminic acid. For example, thio sugars may be selected from the group consisting of 5-Thio-B-D-glucopyranose, Methyl 2,3,4-triacetylthio O-trityl-a-D-glucopyranoside, 4-Thio-B-D-galactopyranose, and ethyl 3,4,6,7-tetraacetyldeoxy-1,5- dithio-a-D-gluco-heptopyranoside.

In certain embodiments, "GalNAc" or "Gal-NAc" refers to 2-(Acetylamino)deoxy—D- galactopyranose, commonly referred to in the literature as N—acetyl galactosamine. In certain embodiments, "N—acetyl osamine" refers to 2-(Acetylamino)deoxy-D-galactopyranose. In certain embodiments, "GalNAc" or "Gal-NAc" refers to 2-(Acetylamino)deoxy-D-galactopyranose. In certain embodiments, "GalNAc" or "Gal-NAc" refers to 2-(Acetylamino)deoxy-D-galactopyranose, which includes both the B- form: 2-(Acetylamino)—2-deoxy-B-D-galactopyranose and : 2-(Acetylamino)deoxy-D- galactopyranose. In certain embodiments, both the B-form: 2-(Acetylamino)deoxy-B-D-galactopyranose and a-form: 2-(Acetylamino)deoxy-D-galactopyranose may be used interchangeably. Accordingly, in structures in which one form is depicted, these ures are ed to include the other form as well. For e, where the structure for an a-form: 2-(Acetylamino)deoxy-D-galactopyranose is shown, this structure is intended to include the other form as well. In n embodiments, In certain preferred embodiments, the B-form 2-(Acetylamino)deoxy-D-galactopyranose is the preferred embodiment. 0 OH HO o ["1] k HO N 2-(Acetylamino)deoxy-D-galactopyranose HO o—§ 2-(Acetylamino)-2 -deoxy-B-D-galactopyranose 2-(Acetylamino)-2 -deoxy-a-D-galactopyranose In certain embodiments one or more ligand has a structure selected from among: Ho?o OH HO 0 0—; Ho OH R and R1 1 R1 0 o R1 wherein each R1 is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected from among: HOOH OH HO HO OH OH o Ho?E;§::§5x/OO O ’0 —o HO \ \ "H ; #3 HO o - HO \ HO , NHAc OH : Hf ’ HO OH OH .0 F; In certain embodiments one or more ligand has a ure selected from among: Hole/Owed NHAc _ i. Certain Conjugates In certain embodiments, ate groups comprise the structural features above. In certain such embodiments, conjugate groups have the following structure: Ho OH o H o HO W"NWNn ) NHAc O n Ho OH O H H H 0 "W" o H W Wc o o )n Ho HN H o o N HO W " NHAc o wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the following ure: Ho OH O H o o HN\/\/N Ho \/\/\H/ NHAc 0 HO OH o O H H H NHAc N\\//A\V//N\WT/\\V//o\\\;E}—~N_1o o HO HN H o o N Ho \\//\\//\\n// In certain such embodiments, conjugate groups have the following structure: HO OH m0O H H O o=F|>—OH NV" 9" c'> n ) = NHAc o BX O n HO OH o o H H 5‘: N N o O N I HO WWW H n O—FI’ZX n n n NHAc o 0 OH o n HO OH o HN HO WHWn NHAc wherein each n is, ndently, from 1 to 20; Z is H or a linked solid t; Q is an antisense compound; Xis Oor S; and Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the following structure: HO OH _ O H H O o=F|>—OH 0 NVN 9H Cl) HO O—FI’ZX NHAc o 0 OH HO OH o H HN O N\/ In certain such embodiments, conjugate groups have the following structure: HO OH _ N I NHAc O o O o H H 5‘: o N N o N 7 o—F'>—o In n such embodiments, conjugate groups have the following structure: HO 0 /(IP?\ ACHN ()6?9 ) NH HoOH n «N 2 HO® WO/llko?hoO O O O O " Q’|| 0 N?NN/J ACHN OH OH o 0‘ Ho OH 9 ? )n HO—ll3=0 O O 1:)\O (I) H0 11 OH |_ In certain such embodiments, conjugate groups have the following structure: HO O\/\/\/\ /\(II? HOOH ()6?0\Lg HOWOWEgmoqko—EHP—:CrN«TI/?Na o 9 o "0 WWW/5X 9 0-P=0 ACHN OH 6H In n such embodiments, conjugate groups have the following structure: H0—1|3=0 HO OH 0 0/ |\O OH 0 HOOH O O O 9 0—1320 HO ONO/lixomo I ACHN 0H OH In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the following structure: O H o H o ('3 HO \/\/\n/N\/\/ K O=F|"O ACHN o H H O O HO \/\/\n/ gim 8 N AcHN o o o In certain such ments, conjugate groups have the following structure: Homo 9 WO’I.)\ In certain such embodiments, conjugate groups have the following structure: HO OH In certain such embodiments, conjugate groups have the following structure: HoOH o O N AcHN $1 Hog/Oo o 4" M "W0 ; AcHN o O /£fj O N HO O HoOH o HO 4 "J1 O L— JOJ\/\/loL O HO O/lir? ? ?/w?:\O—P—§II AcHN o 0 /£fj Ho 4N O In certain such embodiments, conjugate groups have the following structure: In certain such embodiments, conjugate groups have the following structure: In certain such embodiments, conjugate groups have the following structure: OH OH O NH In certain such embodiments, conjugate groups have the following structure: AcHN H o o In certain such ments, conjugate groups have the following structure: HoOH ‘ O 0%" HO 3 O O AcHN | Ozf—OH HO$WOAWo N AcHN | O=T-OH 0 N H0 0m20 E In certain such embodiments, conjugate groups have the following structure: o N HO O/Wi:gl?gio AcHN | CIT-OH Ho?om'" AcHN C|J O=T-OH HO§WOW 20—340 N AcHN ('3 In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: wherein X is a substituted or unsubstituted tether of six to eleven consecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate group has the ing structure: n X is a substituted or unsubstituted tether of ten consecutively bonded atoms.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HO%O\Xo AcHN \ HOOH O O o’X—ngN/i HOOH x/ wherein X is a substituted or unsubstituted tether of four to eleven consecutively bonded atoms and n the tether comprises exactly one amide bond.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOWOWo AcHN \NJ5:Z/o HoOH H Hog/Oo ,O / __ 12 Y\" Z M AcHN H ,N z\ HoOH Y \W O O 0/ o wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a idine, a disulfide, or a thioether.

In n such embodiments, the cell-targeting moiety of the conjugate group has the following ure: HOWOWo AcHN \NJ5:Z/o HoOH H Hog/Oo ,o Y\" / __ 34 Z M AcHN H N z\ HoOH O Y' \W O 0/ o wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl group, or a group comprising exactly one ether or exactly two ethers, an amide, an amine, a piperidine, a phosphate, a phosphodiester, or a orothioate, In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOWOWo AcHN \NJ5:Z/o HoOH H Hog/Oo ,o / __ a Y\M Z M AcHN H IN 2\ HoOH Y \W O O 0/ o wherein Y and Z are independently selected from a C1-C12 substituted or unsubstituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: wherein m and n are independently ed from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

In certain such embodiments, the cell-targeting moiety of the ate group has the following structure: HOOH O Am N/[LtfoH AcHN O " wherein m is 4, 5, 6, 7, or 8, and n is 1, 2, 3, or 4.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: HOOH \X O X HO N371 AcHN H HO o o/X wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and n X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure: wherein X is a substituted or unsubstituted tether of eight consecutively bonded atoms, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the ate group has the following structure: wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein the tether comprises exactly one amide bond, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the ate group has the following structure: HOOH \X O X HO Nfa AcHN H HO o o/X wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms and wherein the tether consists of an amide bond and a substituted or tituted C2-C11 alkyl group.

In certain embodiments, the cell-targeting moiety of the ate group has the following structure: HOOH H O o—YrN O HoOH O O 0/)"\N A HO H g O O—Y/M O wherein Y is ed from a C1-C12 substituted or unsubstituted alkyl, alkenyl, or alkynyl group, or a group comprising an ether, a ketone, an amide, an ester, a carbamate, an amine, a piperidine, a phosphate, a phosphodiester, a phosphorothioate, a triazole, a pyrrolidine, a disulfide, or a thioether.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: O o—YrN O HoOH O O o/JK\N A HO H g O o——Y/11 O wherein Y is selected from a C1-C12 substituted or unsubstituted alkyl group, or a group comprising an ether, an amine, a piperidine, a phosphate, a phosphodiester, or a orothioate.

In certain such embodiments, the argeting moiety of the conjugate group has the following structure: O O—Y—‘N O HoOH O O 0/)"\N 2 HO H g C) o——Y"N O AcHN wherein Y is selected from a C1-C12 substituted or tituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: O o N O HO ?" HoOH O "M "2 Homo n" O Whereinn is 1, 2, 3, 4, 5, 6, 7, 8, 1, or 12.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure: O o N O HoOH O O o6‘N 32 HO nH N O CHEN O HO H wherein n is 4, 5, 6, 7, or 8.

In certain embodiments, conjugates do not comprise a pyrrolidine. a Certain c0n°u ated antisense com ounds In n embodiments, the conjugates are bound to a nucleoside of the nse oligonucleotide at the 2’, 3’, of 5’ position of the nucleoside. In certain embodiments, a conjugated antisense compound has the following structure: A—B—c—D—eE—F) wherein A is the antisense oligonucleotide; B is the cleavable moiety C is the conjugate linker D is the branching group each E is a tether; each F is a ; and q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A—c—D+E—F) wherein A is the antisense oligonucleotide; C is the conjugate linker D is the ing group each E is a tether; each F is a ligand; and q is an integer between 1 and 5.

In certain such embodiments, the ate linker comprises at least one cleavable bond.

In certain such ments, the branching group comprises at least one cleavable bond.

In n embodiments each tether ses at least one cleavable bond.

In certain embodiments, the conjugates are bound to a nucleoside of the antisense oligonucleotide at the 2’, 3 ’, of 5 ’ position of the nucleoside.

In certain embodiments, a conjugated antisense compound has the following structure: A—B—chE—a wherein A is the antisense oligonucleotide; B is the cleavable moiety C is the conjugate linker each E is a tether; each F is a ligand; and q is an integer between 1 and 5.

In certain embodiments, the conjugates are bound to a nucleoside of the nse oligonucleotide at the 2’, 3 ’, of 5 ’ position of the nucleoside. In certain embodiments, a conjugated nse compound has the following structure: A—c+E—F> wherein A is the antisense oligonucleotide; C is the conjugate linker each E is a tether; each F is a ligand; and q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense compound has the following structure: A—B—o+E—F> wherein A is the nse oligonucleotide; B is the cleavable moiety D is the branching group each E is a tether; each F is a ; and q is an integer between 1 and 5.

In certain embodiments, a conjugated antisense nd has the following structure: A D+E—F) wherein A is the antisense oligonucleotide; D is the branching group each E is a tether; each F is a ligand; and q is an integer between 1 and 5.

In certain such embodiments, the conjugate linker comprises at least one cleavable bond.

In certain embodiments each tether comprises at least one cleavable bond.

In n embodiments, a conjugated antisense compound has a structure ed from among the following: Targeting moiety HO OH %W O:P*OH NH2 HN o N K107"f / HOOH s5 NHAC JAN; Linker Cleavable moiety Ligand Tethe :eeww Branching group NHAC In certain embodiments, a ated antisense compound has a structure selected from among the following: Cell targeting moiety HO OH lo& ki— Cleavable moiety (?NNN HOOH Wa HO O/P\O N ACHN Q Tether Ligand "OP:O o l HO OH ,P\ O\/\/\/\O ASO NHAC Branching group In certain embodiments, a conjugated antisense compound has a structure selected from among the following: Cleavable m01ety Cell targeting moiety O}IOI ACHN O 0 1 O HO OH O ())3 Conjugate O linker O ('13? O—l|3_O HO o’éfo o l— O OH NHAC Branching group In certain embodiments, the conjugated antisense compound has the following structure: In n embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc on the 5 ’ end. For instance, in certain embodiments, a compound comprises ISIS 532401 conjugated to GalNAc on the ’ end. . In ?thher embodiments, the compound has the following chemical structure comprising or ting of ISIS 532401 with 5’-X, wherein X is a conjugate group comprising GalNAc as described herein: wherein X is a conjugate group comprising GalNAc.

In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR ated to GalNAc, and wherein each intemucleoside linkage of the oligonucleotide com is a phosphorothioate linkage.

In ?thher ments, the nd comprises the sequence of ISIS 532401 conjugated to GalNAc, and wherein each intemucleoside linkage of the oligonucleotide com is a phosphorothioate linkage. In such embodiments, the chemical structure is as follows: In certain embodiments, a compound comprises an ISIS oligonucleotide targeting GHR conjugated to GalNAc, and wherein each intemucleoside e of the oligonucleotide com is a phosphorothioate linkage or a phosphodiester linkage. In further embodiments, the compound comprises the sequence of ISIS 532401 conjugated to GalNAc, and wherein each intemucleoside linkage of the oligonucleotide com is a phosphorothioate linkage or a phosphodiester e. In such embodiments, the chemical structure is as follows: In certain embodiments, a nd comprises an ISIS oligonucleotide targeting GHR ated to GalNAc. In further such embodiments, the compound comprises the sequence of ISIS 532401 conjugated to GalNAc, and is represented by the following chemical structure: Wherein either R1 is —OCH2CH20CH3 (MOE)and R2 is H; or R1 and R2 together form a bridge, wherein R1 is —O- and R2 is —CH2-, -CH(CH3)-, or -CH2CH2-, and R1 and R2 are directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O-CH2CH2-; And for each pair of R3 and R4 on the same ring, independently for each ring: either R3 is ed from H and -OCH2CH20CH3 and R4 is H; or R3 and R4 er form a bridge, wherein R3 is —O-, and R4 is — CH2-, -CH(CH3)-, or -CH2CH2-and R3 and R4 are directly connected such that the resulting bridge is selected from: -O-CH2-, -O-CH(CH3)-, and —O-CH2CH2-; And R5 is selected from H and —CH3; And Z is selected from S' and O'.

Representative United States patents, United States patent application publications, and international patent application publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, ble moieties as well as other modi?cations include without limitation, US 5,994,517, US 6,300,319, US 720, US 182, US 7,262,177, US 7,491,805, US 8,106,022, US 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and Representative publications that teach the preparation of certain of the above noted conjugates, conjugated antisense compounds, tethers, linkers, branching groups, ligands, cleavable moieties as well as other modifications include t limitation, BIESSEN et al., "The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agen " J. Med. Chem. (1995) 38:1846-1852, BIESSEN et al., "Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor" J. Med. Chem. (1995) 38:1538-1546, LEE et al., "New and more efficient multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes" Bioorganic & Medicinal try (2011) 19:2494-2500, RENSEN et al., "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo" J. Biol. Chem. (2001) 276(40):37577-37584, RENSEN et al., "Design and Synthesis of Novel N—Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor" J. Med. Chem. (2004) 8-5808, EGT et al., "Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of mes to the c Asialoglycoprotein Receptor" J. Med. Chem. (1999) 42:609-618, and Valentijn et al., "Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the glycoprotein Receptor" Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated by reference herein in its entirety.

In certain embodiments, conjugated antisense compounds comprise an RNase H based oligonucleotide (such as a gapmer) or a splice modulating oligonucleotide (such as a ?Jlly modified oligonucleotide) and any conjugate group sing at least one, two, or three GalNAc . In certain embodiments a ated antisense compound comprises any ate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; ly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., edron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., edron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Btoconjug Chem, 1997, 8, 762-765; Kato et al., Glycobtol, 2001, 11, 821-829; Rensen et al., JBz'ol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38- 43; Westerlind et al., Glycoconj J, 2004, 21, 1; Lee et al., Bioorg Med Chem Lett, 2006, 16(19), 5132- 5135; Maierhofer et al., Bioorg Med Chem, 2007, 15, 7661-7676; KhoreV et al., Bioorg Med Chem, 2008, 16, 5216-5231; Lee et al., Bioorg Med Chem, 2011, 19, 2494-2500; Kornilova et al., Analyt Biochem, 2012, 425, 43-46; Pujol et al., Angew Chemie Int Ed Engl, 2012, 51, 7445-7448; Biessen et al., JMed Chem, 1995, 38, 1846-1852; Sliedregt et al., JMed Chem, 1999, 42, 609-618; Rensen et al., JMed Chem, 2004, 47, 5798- 5808; Rensen et al., Arierioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., JAm Chem Soc, 2004, 126, 14013-14022; Lee et al., J Org Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Leii, 2010, 12, 5410-5413; Manoharan, Aniisense Nucleic Acid Dmg Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 281; International applications WOl998/013381; WO2011/038356; /046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; /075035; WO2012/083185; WO2012/083046; /082607; WO2009/134487; WO2010/144740; /148013; WOl997/020563; WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; US. Patents 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812; 6,525,031; 6,660,720; 509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 491; 8,404,862; 7,851,615; Published US. Patent Application Publications US2011/0097264; US2011/0097265; /0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183 886; /0206869; US2011/0269814; /0286973; /0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; /0178512; /0236968; /0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.

In vitro testing ofaniisense oligonucleoiides bed herein are methods for treatment of cells with antisense oligonucleotides, which can be modi?ed appropriately for treatment with other antisense compounds.

Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% con?uency in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, CA). Antisense ucleotides may be mixed with LIPOFECTIN in EM 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of nse 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 nse oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

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

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

Cells are treated with antisense ucleotides by e methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or n levels of target nucleic acids are measured by methods known in the art and bed herein. In general, when ents are performed in multiple replicates, the data are presented as the average of the replicate ents.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the l 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, ad, CA) according to the manufacturer’s recommended protocols. n Indications Certain embodiments provided herein relate to methods of treating, preventing, or rating a disease associated with excess grth 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 speci?c tor, such as an antisense nd or oligonucleotide targeted to GHR. Acromegaly is a disease associated with excess grth e (GH). In over 90 percent of acromegaly patients, the overproduction of grth es is caused by a benign tumor of the ary gland, called an adenoma, which produces excess growth e and compresses surrounding brain tissues.

Expansion of the adenoma can cause headaches and visual impairment that often accompany acromegaly. In some instances, galy 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 e 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, galy often goes undiagnosed until changes in external features become able, such as changes in the face.

Acromegaly is often associated with gigantism.

Features of acromegaly include soft tissue swelling resulting in ement 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 nd or composition targeted to GHR ed 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.

It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is ndent of any modification to a sugar , an intemucleoside linkage, or a nucleobase. As such, antisense nds de?ned by a SEQ ID NO may comprise, independently, one or more modi?cations to a sugar moiety, an intemucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.

The following examples rate certain embodiments of the t disclosure and are not limiting.

Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a ular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are ered suitable, unless ise indicated.

Example 1: General Method for the Preparation of Phosphoramidites, Compounds 1, 1a and 2 Compounds 1, 1a and 2 were prepared as per the procedures well known in the art as described in the specification herein (see Seth et al., Bioorg. Med. Chem., 2011, 21 (4), 1122-1125, J. Org. Chem., 2010, 75(5), 1569-1581, Nucleic Acids Symposium , 2008, 52(1), 553-554); and also see published PCT International Applications ( 2009/006478, and O O Bx DMTOAUBX DMTOAgBx T C $‘ "I /\/OM6 H3C \¢‘\ \: o‘ o O | o o NC\/\O/P\ NC\/\0/P\ NC\/\ /P\ N(1Pr)2 N(1Pr)2 N(1Pr)2 1 1 a 2 Bx is a heterocyclic base; Example 2: Preparation of Compound 7 nds 3 (2-acetamido-1,3,4,6-tetra-0—acetyl-2 -deoxy-B-Dgalactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared ing to published procedures (Weber et al., J. Med. Chem, 1991, 34, 2692).

AcOOAc AcOOAc o o ACO W o , 50 °C HO 0/\© 5 AcO OAc —» N \o CICHzCHzCI AcHN TMSOTf, DCE 3 (93%) 4 (66%) AcOOAc AcO 0Ac ACOWW VG O o o —>AcO MeOH ij A HNc 0 AcHN o (95%) 6 7 Example 3: Preparation of Compound 11 nds 8 and 9 are commercially available.

\H/\\ NC/\\ O 0 HO O Eco WON 9 HCI, EtOH W0 NH2 HO NH —>2 NC/\/O NH —>2 aq. KOH, Reflux, rt, 0 EtO O HO 1,4-dioxane, o (56%) 8 (40%) NC\) 10 ON 11 Example 4: Preparation of Compound 18 Compound 11 was ed as per the procedures illustrated in Example 3. Compound 14 is commercially available. Compound 17 was prepared using similar procedures reported by Rensen er al., J.

Med. Chem, 2004, 47, 5798-5808. \n/\\ O o benzylchloroformate, O ’m'b EtO LiOH, H20 Dioxane, Na2CO3 0 A N O/\© —> H Dioxane 0 EC (86%) 0 EC 0 (91%) ON 11 O HO o m 0 j: *O/IKHMNHZ 14 How/O N O H ’?/Og/NWHTVOZQ'HJLOAQ HBTU, DIEA, DMF (69%) 15 O 13 ’>\OiH/\AH< 0 A00OAc H2N H \/\/\n/\\N MWWO0 OH 0 O ACHN O O CF3COOH HBTU, DIEA, HOBt —>H2N\/\/N\n/\/O\%_NJJ\O/\©H H 95% O O DMF 16 (64%) H2N H o AcOOAc O H o H o AcO WNW K ACHN O AcOOAc H O O H O N N AcO V\/\n/ \/\/ \n/\/O H AcHN o o o AcOOAc HN’CO Example 5: ation of Compound 23 Compounds 19 and 21 are commercially available. 1. H3co)L(‘/):LOH 21 o?/Ob 1. TBDMSCI TBDMSO HBTU,D|EA DMF Imidazode rt(95%) DMF, rt(65%) HO —> —> 2. Pd/C, H2, MeOH, rt 2. TEA.3HF TEA THF -,,OH 87% OTBDMS (72%) HO 0 o 1. DMTCI pyr ) NMOH N OCH3 —» 2. LiOH, Dioxane (97%) : 22 Example 6: Preparation of Compound 24 Compounds 18 and 23 were prepared as per the procedures illustrated in Examples 4 and 5.

AcOOAc ACO \/\/ ACHN \/V\(i)l/ 1. H2, Pd/C, MeOH (93%) AcOOAc K 2. HBTU, DIEA, DMF (76%) O H H O i A00 i O VV\H/ wN 0%") O O (QDMT H .= ACHN 73" 0/\© - o HOJLM’K O a N AcOOAc OH N\/\/H O AcO OM O 18 AcOOAc O H H AcOOAc \/\/\n/N\/\/N\:OO ODMT O O o H H AcO W \/\/N\n/\/ %HO N ACHN O O O HN/‘C OH AcOOAc H O o N\/\/ AcO OM O 24 Example 7: Preparation of Compound 25 Compound 24 was prepared as per the procedures illustrated in e 6.

AcOOAc AcO$Q/O\/\/\n/NO H H WN\EO ACHN o AcOOAc O /ODMT o H 0 O OWNWNwog—HWNQ 1. Succinic anhydride, DMAP, DCE AcHN O O O 2. DMF, HBTU, r)2, PS-SS AcOOAc < HN N\/\/H O A00 o\/\/\n/ AcOOAc O H o H 0 A00 \/\/\n/N\/\/ x: ACHN o AcOOAc o /ODMT H o o ' o H o N O AcO W \/\/N\n/\/ g—HO NJLH’IL8 N NH AcHN o o O ( o AcOOAc HN H O o N\/\/ A00 o\/\/\n/ Example 8: Preparation of Compound 26 Compound 24 is prepared as per the procedures illustrated in Example 6.

AcOOAc O H o H 0 A00 /N\/\/ K ACHN o AcOOAc o /ODMT O O o H H 1" AcO V\/\n/ wNwog—MMNQ Phosphitylation ACHN O O O AcOOAc H "ll/:0 o N\/\/ A00 O\/\/\n/ AcOOAc O H o H 0 A00 \/\/\n/N\/\/ K ACHN o AcOOAc /ODMT O O o H H 1" AcO V\/\n/ \/\/N\n/\/o AcHN O o O "MN: "ll/<3 O NC\/\ O/P‘N IPr( )2 AcOOAc o N\/\/H A00 O\/\/\n/ 0 OR Example 9: General preparation of conjugated ASOs comprising GalNAc3-1 at the 3’ terminus, Compound29 AcOOAc O H o H \/\/\n/N\/\/ K0 A00 ACHN O AcOOAc H O O i O H o O AcO W \/\/N\n/\/ %H0 NWa N NH ACHN O O O 45 o 1. DCA, DCM AcOOAc HN H O 2. DCI, NMI, ACN O O\/\/\n/N Phosphoramidite DNA/RNA 0 ng block 1 ted s nthesizer 3. C ' 4 t-EEISISDJH O BX - DMTO/\<_7’ AcOOAc O H H O I /\/CN ACO \/\/\n/ \/\/ K O=F|"O ACHN O AcOOAc o x0 o H H 0 A00 WW 0%N a NQ NH AcHN o :1)" O 1. DCA, DCM O 2. DCI, NMI, ACN ACOOAc HN H O Phosphoramidite DNA/RNA 0 ng block 1a automated synthesizer A00 O\/\/\n/N\/\/ 3. Capping 27 4. t-BuOOH DMTOWBX (If beMe O:P_O/\/CN AcOOAc H O". o HWN o | AcO Mr O=F|’-O ACHN O AcOOAc ‘2 o x0 O 1 o H H 0 A00 V\/\H/ \/\/ NH H a NQ ACHN 73wog—N O 0 1. DCA, DCM AcOOAc HN4: H\/\/ O 2. DCI, NMI, ACN Phosphoramidite DNA/RNA 0 building blocks automated synthesize 28 3. Capping 4. xanthane hydride or t-BuOOH . Et3N/CH3CN (1:1) 6. Aqueous NHq (cleavage) OLIGO X=R-O' Bx=Heterocyclic base 0". 'beMe X=OorS | O=F<-O o H H o ('3 HO \/\/\n/ W K O=F|"O ACHN O o / O H O H O HO \/\/\n/ WNWO%MMN\Q AcHN O O O Wherein the protected GalNAc3-1 has the structure: o H H o ('3 HO \/\/\n/ \/\/ K O=|:I"O ACHN o o ,0 O O o H H HO W\n/ \/\/N\H/\/O AcHN o "MN: o o HOOH < H o m "J" The GalNAc3 r portion of the conjugate group GalNAc3-1 (GalNAc3-1 a) can be combined with any cleavable moiety to e a variety of conjugate groups. Wherein GalNAc3-1a has the formula: o H H o ACHN O o P o H H HO \/\/\n/ \/\/N\H/\/ %H0 NW1 3 N ACHN O O O The solid support bound protected GalNAcg-l, Compound 25, was ed as per the procedures rated in Example 7. Oligomeric Compound 29 comprising GalNAc3-1 at the 3’ terminus was prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy er al., Angew. Chem. Int. Ed, 2006, 45, 3623-3 627). Phosphoramidite building blocks, Compounds 1 and 1a were prepared as per the procedures rated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare oligomeric compounds haVing a predetermined sequence and composition. The order and quantity of oramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described .

Such gapped oligomeric compounds can have ermined composition and base sequence as dictated by any given target.

Example 10: General preparation conjugated ASOs comprising 3-l at the 5’ terminus, Compound 34 ODMT 1. Capping (A020, NMI, pyr) 1. DCAD DCM -OLIGO 2. PADS or t-BuOOH \_ 3. DCA DCM Q UNL—ODMT\.... 2. DCI, NMI, ACN O oramidite .—UNL—0—1L /\/CN0 :hDCL NMIdACIII ng blocks 05" "an" "6 DNA/RNA DNA/RNA 31 automated thes1zer. automated s thesizer s 1. Cappmg (A020, NMI, pyr). 2. t-BuOOH DMT0:<_7’BX 3. DCA, DCM \/\o—-P 4. DCI, NMI, ACN Phosphoramidite 26 m DNA/RNA I X = O, or S automated s thesizer (I) Bx = Heterocylic base ' AcOOAc O H o H o ACO \/\/\n/N\/\/ \E AcHN O AcO OAc O /ODMT H O O - O H .1 AcHN OWNWNwogiMJR?/KNQOO O AcOOAc HN\/\/ o .\ O o‘ NC\/\ | ACO o\/\/\n/ O O—llDzo 1. Capping (A020, NMI, pyr) (I) 2. t-BuOOH 3. Et3NzCH3CN (1:1 V/V) 4. DCA, DCM . NH4, rt (cleavage) O H o H o HO \/\/\n/N\/\/ K AcHN O HOOH 0 /OH O O : O H H HO \/\/\n/ H/\/ %HO N AcHN o o o _ | O\P\ O BX HOOH 0 Ag H HN/:o HO OW -o—13=O AcHN 34 The UnylinkerTM 30 is commercially available. Oligomeric nd 34 comprising a GalNAc3-1 cluster at the 5 ’ terminus is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy er al., Angew. Chem. Int. Ed, 2006, 45, 3623-3627). oramidite building blocks, Compounds 1 and 1a were prepared as per the procedures illustrated in Example 1. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a predetermined sequence and composition. The order and quantity of oramidites added to the solid support can be ed to prepare gapped oligomeric compounds as described . Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 11: ation of Compound 39 ACOOAC 1. HOWHkO/D AcOOAC A00 35 0 TMSOTf, DCE O AGO$¢OMNH28 2. H2/Pd, MeOH AcHN 36 A00 OAC AC0 1.

HBTU, DMF, EtN(iPr)2 OWH H2, Pd/C, MeOH Compound 13 AcHN 8 2- HBTU DIEA DMF AcO \n/\\O Compound 23 O OWN ACO \H/\O/OO O NH ACO \/\H/\/ ACO OAc Aco¥gvo ODMT WNW Phosphitylation 0ACA(:HN AC0 OMNQOH Ac?w‘WN"wW?" AC0 p o 38 ACO OAc Ammo ODMT ACHN WNW 8 OMNqo AcogOWACO OWN\H/f/O NHAc \n/\\\%NH NC\/\O,PNUPDZ AC0 w o 39 AC0 OWNH nds 4, 13 and 23 were prepared as per the procedures illustrated in Examples 2, 4, and 5.

Compound 35 is prepared using similar procedures published in Rouchaud er al., Eur. J. Org. Chem, 2011, 12, 2346-2353.

Example 12: Preparation of Compound 40 Compound 38 is ed as per the procedures illustrated in Example 1 1.

ACO OAc Ammo ODMT AcHN WNW AcO OMNQOH Acoo§wOWNWV"we?" 1. ic anhydride, DMAP, DCE 0 2. DMF, HBTU, EtN IPr PS—SS AC0 OWNH ( )2 ACO OAc Aco¥gvo /ODMT NHAc o O AC0 W 40 o NH ACO w Example 13: Preparation of Compound 44 ACOOAC HBTU, DMF, EtN(iPr)2 ACQ$WOWNHZo 0 /_© AcHN HO O 36 a" h>LO HOW—f0 ACO OAc Aco¥£yO H O O}... 1. H2,Pd/C,MeOH /\:O 2. HBTU,D|EA, DMF 0 Compound 23 OAc 0 AcOkOWNHA00 0 A00 OAc AcHN 8 N\n/\\ I Phosphitylation :?g‘m(NEW/"3L2 ACO OAc AcHN 8 j/ZIWQ\oH NC8\/\OP\N(iPr)2 ACOk/owE/VpNHOAcACO 44 Compounds 23 and 36 are prepared as per the procedures rated in Examples 5 and 11.

Compound 41 is prepared using similar procedures published in WO 2009082607.

Example 14: Preparation of Compound 45 Compound 43 is prepared as per the procedures rated in Example 13.

A60 OAc AcokowA/Ho _/ODMT AcHN 8 ? O 43 O OMNHp 1. Succinic anhydride, DMAP, DCE AcHN 2. DMF, HBTU, EtN(iPr)2, PS—SS A60 OAc Acok/OMHo _/ODMT AcHN 8 ? o p A60$0M Example 15: ation of Compound 47 Compound 46 is commercially available.

HO DMTO >_ / < > N 1. DMTCI, pyr NH 2. Pd/C, H2, MeOH H0: 46 e 16: Preparation of Compound 53 HBTU, EtN(iPr)21DMF HBCOWNH —>7 2 O /Boc H3COWNO 0 \CBZ H3co\1(1/\<1;\NO OHN/CBzmN/CBZ . 1 TFA ,MeOH 2 HBTU EtN(iPr)Z DMF 2. HBTU, EtN(iPr)2, DMF /CzB Compound 47 O \ DMTO HN 1. H2, Pd/C O —> ,CBZ 2. HBTU, EtN(IPr)21DMF.

HO"" NW" NH I2 Compound 17 EQAAJLNH QM ?wpw iwi ODMT Compounds 48 and 49 are commercially available. Compounds 17 and 47 are prepared as per the procedures illustrated in Examples 4 and 15.

Example 17: Preparation ofCompound 54 32:1:W: ODMT Phosphitylation NHACO32::0"Md: Compound 53 is prepared as per the procedures illustrated in Example 16.

WO 68618 2015/028887 Example 18: Preparation of nd 55 Compound 53 is prepared as per the procedures illustrated in Example 16. 00 0 NH HN N NHACOOWJ\HN O 32$;M»: ODMT 1. Succinic anhydride, DMAP, DCE 2. DMF, HBTU, r)2, PS—SS S§WW1NgngoON gs;w ODMT Example 19: General method for the preparation of conjugated ASOs comprising GalNAc3-1 at the 3’ position via solid phase techniques (preparation of ISIS 647535, 647536 and 651900) Unless otherwise stated, all reagents and solutions used for the synthesis of eric compounds are purchased from commercial sources. Standard phosphoramidite building blocks and solid support are used for incorporation nucleoside residues which include for example T, A, G, and mC residues. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for B-D-Z’-deoxyrib0nucle0side and 2’- MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 umol scale) or on GE care Bioscience AKTA oligopilot synthesizer (40-200 umol scale) by the phosphoramidite coupling method on an GalNAc3-1 loaded VIMAD solid support (110 umol/g, GuzaeV er al., 2003) packed in the column. For the ng step, the phosphoramidites were delivered 4 fold excess over the loading on the solid t and phosphoramidite condensation was carried out for 10 min. All other steps followed rd protocols supplied by the manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing dimethoxytrityl (DMT) group from 5’-hydroxyl group of the nucleotide. 4,5 noimidazole (0.7 M) in anhydrous CH3CN was used as activator during coupling step. Phosphorothioate linkages were introduced by sul?Jrization with 0.1 M solution of xanthane e in 1:1 pyridine/CH3CN for a contact time of 3 minutes.

A solution of 20% tert-butylhydroperoxide in CH3CN containing 6% water was used as an oxidizing agent to provide phosphodiester intemucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate protecting groups were deprotected using a 1:1 (v/v) e of ylamine and itrile with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55 0C for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 um, 2.54 x 8 cm, A = 100 mM ammonium acetate in 30% aqueous CH3CN, B = 1.5 M NaBr in A, 0- 40% ofB in 60 min, ?ow 14 mL min-1, )t = 260 nm). The e was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1 100 MSD system.

Antisense oligonucleotides not comprising a conjugate were synthesized using standard oligonucleotide sis procedures well known in the art.

Using these s, three separate antisense compounds targeting ApoC III were prepared. As summarized in Table 17, below, each of the three antisense compounds targeting ApoC III had the same nucleobase sequence; ISIS 304801 is a 55 MOE gapmer having all phosphorothioate es; ISIS 647535 is the same as ISIS 304801, except that it had a GalNAc3-1 conjugated at its 3’end; and ISIS 647536 is the same as ISIS 647535 except that certain intemucleoside linkages of that compound are phosphodiester linkages. As ?thher summarized in Table 17, two separate antisense compounds targeting SRB-l were synthesized. ISIS 440762 was a 22 cEt gapmer with all phosphorothioate internucleoside linkages; ISIS 651900 is the same as ISIS 440762, except that it included a GalNAc3-l at its 3’-end.

Table 17 Modified ASO tar etin A oC III and SRB-l , , CalCd Observed ISIS A AesGesmCesTesTesm m m C CdsTdsTdsGdsTds Cds GdsmCds TesTesTesAesTe 71 65 ~4 71 64 ~4 2296 3 04801 3(1) ISIS AesGesmCesTesTesmCdsTdsTdsGdsTdsmCdsmCdsAdsGdsmCdsTesTesTesAesTeoAdo" APOC 9239.5 "378 2297 647535 GalNAcs-la III ISIS Aes(}eomCeor-l.‘eoTeomCdsTdsTds(}dsTdsmCdsmCdsAds(}dsmCdsTeoTeoTesAesTeolAdo’' APOC 9140.8 2297 647536 GalNAc3-la -9142.9 Tksm m m m SRB_ CksAdsGdsTds TdsGdsAds Tks Ck 4647.0 4646.4 2298 440762 TkskasAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkskaoAdo"GalNAC3-1 a 65 1900 8111]?" 6721 1 67 194 2299 Subscripts: "e" indicates 2’-MOE modi?ed nucleoside; "d" indicates B-D-2’- ibonucleoside; "k" indicates 6’-(S)-CH3 bicyclic nucleoside (e.g. cEt); "s" indicates phosphorothioate intemucleoside es (PS); "0" indicates phosphodiester intemucleoside linkages (PO); and "0’" indicates -O-P(=O)(OH)-. Superscript "m" indicates 5-methylcytosines. "GalNAc3-1" indicates a conjugate group having the structure shown previously in Example 9. Note that GalNAc3-1 comprises a cleavable adenosine which links the ASO to remainder of the conjugate, which is ated "GalNAc3-l a." This nomenclature is used in the above table to show the ?lll nucleobase sequence, including the adenosine, which is part of the conjugate. Thus, in the above table, the sequences could also be listed as ending with "GalNAcg-l" with the "Ado" omitted. This convention of using the subscript "a" to indicate the portion of a conjugate group lacking a cleavable nucleoside or cleavable moiety is used throughout these Examples. This portion of a conjugate group lacking the cleavable moiety is ed to herein as a "cluster" or "conjugate cluster" or "GalNAc3 cluster." In certain instances it is ient to be a conjugate group by separately providing its cluster and its cleavable moiety.

Example 20: Dose-dependent antisense inhibition of human ApoC III in huApoC III transgenic mice ISIS 304801 and ISIS 647535, each targeting human ApoC III and described above, were separately tested and evaluated in a dose-dependent study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Trealmenl Human ApoCIII transgenic mice were maintained on a r light/dark cycle and fed ad libilum Teklad lab chow. Animals were acclimated for at least 7 days in the research ty before initiation of the experiment. ASOs were prepared in PBS and sterilized by filtering h a 0.2 micron filter. ASOs were ved in 0.9% PBS for ion.

Human ApoC III transgenic mice were injected intraperitoneally once a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25. 0.75, 2.25 or 6.75 umol/kg, or with PBS as a control. Each treatment group consisted of 4 animals. Forty-eight hours after the administration of the last dose, blood was drawn from each mouse and the mice were sacrificed and tissues were collected.

ApoC [H mRNA Analysis ApoC III mRNA levels in the mice’s livers were determined using ime PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using een), prior to ization to PBS-treated control. The results below are ted as the average percent of ApoC III mRNA levels for each treatment group, normalized to PB S-treated control and are denoted as "% PBS". The ximal effective dosage (ED50) of each ASO is also presented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA ve to the PBS control.

Further, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801).

Table 18 Effect ofASO treatment on ApoC III le\A levels in human ApoC III transgenic mice Dose % EDso Intemucleoside SEQ ID ASO 3 , Conjugate. (umol/kg) PBS (umol/kg) linkage/Length No.

PBS 0 100 -- - -- 0.08 95 ISIS 0.75 42 0.77 None PS/20 2296 304801W 6.75 19 0.08 50 ISIS 0.75 15 0.074 GalNAc3-l PS/20 2297 6.75 8 ApoC [[1 Protein Analysis (Turbidomeiric Assay) Plasma ApoC III n analysis was determined using procedures reported by Graham er al, Circulation Research, published online before print March 29, 2013.

Approximately 100 pl of plasma isolated from mice was analyzed without dilution using an Olympus Clinical Analyzer and a cially available turbidometric ApoC III assay (Kamiya, Cat# KAI-006, Kamiya Biomedical, Seattle, WA). The assay protocol was performed as described by the vendor.

As shown in the Table 19 below, both antisense compounds reduced ApoC III protein relative to the PBS control. Further, the antisense compound conjugated to 3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801).

Table 19 Effect ofASO treatment on A oC III lasma rotein levels in human A . oC III trans . enic mice Dose % ED50 Intemucleoside AS0 3, C . SEQ ID on uJ ga et (umol/kg) PBS (umol/kg) Linkage/Length N0- -"-———_ 073‘ None PS/20 2296 304801 019 GalNAc 1 PS/20 2297 647535 ' 3' Plasma triglycerides and terol were extracted by the method of Bligh and Dyer (Bligh, E.G. and Dyer, W.J. Can. J. Biochem. Physiol. 37: 911-917, 1959)(Bligh, E and Dyer, W, Can em Physiol, 37, 7, Bligh, E and Dyer, W, Can JBz'ochem Physiol, 37, 911-917, 1959) and measured by using a nn Coulter clinical analyzer and commercially available reagents.

The triglyceride levels were measured relative to PBS injected mice and are denoted as "% PB S". Results are presented in Table 20. As illustrated, both antisense compounds lowered triglyceride levels. Further, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc3-1 conjugate (ISIS 304801).

Table 20 Effect of ASO treatment on triglyceride levels in transgenic mice Dose % EDso Internucleoside AS0 3’ SEQ ID (umol/kg) PBS (umong) Conjugate Linkage/Length No.

PBS 0 100 -- -- __ 0.08 87 0.63 None PS/20 2296 3 (1)231H 6.75 12 0.08 65 6413;835% 0.13 GalNAc3-1 PS/20 2297 Plasma samples were analyzed by HPLC to determine the amount of total cholesterol and of different fractions of cholesterol (HDL and LDL). Results are presented in Tables 21 and 22. As illustrated, both antisense compounds d total cholesterol levels; both lowered LDL; and both raised HDL. Further, the antisense compound conjugated to GalNAc3-1 (ISIS 647535) was substantially more potent than the nse compound g the GalNAc3-l conjugate (ISIS 304801 ). An increase in HDL and a decrease in LDL levels is a vascular beneficial effect of antisense inhibition ofApoC III.

Table 21 Effect of ASO treatment on total cholesterol levels in transgenic mice Dose Total Cholesterol 3 ’ ucleoside SEQ ( mol/k) (m/dL) Con'uate LinkaHe/Lenth ID No. _—-_——- 0.08 226 352;?" (2):: 1?: None PS/20 2296 6.75 82 0.08 230 6413283 5 (2):: :2 3-1 PS/20 2297 6.75 99 Table 22 Effect ofASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL LDL AS0 3’ Intemucleoside SEQ (umol/kg) (mg/dL) (mg/dL) Conjugate Linkage/Length ID No.

PBS 0 17 28 -- -- 0.08 17 23 None PS/20 2296 3 522801W 6.75 45 2 0.08 21 21 ISIS 0.75 44 2 GalNAc3-1 PS/20 2297 647535W 6.75 58 2 Pharmacokinetics Analysis (PK) The PK of the ASOs was also evaluated. Liver and kidney samples were minced and extracted using standard protocols. Samples were analyzed on MSD1 utilizing C-MS. The tissue level (ug/g) of full-length ISIS 304801 and 647535 was measured and the results are ed in Table 23. As illustrated, liver concentrations of total ?Jll-length antisense compounds were similar for the two antisense compounds.

Thus, even though the GalNAc3-1 -conjugated antisense compound is more active in the liver (as trated by the RNA and protein data above), it is not present at substantially higher concentration in the liver. Indeed, the calculated EC 50 (provided in Table 23) con?rms that the observed increase in potency of the conjugated nd cannot be entirely uted to increased accumulation. This result suggests that the conjugate ed potency by a mechanism other than liver accumulation alone, possibly by improving the productive uptake of the antisense compound into cells.

The results also show that the concentration of GalNAc3-l conjugated antisense nd in the kidney is lower than that of antisense compound lacking the GalNAc conjugate. This has several beneficial eutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding bene?t. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for dney targets, kidney accumulation is undesired. These data suggest that GalNAc3-l ation reduces kidney accumulation.

Table 23 PK analysis ofASO ent in transgenic mice Intemucleoside Dose Liver Kidney Liver EC50 3 ’ . SEQ AS0 Linkage/Length (umong) (98/8) (98/8) (98/8) Conjugate ID No. 0.1 5.2 2.1 ISIS 0.8 62.8 119.6 53 None PS/20 2296 304801 2.3 142.3 191.5 6.8 202.3 337.7 0.1 3.8 0.7 ISIS 0.8 72.7 34.3 3 8' GalNAc3-1 PS/20 2297 647535 2.3 106.8 111.4 6.8 237.2 179.3 Metabolites of ISIS 647535 were also identi?ed and their masses were con?rmed by high resolution mass spectrometry analysis. The ge sites and structures of the observed metabolites are shown below.

The relative % of full length ASO was calculated using standard procedures and the s are presented in Table 23a. The major metabolite of ISIS 647535 was ?Jll-length ASO lacking the entire conjugate (i.e. ISIS 304801), which results from cleavage at cleavage site A, shown below. Further, additional metabolites resulting from other cleavage sites were also observed. These results suggest that introducing other cleavable bonds such as esters, peptides, disul?des, phosphoramidates or ydrazones between the GalNAc3-1 sugar and the ASO, which can be d by enzymes inside the cell, or which may cleave in the reductive environment of the cytosol, or which are labile to the acidic pH inside endosomes and mes, can also be use?Jl.

Table 2321 Observed full length metabolites of ISIS 647535 Metabolite ASO Cleavage site Relative % 1 ISIS 304801 A 36.1 2 ISIS 304801 + dA B 10.5 3 ISIS 647535 minus [3 GalNAc] C 16.1 ISIS 647535 minus 4 D 17 6 [3 GalNAc -- 1 5-hydroxy-pentanoic acid tether] ' ISIS 647535 minus 9 9 [2 GalNAc -- 2 5-hydroxy-pentanoic acid tether] ' ISIS 647535 minus 6 [3 GalNAc -- 3 5-hydroxy-pentanoic acid tether] 9-8 Aso 304801 Cleavage Sltes.

Cleavage site A HO OH Cleavage site C o:F"OH HOHO%OOWHHNWNCleavage site D O (52H KgNNJ Cleavage site C OQO 1:0 ge site B NHAc Cleavage site D H\/\/HN O N:AC\WWCleavageO\N site D Cleavage site C ASO 304801 o:ifOH NH2 Metabolite 1 [T80 304801 Metabolite 2 OH 0&1?" I‘ASO 304801 O:F"*OH NH2 0 N H O OH N : 0 0 <5 H H l N O N O HO WW H F":O Metabolite 3 HN/: ITSO 304801 H O HZNWN 2 o N l/J ; N O o o H H l NW\HA/O N O F":O HO H Metabolite 4 HN/: ASO 304801 O:P*OH NH2 H 0 / \N OH l HZNWN E O g N O o 05 H l WNW u o 5:0 Metabolite 5 HN/: I‘ASO 304801 o N WM \ H 0 / OH i N HZNWN E J g N O o o: H l HZNWN O m 0 F":O Metabolite 6 Example 21: Antisense inhibition of human ApoC III in human ApoC III transgenic mice in single administration study ISIS 304801, 647535 and 647536 each targeting human ApoC III and described in Table 17, were further evaluated in a single administration study for their ability to inhibit human ApoC III in human ApoC III transgenic mice.

Trealmenl Human I transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libilum Teklad lab chow. Animals were acclimated for at least 7 days in the research ty before initiation of the experiment. ASOs were prepared in PBS and ized by filtering through a 0.2 micron filter. ASOs were dissolved in 0.9% PBS for injection.

Human ApoC III transgenic mice were injected intraperitoneally once at the dosage shown below with ISIS 304801, 647535 or 647536 (described above) or with PBS treated l. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III mRNA and protein levels in the liver; plasma triglycerides; and terol, including HDL and LDL fractions were assessed, as described above (Example 20). Data from those es are presented in Tables 24-28, below. Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured ve to saline injected mice using standard protocols. The ALT and AST levels showed that the antisense compounds were well tolerated at all stered doses.

These results show improvement in potency for antisense compounds comprising a GalNAc3-1 conjugate at the 3’ terminus (ISIS 647535 and 647536) compared to the antisense compound lacking a 3-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a GalNAc3-1 conjugate and some odiester linkages was as potent as ISIS 647535, which comprises the same conjugate, and all the ucleoside linkages within the ASO are phosphorothioate.

Table 24 Effect ofASO treatment on AooC III mRNA levels in human A. oC III trans. enic mice 3 ’ Internucleoside linka- e/Len- .32z——';%' 647536 Ac3-1 PS/PO/20 2297 Table 25 Effect ofASO treatment on A oC III lasma rotein levels in human A oC III trans enic mice Dose EDso 3 ’ Intemucleoside ASO 0A) PBS SEQ ID NO' (mg/kg) (mg/kg) Conjugate Linkage/Length PBS 0 99 -- -- __ 1 104 ISIS 3 92 23-2 None PS/20 2296 304801 10 71 40 0.3 98 ISIS 1 70 2-1 GalNAc3-1 PS/20 2297 647535 3 33 20 0.3 103 ISIS 1 60 1.8 GalNAc3-1 PS/PO/20 2297 647536 31 21 Table 26 Effect of ASO treatment on triglyceride levels in transgenic mice 0 EDso Intemucleoside SEQ ID ASO A) PBS 3 , ate~ (mg/kg) (mg/kg) Linkage/Length N0, PBS 0 98 -- -- __ 1 80 ISIS 3 92 29-1 None PS/20 2296 304801 10 70 47 0.3 100 ISIS 1 70 2-2 GalNA03-l PS/20 2297 647535 3 34 23 0.3 95 ISIS 1 66 1.9 GalNAc3-1 PS/PO/20 2297 647536 3 31 23 Table 27 Effect of ASO treatment on total cholesterol levels in trans enic mice 304801 3 96 86 72 ISIS 1 85 GalNAc3-1 PS/20 2297 647535 3 61 53 ISIS 1 79 GalNAc3-1 PS/PO/20 2297 647536 3 51 54 Table 28 Effect ofASO treatment on HDL and LDL cholesterol levels in transgenic mice Dose HDL LDL 3 ’ Internucleoside SEQ ID (mg/kg) % PBS % PBS Conjugate Linkage/Length N0, PBS 0 131 90 __ __ ISIS 3 186 79 None PS/20 2296 304801W 240 46 ISIS 1 214 67 GalNAC3-1 PS/20 2297 647535 3 212 39 218 35 0.3 143 89 ISIS 1 187 56 3-l PS/PO/20 2297 647536 3 213 33 221 34 These results con?rm that the GalNAc3-l conjugate improves potency of an antisense compound.

The results also show equal potency of a GalNAc3-1 conjugated antisense compounds where the antisense oligonucleotides have mixed linkages (ISIS 647536 which has six odiester linkages) and a full phosphorothioate version of the same antisense compound (ISIS 647535).

Phosphorothioate linkages provide several properties to antisense compounds. For example, they resist nuclease digestion and they bind proteins resulting in accumulation of compound in the liver, rather than in the kidney/urine. These are desirable properties, particularly when treating an indication in the liver.

However, phosphorothioate linkages have also been ated with an in?ammatory se. Accordingly, reducing the number of phosphorothioate linkages in a nd is expected to reduce the risk of in?ammation, but also lower concentration of the compound in liver, increase concentration in the kidney and urine, decrease stability in the presence of nucleases, and lower overall potency. The t results show that a GalNAc3-l conjugated antisense compound where n phosphorothioate linkages have been replaced with phosphodiester linkages is as potent against a target in the liver as a counterpart having full phosphorothioate linkages. Such nds are expected to be less proin?ammatory (See Example 24 describing an experiment showing reduction of PS s in reduced in?ammatory effect).

Example 22: Effect of GalNAc3-1 conjugated modi?ed ASO targeting SRB-l in vivo ISIS 440762 and , each ing SRB-l and described in Table 17, were evaluated in a dose- dependent study for their ability to inhibit SRB-1 in Balb/c mice.

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacri?ced 48 hours following the final administration to determine the SRB-1 mRNA levels in liver using real-time PCR and RIBOGREEN® RNA fication reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-l mRNA levels were determined relative to total RNA (using Ribogreen), prior to ization to PB S-treated control. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to PB S-treated control and is denoted as "% PB S".

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNA levels. Further, the antisense compound comprising the GalNAc3-l conjugate (ISIS 651900) was substantially more potent than the antisense compound lacking the GalNAc3-l conjugate (ISIS 440762). These results demonstrate that the potency benefit of GalNAc3-1 conjugates are observed using antisense oligonucleotides complementary to a different target and having different chemically modified sides, in this instance modified nucleosides comprise constrained ethyl sugar moieties (a bicyclic sugar ).

Table 29 Effect ofASO ent on SRB-1 mRNA levels in Balb/c mice Internucleosid Dose Liver ED50 e SE ID ASO 3 , Conjugate.

) % PBS (mg/kg) linkage/Lengt NE).

PBS 0 100 - -- 0.7 85 431722—%i: 2.2 None PS/14 2298 3 0.07 98 0.2 63 0.7 20 0.3 GalNAc3-1 PS/14 2299 651900— 2 6 7 5 Example 23: Human Peripheral Blood Mononuclear Cells (hPBMC) Assay Protocol The hPBMC assay was performed using BD Vautainer CPT tube method. A sample of whole blood from volunteered donors with informed consent at US HealthWorks clinic (Faraday & El Camino Real, Carlsbad) was obtained and collected in 4-15 BD Vacutainer CPT 8 ml tubes (VWR Cat.# BD362753). The approximate starting total whole blood volume in the CPT tubes for each donor was recorded using the PBMC assay data sheet.

The blood sample was remixed immediately prior to centrifugation by gently inverting tubes 8-10 times. CPT tubes were centri?Jged at rt (18-25 0C) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were ved from the buffy coat interface (between Ficoll and r gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical onor. The cells were then washed twice with PBS (Ca++, Mg" free; GIBCO). The tubes were topped up to 50 ml and mixed by inverting several times. The sample was then centri?Jged at 330 x g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much atant as possible without disturbing pellet. The cell pellet was dislodged by gently ng tube and resuspended cells in RPMI+10% FBS+pen/strep (N1 ml/ 10 ml starting whole blood volume). A 60 pl sample was pipette into a sample vial (Beckman Coulter) with 600 pl VersaLyse reagent (Beckman r Cat# A09777) and was gently vortexed for 10-15 sec. The sample was allowed to incubate for 10 min. at rt and being mixed again before counting. The cell sion was d on Vicell XR cell ity analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1 :11 was stored with other parameters). The live cell/ml and viability were ed. The cell suspension was diluted to 1 x 107 live PBMC/ml in RPMI+ 10% FE S+pen/strep.

The cells were plated at 5 x 105 in 50 til/well of 96-well tissue culture plate (Falcon Microtest). 50 til/well of 2x concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100 ill/well total). Plates were placed on the shaker and allowed to mix for approx. 1 min. After being incubated for 24 hrs at 37 °C; 5% C02, the plates were centri?Jged at 400 x g for 10 s before removing the atant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-l).

Example 24: Evaluation of Proin?ammatory Effects in hPBMC Assay for GalNAc3-1 conjugated The antisense oligonucleotides (ASOs) listed in Table 30 were evaluated for ammatory effect in hPBMC assay using the protocol described in Example 23. ISIS 353512 is an internal standard known to be a high responder for IL-6 release in the assay. The hPBMCs were isolated from fresh, volunteered donors and were treated with ASOs at 0, , 0.064, 0.32, 1.6, 8, 40 and 200 "M concentrations. After a 24 hr treatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary t. The EC50 and Emax was ated using standard procedures. Results are expressed as the average ratio of me/Ecso from two donors and is denoted as "Em/Ecso." The lower ratio indicates a relative decrease in the proin?ammatory response and the higher ratio indicates a relative increase in the proin?ammatory response.

With regard to the test compounds, the least proin?ammatory compound was the PS/PO linked ASO (ISIS 616468). The GalNA03-1 conjugated ASO, ISIS 647535 was slightly less proin?ammatory than its non-conjugated counterpart ISIS 304801. These results te that incorporation of some PO linkages reduces proin?ammatory reaction and addition of a GalNAc3-1 conjugate does not make a compound more proin?ammatory and may reduce proin?ammatory response. Accordingly, one would expect that an antisense compound comprising both mixed PS/PO linkages and a GalNAc3-l conjugate would produce lower proin?ammatory responses ve to full PS linked antisense compound with or without a GalNAc3-l conjugate. These results show that GalNAc3_1 conjugated nse compounds, particularly those having reduced PS content are less proin?ammatory.

Together, these results suggest that a GalNAc3-1 conjugated compound, particularly one with reduced PS content, can be administered at a higher dose than a counterpart ?Jll PS antisense nd lacking a GalNAc3-l conjugate. Since half-life is not ed to be ntially different for these compounds, such higher administration would result in less frequent dosing. Indeed such administration could be even less frequent, because the GalNAc3-1 conjugated compounds are more potent (See Examples -22) and re-dosing is necessary once the concentration of a compound has dropped below a desired level, where such desired level is based on potency.

Table 30 Modi?ed ASOS ASO Sequence (5’ to 3’) Target SENCilD ISIS STCSGCSACSTdsTdsAdsGdsAdsGds TNFOL 2300 104838 AdsGdSAdSGdsGesTesmcmmcesmce ISIS Tes Ces Ces CdsAdsTdsTdsTds CdsAdsGds CRP 2301 353512 GdSAdSGdSAdsmCdsmCdSTesGesGe ISIS AeSGESmCmTeSTesmCdSTdSTdSGdSTdS ApoC III 2296 304801 mCdsmCdsAdSGdsmCds TmTesTesAesTe ISIS AeSGESmCmTeSTesmCdSTdSTdSGdSTdS ApoC III 2297 6475 35 IncdsmCdsAdsGdsmCdsTeSTmTesAesTeoAdo"(kllNAC3-1 a ISIS mCeoTeoTeomCdsTdsTdsGdsTds ApoC III 2296 616468 InCasmcdsAdsGasmcdsTeOTeoTesAesTe Subscripts: "e" indicates 2’-MOE modified side; "(1" indicates B-D-2’- deoxyribonucleoside; "k" indicates 6’-(S)-CH3 ic nucleoside (e.g. cEt); a; 99’ S tes phosphorothioate internucleoside linkages (PS); "0" indicates phosphodiester internucleoside linkages (PO); and "0’" indicates -O-P(=O)(OH)-. cript "m" indicates 5-methylcytosines. "AdowGalNAc3-la" indicates a conjugate having the structure GalNAc3-1 shown in Example 9 attached to the 3 ’-end of the antisense oligonucleotide, as ted.

Table 31 Proin?ammatory Effect of ASOs targeting ApoC III in hPBMC assay EC50 Emax 3 ’ Internucleoside SEQ ID ASO O (uM) (uM) Conjugate Linkage/Length No. $18 353512 0.01 265.9 26,590 None PS/20 2301 (high responder) ISIS 304801 0.07 106.55 1,522 None PS/20 2296 ISIS 647535 0.12 138 1,150 GalNAc3-1 PS/20 2297 ISIS 616468 0.32 71.52 224 None 20 2296 Example 25: Effect of GalNAc3-1 conjugated modi?ed ASO targeting human ApoC III in vitro ISIS 304801 and 647535 described above were tested in vitro. Primary hepatocyte cells from transgenic mice at a y of 25,000 cells per well were treated with 003,008, 0.24, 0.74, 2.22, 6.67 and 20 uM concentrations of modi?ed oligonucleotides. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the hApoC III mRNA levels were adjusted according to total RNA content, as measured by EEN.

The IC50 was calculated using the standard methods and the results are presented in Table 32. As illustrated, comparable potency was observed in cells treated with ISIS 647535 as compared to the control, ISIS 304801.

Table 32 Modi?ed ASO tar?etin human A n 0C 111 in rimar heatoc tes Internucleoside SEQ ICso (MM) 3 , ate. linkage/Length We mo 2296 304801 GalNA°3'1 mo 2297 647535 In this experiment, the large potency bene?ts of GalNAc3-1 conjugation that are observed in viva were not observed in vilro. Subsequent free uptake experiments in primary cytes in vitro did show increased potency of oligonucleotides comprising various GalNAc conjugates ve to oligonucleotides that lack the GalNAc conjugate (see Examples 60, 82, and 92).

Example 26: Effect of PO/PS linkages 0n ApoC III ASO ty Human ApoC III transgenic mice were injected intraperitoneally once at 25 mg/kg of ISIS , or ISIS 616468 (both described above) or with PBS treated control once per week for two weeks. The treatment group consisted of 3 animals and the control group consisted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacrificed 72 hours following the last administration.

Samples were collected and analyzed to determine the ApoC III protein levels in the liver as described above (Example 20). Data from those analyses are presented in Table 33, below.

These results show reduction in potency for nse compounds with PO/PS (ISIS 616468) in the wings relative to ?ll PS (ISIS ).

Table 33 Effect ofASO treatment on A 0C 111 rotein levels in human A 0C 111 trans enic mice Dose 3 ’ Intemucleoside ASO 0A) PBS (mg/kg) Conjugate linkage/Length SENQID mg/kg/wk None Full PS 2296 304801 for 2 wks ISIS mg/12 616468 for 2 wks Example 27: Compound 56 Compound 56 is cially available from Glen ch or may be prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

DMTO\/\/O Pr)2 DMTOWOng/P\ /\/CN DMTOMO Example 28: Preparation of Compound 60 Compound 4 was ed as per the procedures illustrated in Example 2. Compound 57 is commercially available. Compound 60 was con?rmed by structural analysis.

Compound 57 is meant to be representative and not intended to be limiting as other monoprotected substituted or unsubstituted aIkyl diols including but not limited to those ted in the speci?cation herein can be used to prepare phosphoramidites having a predetermined composition.

AcO OAc AcO OAc o /\/\/\/OBn 57 AcO H0 O HZ/Pd —>AcO 0\/\/\/\ > OBn MeoH \o TMSOTf, DCE ACHN 58 N? ( uam )q I ( 71%) AcO OAC CNEtO(N(iPr)2)PC1, A00 0A0 N(iPr)2 EDIP o I CN 0 —> O\/\/\/\O/P\O/\/ O Ac0 AGO \/\/\/\OH CH2C12 ACHN 59 (80%) AcHN 60 Example 29: Preparation of nd 63 Compounds 61 and 62 are ed using procedures similar to those reported by Tober er al., Eur. J.

Org. Chem, 2013, 3, 566-577; and Jiang el al., Tetrahedron, 2007, 63(19), 3982-3988. 1. BnCl OH 1. DMTC1,pyr 0 % HO >/ 2. KOH, DMSO 2. Pd/C,HZ CH3 BnO ODMT . 013/0 O OH 3. HCl MeOH 3. Phosphitylatlon I O ’ OH N(iPr) ODMT 4. Ncho3 2 62 63 Alternatively, Compound 63 is ed using procedures similar to those reported in scienti?c and patent literature by Kim er al., Synleii, 2003, 12, 1838-1840; and Kim er al., published PCT International Application, WO 2004063208.

Example 30: Preparation of Compound 63b Compound 63a is prepared using procedures similar to those reported by Hanessian er al., an Journal of Chemistry, 1996, 74(9), 1731-1737.

OH ODMT /_/ CN 0 O TPDBSOonA/OH 1. DMTCl,pyr a 2. TBAF . . O\P/OJE\o/\/ODMT O 3. Phosphitylation I N(iPr)2 63a OH ODMT Example 31: Preparation of Compound 63d Compound 63d is prepared using procedures similar to those reported by Chen er al., Chinese ChemicalLeiierS, 1998, 9(5), 451-453.

DMTO—\—\ HO_\—\ 1' DMTCI’ pyr DMTO\/\/o HO\/\/O 2. 13(1/c,H2 O/\/\ / \1'3 /\/CN O/\/\OBn o o 3. O O itylation —/_/ 630 _/_J 63d Example 32: Preparation of Compound 67 Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 65 is prepared using ures similar to those ed by Or er al., published PCT International Application, W0 2009003009. The protecting groups used for Compound 65 are meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the speci?cation herein can be used.

COZBn AcO OAc AGO OAC O O\/\/\jj\ HZN/K(0TBDMS O COZBH AcO 65 OH R —,AcO ACHN 64 WOMN)\(OTBDMSH HBTU, DIEA ACHN 66 R R : H or CH3 AC0 0AC 1. TEA.3HF, THF o 00an A O OWL O\ /O\/\ 2. Phosphitylation c E 1|) CN AcHN R N(iPr)2 Example 33: Preparation of Compound 70 Compound 64 was prepared as per the procedures illustrated in Example 2. nd 68 is commercially available. The protecting group used for nd 68 is meant to be representative and not intended to be limiting as other protecting groups including but not limited to those ted in the speci?cation herein can be used.

H NyOBn2 AcO OAc 68 160% 0 AcO OAc CH3 O O 0 0M OH HBTU,DIEA 3WOBn TAC0 ACI—IN 64 ACI—IN 69 CH3 A 00AC C 1. Pd/C,H2 [900% o o OWL O\ /O\/\ 2. Phosphitylation Ear PI) CN AcHN CH3 N(iPr)2 Example 34: Preparation of Compound 75a Compound 75 is prepared according to published procedures reported by inov er al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

O CF3 1. TBDMSCI, pyr Y 2. Pd/C,H2 N(iPr) 2 /\/O HNWO I NC 3. CF3COZEt,MeOH H /P\ /\/CN Nc/\/O OH O O NC\/\O 4. TEASHF, THF F3C\[rN\/\/O . Phosphitylation 0 WMO 75 A 75a 0 CF3 Example 35: Preparation of Compound 79 Compound 76 was prepared according to published procedures ed by Shchepinov er al., Nucleic Acids Research, 1997, 25 (22), 4447 -4454.

DMTOWO HOWO DCI NMI ACN 1. BnCl,NaH ’ ’ DMTOWO O .

OH —, OBn Phosphoram1d1te 60.

DMTO/A\//\O 2.DCAJHth HO/"\//\O 76 77 A00 OAC 0 NC\\\ A00 0 (I) \/\/\/\ /P\ AcHN K 1. HZ/Pd, MeOH ACO OAC NC\\\ AcHN 0 A00 ’1‘3\ 0 O O 0 NHAc 78 A00 OAC NC 0 1 A00 O\\/A\¢/\\//A\ 9 Example 36: Preparation of Compound 79a Compound 77 is prepared as per the procedures illustrated in e 35.

HOWO FmocOW0 1 _ FmocCl, pyr I\IT(iPr)2 HO/\/\0 3. Phosphitylation FmocO/\/\O Example 37: General method for the preparation of conjugated oligomeric compound 82 comprising a phosphodiester linked GalNAc3-2 conjugate at 5’ terminus via solid t (Method 1) DMT0:S:7’BX NC\/\O__p—o 1. DCA, DCM :— 2. DCI, NMI, ACN Phosphoramidite 5 6 OLIGO automated synthesizer VIMAD—O-P\O/\/CN X = S' or O' BX = Heterocylic base 80 1. Capping (A0203 NML pyr) 2. t-BuOOH 3. DCA, DCM 4. DCI, NMI, ACN AC0 OAC oramidite 60 O NC\\\ O\/\/\/\ / \l5 AcHN 0 K CN AcO OAC \L I O O O | I O BX \/\/\/\O/P\O/\/\O O_(le W—'O AcHN O NC\\\O NC\/\O—ll3=0 ACO OAC O 0W0 1')\O OLIGO NHAC (I) Q VIMAD—o—PO/\/CN 1. Capping (AcZO, NMI, pyr) 81 2. t-BuOOH 3. 20% EtZNH inToluene (V/V) 4. NH4, 55 0C, NHAC 82 wherein GalNAc3-2 has the structure: HO o 9 \/\/\/\ /P\ AcHN O O=P-O HO OH le O O\/\/\/\Oiio‘0.

The GalNAc3 cluster portion of the conjugate group 3-2 (GalNAc3-2a) can be combined With any cleavable moiety to provide a variety of ate groups. Wherein GalNAc3-2a has the formula: HO OH AcHN O O O\/\/\/\OIPFOO' The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA sis (see Dupouy er al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The phosphoramidite Compounds 56 and 60 were prepared as per the procedures rated in Examples 27 and 28, respectively. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other oramidite building blocks including but not limited those presented in the speci?cation herein can be used to prepare an oligomeric compound haVing a phosphodiester linked conjugate group at the 5 ’ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein haVing any ermined sequence and composition.

Example 38: Alternative method for the preparation of oligomeric nd 82 comprising a phosphodiester linked GalNAc3-2 conjugate at 5’ us (Method 11) NC\/\ Q l. DCA, DCM 013:0 2. DCI, NMI, ACN Phosphoramidite 79 OLIGO DNA/RNA automated synthesizer X = S' or O' BX = Heterocyclic base O O\/\/\/\O’P\O l. Capping 2. t-BuOOH 3. Et3NzCH3CN(1:1V/V) 83 4. NH4, 55 0C Oligomeric Compound 82 The VIMAD-bound oligomeric nd 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy er al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). The GalNA03-2 r phosphoramidite, Compound 79 was prepared as per the procedures illustrated in Example . This alternative method allows a one-step installation of the phosphodiester linked GalNA03-2 conjugate to the oligomeric nd at the ?nal step of the synthesis. The phosphoramidites illustrated are meant to be representative and not intended to be limiting, as other phosphoramidite building blocks including but not limited to those presented in the speci?cation herein can be used to prepare oligomeric compounds having a phosphodiester conjugate at the 5 ’ terminus. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as described herein having any predetermined sequence and composition.

Example 39: l method for the preparation of oligomeric compound 83h comprising a GalNAc3- 3 Conjugate at the 5’ us (GalNAc3-1 modi?ed for 5' end ment) via Solid Support ACO OAC A00 o H H 1. C,MeOH (93%)O AcHN WNth O O BnO OH H H O O 11/»oni WN \n/\/\n/ 838 0A0 0M A00 o/\© o 0 0 o HBTU, DIEA, DMF, 76% NHAc M 3. H2,Pd/C,MeOH 0 Aco A00 A00 o W O OH \COCF3 A00 83b OACO 0M O O O F AGO ‘F— NHAc HNMN 830 H o Pyridine, DMF A00 OAC A00 836 o H 3, 5, N H ? AcHN W \/\/N F \?/\\ F OLIGO o o O—P—O—(CH2)e—NH2 0 ' n H o o N OAc NW 0%NH Borate buffer, DMSO, pH 8.5,!1 0 A00 \n/\’ O O O F F NHAc HNMN H o AGO OAC AcO o \A/VI/ WNWH H o 0 H o I 5' 3' H O N N _ _ _ _ 0A0 OM \/\/N N (CH2)e-O P O OLIGO AGO wogw H H o O O NHAc M HN " 0 OAc OJAO Aqueous ammonia HO OH HO O H H o WNEm M OH O 5' 3 "W" o NH u—(CHm—o—?—o— H0 0H0 OW o 0 HO HNMN nd 18 was prepared as per the procedures illustrated in Example 4. Compounds 83a and 83b are commercially available. Oligomeric Compound 83c comprising a phosphodiester linked hexylamine was ed using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound with aqueous ammonia provided the 5'—GalNAc3-3 ated oligomeric compound (83h).

Wherein 3-3 has the structure: The GalNAc3 cluster portion of the conjugate group GalNAc3-3 (GalNAc3-3 a) can be combined with any cleavable moiety to provide a variety of ate groups. Wherein GalNAc3-3a has the formula: Example 40: General method for the preparation of oligomeric compound 89 comprising a phosphodiester linked GalNAc3-4 conjugate at the 3’ terminus via solid support WODMT 1’ DCA ® MT 0&0/\/\0Fmoc 2. DCI NMI ACN FmocO\/\/O DMTOWO 3. Capping ODMT CN 4. t-BuOOH o\/\/ of OFmoc 1. 2% Piperidine,. . . Fm°c 2% DBU, 96% DMF ’P\O OO/—/— 0 0M0 _/O_/OFmoc 3.DCI,NMI, ACN ._UNL—o—p\O/\/CN O Phosphoramidite 79a DNA/RNA Capping automated synthesizer i. tz-(EufgoHd'o 1per1 me, AcO OAC 2% DBU, 96% DMF ACO 4. DCI, NMI, ACN O Phosphoramidite 60 o DNA/RNA Z 5. Capping AcO OAc v AcO 0 NC P\O 0 CN \/\/\/\I \/\/0 AcHN f 0 av o I O O’P\ 0 / O\/\/ \P20 O I 87 QgerN!‘ t-BuOOHDCAOligo synthesis NA automated synthesizer)CappingOxidationEt3NzCH3CN (1:1, V/V) AcHN \/\/\/\ O o- " O$O\ 88 ??/Ow/w 0 OAC 0' \ -o\ ,0/\/\0 o Aco DMT—-/R\ Q,, | NCN NHAc 0 , 3, UNL—o—g—o HO NH4,55°C AcHN 0 HO OH \1\\\ ,0 o 07P\ Wherein GalNAC3-4 has the structure: HO OH ACHN O HO OH ’0 HO o 075 O o o \/\/0 ACHN \/\/\/\ o 0- Hogoe/OMOH O HO /o/\/\O NHAC E The GalNAc3 cluster portion of the conjugate group 3-4 (GalNAc3-4a) can be combined with any cleavable moiety to provide a variety of ate groups. Wherein GalNAc3-4a has the formula: HO OH ACHN O HO OH [/0 HO o O7_P\ AcHN \/\/\/\ O 0' O’lg\ O /_ 0\/\/ O\P/—O The protected Unylinker ?Jnctionalized solid t Compound 30 is commercially available.

Compound 84 is ed using procedures similar to those reported in the literature (see Shchepinov er al., Nucleic Acids Research, 1997, , 4447-4454; Shchepinov er al., Nucleic Acids ch, 1999, 27, 3035-3041; and Hornet er al., Nucleic Acids Research, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are prepared as per the ures illustrated in Examples 28 and 36. The phosphoramidites illustrated are meant to be representative and not ed to be limiting as other phosphoramidite building blocks can be used to prepare an oligomeric compound having a phosphodiester linked conjugate at the 3 ’ terminus With a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare the oligomeric compounds as bed herein having any predetermined sequence and composition.

Example 41: General method for the preparation of ASOs comprising a phosphodiester linked GalNAc3-2 (see Example 37, Bx is adenine) conjugate at the 5’ position via solid phase techniques (preparation of ISIS 661134) Unless otherwise stated, all reagents and solutions used for the sis of oligomeric compounds are purchased from commercial sources. Standard phosphoramidite ng blocks and solid support are used for incorporation nucleoside residues Which include for example T, A, G, and mC residues.

Phosphoramidite compounds 56 and 60 were used to size the phosphodiester linked GalNAc3-2 conjugate at the 5 ’ terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for [3- D-2’-deoxyribonucleoside and 2’-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 umol scale) or on GE Healthcare Bioscience AKTA oligopilot sizer (40-200 umol scale) by the phosphoramidite ng method on VIMAD solid support (110 umol/g, Guzaev er al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered at a 4 fold excess over the initial loading of the solid support and phosphoramidite coupling was carried out for 10 min. All other steps followed standard protocols supplied by the cturer. A solution of 6% dichloroacetic acid in toluene was used for removing the oxytrityl (DMT) groups from 5’-hydroxyl groups of the nucleotide. 4,5 -Dicyanoimidazole (0.7 M) in anhydrous CH3CN was used as activator during the coupling step. Phosphorothioate linkages were introduced by sul?Jrization with 0.1 M solution of ne hydride in 1:1 pyridine/CH3CN for a contact time of 3 minutes. A solution of 20% tert-butylhydroperoxide in CH3CN containing 6% water was used as an oxidizing agent to provide phosphodiester intemucleoside linkages with a t time of 12 s.

After the d sequence was assembled, the cyanoethyl ate protecting groups were deprotected using a 20% diethylamine in toluene (v/v) with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous a (28-30 wt %) and heated at 55 0C for 6 h.

The unbound ASOs were then filtered and the ammonia was boiled off. The residue was purified by high pressure liquid chromatography on a strong anion exchange column (GE Healthcare Bioscience, Source 30Q, 30 um, 2.54 x 8 cm, A = 100 mM um acetate in 30% aqueous CH3CN, B = 1.5 M NaBr in A, 0- 40% ofB in 60 min, ?ow 14 mL min-1, )t = 260 nm). The residue was desalted by HPLC on a reverse phase column to yield the desired ASOs in an isolated yield of 15-30% based on the initial loading on the solid support. The ASOs were characterized by ion-pair-HPLC coupled MS analysis with Agilent 1 100 MSD system.

Table 34 ASO comprising a odiester linked GalNAc3-2 conjugate at the 5’ position targeting SRB-l Observed SEQ ID ISIS No. ce (5 , to 3 ,) CalCd Mass GalNAC3-2a'o'‘AdOTksmCkslAdsCIdsTdsmCdsAdsTds 661134 6482.2 6481.6 2302 Gds sTdsTkska Subscripts: "e" indicates 2’-MOE modified nucleoside; "d" indicates B-D-2’- deoxyribonucleoside; "k" indicates 6’-(S)-CH3 bicyclic nucleoside (e.g. cEt); "s" indicates phosphorothioate intemucleoside linkages (PS); "0" indicates phosphodiester intemucleoside linkages (PO); and "0’" indicates -O-P(=O)(OH)-. Superscript "m" indicates 5-methylcytosines. The structure of GalNAc3-2a is shown in Example 37.

Example 42: General method for the preparation of ASOs comprising a 3-3 conjugate at the 5’ position via solid phase techniques (preparation of ISIS 661166) The sis for ISIS 661166 was performed using similar procedures as illustrated in Examples 39 and 41.

ISIS 661166 is a 55 MOE gapmer, wherein the 5’ position ses a GalNA03-3 conjugate.

The ASO was characterized by ion-pair-HPLC coupled MS analysis with Agilent 1100 MSD system.

Table 34a ASO comprising a GalNAc3-3 conjugate at the 5’ position via a heiq'lamino phosphodiester e targeting l ISIS Conjugate Calcd Observed S'GalNAc3'3a--o’’mCesGesG?TesGes 661166 mmCdsAdsAdSGdsGdsCdsTdSTdSAdSGds 5’-GalNAc3-3 8992.16 8990.51 2303 GmAeWA TeseT Subscripts: "e" indicates 2’-MOE modi?ed nucleoside; "(1" indicates B-D-2’-deoxyribonucleoside; 6; 599’indicates phosphorothioate internucleoside es (P S); a; 099’indicates phosphodiester intemucleoside linkages (PO); and "o "’ indicates -O-P(=O)(OH)—. Superscriptm"m" indicates 5-methylcytosines. The structure of "5 ’-Ga1NA03-3a" is shownin Example 39.

Example 43: Dose-dependent study of phosphodiester linked GalNAc3-2 (see examples 37 and 41, Bx is adenine) at the 5’ terminus targeting SRB-l in vivo ISIS 661134 (see Example 41) sing a odiester linked GalNA03-2 ate at the 5 ’ us was tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 440762 and 651900 (GalNA03-1 conjugate at 3’ terminus, see Example 9) were included in the study for ison and are described previously in Table 17.

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 661134 or with PBS treated l. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-l mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PB S-treated control. The results below are presented as the average t of SRB-l mRNA levels for each treatment group, normalized to PB S-treated control and is denoted as "% PB S". The EDsos were measured using similar methods as described previously and are presented below.

As illustrated in Table 35, ent with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc3-2 conjugate at the 5 ’ terminus (ISIS 661134) or the GalNAc3-1 ate linked at the 3’ us (ISIS 651900) showed substantial improvement in potency compared to the unconjugated antisense ucleotide (ISIS 440762). Further, ISIS 661134, which comprises the phosphodiester linked GalNAc3-2 conjugate at the 5 ’ terminus was equipotent compared to ISIS 651900, which comprises the GalNAc3-1 conjugate at the 3 ’ terminus.

Table 35 A805 containing GalNAc3-1 0r GalNAc3-2 targeting SRB-l ISIS Dosage SRB-l mRNA ED50 Conjugate SEQ ID NO' No. (mg/kg) levels (% PBS) (mg/kg) PBS 0 100 -- -- 0.2 1 16 0.7 91 440762 2 69 2.58 No conjugate 2298 7 22 5 0.07 95 0.2 77 651900 0.7 28 0.26 3’ GalNAc3-1 2299 2 1 1 7 8 0.07 107 0.2 86 661134 0.7 28 0.25 5’ GalNAc3-2 2302 2 10 7 6 Structures for 3 ’ 3-1 and 5’ GalNAc3-2 were described previously in Examples 9 and 37.

Pharmacokinetics Analysis (PK) The PK of the ASOs from the high dose group (7 mg/kg) was examined and evaluated in the same manner as illustrated in Example 20. Liver sample was minced and extracted using standard protocols. The full length metabolites of 661134 (5 ’ GalNAc3-2) and ISIS 651900 (3 ’ GalNAc3-1) were ?ed and their masses were confirmed by high resolution mass spectrometry analysis. The results showed that the major metabolite detected for the ASO sing a phosphodiester linked GalNAc3-2 ate at the 5 ’ terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a able level, were observed. Unlike its counterpart, additional lites similar to those reported previously in Table 23a ’ terminus (ISIS 651900). These results were observed for the ASO having the GalNAc3-1 conjugate at the 3 t that having the phosphodiester linked GalNAc3-1 or GalNAc3-2 conjugate may improve the PK pro?le ofASOs without compromising their potency.

Example 44: Effect of PO/PS linkages on antisense inhibition of ASOs sing 3-1 conjugate (see Example 9) at the 3’ terminus targeting SRB-l ISIS 655861 and 655862 sing a GalNA03-1 conjugate at the 3 ’ terminus each targeting SRB-l were tested in a single administration study for their ability to inhibit SRB-1 in mice. The parent unconjugated compound, ISIS 353382 was included in the study for ison.

The ASOs are 55 MOE gapmers, wherein the gap region comprises ten 2’-deoxyribonucleosides and each wing region comprises five 2’-MOE ed nucleosides. The ASOs were prepared using similar methods as illustrated previously in Example 19 and are described Table 36, below.

Table 36 Modi?ed ASOs comprising GalNAc3-1 conjugate at the 3’ us targeting SRB-l Chemistry ISIS No. Sequence (5’ to 3’) SEQ oarent) GCdST:TeSInCesmCesTesT ngdsTdsTesCesmCesTesTeoAd0G—alNAc3-1a Full PS With 23 05 655 861G?mC?TeSTeSmCesAdsGdsTdsmCdsAdsTdsGdsAds GalNAc3-1 conjugate - G?mCMTeoTmCeoAdSGdSTdsmCdsAdSTdsGdsAds Mixed PS/PO with 2305 655862 mCdsFl-‘dsFl-‘eomceomCeSTmTeolAdo''GalNAc3'1a GalNAC3-1 conjUgate Subscripts: "e" indicates 2’-MOE modi?ed nucleoside; "d" indicates B-D-2’-deoxyribonucleoside; 6; 599’indicates phosphorothioate internucleoside linkages (P S); a; dicates phosphodiester intemucleoside linkages (PO); and "o "’ indicates -O-P(=O)(OH)—. Superscriptm"m" indicates ylcytosines. The structure of "GalNA03-1" is n e 9.

Trealmenl Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS , 655861, 655 862 or with PBS treated control. Each treatment group ted of 4 animals. Prior to the treatment as well as after the last dose, blood was drawn from each mouse and plasma samples were analyzed. The mice were sacri?ced 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using een), prior to normalization to PB S-treated control. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to PB S-treated control and is denoted as "% PB S". The EDsos were measured using similar methods as described previously and are reported below.

As illustrated in Table 37, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner compared to PBS treated control. Indeed, the antisense oligonucleotides comprising the GalNAc3-1 conjugate at the 3’ terminus (ISIS 655861 and 655862) showed substantial improvement in potency comparing to the unconjugated antisense oligonucleotide (ISIS 353382). Further, ISIS 655862 with mixed PS/PO linkages showed an improvement in y relative to ?ll PS (ISIS 655861).

Table 37 Effect of PO/PS linkages 0n nse inhibition of A805 comprising GalNAc3-1 conjugate at 3’ terminus targeting SRB-l ISIS Dosage SRB-l mRNA EDso Chemistry SEQ ID No.

No. (mg/kg) levels (% PBS) (mg/kg) PBS 0 100 3 76.65 353382 Full PS t 52.40 10.4 2304 (parent) conjugate 24.95 0.5 81.22 Full PS with GalNAc3-1 1'5 63.51 655 861 2.2 conjugate 2305 24.61 14.80 0.5 69.57 1'5 45.78 Mixed PS/PO with 655862 1.3 2305 19.70 GalNAc3-1 conjugate 12.90 Liver minase , alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Organ weights were also evaluated. The results demonstrated that no elevation in transaminase levels (Table 38) or organ weights (data not shown) were observed in mice treated with ASOs compared to PBS control. Further, the ASO with mixed PS/PO linkages (ISIS 655862) showed similar minase levels compared to ?lll PS (ISIS 655861).

Table 38 Effect of PO/PS linkages 0n transaminase levels of A805 sing GalNAc3-1 conjugate at 3’ terminus targeting SRB-l ISIS Dosage ALT AST Chemistry. SEQ ID No.

No. (m /k ) (U/L) (U/L) PBS 0 28.5 65 -- 3 50.25 89 (353:3?) . 27.5 79.3 uw?out 2304 p J g 27.3 97 0.5 28 55.7 1.5 30 78 Full PS with 655861 2305 29 63.5 GalNAc3-1 28.8 67.8 655862 0.5 50 75.5 Mixed PS/PO with 2305 GalNAcs-l Example 45: Preparation ofPFP Ester, Compound 110a HOWNE’ Pd/C H n OAC OAc , 2 OAc OOAC ?OWN3 EtOAc, MeOH 103a; n=1 AcO 103b; n= 7 A00 a ACHN N 104a; n=1 7/0 104b; n= 7 4 OAc ACO%OAcAcHN 0 o OAc OAc OAc Aco?/O OAc WW" 0 WW PFPTFA o n —,AcO AcHN OWNH DMF, pyr AcHN n N02 105a; n=1 Compound 90 0 105b; n= 7 OOACWHN A00 0 O 106a; n=1 106b; n= 7 Aco\%OACAcHN 0 o OAc OAc WM Ra-Ni, H2 0 HBTU, DIEA, DMF MeOH, EtOAc AcHN n 2 OAc OAc HOgCAHJkO’Bn o 2 HN o AcHN 99 Aco\%OAcAcHN 0 o OAc OAc W" A00 OW AcHN n NH OAc OAc Aco?/OO HN O 108a; n=1 O 108b;n=7 | ACO%WOAc Pd/C, H2, A 108a; n=1 EtOAc, MeOH 108b; n= 7 AcO AcHN?/CWnWNHACOW 109a; n= 1 109b; n= 7 ACO%OAcAcHN OW OAc OAc N AcO o\/\/\/\NH PFPTFA, DMF, o OAc OAc O O 109a AcO 110a o F F F F F Compound 4 (9.5g, 28.8 mmoles) was treated with compound 103a or 103b (38 moles), individually, and TMSOTf (0.5 eq.) and molecular sieves in dichloromethane (200 mL), and stirred for 16 hours at room temperature. At that time, the organic layer was filtered thru celite, then washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, ?ltered and reduced under reduced pressure. The resultant oil was purified by silica gel tography (2%-->10% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. LCMS and proton NMR was consistent with the structure. nds 104a and 104b were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 105a and 105b in >90% yield. LCMS and proton NMR was consistent with the structure. nds 105a and 105b were d, individually, with compound 90 under the same conditions as for compounds 901a-d, to give compounds 106a (80%) and 106b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 106a and 106b were treated to the same conditions as for compounds 96a-d (Example 47), to give 107a (60%) and 107b (20%). LCMS and proton NMR was consistent with the structure.

Compounds 107a and 107b were treated to the same conditions as for compounds 97a-d (Example 47), to give compounds 108a and 108b in 40-60% yield. LCMS and proton NMR was consistent with the Compounds 108a (60%) and 108b (40%) were treated to the same conditions as for compounds 100a- d (Example 47), to give compounds 109a and 10% in >80% yields. LCMS and proton NMR was consistent with the structure.

Compound 109a was treated to the same conditions as for compounds 101a-d (Example 47), to give Compound 110a in 30-60% yield. LCMS and proton NMR was consistent with the ure. atively, Compound 110b can be prepared in a similar manner starting with Compound 10%.

Example 46: l Procedure for Conjugation with PFP Esters (Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc3-10) A 5 ’-hexylamino modified oligonucleotide was synthesized and puri?ed using standard solid-phase oligonucleotide procedures. The 5’-hexylamino modi?ed oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 uL) and 3 equivalents of a selected PFP esteri?ed 3 cluster dissolved in DMSO (50 uL) was added. Ifthe PFP ester precipitated upon addition to the A80 on DMSO was added until all PFP ester was in solution. The reaction was complete after about 16 h ofmixing at room temperature. The resulting solution was diluted with water to 12 mL and then spun down at 3000 rpm in a spin ?lter with a mass cut off of 3000 Da. This process was repeated twice to remove small le impurities. The solution was then lyophilized to dryness and redissolved in trated aqueous ammonia and mixed at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia.

The ated oligonucleotide was puri?ed and desalted by RP-HPLC and lyophilized to provide the GalNAc3 conjugated oligonucleotide.

HO OH 0 83a 3, 5, H AcHN 0W4% OLIGO O_(CH2)6'NH2 OH OH 110a _, Ho?/OWNHON 1. Borate buffer, DMSO, pH 8.5, rt AcHN ZNHwan GONZO m-m—o/\W\NH 111 Oligonucleotide 111 is conjugated with 3-10. The GalNAc3 cluster portion of the conjugate group 3-10 (GalNAc3-10a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In n embodiments, the cleavable moiety is -P(=O)(OH)—Ad-P(=O)(OH)- as shown in the ucleotide (ISIS 666881) synthesized with 3-10 below. The structure of GalNAc3-10 (GalNAc3-10a-CM-) is shown below: Following this l procedure ISIS 666881 was prepared. 5’-hexylamino modi?ed oligonucleotide, ISIS 660254, was synthesized and puri?ed using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 umol) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 uL) and 3 equivalents PFP ester und 110a) dissolved in DMSO (50 uL) was added. The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 uL) to ?Jlly dissolve the PFP ester. The reaction was complete after 16 h of mixing at room temperature. The solution was diluted with water to 12 mL total volume and spun down at 3000 rpm in a spin ?lter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was lyophilized to dryness and redissolved in concentrated aqueous a with mixing at room temperature for 2.5 h followed by concentration in vacuo to remove most of the ammonia. The conjugated oligonucleotide was purified and desalted by RP-HPLC and lyophilized to give ISIS 666881 in 90% yield by weight (42 mg, 4.7 umol).

Table 383 3-10 conjugated oligonucleotide NH2(CH2)6'oAdoGesmCesTesTesmCesAdsGdsTds ISIS 660254 Hexylamine~ 2306 mCdsAdsTdsGdsAdsmCdsTdsTesmC?mcesTesTe GalNAc3'1Oa'o’AdoGesmCesTesTesmCesAdsGdsTds ISIS 666881 GalNAc3-10 2306 mCdsAdsTdsGdsAdsmCdsTdsTesmC?mCesTesTe Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE ed nucleoside; "d" indicates a B-D-2’-deoxyribonucleoside; "s" indicates a phosphorothioate internucleoside linkage (PS); "0" indicates a phosphodiester internucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc3-8 a/n\NHBoc BocHNAMA O 91a; n= 1 91b n—2 BocHNMNH N02 M, PFPTFA, DIPEA, DMF BocHNWHNn 92a; n=1 92b,n=2 WNH N02 TMSOTf DCM ; OOAC AcHN 3 HZNWVHN o 93a; n=1 93b,n=2 94a; m=1 94b, m=2 o 0A0 W68" O0Ac 0 HO m AcO —> AcHN OMOHm N 0 7/0 TMSOTf 7-m=1 Pd/C. H2 64 m=2 AACI:%:CWV" N’NAN 0 —.93a(93b) $0AWN"HAN" Ra-Ni, H2 A00 —> HBTU, DIPEA, DMF N02 Aco?/OOAcOMNWHN O AcHN n 96a; n=1, m=1 96b; n=1, m=2 96c; n=2, m=1 96d: n=2. m=2 %?WM 0 HBTU DIEA DMF Aco?/OOAcAWN"HAM-I NH2 AcHN o ODMTr Aco\@04[\\/OOMNWHNOAc HO O )7 AcHN n N o , O "OH 973; n=1, m=1 97b; n=1, m=2 97c; n=2, m=1 97d; n=2 m=2 AACI:%VACOWN Aco‘ggch"MAN: NONEomAc>HNO 00A0 H AcO MN 983; n=1, m=1 98b; n=1, m=2 980; n=2, m=1 98d; n=2 m=2 97a; n=1, m=1 ACKWVo HBTU, DIEA, DMF $0AWN"HAM-1 MOOAc 0 97b; n=1, m=2 —> 97c; n=2, m=1 AcO N O H 0‘ 97d ; n=2, m=2 AcHNO Bn HOZC o’ OAC 99 Aco?/OOMNWHN O AcHN n 100a; n=1, m=1 100b; n=1, m=2 100c; n=2, m=1 OAc 100d; n=2, m=2 Pd(OH)2/C, o 0 H2, EtOAc, OOAC PFPTFA, DMF, —MEQH—> AcO OWN"HAM-11MN OH Jab, AcHNO Aco?/OOAcOMNWHN 0 101a ":1 "‘21 AcHN n 101b; n=1, m=2 O 101c; n=2, m=1 101d; n=2, m=2 ARK:WN Ac0%oW""HANHOAc WN OMF F F m o F AcHNO Aco?/O F OMB]WHN 102a; n=1, m=1 AcHN 102b; n=1, m=2 102c; n=2, m=1 102d; n=2, m=2 The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N-Diisopropylethylamine (12.35 mL, 72 moles). Penta?uorophenyl tri?uoroacetate (8.9 mL, 52 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. Boc-diamine 91a or 91b (68.87 mmol) was added, along with N,N—Diisopropylethylamine (12.35 mL, 72 moles), and the reaction was allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium onate, water and brine. The c layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-->1 0% ol/dichloromethane) to give nds 92a and 92b in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

Compound 92a or 92b (6.7 mmoles) was treated with 20 mL of romethane and 20 mL of tri?uoroacetic acid at room temperature for 16 hours. The resultant solution was evaporated and then dissolved in methanol and treated with DOWEX-OH resin for 30 minutes. The resultant solution was filtered and d to an oil under reduced pressure to give 85-90% yield of compounds 93a and 93b.

Compounds 7 or 64 (9.6 mmoles) were treated with HBTU (3.7g, 9.6 mmoles) and MNDiisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compounds 93a or 93b (3 moles), and allowed to stir at room temperature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, d and reduced to an oil under reduced pressure. The ant oil was puri?ed by silica gel chromatography (5%-->20% methanol/dichloromethane) to give compounds 96a-d in 20-40% yield.

LCMS and proton NMR was consistent with the structure. nds 96a-d (0.75 mmoles), individually, were hydrogenated over Raney Nickel for 3 hours in Ethanol (75 mL). At that time, the catalyst was removed by ?ltration thru celite, and the l removed under reduced pressure to give nds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the structure.

Compound 23 (0.32g, 0.53 mmoles) was treated with HBTU (0.2g, 0.53 mmoles) and N,N— Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30mL) for 15 minutes. To this was added compounds 97a-d (0.38 mmoles), individually, and allowed to stir at room temperature for 16 hours. At that time, the DMF was d by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, ?ltered and reduced to an oil under reduced pressure.

The resultant oil was puri?ed by silica gel chromatography (2%-->20% methanol/dichloromethane) to give compounds 98a-d in 30-40% yield. LCMS and proton NMR was consistent with the structure.

Compound 99 (0.17g, 0.76 mmoles) was treated with HBTU (0.29 g, 0.76 mmoles) and N,N— Diisopropylethylamine (0.35 mL, 2.0 mmoles) in DMF (50mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles), individually, and allowed to stir at room ature for 16 hours. At that time, the DMF was reduced by >75% under reduced pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, ?ltered and reduced to an oil under reduced re.

The resultant oil was puri?ed by silica gel chromatography (5%-->20% ol/ romethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR was consistent with the structure. nds 100a-d (0.16 mmoles), dually, were hydrogenated over 10% Pd(OH)2/C for 3 hours in methanol/ethyl acetate (1 :1, 50 mL). At that time, the catalyst was removed by ion thru celite, and the organics removed under d pressure to give compounds 101a-d in 80-90% yield. LCMS and proton NMR was consistent with the structure.

Compounds 101a-d (0.15 mmoles), individually, were dissolved in DMF (15 mL) and pyridine (0.016 mL, 0.2 mmoles). Penta?uorophenyl tri?uoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the reaction was d to stir at room temperature for 30 minutes. At that time, the DMF was d by >75% under d pressure, and then the mixture was dissolved in dichloromethane. The organic layer was washed with sodium bicarbonate, water and brine. The organic layer was then separated and dried over sodium sulfate, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%-->5% ol/dichloromethane) to give compounds 102a-d in an approximate 80% yield. LCMS and proton NMR were consistent with the structure. 3' 5' ? -o-F|>-o-(CH2)6 NH2 Borate buffer, DMSO, pH 8.5, [1 102d —> 2. aq. ammonia, rt HoOH o o O/\(V)JL N ACHN o o HOOH O O EE JJ\/\/U\ O OMLHVH N NW CM H 4 O .

HoOH o Ho%O OAWLN 0 4 HAM?" Oligomeric Compound 102, comprising a GalNAc3-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The 3 cluster portion of the ate group GalNAc3- 8 (GalNAc3-8a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a preferred embodiment, the cleavable moiety is -P(=O)(OH)—Ad-P(=O)(OH)-.

The structure of GalNAc3-8 (GalNAc3-8a-CM-) is shown below: HO H O O O WAN HO 4 HA$§\M Hg§:$% c/jjkN/f?/\NH 0 w M M/dq:\o END 3 HO 4 H 2 H O HoOH o Homowmwm O Example 48: Preparation of Oligonucleotide 119 Comprising 3-7 ACO OAC A00 OAC O 0 A00 TMSOTf, DCE AcO OWNHCBZ Pd(OH)2/C —> 4 —’ O HowNHCBz ACHN H2, MeOH, EtOAc NW\ 3 4 35b 112 HO\H/\\ HBTU, DIEA ACO OAC O O Aco%¢o\/H\/NH2 DMF HO\n/\/ \gro NHCBZ —.

ACHN 0 1053 3‘; A00 0A0 AcO?Q/OWNHo O A00 OAC ACO%OWNWO%NHCBZo H O O 0 A00 OAC P ACO%Q/o\/P‘Z\/NHO AcO OAC o H 0 A00 W A HNC AcO OAc Pd/C, H2, O O 0 A00 OAC p ACO%OVH4\/NHO A00 OAc A00 OWN 0 HBTU, DIEA, DMF A HNC o o AcO OAc —>ACO$WO\/H4\/NH\H/\/O\%NHOO WOBH O O HO 0Q M O AcO OAc O O 0 H A00 OWNH Compound 112 was synthesized following the procedure bed in the literature (J. Med.

Chem. 2004, 47, 5798-5808).

Compound 112 (5 g, 8.6 mmol) was dissolved in 1:1 methanol/ethyl acetate (22 mL/22 mL).

Palladium hydroxide on carbon (0.5 g) was added. The reaction mixture was stirred at room temperature under hydrogen for 12 h. The reaction mixture was ?ltered through a pad of celite and washed the pad with 1:1 methanol/ethyl acetate. The filtrate and the washings were combined and concentrated to dryness to yield Compound 105a (quantitative). The structure was med by LCMS.

Compound 113 (1.25 g, 2.7 mmol), HBTU (3.2 g, 8.4 mmol) and DIEA (2.8 mL, 16.2 mmol) were ved in anhydrous DMF (17 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 105a (3.77 g, 8.4 mmol) in anhydrous DMF (20 mL) was added. The reaction was stirred at room temperature for 6 h. Solvent was removed under reduced pressure to get an oil. The residue was dissolved in CH2C12 (100 mL) and washed with s saturated NaHCO3 solution (100 mL) and brine (100 mL). The organic phase was ted, dried (NaZSO4), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 10 to 20 % MeOH in dichloromethane to yield Compound 114 (1.45 g, 30%). The structure was con?rmed by LCMS and 1H NMR analysis.

Compound 114 (1.43 g, 0.8 mmol) was dissolved in 1:1 methanol/ethyl acetate (4 mL/4 mL).

Palladium on carbon (wet, 0.14 g) was added. The on mixture was ?ushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction e was ?ltered through a pad of celite. The celite pad was washed with ol/ethyl acetate (1 :1). The e and the washings were combined together and evaporated under reduced pressure to yield Compound 115 (quantitative). The structure was con?rmed by LCMS and 1H NMR analysis.

Compound 83a (0.17 g, 0.75 mmol), HBTU (0.31 g, 0.83 mmol) and DIEA (0.26 mL, 1.5 mmol) were dissolved in anhydrous DMF (5 mL) and the reaction mixture was stirred at room temperature for 5 min. To this a solution of Compound 115 (1.22 g, 0.75 mmol) in anhydrous DMF was added and the reaction was stirred at room temperature for 6 h. The solvent was removed under reduced pressure and the residue was dissolved in CHZClz. The organic layer was washed aqueous saturated NaHCO3 solution and brine and dried over anhydrous NaZSO4 and ?ltered. The organic layer was concentrated to dryness and the residue obtained was purified by silica gel column chromatography and eluted with 3 to 15 % MeOH in romethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and 1H NMR is.

AcO OAc 3 /o\/(,\)4\/ H?o ACHN O Pd/C, H2, AcO 0Ac EtOAC MeOH 116 , o WOH ACO0%OWNHWOf?iNH AcO OAc Q AcO$¢O\/H4\/NH0 117 AcO OAc AcO$¢OWN?OHo F 4 F PFPTFA, DMF, Pyr AcO OAc — MOP F AcomOW/NHWO024w AcO OAc H ACOE\:IQO /O\/H4\/NH 118 Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethyl e (5 mL/S mL).

Palladium on carbon (wet, 0.074 g) was added. The reaction mixture was ?ushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was ?ltered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1 :1). The te and the washings were combined er and evaporated under reduced pressure to yield compound 117 (0.73 g, 98%). The structure was ed by LCMS and 1H NMR analysis.

Compound 117 (0.63 g, 0.36 mmol) was dissolved in anhydrous DMF (3 mL). To this solution N,N- Diisopropylethylamine (70 uL, 0.4 mmol) and penta?uorophenyl tri?uoroacetate (72 uL, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 h and poured into a aqueous saturated NaHC03 solution. The e was extracted with dichloromethane, washed with brine and dried over anhydrous NaZSO4. The dichloromethane solution was concentrated to dryness and purified with silica gel column chromatography and eluted with 5 to 10 % MeOH in dichloromethane to yield compound 118 (0.51 g, 79%). The structure was con?rmed by LCMS and 1H and 1H and 19F NMR. 3' 5' ('3' -O—F|’—O-(CH2)6-NH2 1. Borate buffer, DMSO, pH 8.5, rt 118 —> 2. aq. a, rt HO%HOOH H$¢Wvgw?om HO$¢OA?fQO o 119 Oligomeric Compound 119, comprising a GalNAc3-7 conjugate group, was prepared using the general procedures rated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3- 7 (GalNAc3-7a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In n embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The structure of GalNAc3-7 (GalNAc3-7a-CM-) is shown below: HoOH o HO 4 ")1 Hog/OW"O O M "W0 3 AcHN O O /ij Hog/O M O Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc3-5 k1tldr O Boc\ 30:1;1ij OH Boc\N H HBTU TEA UOH H20 DMF MeOH, THF HN\ 120 122 78%) 123 Compound 120 (14.01 g, 40 mmol) and HBTU (14.06 g, 37 mmol) were dissolved in anhydrous DMF (80 mL). Triethylamine (11.2 mL, 80.35 mmol) was added and stirred for 5 min. The reaction mixture was cooled in an ice bath and a solution of compound 121 (10 g, mmol) in anhydrous DMF (20 mL) was added. Additional triethylamine (4.5 mL, 32.28 mmol) was added and the reaction mixture was d for 18 h under an argon atmosphere. The reaction was red by TLC (ethyl acetate:hexane; 1:1; Rf = 0.47).

The solvent was removed under reduced re. The residue was taken up in EtOAc (300 mL) and washed with 1M NaHSO4 ( 3 x 150 mL), aqueous saturated NaHC03 solution (3 x 150 mL) and brine (2 x 100 mL).

Organic layer was dried with NaZSO4. Drying agent was d by ?ltration and organic layer was concentrated by rotary evaporation. Crude mixture was puri?ed by silica gel column chromatography and eluted by using 35 — 50% EtOAc in hexane to yield a compound 122 (15.50 g, 78.13%). The structure was con?rmed by LCMS and 1H NMR analysis. Mass m/z 589.3 [M + H]+.

A solution of LiOH (92.15 mmol) in water (20 mL) and THF (10 mL) was added to a cooled on of Compound 122 (7.75 g,13.16 mmol) dissolved in methanol (15 mL). The reaction e was stirred at room temperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). The on mixture was concentrated to half the volume under reduced pressure. The ing solution was cooled an ice bath and neutralized by adding concentrated HCl. The reaction mixture was diluted, extracted with EtOAc (120 mL) and washed with brine (100 mL). An emulsion formed and cleared upon standing overnight. The organic layer was separated dried 4), filtered and evaporated to yield Compound 123 (8.42 g). Residual salt is the likely cause of excess mass. LCMS is consistent with structure. Product was used without any ?thher puri?cation. M.W.cal:574.36; M.W.fd:575.3 [M + H]+.

@?-OH' H20(P. ‘9 O o HsNWJxO Toluene, Reflux 6 124 125 126 99.6% Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem.

Soc. 2011, 133, 958-963). 126 BOG op CF3COOH 123 —> \N Obj/W0 —> HOBt DIEA C"'20'2 PyBop, Bop, DMF HN\ 127 CF3COO AcO OAC do %wOH H3N 3W0 AcHN 7 o CF3COO® —> HATU, HOAt, DIEA, DMF CF3COO' @NH3 128 AcO OAc A00 0 AcHN W0 AcOOAcOO:R?ji?wo0J1: Acok MO O AcO OAC Aco%O\/\/\[(NHo 129 AcHN o AcO OAc Aco%wOo o AcHN W Pd/C H M OHe ’ 2’ o A 0 OAcC H 0+ HN N o 0 HM AcO OAC Acog/ow0 NH AcO OAC AcHN o 130 PFPTFA, DMF, Pyr AcO OAC O:SD\KHO Acog/O NWOF AcO OAC AcO OW AcHN O Compound 123 (7.419 g, 12.91 mmol), HOBt (3.49 g, 25.82 mmol) and compound 126 (6.33 g, 16.14 mmol) were dissolved in and DMF (40 mL) and the resulting reaction mixture was cooled in an ice bath. To this isopropylethylamine (4.42 mL, 25.82 mmol), PyBop (8.7 g, 16.7 mmol) followed by Bop coupling reagent (1.17 g, 2.66 mmol) were added under an argon atmosphere. The ice bath was removed and the solution was allowed to warm to room temperature. The reaction was completed after 1 h as determined by TLC (DCM:MeOH:AA; 89:10:1). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc (200 mL) and washed with 1 M NaHSO4 (3x100 mL), aqueous saturated NaHC03 (3x100 mL) and brine (2x100 mL). The organic phase separated dried 4), ?ltered and concentrated. The residue was d by silica gel column chromatography with a gradient of 50% hexanes/EtOAC to 100% EtOAc to yield Compound 127 (9.4 g) as a white foam. LCMS and 1H NMR were consistent with structure. Mass m/z 778.4 [M + H] +.

Tri?uoroacetic acid (12 mL) was added to a solution of compound 127 (1.57 g, 2.02 mmol) in dichloromethane (12 mL) and stirred at room ature for 1 h. The reaction mixture was porated with toluene (30 mL) under reduced pressure to dryness. The residue obtained was co-evaporated twice with acetonitrile (30 mL) and toluene (40 mL) to yield Compound 128 (1.67 g) as tri?uoro acetate salt and used for next step without ?irther purification. LCMS and 1H NMR were consistent with structure. Mass m/z 478.2 [M + H] t.

Compound 7 (0.43 g, 0.963 mmol), HATU (0.35 g, 0.91 mmol), and HOAt (0.035 g, 0.26 mmol) were combined together and dried for 4 h over P205 under reduced pressure in a round bottom ?ask and then dissolved in anhydrous DMF (1 mL) and stirred for 5 min. To this a solution of compound 128 (0.20 g, 0.26 mmol) in anhydrous DMF (0.2 mL) and N,N-Diisopropylethylamine (0.2 mL) was added. The reaction mixture was stirred at room temperature under an argon atmosphere. The reaction was complete after 30 min as determined by LCMS and TLC (7% CM). The on mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO4 (3x20 mL), aqueous saturated NaHCO3 (3 x 20 mL) and brine (3x20 mL). The organic phase was separated, dried over NaZSO4, ?ltered and concentrated. The residue was puri?ed by silica gel column chromatography using 5-15% MeOH in dichloromethane to yield Compound 129 (96.6 mg). LC MS and 1H NMR are consistent with structure. Mass m/z 883.4 [M + 2H]+.

Compound 129 (0.09 g, 0.051 mmol) was ved in methanol (5 mL) in 20 mL scintillation vial.

To this was added a small amount of 10% Pd/C (0.015 mg) and the on vessel was ?ushed with H2 gas.

The on mixture was stirred at room ature under H2 atmosphere for 18 h. The reaction mixture was filtered through a pad of Celite and the Celite pad was washed with methanol. The filtrate washings were pooled together and concentrated under reduced pressure to yield Compound 130 (0.08 g). LCMS and 1H NMR were consistent with structure. The product was used without further purification. Mass m/z 838.3 [M + 2H]+.

To a 10 mL pointed round bottom ?ask were added compound 130 (75.8 mg, 0.046 mmol), 0.37 M pyridine/DMF (200 uL) and a stir bar. To this solution was added 0.7 M uorophenyl trifluoroacetate/DMF (100 uL) drop wise with stirring. The reaction was ted after 1 h as determined by LC MS. The solvent was removed under reduced pressure and the residue was dissolved in CHC13 (N 10 mL). The organic layer was partitioned t NaHSO4 (1 M, 10 mL) saturated NaHC03 (10 mL) , aqueous and brine (10 mL) three times each. The organic phase separated and dried over NaZSO4, filtered and trated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used without ?irther purification. Mass m/z 921.3 [M + 2H]+.

HO OH HO 0 . 5 . 3 H -O-F|’-O-(CH2)e-NH2 AcHN W0 1. Borate buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt HO$¢HOOH HDY HOOH NAMfOH Oligomeric nd 132, comprising a GalNAc3-5 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAcg- (GalNAc3-5a) can be combined with any cleavable moiety to provide a variety of conjugate . In certain ments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The structure of GalNAc3-5 (GalNAcg-Sa-CM-) is shown below: HO OH Ho%w00 o AcHN W HO OH N "N NH HO OM O HO OH 0 NH HO W NAM/\o—I—E O H 4 AcHN o e 50: Preparation of Oligonucleotide 144 Comprising GalNAc4-11 N ACN, VIMAD Resin N pIpZDBUiDMF —> —, O O 2. A020 Capping (222296) a,OUOH Kaiser: Negetive HN/Fmoc KC}H FmOC\N/\/\/\n/OHH O O DMTr\ 136 O HBTU, DIEA, DMF NH-Fmoc DMTr| 1. Ep_:DBUF:JDi\_/It_F i O 1. 2% hydrazine/DMF aiser: OSI Ive (CH2)5/N Nw—>Kaiser: Positive N 2. Dde-Lys(Fmoc)—OH (138) . O 2. Fmoc-Lys(Fmoc)—OH (140) HATU, DIEA, DMF O HATU, DIEA, DMF Kaiser: ve Kaiser: Negative O /Fmoc N \Fmoc AcO OAC ACHN OWNH A00 OAC ACHN OVV>\N N 1. pip:DBU:DMF O Kaiser: Posntlve 2. 7, HATU, DIEA, ACO OAC K ' alser:Nega Ivet' O H AcO WN O 0 A00 OAC ACHN O\/\/\n/NH Synthesis of Compound 134: To a Merrifield ?ask was added aminomethyl VIMAD resin (2.5 g, 450 umol/g) that was washed with acetonitrile, dimethylformamide, dichloromethane and acetonitrile. The resin was swelled in acetonitrile (4 mL). Compound 133 was pre-activated in a 100 mL round bottom ?ask by adding 20 (1.0 mmol, 0.747 g), TBTU (1.0 mmol, 0.321 g), acetonitrile (5 mL) and DIEA (3.0 mmol, 0.5 mL). This on was d to stir for 5 min and was then added to the Merrifield ?ask with shaking.

The suspension was allowed to shake for 3 h. The on mixture was drained and the resin was washed with acetonitrile, DMF and DCM. New resin loading was quantitated by measuring the absorbance of the DMT cation at 500 nm (extinction ient = 76000) in DCM and determined to be 238 umol/g. The resin was capped by suspending in an acetic anhydride solution for ten minutes three times.

The solid support bound compound 141 was synthesized using iterative Fmoc-based solid phase peptide sis methods. A small amount of solid support was withdrawn and suspended in aqueous ammonia (28-30 wt%) for 6 h. The cleaved compound was analyzed by LC-MS and the ed mass was consistent with structure. Mass m/z 1063.8 [M + 2H]+.

The solid support bound compound 142 was synthesized using solid phase peptide synthesis methods.

AcO OAC AcHN OV\/>\NH A00 OAC A00 OW H N o H DNA syntesizer O 142—> AcO OAC HO OH o H pH H ‘ aqueous NH3 0 —, 3 N HO OH 0 é o HO H NH | .cm ASO o o HO OH HO NH The solid support bound compound 143 was synthesized using standard solid phase synthesis on a DNA synthesizer.

The solid support bound compound 143 was suspended in aqueous ammonia (28-30 wt%) and heated at 55 0C for 16 h. The solution was cooled and the solid support was ?ltered. The e was concentrated and the residue dissolved in water and puri?ed by HPLC on a strong anion exchange column. The fractions ning ?Jll length compound 144 were pooled together and ed. The resulting GalNAc4-11 conjugated oligomeric compound was analyzed by LC-MS and the observed mass was consistent with structure.

The GalNAc4 r portion of the conjugate group 4-11 (GalNAc4-11a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)—Ad-P(=O)(OH)-.

The structure of GalNAc4-11 (GalNAc4-11a-CM) is shown below: HO OH AcHN O\/\/>\ HO OH \/\/O>\N O W»?H HO 0W -—§CM o o HO OH HO$WWNHAcHN Example 51: Preparation of Oligonucleotide 155 sing GalNAc3-6 ©Vo o H o o N NH2 BFQkOH \g/ W/j: ©/ OTNW/ZNQLOHH O OH 0 2M NaOH 0 Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54- H 0 35b 0 o i TMS—OTf, 4 A molecular sieves, , rt H Q 0 OTNdLOHH A00 OAC H2, Pd(OH)2 /C O O 147 —>ACO WNHZ EtOAc/MeOH ACHN 105a HBTU, DIEA, DMF, rt AcO OAC Acog/OWNJK/ \n/O\/© —>o H o H2, Pd(OH)2 /C, EtOAc/MeOH 148 0 A00 OAC O O\/\/\/\NJJ\/NH2 ACO Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH2C12 (200 ml). Activated molecular sieves (4 A. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under nitrogen atmosphere. TMS-OTfwas added (4.1 ml, 22.77 mmol) and the reaction was allowed to stir at room temp overnight. Upon completion, the reaction was quenched by pouring into solution of saturated aqueous NaHC03 (500 ml) and crushed ice (N 150 g). The organic layer was ted, washed with brine, dried over MgSO4, filtered, and was concentrated to an orange oil under d pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10 % MeOH in CH2C12 to yield Compound 112 (16.53 g, 63 %). LCMS and 1H NMR were consistent with the expected compound.

Compound 112 (4.27 g, 7.35 mmol) was dissolved in 1:1 MeOH/EtOAc (40 ml). The reaction mixture was purged by ng a stream of argon through the solution for 15 minutes. Pearlman’s catalyst dium hydroxide on carbon, 400 mg) was added, and en gas was bubbled through the solution for minutes. Upon completion (TLC 10% MeOH in CH2C12, and LCMS), the catalyst was removed by ?ltration through a pad of . The filtrate was concentrated by rotary evaporation, and was dried brie?y under high vacuum to yield Compound 105a (3.28 g). LCMS and 1H NMR were consistent with desired product.

Compound 147 (2.31 g, 11 mmol) was dissolved in anhydrous DMF (100 mL). N,N— ropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was d to stir for N 15 minutes under en. To this a solution of compound 105a (3.3 g, 7.4 mmol) in dry DMF was added and stirred for 2 h under nitrogen atmosphere. The reaction was diluted with EtOAc and washed with saturated aqueous NaHCO3 and brine. The organics phase was separated, dried (MgSO4), filtered, and concentrated to an orange syrup. The crude material was puri?ed by column chromatography 2-5 % MeOH in CH2C12 to yield Compound 148 (3.44 g, 73 %). LCMS and 1H NMR were consistent with the expected product.

Compound 148 (3.3 g, 5.2 mmol) was dissolved in 1:1 tOAc (75 ml). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman’s catalyst (palladium hydroxide on carbon) was added (350 mg). Hydrogen gas was bubbled through the solution for minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by ?ltration h a pad of celite. The te was concentrated by rotary evaporation, and was dried brie?y under high vacuum to yield Compound 149 (2.6 g). LCMS was consistent with desired product. The residue was dissolved in dry DMF (10 ml) was used immediately in the next step.

ACO OAC o o o NJK/H ACO W N A 0 OAc o C AcHN 3 H /U\ 0 0 O N A00 WNL"\p H AcHN 3 O 146 —> AGO OAc o HBTU DIEA DMF NH ' ' 0 OWN" A00 3 H A00 OAC o o o H ACO wNJK/N AcO OAc Pd(OH)2/C, H2 AcHN 3 —’ o III—I O N NH2 MeOH, EtOAc ACO$W \/\(«)/\NJ\/ \n/\NO AcHN 3 H A00 0A0 o O OWNM nd 146 (0.68 g, 1.73 mmol) was ved in dry DMF (20 ml). To this DIEA (450 uL, 2.6 mmol, 1.5 eq.) and HBTU (1.96 g, 0.5.2 mmol) were added. The reaction mixture was allowed to stir for 15 minutes at room temperature under nitrogen. A solution of nd 149 (2.6 g) in anhydrous DMF (10 mL) was added. The pH of the reaction was adjusted to pH = 9-10 by addition of DIEA (if necessary). The reaction was allowed to stir at room temperature under nitrogen for 2 h. Upon completion the reaction was diluted with EtOAc (100 mL), and washed with aqueous saturated aqueous NaHC03, followed by brine. The organic phase was separated, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography and eluted with 2-10 % MeOH in CH2C12 to yield Compound 150 (0.62 g, %). LCMS and 1H NMR were consistent with the d t.

Compound 150 (0.62 g) was dissolved in 1:1 MeOH/ EtOAc (5 L). The reaction mixture was purged by bubbling a stream of argon through the solution for 15 minutes. Pearlman’s catalyst (palladium hydroxide on carbon) was added (60 mg). Hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by ?ltration (syringe-tip Te?on ?lter, 0.45 um). The ?ltrate was concentrated by rotary evaporation, and was dried brie?y under high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent with the d product. The product was dissolved in 4 mL dry DMF and was used immediately in the next step. 3 H OBn 151 —, AcHN 3 H O PFP-TFA DIEA DMF ’ ’ AcO OAC o O OWN" AcO 3 H ACO OAC o o o H ACO m N AGO C AcHN 3 H O O o H Pd(OH)2/C, H2 0 N M MeOH, EtOAc AcHN 3 H O AcO OAC o Lfo Aco OAc AcHN 3 N o o F o i; H PFP-TFA, DIEA N M —>c 3 DMF AcHN 3 H O L’Co F AcO OAC o O /u\/ AcO o\/\M’\N Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and N,N- Diisopropylethylamine (75 uL, 1 mmol) and PFP-TFA (90 uL, 0.76 mmol) were added. The reaction mixture turned magenta upon contact, and gradually turned orange over the next 30 minutes. Progress of reaction was monitored by TLC and LCMS. Upon completion (formation of the PFP ester), a solution of nd 151 (0.57 g, 0.33 mmol) in DMF was added. The pH of the reaction was adjusted to pH = 9-10 by addition of isopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for N min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was d with CH2C12 and washed with aqueous saturated NaHC03, followed by brine. The organic phase separated, dried over MgSO4, ed, and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10 % MeOH in CH2C12) to yield Compound 152 (0.35 g, 55 %). LCMS and 1H NMR were consistent with the desired product.

Compound 152 (0.35 g, 0.182 mmol) was dissolved in 1:1 MeOH/EtOAc (10 mL). The reaction mixture was purged by bubbling a stream of argon thru the solution for 15 minutes. Pearlman’s catalyst dium hydroxide on ) was added (35 mg). Hydrogen gas was bubbled thru the solution for 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration (syringe-tip Te?on filter, 0.45 um). The filtrate was trated by rotary evaporation, and was dried brie?y under high vacuum to yield Compound 153 (0.33 g, quantitative). The LCMS was consistent with desired product.

Compound 153 (0.33 g, 0.18 mmol) was dissolved in anhydrous DMF (5 mL) with stirring under nitrogen. To this N,N-Diisopropylethylamine (65 uL, 0.37 mmol) and PFP-TFA (35 uL, 0.28 mmol) were added. The reaction e was stirred under nitrogen for N 30 min. The reaction mixture turned magenta upon t, and gradually turned orange. The pH of the reaction mixture was maintained at pH = 9-10 by adding more N,-Diisopropylethylamine. The progress of the reaction was monitored by TLC and LCMS.

Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH2C12 (50 mL), and washed with saturated aqueous NaHC03, followed by brine. The organic layer was dried over MgSO4, filtered, and trated to an orange syrup. The e was purified by column chromatography and eluted with 2-10 % MeOH in CH2C12 to yield Compound 154 (0.29 g, 79 %). LCMS and 1H NMR were consistent with the desired product. 3- 5' II HOOH O O-P-O-(CH ) NH 0 26 2 I HO OAHfN OH H A HNC HN 1. Borate buffer DMSO HOOH O 154 ' H pH 8.5 rt 0 ’ JV" ‘?/\N H Ho 041?" MNWO 2. aq. ammonia, rt 0 4 5 AcHN O O HoOH LN o HO oA92}N 0 AcHN Oligomeric Compound 155, comprising a GalNAC3-6 conjugate group, was prepared using the general procedures rated in e 46. The GalNAC3 cluster portion of the conjugate group GalNAC3- 6 (GalNAc3-6a) can be combined with any cleavable moiety to provide a y of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.

The ure of GalNAc3-6 (GalNAc3-6a-CM-) is shown below: Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc3-9 AcOOAc AcOOAc NOISE TMSOTf 50 oC ACO% AG AcHN CICHZCHZCI rt 93% TMSOTf DCE 66% 3 4W AcO OAC Aco$¢0WW —’ AcO OAc H2, Pd/C o MeOH, 95% Ac:%wOWOH10 AcHN AcHN o 156 157 HBTU, DMF, EtN(iPr)2 ' AcOOAC Phosphitylation ACOWWN DMTO 81% AcHN ODMT H5 47 NC .~ _P/ \N(iPr)2 ACOWWNAcOOAc AcHN ODMT Compound 156 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808). nd 156, (18.60 g, 29.28 mmol) was dissolved in methanol (200 mL). Palladium on carbon (6.15 g, 10 wt%, loading (dry basis), matrix carbon powder, wet) was added. The reaction mixture was d at room temperature under hydrogen for 18 h. The reaction mixture was ?ltered through a pad of celite and the celite pad was washed thoroughly with methanol. The combined filtrate was washed and concentrated to dryness. The residue was puri?ed by silica gel column chromatography and eluted with 5-10 % ol in dichloromethane to yield Compound 157 (14.26 g, 89%). Mass m/z 544.1 [M-H]'.

Compound 157 (5 g, 9.17 mmol) was dissolved in anhydrous DMF (30 mL). HBTU (3.65 g, 9.61 mmol) and N,N-Diisopropylethylamine (13.73 mL, 78.81 mmol) were added and the reaction mixture was stirred at room temperature for 5 minutes. To this a solution of compound 47 (2.96 g, 7.04 mmol) was added.

The reaction was stirred at room temperature for 8 h. The on mixture was poured into a saturated NaHC03 s on. The mixture was extracted with ethyl acetate and the c layer was washed with brine and dried (NaZSO4), d and evaporated. The residue obtained was puri?ed by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25g, 73.3%).

The structure was con?rmed by MS and 1H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P205 under reduced pressure. The dried compound was dissolved in ous DMF (50 mL). To this 1H-tetrazole (0.43 g, 6.09 mmol) and N- imidazole (0.3 mL, 3.81 mmol) and 2-cyanoethyl-N,N,N’ ,N’-tetraisopropyl phosphorodiamidite (3.65 mL, 11.50 mmol) were added. The reaction mixture was stirred t under an argon atmosphere for 4 h. The reaction mixture was diluted with ethyl acetate (200 mL). The reaction mixture was washed with saturated NaHC03 and brine. The organic phase was separated, dried (Na2804), filtered and evaporated. The residue was purified by silica gel column chromatography and eluted with 50-90 % ethyl acetate in hexane to yield Compound 159 (7.82 g, 80.5%). The structure was con?rmed by LCMS and 31P NMR analysis. 0 CW"? HO 9 O O ACHN O—Fl> OH 1.DNASynthesizer 159 oAWN? 2. aq. NH4OH Ho?‘vo 9 o o ACHN O‘FI’ OH HOMowqo9 . - Oligomeric Compound 160, comprising a 3-9 conjugate group, was prepared using standard ucleotide synthesis procedures. Three units of compound 159 were coupled to the solid support, followed by nucleotide oramidites. Treatment of the protected oligomeric compound with aqueous ammonia d compound 160. The GalNAc3 cluster portion of the conjugate group GalNAc3-9 (GalNAc3- 9,) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)—Ad-P(=O)(OH)-. The structure of GalNAc3-9 (GalNAcg- 9a-CM) is shown below: HoOH ‘ HOWOAWN?O o ACHN O—FI’ OH ACHN 0‘: OH Ho%/o 0%N9 0 20_-_‘,CM 3 Example 53: Alternate procedure for preparation of Compound 18 (GalNAcg-la and GalNAc3-3a) Lactone 161 was d with diamino propane (3 -5 eq) or Mono-Boc protected o propane (1 eq) to provide alcohol 162a or 162b. When unprotected propanediamine was used for the above reaction, the excess diamine was removed by evaporation under high vacuum and the free amino group in 162a was protected using Csz1 to e 162b as a white solid after puri?cation by column chromatography.

Alcohol 162b was further reacted with compound 4 in the ce of TMSOTf to provide 163a which was converted to 163b by removal of the Cbz group using catalytic hydrogenation. The penta?uorophenyl (PFP) ester 164 was prepared by reacting d 1 13 (see Example 48) with PFPTFA (3.5 eq) and pyridine (3 .5 eq) in DMF (0.1 to 0.5 M). The triester 164 was directly reacted with the amine 163b (3—4 eq) and DIPEA (3—4 eq) to provide Compound 18. The above method greatly facilitates purification of intermediates and zes the formation of byproducts which are formed using the procedure described in Example 4. é /\/\ H2N NHR H TMSOTf HO NwNHR R=Hoerz OAc 0 OAc 161 0 CszI, Et3N E, E: ga A00% 4 >\/o OAc PFPOh OAc o o AcO O\/\/\n/N\/\/NHR + NHAc PFPOjl/VOQ‘NHCBZ —> o o o o R = Cbz’ 163a Pd/C, H2 l— PFPOM —> R = H, 163b AC0 Olv?kl—IN H NHAc W OAc 77/\\ AcogQ/oOAc O O O J_H H NHCBZ 4 N\/\/Nm/\/O O o 0 Example 54: Alternate procedure for preparation of Compound 18 (GalNAcg-la and GalNAc3-3a) The triPFP ester 164 was prepared from acid 113 using the ure outlined in example 53 above and reacted with mono-Boc protected diamine to provide 165 in essentially quantitative yield. The Boc groups were removed with hydrochloric acid or tri?uoroacetic acid to provide the triamine which was reacted with the PFP ted acid 166 in the presence of a suitable base such as DIPEA to provide nd 18.

The PFP protected Gal-NAc acid 166 was prepared from the ponding acid by treatment with PFPTFA (1 -1 .2 eq) and pyridine (1 -1 .2 eq) in DMF. The precursor acid in turn was prepared from the corresponding alcohol by oxidation using TEMPO (0.2 eq) and BAIB in acetonitrile and water. The precursor alcohol was prepared from sugar intermediate 4 by reaction with 1,6-hexanediol (or 1,5-pentanediol or other diol for other n values) (2-4 eq) and TMSOTf using conditions described previously in example 47.

\ PFPTFA O PFPOWo DMF,pyr O O NHCBZ O NHCBZ HOZCN Jf "POW Jf o O O HO C2 \) PFPOM 113 H 164 /\/NW/\\O 1. HCI or TFA —. NHCBZ BocHN N —, W \n/V0% DIPEA M °A° BOCHN N A600% H 0% 165 NHAc Q: 166 O 0% 1. 1 6-hexanediol AcO H ’ 4 HN N or 1,5-pentane-dlol NHAc W TMSOTf + compound 4 OAc 77/\\ 2. TEMPO OAc O O O 3. PFPTFA, pyr o H H NHCBZ o o o Example 55: Dose-dependent study of oligonucleotides comprising either a 3' or 5'—conjugate group (comparison of GalNAc3-1, 3, 8 and 9) targeting SRB-l in vivo The ucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a standard. Each of the s GalNAc3 conjugate groups was attached at either the 3' or 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'—de0xyaden0sine nucleoside (cleavable moiety).

Table 39 Modi?ed ASO targeting SRB-l , , . . SEQ eeeeeeee eee> ISIS 3 5 3 3 82 GesmCesTesTesmCeSAdSGdSTdsmCdsAdsTdsGdSAds /10/5 2304 mCdsTdsTesmCmmCmTesTe (iesmCesTesTesmCesAdsGdsTdsmCdslAdsTdsGdsAds ISIS 655861 5/10/5 GalNAc3-l 2305 mCdsTdSTesmC?mCmTeSTeoAd,,,-G211NAc3-1 a CiesmCesTesTesmCesAdsGdsTdsmCdslAdsTdsGdsAds ISIS 664078 5/10/5 GalNAc3-9 2305 mCdsTdsTesmC?mCmTesTeoAAdo’-(;31N1§C3-9a GalNAc3-3a-O,Ad0 ISIS 661 161 GesmCesTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds 5/10/5 GalNAc3-3 2304 mCdsTdSTesmC?mCmTESTe GalNAc3-8a—yAdo ISIS 665 001 GesmCesTesTesmCeSAdsGdSTdsmCdsAdsTdsGdSAds 5/1 0/5 GalNA03-8 2304 InCdsTdSTesmC?mCmTESTe Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "d" tes a B-D-2’-deoxyribonucleoside; "s" indicates a orothioate intemucleoside linkage (PS); "0" indicates a phosphodiester cleoside e (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The ure of GalNAc3-1a was shown previously in Example 9. The structure of GalNAc3-9 was shown previously in Example 52. The structure of GalNAc3-3 was shown previously in Example 39. The structure of GalNAc3-8 was shown previously in Example 47.

Trealmenl Six week old male Balb/c mice on Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 664078, 661161, 665001 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA fication reagent (Molecular , Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 40, ent with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. Indeed, the antisense oligonucleotides comprising the phosphodiester linked GalNAc3-1 and GalNAc3-9 conjugates at the 3’ terminus (ISIS 655861 and ISIS 664078) and the 3-3 and GalNAc3-8 conjugates linked at the 5 ’ terminus (ISIS 661161 and ISIS 665001) showed substantial improvement in potency compared to the ugated antisense oligonucleotide (ISIS 353382).

Furthermore, ISIS 664078, comprising a GalNAc3-9 conjugate at the 3' terminus was essentially equipotent compared to ISIS 655861, which comprises a 3-1 ate at the 3’ terminus. The 5' ated antisense oligonucleotides, ISIS 661161 and ISIS 665001, comprising a GalNAc3-3 or GalNAc3-9, respectively, had increased potency compared to the 3' conjugated antisense oligonucleotides (ISIS 655 861 and ISIS 664078).

Table 40 ASOs containing GalNAc3-1, 3, 8 or 9 targeting SRB-l Dosage SRB-l mRNA ISIS No. ConJugate. (m /k ) (% Saline) Saline n/a 100 3 88 353382 10 68 none 36 0.5 98 655861 —155;? GalNAc; -1 (3') 20 0.5 88 1.5 85 664078 —5 GalNAc3-9 (3), 20 0.5 92 661161 —1'5 GalNAc3-3 (5') 19 11 0.5 100 1.5 73 665001 3-8 (5), 29 13 Liver transaminase levels, alanine aminotransferase (ALT) and ate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no signi?cant change from the saline group.

ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

Table 41 Dosa e Total ISIS No. m /kg ALT AST BUN ConJugate.

Bilirubin Saline 24 59 0.1 37.52 3 21 66 0.2 34.65 353382 10 22 54 0.2 34.2 none 22 49 0.2 33.72 0.5 25 62 0.2 30.65 1.5 23 48 0.2 30.97 655861 —5GalNAc3-1 (3 ), 28 49 0.1 32.92 40 97 0.1 31.62 0.5 40 74 0.1 35.3 1.5 47 104 0.1 32.75 664078 3-9 (3 ), 20 43 0.1 30.62 38 92 0.1 26.2 0.5 101 162 0.1 34.17 661161 1.5 g 42 100 0.1 33.37 GalNAc3-3 (5') g 23 99 0.1 34.97 ______— 665001 GalNAc3_8(5.) Example 56: Dose-dependent study of oligonucleotides comprising either a 3' or 5'-conjugate group (comparison of GalNAc3-1, 2, 3, 5, 6, 7 and 10) targeting SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a rd. Each of the s GalNAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'-de0xyaden0sine nucleoside (cleavable moiety) except for ISIS 655861 which had the GalNAc3 conjugate ’ terminus. group attached at the 3 Table 42 Modi?ed ASO targeting SRB-l Motif Conjugate SEQ ASO Sequence (5’ t0 3 ’) ID NO.

ISIS 353382 GesmCesTesTesmCesAdsGdSTdsmCdsAdsTdsGdSAds 5/10/5 . 2304 "0 conjugate (parent) "1c,TdsTesmCmcmeTesT GesmCeSTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds 5/10/5 2305 ISIS 655 861 3-1 chsTdsTesmCwmCTesTeoAdo-GalNAc3-la GalNAcg-Za-0AdoG?mCesTesT?mCeSAdSGdSTdS 5/10/5 GalNAc3-2 2306 ISIS 664507 mCcslAdsTdsC}dslAdsmCdsTdsTesmCmmcesTesTe GalNAC3-3a-yAd0 5/10/5 GalNAC3-3 2304 ISIS 661 161 GesmCesTesTesmCesAdsGdSTdsmCdsAdsTdsGdSAds mCcmsTdsTesC6mC"TEST GalNAcg-Sa-0,?AdoG?mCGSTCSTmCeSAdSGdSTdS 5/10/5 GalNAc3-5 2306 ISIS 666224 mcSAdsTdsGdSAdsmcdsTdsTesmcmcesTesT GalNAc3-6a-0AdoG?mCesTesT?mCeSAdSGdSTdS 5/10/5 3-6 2306 ISIS 666961 mCcslAdsTdsCidslAdsmCdsTdsTesmC?mcesTesTe GalNAC3-7a-o’AdoG?mCesTesT?mCeSAdSGdSTdS 5/1 0/5 GalNAC3-7 2306 ISIS 666981 mCcslAdsTdsC}dslAdsmCdsTdsTesmCmmcesTesTe ISIS 666881 galNAc3'10a'o’AntlloGes CesTnesTe;1 GdsTds 5/10/5 3'10 CcsAdsTdsGdsAds CdsTdsTes Cw Te Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "(1" indicates aB-D-2’-de0xyrib0nucle0side; 6; s99 indicates a phosphorothioate internucleoside linkage (PS); a; 099’indicates a phosphodiester internucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of 3-1a was shown usly in Example 9. The structure of GalNA03-2a was shown previously in Example 37. The structure of GalNAcg-3a was shown usly in Example 39. The structure of GalNAcg-Sa was shown previously in Example 49. The structure of GalNAcg-6a was shown usly in Example 51. The structure of GalNAc3-7a was shown previously in Example 48. The structure of GalNAc3-10a was shown previously in Example 46.

Trealmenl Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected aneously once at the dosage shown below with ISIS 3533 82, 655861, 664507, 661161, 666224, 666961, 666981 666881 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular , Inc. Eugene, OR) according to standard ols. The s below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline l.

As illustrated in Table 43, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. Indeed, the conjugated antisense oligonucleotides showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS ). The 5' conjugated antisense oligonucleotides showed a slight increase in potency compared to the 3' conjugated antisense ucleotide.

Table 43 Dosage SRB-l mRNA ISIS No. Conjugate. (m /k ) (% Saline) Saline n/a 100.0 3 96.0 353382 10 73.1 none 36.1 0.5 99.4 1.5 81.2 655861 —5 GalNAc3-1 (3), 15.2 0.5 102.0 1.5 73.2 664507 —5 GalNAc3-2 (5 ), 10.8 0.5 90.7 1.5 67.6 661161 —5 GalNAC3-3 (5), 11.5 0.5 96.1 1.5 61.6 666224 GalNAc3-5 (5 ), 25.6 11.7 0.5 85.5 1.5 56.3 666961 —5 GalNAc3-6 (5 ). 13.1 666981 0.5 84.7 GalNAc3-7 (5') 666881 GalNAc3-10 (5') Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no signi?cant change from the saline group.

ALTs, ASTs, total bilirubin and BUN values are shown in Table 44 below.

Table 44 Dosage Total ISIS N0. ALT AST BUN ConJugate. m /k bin Saline 26 57 0-2 27 3 25 92 0.2 27 353382 10 23 40 0.2 25 none 29 54 0.1 28 0.5 25 71 0.2 34 1.5 28 60 0.2 26 655861 GalNAc3-1 (3), 26 63 0.2 28 25 61 0.2 28 0.5 25 62 0.2 25 1.5 24 49 0.2 26 664507 GalNAc3-2 (5 ), 21 50 0‘2 26 59 84 0.1 22 0.5 20 42 0.2 29 1.5 g 37 74 0.2 25 661161 ; 3 (5),_ 28 61 0‘2 29 21 41 0.2 25 0.5 34 48 0.2 21 1.5 23 46 0.2 26 666224 GalNAC3-5 (5 ), 24 47 0.2 23 32 49 0.1 26 0.5 17 63 0.2 26 1.5 23 68 0.2 26 666961 GalNAc3-6 (5 ), 25 66 0‘2 26 29 107 0.2 28 0.5 24 48 0.2 26 1.5 30 55 0.2 24 666981 GalNAc3-7 (5 ), 46 74 0‘1 24 29 58 0.1 26 0.5 20 65 0.2 27 1.5 23 59 0.2 24 666881 GalNAc3-10 (5 ), 45 70 0.2 26 21 57 0.2 24 Example 57: Duration of action study of oligonucleotides comprising a 3'—conjugate group targeting ApoC III in vivo Mice were injected once with the doses indicated below and monitored over the course of 42 days for ApoC-III and plasma triglycerides (Plasma TG) . The study was performed using 3 transgenic mice that s human APOC-III in each group.

Table 45 Modi?ed ASO targeting ApoC III ISIS AesGesmCmTesTmmCdsTdSTdsGdSTds PS 2296 304801 mCdsmcdsAdsGdsmCdsTesTesTesAesTe 647535 AdsGdsmCdsTesT?TesA?TeoAdowGalNAcg-la AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmCds 6475 36 AdSGdsmCdSTeOT60TeSAmTeoAdoa-GalNAc3-1 a Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "(1" indicates a B-D-2’-deoxyribonucleoside; "5" indicates a phosphorothioate internucleoside e (PS); "0" indicates a phosphodiester internucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of 3-1a was shown usly in Example 9.

Table 46 ApoC III mRNA (% Saline on Day 1) and Plasma TG Levels (% Saline on Day 1) ASO Dose Target Day 3 Day 7 Day 14 Day 35 Day 42 Saline 0 mg/kg ApoC-III 98 100 100 95 1 16 ISIS 304801 30 mg/kg ApoC-III 28 30 41 65 74 ISIS 647535 10 mg/kg ApoC-III 16 19 25 74 94 ISIS 647536 10 mg/kg ApoC-III 18 16 17 35 51 Saline 0 mg/kg Plasma TG 121 130 123 105 109 m—g/kgPlasmaTG3437506969 ISIS 647535 10 mg/kg Plasma TG 18 14 24 18 71 As can be seen in the table above the duration of action increased with addition of the jugate group ed to the unconjugated oligonucleotide. There was a further increase in the duration of action for the conjugated mixed PO/PS oligonucleotide 647536 as compared to the conjugated full PS oligonucleotide 647535.

Example 58: Dose-dependent study of oligonucleotides comprising a 3'-c0njugate group (comparison of GalNAc3-l and GalNAc4-11) ing SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. ugated ISIS 440762 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside cleavable moiety.

The structure of GalNAc3-1a was shown previously in Example 9. The structure of GalNAc3-11a was shown previously in Example 50.

Trealmenl Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 440762, 651900, 663748 or with . Each treatment group consisted of 4 animals. The mice were ced 72 hours following the final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA ?cation reagent (Molecular , Inc. Eugene, OR) according to rd protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each ent group, normalized to the saline control.

As illustrated in Table 47, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising the phosphodiester linked GalNAc3-1 and GalNAc4-11 conjugates at the 3 ’ terminus (ISIS 651900 and ISIS 663748) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762). The two ated oligonucleotides, 3-1 and GalNAc4-11, were equipotent.

Table 47 Modi?ed ASO targeting SRB-l % Saline SEQ ID ASO Sequence (5 , t0 3 , ) Dose mg/kg control No.

Saline 100 m m 0.6 73.45 ISIS 440762 CksAdifédSdeS TcdglédsTdsGdsAdS 2 59.66 2298 ds ds ks k 6 2350 0.2 62.75 TkskasAdsGdsTdsmCdSAdsTdSGdsAds 0.6 29.14 ISIS 651900 2299 mCdsTdSTkskaoAdo.-GalNAc3-1a 2 8.61 6 5.62 sAdsGdsTdsmCdsAdsTdsGdsAds ISIS 663748 mCdsTdsTkSmCkoAdov-GalNAc4-1 1 a Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine. ipts: "e" indicates a 2’-MOE modified nucleoside; "k" indicates -CH3 bicyclic nucleoside; "d" indicates a B-D-2’-deoxyribonucleoside; "s" indicates a phosphorothioate intemucleoside linkage (PS); "0" indicates a phosphodiester intemucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. ate groups are in bold.

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no signi?cant change from the saline group.

ALTs, ASTs, total bilirubin and BUN values are shown in Table 48 below.

Table 48 Dosage Total ISIS No. ALT AST BUN Conjugate mg/kg bin Saline 30 76 0.2 40 0.60 32 70 0.1 35 440762 2 26 57 0.1 35 1’101’16 6 31 48 0.1 39 0.2 32 115 0.2 39 0.6 33 61 0.1 35 651900 GalNAc3-1 (3') 2 30 50 0.1 37 6 34 52 0.1 36 0.2 28 56 0.2 36 0.6 34 60 0.1 35 663748 GalNAc4-11 (3') 2 44 62 0.1 36 6 38 71 0.1 33 Example 59: Effects of 3-1 conjugated ASOs targeting FXI in vivo The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of FXI in mice. ISIS 404071 was included as an unconjugated standard. Each of the conjugate groups was attached at the 3' terminus of the respective oligonucleotide by a phosphodiester linked 2'-deoxyadenosine nucleoside cleavable moiety.

Table 49 d ASOs targeting FXI ISIS TesGesGesTesAesAdsTdsmCdsmCdsAdsmCds 404071 TdsTdsTdsmCdsAmGesAesGesGe ISIS esTesAesAdsTdsmCdsmCdsAdsmCds PS 23 08 656172 TdSTdsTdSmCdSA?GGSA?GGSGmAdOa-GalNAcg-l a ISIS GeoT60Ae(,AdSTdsmCdsmCdsAdsmCdS PO/PS 2308 65 6173 TdsTdsTdsmCdsAeoGeersGesGeoAdo"GalNAc3'1 a Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "d" indicates a B-D-2’-deoxyribonucleoside; "s" indicates a phosphorothioate intemucleoside e (PS); "0" indicates a phosphodiester intemucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNAc3-1a was shown previously in e 9.

Trealmenl Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously twice a week for 3 weeks at the dosage shown below with ISIS 404071, 656172, 656173 or with PBS treated control. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration to determine the liver FXI mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. Plasma FXI protein levels were also measured using ELISA. FXI mRNA levels were determined relative to total RNA (using EEN®), prior to normalization to PB ted control. The results below are presented as the average percent of FXI mRNA levels for each ent group. The data was normalized to PBS-treated control and is denoted as "% PB S". The ED50s were measured using similar methods as described previously and are presented below.

Table 50 Factor XI mRNA (% Saline) ASO 0A) Control Conjugate. Linkages. mg/kg Saline 100 none 3 92 $15071 10 40 none PS 15 0.7 74 ISIS — 2 33 3-1 PS 656172 6 9 0.7 49 ISIS — GalNAc3-1 PO/PS 656173 22—2 As illustrated in Table 50, treatment with nse oligonucleotides lowered FXI mRNA levels in a dose-dependent manner. The oligonucleotides comprising a 3'—GalNAc3-1 conjugate group showed ntial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071).

Between the two conjugated oligonucleotides an improvement in potency was ?thher provided by substituting some of the PS es with P0 (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotides lowered FXI protein levels in a dose-dependent manner. The oligonucleotides sing a 3'—GalNAc3-1 conjugate group showed ntial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071).

Between the two conjugated oligonucleotides an improvement in potency was ?thher ed by substituting some of the PS linkages with P0 (ISIS 656173).

Table 50a Factor XI protein (% Saline) 113102: 2:36:21) (% ASO Conjugate Linkages Saline 100 none $28071% none PS 3 GalNAc3'1 PS 656172 2 23 6 1 3-1 PO/PS 656173 2 6 6 0 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using rd protocols. Total bilirubin, total albumin, CRE and BUN were also evaluated. The change in body weights was evaluated with no signi?cant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

Table 51 Dosage Total Total ISIS No. ALT AST CRE BUN Conjugate. m /k Albumin Bilimbm Saline 71.8 84.0 3.1 0.2 0.2 22.9 3 152.8 176.0 3.1 0.3 0.2 23.0 404071 10 73.3 121.5 3.0 0.2 0.2 21.4 none 82.5 92.3 3.0 0.2 0.2 23.0 0.7 62.5 111.5 3.1 0.2 0.2 23.8 656172 2 33.0 51.8 2.9 0.2 0.2 22.0 @118:ch 6 65.0 71.5 3.2 0.2 0.2 23.9 0.7 54.8 90.5 3.0 0.2 0.2 24.9 656173 2 85.8 71.5 3.2 0.2 0.2 21.0 @118:ch 6 114.0 101.8 3.3 0.2 0.2 22.7 Example 60: Effects of conjugated ASOs ing SRB-l in vitro The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-l in primary mouse hepatocytes. ISIS 353382 was included as an unconjugated rd. Each of the conjugate groups were attached at the 3' 0r 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'-de0xyadenosine side cleavable moiety.

Table 52 Modi?ed ASO targeting SRB-l ASO ce (5’ t0 3 ’) Motif Conjugate ID No.

GesmcesTesTesmCesAdsGdsTdsmCdslAdsTdsGdsAds ISIS 353382 5/10/5 none 2304 mCdsTcSTesmC?mCmTeSTe GesmCesTesTesmCesAdsGdSTdsmCdsAdsTdsGdSAds ISIS 655 861 5/10/5 GaL‘IAc3-l 2305 mCdsTcSTesmC?mCmTesTe.,AdO'-GalNAc3-1 a GesmceoTeoTeomCeoAdsGdsTdsmCdsAdsTdsGdsAds ISIS 655862 5/10/5 GaL‘IAc3-l 2305 mCdsTcSTeOmCmmCmTesTeoAdou-GalNAcg-l a GalNAc3-3a_.,,AdonmCmTesTesmCmAdSGds ISIS 661161 5/10/5 GaL‘IAcg-3 2306 TdsmCCsAdsTdsGdsAdSmCdsTdsTesmCesmCesTmTe GalNAc3-8a_.,,AdonmCmTesTesmCmAdSGds ISIS 665001 5/10/5 GaNAc3-8 2306 TdsmCCsAdsTdsGdsAdSmCdsTdsTesmCesmCesTmTe GesmcesTesTesmCesAdsGdsTdsmCdslAdsTdsGdsAds ISIS 664078 5/10/5 GaNAc3-9 2305 mCdsTcsTesmC?mCmTEST€014"!-(hilNAAC3'9-a GalNAc3-6a-O:Ad0G?mCesTEST?mCeSAdSGdS ISIS 666961 5/10/5 GaNAc3-6 2306 TdsmCCsAdsTdsGdsAdSmCdsTdsTesmCesmCesTmTe GalNAc3-2a-O’AdoG?mCesTesT?mCesAdsGdsTds ISIS 664507 5/10/5 GaNAc3-2 2306 mCdsAdSTdsGdsAdsmCdSTdSTesmC?mCesTesTe 3-1Oa-o’AdoGesmCesTesTesmCesAdsGdsTds ISIS 666881 5/10/5 GaNAc3-10 2306 mCdsAdSTdsGdsAdsmCdSTdSTesmC?mCesTCSTe GalNAc3-5a-O’AdoG?mCesTesT?mCesAdsGdsTds ISIS 666224 5/10/5 GaNAc3-5 2306 mCdsAdSTdsGdsAdsmCdSTdSTesmC?mCesTesTe GalNAc3-7a-O’AdoG?mCesTesT?mCesAdsGdsTds ISIS 666981 5/10/5 GaL‘IAcg-7 2306 mCdsAdSTdsGdsAdsmCdSTdSTesmC?mCesTesTe Capital letters indicate the nucleobase for each nucleoside and mC indicates a yl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "(1" indicates a B-D-2’-de0xyrib0nucleoside; "5" indicates a phosphorothioate cleoside linkage (PS); "0" indicates a phosphodiester intemucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNA03-1a was shown previously in Example 9. The structure of GalNA03-3a was shown previously in Example 39. The ure of GalNAcg-8a was shown usly in e 47. The structure of GalNAcg-9a was shown previously in Example 52. The structure of GalNAcg-6a was shown previously in Example 51. The structure of GalNAcg-2a was shown previously in Example 37. The structure of GalNAc3-10a was shown previously in Example 46. The structure of GalNAcg-Sa was shown previously in Example 49. The structure of GalNAcg-7a was shown previously in Example 48.

Trealmenl The ucleotides listed above were tested in vitro in primary mouse hepatocyte cells plated at a density of 25,000 cells per well and treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 or 20 nM modi?ed oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and mRNA levels were measured by quantitative real-time PCR and the SRB-l mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.

The IC50 was calculated using standard methods and the results are presented in Table 53. The results show that, under free uptake ions in which no reagents or electroporation techniques are used to arti?cially promote entry of the oligonucleotides into cells, the oligonucleotides sing a GalNAc conjugate were significantly more potent in hepatocytes than the parent oligonucleotide (ISIS 353382) that does not comprise a GalNAc conjugate.

Table 53 A80 ICso (nM) Inte?nnigleé’smde Conjugate SEQ ID No.

ISIS 353382 190a PS none 2304 ISIS 655861 11a PS GalNAcg-l 2305 ISIS 655862 3 PO/PS GalNAcg-l 2305 ISIS 661161 15a PS 3-3 2306 ISIS 665001 20 PS GalNAc3-8 2306 ISIS 664078 55 PS 3-9 2305 ISIS 666961 22a PS GalNAc3-6 2306 ISIS 664507 30 PS GalNAc3-2 2306 ISIS 666881 30 PS GalNAc3-10 2306 ISIS 666224 30a PS GalNAc3-5 2306 ISIS 666981 40 PS GalNAc3-7 2306 21Average of multiple runs.

Example 61: Preparation of oligomeric compound 175 comprising GalNAc3-12 0 OAc BOC\H/\/\NH2 0 0 A0c HN OC\N/\/\N 0 \Ac H H OAc 166 HN 167 \Ac /N N AcO CBZ o LCOOH TFA COOH /\/\ /U\/\/\/O O 169 —* HZN H OAc DOM HN\AC HBTU DIEA DMF A00 OAC JokA/VO O OAC HN HN\ O H A ©\/O\n/NH KLNW O AOC N O OAc L /\/\ We 0 0 N N O HN H H HN ACO 170 HN \Ac AcO OAC JokA/VO O OAC Pd(OH)2/C,H2 HN O HN\AC MeOH/EtOAc N\/\/H O A0C HZN N O \/\/:L/ ANMNWO OAc o HN H H \\\\ HN\ HN ACO 171 HN\ benzyl uorophenyl) glutarate AcO OAc SKA/V0 0 OAc O HN\A o o N N OAc o "N H H HN AcO ACO OAc HN HN\ 0 NW AC Pd(OH>2/C,H2 172 —> H 9/ MeOH/EtOAc o ACO HOMN N o OAC /\/\ W0 0 O O \/\/O:L/ LU" H V/gOAc HN ACO 173 HN\ ACO OAc PFP-TFA —> o o DIEA DMF W0 OAC HN HN O H \AC F F H RLNW O ACO N O HN AcO 174 HN\ 3' 5' || OLIGO O—F|’—O—(CH2)e—NH2 1. Borate rt 174 buffer, DMSO, pH 8.5, 2. aq. ammonia, rt HO O ACHN M ZI«‘3.- Compound 169 is commercially available. Compound 172 was prepared by addition of benzyl (per?uorophenyl) glutarate to compound 171. The benzyl (per?uorophenyl) glutarate was prepared by adding PFP-TFA and DIEA to 5-(benzyloxy)-5 -oxopentanoic acid in DMF. Oligomeric compound 175, comprising a GalNAc3-12 conjugate group, was ed from compound 174 using the general procedures illustrated in Example 46. The 3 cluster portion of the conjugate group GalNAc3-12 (GalNAc3-12a) can be ed with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is -P(=O)(OH)—Ad-P(=O)(OH)-. The ure of GalNAc3-12 (GalNAc3-12a-CM-) is shown below: OH OH ngowowACHN Ol‘bH in ACHN W MNJK—N N H Fr O f/j’NH e 62: Preparation of oligomeric compound 180 comprising GalNAc3-13 AcHN OWLOH ONWO HATU, HOAt DIEA, DMF OAc OAc A00 0M oOAc H2, Pd/C AGO OWEN —> AcHN (DH/MO OAc OAc o 177 A00 OW AcHN 0 OAc OAc AcO 0M "0&0OAcOWN PFPTFA TEA AcHN (DH/MW OAc OAc 178 ACO OW AcHN o OAC OAc Aco?/OMOO AcHN NH OAc 0Ac Aco?/OW0 0 : OAc OAc AcO 0W AcHN o 3' 5' || -O-(CH2)e-NH2 1. Borate buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt OH OH HO 0M OH OH HO OW AcHN 0 Compound 176 was prepared using the general procedure shown in Example 2. Oligomeric compound 180, comprising a GalNAc3-13 conjugate group, was prepared from compound 177 using the general procedures illustrated in Example 49. The GalNAc3 cluster portion of the conjugate group g- 13 (GalNAc3-13a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certainembodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAC3-1 3 (GalNAc3-13a-CM-) is shown below: OH OH How \MJOLO NH HO o H O H O O\WL N N\9/6 3 N N/W\n/ a AcHN H O H O Example 63: Preparation of oligomeric compound 188 comprising GalNAc3-14 H OAc HO\n/\\O HONNH2 HONBNWO A:O% HOWongHCBz 181 Nm/f/OQVNHCBZ 4 N7/O HBTU DIEA 0p DMF HO 0V H0 Mm 13 182 OAC 0A0 A00 AcO H A000%,ON6NWO ACO&/ON6N\"/\\ OAc NHAC OAc NHAc O 0 A00 A00 ONN Pd/C H N NHCBz 2 ON 71/2/0 N H2 AcO \?/\/O A00 6 I—IN*<—/O o o NHAc NHAc A:&/083H"" :ZwWNW Op ACO&Nm"AcO ON 1. Pd/C H2 HO\n/\/\n/ OAC NHAC 2- PFP.TFA pyr 0 O DMF —> AcO NEHW/Z/ HBTU, DIEA, ACNH/gcAc DMF oWNW2%:210 AGO H 0N6 W0 F A00 "M: F OAc NNNC A00 F OAcWNW/\JOO 3&1/11" 83e HO . O ' H '1|:||"O'(CH2)6_NH2 351, M H0'1ngT6 187 1. Borate buffed—IDMSO, pH 8.5, rt /ZNENHO H Mia/6 2. aq. a, rt HO H Ho?/O 6" 188 nds 181 and 185 are commercially available. Oligomeric compound 188, comprising a GalNAc3-14 conjugate group, was prepared from compound 187 using the general ures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3-14 (GalNAc3-14a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)—Ad-P(=O)(OH)-. The structure of GalNAc3-14 (GalNAc3-14a-CM-) is shown below: HOOH O O o N H0 10 HR AcHN o HOOH O O O O O NJK/‘O JJ\/\/U\ § H0 10 H M "WC a AcHN O O Z o N O H0 10 H Example 64: Preparation of oligomeric compound 197 comprising GalNAc3-15 AcO 0A0 OTBS OTBS OMOH AcO OAc O 189 OM A HNC N HBTU, DIEA AcHN 7 N NH3/MeOH OTBS —. (3/ 8220, DMAP HO OH N —> HO 0 BzO OBZ CO 820%0192 AcHN O ,N \ \( Phosphitylation 820 082 O C BZO§¢OMNN \/\/o DMTO /N(iPr)2 \o DMTO /\/\0 DMTO _\_CN \/\/o . 3. 195 \/\/Clo-I.

/\/\O 88, DNA synthesizer 1 96 HO4%? 1. 194, DNA synthesizer AcHN N O\P// 2.Aq NH3 55 C,18h0 O |\OH O O O\ // \/\/O HO O U \/\/\n/ OH NHAc O OH 0 Compound 189 is commercially available. Compound 195 was prepared using the l procedure shown in Example 31. Oligomeric compound 197, comprising a GalNAc3-15 conjugate group, was prepared from compounds 194 and 195 using standard oligonucleotide synthesis ures. The GalNAc3 cluster portion of the conjugate group GalNAc3-15 (GalNAc3-15a) can be ed with any cleavable moiety to e a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)—Ad- P(=O)(OH)-. The structure of GalNAc3-15 (GalNAc3-15a-CM-) is shown below: HOOH 0’13\ HO o N0/ "Pk AcHN W O o HoOH o’gpmo O o 0/ o HO O\/\/\n/N AcHN o (135 Example 65: Dose-dependent study of oligonucleotides comprising a 5’-conjugate group rison of GalNAc3-3, 12, 13, 14, and 15) targeting SRB-l in vivo The oligonucleotides listed below were tested in a dose-dependent study for nse inhibition of SRB-l in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GalNAc3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked 2'- deoxyadenosine nucleoside (cleavable moiety).

Table 54 d ASOs targeting SRB-l ISIS , , . SEQ Sequences (5 t0 3 ) Conjugate N0. ID No. 3 5 3 3 82 CiesmCesT?TesmCesAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe none 23 04 GaiNAIE3'3a'o’AdoGes CesTesTes CesAdsGdsTdS CdsAdsTdsGdsAds CdsTds 661 161 Gal\AC3_3 23 06 Tes Ces CesTesTe GaiNAIETlZa'o’Adon C?TesTes CesAdsGdsTds CdsAdSTdsGdsAds CdsTds 671 144 Gal\A03-12 23 06 Tes Ces CesTesTe 670061 GaiNAIE3'13a'o’AdoG? C?TesTes CesAdsGdsTds CdsAdSTdsGdsAds CdsTds Gal\AC3_13 23 06 Tes Ces CesTesTe 671261 GaiNAIE3'14a'o’Adon C?TesTes CesAdsGdsTds CdsAdSTdsGdsAds CdsTds Gal\A03-14 23 06 Tes Ces CesTesTe 671262 sé'liangdon C?TesTes CesAdsGdsTds CdsAdSTdsGdsAds CdsTds Gal\A03-15 23 06 Capital letters indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified side; "(1" indicates a B-D-Z’-de0xyrib0nucle0side; "s" tes a phosphorothioate internucleoside linkage (PS); "0" tes a phosphodiester internucleoside linkage (PO); and "0’" indicates -O-P(=O)(OH)-. Conjugate groups are in bold.

The structure of GalNAC3-3a was shown previously in e 39. The structure of GalNAC3-12a was shown previously in Example 61. The structure of GalNAC3-13a was shown previously in Example 62.

The structure of GalNAc3-14a was shown usly in Example 63. The structure of GalNAc3-15a was shown previously in Example 64.

Trealmenl Six to eight week old C57bl6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once or twice at the dosage shown below with ISIS 3533 82, 661161, 671144, 670061, 671261, , or with saline. Mice that were dosed twice received the second dose three days after the first dose. Each treatment group consisted of 4 animals. The mice were ced 72 hours following the final stration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) ing to standard protocols. The results below are presented as the e percent of SRB-l mRNA levels for each treatment group, normalized to the saline l.

As illustrated in Table 55, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. No signi?cant differences in target knockdown were observed between animals that received a single dose and animals that received two doses (see ISIS 353382 dosages 30 and 2 x 15 mg/kg; and ISIS 661161 dosages 5 and 2 x 2.5 mg/kg). The antisense oligonucleotides comprising the phosphodiester linked 3-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).

Table 55 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA (% ED50 (mg/kg) Conjugate Saline) Saline n/a 100.0 n/a n/a 3 85.0 69.2 353382 —30 22.4 none 2 x 15 36.0 0.5 87.4 1.5 59.0 661161 5 25.6 2.2 GalNAC3-3 2 x 2.5 27.5 17.4 0.5 101.2 1.5 76.1 671144 3.4 3-12 32.0 17.6 0.5 94.8 1.5 57.8 670061 —52.1 GalNAc3-13 13.3 671261 4'1 Gamer" 109.4 671262 Gamer" Liver transaminase levels, alanine aminotransferase (ALT) and ate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no signi?cant differences from the saline group (data not . ALTs, ASTs, total bin and BU\I values are shown in Table 56 below.

Table 56 Total Dosage ALT ISIS No. AST (U/L) Bilirubin. . . BUN Conjugate. (mg/kg) (U/L) (mg/dL) (1n /dL) Saline n/a 28 60 0.1 39 n/a 3 30 77 0.2 36 25 78 0.2 36 353382 "one 28 62 0.2 35 2 x 15 22 59 0.2 33 0.5 39 72 0.2 34 1.5 26 50 0.2 33 661161 5 41 80 0.2 32 GalNAc3-3 2 x 2.5 24 72 0.2 28 32 69 0.2 36 0.5 25 39 0.2 34 1.5 26 55 0.2 28 671144 GalNAc3-12 48 82 0.2 34 23 46 0.2 32 0.5 27 53 0.2 33 1.5 24 45 0.2 35 670061 GalNAc3-13 23 58 0'1 34 24 72 0.1 31 0.5 69 99 0.1 33 1.5 34 62 0.1 33 671261 —5GalNAc3-14 43 73 0'1 32 32 53 0.2 30 0.5 24 51 0.2 29 1.5 32 62 0.1 31 671262 GalNAc3-15 30 76 0‘2 32 31 64 0.1 32 Example 66: Effect of various cleavable moieties on antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a 5’-GalNAc3 cluster The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice. Each of the 3 conjugate groups was attached at the 5' terminus of the respective oligonucleotide by a phosphodiester linked nucleoside (cleavable moiety (CM)).

Table 57 Modi?ed ASOs ing SRB-l ISISSequem:es(5’to3’)—Gal\A03Cl\/ISEQ No. Cluster ID No. 661 161 GalNAC3'3a'o’AdoG?mCesTesT?mC?AdsGdsTdsmCdsAds Tds Gal\A03-3a Ad 2306 GCSACSmCCSTCST?mC:CesTesTe 670699 GalNAc3-3..-.,,Td.,G:ceonTeomceoAdsGdsTdsmcdSAdSTds Gal\ACs-3a Td 2309 GCsAcsmCcsTcsTeomCeomCesTesTe 670700 GalNAc3-3..-.,,Ae.,G:ceonTeomceoAdsGdsTdsmcdSAdSTds Gal\ACs-3a Ac 2306 GCSACSmCCST:STe()H1Ce()mCesTesTe 670701 GalNAc3-3a'O’TeoGesmCeoTeoTeomCeoAdsGdsTdsmCdsAdsTds Gal\A03-3a TC 2309 mCcsTcsTeomCeomCesTesTe 671 165 GalNAc3-13 oGesmCeoTeoTeomCeoAdsGdsTdsmCdsAdsTds Gal\A03-13 a Ad 23 06 GCSACSmCCST:STe()H1Ce()mCesTesTe Capital s indicate the nucleobase for each nucleoside and mC indicates a 5-methyl cytosine.

Subscripts: "e" indicates a 2’-MOE modified nucleoside; "d" indicates a B-D-2’-deoxyribonucleoside; "5" indicates a phosphorothioate internucleoside linkage (PS); "0" indicates a phosphodiester internucleoside e (PO); and "0’" indicates O)(OH)-. Conjugate groups are in bold.

The structure of GalNA03-3a was shown previously in Example 39. The structure of GalNAcg-13a was shown previously in Example 62.

Trealmenl Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with ISIS 661161, 670699, 670700, 670701, 671165, or with . Each treatment group ted of 4 animals. The mice were sacri?ced 72 hours ing the ?nal administration to determine the liver SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 58, treatment with antisense oligonucleotides d SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides sing various cleavable moieties all demonstrated similar potencies.

Table 58 SRB-l mRNA (% ) ISIS No. Dosage (mg/kg) SRB-1 mRNA GalNAc3 CM 1% Saline) Cluster Saline n/a 100.0 n/a n/a 0.5 87.8 1.5 61.3 661161 GalNAc3-3a Ad 33.8 14.0 0.5 89.4 1.5 59.4 670699 GalNAc3-3a Td 31.3 17.1 0.5 79.0 1.5 63.3 670700 GalNAc3-3a A6 32.8 17.9 0.5 79.1 1.5 59.2 670701 GalNAc3-3a Te 35.8 17.7 0.5 76.4 1.5 43.2 671165 GalNAc3-13a Ad 22.6 10.0 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin and BUN were also evaluated. The changes in body weights were evaluated with no signi?cant differences from the saline group (data not shown). ALTs, ASTs, total bin and BUN values are shown in Table 59 below.

Table 59 Total Dosage ALT AST . . . BUN GalNAc3 ISIS No. Bilirubin CM (mg/kg) (U/L) (U/L) (mg/dL) Cluster (m /dL) Saline n/a 24 64 0.2 31 n/a n/a 0.5 25 64 0.2 31 1.5 24 50 0.2 32 661161 3-3a Ad 26 55 0.2 28 27 52 0.2 31 0.5 42 83 0.2 31 1.5 33 58 0.2 32 670699 GalNAc3-3a Td 26 70 0.2 29 25 67 0.2 29 0.5 40 74 0.2 27 1.5 23 62 0.2 27 670700 GalNA03-3a A6 24 49 0‘2 29 25 87 0.1 25 0.5 30 77 0.2 27 1.5 22 55 0.2 30 670701 GalNA03-3a Te 81 101 0‘2 25 31 82 0.2 24 0.5 44 84 0.2 26 1.5 47 71 0.1 24 671165 GalNA03-13a Ad 33 91 0‘2 26 33 56 0.2 29 Example 67: Preparation of oligomeric compound 199 comprising 3-16 0;:ACI:%:CWNNW OOAc:OWNWHANH /ODMTr AcO 1. Succinic anhydride O DMAPDCE Aco?/OOMB]WHNOOAC N"<(f:>)-N{O 2. DMF HBTU DIEA PS-88 ACHN 2 AcOOAc ACHN ODMT AcOOAc O ./ NW " 1.DNA Synthesizer AcOOAc c 2 ACHN 198 o H H HO OMNWN o 2 2 / CM HOOH ACHN O O - M o 4 HO0&0W"WHO QMN: ACHN Z H o 0 CM W0N Ho 2 Oligomeric compound 199, comprising a GalNAc3-16 conjugate group, is ed using the general procedures illustrated in Examples 7 and 9. The GalNA03 cluster portion of the conjugate group GalNA03-16 (GalNA03-16a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the ble moiety is -P(=O)(OH)-Ad-P(=O)(OH)-.The structure of GalNA03-16 (GalNAc3-16a-CM-) is shown below: HoOH o o "0%O o/\(v))l\ N N 4 HAH? ,‘g H o o ’0 HOo 0 N : HO NA?/ 4 H 2 o "WE? AcHN OH HoOH o oAWLN 0 4 HAM?" A00 OOAc 3- 5' ? AcHN L OLIGO o—F|>—0—(CH2)6—NH2 0A0 0A0 OH 0 OWLN/\H/\NHH A00 NM: 1. Borate buffer DMSO pH8.5 rt 2. aq. ammonia rt HOQOHOo/\(")JLN’\/\N OAcHN 3 H H o o OHoAfjJLNMN "WHWO 3 H H Oligomeric compound 200, comprising a GalNAc3-17 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group GalNAc3- 17 (GalNAc3-17a) can be combined with any cleavable moiety to provide a variety of ate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-17 (GalNAc3-17a-CM-) is shown below: HoOH o o O W /\/\ O N HO 3 H N AcHN H o o HO O O /\/ O N HNJJ\/\/U\N/\(v)/\O_-_gH HO 3 H O HoOH o O N/\/\ O HO 3 H N AcO OOAc O AcHN O N’\/\N O . .

OAC O0Ac O H o F F OLIGO O-(CH2)6-NH2 AcO N O F AcHN H 1. Borate buffer, DMSO, pH 8.5, rt OAc OAC O O .

A00 0 NWHN 2 rt O . aq. ammonia, AcHN W HoOH o o HO O N/\/\N 4 H H AcHN O O HO O O Ho§wo W O N W"*-4O OLIGO H H 4 HM" O NMN O HO 4 H H Oligomeric compound 201, comprising a GalNAc3-18 conjugate group, was ed using the general procedures illustrated in Example 46. The GalNAc3 cluster portion of the conjugate group g- 18 (GalNAc3-18a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-1 8 (GalNAc3-18a-CM-) is shown below: HoOH o o O/\(V)JL /\/\N 4 H N AcHN H o o HO O o x\/ NWNWO O N H H HO 4 H o HoOH o O /\/\ O N O 4 H N Example 70: Preparation of oligomeric compound 204 comprising GalNAc3-19 AcOOAc AcOOAc o o o O\v/\¢/\VJL\ HBTU,DMF,DEA O o\/»\/»\/J\ AcO A00 OH —> N ....OH AcHN DMTO AcHN 64 NH 202 ‘ 47 AcOOAc PhosphityIation O\/\/\)J\ ) AcO$W N ----IO NC 1. DNA synthesizer AcHN \ O —’ P/ \J l 2. aq. NH3 DMTO UPDZN O N O o AcHN | o=Fl>—OH O o AcHN | O—T—OH HO 3 O 0 II.CM I'll.OLIGO Oligomeric compound 204, comprising a g-19 conjugate group, was prepared from compound 64 using the general ures illustrated in Example 52. The GalNA03 cluster portion of the conjugate group GalNA03-19 (GalNA03-19a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAC3-1 9 (GalNAC3-19a-CM-) is shown below: HoOH ‘ o N O o ACHN O—FI’ OH Ho§wowo N ACHN O‘FI’ OH 0 N Ho 0% Example 71: Preparation of oligomeric compound 210 sing GalNAc3-20 NM: F H EtN(iPr)2, CHscN F?N F NMN -""OH DMTO 0 206 DMTO AcOOAc 0 A00wow K2003/Methanol H2NMN AcHN 166 .... OH ACOOAC O\/\/\)OI\NH(MN Phosphitylation IIIOH A000&0 208 Op AcOOAcO 1. DNA synthesizer OMNH "IO NC AcO \P/OV 2. aq. NH3 AcHN I DMTO (IPr)2N_ 0 "MgN HO 3 O o AcHN | o=Fl>—OH OH : o "MN HO 3 AcHN (I) o=F|>—OH OH 0 _.~‘ Compound 205 was prepared by adding PFP-TFA and DIEA to ,2 -tri?uoroacetamido)hexanoic acid in acetonitrile ,which was prepared by adding tri?ic anhydride to 6-aminohexanoic acid. The reaction mixture was heated to 80 0C, then lowered to rt. Oligomeric compound 210, comprising a GalNAc3-20 conjugate group, was prepared from compound 208 using the general ures illustrated in Example 52.

The GalNAc3 cluster portion of the conjugate group GalNAc3-20 (GalNAc3-20a) can be combined with any ble moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is (OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-20 (GalNAc3-20a-CM-) is shown below: OH " HO o o NMN AcHN (I) OZT—OH 0 39 O NMN AcHN (I) o:Fl>—OH Example 72: Preparation of oligomeric nd 215 comprising GalNAc3-21 1 A00 OAc O OH NH O AcO$¢OM H AcO OAc OH O AcHN 176 0 r/ A00 \MJK BOP, EtN(iPr)2, 1,2-dichloroethane AcHN \\\ 212 0H AcOOAc DMTCI Pyridine rt r/ Phosphitylation —>AcO $0: 0M —> 1. DNA synthesizer ACOOAC ACO%O o N(iPr)2 —> O Mr/ 2. a .NHq 3 AcHN \\\ 0 N HO OW \\\ AcHN Cl) CIT—OH HO F1 o N HO OW L AcHN Cl) o=F|>—0H Compound 211 is commercially available. eric compound 215, comprising a GalNAc3-21 conjugate group, was prepared from nd 213 using the general procedures illustrated in Example 52.

The 3 cluster portion of the conjugate group GalNAc3-21 (GalNAc3-21a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)-Ad-P(=O)(OH)-. The structure of GalNAc3-21 (GalNAc3-21a-CM-) is shown below: HO H o N AcHN Cl) OZT—OH HO H o N Ho "W? L AcHN Cl) o:Fl>—0H 0 N OW L CM AcHN O—-_§ Example 73: Preparation of oligomeric compound 221 sing GalNAc3-22 O O H H\ /\/OH H F C NM3 jr N F C \WL 0 3 er N/\\/OH o F F 211 o OH 5 205 F F 216 OH DIEA ACN O K2C03 DMT-CI FscTNMNNODMTr —> pyridine o % MeOH / H20 217 OH HZNWLNNODMTr 0A0 F O/\V/\\/Ajro F % O 218 NHAc F F OH 166 OAc H A00goe/OWNWLN/\/ODMTr Phosphitylation A00 0 1. DNA Synthesizer ’ OH OH "MLN/\/0’ 3OH 2. Aq. NH3 QSA/O/W?l/ 221 m Compound 220 was prepared from compound 219 using diisopropylammonium tetrazolide.

Oligomeric compound 221, comprising a GalNAc3-21 conjugate group, is prepared from compound 220 using the general procedure illustrated in Example 52. The GalNAc3 cluster portion of the conjugate group GalNAc3-22 (GalNAc3-22a) can be ed with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is -P(=O)(OH)—Ad-P(=O)(OH)-. The ure of GalNAc3-22 (GalNAc3-22a-CM-) is shown below: gym M HO O OH HM ’P< Q‘MW" NNO 0" HO o NHAc R O Lo OH H gym O/P HO O Example 74: Effect of various cleavable moieties on antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a 5’-GalNAc3 conjugate The oligonucleotides listed below were tested in a ependent study for nse inhibition of SRB-l in mice. Each of the GalNAc3 conjugate groups was attached at the 5' us of the respective oligonucleotide.

Table 60 Modi?ed ASOs targeting SRB-l ISIS GalNAc3 SEQ sequences (5 , to3), CM No. Cluster IDNo.

GmmCTT mCAGT mdsdsCATGAmdSCTT 353382 es es es es dsm ds ds ds ds ds ds es n/a n/a 2304 GSmCCSTGSTe GalNAc-33 S0’AG CTT CAGT CAT‘10 661161 es 63,, es es ‘13 ds ‘13 ds ‘13 GalNAc3-3a Ad 2306 GdsAdsmCdsTdsTesmfesmCesTmTe 3-3a-o’GmmC T T mC d dedAdT 666904 S SS SmS SS S S S S SS GalNAc3-3a PO 2304 GdSAdsmCdSTdsTesmCesmC STmeT GalNAC3-17a-0’AdoG mC T T mCmAd Gde mdstAdT 675441 SS SS SS S S S SS S S 3-17a Ad 2306 GdsAdsmCdSS-[Sds'l-‘esmCesmTCesTese -l8-,A G mc T T mc A G T mc A T3 S0 675442 ‘12,, ES 65 enema ‘15 d5 d5 d5 ‘15 ds GalNAc3-18a Ad 2306 GdsAds CdsTdsTes Ces CesTmTe In all tables, capital letters te the nucleobase for each nucleoside and InC indicates a 5-methyl cytosine Subscripts: "e" indicates a 2’-MOE modified nucleoside; "(1" indicates a B-D-2’- deoxyribonucleoside; 6; s99’indicates a phosphorothioate intemucleoside linkage (PS); a; 099’indicates a phosphodiester intemucleoside linkage (PO); and "o "’ indicates -O-P(=O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc3-3a was shown previously in Example 39. The structure of GalNAc3-17a was shown previously in Example 68, and the structure of GalNAc3-18a was shown in Example 69.

Trealmenl Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 60 or with saline.

Each treatment group consisted of 4 animals. The mice were sacri?ced 72 hours following the ?nal administration to determine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are ted as the average percent of SRB-l mRNA levels for each treatment group, normalized to the saline control.

As rated in Table 61, treatment with nse oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising a GalNAc ate showed similar potencies and were significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

Table 61 SRB-l mRNA (% Saline) ISIS No. Dosage (mg/kg) SRB-l mRNA GalNAc3 CM (% Saline) Cluster Saline n/a 100.0 n/a n/a 3 79.38 353382 10 68.67 n/a n/a 40.70 0.5 79.18 1.5 75.96 661161 —5 3-3a Ad 12.52 0.5 91.30 1.5 57.88 666904 —5 GalNAc3-3a P0 21.22 16.49 0.5 76.71 1.5 63.63 675441 3-17a Ad 2957 13.49 0.5 95.03 1.5 60.06 675442 GalNAc3-18a Ad 31‘04 19.40 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using rd protocols. Total bilirubin and BUN were also evaluated. The change in body weights was evaluated with no signi?cant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 62 below.

Table62 Dosage ALT AST BUN GalNAc ISIS NO' Blhmbm.. . 3 CM (mg/kg) (U/L) (U/L) (mg/dL) Cluster (mg/dL) Saline n/a 26 59 0.16 42 n/a n/a 3 23 58 0.18 39 353382 10 28 58 0.16 43 n/a n/a 20 48 0.12 34 0.5 30 47 0.13 35 1.5 23 53 0.14 37 661161 GalNAc3-3a Ad 26 48 0.15 39 32 57 0.15 42 0.5 24 73 0.13 36 1.5 21 48 0.12 32 666904 —5 GalNAc3-3a P0 19 49 0‘14 33 20 52 0.15 26 0.5 42 148 0.21 36 675441 1.5 60 95 0.16 34 GalNAc3-17a Ad 27 75 0.14 37 ——-———- 675442Ga1NAC_18a A Example 75: Pharmacokinetic analysis of oligonucleotides comprising a 5’-conjugate group The PK of the ASOs in Tables 54, 57 and 60 above was evaluated using liver samples that were obtained ing the treatment procedures described in Examples 65, 66, and 74. The liver s were minced and extracted using standard protocols and analyzed by lP-HPLC-MS alongside an internal rd.

The combined tissue level (ug/g) of all metabolites was measured by integrating the appropriate UV peaks, and the tissue level of the full-length ASO missing the conjugate nt," which is Isis No. 353382 in this case) was measured using the appropriate extracted ion tograms (EIC).

Table63 PK Analysis in Liver (mg/kg) byUVmg/g) Level by EIC (pg/g) muster 353382% 54.2 442 661161 5 32.4 207 G31\AC3-3a Ad 632 44.1 671144 5 205 19.2 Gal\AC3-123 Ad 486 41.5 670061 5 31.6 280 Gal\A03-l3a Ad 67.6 55.5 671261 5 19.8 163 G31\A03-14a Ad 64.7 49.1 671262 5 185 7.4 G31\A03-15a Ad 52.3 24.2 670699 5 16.4 104 Gal\AC3-3a Td 31.5 225 670700 5 19.3 109 Gal\AC3-3a Ae 38.1 200 670701 5 218 8.8 Gal\Ac3_3a T. 35.2 16.1 671165 5 27.1 26.5 Gal\ACg-13a Ad 48.3 443 666904 5 30.8 240 Gal\Ac3—3a p0 52.6 37.6 675441 5 25.4 190 G31\AC3-17a Ad 542 421 675442 5 22.2 207 Gal\A03-18a Ad 39.6 290 The results in Table 63 above show that there were greater liver tissue levels of the oligonucleotides comprising a GalNAc3 conjugate group than of the parent ucleotide that does not comprise a GalNAc3 conjugate group (ISIS 353382) 72 hours ing oligonucleotide administration, particularly when taking into consideration the differences in dosing n the oligonucleotides with and t a GalNAc3 conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a GalNAc3 conjugate group was metabolized to the parent compound, indicating that the GalNAc3 conjugate groups were cleaved from the oligonucleotides.

Example 76: Preparation of oligomeric compound 230 comprising GalNAc3-23 ToSCI NaN3 HO/\/O\/\O/\/OH —> HO/\/O\/\O/\/OTS 222 223 4 TMSOTf HO/\/ \/\O/\/0 N 0Ac O o/\/O\/\O/\/N3 2 OACOAC ACN O O/\/ \/\o/\/0 NH —,2 H2,EtOAc,MeOH OAc F F 226 F F F o c—No2 OAC H OAc N O O O/\/O\/\O/\/ OAcOAc NHAc H No2 1)Reduce o No\/\O/\/N 2) Couple Diacid OAc 3) Pd/C o o OAc 4) PFPTFA NHAc OAc NH —> O O/\/O\/\O/\/ OAc H N O O O/\/O\/\O/\/ OACOAC NHAc H NHMO F OAc O O O O F F NHAc OAc F 0 O/\/O\/\O/\/ NHAc 229 3' 5' ll -O-F|’-O-(CH2)e-NH2 1. Borate buffer, DMSO, pH 8.5, rt 2. aq. ammonia, rt OH H N 0 OHOH NHAc H NH N M"W0 OONowow 4-m- OH O O O O NHAc OH Compound 222 is commercially available. 44.48 ml (0.33 mol) of compound 222 was treated with tosyl chloride (25.39 g, 0.13 mol) in pyridine (5 00mL) for 16 hours. The reaction was then evaporated to an oil, dissolved in EtOAc and washed with water, sat. NaHC03, brine, and dried over NaZSO4. The ethyl acetate was concentrated to dryness and purified by column chromatography, eluted with EtOAc/hexanes (1 :1) followed by 10% methanol in CH2C12 to give nd 223 as a colorless oil. LCMS and NMR were consistent with the structure. 10 g (32.86 mmol) of 1-Tosyltriethylene glycol (compound 223) was treated with sodium azide (10.68 g, 164.28 mmol) in DMSO (100mL) at room temperature for 17 hours. The reaction mixture was then poured onto water, and extracted with EtOAc. The organic layer was washed with water three times and dried over NaZSO4. The organic layer was concentrated to dryness to give 5.3g of compound 224 (92%). LCMS and NMR were consistent with the structure. 1-Azidotriethylene glycol und 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were treated with 4A lar sieves (5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100mL) under an inert atmosphere.

After 14 hours, the reaction was filtered to remove the sieves, and the organic layer was washed with sat.

NaHC03, water, brine, and dried over NaZSO4. The c layer was concentrated to dryness and purified by column chromatography, eluted with a gradient of 2 to 4% methanol in dichloromethane to give compound 225. LCMS and NMR were tent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in Methanol (4:1, 250mL) over Pearlman's catalyst. After 8 hours, the catalyst was removed by filtration and the solvents removed to dryness to give compound 226. LCMS and NMR were consistent with the structure.

In order to generate compound 227, a solution of nitromethanetrispropionic acid (4.17 g, 15.04 mmol) and Hunig’s base (10.3 ml, 60.17 mmol) in DMF ) were treated dropwise with penta?ourotri?uoro acetate (9.05 ml, 52.65 mmol). After 30 minutes, the reaction was poured onto ice water and extracted with EtOAc. The organic layer was washed with water, brine, and dried over NaZSO4. The organic layer was concentrated to dryness and then recrystallized from heptane to give nd 227 as a white solid. LCMS and NMR were consistent with the structure. Compound 227 (1.5 g, 1.93 mmol) and compound 226 (3.7 g, 7.74 mmol) were stirred at room temperature in acetonitrile (15 mL) for 2 hours. The reaction was then evaporated to dryness and purified by column chromatography, eluting with a gradient of 2 t010% methanol in dichloromethane to give compound 228. LCMS and NMR were consistent with the ure. Compound 228 (1.7 g, 1.02 mmol) was treated with Raney Nickel (about 2g wet) in ethanol (100mL) in an atmosphere of hydrogen. After 12 hours, the catalyst was removed by filtration and the organic layer was evaporated to a solid that was used directly in the next step. LCMS and NMR were consistent with the ure. This solid (0.87 g, 0.53 mmol) was treated with glutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 ul, 1.6 mmol) in DMF (5mL). After 16 hours, the DMF was removed under reduced pressure at 65°C to an oil, and the oil was dissolved in dichloromethane. The organic layer was washed with sat. , brine, and dried over Na2804. After evaporation of the organic layer, the compound was purified by column chromatography and eluted with a gradient of 2 to 20% methanol in dichloromethane to give the coupled product. LCMS and NMR were consistent with the structure. The benzyl ester was deprotected with Pearlman’s catalyst under a hydrogen atmosphere for 1 hour. The catalyst was them removed by ?ltration and the solvents removed to dryness to give the acid.

LCMS and NMR were consistent with the structure. The acid (486 mg, 0.27 mmol) was dissolved in dry DMF (3 mL). Pyridine (53.61 ul, 0.66 mmol) was added and the reaction was purged with argon.

Penta?ourotriflouro acetate (46.39 ul, 0.4 mmol) was slowly added to the reaction mixture. The color of the on changed from pale yellow to burgundy, and gave off a light smoke which was blown away with a stream of argon. The on was allowed to stir at room ature for one hour (completion of reaction was confirmed by LCMS). The solvent was removed under reduced pressure ap) at 70 °C. The residue was diluted with DCM and washed with 1N , brine, saturated sodium bicarbonate and brine again. The organics were dried over NaZSO4, filtered, and were concentrated to dryness to give 225 mg of compound 229 as a brittle yellow foam. LCMS and NMR were consistent with the structure.

Oligomeric compound 230, comprising a GalNAc3-23 conjugate group, was prepared from compound 229 using the general procedure rated in Example 46. The GalNAc3 cluster portion of the GalNAC3-23 conjugate group (GalNAC3-23a) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of 3-23 (GalNAC3-23a-CM) is shown below: N 0 OH NHAc H o wowowN OH ""va"46 NHAc OHOH O O/\/O\/\O/\/ Example 77: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a GalNAc3 conjugate The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.

Table 64 Modi?ed ASOs tar etin SRB-l ISIS , , GalNAc3 SEQ sequences (5 303) CM No. Cluster IDNo.

GalNAc-33 3°,A G mCTT mCAGT mdSCAT3° 661161 6° 6°m 6° 6° 6° 6° 6° 6° 6° GalNAC3-3a Ad 2306 GCSACSmCCSTCSTCSmCCSmCCSTCSTC GalNAC3-3a-0’G mc T T mc d dedAdT 666904 6; 6° 6° 6° 6° ° ° ° ° 6° GalNAc3-3a PO 2304 GCSACS CCSTCWTmcsmc TmeT GalNAc,A GmC3 3° T T Inc A G 673502 d°m6° 6° 6° 6° 6° 6° 6°Tdsmc A6° 6°Tds GalNAc3-10a AC 2306 GCSACS CCSTCSTCOmCCOmCCSTCSTC ’A GmCTTmCAGT mdSCAT3 3° 677844 d°m6° 6° 6°m 6° 6° 6° 6° 6° 6° 6° GalNAc3-9a AC 2306 GCSACS CCSTCSTCSmCCSmCCSTCSTC GalNAC3-23a-0aAd0G mc T T mc AdeT mdstAdT 677843 6° 6° 6° 6° 6° ° ° 6° ° ° m GalNAc3-23a AC 2306 mGCSACS CCSTCSTCSmCCSmCCSTCSTC G mC T T mC A 6° GdsTd mcMAdSTdGASmCdsTdT mc 655861 6° 6° m6° 6° ° ° 6° GalNAcg-la AC 2305 CesTesTeoAdo"GalNAc3'1 GmCTTmCAGTmCATGAmCTTmC 677841 6 6 es es mm cs ds ds ds ds ds ds ds ds ds es es GalNA03-l9a Ac 2305 CesT?TmAdealNAcg-wa GmCTTmCAGTmCAT GAmCTTmC 677842 6 6 es es mes ds ds ds ds ds ds ds ds ds ds es 6 GalNA03-20a Ac 2305 eotA(10"(;31NAAC3-20a The ure of GalNAc3-1a was shown preViously in Example 9, GalNAc3-3a was shown in Example 39, GalNAC3-9a was shown in e 52, GalNAC3-10a was shown in Example 46, GalNAC3-19a was shown in Example 70, GalNAc3-20a was shown in Example 71, and GalNAc3-23a was shown in Example Trealmenl Six to eight week old C57BL/6 mice (Jackson tory, Bar Harbor, ME) were each ed subcutaneously once at a dosage shown below with an oligonucleotide listed in Table 64 or with saline. Each treatment group consisted of 4 animals. The mice were iced 72 hours following the final administration to ine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the average percent of SRB-l mRNA levels for each ent group, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotides d SRB-l mRNA levels in a dose-dependent manner.

Table 65 SRB-l mRNA (% Saline) SRB-l mRNA GalNAc3 ISIS No. Dosage (mg/kg) CM (% Saline) Cluster Saline n/a 100.0 n/a n/a 0.5 89.18 1.5 77.02 661161 GalNAc3-3a Ad 29.10 12.64 0.5 93.11 1.5 55.85 666904 GalNAc3-3a P0 21.29 13.43 0.5 77.75 1.5 41.05 673502 GalNAc3-10a Ad 19-27 14.41 0.5 87.65 1.5 93.04 677844 —5 GalNAc3-9a Ad 40.77 16.95 0.5 102.28 1.5 70.51 677843 GalNAc3-23a Ad 30.68 13.26 0.5 79.72 1.5 55.48 655861 GalNAc3-1a Ad 26.99 17.58 0.5 67.43 1.5 45.13 677841 GalNAc3-19a Ad 27.02 12.41 677842 0.5 64.13 GalNAc3-20a Ad Liver minase levels, alanine aminotransferase (ALT) and aspartate ransferase (AST), in serum were also measured using standard protocols. Total bilirubin and BUN were also evaluated. Changes in body weights were evaluated, with no signi?cant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 66 below.

Table66 Total Dosage ALT AST . . . BUN GalNAc3 ISIS No. Bilirubin CM ) (U/L) (U/L) (mg/dL) Cluster (mg/dL) Saline n/a 21 45 0.13 34 n/a n/a 0.5 28 51 0.14 39 1.5 23 42 0.13 39 661161 GalNAc3-3a Ad 22 59 0.13 37 21 56 0.15 35 0.5 24 56 0.14 37 1.5 26 68 0.15 35 666904 GalNAc3-3a P0 23 77 0‘14 34 24 60 0.13 35 0.5 24 59 0.16 34 1.5 20 46 0.17 32 673502 —5 GalNAc3-10a Ad 24 45 0‘12 31 24 47 0.13 34 0.5 25 61 0.14 37 1.5 23 64 0.17 33 677844 GalNAc3-9a Ad 25 58 0.13 35 22 65 0.14 34 0.5 53 53 0.13 35 1.5 25 54 0.13 34 677843 GalNAc3-23a Ad 21 60 0.15 34 22 43 0.12 38 0.5 21 48 0.15 33 1.5 28 54 0.12 35 655861 GalNAc3-1a Ad 22 60 0.13 36 21 55 0.17 30 0.5 32 54 0.13 34 1.5 24 56 0.14 34 677841 GalNAc3-19a Ad 23 92 0.18 31 24 58 0.15 31 0.5 23 61 0.15 35 1.5 24 57 0.14 34 677842 GalNAc3-20a Ad 41 62 0.15 35 24 37 0.14 32 Example 78: Antisense tion in vivo by oligonucleotides targeting Angiotensinogen comprising a ; conjugate The oligonucleotides listed below were tested in a dose-dependent study for nse inhibition of ensinogen (AGT) in normotensive Sprague Dawley rats.

Table 67 Modi?ed ASOs targeting AGT , , GalNA03 SEQ CesA? ?AdsTdsTgTXT$TdsGds Cds Cds CdsAesGes 552668 2310 CesA? C?TeSG?AdSTdSTdSTdSTdSTdSGdS Cds Cds CdsAesGes 669509 GalNAC3-1a Ad 231 1 GesAesTeoAdo"GalNAc3'1 a The structure of GalNA03-1a was shown previously in Example 9. 1 0 Trealmenl Six week old, male Sprague Dawley rats were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 67 or with PBS. Each treatment group consisted of 4 animals. The rats were sacri?ced 72 hours ing the ?nal dose. AGT liver mRNA levels were measured using real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. AGT plasma protein levels were measured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBL International, Toronto, ON) with plasma diluted 120,000. The results below are presented as the average percent ofAGT mRNA levels in liver or AGT protein levels in plasma for each treatment group, normalized to the PBS control.

As illustrated in Table 68, treatment with antisense oligonucleotides lowered AGT liver mRNA and plasma n levels in a dose-dependent , and the oligonucleotide comprising a GalNAc conjugate was signi?cantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

Table 68 AGT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) AGT liver mRNA AGT plasma GalNA03 CM No. (% PBS) protein (% PBS) Cluster PBS n/a 100 100 n/a n/a 3 95 122 85 97 552668 n/a n/a 46 79 90 8 1 1 0.3 95 70 1 95 129 669509 GalNAcg-la Ad 3 62 97 9 23 Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in plasma and body weights were also measured at time of sacrifice using standard protocols. The results are shown in Table 69 below.

Table 69 Liver transaminase levels and rat body weights Dosage GalNAC3 ISIS No. ALT (U/L) AST (U/L) Weight (% CM (mg/kg) r of baseline).

PBS n/a 51 81 186 n/a n/a 3 54 93 183 51 93 194 552668 n/a n/a 59 99 182 90 56 78 170 0.3 53 90 190 1 51 93 192 669509 GalNAc3-1a Ad 3 48 85 189 56 95 189 Example 79: Duration of action in vivo of ucleotides targeting APOC-III comprising a 3 The oligonucleotides listed in Table 70 below were tested in a single dose study for duration of action in mice.

Table 70 Modi?ed ASOs targeting APOC-III sequences (5 , to 3 , GalNA03 SEQ ) CM No. Cluster ID No.

AmGes CesTesTes CdsTdsT%sG:sTil_‘s Cds CdsAdsGds CdsTesTes 304801 n/a n/a 2296 AmGes CesTesTes CdsTdsTdsGdsTds Cds Gds CdsTesTes 647535 GalNA03-1a Ad 2297 TesAesTeoAdo"GalNAc3'1a 663083 GalNAc3-33E’AderSG: C?TesT? CdsTdsTdsGdsTds Cds GalNAC3-3a Ad 2312 CdsAdsGds CdsTesTes TesAesTe GalNAc3'7a'o’AderSG?mCmTesTesmCdsTdsTdsGdsTdsmCds 674449 G lNAa 03-7a Ad 2312 mCdslAdsC}dsmCdsTesTes TesAesTe 674450 GalNAc3'10ago’AdersG: CesTesTes CdsTdsTdsGdsTds GalNAC3-103 Ad 2312 CdsAdsGds CdsTesTes TesAesTe 674451 GalNAc3'13ago’AdersG: Tes TdsGdsTds Cds GalNAC3-1 3a Ad 2312 CdsAdsGds CdsTesTes TesAesTe The ure of GalNAc3-1a was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in Example 46, and GalNAcg- 13a was shown in Example 62.

Trealmenl Six to eight week old transgenic mice that express human APOC-HI were each injected subcutaneously once with an oligonucleotide listed in Table 70 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to determine baseline and at 72 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the dose. Plasma triglyceride and APOC-III protein levels were ed as described in Example 20. The s below are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels, showing that the oligonucleotides comprising a GalNAc conjugate group exhibited a longer duration of action than the parent oligonucleotide t a conjugate group (ISIS 304801) even though the dosage of the parent was three times the dosage of the oligonucleotides comprising a GalNAc conjugate group.

Table 71 Plasma triglyceride and APOC-III protein levels in transgenic mice Tune pomt AP0cm ISIS Dosage Triglycerides 3 CM (days post- No (mg/kg) (% baseline) pmtem (% ' Cluster dose) baseline) 3 97 102 7 101 98 14 108 9 8 PBS n/a 21 107 107 n/a n/a 28 94 91 88 90 42 91 105 3 40 34 7 41 37 14 50 57 304801 30 21 50 50 n/a n/a 28 57 73 68 70 42 75 93 3 36 37 7 39 47 14 40 45 647535 10 21 41 41 GalNAc3-1a Ad 28 42 62 69 69 42 85 102 3 24 18 7 28 23 14 25 27 663083 10 21 28 28 GalNAc3-3a Ad 28 37 44 55 57 42 60 78 674449 10 3 29 26 GalNAc3-7a Ad 7 32 31 14 38 41 21 44 44 28 53 63 69 77 42 78 99 3 33 30 7 35 34 14 31 34 674450 10 21 44 44 GalNAc3- 1 0a 28 56 61 68 70 42 83 95 3 35 33 7 24 32 14 40 34 674451 10 21 48 48 GalNAc3- 1 3 a 28 54 67 65 75 42 74 97 Example 80: Antisense inhibition in vivo by oligonucleotides targeting Alpha-1 Antitrypsin (AlAT) comprising a GalNAc; conjugate The oligonucleotides listed in Table 72 below were tested in a study for dose-dependent inhibition of A1AT in mice.

Table 72 Modi?ed ASOs targeting AlAT Sequences (5 , , GalNA03 SEQ ID t0 3 ) CM No. r N0.

Aes Ces Ces CesAmAdsTdsTds CdsAdsGdsAdsAdsGdsGdsAesAes 476366 n/a n/a 23 1 3 AesmcesmCesmCesAesAdsTdsTdsmCdsAdsGdsAdsAdsGdsGdsAesAes 656326 G lNAa 03 -1 a Ad 23 14 G?GesAmAdowGalNAg-la GalNAc3'3a'o’AderS Ces Ces CesAesAdsTdsTds CdsAdsGdsAds 67 83 81 3-3a Ad 23 1 5 AdsGdsGdsAesAes GesGesAe GalNAc3'7a'o’AderS Ces Ces CesAesAdsTdsTds CdsAdsGdsAds 67 83 82 GalNA03-7a Ad 2315 AdsGdsGdsAesAes GesGesAe 3'1Oa'o’AderSmCeSmCesmcesAesAdsTdsTdsmCdsAdsGds 678383 G lNAa 03-10a Ad 2315 AdsAdsGdsGdsAesAes GesGesAe GalNAc3-13a-0’Ad0Aes Ces Ces CeSAesAdsTdsTds Gds 67 83 84 GalNAC3-13 a Ad 2315 AdsAdsGdsGdsAesAes GesGesAe The structure of GalNAc3-1a was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in e 46, and GalNAc3- 13a was shown in Example 62.

Trealmenl Six week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed in Table 72 or with PBS. Each treatment group ted of 4 animals. The mice were sacrificed 72 hours following the ?nal administration. A1AT liver mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. AlAT plasma protein levels were ined using the Mouse Alpha 1-Antitrypsin ELISA (catalog # 41 -A1AMS-E01 , Alpco, Salem, NH). The results below are presented as the average percent of A1AT liver mRNA and plasma protein levels for each ent group, normalized to the PBS control.

As illustrated in Table 73, ent with antisense oligonucleotides lowered A1AT liver mRNA and A1AT plasma protein levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent (ISIS 476366).

Table 73 AlAT liver mRNA and plasma protein levels ISIS Dosage (mg/kg) A1AT liver A1AT plasma GalNAc3 Cluster CM No. mRNA (% PBS) n (% PB S) PBS n/a 100 100 n/a n/a 86 78 476366 73 61 n/a n/a 45 30 38 0.6 99 90 2 61 70 656326 GalNAc3-1a Ad 6 15 30 18 6 10 0.6 105 90 678381 2 53 60 GalNAc3-3a Ad 6 16 20 18 7 13 0.6 90 79 2 49 57 678382 GalNAc3-7a Ad 6 21 27 18 8 11 0.6 94 84 2 44 53 678383 GalNAc3-10a Ad 6 13 24 18 6 10 0.6 106 91 2 65 59 678384 GalNAc3-13a Ad 6 26 31 18 11 15 Liver transaminase and BUN levels in plasma were measured at time of sacrifice using standard protocols. Body s and organ weights were also measured. The results are shown in Table 74 below.

Body weight is shown as % relative to baseline. Organ weights are shown as % of body weight relative to the PBS control group.

Table 74 ISIS Dosage ALT AST BUN BOdy .Lwer K‘dney Spleen we1ght (% weight (Rel weight (Rel weight (Rel No ' (m /kg g) (U/L) (U/L) (m /dL)g baseline) % BVW % BW) % BW) PBS n/a 25 51 37 119 100 100 100 34 68 35 116 91 98 106 476366 15 37 74 30 122 92 101 128 45 30 47 31 118 99 108 123 0.6 29 57 40 123 100 103 119 2 36 75 39 114 98 111 106 656326 6 32 67 39 125 99 97 122 18 46 77 36 116 102 109 101 0.6 26 57 32 117 93 109 110 2 26 52 33 121 96 106 125 678381 6 40 78 32 124 92 106 126 18 31 54 28 118 94 103 120 0.6 26 42 35 114 100 103 103 2 25 50 31 117 91 104 117 678382 6 30 79 29 117 89 102 107 18 65 112 31 120 89 104 113 . 30 67 38 121 91 100 123 2 33 53 33 118 98 102 121 678383 6 32 63 32 117 97 105 105 18 36 68 31 118 99 103 108 . 36 63 31 118 98 103 98 2 32 61 32 119 93 102 114 678384 6 34 69 34 122 100 100 96 18 28 54 30 117 98 101 104 Example 81: Duration of action in vivo of oligonucleotides targeting AlAT comprising a GalNAc; conjugate The ucleotides listed in Table 72 were tested in a single dose study for duration of action in mice.

Trealmenl Six week old, male C57BL/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 72 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn the day before dosing to determine baseline and at 5, 12, 19, and 25 days following the dose. Plasma AlAT protein levels were measured via ELISA (see Example 80). The results below are ted as the average percent of plasma AlAT protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent and had longer duration of action than the parent g a GalNAc conjugate (ISIS 476366). Furthermore, the oligonucleotides comprising a 5’- GalNAc conjugate (ISIS , 678382, 6783 83, and 678384) were generally even more potent with even longer duration of action than the oligonucleotide comprising a 3’-GalNAc conjugate (ISIS 656326).

Table 75 Plasma AlAT protein levels in mice ISIS Dosage Time point AlAT (% GalNAc3 CM No. (mg/kg) (days post- baseline) Cluster dose) 93 12 93 PBS n/a n/a n/a 1 9 90 97 38 12 46 476366 100 n/a n/a 19 62 77 33 12 36 656326 18 —1 GalNAC3- 1 a Ad 9 51 72 21 12 21 678381 18 —19GalNAc3-3a Ad 48 21 12 21 6783 82 18 —1 GalNAc3-7a Ad 9 39 60 24 12 21 678383 18 GalNAc3-10a Ad 19 45 73 29 12 34 67 83 84 18 —1GalNAC3- 1 3a Ad 9 57 76 Example 82: Antisense inhibition in vitro by oligonucleotides targeting SRB-l comprising a GalNAc3 conjugate Primary mouse liver hepatocytes were seeded in 96 well plates at 15,000 cells/well 2 hours prior to treatment. The oligonucleotides listed in Table 76 were added at 2, 10, 50, or 250 nM in ms E medium and cells were incubated overnight at 37 0C in 5% C02. Cells were lysed 16 hours following ucleotide addition, and total RNA was puri?ed using RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular Probes, Inc.

Eugene, OR) according to rd protocols. IC50 values were ined using Prism 4 software (GraphPad). The results show that oligonucleotides comprising a variety of different GalNAc conjugate groups and a variety of different cleavable moieties are significantly more potent in an in vitro free uptake experiment than the parent oligonucleotides lacking a GalNAc conjugate group (ISIS 353382 and 666841).

Table 76 Inhibition of SRB-l sion in vitro Sequence (5 , to3 , . GalNAc IC50 SEQ ) Linkages CM No. cluster nM IDNo. 353382 CSSTSSECSS gSSTASSnCEESC CTSSédSTSSGdSASS PS n/a n/a 250 2304 ds ds es es es es e GesmCeSTesT?mCesAdsGdsTdsmCdsAdsTdsGdsAds Gal\AC3 633861 PS AS 40 3305 mcdsTdsT?mcesmcesTesTekoGalNAcs-la -la GglNAC3-3a-03Adognes THels AdSGdSTdS Ga1\Ac3 661161 PS Ac 40 2306 CdsAdsTdsGdsAds CdsTds Tes Ces C?TeSTe '33. 661162 GglNAcg-3a-0AdoGnes CeoTeoTIrelo CneloAdsGdsTdS Ga1\A03 P0/PS Ac 8 23 06 CdsAdsTdsGdsAds CdsTds Teo Ceo C?TesTe '33.

GesmCeSTesT?mCesAdsGdsTdsmCdsAdsTdsGdsAds Gal\AC3 664078 PS AS 20 2305 mcdsTdsT?mcesmcesTesTwAdo,-Ga1NAc3-9a 91 GalNAC30’AdoGesmCesTesTesmCesAdsGdsTds 3 665001 PS AC 70 23 06 IncdsAdsTdsGdsAdsmCdsTdsTesmCesmCesTesTe ' 8a GalNAC3-sa-0’AdoGesmCesTesTesmCesAdsGdsTds Ga1\Ac3 666224 PS AC 80 2306 mCdslAdsTdSChISIAdsmCdsTdsTesmCeSmCesTesTe 'Sa 666841 CSSTSgnTCSS TCSfSSnCESSTmSSC CTSSlfdeTSSGdSASS PO/PS n/a n/a >250 2304 ds ds e0 e0 6 es e ,AdonmCmTesTmmCesAdsGdsTdS Gal\A03 6668 81 PS Ad 30 23 06 mCdslAdsTdsCidslAdsmCdsTdsTesmCeSmCesTesTe '10a GalNA030,G?m CesTesTesmCes?dsGdsTds Cds 3 666904 PS PO 9 2304 AdsTdsGdsAds CdsTds Tes Ces CesTesTe '33, GalNAc3'3-a'o’TdoC}esmCesTesTesmCesAdsGdsTds Gal\AC3 666924 PS Td 15 2309 mmCdslAdsTdsCicslAcsCcsTcs smCmTesTe '33. c;alNlAc3'621'o’lAdoC}esm esTesTesmCesAdsGdsTds Gal\AC3 666961 PS Ad 150 2306 mCdsAdsTdchsAcschs csTesmCeSmCesTeSTe '6a c;alNlAc3'37a'o’lAdoC}esm esTesTesmCesAdsGdsTds Gal\AC3 6669 81 PS Ad 20 23 06 mCdslAdsTdsChslAcschsTcsTesmCeSmCesTeSTe '7a 670061 ngNAc3-13a-O3Adog? CmTesTmm gesAdsGdsTds Ga1\A03 PS Ad 30 2306 CdsAdsTdchsAcs CcsTcs Tes Ces CmTesTe '133.

GalNAcTOG mc T T mc A G T3 ‘3 670699 USS S0 SS US: $1" SS S SS G31\ACS PO/PS Td 15 2309 IncdsAdsTdchsAcs CcsTcsTeo CEneo C?TesTe '33.

GalNAc-3aMAG mC T T mC A G T3 670700 S S0 S0 S0 S0 SS S SS Gal\ACS PO/PS AC 30 2306 IncdsddsAdsTdSGSAmcdSTdSTmmcemcmeT T -3a GalNAc -3a-0:Te0G mC T T mC A G T3 Gal\ACS 670701 I35 S0 S0 I? I? SS S SS m PO/PS Te 25 2306 CdsAdsTds GdsAds CdsTdsTeo Ce0 C?TesTe '33. 671144 Acg-IZa-OAdOICn}m CmTesTmm GdsTds 3 PS Ad 40 2306 CdsAdsTdsGdsAds CdsTds Tes Ces C?TeSTe _123' GalNAc -13,-0A 0Gmc T T3 d Inc A G T 6° 6° 6° 6° 6° 6° G31\AC3 671165 m6° PO/PS A, 8 2306 IncdSAdSTdSGsdsAmcdSTdSTmmcmeomC?TeST -13a 671261 GarlNAcg0:AdoICn}mmmCmTeSTmmgesAdsGdsTds 3 PS Ac >250 2306 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '143.

Gal\AC3 671262 rlS-a-O’Adog? C?TesTn? gleSAdsGdsTds PS Ac >250 23 06 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '153.

Gal\Ac3 6735 01 GanlNA03-7a-0sAdoCines CeOTeoTU?0 CIEOAdSGdsTdS PO/PS Ac 30 23 06 CdsAdsTdsGdsAds CdsTdsTeo Ceo CesTesTe '73.

Gal\A03 673502 GillNAcTIOa'o’Adogm CeoTeo'Eeo geoAdsGdsTds PO/PS Ac 8 23 06 CdsAdsTdsGdsAds CdsTds Teo Ceo C?TesTe '103. 675441 '17a'o’Adogm C?TesTnm CtileslAdsChls'l-‘ds Gal\AC3 PS Ac 30 23 06 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '173. 675442 ngNAc3O’Ad0g? C?TeSF‘IEI? (EneSAdSGdSTdS PS AC 20 2306 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe _18a GesmCeSTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Gal\A03 677841 PS A6 40 23 05 mcdsTdsTesmc,Jnc,sTesTmAdo,—GalNAc3-19a —19, GesmCeSTesTesmCesAdsGdsTdsmCdsAdsTdsGdsAds Gal\A03 677842 PS A6 30 23 05 mcdsTdsTesmc,Jnc,sTesTmAdo,—GalNAc3-20a —20a Gal\Ac3 677843 ngNAc3O,Ad°g? CmTeS?mm gesAdsGdsTds PS Ac 40 2306 CdsAdsTdsGdsAds CdsTds Tes Ces CesTesTe '233.

The structure of GalNAc3-1a was shown usly in Example 9, 3-3a was shown in Example 39, GalNAC3-5a was shown in Example 49, GalNAC3-6a was shown in Example 51, GalNAC3-7a was shown in Example 48, GalNAc3-8a was shown in Example 47, GalNAc3-9a was shown in Example 52, GalNAc3-10a was shown in e 46, GalNAc3-12a was shown in Example 61, GalNAc3-13a was shown in Example 62, GalNAc3-14a was shown in Example 63, GalNAc3-15a was shown in Example 64, GalNAc3-17a was shown in Example 68, GalNAc3-18a was shown in Example 69, GalNAc3-19a was shown in e 70, GalNAc3-20a was shown in Example 71, and GalNAc3-23a was shown in Example 76.

Example 83: Antisense inhibition in vivo by oligonucleotides ing Factor XI sing a GalNAc; conjugate The oligonucleotides listed in Table 77 below were tested in a study for dose-dependent inhibition of Factor XI in mice.

Table 77 Modi?ed oli onucleotides tar ' etin Factor XI ISIS GalNAc SEQ TesGesGesTAesAdsTds c: 3:31:13 cdsTdsTdsTds CdsAesG 404071 2307 TesGeoGeoTeeroAdsTdsmCdsmCdsAdsmCdsTdsTdsTdsmCdsAeoGeo 656173 GalNAcg-la 2308 A?GesGmAdos-GalNAc3-1, 663086 GalNAc-3a-oAdoT?GeonTCOAeoAdSTdsmcdsmcdsAdsmcdsTds GalNAca-sa 2316 _ TdsTdsmCdsAeoGeersG?Ge —-_ GalNAc3'7a'o’AdoT?GeoGeoTeeroAdsTdsmCdsmCdsAdsmCdsTds 678347 GalNAc3-7a 2316 TdsTdsmCdsA60GeoA65G?Ge 3'10a'o’AdoT?GeoGeoTeeroAdsTdsmCdsmCdsAdsmCds 67 8348 GalNAC3-10a Ad 2316 TdsTdsTdsmCdslAeoC}eolAesC}®SC}e GalNAC3-13a'o’AdonGeoGeoTeeroAdsTdsmCdsmCdsAdsmCds 67 8349 GalNAc3-13a Ad 2316 TdsTdsTdsmCdslAeoC}eolAesC}®SC}e The structure of GalNAc3-1a was shown previously in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in Example 48, GalNAc3-10a was shown in Example 46, and GalNAc3- 13a was shown in Example 62.

Trealmenl Six to eight week old mice were each ed subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide listed below or with PBS. Each treatment group consisted of 4 animals. The mice were sacri?ced 72 hours following the final dose. Factor XI liver mRNA levels were measured using real-time PCR and normalized to cyclophilin ing to rd protocols.

Liver transaminases, BUN, and bilirubin were also measured. The results below are presented as the average percent for each treatment group, normalized to the PBS control.

As illustrated in Table 78, treatment with antisense oligonucleotides lowered Factor XI liver mRNA in a dose-dependent manner. The results show that the oligonucleotides sing a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5’-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent than the oligonucleotide sing a 3’-GalNAc conjugate (ISIS 656173).

Table 78 Factor XI liver mR‘IA, liver transaminase, BUN, and bilirubin levels ISIS Dosage Factor XI ALT AST BUN bin GalNAc3 SEQ No. (mg/kg) mRNA (% PBS) (U/L) (U/L) (mg/dL) (mg/dL) Cluster ID No.

PBS n/a 100 63 70 21 0.18 n/a n/a 3 65 41 58 21 0.15 404071 33 49 53 23 0.15 n/a 2307 17 43 57 22 0.14 0.7 43 90 89 21 0.16 656173 2 9 36 58 26 0.17 GalNAc3-1a 2308 6 3 50 63 25 0.15 0.7 33 91 169 25 0.16 663086 2 7 38 55 21 0.16 GalNAc3-3a 2316 6 1 34 40 23 0.14 0.7 35 28 49 20 0.14 678347 2 10 180 149 21 0.18 GalNAc3-7a 2316 6 1 44 76 19 0.15 0.7 39 43 54 21 0.16 678348 2 5 38 55 22 0.17 GalNAc3-10a 2316 6 2 25 38 20 0.14 678349_-___GalNA03-13a 2316 _—-__ Example 84: on of action in vivo of oligonucleotides ing Factor XI comprising a GalNAc3 conjugate The oligonucleotides listed in Table 77 were tested in a single dose study for duration of action in mice.

Trealmenl Six to eight week old mice were each injected aneously once with an oligonucleotide listed in Table 77 or with PBS. Each treatment group consisted of 4 animals. Blood was drawn by tail bleeds the day before dosing to determine baseline and at 3, 10, and 17 days following the dose. Plasma Factor XI protein levels were measured by ELISA using Factor XI capture and biotinylated detection antibodies from R & D Systems, Minneapolis, MN (catalog # AF2460 and # BAF2460, respectively) and the OptEIA Reagent Set B (Catalog # 550534, BD Biosciences, San Jose, CA). The results below are presented as the average t ofplasma Factor XI protein levels for each treatment group, normalized to baseline . The results show that the oligonucleotides comprising a GalNAc conjugate were more potent with longer duration of action than the parent g a GalNAc conjugate (ISIS 404071). Furthermore, the oligonucleotides comprising a 5 ’-GalNAc conjugate (ISIS 663086, 678347, 678348, and 678349) were even more potent with an even longer duration of action than the oligonucleotide comprising a 3’-GalNAc conjugate (ISIS ).

Table 79 Plasma Factor XI protein levels in mice ISIS Dosa e Time oint (da 5 Factor XI (% CM SEQ ID No. (mg/kgg) poEt-dose) Y GalNA03 r baseline) No. 3 123 PBS n/a 10 56 n/a n/a n/a 17 100 3 1 1 404071 30 10 47 n/a n/a 2307 17 52 3 1 656173 6 10 3 GalNAcg-la Ad 2308 17 21 3 1 663086 6 10 2 GalNA03-3a Ad 2316 17 9 3 1 678347 6 10 1 GalNA03-7a Ad 2316 17 8 678348 6 130 i GalNA03-10a Ad 2316 _——"—-_ 678349-== GalNACs- 13a 2316 Example 85: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising a GalNAc3 conjugate Oligonucleotides listed in Table 76 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.

Trealmenl Six to eight week old C57BL/6 mice were each ed subcutaneously once per week at a dosage shown below, for a total of three doses, with an ucleotide listed in Table 76 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 48 hours following the final administration to determine the SRB-l mRNA levels using real-time PCR and RIBOGREEN® RNA quanti?cation t (Molecular Probes, Inc. Eugene, OR) according to standard protocols. The results below are presented as the e percent of liver SRB-l mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner.

Table 80 SRB-l mRNA in liver ISIS No. Dosage (mg/kg) SRB-l mRNA (% GalNAc3 Cluster CM Saline) Saline n/a 100 n/a n/a 0.1 94 655861 —0'3 GalNAc3-1a Ad 1 68 3 32 0.1 120 661161 —0'3 GalNAc3-3a Ad 1 68 3 26 0.1 107 666881 :37 GalNAc3-10a Ad 3 27 0.1 120 666981 —0'3 3-7a Ad 1 54 3 21 0.1 1 18 670061 —(1)'3:3 GalNAc3-13a Ad 3 18 677842 —— GaINACS'ZOa Ad Table 81 SRB-l mRNA in liver ISIS No. Dosage (mg/kg) SRB-1 mRNA (% GalNA03 Cluster CM Saline) 0.1 107 661161 %GalNA03-3a Ad 3 18 0.1 1 10 677841 %GalNA03-19a Ad 3 25 Liver transaminase levels, total bilirubin, BUN, and body weights were also measured using standard ols. Average values for each treatment group are shown in Table 82 below.

Table 82 ISIS Dosage ALT AST Bilirubin BUN Body Weight GalNA03 CM No. (mg/kg) (U/L) (U/L) (mg/dL) (mg/dL) (% baseline) Cluster Saline n/a 19 39 0.17 26 118 n/a n/a 0.1 25 47 0.17 27 114 0.3 29 56 0.15 27 118 655861WGalNA03-la Ad 3 27 54 0.14 24 115 0.1 35 83 0.13 24 113 0.3 42 61 0.15 23 117 661161 3-3a Ad 1 34 60 0.18 22 116 3 29 52 0.13 25 117 0.1 30 51 0.15 23 118 0.3 49 82 0.16 25 119 666881—1GalNA03-10a Ad 23 45 0.14 24 117 3 20 38 0.15 21 112 0.1 21 41 0.14 22 113 0.3 29 49 0.16 24 112 666981 GalNA03-7a Ad 1 19 34 0.15 22 111 3 77 78 0.18 25 115 0.1 20 63 0.18 24 111 0.3 20 57 0.15 21 115 670061 GalNA03-13a Ad 1 20 35 0‘14 20 115 3 27 42 0.12 20 116 0.1 20 38 0.17 24 114 0.3 31 46 0.17 21 117 677842 GalNA03-20a Ad 1 22 34 0.15 21 119 3 41 57 0.14 23 118 e 86: Antisense inhibition in vivo by oligonucleotides targeting TTR comprising a GalNAc; conjugate Oligonucleotides listed in Table 83 below were tested in a dose-dependent study for nse inhibition ofhuman transthyretin (TTR) in transgenic mice that express the human TTR gene.

Trealmenl Eight week old TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an ucleotide and dosage listed in the tables below or with PBS.

Each treatment group ted of 4 animals. The mice were sacri?ced 72 hours following the ?nal administration. Tail bleeds were performed at various time points throughout the experiment, and plasma TTR protein, ALT, and AST levels were measured and reported in Tables 84-87. After the animals were sacri?ced, plasma ALT, AST, and human TTR levels were measured, as were body weights, organ weights, and liver human TTR mRNA levels. TTR protein levels were measured using a clinical analyzer (AU4 80, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular Probes, Inc. Eugene, OR) were used according to standard protocols to determine liver human TTR mRNA levels.

The s presented in Tables 84-87 are the e values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma n levels are the average values relative to the average value for the PBS group at ne. Body weights are the average percent weight change from baseline until sacri?ce for each individual treatment group. Organ weights shown are normalized to the animal’s body weight, and the average normalized organ weight for each treatment group is then presented relative to the average normalized organ weight for the PBS group.

In Tables 84-87, "BL" indicates baseline, measurements that were taken just prior to the ?rst dose.

As illustrated in Tables 84 and 85, treatment with antisense ucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc ate (ISIS 420915). rmore, the oligonucleotides comprising a GalNAc conjugate and mixed PS/PO internucleoside linkages were even more potent than the oligonucleotide sing a GalNAc conjugate and full PS linkages.

Table 83 Oligonucleotides targeting human TTR ISlS No.. Sequence 5 , , Linkages.

GalNAc SEQ to 3 CM cluster ID No. 420915 TesGefdsTTdtqudség SESAdsTdsGdsAdsAds PS n/a n/a 23 17 T?mCesTesTesGesGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 660261 PS GalNAcg-la Ad 2318 AmTesmCesmCesmCeoAdowGalNAcg-1a GalNAc3'3a-o’TesmCeoTeoTeoGeoGdsTdsTdsAdsmCdsAds 682883 PS/PO GalNAc3-3a PO 2317 TdsGdsAdsAdsAeoTeomCesmcesmce GalNAc3'7a-O’TesmceoTeoTeoGeoGdsTdsTdsAdsmCdsAds 682884 PS/PO GalNAc3-7a PO 2317 TdsGdsAdsAdsAeoTeomCesmcesmce GalNAc3'10a-O’TesmceoTGOTGOGGOGdSTdsTdsAdsmCds 682885 PS/PO GalNAc3-10a m 2317 AdsTdsGdsAdsAdsAeoTeomCesmC?mc e GalNAc3'13a-O’TesmceoTeoTeoC}eoC}dsTdsTdsAdsmCds 682886 PS/PO GalNAc3-13a m 2317 AdsTdsGdsAdsAdsAeoTeomCesmC?mc e T65 CeoTeoTeoGeoGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 684057 PS/PO GalNAc3-19a 2318 [AeoTeoInCestnCesmCeoAdo"GalNAc3'19a The legend for Table 85 can be found in Example 74. The ure of GalNAC3-1 was shown in Example 9. The structure of 3-3a was shown in Example 39. The structure of GalNAC3-7a was shown in Example 48. The structure of GalNAc3-10a was shown in Example 46. The structure of GalNAc3-13a was shown in Example 62. The ure of GalNAc3-19a was shown in Example 70.

Table 84 nse inhibition of human TTR in vivo Isis Dosage TTR mRNA (% Plasma TTR protein SEQ ID GalNAc cluster CM No. (mg/kg) PB S) (% PB S) No.

PBS n/a 100 100 n/a n/a 6 99 95 420915 20 48 65 n/a n/a 2317 60 18 28 0.6 1 13 87 2 40 56 660261 GalNAc3-1 a 2318 6 20 27 9 1 1 Table 85 Antisense inhibition of human TTR in vivo TTR Plasma TTR protein g A) PBS at BL}0 SEQ Dosage GalNAc Isis No. mRNA Day 17 CM ID (mg/kg) BL Day 3 Day 10 cluster (% PBS) (After sac) No.

PBS n/a 100 100 96 90 1 14 n/a n/a 6 74 106 86 76 83 420915 20 43 102 66 61 58 n/a n/a 2317 60 24 92 43 29 32 0.6 60 88 73 63 68 682883 2 18 75 38 23 23 Gan??? 2317 6 10 80 35 1 1 9 0.6 56 88 78 63 67 682884 2 19 76 44 25 23 Gauge? 2317 6 15 82 35 21 24 0.6 60 92 77 68 76 682885 2 22 93 58 32 32 (13%:03' 2317 6 17 85 37 25 20 0.6 57 91 70 64 69 GalNAc3- 682886 P0 2317 2 21 89 5 0 31 30 13a —.-——————-- "__"n G INA 684057 319303" Ad 2318 Table 86 Transaminase levels, body weight changes, and relative organ weights Dos ALT (U/L) AST (U/L) Body L1ver~ Spleen K1dne~ age SEQ IsisN0~ % BL Day Day Day BL Day Day Day (% (% (% y(% IDNO. 3 10 17 3 10 17 BL) PBS) PBS) PBS) PBS n/a 33 34 33 24 58 62 67 52 105 100 100 100 n/a 6 34 33 27 21 64 59 73 47 115 99 89 91 420915 20 34 30 28 19 64 54 56 42 111 97 83 89 2317 60 34 35 31 24 61 58 71 58 113 102 98 95 0.6 33 38 28 26 70 71 63 59 111 96 99 92 2 29 32 31 34 61 60 68 61 118 100 92 90 660261 2318 6 29 29 28 34 58 59 70 90 114 99 97 95 33 32 28 33 64 54 68 95 114 101 106 92 Table 87 Transaminase levels, body weight changes, and relative organ weights Dos ALT (U/L) AST (U/L) ~ ~ —Body L1ver Spleen K1dne age SEQ IsisN0~ Day Day Day Day Day Day (% (% (% y(% 5k;1’1’1 BL BL IDNO. 3 10 17 3 10 17 BL) PBS) PBS) PBS) PBS n/a 32 34 37 41 62 78 76 77 104 100 100 100 n/a 6 32 30 34 34 61 71 72 66 102 103 102 105 420915 20 41 34 37 33 80 76 63 54 106 107 135 101 2317 60 36 30 32 34 58 81 57 60 106 105 104 99 0.6 32 35 38 40 53 81 74 76 104 101 112 95 682883 2 38 39 42 43 71 84 70 77 107 98 116 99 2317 6 35 35 41 38 62 79 103 65 105 103 143 97 0.6 33 32 35 34 70 74 75 67 101 100 130 99 682884 2 31 32 38 38 63 77 66 55 104 103 122 100 2317 6 38 32 36 34 65 85 80 62 99 105 129 95 0.6 39 26 37 35 63 63 77 59 100 109 109 112 682885 2 30 26 38 40 54 56 71 72 102 98 111 102 2317 6 27 27 34 35 46 52 56 64 102 98 113 96 0.6 30 40 34 36 58 87 54 61 104 99 120 101 682886 2 27 26 34 36 51 55 55 69 103 91 105 92 2317 6 40 28 34 37 107 54 61 69 109 100 102 99 0.6 35 26 33 39 56 51 51 69 104 99 110 102 684057 2 33 32 31 40 54 57 56 87 103 100 112 97 2318 6 39 33 35 40 67 52 55 92 98 104 121 108 Example 87: Duration of action in vivo by single doses of oligonucleotides targeting TTR sing a GalNAc; conjugate ISIS numbers 420915 and 660261 (see Table 83) were tested in a single dose study for duration of action in mice. ISIS s 420915, 682883, and 682885 (see Table 83) were also tested in a single dose study for duration of action in mice.

Treatment Eight week old, male transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915 or 13.5 mg/kg ISIS No. 660261. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86. The s below are presented as the average percent of plasma TTR levels for each treatment group, normalized to baseline levels.

Table 88 Plasma TTR protein levels ISIS Dosage Time point TTR (A) ne)0 . GalNAc3 CM SEQ ID NO' No. (mg/kg) (days post-dose) Cluster 3 30 7 23 35 420915 100 n/a n/a 2317 17 53 24 75 39 100 3 27 7 21 660261 13.5 —10GalNAcg-la Ad 2318 17 36 24 48 39 69 Treatment Female transgenic mice that express human TTR were each injected subcutaneously once with 100 mg/kg ISIS No. 420915, 10.0 mg/kg ISIS No. 682883, or 10.0 mg/kg 682885. Each treatment group consisted of 4 animals. Tail bleeds were performed before dosing to determine baseline and at days 3, 7, 10, 17, 24, and 39 following the dose. Plasma TTR protein levels were measured as described in Example 86.

The results below are presented as the average t of plasma TTR levels for each treatment group, normalized to ne levels.

Table 89 Plasma TTR protein levels ISIS Dosage Time point 0 GalNAc3 420915 100 31 80 3 45 7 37 682883 10.0 10 38 GalNAc3-3a PO 2317 17 42 31 65 3 40 7 33 682885 10.0 10 34 GalNAc3-10a PO 2317 17 40 31 64 The results in Tables 88 and 89 show that the oligonucleotides comprising a GalNAc conjugate are more potent with a longer duration of action than the parent oligonucleotide lacking a conjugate (ISIS 420915) Example 88: Splicing modulation in vivo by oligonucleotides targeting SMN comprising a GalNAc; conjugate The oligonucleotides listed in Table 90 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.

Table 90 Modi?ed ASOs targeting SMN , , GalNA03 SEQ sequences (5 to 3 ) CM No. r ID No.

AesTesTes CesAes CesTesTesTes CSesAmTesAesAesTeSCIes CesTesGes 387954 n/a n/a 2319 GalNAC3-7a'o’AeSTmTesmCesAmmC?TesTesTesmCesAesTesAesAes 699819 GaINA03 7a- P0 23 1 9 TesGesmcesTtsGesGe GalNAc3'7a'0’A?TeoTeomCeeromCeoTeoTeoTeomCeeroTeero 699821 GaINA03 7a- P0 23 1 9 ABOTBoGeOmcmTaGesGe AesTesTes CesAes CesTesTesTes TesAwAesTesGes CesTmGes 700000 GalNAc3-1a Ad 2320 GmAdowGalNAc3-la 703421 X-ATTmCAmCTTTmCATAATGmCTGG n/a n/a 2319 703422 GalNAc3-7b—X-ATTmCAmCTTTmCATAATGmCTGG GalNA03-7b n/a 2319 The structure of 3-7a was shown previously in Example 48. "X" tes a 5 ’ primary amine generated by Gene Tools (Philomath, OR), and GalNAc3-7b indicates the structure of GalNAc3-7a lacking the —NH-C6-O portion of the linker, as shown below: HoOH o o N HO 4 HR HoOH o o o HO .. 4 H H AcHN o o t o N HO O ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two oligonucleotides is a morpholino nucleotide.

Trealmenl Six week old transgenic mice that express human SMN were injected subcutaneously once with an oligonucleotide listed in Table 91 or with saline. Each treatment group consisted of 2 males and 2 females.

The mice were sacrificed 3 days following the dose to determine the liver human SMN mRNA levels both with and without exon 7 using real-time PCR ing to rd protocols. Total RNA was measured using Ribogreen reagent. The SMN mRNA levels were normalized to total mRNA, and ?thher ized to the averages for the saline treatment group. The resulting e ratios of SMN mRNA including exon 7 to SMN mRNA missing exon 7 are shown in Table 91. The results show that fully modified oligonucleotides that modulate splicing and comprise a GalNAc conjugate are significantly more potent in altering splicing in the liver than the parent oligonucleotides lacking a GlaNAc conjugate. Furthermore, this trend is ined for multiple cation chemistries, including 2 ’-MOE and morpholino modified oligonucleotides.

Table 91 Effect of oligonucleotides targeting human SMN in vivo 113:8 Dose (mg/kg) +Exon 7 / -Exon 7 G813::133 CM ISBN?) Saline n/a 1.00 n/a n/a n/a 387954 32 1.65 n/a n/a 2319 387954 288 5.00 n/a n/a 2319 699819 32 7.84 GalNAc3-7a PO 2319 699821 32 7.22 GalNAc3-7a PO 2319 700000 32 6.91 GalNAcg-la Ad 2320 703421 32 1.27 n/a n/a 2319 703422 32 4.12 GalNAc3-7b n/a 2319 Example 89: Antisense inhibition in vivo by oligonucleotides targeting Apolipoprotein A (Ap0(a)) comprising a GalNAc; conjugate The oligonucleotides listed in Table 92 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.

Table 92 Modi?ed ASOs targeting Apo(a) ISIS GalNAc3 SEQ ID Sequences (5 , to 3 , ) CM No. Cluster No.

TesCIes CesTes Ces CdsGdsTdsTdsGdsGdsTdsGds Cds 494372 TdsTesCiesTesTesmCe 681257 232 1 GalNAc3'7a'0’TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds TdsGdsmCds TdsTeoGeoTesTesmCe iam-n/a 2321 G lNA -7 The structure of GalNAc3-7a was shown in Example 48.

Trealmenl Eight week old, female C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were each injected subcutaneously once per week at a dosage shown below, for a total of six doses, with an oligonucleotide listed in Table 92 or with PBS. Each treatment group consisted of 3-4 s. Tail bleeds were performed the day before the first dose and weekly following each dose to determine plasma Apo(a) protein levels. The mice were sacrificed two days following the final administration. Apo(a) liver mRNA levels were determined using real-time PCR and EEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, OR) according to standard protocols. Apo(a) plasma protein levels were determined using ELISA, and liver transaminase levels were determined. The mRNA and plasma n s in Table 93 are presented as the treatment group average percent relative to the PBS treated group. Plasma protein levels were ?thher normalized to the baseline (BL) value for the PBS group. Average absolute transaminase levels and body weights (% relative to baseline averages) are reported in Table 94.

As illustrated in Table 93, treatment with the ucleotides lowered Apo(a) liver mRNA and plasma protein levels in a dose-dependent manner. Furthermore, the oligonucleotide comprising the GalNAc conjugate was significantly more potent with a longer duration of action than the parent ucleotide g a GalNAc ate. As illustrated in Table 94, transaminase levels and body weights were unaffected by the oligonucleotides, indicating that the oligonucleotides were well tolerated.

Table 93 Apo(a) liver mRNA and plasma protein levels ISIS Dosage Apo(a) mRNA Apo(a) plasma protein (% PBS) No. (mg/kg) (% PBS) BL Weekl Week2 Week3 Week4 Week5 PBS 100 120119 113 121 Wee7k6 494372 _—-_"_M ——-—————n Table 94 ISIS No. Dosa_e (mu/k) ALT (U/L) AST (U/L) Bod wei_ht (% baseline) EEIIIIIIIIIIIHEIIIIIIIIIIIIEiIIIIIIIIIIIIEIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIIEEIIIIIIIIIIEIIIIIIIIIIIIIIIIIIEIIIIIIIIIII 494372 22 55 19 48 IIIIIIIEIIIIIIIIIIIMIIIIIIIIIIEEIIIIIIIIIIIIIIIIIIMIIIIIIIIIII IIIIIIIIIIIIIIIIIIEEIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIEIIIIIIIIIII 681257 lilllllllllli?llllIIIIIEEIIIIIIIIIIIIIIIIEEIIIIIIIIII IIIIIIIIEIIIIIIIIIIIIEIIIIIIIIIIIIIEZIIIIIIIIIIIIIIIIIIlIiiIIIIIIIIIIII Example 90: Antisense tion in vivo by oligonucleotides targeting TTR sing a GalNAc3 Oligonucleotides listed in Table 95 below were tested in a dose-dependent study for antisense inhibition ofhuman hyretin (TTR) in transgenic mice that express the human TTR gene.

Trealmenl TTR transgenic mice were each injected subcutaneously once per week for three weeks, for a total of three doses, with an oligonucleotide and dosage listed in Table 96 or with PBS. Each treatment group consisted of 4 animals. Prior to the ?rst dose, a tail bleed was performed to determine plasma TTR protein levels at baseline (BL). The mice were sacrificed 72 hours following the final administration. TTR protein levels were measured using a clinical analyzer (AU480, Beckman Coulter, CA). Real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular , Inc. Eugene, OR) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Table 96 are the average values for each treatment group. The mRNA levels are the average values relative to the average for the PBS group. Plasma protein levels are the average values relative to the average value for the PBS group at baseline. "BL" indicates baseline, measurements that were taken just prior to the ?rst dose. As illustrated in Table 96, treatment with nse oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent g a GalNAc conjugate (ISIS 420915), and oligonucleotides comprising a phosphodiester or deoxyadenosine cleavable moiety showed signi?cant improvements in potency compared to the parent lacking a conjugate (see ISIS numbers 682883 and 666943 vs 420915 and see es 86 and 87).

Table 95 Oligonucleotides targeting human TTR . , , . GalNAc SEQ TesmcesTesTesGesGdsTdsTdsAdsmCdsAdsTdsGdsAdsAds 682883 GalNAc30,TesmCeoTeoTeoGeoGdsTdsTdsAdsmCdsAds PS/PO GalNA03-3a II" 2317 _ TdsGdsAdsAdsAeoTeomCesmCesmCe —-- GalNAc3'3a-O’AdoT?mCeoTeoTeoGeoGdsTdsTdsAds 666943 GalNAc3-3a 2322 mCdsAdsTdsGdsAdsAds AeoTeomC?mCesmCe GalNAc3'7a-O’AdoT?mCeoTeoTeoGeoGdsTdsTdsAds 682887 PS/PO GalNA03-7a Ad 2322 mCdsAdsTdsGdsAdsAdsAeoTeomC?mC?mCe GalNAc3'1Oa-O’AdoTesmCeoTeoTeoGeoGdsTdsTdsAds 682888 PS/PO GalNAc3-10a Ad 2322 sTdsGdsAdsAdsAeoTeomC?mC?mCe GalNAc3'13a-o’AdoTesmCeoTeoTeoGeoGdsTdsTdsAds 682889 GalNAc3-l3a 2322 mCdsAdsTdsGdsAdsAdsAeoTeomC?mC?mCe The legend for Table 95 can be found in Example 74. The structure of c3-3a was shown in Example 39. The structure of GalNAc3-7a was shown in Example 48. The structure of GalNAc3-10a was shown in Example 46. The structure of GalNAc3-13a was shown in Example 62.

Table 96 Antisense inhibition of human TTR in vivo Isis No. Dosage (mg/kg) TTR mRNA (% PBS) TTR protein (% BL) GalNAc cluster CM PBS n/a 100 124 n/a n/a 6 69 114 420915 20 71 86 n/a n/a 60 21 36 0.6 61 73 682883 2 23 36 GalNAc3-3a PO 6 18 23 0.6 74 93 666943 2 33 57 GalNAc3-3a AC 6 17 22 0.6 60 97 682887 2 36 49 GalNAc3-7a AC 6 12 19 0.6 65 92 682888 2 32 46 GalNAc3-10a AC 6 17 22 0.6 72 74 682889 2 38 45 GalNAc3-13a AC 6 16 18 Example 91: Antisense tion in vivo by oligonucleotides targeting Factor VII comprising a GalNAc; conjugate in non-human primates Oligonucleotides listed in Table 97 below were tested in a non-terminal, dose escalation study for antisense inhibition of Factor VII in s.

Trealmenl Non-naive monkeys were each injected aneously on days 0, 15, and 29 with escalating doses of an oligonucleotide listed in Table 97 or with PBS. Each treatment group consisted of 4 males and 1 female. Prior to the first dose and at various time points thereafter, blood draws were performed to determine plasma Factor VII protein levels. Factor VII protein levels were measured by ELISA. The results presented in Table 98 are the average values for each ent group relative to the average value for the PBS group at baseline (BL), the ements taken just prior to the first dose. As illustrated in Table 98, treatment with antisense oligonucleotides lowered Factor VII expression levels in a dose-dependent manner, and the oligonucleotide comprising the GalNAc conjugate was significantly more potent in monkeys compared to the ucleotide lacking a GalNAc ate.

Table 97 Oligonucleotides targeting Factor VII GalNAc SEQ Is1s No. Sequence 5’ to 3 ’ es cluster ID NO A?TesGmmCesAesTdsGdsGdsTdsGdsAdsTdsGdsmCdsTds 3-10a-o’lAesTeSChSmCesAesTdsGdsGdsTdsGds 686892 AdsTdsGdsmCdsTdS TesmCeSTCSG?Ae The legend for Table 97 can be found in Example 74. The structure of GalNAc3-10a was shown in Example Table 98 Factor VII plasma protein levels ISIS No. Da Dose (m /k ) Factor VII (% BL) 0 n/a 100 10 87 22 n/a 92 407935 29 30 77 36 n/a 46 43 n/a 43 0 3 100 10 56 22 n/a 29 686892 29 30 19 36 n/a 15 43 n/a 11 Example 92: Antisense inhibition in primary hepatocytes by antisense oligonucleotides targeting Apo- CIII comprising a GalNAc3 conjugate Primary mouse hepatocytes were seeded in 96-well plates at 15,000 cells per well, and the oligonucleotides listed in Table 99, targeting mouse ApoC-III, were added at 0.46, 1.37, 4.12, or 12.35, 37.04, 111.11, or 333.33 nM or 1.00 uM. After tion with the oligonucleotides for 24 hours, the cells were lysed and total RNA was purified using RNeasy (Qiagen). ApoC-III mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quanti?cation reagent (Molecular , Inc.) according to standard protocols. IC50 values were determined using Prism 4 software (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a deoxyadensoine, the oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent oligonucleotide g a conjugate.

Table 99 Inhibition of mouse II expression in mouse primary hepatocytes ISIS , , 1c50 SEQ Sequence (5 to 3 ) CM No. (nM) ID No. 440670 mCesAesGesmCmTesTdsTdsAdsTdsTdSAdsGdSGdsGdsAdsmCesAesGesmCesAe n/a 1 3 .20 2324 mCeslAes(}esmC?TeSTdsTdsAdsTdsTdsAdsGdsGdsGdsAdsmCes 661180 Ad 1.40 2325 mCesAeo Ad0,-GalNAc3-la GalNAc3'3a-o’ CesAesGes 680771 CesT?TgrsTdsAdsTdsTdsAdsGdsGdsGdsAds Ces PO 070 2324 AesCies CesAe GalNAc3'7a-o’ CesAesGes 680772 CesT?TgsTdsAdsTdsTdsAdsGdsGdsGdsAds Ces PO 1 70 2324 AesCies CesAe GalNAc3'10a-O’ CesAesGes 680773 C?TesTntllsTdsAdsTdsTdsAdsGdsGdsGdsAds Ces PO 200 2324 AesCIes CesAe GalNAc3'13a-O’ CesAesGes 680774 C?TesTntllsTdsAdsTdsTdsAdsGdsGdsGdsAds Ces PO 150 2324 AesCies CesAe GalNAc3'3a-O’ CesAeoGeo 681272 CXTnglsgdsAAdsTdsTdsAdsGdsGdsGdsAds Ceo PO < 046 2324 GalNAC3-3a'o’AdomCesAesGesmCesTesTdsTdsAdsTdsTdsAdsGdsGdsGdsAds 681273 Ad 1.10 2324 mCesAesGeSmCesAe mCeslAesC}esmC?TeSTdsTdsAdsTdsTdsAdsGdsGdsGdsAdsmCes 683733 Ad 2.50 2325 AesGesmC?AeoAdowGalNAcg-l9a The structure of GalNAc3-1a was shown usly in Example 9, GalNAc3-3a was shown in Example 39, GalNAc3-7a was shown in e 48, GalNAc3-10a was shown in Example 46, GalNAc3-13a was shown in Example 62, and GalNAc3-19a was shown in Example 70.

Example 93: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising mixed wings and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 100 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.

Table 100 Modi?ed ASOs targeting SRB-l ISIS Sequences (5’ to 3’) GalNAc3 CM SEQ No. Cluster ID No. 449093 kasAdsGdsTdsmCds AdsTds Gds AdsmcdsTdsTkskaska 2326 TdsTkskaska GalNAC3-7a-o’TksTkskasAdsGdsTdsmCds AdsTds mCdS 'n- TdsTkskaska TBTmmCmmCe GalNAC3-7a-oaT?TesmCmAdsGdsTdsmCds AdsTds GdSAdSmCds "a TdsTkskaska GalNAC3-7a-o’TksTdskasAdsGdsTdsmCds AdsTds GdSAdSmCdS 'n- TdsTksmCdska GalNAC3-7a—yT?TkSkaSAdSGdSTdSmCdS AdSTdS GdSAdsmCds "a kasmCe The structure of GalNAc3-3a was shown previously in Example 39, and the structure of GalNAc3-7a was shown previously in Example 48. Subscripts: "e" tes 2’-MOE modi?ed nucleoside; "d" indicates B-D- 2’-deoxyribonucleoside; "k" indicates 6’-(S)-CH3 bicyclic nucleoside (cEt); a; 99 S indicates orothioate internucleoside linkages (PS); "0" indicates odiester internucleoside linkages (PO). Supersript "m" indicates 5-methylcytosines.

Trealmenl Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar , ME) were injected subcutaneously once at the dosage shown below with an oligonucleotide listed in Table 100 or with saline.

Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-l mRNA levels were measured using real-time PCR. SRB-l mRNA levels were normalized to cyclophilin mRNA levels ing to standard protocols. The results are presented as the average t of SRB-l mRNA levels for each treatment group relative to the saline control group. As illustrated in Table 101, treatment with antisense oligonucleotides lowered SRB-l mRNA levels in a dose- dependent manner, and the gapmer oligonucleotides comprising a GalNAc ate and having wings that are either full cEt or mixed sugar modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising ?Jll cEt modified wings.

Body weights, liver transaminases, total bin, and BUN were also measured, and the average values for each treatment group are shown in Table 101. Body weight is shown as the average percent body weight relative to the baseline body weight (% BL) measured just prior to the oligonucleotide dose.

Table 101 SRB-l mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS Dosage SRB-l mRNA ALT AST Body weight Bil BUN No. (mu/k) (% PBS) (U/L) (U/L) (% BL) 449093 699806 0.1 121 19 41 0.14 25 100 699807 0.3 73 23 56 0.13 26 105 1 24 22 69 0.14 25 102 0.1 125 23 57 0.14 26 104 699809 0.3 70 20 49 0.10 25 105 1 33 34 62 0.17 25 107 0.1 123 48 77 0.14 24 106 699811 0.3 94 20 45 0.13 25 101 1 66 57 104 0.14 24 107 0.1 95 20 58 0.13 28 104 699813 0.3 98 22 61 0.17 28 105 1 49 19 47 0.11 27 106 0.1 93 30 79 0.17 25 105 699815 0.3 64 30 61 0.12 26 105 1 24 18 41 0.14 25 106 Example 94: nse inhibition in vivo by oligonucleotides targeting SRB-l comprising 2’-sugar modi?cations and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 102 were tested in a dose-dependent study for antisense inhibition of SRB-l in mice.

Table 102 Modi?ed ASOs targeting SRB-l ISIS Sequences (5 ’ t0 3 ’) GalNA03 CM SEQ N0. Cluster ID No. 3533 82 GesmCCST?TGSmCGSAdSGdSTdSmCdsAdsTdsGdSAdsmCdSTdSTesmCesmCes n/a n/a 2304 TesTe 700989 GmscmsUmsUmsCmsAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsUmsCmsCms n/a n/a 2327 UmSUm 666904 GalNAC3-3a'o’GesmCesTesT?mCesAdsGdsTdsmCdsAdsTdsGdsAds GalNAC3‘3 a P0 23 04 IncdsTdsTmmCmmCmTesTe 700991 3'7a'o’GmscmsUmsUmsCmsAdsGdsTdsmCdsAdsTdsGds GalNAC3-7a PO 2327 [AdstncdsTdsUmsCmsCmsUmsUm structure of GalNAcg-3a was shown usly in Example 39, and the structure of GalNA03-7a was shown previously in Example 48.

The study was completed using the protocol described in Example 93. Results are shown in Table 103 below and show that both the 2’-MOE and 2’-OMe d oligonucleotides comprising a GalNAc conjugate were significantly more potent than the respective parent oligonucleotides g a conjugate. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

Tabh103 SIKB-linIUst EENQ Dwg?myg) OMBS PBS 10a 100 116 353382 15 58 45 27 120 700989 15 92 45 46 1 98 666904 3 45 17 1 118 700991 3 63 14 Example 95: Antisense inhibition in vivo by oligonucleotides targeting SRB-l comprising bicyclic nucleosides and a 5’-GalNAc3 conjugate The oligonucleotides listed in Table 104 were tested in a dose-dependent study for nse inhibition of SRB-l in mice.

Table 104 Modi?ed ASOs targeting SRB-l 1:1? Sequences (5 ’ to 3 ’) (gig? CM ITS)E1\C120 440762 TkskasAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska l’l/a n/a 2298 666905 GalNA03-3a-oaTkskasAdsGdSTdSmCdSAdSTdSGdSAdsmCdSTdSTkSka GalNA03-3a PO 2298 6997 82 G31NAC3'7a-O’TkskaSAdsGdsTdsmCdsAdSTdsGdsAdsmCdsTdsTkska GalNA03'7 a P0 2298 6997 83 GalNAc3-3a-o’TlsmC1sAdsGdsTdsmCdsAdSTdsGdsAdsmCdsTdsTjsmC1 GalNA03'3 a P0 2298 653621 TlsmC1sAdsGdsTdsmCdsAdSTdsGdsAdsmCdsTdSTjsmCmAdo’-G31NAC3-1a GalNACB'l 3. Ad 2299 439879 Tg§mCgsAdsGdsTdsmCdsAdsTd GdsAdsmCdsTdsTngg n/a n/a 2298 699789 30,Tg§mC g§AdsCidsTdsmCdsAdSTd GdsAdsmCdsTdsTngg 3'3a PO 2298 Subscript "g" indicates a ?uoro-HNA nucleoside, subscript "1" indicates a locked nucleoside sing a 2’- O-CH2-4’ bridge. See the Example 74 table legend for other abbreviations. The structure of GalNAc3-1a was shown previously in Example 9, the structure of 3-3a was shown previously in Example 39, and the structure of GalNA03-7a was shown previously in Example 48.

Trealmenl The study was completed using the ol described in e 93. Results are shown in Table 105 below and show that oligonucleotides comprising a GalNAc conjugate and s bicyclic nucleoside modi?cations were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising bicyclic nucleoside modi?cations. Furthermore, the oligonucleotide comprising a GalNAc conjugate and ?uoro-HNA modi?cations was signi?cantly more potent than the parent lacking a conjugate and comprising ?uoro-HNA modifications. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements ted that the compounds were all well tolerated.

Table 105 SRB-l mRNA, ALT, AST, BUN, and total bilirubin levels and body weights ISIS No. Dosage (mg/kg) SRB-1 mRNA (% PBS) PBS n/a 100 1 104 440762 3 65 35 0.1 105 666905 0.3 56 1 18 0.1 93 699782 0.3 63 1 15 0.1 105 699783 0.3 53 1 12 0.1 109 653621 0.3 82 1 27 1 96 439879 3 77 37 0.1 82 699789 0.3 69 1 26 Example 96: Plasma protein binding of antisense ucleotides comprising a GalNAc; conjugate group ucleotides listed in Table 70 targeting ApoC-III and oligonucleotides in Table 106 targeting Apo(a) were tested in an ultra-?ltration assay in order to assess plasma protein binding.

Table 106 Modi?ed oligonucleotides targeting Apo(a) sequences (5 , GalNA03 SEQ t0 3 , ) CM No. Cluster ID No TesGes CesTes Ces CdSGdSTL$TdI§gdSGdSTdSGdS CdsTdsTeSGesTes 494372 n/a n/a 2321 TesC}eo CeoTeo Ce0 CdSGdST$T$gdSGdSTdSGdS CdsTdsTeoGeoTes 693401 n/a n/a 2321 GalNAc3-7a-O’T?Ges CesT? Ces 681251 CdsgdsTdsTdsGdsGdsTdsGds Cds GalNAC3-7a PO 2321 TdST?GeST?TeS Ce GalNAc3'7a'o’TeSGeo CeoTeo Ce0 681257 TdsTdsGdsGdsTdsGds Cds 3-7a PO 2321 TdsTeoGeonTes Ce See the Example 74 for table legend. The structure of 3-7a was shown previously in Example 48.

Ultrafree-MC iltration units (30,000 NMWL, nding regenerated cellulose membrane, Millipore, Bedford, MA) were pre-conditioned with 300 uL of 0.5% Tween 80 and centrifuged at 2000 g for minutes, then with 300uL of a 300 ug/mL solution of a control oligonucleotide in H20 and centrifuged at 2000 g for 16 minutes. In order to assess non-specific binding to the ?lters of each test oligonucleotide from Tables 70 and 106 to be used in the studies, 300 uL of a 250 ng/mL solution of oligonucleotide in H20 at pH 7.4 was placed in the pre-conditioned ?lters and centrifuged at 2000 g for 16 minutes. The unfiltered and filtered samples were analyzed by an ELISA assay to determine the oligonucleotide concentrations. Three replicates were used to obtain an average concentration for each sample. The average concentration of the filtered sample relative to the unfiltered sample is used to determine the percent of oligonucleotide that is recovered through the filter in the absence of plasma (% recovery).

Frozen whole plasma samples collected in K3-EDTA from normal, drug-free human eers, cynomolgus monkeys, and CD-1 mice, were purchased from lamation LLC (Westbury, NY). The test oligonucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 . An aliquot (300 uL) of each spiked plasma sample was placed in a pre-conditioned filter unit and incubated at 37°C for 30 minutes, immediately followed by centrifugation at 2000 g for 16 minutes. ts of filtered and unfiltered spiked plasma samples were analyzed by an ELISA to determine the oligonucleotide concentration in each sample. Three replicates per concentration were used to determine the average percentage ofbound and unbound ucleotide in each sample. The average concentration of the filtered sample relative to the concentration of the unfiltered sample is used to ine the percent of oligonucleotide in the plasma that is not bound to plasma proteins (% unbound). The final unbound oligonucleotide values are corrected for non-speci?c binding by dividing the % unbound by the % ry for each oligonucleotide. The final % bound oligonucleotide values are determined by subtracting the final % unbound values from 100. The results are shown in Table 107 for the two concentrations of oligonucleotide tested (5 and 150 ug/mL) in each species of plasma. The results show that GalNAc conjugate groups do not have a icant impact on plasma protein binding. Furthermore, oligonucleotides with ?lll PS internucleoside es and mixed PO/PS es both bind plasma proteins, and those with full PS linkages bind plasma proteins to a somewhat greater extent than those with mixed PO/PS linkages.

Table 107 Percent of modi?ed oligonucleotide bound to plasma proteins ISIS Monkey plasma No. 5 ug/mL 150 ug/mL 5 ug/mL 150 ug/mL 5 ug/mL 150 ug/mL 304801 666666"m 674466 WO 68618 2015/028887 494372 693401 93.6 89.9 96.7 92.0 94.6 90.2 681251 95.4 93.9 99.1 98.2 97.8 96.1 Example 97: Modi?ed oligonucleotides targeting TTR comprising a 3 conjugate group The oligonucleotides shown in Table 108 comprising a GalNAc conjugate were ed to target Table 108 Modi?ed oligonucleotides targeting TTR ISIS N0. Sequences (5’ t0 3 ,) (31111133 CM SEISOH) 666941 GalNAczindeTAjidaTTSCGSISCTSECSASS mCdS GalNA03-3 Ad 2322 ASCiSmTcS:::S::%;i‘NS:$:i£S AS AS W... A. SS 18 682876 Gaming-34,31 22%;:EmCéd33:33" mCSS ASS TSS GalNA03-3 PO 2317 682877 SJSS’CT}: :Cdfgi??mfédgdégéS mCSS ASS TSS GalNA03-7 PO 2317 682878 GalNA03-119d:..él;es:dCA;f:T1:de EcdsesTSgsclde mCSS ASS GalNA03-10 PO 2317 682879 GalNA03-113d:..é1;es:dCA;f:T1:de ECSSCSTJSCTS mCdS ASS GalNA03-13 PO 2317 682880 GaINA"Xiagfdédk:sizkesTiC?ch?cT:ECSSASS mCdS GalNA03-7 Ad 2322 682881 GalNAc?fngédadmizS 11:2; ?éGgsCTfmTcd: ASS mCSS GalNA03-10 Ad 2322 682882 GalNAc?deAédadmgd1:2;chGgSCTjSmTCS: ASS mCSS GalNA03-13 Ad 2322 TSS mCSS i: Tr: ECGQSCEmgd2112;112:3131 ASS ASS 684056 GalNA03-19 Ad 2318 The legend for Table 108 can be found in Example 74. The structure of GalNA03-1 was Shown in e 9. The structure of GalNA03-3a was Shown in Example 39. The structure of GalNA03-7a was Shown in Example 48. The structure of GalNAc3-10a was shown in Example 46. The structure of GalNA03-13a was shown in Example 62. The structure of GalNAcg-19a was shown in Example 70.

Example 98: Evaluation of pro-in?ammatory effects of ucleotides comprising a GalNAc conjugate in hPMBC assay The oligonucleotides listed in Table 109 and were tested for pro-in?ammatory effects in an hPMBC assay as described in Examples 23 and 24. (See Tables 30, 83, 95, and 108 for descriptions of the oligonucleotides.) ISIS 353512 is a high responder used as a positive l, and the other oligonucleotides are described in Tables 83, 95, and 108. The s shown in Table 109 were obtained using blood from one volunteer donor. The results show that the oligonucleotides comprising mixed PO/PS internucleoside linkages produced signi?cantly lower pro-in?ammatory responses compared to the same oligonucleotides having ?Jll PS es. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.

Table 109 ISIS N0. Cso 3 cluster Linkages CM 353512 3630 n/a PS n/a 684057 GalNAC3-19 PO/PS Example 99: Binding af?nities of oligonucleotides comprising a GalNAc conjugate for the asialoglycoprotein receptor The binding affinities of the ucleotides listed in Table 110 (see Table 76 for descriptions of the oligonucleotides) for the asialoglycoprotein receptor were tested in a competitive receptor binding assay. The competitor ligand, (11 -acid rotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 U neuraminidase-agarose for 16 hours at 37°C, and > 90% desialylation was confirmed by either sialic acid assay or size exclusion chromatography (SEC). Iodine monochloride was used to iodinate the AGP according to the procedure by Atsma et al. (see J Lipid Res. 1991 Jan; 32(1):173-81.) In this method, desialylated a1- acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Nalzsl, and 1 M glycine in 0.25 M NaOH.

After incubation for 10 minutes at room temperature, 1251 -labeled de-AGP was separated from free 1251 by concentrating the mixture twice utilizing a 3 KDMWCO spin column. The protein was tested for labeling efficiency and purity on a HPLC system equipped with an Agilent SEC-3 column (7.8x300mm) and a 13- RAM counter. Competition experiments utilizing 1251 ed de-AGP and various GalNAc-cluster containing ASOs were performed as follows. Human HepG2 cells (106 cells/ml) were plated on 6-well plates in 2 ml of appropriate grth media. MEM media supplemented with 10% fetal bovine serum (FB S), 2 mM amine and 10mM HEPES was used. Cells were incubated 16-20 hours @ 37°C with 5% and 10% C02 respectively. Cells were washed with media without FBS prior to the experiment. Cells were incubated for 30 min @37°C with 1ml competition mix containing appropriate grth media with 2% FBS, 10'8 M 1251 - labeled de-AGP and GalNAc-cluster containing ASOs at concentrations ranging from 10'11 to 10'5 M. Non- specific binding was determined in the ce of 10'2 M GalNAc sugar. Cells were washed twice with media t FBS to remove unbound 125I -labeled de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen’s RLT buffer containing 1% B-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min /thaw cycle and assayed on a y-counter. Non-specific binding was subtracted before dividing 125 I n counts by the value of the lowest -ASO concentration .

The inhibition curves were fitted according to a single site competition binding equation using a nonlinear regression algorithm to calculate the binding affinities (KD’s).

The results in Table 110 were obtained from experiments performed on five different days. Results for oligonucleotides marked with superscripta6;a" are the average of experiments run on two different days.

The results show that the oligonucleotides comprising a GalNAc conjugate group on the 5’-end bound the asialoglycoprotein receptor on human HepG2 cells with 1.5 to 16-fold greater affinity than the oligonucleotides comprising a GalNAc conjugate group on the 3 ’-end.

Table 110 Asialoglycoprotein receptor binding assay results Oligonucleotide end to GalNAc ISIS No. . which GalNAc ate KD (nM) conjugate . is attached 66116" GalNACs-3 66688" s-IO 666981 GalNA63-7 —-1_ 670061 3-13 65586" GalNACs-l 677841a GalNA63-19 e 100: Antisense inhibition in vivo by ucleotides comprising a GalNAc conjugate group targeting Apo(a) in vivo The ucleotides listed in Table 111a below were tested in a single dose study for duration of action in mice.

Table 111a Modi?ed ASOs targeting APO(a) ISIS GalNAc3 SEQ GalNAc3-7MTeSG cTes Ces cdsGdsTdsTdsGdsGds 681251 GalN1103-721 ? 2321 TdsC}dsmCdsTdsTesGes TesTesmCe GalNAC3'7a'o’TesGeomCeoTeomCeoInCdsGdsTdsTdsGdsGdS 681257 G 1NA _7 2321 The structure of GalNAc3-7a was shown in Example 48.

Trealmenl Female transgenic mice that express human Apo(a) were each injected subcutaneously once per week, for a total of 6 doses, with an oligonucleotide and dosage listed in Table 111b or with PBS. Each treatment group consisted of 3 animals. Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 72 hours, 1 week, and 2 weeks following the first dose. Additional blood draws will occur at 3 weeks, 4 weeks, 5 weeks, and 6 weeks following the first dose. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 111b are ted as the average percent of plasma Apo(a) protein levels for each ent group, normalized to baseline levels (% BL), The results show that the oligonucleotides comprising a GalNAc conjugate group exhibited potent reduction in Apo(a) expression. This potent effect was observed for the oligonucleotide that comprises ?lll PS cleoside linkages and the oligonucleotide that comprises mixed PO and PS linkages.

Table 111b Apo(a) plasma protein levels Apo(a) at 72 hours Apo(a) at 1 week Apo(a) at 3 weeks ISIS No. Dosage (mg/kg) (% BL) (% BL) (% BL) PBS n/a 116 104 107 0.3 97 108 93 1.0 85 77 57 681251 3.0 54 49 11 .0 23 15 4 0.3 114 138 104 1.0 91 98 54 681257 3.0 69 40 6 .0 30 21 4 Example 101: Antisense inhibition by oligonucleotides comprising a GalNAc cluster linked via a stable moiety The oligonucleotides listed in Table 112 were tested for inhibition of mouse APOC-III expression in viva. C57Bl/6 mice were each injected subcutaneously once with an oligonucleotide listed in Table 112 or with PBS. Each treatment group consisted of 4 animals. Each mouse d with ISIS 440670 received a dose of 2, 6, 20, or 60 mg/kg. Each mouse treated with ISIS 680772 or 696847 received 0.6, 2, 6, or 20 mg/kg. The GalNAc conjugate group of ISIS 696847 is linked via a stable moiety, a phosphorothioate linkage instead of a y cleavable phosphodiester containing linkage. The animals were sacrificed 72 hours after the dose. Liver APOC-III mRNA levels were measured using ime PCR. APOC-III mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are ted in Table 112 as the average percent of APOC-III mRNA levels for each treatment group ve to the saline control group. The results show that the oligonucleotides comprising a GalNAc conjugate group were significantly more potent than the oligonucleotide lacking a conjugate group. Furthermore, the oligonucleotide comprising a GalNAc conjugate group linked to the ucleotide via a cleavable moiety (ISIS 680772) was even more potent than the oligonucleotide comprising a GalNAc conjugate group linked to the oligonucleotide via a stable moiety (ISIS 696847).

Table 112 Modi?ed oligonucleotides targeting mouse APOC-III ApoC-III SEQ Isis Sequences (5’ t0 3’) CM (aim/1g: mRNA ID ‘ g g (% PBS N0.

InCeslAesCTesmCesTesTdsTdsAdsTdsTds‘AdS & 440670 M 2324 GdsGdsGdSAdsmcesA?GesmcesA, M 60 37 GalNAC3-7a?fncesAesGesmCeSTeSTdsTdsAds TdsTdsAdsGds 2 5 8 680772 PO 2324 AdsmCes AesGesmcmAe # 13 GalNAC3'7a-s’mcesAesGesmCesTdesTdsAdsTdsTdsAdSGdS n/a# 696847 2324 GdSGdsAdsmCes Aeseesmc?Ae (PS)& 28 The structure of GalNAc3-7a was shown in Example 48.

Example 102: bution in liver of antisense oligonucleotides comprising a GalNAc ate The liver distribution of ISIS 353382 (see Table 36) that does not comprise a GalNAc conjugate and ISIS 655861 (see Table 36) that does comprise a GalNAc conjugate was evaluated. Male Balb/c mice were subcutaneously injected once with ISIS 353382 or 655 861 at a dosage listed in Table 113. Each treatment group consisted of 3 animals except for the 18 mg/kg group for ISIS 655 861, which consisted of 2 animals.

The animals were ced 48 hours following the dose to determine the liver distribution of the oligonucleotides. In order to measure the number of antisense oligonucleotide les per cell, a Ruthenium (II) tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to an ucleotide probe used to detect the antisense oligonucleotides. The s presented in Table 113 are the average concentrations of oligonucleotide for each treatment group in units of millions of oligonucleotide molecules per cell. The results show that at equivalent doses, the oligonucleotide sing a GalNAc conjugate was present at higher concentrations in the total liver and in hepatocytes than the oligonucleotide that does not comprise a GalNAc conjugate. Furthermore, the oligonucleotide comprising a GalNAc conjugate was present at lower concentrations in renchymal liver cells than the oligonucleotide that does not comprise a GalNAc conjugate. And while the concentrations of ISIS 655861 in hepatocytes and non-parenchymal liver cells were similar per cell, the liver is approximately 80% hepatocytes by volume. Thus, the ty of the ISIS 655 861 oligonucleotide that was present in the liver was found in hepatocytes, whereas the majority of the ISIS 353382 oligonucleotide that was present in the liver was found in non-parenchymal liver cells.

Table 113 ISIS Dosage Concentration in whole Concentration in Concentration in non- liver (molecules*10A6 hepatocytes hymal liver cells No. (mg/kg) per cell) (molecules*10A6 per cell) (molecules*10A6 per cell) 3 9.7 1.2 37.2 17.3 4.5 34.0 23.6 6.6 65.6 353382 29.1 11.7 80.0 60 73.4 14.8 98.0 90 89.6 18.5 119.9 0.5 2.6 2.9 3.2 1 6.2 7.0 8.8 655861 3 19.1 25.1 28.5 6 44.1 48.7 55.0 18 76.6 82.3 77.1 Example 103: Duration of action in vivo of oligonucleotides targeting APOC-III comprising a GalNAc3 conjugate The oligonucleotides listed in Table 1 14 below were tested in a single dose study for duration of action in mice.

Table 114 Modi?ed ASOs targeting II ISIS ces (5’ to 3’) GalNAc3 CM SEQ No. Cluster ID No. 304801 AesGesmcasTeSTmmCdsTdSTdsGdSTdsmCdsmCdsAdSGdsmCdSTesTes n/a 2296 TesAesTe 663084 GalNAc3'3a'o’AdersGeomCeoTeoTeomCdsTdsTdsGdsTdsmCds GalNAC3—3a Ad 2312 IncdsAdsGdsmCdsTeoTeo TesAesTe 679241 AesGeomCeoTeoTeomCdsTdsTdsGdsTdsmCdsmcdsAdsGdsmCdsTeoTeo GalNAC3—19a Ad 2297 TesAesTeoAdo"GalNAc3'1 9a The structure of GalNAc3-3a was shown in Example 39, and 3-19a was shown in Example 70.

Trealmenl Female transgenic mice that express human APOC-III were each injected subcutaneously once with an oligonucleotide listed in Table 114 or with PBS. Each treatment group consisted of 3 animals. Blood was drawn before dosing to ine baseline and at 3, 7, 14, 21, 28, 35, and 42 days following the dose. Plasma ceride and APOC-III protein levels were measured as described in Example 20. The results in Table 1 15 are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, ized to baseline . A comparison of the results in Table 71 of example 79 with the results in Table 115 below show that oligonucleotides comprising a mixture of phosphodiester and phosphorothioate internucleoside linkages ted increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.

Table 115 Plasma triglyceride and APOC-III protein levels in transgenic mice Tlme p01nt ISIS Dosage Triglycerides AFDC-1P GalNA03 CM (days post- N0 (mg/kg) (% baseline) pmtem (A) ' Cluster dose) e) 3 96 101 7 88 98 14 91 103 PBS n/a 21 69 92 n/a n/a 28 83 81 65 86 42 72 88 3 42 46 7 42 51 14 59 69 304801 30 21 67 81 n/a n/a 28 79 76 72 95 42 82 92 3 35 28 7 23 24 14 23 26 663084 10 21 23 29 GalNA03-3a 28 30 22 32 36 42 37 47 3 38 30 7 31 28 14 30 22 679241 10 21 36 34 Galgi"? 28 48 34 50 45 42 72 64 Example 104: Synthesis of oligonucleotides comprising a 5’-GalNAc2 conjugate HN.Boc HN'BOC HBTU, HOBt o H TFA Boc\ OH /\)LO —> 800 \ NM —> H DIEA, DMF ? 0 O O 120 126 85% 231 ZI o H2N O + AcO O\/\/\/?\ o AcHN O 232 166 F AcogbOACOAc\/\/\/ENH 0A0 Aco?voO\/\/\)LNH0A0 ACHN 1. H2, Pd/C, MeOH OOAC 2 PFPTFA, DMF 0A0 A00%OWN 0A0 F F 1 go o 0 A00 OW NM ACHN OH\/\/\)OLO/\© AcHN o F o 83e 3| 5' II OLIGO O-F|’-o- (CH2)6-NH2 H/ScHNmo\AA/ENH 1. Borate buffer, DMSO, pH 8.5, r1 OH —> OOH 2. aq. ammonia,' HAOcHN 0W "mil r1 N/‘(V)’\om-O_IGO Compound 120 is cially available, and the synthesis of compound 126 is described in Example 49. Compound 120 (1 g, 2.89 mmol), HBTU (0.39 g, 2.89 mmol), and HOBt (1.64 g, 4.33 mol) were dissolved in DMF (10 mL. and N,N—diisopropylethylamine (1.75 mL, 10.1 mmol) were added. After about 5 min, aminohexanoic acid benzyl ester (1.36 g, 3.46 mmol) was added to the reaction. After 3h, the reaction e was poured into 100 mL of 1 M NaHSO4 and extracted with 2 x 50 mL ethyl acetate.

Organic layers were ed and washed with 3 x 40 mL sat NaHC03 and 2 x brine, dried with NaZSO4, ?ltered and concentrated. The product was purified by silica gel column chromatography (DCM:EA:Hex 1:1 :1) to yield compound 231. LCMS and NMR were consistent with the structure. Compounds 231 (1.34 g, 2.438 mmol) was dissolved in dichloromethane (10 mL) and tri?uoracetic acid (10 mL) was added. After stirring at room temperature for 2h, the on e was concentrated under reduced pressure and co- evaporated with toluene ( 3 x 10 mL). The residue was dried under reduced pressure to yield compound 232 as the oracetate salt. The synthesis of compound 166 is described in Example 54. Compound 166 (3.39 g, 5.40 mmol) was dissolved in DMF (3 mL). A solution of compound 232 (1.3 g, 2.25 mmol) was dissolved in DMF (3 mL) and N,N—diisopropylethylamine (1.55 mL) was added. The reaction was stirred at room temperature for 30 minutes, then poured into water (80 mL) and the aqueous layer was extracted with EtOAc (2x100 mL). The organic phase was separated and washed with sat. aqueous NaHC03 (3 x 80 mL), 1 M NaHSO4 (3 x 80 mL) and brine (2 x 80 mL), then dried (NaZSO4), filtered, and concentrated. The residue was puri?ed by silica gel column chromatography to yield compound 233. LCMS and NMR were consistent with the structure. Compound 233 (0.59 g, 0.48 mmol) was dissolved in methanol (2.2 mL) and ethyl e (2.2 mL). Palladium on carbon (10 wt% Pd/C, wet 0.07 g) was added, and the reaction mixture was stirred under hydrogen atmosphere for 3 h. The reaction mixture was ?ltered through a pad of Celite and concentrated to yield the carboxylic acid. The carboxylic acid (1.32 g, 1.15 mmol, cluster free acid) was dissolved in DMF (3.2 mL). To this N,N-diisopropylehtylamine (0.3 mL, 1.73 mmol) and PFPTFA (0.30 mL, 1.73 mmol) were added. After 30 min stirring at room ature the reaction mixture was poured into water (40 mL) and extracted with EtOAc (2 x 50 mL). A standard p was completed as described above to yield compound 234. LCMS and NMR were tent with the structure. Oligonucleotide 235 was prepared using the general procedure described in Example 46. The GalNAcz cluster portion (GalNAc2-24a) of the conjugate group GalNAc2-24 can be combined with any cleavable moiety t on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc2-24 c2-24a-CM) is shown below: OH OH E52» 0 HO OW AcHN NH HECHN CONN N\/\/\ji NAM/\Omg Example 105: Synthesis of oligonucleotides comprising a GalNAc1-25 conjugate ' ? --0F|>—0-e-NH2 0A0 OAc Amok/Owig;1. Borate OH buffer, DMSO, pH8.5, rt 2. aq. ammonia, rt OH OH O\/\/\)J\NMCM O_IGO The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was ed using the general procedure described in e 46. Alternatively, oligonucleotide 236 was synthesized using the scheme shown below, and compound 238 was used to form the oligonucleotide 236 using procedures described in Example 10.

WoH GAO H2N 0 o O o 0 A00(?;0 A00 0 + PFPTFA NHAc NHAc /\/\/\/OH TEA, Acetonltrlle_ _ H ole, 1-Methylimidazole, DMF O O AGO OW Y 2-cyanoethyltetraisopropyl phosphorodiamidite NHAc N/\/\/\/ \ 238 1 OH OH Oligonucleotide synthesis HO O ’ OMN?O/- OLIGO AcHN H 6 The GalNAc1 cluster portion (GalNAc1-25a) of the conjugate group GalNAc1-25 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc1-25 (GalNAc1-25a-CM) is shown below: OH OH OM?/Hgo/- E ACHN Example 106: Antisense inhibition in vivo by oligonucleotides ing SRB-l comprising a 5’- GalNAc; or a 5’-GalNAc3 conjugate Oligonucleotides listed in Tables 116 and 117 were tested in ependent studies for nse inhibition of SRB-l in mice.

Trealmenl Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar , ME) were ed subcutaneously once with 2, 7, or 20 mg/kg of ISIS No. 440762; or with 0.2, 0.6, 2, 6, or 20 mg/kg of ISIS No. 686221, 686222, or 708561; or with saline. Each treatment group consisted of 4 animals. The mice were sacri?ced 72 hours following the final administration. Liver SRB-l mRNA levels were measured using real- time PCR. SRB-l mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The nse oligonucleotides lowered SRB-l mRNA levels in a dose-dependent manner, and the ED50 results are presented in Tables 116 and 117. Although previous studies showed that trivalent - conjugated oligonucleotides were significantly more potent than divalent GalNAc-conjugated oligonucleotides, which were in turn significantly more potent than lent GalNAc conjugated oligonucleotides (see, e.g., Khorev et al., Bioorg. & Med. Chem, Vol. 16, 5216-5231 (2008)), treatment with antisense oligonucleotides comprising monovalent, divalent, and trivalent GalNAc clusters lowered SRB-l mRNA levels with similar potencies as shown in Tables 116 and 117.

Table 116 Modi?ed oligonucleotides targeting SRB-l INSIS ED50 SEQ ces (5’ to 3’) GalNAc Cluster 44N0762 ) ID No TkskasAdsGdsTdsmCdSAdSTdSGdSAdSmCdSTdsTkska 2298 GalNAc2-24a-0’mAdOEkSCkSAdSEdSTdSmCdsAdsTdsGdsAds 686221 GalNACZ-24a, 039 2302 CdsTdsTks Ck GalNAc3'13a'0AdOEkS 686222 CkSAdsgdSTdS CdSAdSTdSGdSAdS GalNAC3-13 a 0.41 2302 CdsTdsTks Ck See e 93 for table legend. The structure of GalNAc3-13a was shown in Example 62, and the structure of GalNAc2-24a was shown in Example 104.

Table 117 Modi?ed oligonucleotides targeting SRB-l , , ED50 SEQ Sequences (5 to 3 ) GalNAc Cluster (mg/kg) 440762 TkskasAdsGdsTdsmCdsAdsTdsGdsAdsmCdsTdsTkska 2298 708561 GalNAcl'253'0’Tk; CkSAdSGngdS CdSAdSTdSGdSAdS GalNAc1-25a 0.4 2298 CdsTdsTks Ck See Example 93 for table legend. The structure of GalNAc1-25a was shown in Example 105.

The trations of the oligonucleotides in Tables 116 and 117 in liver were also assessed, using procedures described in Example 75. The results shown in Tables 117a and 117b below are the average total nse oligonucleotide tissues levels for each treatment group, as measured by UV in units of ug oligonucleotide per gram of liver tissue. The results show that the oligonucleotides comprising a GalNAc conjugate group accumulated in the liver at signi?cantly higher levels than the same dose of the ucleotide lacking a GalNAc conjugate group. Furthermore, the antisense oligonucleotides comprising one, two, or three GalNAc s in their respective conjugate groups all accumulated in the liver at similar levels. This result is sing in view of the Khorev et al. ture reference cited above and is consistent with the activity data shown in Tables 116 and 117 above.

Table 117a Liver concentrations of oligonucleotides comprising a GalNAc; or GalNAc; conjugate group ISIS No. ?s/ige) Antisense oligonucleotide (ug/g) GalNAc cluster 440762 Na Na 686221_ GalNA02-24a Ad 686222 G Ma 03' 13 a Ad Table 117b Liver concentrations of oligonucleotides comprising a GalNAc1 ate group Dosage ISIS N0. Antisense 011. . onucleotide. / GalNAc cluster CM (mg/kg) g (lug g) 2 2.3 440762 7 8.9 n/a n/a 23.7 0.2 0.4 0.6 1.1 708561 2 5.9 1-25a PO 6 23.7 53.9 Example 107: Synthesis of oligonucleotides comprising a GalNAc1-26 or 1-27 conjugate O OM ""0 Oligonucleotide 239 is synthesized via coupling of compound 47 (see Example 15) to acid 64 (see e 32) using HBTU and DIEA in DMF. The resulting amide containing compound is phosphitylated, then added to the 5 ’-end of an oligonucleotide using procedures described in Example 10. The GalNAc1 cluster portion (GalNAc1-26a) 0f the conjugate group GalNAc1-26 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of ate groups. The structure of GalNAc1-26 (GalNAc1-26a-CM) is shown below: OH E HO$WOWKNHO o " ‘0I In order to add the GalNAc1 conjugate group to the 3’-end of an oligonucleotide, the amide formed from the reaction of compounds 47 and 64 is added to a solid support using procedures described in e 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form ucleotide 240.

Q ..:IOH HO \/\/\/U\ 240 3' 5' om The GalNAc1 cluster portion (GalNAc1-27a) of the conjugate group GalNAc1-27 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc1-27 (GalNAc1-27a-CM) is shown below: HokoHO O M "I OH‘ Example 108: Antisense inhibition in vivo by oligonucleotides comprising a GalNAc ate group targeting Ap0(a) in vivo The oligonucleotides listed in Table 1 18 below were tested in a single dose study in mice.

Table 118 Modi?ed ASOs targeting APO(a) ISIS SEQ Sequences (5 , to 3 ,) GalNAc3 Cluster CM No. ID No.

TesGes CesTes Ces TdsTdsGdsGdsTdsGds Cds 494372 n/a n/a 2321 TdsTesGesTesTesmCe GalNAc3'7a'o’T?GeSmCesTesmCesmCdsGdsTdsTdsGdsGds 681251 G lNAa 03-7a PO 2321 mCdsTdsTesGes T?TesmCe GalNAc3'3a'o’TesGeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681255 G lNAa 03-3a PO 2321 TdsGdsmCdsTdsTeoGeo TesTesmCe CCOTCO C60 681256 30’T?geo CdsgdsTdsTdsGdsGds GalNA03-1Oa PO 2321 TdsGds CdsTdsTeoGeo TesTes Ce 68125 7 GalNAc3'7a'o’Tesgeo CeoTeo Ce0 TdsTdsGdsGds GalNAc3-7a 2321 TdsGds CdsTdsTeoCieo TesTes Ce -GalNAc3'13a'o’T?GeomCeoTeomCeomCdsGdsTdsTdsGdsGds 681258 G INA -13 2321 mCdsTdsTeoGeo TesTesmCe "-- TesC}eo CeoTeo Ce0 CESGdsTdsTdsGdsGds s CdsTdsTeoGeo 681260 GalNAc3-19a Ad 2328 TesTm CmAd0,-GalNA03-19 The structure of GalNAc3-7a was shown in Example 48.

Male transgenic mice that express human Apo(a) were each injected subcutaneously once with an oligonucleotide and dosage listed in Table 119 or with PBS. Each treatment group consisted of 4 s.

Blood was drawn the day before dosing to determine baseline levels of Apo(a) protein in plasma and at 1 week following the first dose. Additional blood draws will occur weekly for approximately 8 weeks. Plasma Apo(a) protein levels were measured using an ELISA. The results in Table 1 19 are presented as the average t of plasma Apo(a) protein levels for each treatment group, normalized to baseline levels (% BL), The results show that the antisense oligonucleotides reduced Apo(a) protein expression. Furthermore, the oligonucleotides comprising a GalNAc conjugate group ted even more potent reduction in Apo(a) expression than the oligonucleotide that does not comprise a conjugate group.

Table 119 Apo(a) plasma protein levels ISIS No. Dosage (mg/kg) Apo((aaéllyeek PBS n/a 143 494372 50 58 681251 10 15 681255 10 14 681256 10 17 681257 10 24 681258 10 22 681260 10 26 Example 109: Synthesis of oligonucleotides comprising a GalNAc1-28 or GalNAc1-29 conjugate OH 5' 3' Hog?) OMHO \.0\ W AcHN NW 241 OH Oligonucleotide 241 is synthesized using procedures similar to those described in Example 71 to form the phosphoramidite intermediate, followed by procedures described in e 10 to synthesize the oligonucleotide. The GalNAc1 cluster portion (GalNAc1-28a) of the conjugate group GalNAc1-28 can be combined with any cleavable moiety present on the oligonucleotide to e a variety of conjugate groups.

The ure of GalNAcl-28 (GalNAcl-28a-CM) is shown below: In order to add the GalNAc1 conjugate group to the 3’-end of an oligonucleotide, procedures r to those described in Example 71 are used to form the hydroxyl intermediate, which is then added to the solid support using procedures described in Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 242.

HogvomHO .\\OH AcHN MW 3. 5.

The GalNAc1 cluster portion (GalNAc1-29a) of the conjugate group GalNAc1-29 can be combined with any ble moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of 1-29 (GalNAc1-29a-CM) is shown below: o "\OH HO oMN AcHN HWN Example 110: Synthesis of oligonucleotides comprising a GalNAc1-30 conjugate OAc OAc AcO AcO /\/\/\ O HO OTBDPS AcO OMOTBDPS TMSOTf N ACHN 7&0 243 1. NH /MeOH ODMTr 2_ DM3TrCI AcO 1. TBAF 3_ AczO, pyr O 2. Phosphitilation ACO O\/\/\/OTBDPS ODMTr 1. Couple to 5'-end of A80 ACO O\/\/\/O\P/OCE ACHN N DMT-on puri?cation methods HO&/O 5' 3' HO O\/\/\/O\ /O\ AcHN R Oligonucleotide 246 comprising a 1-30 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc1 cluster portion (GalNAc1-3 0a) of the conjugate group GalNAc1-30 can be combined with any cleavable moiety to provide a variety of conjugate groups. In n embodiments, Y is part of the ble moiety. In certain embodiments, Y is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc1-30a is shown below: HO&O\/\/\/O\fHO Example 111: Synthesis of oligonucleotides comprising a GalNACZ-Sl 0r GalNACZ-SZ conjugate HO 1- DMTICI DMTrO OCE Couple to 5'-end of A80 2. Phosphitilation I OH —» P\ —> N(iPr)2 DMTrO H0 247 248 Bx 1. Remove DMTr groups DMTFO 2. Couple amidite 245 O O "'X —’ 7p; 3. Deprotect and purify ASO usmg , - \: DMT-on purification s 0 O Y OH ?(5’ Y HQAcHN Oligonucleotide 250 comprising a GalNAc2-31 conjugate group, wherein Y is selected from O, S, a substituted or tituted C1-C10 alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAcz cluster portion (GalNAc2-31a) of the conjugate group GalNAc2-31 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y- containing group directly adjacent to the 5 ’-end of the ucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5 ’-end of the ucleotide is part of a stable moiety, and the ble moiety is present on the oligonucleotide. The structure of GalNAc2-3 1a is shown below: The synthesis of an oligonucleotide comprising a GalNAc 2-32 conjugate is shown below. 1. DMTrCI 2. Allyl Br 3. 0304, Na|O4 1 . COL] DMTrO pie to 5-end of A80' HO 2 g?Bl—hh't'l 2. Remove DMTr groups . OSP I la Iont' 3. Cou Ie e 245 0H —. OX0 P—. 4. Deprotect and purify ASO using DMTrO HO :‘P N(|Pr)2 DMT-on purification methods 247 CEO o O Y OH ?(5’ Y Oligonucleotide 252 comprising a GalNAc2-32 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C 1-C10 alkyl, amino, substituted amino, azido, l or alkynyl, is synthesized as shown above. The GalNAcz r portion c2-32a) of the conjugate group GalNAc2-32 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the Y- containing group directly adjacent to the 5 ’-end of the oligonucleotide is part of the cleavable moiety. In certain embodiments, the Y-containing group directly adjacent to the 5 ’-end of the oligonucleotide is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc 2-32a is shown below: Example 112: Modi?ed ucleotides comprising a GalNAc1 conjugate The oligonucleotides in Table 120 targeting SRB-l were synthesized with a GalNA01 conjugate group in order to ?thher test the potency of oligonucleotides comprising conjugate groups that contain one GalNAc ligand.

Table 120 GalNAc SEQ ISIS No. Sequence (5 , to 3 , ) CM cluster ID NO. 71 1461 GalNAcl'Zsa-O’Ado Ges mCes Tes Tes mCes Ads Gds Tds Incds Ads Tds Gal\AC '253, Ad 2306 Gcs Ads mcds Tds Tes mcm mCes Tes Te 71 1462 GalNAcl'Zsa-O’Ges mCm Tes Tes mCes Ads Gds Tds Incds Ads Tds Gds Gal\AC '253. PO 2304 Acs mCds Tds Tes mCes mCes Tes Te 71 1463 GalNAcl'Zsa-O’Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gal\AC '253, PO 2304 Gcs Ads mcds Tds Teo mCeo mCes Tes Te 71 1465 GalNAcl'26a-O’Ado Ges mCes Tes Tes mCes Ads Gds Tds mcds Ads Tds Gal\AC -26a Ad 2306 Gcs Ads mcds Tds Tes mcm mCes Tes Te 71 1466 GalNAcl'26a-O’Ges mCm Tes Tes mCes Ads Gds Tds mcds Ads Tds Gds Gal\AC '263, P0 2304 Acs mCds Tds Tes mCes mCes Tes Te 711467 GalNAc1-26MGes InCCO T60 T60 InCCO Ads Gds Tds mCdS Ads Tds Gal\Ac -26a PO 2304 Gcs Ads Incds Tds Teo mCeo mCes Tes Te 71 1468 GalNAcl'Zsa-O’Ado Ges mCes Tes Tes mCes Ads Gds Tds mcds Ads Tds Gal\AC -28a Ad 2306 Gcs Ads Incds Tds Tes mcm mCes Tes Te 71 1469 GalNAcl'Zsa-O’Ges mCm Tes Tes mCes Ads Gds Tds mcds Ads Tds Gds Gal\AC '283, P0 2304 Acs mCds Tds Tes mCes mCes Tes Te 71 1470 l'Zsa-O’Ges mCeo Teo Teo mCeo Ads Gds Tds mCds Ads Tds Gal\AC '283, P0 2304 Gcs Ads mcds Tds Teo mCeo mCes Tes Te 713844 Gas mCes Tee Tes Incas Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Gal\AC '273. PO 2304 T6 InCes InCes Tes Teoa_GalNAcl-27a 713845 Gee mCeo Teo Teo mCeo Ads Gds Tds mcds Ads Tds Gds Ads mCds Tds Gal\AC '273. PO 2304 Too mCeo mCes Tes Teo’.GalNAcl-27a 713846 Gee mCeo Teo Teo mCeo Ads Gds Tds mcds Ads Tds Gds Ads mCds Tds Gal\AC '273, Ad 2305 Too mCeo mCes Tes Teo Ado’-GalNAcl'27a 713847 Gee mCes Tee Tes Incas Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds Gal\AC '293, P0 2304 T6 mCes mCes Tes Teoa_GalNAcl-29a 713848 Gas mCeo Teo Teo mCeo Ads Gds Tds mcds Ads Tds Gds Ads mCds Tds GalNACl'29a 2304 T60 mCeo mCes Tes Teo’.GalNAc1-29a 713849 Gas mCes T? Tes mC? Ads Gds Tds mCds Ads Tds Gds Ads mCds Tds GalNACl-29a Ad 2305 T? mCes mCes Tes Teo Ado’-GalNAcl'29a 713850 Gas mCeo Teo Teo mCeo Ads Gds Tds mcds Ads Tds Gds Ads mCds Tds GalNACl'29a Ad 2305 T60 mCeo mCes Tes Teo Ado’-GalNAcl'29a Example 113: Antisense oligonucleotides targeting growth e receptor and comprising a GalNAc cluster The oligonucleotides in Table 121 were designed to target human growth hormone receptor (GHR).

Table 121 Se uences (5’ t0 3’) SEQ ID No.

GalNAc3mcesmC,sA?mCGSmCmTdSTdsTdSGdSGdSGdSTdSGdsAdSAdSTesAesGesmC,SAe 703 GalNAc3 mCesmCeeromCmmCGOTdsTdsTdsGdsGdsGdsTasGdSAdSAdsTeeroGesmC65Ae 703 GalNAc3 mcesmCesAesmC,smCCSTdsTdSTdSGdSGdSGdSTdsGdsAdsAdST?AesG?mCesAe 703 GalNAc3 mCesmCeeromCmmCGOTdsTdsTdsGdsGdsGdsTasGdSAdSAdsTeeroGesmC65Ae 703 3 mCesmCesAesmCmmC,sTdSTdsTdsGdsGdsGdsTdsGdsAdSAdsTesAesGesmC,SAe 703 GalNAc3 mCesmCmAmmC,0cheonsTdsTdSGdsGdSGdSTdSGdSAdsAdSTeeronmC65Ae 703 GalNAc3 mCesmCesAesmCmmC,sTdSTdsTdsGdsGdsGdsTdsGdsAdSAdsTesAesGesmC,SAe 703 GalNAc3 mCesmCmAmmC,0cheonsTdsTdSGdsGdSGdSTdSGdSAdsAdSTeeronmC65Ae 703 CesAmmC?mC,STdSTdsTdsGdsGdSGdsTdSGdsAdSAdSTCSAesGesmC?A, -GalNAc3-19 703 mC,SmCmAmmC,0cheoTdsTdSTdSGdSGdsGdSTdsGdsAdsAdsTeeroGesmCesAe-GalNAc3-19 703 e 114: 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 e conditions. The s 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 ucleotide. After a ent 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: 2329; reverse sequence AAGAGCCATGGAAAGTAGAAATCTTC, ated herein as SEQ ID NO: 2330; probe sequence TTCCTCAGATGAGCCAATT, designated herein as SEQ ID NO: 2331) was used to measure mRNA levels. GHR mRNA levels were ed 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 55 MOE or 34 MOE gapmers. The 55 MOE s are 20 nucleosides in length, wherein the central ’ ion and gap segment comprises of ten 2’-deoxynucleosides and is ?anked by wing segments on the 5 the 3 ’ direction comprising ?ve nucleosides each. The 34 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of ten 2’-deoxynucleosides and is ?anked by wing segments on the 5 ’ direction and the 3 ’ direction comprising three and four nucleosides respectively. Each side in the 5 ’ wing segment and each nucleoside in the 3’ wing segment has a 2’-MOE modi?cation. 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 ed 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. 576.16 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 ce alignment for a target gene in a ular 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 122 Inhibition of GHR mRNA by 55 MOE gapmers targeting exonic s of SEQ ID NO: 1 and 2 SE? SEQ SEQ SEQ ID ID ID SEQ ISIS NO NO: 1 Target ce . .4?0 . NO: 2 NO: 2 ID 1 Region inhibition Stop Start Stop NO Start . . .

. Site Site Site 523266 164 183 Exonl ACCTCCGAGCTTCGCCTCTG 64 3040 3059 20 Exon- 523267 171 190 exon CTGTAGGACCTCCGAGCTTC 31 n/a n/a junction 21 Exon- 523268 178 197 exon TCCATACCTGTAGGACCTCC 37 n/a n/a junction 22 523271 206 225 Exon 2 TGCCAAGGTCAACAGCAGCT 80 144990 145 009 23 523272 213 232 Exon 2 CTGCCAGTGCCAAGGTCAAC 53 144997 145016 24 523273 220 239 Exon 2 CTTGATCCTGCCAGTGCCAA 49 145004 145023 25 523274 227 246 Exon 2 AGCATCACTTGATCCTGCCA 67 145011 145030 26 523275 234 253 Exon 2 CAGAAAAAGCATCACTTGAT 0 145018 145037 27 523276 241 260 Exon 2 TCACTTCCAGAAAAAGCATC 1 145025 145044 28 523284 361 380 Exon 4 GTCTCTCGCTCAGGTGAACG 48 268024 268043 29 523285 368 387 Exon 4 TGAAAAAGTCTCTCGCTCAG 15 268031 268050 30 523286 375 394 Exon 4 AGTGGCATGAAAAAGTCTCT 14 268038 268057 31 523287 382 401 Exon 4 CAGTGGCATGAAAA 4 268045 268064 32 523301 625 644 Exon 6 GGATCTGGTTGCACTATTTC 36 n/a n/a 33 523302 632 651 Exon 6 AATGGGTGGATCTGGTTGCA 28 278926 278945 34 523303 647 666 Exon 6 AGTCCAGTTGAGGGCAATGG 26 278941 278960 35 523304 654 673 Exon 6 TCAGTAAAGTCCAGTTGAGG 0 278948 278967 36 523305 675 694 Exon 6 GAATCCCAGTTAAACTGACG 19 278969 278988 37 523306 682 701 Exon 6 TCTGCATGAATCCCAGTTAA 39 278976 278995 38 523309 736 755 Exon 6 CCTTTCTGAATATC 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 CTTTGTATTGAAGT 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 81 1 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 GAAGTGTTACATAG 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 123 Inhibition of GHR mRNA by 55 MOE gapmers targeting exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ ID ID SEQ ID SEQ ID ISIS NO: NO: Target % NO: 2 NO: 2 Sequence . ID NO 1 1 Region inhibition. Start Stop N0 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 53 145 009 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 AGTGAGACCATTTC 65 298164 298183 79 533050 1748 1767 exon 10 TTGGCAGAGTGAGACCATTT 47 298165 298184 80 533051 1749 1768 exon 10 AGAGTGAGACCATT 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 TGTGTAGTGTAATA 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 124 Inhibition of GHR mRNA by 55 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SE? SEDQ SE3 SE3 1:188 Ni3 ‘. NO‘ 1 1:23:12 sequence inhilfition0 NO‘ 2 NO‘ 2 118330 Start Stop Start Stop 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 AAGGCCACTCTTCC 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 CAAACCAACACTAA 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 67 133 113034 113053 121991 122010 53313 8 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 5 3 39722 39741 139 533144 n/a n/a Intron 1 TTTTAGTATGAACCTTAAAA 0 42139 4215 8 140 533145 n/a n/a Intron 1 CTTTTAGTATGAACCTTAAA 3 8 42140 42159 141 533146 n/a n/a Intron 1 AGTATGAACCTTAA 57 42141 42160 142 533147 n/a n/a Intron 1 AATCTTTTAGTATGAACCTT 60 42143 42162 143 533 148 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 53315 6 n/a n/a Intron 1 AAGCAGGTTGGCAGACAGAC 79 53467 53486 152 53315 7 n/a n/a Intron 1 GAAGCAGGTTGGCAGACAGA 60 53468 53487 153 53315 8 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 125 Inhibition of GHR mRNA by 55 MOE gapmers targeting intronic and exonic s of SEQ ID NO: 1 and 2 SE? SEDQ SEQ ID sIII)Q SEQ 113% N10. N10. 1:23:12 0 sequence inhilfition NO‘z. NO‘ 2 ID Start Site Stop NO Start Stop Site Site Site 26495 26514 533133 n/a n/a Intron 1 TGGAAAACCAAATA 76 121986 122005 129 26496 26515 533 134 n/a n/a Intron 1 TCAGGAATGGAAAACCAAAT 83 13 0 121987 122006 533174 n/a n/a Intron 1 TAAGTCTTCTTGTGAGCTGG 73 64886 64905 15 8 533175 n/a n/a Intron 1 TTAAGTCTTCTTGTGAGCTG 5 8 64887 64906 159 533176 n/a n/a Intron 1 TCTTCTTGTGAGCT 51 64888 64907 160 533177 n/a n/a Intron 1 TCTCTTCCACTCACATCCAT 72 65989 66008 161 533178 n/a n/a Intron 1 GTCTCTTCCACTCACATCCA 86 65990 66009 162 533179 n/a n/a Intron 1 AGTCTCTTCCACTCACATCC 80 65991 66010 163 533180 n/a n/a Intron 1 TTTGTAGCAGTTGC 31 78195 78214 164 533181 n/a n/a Intron 1 CTAAGTATTTGTAGCAGTTG 14 78196 78215 165 533182 n/a n/a Intron 1 GCTAAGTATTTGTAGCAGTT 59 78197 78216 166 533183 n/a n/a Intron 1 TGGCTAAGTATTTGTAGCAG 34 78199 78218 167 533184 n/a n/a Intron 1 TTGGCTAAGTATTTGTAGCA 18 78200 78219 168 533185 n/a n/a Intron 1 TTTGGCTAAGTATTTGTAGC 21 7 8201 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 AATGTCAACAGTGC 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 CTTCTCTTTGGCCA 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 CTGAATTCTGAGCT 81 145434 145453 221 53323 8 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 GATCTCAGCTGCAA 76 146240 146259 226 533243 230 249 Exon 2 AAAAGCATCACTTGATCCTG 23 145014 145033 227 Table 126 Inhibition of GHR mRNA by 34 MOE gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SEQ SEQ ID ID SEQ SEQ ID ISIS NO: NO: Target % ID NO: NO: 2 SEQ Sequence NO 1 1 Region inhibition. . 2 Start Stop ID NO Start Stop Site Site Site Site 539284 206 222 Exon 2 CAACAGCAGCT 62 144990 145006 228 539285 207 223 Exon 2 CCAAGGTCAACAGCAGC 74 144991 145007 229 539286 208 224 Exon 2 GCCAAGGTCAACAGCAG 73 144992 145008 230 539290 869 885 Exon 7 TCTCACACGCACTTCAT 29 290367 290383 231 539291 870 886 Exon 7 ATCTCACACGCACTTCA 51 290368 290384 232 539292 871 887 Exon 7 GATCTCACACGCACTTC 56 290369 290385 233 539299 n/a n/a Intron 1 CTTTCATGAATCAAGCT 63 17927 17943 234 539300 n/a n/a Intron 1 TCTTTCATGAATCAAGC 49 17928 17944 235 539301 n/a n/a Intron 1 GTCTTTCATGAATCAAG 61 17929 17945 236 539302 n/a n/a Intron 1 GGTCTTTCATGAATCAA 93 17930 17946 237 539303 n/a n/a Intron 1 ATGGTCTTTCATGAATC 74 17932 17948 238 539304 n/a n/a Intron 1 GATGGTCTTTCATGAAT 56 17933 17949 239 539305 n/a n/a Intron 1 TATATCAATATTCTCCC 42 21820 21836 240 539306 n/a n/a Intron 1 TTATATCAATATTCTCC 33 21821 21837 241 539307 n/a n/a Intron 1 GTTATATCAATATTCTC 12 21822 21838 242 539308 n/a n/a Intron 1 TTTCTTTAGCAATAGTT 21 22518 22534 243 539309 n/a n/a Intron 1 CTTTCTTTAGCAATAGT 38 22519 22535 244 539310 n/a n/a Intron 1 GCTTTCTTTAGCAATAG 39 22520 22536 245 26497 26513 539311 n/a n/a Intronl AGGAATGGAAAACCAAA 18 113031 113047 246 26498 26514 539312 n/a n/a l 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 5 3466 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 TTGTGAGCTGG 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 TCCACTCACAT 74 65993 66009 262 539328 n/a n/a Intron 1 ATAGATTTTGACTTCCC 57 72107 72123 263 539329 n/a n/a Intron 1 TTTTGACTTCC 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 8065 8 269 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 TCACCAAACAC 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 127 Inhibition of GHR mRNA by 55 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ SEQ ID ID SE 113% Sequence inhi:)/i)ti0n NO: 2 NO: 2 IDQ 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 AAATAAAGTGAGGA 0 135 81 13600 299 523566 TAACCTGCTAATAA 40 14451 14470 300 523567 ATGTGCCTTACAGTTATCAG 36 15251 15270 301 523568 ATTTAGAATTATAG 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 303 67 315 523582 GCACTACAAGAACTGCTTAA 40 31172 31191 316 523583 TTTCCAGACAAAGAATTCAG 6 31978 31997 317 523584 GTAGACAGCCTTTCTGGAAC 20 32827 32846 318 523585 ACATAGTGGCTGTG 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 3 8094 3 8113 324 523591 TTTGAATTTCAAAG 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 AATGTAATGAGTGT 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 52361 1 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 GAATTAATCCTCAG 49 70114 70133 354 523621 AATGCCCTCTCCCTGTGCCT 48 70925 70944 355 523622 ATCAACCTTTGCTA 9 71741 71760 356 523623 CTACTTAAAATAAT 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 128 Inhibition of GHR mRNA by 55 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ SEQ ID ID 0 SEQ 11338 Sequence inhilfition NO: 2 NO: 2 ID 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 3 80 532155 CTGCTGATTTTGGAATGGAG 68 10660 10679 3 81 532156 ACTGAACACCTATTCTATGG 51 11968 11987 382 532157 TTTACTGAACACCTATTCTA 23 11971 11990 383 532158 AATTATCCACAAAC 89 12053 12072 384 532159 AATGTTTCCAAGGC 63 12186 12205 385 532160 TTACATCCTGTAGGCTAATT 82 12469 12488 3 86 532161 CCACTAGCCTGGCCAGACTT 73 12487 12506 3 87 532162 CTGGTAGATGATCTCAAGTT 84 13351 13370 3 88 532163 AAAGAATTGAGTTATAAATC 23 13670 13689 3 89 532164 AACTCATCTCTGGCCAGCAG 89 14361 14380 390 532165 CAACATCATTGTATTTTCTG 33 14965 14984 391 532166 CTTACCAATGAGGA 81 15085 15104 392 532167 TTCCCAGAGCCAAAGCTCAA 77 15982 16001 393 532168 TTTGGCCAATCCCAGCTTAT 59 16253 16272 394 532169 AAATCTTCATTCAC 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 TCATCTTTATATTC 25 19106 19125 402 532178 ATTGCTCTTAAGATAAGTAA 41 19661 19680 403 532179 CAGCTCCCTGAATATCTCTT 74 19783 19802 404 532180 ACTTCACAAATATATTATAA 0 19885 19904 405 532181 TCAACTTTACTTCA 89 19899 19918 406 532182 CAATTCCCACTCTTGTCAAC 55 20288 203 07 407 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 2203 8 22057 412 32188 GGCTTTCTTTAGCAATAGTT 86 22518 225 37 413 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 3711 1 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 3221 1 TTATTTCATATTTCCAGAAA 0 42675 42694 436 532212 CATGAATTACAGCTAAAGAT 20 42741 42760 437 532213 TTGCATGTATGTGTTTCTGA 62 43518 43537 438 532214 TCAATCTCTTTATACCCTTA 75 43765 43784 439 532215 ATCTCTTTATACCC 5 8 43768 43787 440 532216 CTATGCCCTAGTTACTTCTA 47 43972 43991 441 532217 AAAGAGAATCTCTTCCTTTT 27 44070 44089 442 32218 TCATTAAAGATTATTATAAC 0 44222 44241 443 532219 TTTGGATGAGTGGAAGGCTA 0 44528 44547 444 532220 GGAAATGGCCTTTTTCCTTA 72 45400 45419 445 532221 GGAGAAGCCCTCTGCCTGTA 60 46477 46496 446 32222 AAACCATATTGTCCACCAGA 84 46510 46529 447 Table 129 Inhibition of GHR mRNA bx 55 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ SEQ ID ID 0 SEQ 11338 Sequence inhilfition NO: 2 NO: 2 ID 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 32227 TTACTTGCTGACTAAGCCAT 69 51071 51090 452 532228 CAACTCAACATCAA 73 51215 51234 453 32229 TTGTATATATATAC 3 3 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 GAGTAGAAGGCAAA 43 55926 55945 461 532237 TAGGAAAGGAAGAATACACT 33 63 881 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 5 8 69223 69242 472 532248 TTTGCATTCAAAGCCCTGGG 91 69565 695 84 473 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 7763 8 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 AGCAAACAAACATA 42 81524 81543 506 532282 TATAAATTCCTTGGTCTGAT 33 82835 82854 507 532283 AAAATATAAATTCCTTGGTC 13 82839 82858 508 532284 AACAGCCTCTGACA 3 8 82959 82978 509 532285 AAAAGACCATGTTGCTTATT 72 83179 83198 510 532286 AGTCAGAATGTGGT 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 32293 GATGTACACCATTTTGAATT 15 86629 86648 518 532294 TGCTCTGGCCTAGCCTATGT 62 86901 86920 519 532295 CAGAGGTGTCTCCCAAGAAA 60 89940 89959 520 532296 AAAGAGAATGGATCAAAGCT 36 91930 91949 521 532297 CAGAACAAATCTTG 37 93332 93351 522 532298 TGGTTATGAAGGTTGGACCA 52 94839 94858 523 532299 TGGCTAATTAATGGGCAATT 63 95292 95311 524 Table 130 Inhibition of GHR mRNA bx 55 MOE gapmers targeting intron 1 of SEQ ID NO: 2 SEQ SEQ ID ID 0 SEQ If}? Sequence inhilfition NO: 2 NO: 2 ID 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 84 962 82 96301 529 532305 TTTCAATACAATAAGATTTA 34 96793 96812 530 532306 GTCCTTAGACCCCTCAATGG 62 96987 97006 531 532307 GAGGATTTATTCATCTAGGC 68 97806 97825 532 532308 GAGGATCAGATATC 46 97870 97889 533 532309 ATCCCATCCAGCAGCTGGAC 67 98132 98151 534 532310 AACTTGGGATGAGTTACTGA 56 98653 98672 535 532311 TACCTAAAAGAAAT 43 98810 98829 536 532312 ATATTCACAACATT 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 28 103 034 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 TGGTCTGTGTATTT 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 AGCAATGAATGGGT 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 TGCGATCACTTCTT 76 120826 120845 570 532346 CCAGAAATTATCCTCCTCTC 70 121209 121228 571 532347 AGGGAAATGCAAATTAAAAC 20 122479 122498 572 532348 AGATACAGAAAAAT 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 TTATAGAGCTTGAG 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 CCTTTACTTTCCCA 36 134598 134617 601 Table 131 Inhibition of GHR mRNA by 55 MOE gapmers targeting introns 1 and 2 of SEQ ID NO: 2 SEQ SEQ ID ID SEQ SEQ ISIS NO: NO: % ID NO: ID NO: Sequence Target 1 . ID NO 1 . region tion 2 Start 2 Stop 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 ACTTCTTGAATCAA Intron 1 38 86373 863 92 604 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 54 945 81 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 1 10403 1 10422 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 l 28 113646 113665 637 523674 n/a n/a ATAAAACTGGATCCTCATCT Intronl 13 114446 114465 638 523675 n/a n/a ATTAAAACTCTCAGCAAAAT Intron 1 0 115450 115469 639 523676 n/a n/a TGAAAGAACACAAA 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 GACTATGTTCTGGC 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 TTCAAACCCCCCAG 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 34 133 85 8 133 877 661 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 138035 138054 666 523703 n/a n/a AATTTATTTCCTTCCTATGA Intron 1 138857 138876 667 523704 n/a n/a TTTTGGAAATGTGAGAAACG Intron 1 139771 139790 668 523705 n/a n/a AAACACATGAGAAAAGATGA Intron 1 0000 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 0 143 829 143 848 673 523710 n/a n/a AGGGCCAGTAAGGTTTGCTT Intron 1 12 144631 144650 674 523711 n/a n/a AATTCTGAGCTCTG Intron 2 88 145431 145450 675 523712 n/a n/a AGAGGATCTCAGCTGCAATT Intron 2 71 14623 8 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 132 Inhibition of GHR mRNA by 55 MOE s targeting introns 1 and 2 of SEQ ID \10: 2 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 1362 82 136301 682 532381 TCAAGCACTTACATCATATG Intron 1 42 136377 136396 683 532382 AGGGTTAGTTATTACACTTA Intron 1 60 136576 136595 684 532383 AGGCTTCATGTGAGGTAACA Intron 1 5 8 136996 137015 685 532384 TGAAAGCTTAGTACAAGAAG Intron 1 51 138048 138067 686 532385 TCTTGGAGATCCAG Intron 1 58 138782 138801 687 532386 GCTGAGATTTCTCTCCTCTT Intron 1 78 138792 138811 688 532387 CTTTTGCTGAGATTTCTCTC Intron 1 5 8 13 8797 13 8816 689 532388 GAACATATGTCCATAGAATG Intron 1 57 141700 141719 690 532389 GCTATGTAATCAAA 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 1463 67 1463 86 695 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 5 7 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 1541 12 705 532404 TTCTAATAATTCAC Intron 2 46 1545 02 154521 706 532405 ACCTGCACTTGGACAACTGA Intron 2 60 154727 154746 707 532406 GTCAGTGCTTTGGTGATGTA Intron 2 1 1 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 AGTGACAGTACAGT Intron 2 65 1615 88 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 ACTTCTCTGAGGTC Intron 2 68 165040 165059 724 532423 GCCTGGCAAGAAGGGCCCTT Intron 2 56 165856 165875 725 532424 ACACATGTTTTTAAATTTAT Intron 2 21 166241 166260 726 532425 TGCACTAAAAGAAA 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 73 3 532432 GAACTTTGGAGGCTTTTAGA Intron 2 5 5 170703 170722 734 532433 TGTATGCCCCAAACTCCCAT Intron 2 83 171431 171450 735 532434 ACACAAATAAGGGAATAATA Intron 2 24 171549 1715 68 73 6 532435 TAGTTCAGCCACTATGGAAA Intron 2 47 171926 171945 737 532436 CTCCAAATTCCAGTCCTAGG Intron 2 93 172746 172765 73 8 532437 AGTTGGCACTGCTATATCAG Intron 2 66 173668 173687 739 532438 GGCCTTAGATTGTAAGTTTT Intron 2 69 174122 174141 740 532439 TTTTAGTATTATTGTAGGAA Intron 2 16 1741 88 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 3 5 1765 92 17661 1 745 532444 GTGACTATGTCACTGAATTT Intron 2 14 176918 176937 746 532445 GCCCTACCCAGCAGCCTGTG Intron 2 79 177540 177559 747 532446 CAAACATAAAGAGAGTTCCA Intron 2 79 177811 177830 748 _—--_- Table 133 Inhibition of GHR mRNA bx 55 MOE gapmers targeting intron 2 of SEQ ID NO: 2 SEQ SEQ ID ID SEQ 113% Sequence inhib/(iti n0 NO: 2 NO: 2 ID Start Stop NO Site Site 533249 AGCAGAGGATCTCAGCTGCA 84 146241 146260 756 533250 TGCTCAAGTGCTAC 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 ATTGTAATTCTTGG 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 TTATGTGCTTTGCC 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 AAGAAAGGAACCTT 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 CAGTATCCTAGCCT 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 TTTCTGCAGTATCC 80 166356 166375 826 533320 GTTTCCATTTTCTTGATTCC 70 169601 169620 827 Table 134 Inhibition of GHR mRNA bx 55 MOE gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ SEQ ID ID 11% 0 sequence 35%: inhilfition NO‘ 2 NO‘ 2 g SEIEOID Start Stop Site Site 533326 AACCCATTTCATCCATTTAA Intron 2 93 175369 175388 833 533327 GAACCCATTTCATCCATTTA Intron 2 83 175370 175389 834 533328 CATTTCATCCATTT 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 AGGGATTGCCTCAG 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 845 333 39 TAACTTAGGACTTGGGACAT Intron 2 54 184576 1 845 95 846 533340 CTAACTTAGGACTTGGGACA Intron 2 63 184577 184596 847 533341 CACTAACTTAGGACTTGGGA Intron 2 82 184579 184598 848 33342 TCACTAACTTAGGACTTGGG Intron 2 77 1845 80 1845 99 849 33343 GTCACTAACTTAGGACTTGG Intron 2 83 1845 81 184600 850 33344 TGGGCTAGATCAGGATTGGT Intron 2 81 18 8617 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 TGGGCTAGATCAGG 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 1902 84 190303 861 533355 ATGTCTCAGCAAACCTGGGA Intron 2 81 190285 190304 862 533356 GAATGTCTCAGCAAACCTGG Intron 2 76 190287 190306 863 533357 TCTCAGCAAACCTG Intron 2 82 190288 1903 07 864 533358 AGGAATGTCTCAGCAAACCT Intron 2 85 190289 190308 865 333 59 TACAGACATAGCTCTAACCT Intron 2 79 191139 19115 8 866 333 60 ATACAGACATAGCTCTAACC Intron 2 79 191 140 191 159 867 333 61 GATACAGACATAGCTCTAAC Intron 2 71 191 141 191 160 868 333 62 TGGATACAGACATAGCTCTA Intron 2 79 191 143 191 162 869 333 63 CTGGATACAGACATAGCTCT Intron 2 82 191 144 191 163 870 333 64 GCTGGATACAGACATAGCTC Intron 2 95 191 145 191 164 871 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 875 333 69 AAACAACACTGTTTGTGAGG Intron 2 76 191944 191963 876 533370 ACACTGTTTGTGAG 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 5333 80 GGTATGTGGCTTTAATTCAC Intron 2 83 198149 198168 887 5333 81 TGGTATGTGGCTTTAATTCA Intron 2 81 198150 198169 888 5333 82 GTGGTATGTGGCTTTAATTC Intron 2 86 198151 198170 889 533383 TTCAGTTGCATCAC Intron 2 75 199817 199836 890 5333 84 TTCTGTGTTCAGTTGCATCA Intron 2 82 199818 199837 891 5333 85 GTTCTGTGTTCAGTTGCATC Intron 2 86 199819 199838 892 5333 86 ATGAGGAGGCACTT Intron 2 81 201413 201432 893 5333 87 GGTACTCATGAGGAGGCACT Intron 2 82 201414 201433 894 533388 TGGTACTCATGAGGAGGCAC Intron 2 78 201415 201434 895 5333 89 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 Table 135 tion of GHR mRNA bx 55 MOE gapmers ing introns 2 and 3 of SEQ ID NO: 2 SE? SEQ ID ISIS Target % NO: 2 sequence NO' 2. ID NO region inhibition Stop Start . NO 523715 GTCAATTATGTGCTTTGCCT Intron 2 91 148907 148926 910 523716 ACATTCAAAATTCTTCCTTG Intron 2 50 149787 149806 911 523717 ATCCTGCATATATTTTATTG Intron 2 20 150588 150607 912 523718 CTGCTGGTGACTCTGCCTGA Intron 2 77 151388 151407 913 523719 AATGCTGAAGGATGGGCATC Intron 2 66 152204 152223 914 523720 TTATCCAGTAGTCAATATTA Intron 2 71 153 004 15 3023 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 15 6413 919 523725 TGCTACAATGCACAGGACAC Intron 2 80 157201 157220 920 523726 TCTGATATTTATTGCTGTAC Intron 2 73 158007 15 8026 921 523727 CCTTTAATAAATGT Intron 2 0 158807 15 8826 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 4 163 943 163962 928 523734 GTTTTCTTTAAAAA Intron 2 0 164746 164765 929 523735 CTTCCAGAAGAATTACCCAT Intron 2 56 165553 165572 930 523736 CAGTTTCTGCAGTATCCTAG Intron 2 77 166353 1663 72 931 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 1762 82 943 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 AGGAAGACAGTCTC Intron 2 16 182962 182981 951 523757 TGGCTCATAACTTCCTTAGC Intron 2 43 183762 183781 952 523758 ACTAACTTAGGACTTGGGAC Intron 2 72 184578 184597 953 52375 9 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 TAGCAGTCACTTAG Intron 2 62 189481 189500 959 523765 AATGTCTCAGCAAACCTGGG Intron 2 62 190286 1903 05 960 523766 AGACATAGCTCTAA Intron 2 75 191 142 191 161 961 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 CTCTGCAACTCCAA 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 2105 80 984 523790 GACTGAAACTTAAAGGTGGG Intron 3 7 211399 211418 985 Table 136 Inhibition of GHR mRNA by 34 MOE gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ SEQ Sequence Target % ID NO: ID NO: NO . . region tion 2 Start 2 Stop Site Site 539360 GACTCTGCCTG Intron 2 95 151389 151405 987 539361 TGCTGGTGACTCTGCCT Intron 2 95 151390 151406 988 539362 GTGACTCTGCC 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 TGAGGACAATA 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 5393 80 CTTGGTTTGTCAATCCT Intron 2 97 156891 156907 1007 5393 81 TACAATGCACAGGACAC Intron 2 65 157201 157217 1008 53 93 82 CTACAATGCACAGGACA Intron 2 85 157202 157218 1009 539383 GCTACAATGCACAGGAC Intron 2 96 157203 157219 1010 539384 GATATTTATTGCTGTAC Intron 2 43 158007 158023 1011 5393 85 TGATATTTATTGCTGTA Intron 2 35 158008 158024 1012 5393 86 CTGATATTTATTGCTGT Intron 2 38 158009 158025 1013 5393 87 AGGGTCTTTACAAAGTT Intron 2 61 162751 162767 1014 5393 88 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 CATAGCTCTAA Intron 2 45 191 142 191 15 8 1033 539407 ATACAGACATAGCTCTA Intron 2 53 191 143 191 159 1034 539408 GATACAGACATAGCTCT Intron 2 67 191 144 191 160 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 GATGTGAGGCT Intron 3 82 210561 210577 1042 539416 ACATCTGGATGTGAGGC Intron 3 86 210562 210578 1043 539417 CACATCTGGATGTGAGG Intron 3 55 210563 210579 1044 539418 AATTTCTGGAA Intron 3 35 219019 219035 1045 539419 TAATTTCTGGA 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 53 9431 CTCAATTGCAGATGCTC Intron 3 82 237676 237692 105 8 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 137 Inhibition of GHR mRNA by 55 MOE gapmers targeting introns 1 and 3 of SEQ ID NO: 2 SE(Q SEKQ H) H) ISIS Target 96 SEQ ID Sequence DMD:2 DMD:2 FR) 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 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 143293 143312 1070 143321 143340 143349 143368 143377 143396 /168618 143405 143424 143433 143452 143461 143480 143210 143229 143238 143257 143266 143285 143294 143313 143322 143341 533410 TGTGAGATTTCAAATAAATC Intron 1 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 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 533415 CACTTTGTGAGATTTCAAAT Intron 1 69 —143299143318 1076 143327 143346 143355 143374 143383 143402 143467 143486 533 895 AGTATTTCAGGCTGGAAAAA Intron 3 35 214622 214641 1077 33 896 TGAGTATTTCAGGCTGGAAA Intron 3 55 214624 214643 1078 33 897 TCTGAGTATTTCAGGCTGGA Intron 3 71 214626 214645 1079 533 898 ATCTGAGTATTTCAGGCTGG Intron 3 77 214627 214646 1080 33 899 TATCTGAGTATTTCAGGCTG Intron 3 5 8 21462 8 214647 1081 33 900 TTTTGTGTTATGCCTTGAGG Intron 3 51 221483 221502 1082 533901 TTTTTGTGTTATGCCTTGAG Intron 3 55 221484 221503 1083 33 902 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 ATTTACCTGGGTAA 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 TGCTTATTTACCTG Intron 3 69 225570 225589 1092 33 911 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 22963 8 1097 533916 TTCCAATGATGTTACTACTA Intron 3 74 229620 229639 1098 33 917 ATTCCAATGATGTTACTACT Intron 3 74 229621 229640 1099 533918 GAGCAATGGTCTAG Intron 3 71 232823 232842 1 100 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 1 106 533925 CATTTGGAAGACAG Intron 3 74 233624 233643 1 107 533926 AGGCTCACATTTGGAAGACA Intron 3 56 233625 233644 1 108 533927 AGAGGCTCACATTTGGAAGA Intron 3 34 233627 233646 1 109 533928 TAGAGGCTCACATTTGGAAG Intron 3 18 233628 233647 1 110 533929 TTAGAGGCTCACATTTGGAA Intron 3 19 233629 233648 1 1 1 1 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 1 115 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 1 123 533942 AATATTGAAAGGCCCATCAA Intron 3 31 246499 246518 1 124 533943 AAATATTGAAAGGCCCATCA Intron 3 51 246500 246519 1 125 533944 GAAAATATTGAAAGGCCCAT Intron 3 22 246502 246521 1 126 533945 GGAAAATATTGAAAGGCCCA Intron 3 42 246503 246522 1 127 533946 AGGAAAATATTGAAAGGCCC Intron 3 28 246504 246523 1128 533947 GTATATTCAGTCCAAGGATC Intron 3 65 248228 248247 1129 533948 AGTATATTCAGTCCAAGGAT Intron 3 63 248229 248248 1 130 533949 CAGTATATTCAGTCCAAGGA Intron 3 67 248230 248249 1 131 533950 AACAGTATATTCAGTCCAAG Intron 3 56 248232 248251 1 132 533951 AAACAGTATATTCAGTCCAA Intron 3 60 248233 248252 1 133 533952 AAAACAGTATATTCAGTCCA Intron 3 59 248234 248253 1 134 533953 TCTATTGTTGCCACCTTTAT Intron 3 45 25283 8 252857 1135 533954 TTCTATTGTTGCCACCTTTA Intron 3 52 252839 252858 1136 533955 TTTCTATTGTTGCCACCTTT Intron 3 46 252840 252859 1137 533956 TATTGTTGCCACCT Intron 3 59 252842 252861 1138 533957 CAGTTTCTATTGTTGCCACC Intron 3 41 252843 252862 1139 533958 CCAGTTTCTATTGTTGCCAC Intron 3 48 252844 252863 1140 Table 138 Inhibition of GHR mRNA b 5- 10-5 MOE wamers tar NO inhibition -————- -——-- 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 AAATTTGATCCCCA 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 TCTAACTGCCCAAT 77 199544 199563 1171 532485 TTCTGCAAAGCTTCCCAGTA 72 200314 200333 1172 532486 ACAACTTCAAGCTTCACATA 65 200599 200618 1173 532487 ATGTTCTGGCAAGA 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 CTATTTATGATTCC 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 ACCTCAGAGACATG 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 TATTAAATTTTAAA 0 223001 223020 1199 532514 TAATGTAGTGATTT 19 223156 223175 1200 532515 AAATATTTGATAGCTCACAT 18 224409 224428 1201 532516 AGAAATATTTGATAGCTCAC 57 22441 1 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 139 Inhibition of GHR mRNA by 55 MOE gapmers targeting introns 3-8 and intron-exonic regions of SEQ ID NO: 2 SEQ SEQ ISIS % ID NO: ID NO: Sequence Targetreg 0i n ID NO inhibition 2 Start 2 Stop Site.

Site. ——4_--523792AAAGCTTTGTGGATAAAGTT Intron 3 213025 213044 1217 523794 CTGAGTATTTCAGGCTGGAA—_ 214625 214644 1219 523795 TTGAATTATCCCTTTAAAAA Intron 3 38 215446 215465 1220 523796 TTTAGAATGGTTTGGCATAC Intron 3 66 216365 2163 84 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 2231 10 223129 1227 523803 GATTATTCTATTTTTATTTT Intron 3 33 223948 223967 1228 523804 AGGAAGAGTGGCATGGACAT Intron 3 43 224748 224767 1229 523805 CTTTTGCTTATTTACCTGGG Intron 3 84 225568 2255 87 1230 523806 TTTATATTATTAATATCATT Intron 3 31 226371 226390 1231 523807 TGGCTTTTAAGTGG 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 CTAGAAGAAGATTT 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 AATTGCAGATGCTC 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 25 8774 25 8793 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 Intron3 43 267375 267394 1263 523839 TGGTACCTATATTGAGAGGT Intr0n4 46 269052 269071 1264 523840 AAAAATATAGTATA Intr0n4 7 269854 269873 1265 523841 TTATTTATGTGTCAGGGATG Intr0n4 28 270668 270687 1266 523842 CAAAAGTTAAGTGCTTTAGG Intr0n4 10 271468 271487 1267 523843 TTCATAGATGTCTAAGGAAT Intr0n4 32 273341 273360 1268 523844 ACCTGTGATTTACCTATTTC Exoiiétl?gons 18 274185 274204 1269 523845 TGCCTAGAAAACCACATAAA Intr0n5 38 274985 275004 1270 523846 AAACATCCTCAAAGGTACCT Intr0n5 64 275808 275827 1271 523847 CTTCCCTGAGACACACACAT Intron5 35 276617 276636 1272 523848 CTTCTTCAATCTTCTCATAC 5 33 278288 278307 1273 523849 TACCATTTTCCATTTAGTTT Exo?ug;:;:°n6 7 279088 279107 1274 523850 ATTGGCATCTTTTTCAGTGG Intr0n6 34 279902 279921 523851 TCACGGTTGGAGAC Intr0n6 36 280799 280818 523852 AAATGAAATCAGTATGTTGA Intr0n6 0 281622 281641 523853 TGATTTATCACAAAGGTGCT Intr0n6 29 282437 282456 523854 AAAACAGTAGAAAAGATTAA 6 14 284073 284092 523855 CTACATCACAGCAGTCAGAA Intr0n6 23 285187 285206 286349 286368 523856 AAAAGATGTAAGTGTGACAT Intr0n6 28 1281 286919 286938 523857 TTACAAGAACTGCTAAAGGG Intr0n6 15 287151 287170 1282 523858 ATAAAGAAAAAGTTAACTGA Intr0n6 9 287982 288001 1283 523859 TATACTTCTTCTAT Intr0n6 4 288809 288828 1284 523860 CCTTCTTCACATGTAAATTG EXOLLLEEEOM 19 290456 290475 1285 523861 TTTCTATGTAGCTTGTGGTT Intr0n7 30 291258 291277 1286 523862 AGGCAGAGTTTTTATTGATA Intr0n7 19 292058 292077 1287 523863 ACCAGCCTAAGCCT Intr0n8 28 292858 292877 1288 523864 AGACTTTTAGCATGCTTGAC Intr0n8 56 293658 293677 1289 523865 TTTACAGCCCTACAGTTCTA Intr0n8 7 294464 294483 1290 523866 CCAGAGAACCTGACTCCAAA Intr0n8 6 295330 295349 1291 523867 CAGAAGAAAATATTAGACAG Intr0n8 10 296993 297012 1292 Table 140 Inhibition of GHR mRNA b 55 MOE wamers tar ISIS Target Se uenceq NO Region 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 23 8186 23 8205 1297 532536 AAATAAATCAGATTATTATA Intron 3 52 23 8242 23 8261 1298 532537 CTTAGATCTGTGCTGTCCAA Intron 3 81 24575 8 245777 1299 53253 8 GTTAGTGTTAGATTCTTTGA Intron 3 67 246152 246171 13 00 532539 CATGCTCACGGCTGTGTTAC Intron 3 66 246248 246267 1301 532540 CCCATCAAATACTGAGTTCT Intron 3 86 246487 246506 1302 532541 AGTGATTAATGAGA Intron 3 38 247012 247031 1303 532542 ATTAATCAACAAGTGGCATT Intron 3 72 247203 247222 13 04 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 53255 5 TTTCACAAGATTTACTTGGT Intron 3 77 254641 254660 1317 532556 CAGTTGTGATTGTCAACCTA Intron 3 77 257073 257092 1318 532557 AATCTTGCCTCGATGAAAGT Intron 3 57 257819 257838 1319 53255 8 TGGCCTAAATGTATCAGTTA Intron 3 66 259157 259176 1320 532559 TGGGTAAAATCTTT Intron 3 67 259184 259203 1321 532560 TATGATTTTTAAAGATTAAA Intron 3 20 261419 26143 8 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 AGTTGTACACAAAC 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 13 30 532569 TAATGCCCTTTCTCCCTACT Intron 4 60 271215 271234 1331 532570 TAGATTATCCCAAA 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 AGTCTCCTCGCTCA Intron 5 0 27673 8 276757 1343 277045 277064 532582 ATATAACAGAATCCAACCAT Intron 5 —47 1344 278361 278380 532583 TGCAAAATGGCCAAACTACA Intron 5 56 277577 277596 1345 532584 TCTTCCTAGCCACATGTGAT Intron 5 32 278227 278246 1346 532585 GCTCTCTAATTGCC 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 AAAAGTCTTCAAAT Intron 6 24 280831 280850 1352 532591 GGATCATTTCCCCATGCATG Intron 6 52 280982 281001 13 53 532592 ATATTATATTAAGAAAAATG Intron 6 4 281422 281441 13 54 532593 CTCCCATGTTCATTACTTAT Intron 6 49 281587 281606 1355 532594 CATGACATTGGTTTGGGCAA Intron 6 43 282229 282248 13 56 532595 AATGTTGTTGGGAAAATTGG Intron 6 42 282383 282402 1357 532596 AGCTGCAGGATACAAAGTCA Intron 6 49 282986 283005 1358 532597 ATATCCTTTCATGATAAAAA lntron 6 31 283354 283373 1359 532598 TAATATCTCTGATA lntron 6 50 283590 283609 1360 532599 ACATTACTAATAATTAGAGA lntron 6 0 285236 285255 1361 532600 ATAAAAACATATGAAAGTAT lntron 6 12 287093 287112 1362 532601 TTCTGAATTAAATCTATTAG lntron 6 16 287408 287427 1363 532602 TTACATTTTTGCAAATTTAT lntron 6 31 287472 287491 13 64 532603 TGAACAGTTGATTAACAAAG lntron 6 15 287887 287906 1365 532604 AAGTTATTGGTTTACTAGAT lntron 6 0 288598 288617 1366 532605 TTGGAAAAGGTCCTAGAAAA lntron 6 24 289808 289827 1367 532606 AGAAACTTCTTAGA lntron 7 25 292035 292054 1368 532607 CCATACTTGCTGACAAATAT lntron 8 39 294389 294408 1369 Example 115: 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 0.625 uM, 1.25 uM, 2.50 uM, 5.00 uM and 10.00 uM trations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of imately 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 e 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 141 ----0.625 1.250 2.50 5.00 10.00 ICso ISIS No mum-n Table 142 mm?nmmnHM HM HM HM HM (11M) -"--__ ——————m ----n Table 143 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 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 Table 144 ISIS No "BA (HM) 523655 60 2.1 523656 45 2.4 523658 62 3.1 523715 92 <0.6 523718 67 1.4 523723 83 0.3 523725 79 0.6 523726 77 1.2 523736 75 1.5 523747 80 0.6 523758 61 1.9 523766 66 2.0 523776 72 1.3 523789 81 0.2 Table 145 0625 1250 250 500 1000 ICso ISIS No "BA (HM) 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 42 63 81 93 2.0 523740 37 58 72 83 88 1.0 523752 29 52 72 86 2.5 523763 32 57 70 80 2.6 523764 43 52 67 77 79 0.9 523765 24 48 62 88 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 ——————n Table 146 ISIS No 0.625 1.250 2.50 5.00 10.00 IC50 M M M M M M 532151 57 69 76 85 88 <0.6 532153 23 43 54 80 86 1.8 532158 46 58 81 87 87 0.6 532160 17 26 55 76 92 2.2 532162 14 46 71 83 93 1.7 532164 37 76 82 90 93 0.6 532171 41 81 67 81 83 <0.6 532181 56 81 84 89 93 0.2 532186 26 65 75 83 91 1.1 532188 51 68 80 89 93 <0.6 532189 24 31 52 75 86 2.1 532197 0 40 66 85 93 2.1 532199 24 37 50 73 87 2.1 532222 12 41 67 84 94 1.8 Table 147 ISIS No 0.625 1.250 2.50 5.00 10.00 IC50 "M "M "M "M "M (HM) 532175 41 54 76 84 89 0.9 532223 53 69 75 88 94 <0.6 532235 43 58 67 77 82 0.8 532241 39 53 62 73 87 1.2 532248 49 65 72 85 93 0.6 532254 52 62 85 87 92 <0.6 532300 20 29 49 66 78 2.7 532304 26 39 66 78 90 1.7 532316 41 66 76 86 94 0.7 532395 32 56 84 93 97 1.0 532401 47 80 92 96 98 <0.6 532411 73 90 94 97 98 <0.6 532420 38 49 82 85 97 1.0 532436 37 58 75 90 96 0.9 wo 68618 Table 148 ISIS No 0.625 1.250 2.50 5.00 10.00 ICso "M "M "M "M NM (NM) 532410 66 83 92 94 97 <0.6 532468 45 68 78 93 94 0.6 532469 0 17 56 76 92 2.8 532470 10 34 62 84 94 2.0 532475 13 36 52 64 87 2.5 532476 34 64 73 79 93 0.9 532480 28 54 67 78 87 1.4 532482 21 39 69 83 92 1.7 532490 42 60 68 84 93 0.9 532500 37 50 63 81 87 1.2 532506 13 41 66 75 89 1.9 532507 47 59 71 86 89 0.7 532508 0 31 73 83 89 2.2 532526 31 56 78 79 88 1.1 Table 149 ISIS No 0.625 1.250 2.50 5.00 10.00 1C50 HM HM HM HM HM (11M) 532495 59 74 81 87 95 <0.6 532501 49 53 71 83 84 0.7 532534 53 75 85 91 97 <0.6 532535 0 34 61 84 92 2.6 532537 49 67 80 90 94 <0.6 532540 59 70 87 93 95 <0.6 532547 57 71 81 91 92 <0.6 532555 48 36 61 72 85 1.3 532556 33 57 67 86 90 1.1 Table 150 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 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 wo 68618 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 <06 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 151 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 11M HM 11M 11M HM (11M) 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 152 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 11M HM 11M 11M HM (11M) 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 "mum- ——————m Table 153 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 533364 71 77 92 90 94 <0.6 533925 26 55 61 85 91 1.4 533326 54 77 80 93 95 <0.6 533916 18 62 69 83 93 1.4 533328 52 68 89 94 98 <0.6 533932 42 49 80 86 92 0.9 533352 42 82 88 93 94 <0.6 533917 20 37 57 78 84 2.0 533331 54 83 89 93 96 <0.6 533936 21 46 73 84 88 1.5 533329 56 73 84 92 98 <0.6 533937 26 32 79 86 94 1.5 533908 58 66 81 88 94 <0.6 533898 61 64 84 90 92 <0.6 Table 154 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 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 116: Dose-dependent antisense inhibition of human GHR in Hep3B cells by MOE s Gapmers from the studies described above exhibiting significant in vitro tion 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 r culture ions. The results for each ment 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 uM, 0.625 "M, 1.25 "M, 2.50 "M, 5.00 uM and 10.00 uM 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 ucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

Table 155 mmmmmmWwM M M M M M ( M) 523814 0 24 48 52 68 82 2.2 523805 13 29 55 0 79 85 1.5 523822 0 19 26 41 65 85 2.8 523820 0 19 29 58 74 86 2.3 523815 3 6 19 37 45 71 4.8 523828 12 19 32 51 64 74 2.7 523801 3 9 31 43 59 76 3.3 523824 12 28 44 63 77 85 1.7 523794 13 21 30 51 66 78 2.5 523810 15 34 55 72 78 86 1.3 523819 0 24 40 60 66 75 2.4 Table 156 Mmm??mmnHM HM HM HM HM HM (HM) mum-m- —————m ———————m —————m manna-n Table 157 0.3125 0.625 1.250 2.50 5.00 10.00 ICso ISIS N0 HM HM HM HM HM HM (HM) 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 158 0.3125 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM HM (HM) 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 117: Dose-dependent antisense inhibition of human GHR in Hep3B cells by MOE gapmers s from studies described above exhibiting signi?cant 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 ment are presented in te tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 uM, 1.25 uM, 2.50 uM, 5.00 uM and 10.00 uM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of imately 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 EEN®. 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 159 M M M M ( M) 523271 mm 523274 ----— 523324 ---- 523577 523604 523614 Table 160 ISIS No 0.625 1.250 2.50 5.00 10.00 1C50 HM HM HM HM HM (11M) 523564 16 48 69 75 91 1.7 523570 24 52 65 71 88 1.6 523592 6 31 52 65 81 2.8 523595 13 49 60 79 92 1.8 523596 20 49 62 71 75 1.9 523607 38 63 66 74 76 0.8 523615 17 48 60 80 92 1.8 523630 19 42 42 67 80 2.5 523633 41 69 78 79 80 0.6 523665 16 45 56 71 80 2.1 523687 37 59 73 75 78 0.9 523711 33 63 78 91 93 0.9 523712 13 36 61 78 87 2.1 523714 63 85 91 96 96 <0.6 wo 68618 Table 161 ISIS No 0.625 1.250 2.50 5.00 10.00 1C50 HM HM HM HM HM (11M) 523655 28 42 57 74 76 1.9 523656 33 43 53 74 88 1.7 523661 29 29 66 79 82 1.9 523713 35 45 64 83 87 1.3 523715 83 86 92 93 94 <0.6 523718 27 52 69 84 95 1.3 523723 65 74 86 85 94 <0.6 523725 37 63 78 78 92 0.8 523726 43 57 72 86 89 0.8 523736 39 65 80 88 95 0.8 523747 51 71 83 86 93 <0.6 523766 30 50 70 82 89 1.3 523776 45 59 67 79 84 0.7 523789 63 75 76 83 83 <0.6 Table 162 ISIS No 0.625 1.250 2.50 5.00 10.00 1C50 "M "M "M "M NM (NM) 523719 18 40 56 73 83 2.1 523720 36 46 59 64 89 1.5 523724 44 60 75 81 87 0.7 523735 11 40 60 78 84 2.1 523740 17 47 61 80 81 1.8 523752 25 31 38 70 84 2.5 523758 23 48 58 72 80 1.8 523763 2 24 48 64 75 3.3 523764 22 49 45 73 75 2.1 523765 42 40 57 79 87 1.4 523767 43 53 56 69 79 1.2 523774 36 52 71 81 89 1.1 523778 15 45 59 75 79 2.0 523783 5 30 48 66 83 2.9 Table 163 0.625 1.250 2.50 5.00 10.00 IC50 ISIS N0 "M "M "M "M "M (HM) 532151 40 45 64 71 82 1.3 532158 28 47 63 70 87 1.6 532164 36 47 64 75 89 1.3 532171 35 47 50 69 89 1.6 532175 27 38 43 75 87 2.1 532181 21 56 63 69 80 1.7 532186 28 49 62 73 91 1.5 532188 40 52 73 75 90 1.0 532223 22 34 53 71 90 2.2 532235 35 31 48 68 73 2.3 532241 6 24 29 51 72 4.5 532248 19 37 47 73 84 2.3 532254 56 56 72 85 90 0.5 532316 32 55 50 78 90 1.5 Table 164 0.625 1.250 2.50 5.00 10.00 IC50 ISIS N0 "M "M "M "M "M (uM) 532304 44 57 68 78 73 0.7 532395 47 62 82 91 96 0.6 532401 70 83 91 94 96 <0.6 532410 56 71 85 90 96 <0.6 532411 88 93 96 97 98 <0.6 532420 61 67 82 85 96 <0.6 532436 48 49 77 90 97 0.8 532468 42 67 82 89 94 0.6 532476 32 58 75 84 90 1.1 532482 5 26 56 71 87 2.6 532490 18 47 55 69 86 2.0 532501 4 22 43 59 77 3.5 532507 39 63 66 83 89 0.9 532526 30 48 67 82 88 1.4 Table 165 0.625 1.250 2.50 5.00 10.00 1c50 ISIS N ------m wo 68618 533125 47 61 74 89 89 0.6 533136 5 25 58 79 90 2.4 533156 37 48 69 77 87 1.2 533161 28 67 77 89 90 1.0 533178 30 60 72 90 92 1.1 533179 37 66 76 76 87 0.8 533188 32 64 74 80 90 1.0 533189 49 66 77 81 81 0.4 533193 26 48 69 75 85 1.5 533233 39 60 59 84 93 1.0 533234 45 69 84 91 94 0.5 533235 28 49 69 82 90 1.4 Table 166 0.625 1.250 2.50 5.00 10.00 ICso ISIS N0 11M HM 11M 11M HM (11M) 533256 47 72 86 90 94 <0.6 533257 63 77 88 91 96 <0.6 533258 66 81 88 95 95 <0.6 533259 48 70 84 90 93 <0.6 533262 44 66 79 90 96 0.7 533265 59 74 85 93 96 <0.6 533269 25 55 74 86 87 1.2 533270 34 59 73 86 95 1.0 533271 63 82 88 92 92 <0.6 533291 14 46 64 84 89 1.8 533301 49 61 75 83 91 0.6 533315 22 39 73 76 91 1.7 533317 26 53 68 85 94 1.3 533318 29 40 46 77 91 1.9 Table 167 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 11M HM 11M 11M HM (11M) 533280 58 64 77 82 87 0.3 533316 35 55 68 87 91 1.1 533326 34 68 76 89 96 0.8 533328 54 55 79 83 92 0.5 533329 46 62 72 83 95 0.7 533330 56 75 83 91 94 0.3 533331 54 61 80 86 89 0.4 ——————m Example 118: Dose-dependent antisense inhibition of human GHR in Hep3B cells by MOE gapmers Gapmers from s described above exhibiting signi?cant 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 uM, 0.625 "M, 1.25 "M, 2.50 "M, 5.00 uM and 10.00 uM concentrations of antisense ucleotide, 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 ime 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 t inhibition of GHR, relative to ted 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 ucleotide treated cells.

Table 168 0.3125 0.625 1.250 2.50 5.00 10.00 IC50 ISIS N0 "M "M "M "M "M "M (HM) 523577 0 16 33 59 72 94 2.2 523633 15 33 66 73 82 86 1.1 523764 11 33 50 68 78 83 1.5 523794 12 30 33 56 76 82 1.9 523805 21 48 66 78 85 92 0.8 523810 18 36 61 80 89 90 1.0 523814 13 35 52 67 81 88 1.3 523819 11 30 57 72 81 89 1.3 523820 0 15 43 61 84 92 1.8 523824 21 27 59 72 84 90 1.2 Table 169 0.3125 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM HM (HM) 539302 34 41 56 83 83 96 0.8 539321 30 32 76 73 80 94 0.8 539322 22 36 57 72 78 94 1.1 539355 23 42 48 72 71 88 1.2 539359 21 38 48 73 78 92 1.2 539320 14 32 53 72 82 91 1.3 539341 3 19 35 56 78 89 2.0 539342 6 18 33 51 70 83 2.3 539356 0 0 21 45 73 94 2.7 539358 0 15 23 50 52 91 2.9 Table 170 0.3125 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM HM (HM) 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 171 0.3125 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM HM (HM) 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 ----m Example 119: Antisense inhibition of human growth hormone receptor in Hep3B cells by dequ, MOE and (S)-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 ments that had similar culture conditions. The s 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 nse 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 7_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 tion of GHR, relative to untreated l cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE, and (S)—cEt gapmers. The deoxy, MOE and (S)-cEt ucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modi?cation, an (S)—cEt sugar modification, or a deoxy modi?cation. The stry’ column bes the sugar ations of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modi?cation; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE ation. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. A11 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 ed 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 172 Inhibition of GHR mRNA by deoxy, MOE and t gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SE? SE? SEQ 113% N? : Target Region ce Chemistry fition NO: 2 ID Start 88:1: NO 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 84 156891 1370 541263 164 Intron 1 CCGAGCTTCGCCTCTG eekddddddddddkke 89 3040 1371 541264 167 Intron 1 CCTCCGAGCTTCGCCT eekddddddddddkke 90 3043 1372 Junction 541265 170 spanning two GGACCTCCGAGCTTCG eekddddddddddkke 89 n/a 1373 exons Junction 541266 176 spanning two CCTGTAGGACCTCCGA eekddddddddddkke 83 n/a 1374 exons 541268 214 Exon 2 CCAGTGCCAAGGTCAA dddddddkke 87 144998 1375 541269 226 Exon 2 CACTTGATCCTGCCAG eekddddddddddkke 67 145010 1376 541270 244 Exon 2 CAGAAAAAGC eekddddddddddkke 34 145028 1377 54127 8 365 Exon 4/Intron 3 GTCTCTCGCTCAGGTG eekddddddddddkke 77 268028 1378 541279 368 Exon 4/Intron 3 AAAGTCTCTCGCTCAG eekddddddddddkke 76 268031 1379 541280 373 Exon 4/Intron 3 ATGAAAAAGTCTCTCG eekddddddddddkke 66 268036 13 80 541283 445 6X931120:1212" 3 TCCTTCTGGTATAGAA eekddddddddddkke 37 n/a 1381 54128 8 554 Exon 5 CAATAAGGTATCCAGA eekddddddddddkke 49 2741 14 13 82 541289 561 Exon 5 CTTGATACAATAAGGT eekddddddddddkke 66 274121 13 83 541290 569 Exon 5 CTAGTTAGCTTGATAC eekddddddddddkke 61 274129 13 84 541293 628 "93113033" 4 GATCTGGTTGCACTAT eekddddddddddkke 57 n/a 1385 541294 639 Exon 6 GGCAATGGGTGGATCT eekddddddddddkke 3 8 278933 13 86 541295 648 Exon 6 CCAGTTGAGGGCAATG dddddddkke 67 278942 13 87 541296 654 Exon 6 TAAAGTCCAGTTGAGG eekddddddddddkke 43 278948 13 88 541301 924 Exon 7 TACATAGAGCACCTCA eekddddddddddkke 86 290422 13 89 541302 927 Exon 7 TGTTACATAGAGCACC eekddddddddddkke 7 8 290425 1390 541303 930 Exon 7 AAGTGTTACATAGAGC eekddddddddddkke 5 9 290428 1391 541304 95 8 Exon 7 CTTCACATGTAAATTG eekddddddddddkke 26 290456 1392 541305 981 Exon 8 GAGCCATGGAAAGTAG eekddddddddddkke 66 292535 1393 541310 1127 $931123?" 8 CCTTCCTTGAGGAGAT eekddddddddddkke 26 n/a 1394 541320 1317 Exon 10 CTTCACCCCTAGGTTA eekddddddddddkke 3 8 297734 1395 541321 1322 Exon 10 CCATCCTTCACCCCTA eekddddddddddkke 81 297739 1396 541322 1326 Exon 10 GTCGCCATCCTTCACC eekddddddddddkke 79 297743 1397 541323 13 31 Exon 10 CCAGAGTCGCCATCCT dddddddkke 64 297748 1398 541325 1420 Exon 10 GTGGCTGAGCAACCTC eekddddddddddkke 79 297837 1399 541326 1434 Exon 10 CCCTTTTAACCTCTGT eekddddddddddkke 67 297851 1400 54133 1 1492 Exon 10 CATCATGATAAGGTGA eekddddddddddkke 16 297909 1401 541332 1526 Exon 10 TGGATAACACTGGGCT eekddddddddddkke 3 0 297943 1402 54133 3 15 32 Exon 10 TCTGCTTGGATAACAC eekddddddddddkke 63 297949 1403 54133 5 15 97 Exon 10 GAATATGGGCAGCTTG eekddddddddddkke 3 3 298014 1404 54133 6 1601 Exon 10 AGCTGAATATGGGCAG eekddddddddddkke 34 298018 1405 54133 7 1607 Exon 10 TTGCTTAGCTGAATAT eekddddddddddkke 3 9 298024 1406 54133 8 161 1 Exon 10 TGGATTGCTTAGCTGA dddddddkke 79 298028 1407 54133 9 1614 Exon 10 ACTTGGATTGCTTAGC eekddddddddddkke 73 298031 1408 Example 120: Antisense inhibition of human growth hormone receptor in Hep3B cells by dequ, MOE and (S)-cEt gapmers Additional nse 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 s for each experiment are presented in separate tables shown below. ed Hep3B cells at a density of 20,000 cells per well were transfected using electroporation with 4,5 00 nM antisense oligonucleotide. After a treatment period of imately 24 hours, RNA was ed 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 t, 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 (S)—cEt gapmers. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)—cEt sugar modification, or a deoxy cation. The ‘Chemistry’ column bes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modi?cation; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) es. A11 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 175 do not target SEQ ID NOs: 1 or 2, but d 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 173 Inhibition of GHR mRNA by deoxy, MOE and (S)—cEt gapmers targeting intronic and exonic regions of SEQ ID NO: 1 and 2 SE? SE)Q 11328 N? '. Eggs; 0 Sequence Chemistry inhilfition NO: 2 SENQOID Start 882:1: 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 86 156891 1370 541340 1619 Exon 10 AGTGAACTTGGATTGC eekddddddddddkke 73 298036 1409 541341 1641 Exon 10 AAAGTCGATG 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 AGGAGCCACA eekddddddddddkke 54 298244 1414 541346 1830 Exon 10 GTGAGGAGCC dddddddkke 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 dddddddkke 41 298366 1420 541352 1965 Exon 10 GTCTGGGACA 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 GTACACAGTG 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 dddddddkke 52 299282 1441 541386 2941 Exon 10 GTTGATCTGTGCAAAC dddddddkke 74 29935 8 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 33 05 Exon 10 GGTTATAGGCTGTGAA eekddddddddddkke 0 299722 1446 541396 33 08 Exon 10 TCTGGTTATAGGCTGT eekddddddddddkke 88 299725 1447 541397 331 1 Exon 10 GTGTCTGGTTATAGGC eekddddddddddkke 56 29972 8 1448 54139 8 33 16 Exon 10 TGTCTGGTTA eekddddddddddkke 76 29973 3 1449 541399 33 71 Exon 10 GGGACTGAAAACCTTG eekddddddddddkke 50 29978 8 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 174 Inhibition of GHR mRNA by deoxy, MOE and (S)—cEt gapmers ing intronic and exonic s of SEQ ID NO: 1 and 2 SEDQ SEQ ISIS NO: Target Region Sequence Chemistry . .% . NE): 2 811%? NO 1 inhibition Start NO St?rt Site 541262 n/a Intron 2 TGTCAATCCT eekddddddddddkke 85 156891 13 70 541421 4418 Exon 10 CACAACTAGTCATACT eekddddddddddkke 42 30083 5 1456 541422 4428 Exon 10 AACTGCCAGACACAAC eekddddddddddkke 68 300845 1457 541423 4431 Exon 10 ATAAACTGCCAGACAC eekddddddddddkke 86 300848 145 8 541424 4503 Exon 10 TATCAGGAATCCAAGA eekddddddddddkke 1 1 300920 1459 541425 4521 Exon 10 TTGATAACAGAAGCAC eekddddddddddkke 16 30093 8 1460 541426 4528 Exon 10 TTGGTGTTTGATAACA eekddddddddddkke 31 300945 1461 541427 4531 Exon 10 ATGTTGGTGTTTGATA eekddddddddddkke 32 300948 1462 541429 30 Exon 1 CCGCCACTGTAGCAGC eekddddddddddkke 77 2906 1463 541430 35 Exon 1 CGCCACTGTA eekddddddddddkke 88 291 1 1464 541431 63 Exon 1 GCCGCCCGGGCTCAGC dddddddkke 86 2939 1465 541432 67 Exon 1 CGCCGCCGCCCGGGCT eekddddddddddkke 61 2943 1466 541433 144 Exon 1 GAGAGCGCGGGTTCGC eekddddddddddkke 57 3020 1467 541434 n/a Exon 1/Intron 1 CTACTGACCCCAGTTC eekddddddddddkke 80 3655 1468 541435 n/a Exon 1/Intron 1 TCACTCTACTGACCCC eekddddddddddkke 90 3660 1469 541436 n/a Exon 1/Intron 1 TCATGCGGACTGGTGG eekddddddddddkke 56 3679 1470 541437 n/a Exon 3/Intron 3 ATGTGAGCATGGACCC eekddddddddddkke 82 22543 8 1471 541438 n/a Exon 3/Intron 3 TCTTGATATGTGAGCA eekddddddddddkke 93 225445 1472 541439 n/a Ex0n 3/Intr0n 3 TTGGTGAGCT eekddddddddddkke 72 22678 8 1473 541440 n/a Exon 3/Intr0n 3 TGCTTCCTTCAAGTTG eekddddddddddkke 68 226795 1474 541441 n/a Ex0n 3/Intr0n 3 TGTAATTTCATTCATG eekddddddddddkke 62 226809 1475 541442 n/a Ex0n 3/Intr0n 3 CCTTTTGCCAAGAGCA eekddddddddddkke 85 226876 1476 541443 n/a Ex0n 3/Intr0n 3 GATCCTTTTGCCAAGA eekddddddddddkke 77 226879 1477 541444 n/a Ex0n 3/Intr0n 3 GCTAGTAATGTTACAT eekddddddddddkke 68 23 833 1 1478 541445 n/a Ex0n 3/Intr0n 3 GCAACTTGCTAGTAAT eekddddddddddkke 65 23 833 8 1479 541446 n/a Ex0n 3/Intr0n 3 TGTGCAACTTGCTAGT eekddddddddddkke 44 23 8341 1480 541447 n/a Ex0n 3/Intr0n 3 GGATTTCAGTTTGAAT eekddddddddddkke 0 23 8 3 63 1481 541448 n/a Ex0n 3/Intr0n 3 CTCAGAGCCTTGGTAG eekddddddddddkke 65 23 842 8 1482 541449 n/a Ex0n 0n 1 CAAACGCGCAAAAGAC dddddddkke 1 3608 1483 541450 n/a Exon 1/Intr0n 1 GCCCGCACAAACGCGC dddddddkke 1 1 3615 1484 54145 1 n/a Ex0n 1/Intr0n 1 GGTTAAAGAAGTTGCT eekddddddddddkke 60 93 190 1485 541452 n/a Ex0n 1/Intr0n 1 CCCAGTGAATTCAGCA eekddddddddddkke 85 93245 1486 541453 n/a Exon 1/Intr0n 1 GCGCCCAGTGAATTCA eekddddddddddkke 74 93248 1487 541454 n/a Ex0n 1/Intr0n 1 AAGATGCGCCCAGTGA eekddddddddddkke 71 93253 1488 541455 n/a Ex0n 1/Intr0n 1 TGTAAGATGCGCCCAG eekddddddddddkke 75 93256 1489 541456 n/a Ex0n 1/Intr0n 1 AATTACTTGTAAGATG dddddddkke 15 93263 1490 541457 n/a Ex0n 1/Intr0n 1 CCCAGAAGGCACTTGT eekddddddddddkke 61 93302 1491 54145 8 n/a Ex0n 1/Intr0n 1 TTGCAGAACAAATCTT eekddddddddddkke 3 933 33 1492 541459 n/a Ex0n 1/Intr0n 1 CATGGAAGATTTGCAG eekddddddddddkke 17 93343 1493 541460 n/a Ex0n 1/Intr0n 1 GGTCATGGAAGATTTG eekddddddddddkke 57 93346 1494 541461 n/a Ex0n 1/Intr0n 1 GACCTTGGTCATGGAA eekddddddddddkke 51 93352 1495 541462 n/a Ex0n 1/Intr0n 1 TGCCAATCCAAAGAGG eekddddddddddkke 34 93369 1496 541463 n/a Ex0n 1/Intr0n 1 GGGTCTGCCAATCCAA eekddddddddddkke 67 93374 1497 541464 n/a Exon 0n 1 GGTCTGCCAA eekddddddddddkke 82 933 79 1498 541465 n/a Ex0n 1/Intr0n 1 GAATTTATCT eekddddddddddkke 16 93408 1499 541466 n/a Ex0n 1/Intr0n 1 GGAGATCTCAACAAGG eekddddddddddkke 3 8 93428 1 5 00 541468 n/a Ex0n 1/Intr0n 1 TCGCCCATCACTCTTC eekddddddddddkke 43 93989 15 01 541469 n/a Ex0n 1/Intr0n 1 CCTGTCGCCCATCACT dddddddkke 61 93993 15 02 541470 n/a Exon 1/Intr0n 1 GTCGCCCATC eekddddddddddkke 70 93996 15 03 541471 n/a Exon 1/Intr0n 1 CCATCACCTGTCGCCC eekddddddddddkke 89 93999 1504 541472 n/a Ex0n 1/Intr0n 1 TCACCATCACCTGTCG eekddddddddddkke 72 94002 15 05 541473 n/a Ex0n 1/Intr0n 1 TAATAGTTGTCACCAT eekddddddddddkke 42 9401 1 15 06 541474 n/a Ex0n 1/Intr0n 1 TTCAGATCTTATTAAT eekddddddddddkke 0 94023 1 5 07 541475 n/a Ex0n 0n 1 TTGCAAATTCAGTCTG dddddddkke 32 94096 15 08 541477 n/a Ex0n 2/Intr0n 2 CGTTCTCTTGGAAGTA eekddddddddddkke 78 198766 15 09 541478 n/a Ex0n 2/Intr0n 2 TCTTGAATAAATTTCG eekddddddddddkke 25 198780 15 10 541479 n/a Ex0n 2/Intr0n 2 AAGCTCACTCTTCAAT eekddddddddddkke 60 198 810 15 1 1 541480 n/a Ex0n 2/Intr0n 2 TCCAAGCTCACTCTTC eekddddddddddkke 49 198 813 15 12 541481 n/a Exon 2/Intr0n 2 GCTCCTGCCACTCTGT eekddddddddddkke 75 198837 1513 541482 n/a Ex0n 2/Intr0n 2 ATGGGCAAAGGCATCT eekddddddddddkke 60 198 874 15 14 541483 n/a 5' UTR AGTCTTCCCGGCGAGG eekddddddddddkke 32 25 71 15 15 gplplg 541484 n/a CCGCCGCTCCCTAGCC eekddddddddddkke 73 2867 1516 541485 n/a Intron 1 GCCCGCAACTCCCTGC eekddddddddddkke 37 3341 1517 541486 n/a Intron 1 CGCCTCCCCAGGCGCA eekddddddddddkke 34 4024 1518 541487 n/a Intron 1 GAGTGTCTTCCCAGGC dddddddkke 86 4446 1519 541488 n/a Intron 1 CTGAAGACTCCTTGAA eekddddddddddkke 39 4721 1520 541489 n/a 111116111 GGCTAGCCAAGTTGGA eekddddddddddkke 54 5392 1521 541490 n/a Intron 1 TGACTCCAGTCTTACC eekddddddddddkke 76 5802 1522 541491 n/a Intron 1 TGTGGTCAGC eekddddddddddkke 91 6128 1523 541492 n/a Intron 1 GAAGTGGGTTTTTCCC eekddddddddddkke 86 6543 1524 541493 n/a Intron 1 GCCTTGGTTCAGGTGA eekddddddddddkke 79 6786 1525 Table 175 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting SEQ ID NO: 4 and 7 Sequence Chemistry inhibition —m-———- Table 176 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers ing intronic regions of SEQ ID NO: 2 SEQ SEQ ID ID SE 1:3 NO: 1 NO: 2 1:23:12 Sequence Chemistry inhi‘ffition IDQ Start Start NO Site Site 541262 156891 541277 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 80 13 70 541494 723 1 541509 Intron 1 GTCCAGGCAGAGTTGT eekddddddddddkke 30 1528 541495 7570 541510 Intron 1 AGCCAAATGTTGGTCA eekddddddddddkke 19 1529 541496 8395 54151 1 Intron 1 GAGGGCGAGTTTTTCC eekddddddddddkke 71 15 30 541497 915 3 541512 Intron 1 GTGGCATTGGCAAGCC eekddddddddddkke 81 15 31 541498 9554 541513 Intron 1 ACCCCACTGCACCAAG eekddddddddddkke 67 15 32 541499 993 1 541514 Intron 1 TCCAAGTACTTGCCAA eekddddddddddkke 83 15 33 541500 10549 541515 Intron 1 AGTGCCTGGCCTAAGG eekddddddddddkke 75 15 34 5415 01 1 1020 541516 Intron 1 GCGCTTCTTCCCTAGG eekddddddddddkke 71 15 35 541502 1 1793 541517 Intron 1 CATCTTGCCCAGGGAT eekddddddddddkke 84 15 36 5415 03 12214 541518 Intron 1 CCATCTTGCTCCAAGT dddddddkke 93 15 37 541504 12474 541519 Intron 1 CTTACATCCTGTAGGC eekddddddddddkke 71 15 3 8 541505 12905 541520 Intron 1 CGCCTCCTGGTCCTCA eekddddddddddkke 97 1539 541506 13400 541521 Intron 1 CCCTATGCACTACCTA eekddddddddddkke 49 1540 541507 13 717 541522 Intron 1 GAGGGACTGTGGTGCT eekddddddddddkke 65 1541 541508 14149 541523 Intron 1 TATGTGCCAG eekddddddddddkke 60 1542 541509 14540 541524 Intron 1 GCTCTCTCATCGCTGG eekddddddddddkke 90 1543 5415 10 15264 541525 Intron 1 CTCAAGGCTATGTGCC eekddddddddddkke 67 1544 541511 15849 541526 Intron 1 TCCACATCCCTCATGT eekddddddddddkke 68 1545 5415 12 1653 0 541527 Intron 1 AGGACTGAAGGCCCAT eekddddddddddkke 49 1546 5415 13 17377 541528 Intron 1 GTGCGACTTACCAGCT eekddddddddddkke 85 1547 5415 14 175 81 541529 Intron 1 TCGCTAAAGCCACACA eekddddddddddkke 89 1548 541515 17943 541530 Intron 1 GCTCTGGCTGATGGTC eekddddddddddkke 92 1549 5415 16 1835 3 541531 Intron 1 TTCCCATGAGGATTTC eekddddddddddkke 70 15 50 5415 17 1863 6 541532 Intron 1 TTGGGCTTAAGCACTA eekddddddddddkke 71 15 51 5415 18 1925 6 541533 Intron 1 ACCTAGTCCA eekddddddddddkke 71 15 52 5415 19 19 814 541534 Intron 1 CCTCTGGCCTACAACA eekddddddddddkke 64 15 5 3 541520 20365 541535 Intron 1 CATCAGCACC eekddddddddddkke 93 15 54 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 15 5 8 541525 29049 541 540 Intron 1 GGGATGTAGGTTTACC eekddddddddddkke 74 15 5 9 541526 29554 541541 Intron 1 GCAACCGATATCACAG eekddddddddddkke 60 15 60 541527 30245 541542 Intron 1 TGCCCTGGAACAAATT eekddddddddddkke 13 15 61 541528 3055 0 541543 Intron 1 AGTCTAGGAGTAGCTA dddddddkke 50 15 62 541529 30915 541544 Intron 1 GCTGTTGTCAAGAGAC eekddddddddddkke 55 15 63 5415 30 31468 541545 Intron 1 CACCTAGACACTCAGT eekddddddddddkke 47 15 64 5415 31 32366 541546 Intron 1 GTCAAGGGATCCCTGC eekddddddddddkke 34 15 65 541532 32897 541547 Intron 1 TCCCCCTGGCACTCCA eekddddddddddkke 79 1566 5415 33 33187 541548 Intron 1 GCCTGGTAACTCCATT eekddddddddddkke 56 15 67 541534 33 7 8 0 541549 Intron 1 GGGCTCACCAACTGTG eekddddddddddkke 39 15 68 5415 35 34407 541550 Intron 1 CCACAGGATCATATCA eekddddddddddkke 37 15 69 5415 36 34846 541551 Intron 1 CTCCAGCAGAAGTGTC eekddddddddddkke 10 15 70 541537 35669 541552 Intron 1 AGCCCAACTGTTGCCT eekddddddddddkke 79 1571 5415 3 8 36312 541553 Intron 1 TGCCAGGCAGTTGCCA eekddddddddddkke 75 15 72 5415 39 36812 541554 Intron 1 GCCAGTAAGCACCTTG eekddddddddddkke 93 15 73 541540 37504 541555 Intron 1 CTAGCTTCCCAGCCCC dddddddkke 46 1574 541541 3 8 841 541556 Intron 1 TCAAGCCCAGCTAGCA eekddddddddddkke 39 15 75 541542 39108 541557 Intron 1 CCTCACAGGCCCTAAT eekddddddddddkke 4 15 76 541543 39408 54155 8 Intron 1 ACCTGCTTACATGGTA eekddddddddddkke 21 15 77 541544 4025 0 541559 Intron 1 CCTTTGCTAGGACCCA eekddddddddddkke 52 15 7 8 541545 40706 541560 Intron 1 GGGACTGCCACCAAGG eekddddddddddkke 27 15 79 541546 40922 541561 Intron 1 TGTTCAGGCC eekddddddddddkke 34 15 80 541547 41424 541562 Intron 1 CCTATGGCCATGCTGA eekddddddddddkke 32 15 81 541548 41999 541563 Intron 1 GTATGCTAGTTCCCAT eekddddddddddkke 83 15 82 541549 42481 541564 Intron 1 CCCTCATAATCTTGGG eekddddddddddkke 13 15 83 5415 50 42700 541565 Intron 1 CCACTACCAC dddddddkke 74 15 84 5415 51 43291 541566 Intron 1 AGATAGCTGA eekddddddddddkke 73 15 85 541552 43500 541567 Intron 1 GCATGACCCCACTGCC eekddddddddddkke 72 1586 5415 53 43947 541568 Intron 1 GAGGGTCACATTCCCT eekddddddddddkke 23 15 87 541554 44448 541569 Intron 1 TCTCTTACTGGTGGGT eekddddddddddkke 90 15 8 8 541555 45162 541570 Intron 1 GCCCCCTTCCTGGATA dddddddkke 28 1589 5415 56 46010 541571 Intron 1 CCTCATGCGACACCAC eekddddddddddkke 71 15 90 541557 46476 541572 Intron 1 AGCCCTCTGCCTGTAA eekddddddddddkke 67 15 91 5415 5 8 47447 541573 Intron 1 CTCCCAGCTATAGGCG eekddddddddddkke 3 8 15 92 5415 59 47752 541574 Intron 1 GCTAGCTGCGCAAGGA eekddddddddddkke 5 15 93 541560 48001 541575 Intron 1 GCGCAGCCCGCTGCAA eekddddddddddkke 18 1594 5415 61 48423 541576 Intron 1 TGCATGATCCACCCCA eekddddddddddkke 65 15 95 541562 50195 541577 Intron 1 GCTTAGTGCTGGCCCA eekddddddddddkke 72 15 96 5415 63 50470 541578 Intron 1 CCTTCCAGTCCTCATA eekddddddddddkke 81 15 97 541564 51 104 541579 Intron 1 ATAGTGTCAAGGCCCA eekddddddddddkke 91 15 9 8 5415 65 5175 6 5415 80 Intron 1 TAGTCACCCA dddddddkke 88 15 99 541566 52015 5415 81 Intron 1 TAACCAACCTAAGGGA eekddddddddddkke 1 1 1600 541567 5223 0 541582 Intron 1 ATTCTGGTGATGCCCT eekddddddddddkke 66 1601 541568 525 8 8 5415 83 Intron 1 ACTGCCATGA eekddddddddddkke 67 1602 541569 53 5 3 2 5415 84 Intron 1 GGTAGAGCACACTGCC eekddddddddddkke 47 1603 541570 54645 5415 85 Intron 1 CCACTTTAATGCCACC eekddddddddddkke 76 1604 Table 177 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers targeting intronic s of SEQ ID NO: 2 SEQ SEQ ID ID gigs NO: 2 NO: 2 Eggs; ce Chemistry inhi‘ffition SEIEOID Start Stop Site Site 541262 156891 15 6906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 88 1370 541571 54886 54901 Intron 1 GTCAAATGCTGTTGGG eekddddddddddkke 91 1605 541572 5 5900 5 5915 Intron 1 CATCCCCTATCAGGGT eekddddddddddkke 53 1606 541573 62266 622 81 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 GCCTCAGTGG eekddddddddddkke 81 1610 541577 63964 63979 Intron 1 CAGAAGGACTGCCTCT eekddddddddddkke 50 161 1 541578 64213 64228 Intron 1 ACAATGCTCAACAGCC eekddddddddddkke 75 1612 541579 64576 645 91 Intron 1 TCTGGCATGC eekddddddddddkke 80 1613 541580 65027 65042 Intron 1 CGGCTGAGAGCAAGGG eekddddddddddkke 88 1614 5415 81 653 63 653 78 Intron 1 GAGAGGGTTCAGCCTG eekddddddddddkke 62 1615 541582 65600 65615 Intron 1 ACTTAGTTCCTAGCCA eekddddddddddkke 91 1616 5415 83 66087 66102 Intron 1 GTGAACCAGATGTGCT eekddddddddddkke 86 1617 541 5 84 66566 665 81 Intron 1 GGAGTGACAGCTAAGT eekddddddddddkke 98 1 61 8 5415 85 66978 66993 Intron 1 AAGTGTTCAGAGCCAC eekddddddddddkke 97 1619 541586 67662 67677 Intron 1 AACCCTGCCAAGGTAC eekddddddddddkke 45 1620 5415 87 67914 67929 Intron 1 GAGCACTACC eekddddddddddkke 78 1621 541 5 88 68278 68293 Intron 1 GGCAGGATAGGACAGA dddddddkke 1 1 1 622 541589 68727 68742 Intron 1 GCAAAGTGATGAGCCT dddddddkke 81 1623 541590 69207 69222 Intron 1 CTATCCACACCATTCC eekddddddddddkke 93 1624 541591 69605 69620 Intron 1 TGGGCCCCTA eekddddddddddkke 70 1625 541592 70130 70145 Intron 1 GTGAATTTGCTGGGCC eekddddddddddkke 94 1626 541593 705 69 705 84 Intron 1 GTGATGGGCCCAAGGC eekddddddddddkke 67 1627 541594 71056 71071 Intron 1 TCCTCAGTCGGCTTGC eekddddddddddkke 69 162 8 541595 713 14 71329 Intron 1 CAGCCTTTTGCCAGAT eekddddddddddkke 93 1629 541596 71620 71635 Intron 1 CCTCCCTAGGATTACC eekddddddddddkke 42 163 0 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 83 163 3 541600 73708 73723 Intron 1 GATGCCTGTGGCTGAC eekddddddddddkke 84 1634 541601 74107 74122 Intron 1 GGCTAGCATGTTGCCT eekddddddddddkke 19 163 5 541602 74542 745 57 Intron 1 TAACCCACTAGGCTGG eekddddddddddkke 84 163 6 541603 74947 74962 Intron 1 TGGCCCAAAACTAATC eekddddddddddkke 34 163 7 541604 75192 75207 Intron 1 GGAGCAGTCTGGCACC eekddddddddddkke 85 163 8 541605 75699 75714 Intron 1 TATTCTGTGGGACAAG eekddddddddddkke 51 163 9 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 773 07 Intron 1 GCTCTAGGCATATGAA eekddddddddddkke 63 1644 54161 1 775 55 77570 Intron 1 TACCTCCCTTGTAGGG eekddddddddddkke 27 1645 541612 77854 77869 Intron 1 GGTTCCCTTGCAGAGA eekddddddddddkke 62 1646 541613 7 8311 7 8326 Intron 1 GTGCCCTCTTCATGCC eekddddddddddkke 68 1647 541614 79006 79021 Intron 1 CCTGTGTGCAACTGGC eekddddddddddkke 85 1648 541615 79490 795 05 Intron 1 CTGAGTCATTTGCCTG eekddddddddddkke 93 1649 541616 79829 79844 Intron 1 GGCCTTAGTAGGCCAG eekddddddddddkke 0 165 0 541617 80277 80292 Intron 1 GTCCTTGCAGTCAACC eekddddddddddkke 77 165 1 541618 805 75 80590 Intron 1 CCAAGTCCAT dddddddkke 77 1652 541619 80895 80910 Intron 1 TAGGGCACTTTTTGCC eekddddddddddkke 31 165 3 541620 81207 81222 Intron 1 GCTGAGGTCCCTCTCT eekddddddddddkke 34 1654 541621 81761 81776 Intron 1 CTTTGGTCCCATTGCC eekddddddddddkke 83 165 5 541622 82233 82248 Intron 1 GGAACATGCCAAGGGC eekddddddddddkke 91 165 6 541623 8273 8 82753 Intron 1 AGGTGGTCTCCCTTCA dddddddkke 74 165 7 541624 83056 83071 Intron 1 TCCCAAAGCTCCCCTC eekddddddddddkke 53 165 8 541625 83401 83416 Intron 1 CCTGGCCTAGCAAGCT dddddddkke 47 165 9 541626 84048 84063 Intron 1 TCTTAGCCCTGGGCTA eekddddddddddkke 12 1660 541627 843 88 84403 Intron 1 GACTTGGACTGGGCTC eekddddddddddkke 81 1661 541 628 85261 85276 Intron 1 GGCCTAGGATCTAGGA eekddddddddddkke 0 1 662 541629 85714 85729 Intron 1 GTCAGGCTAGAGGGAC dddddddkke 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 TGTCAGATGC eekddddddddddkke 79 1667 541634 8 8293 8 83 08 Intron 1 TCTCAGGTGTAGGCAG eekddddddddddkke 59 1668 541635 8 8605 8 8620 Intron 1 GGTCACTGAGACTGGG dddddddkke 88 1669 541636 8 8952 8 8967 Intron 1 ACCCACTAGCAGCTAG eekddddddddddkke 61 1670 541 637 891 60 891 75 Intron 1 CGGATGAGGCAGTTAG eekddddddddddkke 42 1 67 1 54163 8 89855 89870 Intron 1 TGGTAGGCCCTCTGGC eekddddddddddkke 28 1672 541639 90240 90255 Intron 1 GTCACAAGGTGGGTGC eekddddddddddkke 28 1673 541640 905 13 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 dddddddkke 68 1677 541 644 92356 923 71 Intron 1 AGAGGGCACT eekddddddddddkke 75 1 67 8 541645 92904 92919 Intron 1 GTTTTAACCTCACCCT eekddddddddddkke 0 1679 541646 93846 93 861 Intron 1 CCTTCCACTGACCTTC eekddddddddddkke 56 1680 541647 94374 943 89 Intron 1 GACACTAGCCTAAGCC eekddddddddddkke 37 1681 Table 178 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt gapmers ing 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 13 70 541648 9463 8 94653 Intron 1 GGTTAGCCCTCAGCCT eekddddddddddkke 61 1682 541649 9483 9 94854 Intron 1 TATGAAGGTTGGACCA eekddddddddddkke 69 1683 54165 0 95 509 95524 Intron 1 CAACCAGCTCACCTGA eekddddddddddkke 37 1684 54165 1 95 829 95844 Intron 1 GGGCTCCAAGGCTCTC dddddddkke 75 1685 541652 9615 8 96173 Intron 1 AGCTGTTACATGCCAA eekddddddddddkke 93 1686 54165 3 9648 8 965 03 Intron 1 GGCCCAGAGGTTATAG eekddddddddddkke 30 1687 541654 96991 97006 Intron 1 GTCCTTAGACCCCTCA eekddddddddddkke 70 1688 54165 5 9753 9 97554 Intron 1 GCCCTGGCTAGAGACA eekddddddddddkke 39 1689 54165 6 98132 9 8147 Intron 1 CATCCAGCAGCTGGAC eekddddddddddkke 35 1690 54165 7 98 83 3 9 8848 Intron 1 GACTGAGGTCATCACA eekddddddddddkke 60 1691 54165 8 9925 8 99273 Intron 1 GGCCAGGCACATCATG eekddddddddddkke 45 1692 54165 9 99 843 9985 8 Intron 1 GGAGCTCATTGAGCCA eekddddddddddkke 36 1693 541660 100406 100421 Intron 1 GTGCCCATTGCTGTGT eekddddddddddkke 70 1694 541661 100742 10075 7 Intron 1 CCAAGTGTGGCTTCAG eekddddddddddkke 54 1695 541662 1013 05 101320 Intron 1 CCACCCTTTATACGCA eekddddddddddkke 87 1696 541663 1017 88 101 803 Intron 1 CAGTAACCCCAAGGGA eekddddddddddkke 12 1697 541664 102649 102664 Intron 1 CCCCACCTTATATGGG eekddddddddddkke 9 1698 541665 103034 103 049 Intron 1 AGGCCCTTTTTACATG eekddddddddddkke 9 1699 541666 1033 16 103 33 1 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 10878 8 Intron 1 GTGACCCACCCAAGAG eekddddddddddkke 34 1707 541674 109336 10935 1 Intron 1 GTTGTAGCTAGTGTTC eekddddddddddkke 74 1708 541675 109849 109 864 Intron 1 GCCTTAGTTTGTGCCA eekddddddddddkke 78 1709 541676 1 10427 1 10442 Intron 1 GCCCCAGCTGAGAATT eekddddddddddkke 29 17 10 541677 1 10701 1 10716 Intron 1 ACAACAATCCAGGGTG eekddddddddddkke 61 17 1 1 54167 8 1 10959 1 10974 Intron 1 CTCCCCTGGAAGTCAC eekddddddddddkke 59 1712 541679 1 1 13 07 1 1 1322 Intron 1 GCCCTCATGGCTCAAG eekddddddddddkke 60 1713 541680 1 12499 1 12514 Intron 1 TCAGCAGATAGGGAGC eekddddddddddkke 61 1714 541681 1 13 896 1 13 91 1 Intron 1 GAATGCGGTGATCAGG dddddddkke 29 17 15 541682 1 17477 1 17492 Intron 1 CTGAGAGAATTGGCCC eekddddddddddkke 5 1716 541683 1 17740 1 1775 5 Intron 1 AGGCACATTGTTACCA eekddddddddddkke 26 17 17 541684 1 18229 1 18244 Intron 1 GGGAGGCACTAGAGAA eekddddddddddkke 13 17 18 541685 1 19269 1 19284 Intron 1 TACAGTAACACATCCC eekddddddddddkke 78 1719 541686 1 19688 1 19703 Intron 1 GAAGCTCAGCCTGATC eekddddddddddkke 45 1720 541687 120376 120391 Intron 1 CTTGCCTGACAACCTA dddddddkke 53 1721 541688 120738 120753 Intron 1 GCCTACCTGCTTTTGC eekddddddddddkke 10 1722 541689 121242 12125 7 Intron 1 AACCACTTAG eekddddddddddkke 7 1723 541690 121615 12163 0 Intron 1 TCTCCTATTTCAGTTA eekddddddddddkke 23 1724 541691 121823 121 83 8 Intron 1 GGGTGATGGATGAACT eekddddddddddkke 40 1725 541692 122345 122360 Intron 1 ACACTGCTGGTAGTGA dddddddkke 0 1726 541693 1225 88 122603 Intron 1 ACCCAACTAGCCTGTC eekddddddddddkke 35 1727 541694 123152 123167 Intron 1 TGCTGCCTGA eekddddddddddkke 80 1728 541695 123671 123 686 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 125 047 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 dddddddkke 79 1736 541703 127179 127194 Intron 1 GTCTCATGAGGCCTAT eekddddddddddkke 60 1737 541704 127454 127469 Intron 1 GGAGGTGGGATCCCAC dddddddkke 35 173 8 541705 128467 128482 Intron 1 GCCCACTACCTAGCAC dddddddkke 30 1739 541706 129096 1291 1 1 Intron 1 CCCAGCTGGCTGGTCG eekddddddddddkke 50 1740 541707 129312 129327 Intron 1 GCACCAGGTCTCCTGT eekddddddddddkke 7 1741 541708 1295 16 12953 1 Intron 1 GTCTAGAAGCCTAGGG eekddddddddddkke 23 1742 541709 129976 129991 Intron 1 GCCGGGTGTTGGTGCA eekddddddddddkke 50 1743 541710 1303 08 130323 Intron 1 CCTGTGTTGC eekddddddddddkke 49 1744 54171 1 130767 130782 Intron 1 TGCTTCTGATCCCTAC eekddddddddddkke 18 1745 541712 1312 86 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 133 53 7 Intron 1 TAGGGTGTTCCAGATC eekddddddddddkke 44 1751 54171 8 133724 133 73 9 Intron 1 GTACCATGAAGCTCTG eekddddddddddkke 67 1752 541719 134086 134101 Intron 1 CTTGGACTTGGACCAT eekddddddddddkke 42 1753 541720 134441 13445 6 Intron 1 GTGCATAGGGCCTGTC eekddddddddddkke 42 1754 541721 135015 135 03 0 Intron 1 CCTCACCTGAACACCC eekddddddddddkke 23 1755 541722 135859 135 874 Intron 1 CCCCGCAACT eekddddddddddkke 27 1756 541723 1362 87 136302 Intron 1 TTGTGCTTGGGTGTAC eekddddddddddkke 39 1757 541724 137000 137015 Intron 1 AGGCTTCATGTGAGGT eekddddddddddkke 86 175 8 Table 179 Inhibition of GHR mRNA by deoxy, MOE and (S)-cEt s targeting s 1 and 2 of SEQ ID NO: 2 SEQ SEQ 11328 135353. $213. E2333 0 Seeeeeee Chemistry Miami 81% Site Site 541262 15 6891 15 6906 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 TAGGACTGCT eekddddddddddkke 73 1762 541729 140343 1403 5 8 Intron 1 CAGACTAGGG dddddddkke 5 3 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 1431 18 143133 Intron 1 GGCCTTGAGCTGATGG eekddddddddddkke 1 1 1769 541736 143836 143851 Intron 1 TATCCCTAATCAGGCT dddddddkke 40 1770 541737 144094 144109 Intron 1 GGTGTCCACATCCCGG eekddddddddddkke 5 8 1771 541738 144558 144573 Intron 1 AGCTGGACAGGCCATA eekddddddddddkke 27 1772 541740 1455 10 145525 Intron 2 GGTAATCACCCAGAGA eekddddddddddkke 90 1773 541741 145937 145952 Intron 2 GCGCTAAGTCTGCTGT eekddddddddddkke 92 1774 541742 146320 1463 35 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 93 177 8 541746 148139 148154 Intron 2 AGCTGAGGTATCCCTC eekddddddddddkke 94 1779 541747 1485 64 1485 79 Intron 2 GGGCCCAGCAAGTCTT eekddddddddddkke 3 3 1780 541748 149069 149084 Intron 2 GTTTTGTCAGTGTGCA eekddddddddddkke 9 8 1781 541749 149491 149506 Intron 2 GTGACCTGCTGAACTC eekddddddddddkke 95 1782 541750 15 02 36 15 0251 Intron 2 GGCTGAACTGTGCACC eekddddddddddkke 95 1783 541751 15 0748 15 0763 Intron 2 GGGTGGTCCCACTCCT eekddddddddddkke 91 1784 541752 15 1 124 15 1 139 Intron 2 GAGGAATCCTGGGCCC eekddddddddddkke 94 1785 541753 15 13 73 15 13 88 Intron 2 ATGACAAGCTAGGTGC eekddddddddddkke 81 1786 541754 15 1644 15 1659 Intron 2 TTGCCAGACAGGGCAC dddddddkke 1 8 1787 541755 152373 152388 Intron 2 CTCCCACTAT dddddddkke 43 1788 541756 152617 152632 Intron 2 GGTGCTGGGTGACCGG dddddddkke 91 1789 541757 15 3349 15 33 64 Intron 2 ACGGTGCCCT eekddddddddddkke 23 1790 54175 8 15 3918 15 3 9 3 3 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 15 52 89 15 5 3 04 Intron 2 CTGTCAGTGCTTTGGT eekddddddddddkke 52 1794 541762 15 62 3 3 15 6248 Intron 2 GAGTACCCTGGCAGGT eekddddddddddkke 5 8 1795 541763 15 6847 15 6862 Intron 2 TAGCTAGCACCTGGGT eekddddddddddkke 90 1796 541764 15 75 52 15 75 67 Intron 2 GGCAAACCTTTGAGCC eekddddddddddkke 27 1797 541765 15 7927 15 7942 Intron 2 GCTATCATTGGAGCAG eekddddddddddkke 94 179 8 541766 15 8542 15 85 5 7 Intron 2 CCTCTGAGTACTCCCT eekddddddddddkke 96 1799 541767 15 9252 15 9267 Intron 2 AGCTGAAGGCAACCAG eekddddddddddkke 97 1 800 541768 15 95 3 9 15 95 54 Intron 2 GGGCAGTTTTCCATAG eekddddddddddkke 89 1 801 541769 15 9778 15 9793 Intron 2 GGTCCTACCTCTGACA eekddddddddddkke 82 1802 541770 1603 52 1603 67 Intron 2 GGCTGCCTTAGGGTGG dddddddkke 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 3 3 1807 541775 162639 162654 Intron 2 GGGACCTGCGCTGACT eekddddddddddkke 86 1 80 8 541776 163024 163039 Intron 2 CTTTCACCTGGTGACT eekddddddddddkke 83 1809 541777 163542 1635 57 Intron 2 AGCTTGAGGGAGTATA dddddddkke 52 1 81 0 541778 164144 164159 Intron 2 GCCTGCTCAATTGAGG eekddddddddddkke 32 1 81 1 541779 164570 1645 85 Intron 2 ATAGCAGCTGGCTGCC eekddddddddddkke 24 1 812 541780 165419 165434 Intron 2 AAAAGCTTGGCACCCC dddddddkke 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 5417 85 168004 168019 Intron 2 TTACTGTGGCCCGAGT eekddddddddddkke 94 1 81 8 541786 169062 169077 Intron 2 GTAGACTCCTAGGGTC eekddddddddddkke 90 1 819 541787 169696 16971 1 Intron 2 CCTCCAGTTAGTGTGC eekddddddddddkke 91 1 820 541788 170081 170096 Intron 2 GTGGGTGGCCAACAGG eekddddddddddkke 91 1 821 541789 170799 170814 Intron 2 GGGATTCCCTGGTAGC eekddddddddddkke 77 1 822 541790 171021 171036 Intron 2 GTGAGACCGGCCTTTG eekddddddddddkke 23 1 823 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 1 826 541794 173045 173060 Intron 2 GGTGGCAGCTAGGTGG eekddddddddddkke 80 1 827 541795 173677 173692 Intron 2 TCCACAGTTGGCACTG eekddddddddddkke 77 1 82 8 541796 174128 174143 Intron 2 TGGGCCTTAGATTGTA eekddddddddddkke 69 1 829 541797 174521 174536 Intron 2 CCTGGTGGCC eekddddddddddkke 97 183 0 541798 174870 174885 Intron 2 CCCGCCTCTCCAGCAA eekddddddddddkke 89 1831 541799 175275 175290 Intron 2 GCAGCAGCCAATAAGT eekddddddddddkke 76 1 832 541800 175691 175706 Intron 2 CCTGGCCCCT eekddddddddddkke 80 183 3 541801 17603 8 176053 Intron 2 GCCTCATGGGCCTTAC dddddddkke 66 1834 Table 180 Inhibition of GHR mRNA by deoxy, MOE and t gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ SEQ ID ISIS ID NO: NO: 2 Target Sequence Chemistry .% ID NO 2 Start Stop Region inhibition. .

Site Site 541262 15 6891 156906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 97 1370 541802 176619 176634 Intron 2 GGATGCCAGTCTTGGC eekddddddddddkke 48 1835 541803 17683 5 176850 Intron 2 TCAGTACCTC eekddddddddddkke 87 1836 541804 177300 177315 Intron 2 ACCCAAGAAGTCACCT eekddddddddddkke 93 1837 541805 177551 177566 Intron 2 GCCTCAAGCCCTACCC eekddddddddddkke 73 1838 541806 17 8066 178081 Intron 2 AGCTCCAGCCTATAGA eekddddddddddkke 81 1839 541807 17 8361 178376 Intron 2 GGTCCACATGGCCCTA eekddddddddddkke 90 1840 541808 17 8895 178910 Intron 2 CAGGCCCAGGATTGTC eekddddddddddkke 81 1 841 541809 179444 179459 Intron 2 GGGCCTGCTTTGCAGC eekddddddddddkke 81 1842 541810 179863 179 878 Intron 2 ACTCCTCTCTTTAGGC eekddddddddddkke 87 1843 54181 1 180524 180539 Intron 2 CTGGGTAACAGTCCTC eekddddddddddkke 98 1844 541812 18152 8 181543 Intron 2 ACTGTATGGTTTCCAC eekddddddddddkke 83 1845 541813 182103 1 821 18 Intron 2 GCCAAAGATAGCTCTT eekddddddddddkke 94 1 846 541814 18297 8 1 82993 Intron 2 GGCATTGGAAGTTGGT eekddddddddddkke 87 1 847 541815 183193 183208 Intron 2 CCCTTCCTGACCTTAC eekddddddddddkke 55 1848 541816 18365 8 183 673 Intron 2 TTACCCTCTATTCACC eekddddddddddkke 65 1849 541818 184501 184516 Intron 2 GGCACCCCAGGCCGGG dddddddkke 25 1850 541819 185080 185 095 Intron 2 CAGCAGCTAGTTCCCC dddddddkke 96 1851 541820 185327 1 85 342 Intron 2 GTGGGCACTAGTGTGT eekddddddddddkke 75 1 852 541821 185682 185 697 Intron 2 TGCCCTTGTCAGGGCA eekddddddddddkke 20 1853 541822 186025 1 86040 Intron 2 GCAGATAGGCTCAGCA eekddddddddddkke 98 1 854 541823 186570 1 865 85 Intron 2 CCCTAGCCCTTAGCAC eekddddddddddkke 44 1 85 5 541824 186841 186856 Intron 2 ACTGGAATGGCCCTCT eekddddddddddkke 86 1856 541825 187176 187191 Intron 2 CATGCTCACA eekddddddddddkke 96 1857 541826 187629 1 87644 Intron 2 GTGTGTCACT eekddddddddddkke 99 1 85 8 541827 18785 7 1 87 872 Intron 2 TATGTGGTAGCATGTC dddddddkke 96 1 859 54182 8 18 8442 188457 Intron 2 CCCCAGGAAGTTGGCC eekddddddddddkke 68 1860 541829 189086 189101 Intron 2 TAGCTGTCAAGGCCCT eekddddddddddkke 90 1861 54183 0 189534 1 89549 Intron 2 CCTAGTCAGCCACTAG eekddddddddddkke 20 1 862 541831 189889 189904 Intron 2 AGACTCCCCATCAGCC eekddddddddddkke 74 1863 541832 190172 190187 Intron 2 GTGAAGGGCCTTCATC dddddddkke 68 1864 54183 3 190961 190976 Intron 2 GAGTCCAATG dddddddkke 95 1 865 541834 191404 191419 Intron 2 CAGCTAATTCCCTCAT eekddddddddddkke 79 1866 54183 5 191614 191629 Intron 2 TTGTGTCTCAACCCAC eekddddddddddkke 95 1867 54183 6 191999 192014 Intron 2 GGCTATGCTGCATGCT eekddddddddddkke 91 1868 54183 7 192860 192 875 Intron 2 CCCCATACCCAGTGGA eekddddddddddkke 71 1869 54183 8 193460 193475 Intron 2 GGTGGTTTTCCTCCCT eekddddddddddkke 95 1870 54183 9 194144 194159 Intron 2 GAGCCTGCCCAACTTT eekddddddddddkke 90 1871 541840 194425 194440 Intron 2 TGATGCCCAAGAGTGA eekddddddddddkke 85 1 872 541841 19495 3 194968 Intron 2 TTCCCTCTGCGAACAT dddddddkke 96 1873 541842 19542 8 195443 Intron 2 GTTCCATCTCAATCCA eekddddddddddkke 94 1874 541843 19685 8 196873 Intron 2 ACGGCCACTCCACTGG eekddddddddddkke 44 1 875 541844 197326 197341 Intron 2 TGGAAGTGGTTCCAGA eekddddddddddkke 90 1 876 541845 197946 197961 Intron 2 TTGCCCCAGACCAACA eekddddddddddkke 47 1877 541846 19 8366 1983 81 Intron 2 GAGGTTGTGGAGGTGC eekddddddddddkke 26 1 87 8 541847 19 8715 198730 Intron 2 GAGTTGCTGTGTGTGA eekddddddddddkke 83 1879 541848 19 893 9 198954 Intron 2 CATGTCAGAGGTGTCC eekddddddddddkke 93 1 880 541849 199506 199521 Intron 2 AGGTAAGGATCATGGC dddddddkke 87 1881 54185 0 199816 199 831 Intron 2 GTTCAGTTGCATCACG eekddddddddddkke 90 1882 541851 200249 200264 Intron 2 GCCCAGCTAGCCACCC eekddddddddddkke 68 1883 541852 20125 8 201273 Intron 2 CCTTAGCAGCCAGGCC eekddddddddddkke 86 1884 54185 3 202079 202094 Intron 2 GCACTTAGGGTTTTGC eekddddddddddkke 94 1 885 541854 202382 202397 Intron 2 GTTGAACTTTCCCTAC eekddddddddddkke 53 1 886 54185 5 202702 202717 Intron 2 TGACTCCTTGAGACAG eekddddddddddkke 83 1 887 54185 6 20309 8 2031 13 Intron 2 TGCGCTGGCTTAGCAA eekddddddddddkke 59 1888 54185 7 203464 203479 Intron 2 GGCCTAACATCAGCAG eekddddddddddkke 88 1 889 54185 8 204212 204227 Intron 2 ACTCCTCCCAGTTAGC eekddddddddddkke 70 1890 54185 9 20563 0 205645 Intron 2 ACCAGTGGCCAATGTC eekddddddddddkke 92 1891 541861 206422 206437 Intron 2 GCCTAGACACAGTAGG eekddddddddddkke 70 1 892 541862 206749 206764 Intron 2 TATTCTCCCCCTAGGG eekddddddddddkke 42 1 893 207517 207532 541863 Intron 2 GACGGCCTTGGGCACA eekddddddddddkke 96 1894 210196 21021 1 541865 20865 9 208674 Intron 3 GCAGGCTGTATTAGCA eekddddddddddkke 15 1 895 541867 209999 210014 Intron 3 ACCCCCTATCCTGCAC eekddddddddddkke 58 1896 210281 210296 541868 Intron 3 TCCTCCATACCTAGAG eekddddddddddkke 61 1 897 21 103 3 21 1048 541869 210502 210517 Intron 3 GATAGGTGCCCACTGT eekddddddddddkke 80 1 898 541870 210920 210935 Intron 3 GTCAGTTCTGGCTAGG eekddddddddddkke 97 1 899 541871 21 1269 21 1284 Intron 3 GCCTGAACTTACAAGC eekddddddddddkke 68 1900 541872 21 183 6 211851 Intron 3 ACCCTGGGCTGACCTT eekddddddddddkke 92 1901 541873 212606 212621 Intron 3 GGACCTGGACAAGCAA eekddddddddddkke 97 1902 541874 213099 213 1 14 Intron 3 CTCCTTGCGAGAGAGG eekddddddddddkke 7 1903 541875 213425 213440 Intron 3 AGAGTTGACATGGGCA eekddddddddddkke 96 1904 541876 213846 213 861 Intron 3 CACTAGGTCCCTGACC eekddddddddddkke 37 1905 541877 214483 214498 Intron 3 CACTCTCTTGGGCTGT eekddddddddddkke 94 1906 54187 8 214884 214899 Intron 3 AGGGACCTGCATTCCA eekddddddddddkke 72 1907 Table 181 Inhibition of GHR mRNA by deoxy, VIOE and (S)-cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2 SEQ ID ISIS ID NO: Tar et % SE ID .

NO 82191816 2 581th Regzigon sequence Chemistry tion 15120 541262 156891 15 6906 Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 91 13 70 541879 215493 215508 Intron 3 TTCACCACCCATTGGG eekddddddddddkke 63 1908 541880 216192 216207 Intron 3 ATCTGGTCTGAGGGCC eekddddddddddkke 92 1909 541881 21645 8 216473 Intron 3 GACATGCAATTGACCC eekddddddddddkke 98 1910 541882 217580 217595 Intron 3 GTGTGCAGCAGACTGT eekddddddddddkke 92 191 1 541883 218233 218248 Intron 3 GACAGTCCAGCTGCAA eekddddddddddkke 84 1912 541884 218526 218541 Intron 3 GCAGTGAAGA eekddddddddddkke 85 1913 541885 218734 21 8749 Intron 3 CTCTGAGGATAACCCT dddddddkke 76 1914 541886 219342 219357 Intron 3 GTTCCCAGCTCCCCAA eekddddddddddkke 68 1915 541887 219618 219633 Intron 3 TAGGGTCAGTGTCCCA eekddddddddddkke 79 1916 54188 8 220039 220054 Intron 3 CCTCTCAGCC eekddddddddddkke 52 1917 541889 220393 220408 Intron 3 TCCAGGCAGT eekddddddddddkke 91 1918 541890 220665 220680 Intron 3 TCCCTCCCTTAGGCAC eekddddddddddkke 71 1919 541 89 1 221 044 22 1059 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 2225 84 Intron 3 GCCCACACCATTGAGC eekddddddddddkke 70 1923 541895 222983 222998 Intron 3 AGTGAGATGCCCTGGT eekddddddddddkke 92 1924 541896 223436 223451 Intron 3 CAGTTAGACC dddddddkke 88 1925 541897 224107 224122 Intron 3 ACTCTGGCCACTAGTA dddddddkke 80 1926 54189 8 224731 224746 Intron 3 GGTAGGGTGGCCACAT eekddddddddddkke 78 1927 541899 225 133 225148 Intron 3 GAGCCATGTCTAGGCA eekddddddddddkke 18 1928 541900 225465 225480 Intron 3 CAGACTGAAACCCACC eekddddddddddkke 86 1929 541901 225 671 225686 Intron 3 TATGGTCCAGCCACCA eekddddddddddkke 76 1930 541902 2261 10 226125 Intron 3 CTCTGTTGGT eekddddddddddkke 36 1931 541903 227025 227040 Intron 3 ACACCTCAGTCATGAT eekddddddddddkke 92 1932 541904 227236 227251 Intron 3 AACAGGCTTCAAGAGG eekddddddddddkke 91 1933 541905 227485 2275 00 Intron 3 GTACTACTGGCCATGT eekddddddddddkke 73 1934 541906 227914 227929 Intron 3 GCGGTTGCTA eekddddddddddkke 60 1935 541907 228718 228733 Intron 3 GTCTGTTGCCAAGAGC eekddddddddddkke 95 1936 541908 229174 2291 89 Intron 3 CCCTGGGTCACTTAAG eekddddddddddkke 44 1937 541909 229423 229438 Intron 3 CCTGTCCTTGCTTGCA eekddddddddddkke 96 1938 541910 230042 230057 Intron 3 GCCCAGCTTATCCTAA dddddddkke 78 1939 54191 1 230313 23 0328 Intron 3 AGTAGAGCCTTTGCCT eekddddddddddkke 75 1940 541912 230580 230595 Intron 3 CTGTCTCTTGGCCCAT eekddddddddddkke 80 1941 541913 231330 23 1345 Intron 3 GGCCCAAATCTTGAGT eekddddddddddkke 67 1942 541914 231 817 23 1832 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 2343 1 1 Intron 3 GGAAGGGCCCAAATAT dddddddkke 15 1949 541921 234903 234918 Intron 3 GATCTACTCCTACTGC eekddddddddddkke 65 1950 541922 235313 235328 Intron 3 GTCAGCCTGTGTCTGA eekddddddddddkke 45 1951 541923 235770 23 57 85 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 2375 85 237600 Intron 3 GATTGATTGAGCTCCT eekddddddddddkke 86 1956 54192 8 237949 23 7964 Intron 3 ATGGACTCCCTAGGCT eekddddddddddkke 61 1957 541929 238542 238557 Intron 3 TACTCAAGGGCCCCTC eekddddddddddkke 67 1958 54193 0 245319 2453 34 Intron 3 GGCATATGTAGCTTGC eekddddddddddkke 91 1959 54193 1 245765 2457 80 Intron 3 GAGCTTAGATCTGTGC dddddddkke 73 1960 541932 246251 246266 Intron 3 ACGGCTGTGT eekddddddddddkke 81 1961 54193 3 246500 2465 15 Intron 3 ATTGAAAGGCCCATCA eekddddddddddkke 45 1962 541934 246936 246951 Intron 3 CAACCCAGTTTGCCGG eekddddddddddkke 71 1963 54193 5 247225 247240 Intron 3 CAGCTATTCCCTGTTT eekddddddddddkke 53 1964 54193 6 247644 247659 Intron 3 GCTGTGTCACACTTCC eekddddddddddkke 98 1965 54193 7 248223 24823 8 Intron 3 GTCCAAGGATCACAGC eekddddddddddkke 86 1966 54193 8 248695 248710 Intron 3 GCTACCACTAGAGCCT eekddddddddddkke 81 1967 54193 9 249494 249509 Intron 3 GTTTCAGGGCTTATGT eekddddddddddkke 63 1968 541940 250693 25 0708 Intron 3 TCCCACACCTATTGAA eekddddddddddkke 51 1969 541941 251622 25 1637 Intron 3 ACTGACTAGAGAGTCC eekddddddddddkke 81 1970 541942 251950 251965 Intron 3 TCCAAGGCTGATGTCC dddddddkke 85 1971 541943 252665 252680 Intron 3 TCCCATGGTGGACATG eekddddddddddkke 39 1972 541944 253 140 25 3155 Intron 3 AGTAGCTGGCAGAAGG eekddddddddddkke 85 1973 541945 253 594 25 3609 Intron 3 CTGGGAGTGACTACTA dddddddkke 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 ACCTGAACTA eekddddddddddkke 65 1977 541949 255 618 25 5633 Intron 3 ATAGGCTACTGGCAGG eekddddddddddkke 89 1978 54195 0 255 992 256007 Intron 3 CCCAGCTAGCTGGAGT eekddddddddddkke 50 1979 54195 1 256428 256443 Intron 3 GGCTGGCTCTCAAAGG eekddddddddddkke 61 19 80 541952 256689 25 6704 Intron 3 TGGTGATACTGTGGCA eekddddddddddkke 94 19 81 541953 257317 257332 Intron 3 GCTGATTTTGGTGCCA eekddddddddddkke 92 1982 541954 257826 257841 Intron 3 GCTAATCTTGCCTCGA eekddddddddddkke 52 1983 54195 5 25 8407 25 8422 Intron 3 CACTGGTGGCTTTCAA eekddddddddddkke 31 19 84 Table 182 Inhibition of GHR mRNA by deoxy, MOE and (S)—cEt gapmers targeting intronic and exonic regions of SEQ ID NOs: 1 and 2 SEQ SEQ ID . SEQ 113138 I1DS1113 1:23:12 ce Chem" inhib/ition0 1821112. ID 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 dddddddkke 75 266861 1995 541967 n/a Intron 3 GCAGAATGCC 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 CACTCTCCTC eekddddddddddkke 79 268786 2000 541972 n/a Intron 4 CGAACACCTTGAGCCA eekddddddddddkke 90 269252 2001 541973 n/a Intron 4 GGCCCAGCTTAAGAGG dddddddkke 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 TTCTCCCTAC 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 GACCATCCCT eekddddddddddkke 41 272 834 2008 541980 n/a Intron 4 AGTGCTCTCTAGGGTC eekddddddddddkke 87 273257 2009 5419 81 n/a Intron 4 TACAGAGAATCACCCC eekddddddddddkke 82 273 651 2010 541982 n/a Intron 4 GTCCAAGTAAGGTGCT eekddddddddddkke 57 273 947 201 1 5419 83 n/a Intron 5 GACCTTGCAGGCTTCC eekddddddddddkke 87 274244 2012 541984 n/a Intron 5 GGGCAAAGGATCCTCT eekddddddddddkke 71 27475 8 2013 5419 85 n/a Intron 5 CCCATTCTGCTATCCC eekddddddddddkke 92 275198 2014 541986 n/a Intron 5 GCTGACTAGGAGGGCT eekddddddddddkke 62 275 732 2015 541987 n/a Intron 5 AGGTAGTACC eekddddddddddkke 83 276309 2016 5419 88 n/a Intron 5 GTCCCCCTCCAGTCTA eekddddddddddkke 50 276932 2017 541989 n/a Intron 5 GAGGACTCAATTCCTC eekddddddddddkke 0 277 149 201 8 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 3 8 2 80157 2025 541997 n/a Intron 6 GCTCACGGTTGGAGAC eekddddddddddkke 42 2 80799 2026 541998 n/a Intron 6 CCCATGTTCA eekddddddddddkke 45 2 81595 2027 541999 n/a Intron 6 TCACTCTACCAACCTC eekddddddddddkke 33 2 82 572 2028 542000 n/a Intron 6 TCCTTGCTTACAGATG eekddddddddddkke 33 2 83 079 2029 542001 n/a Intron 6 TAGCATTACC eekddddddddddkke 37 2 83 65 3 2030 542002 n/a Intron 6 TGGGTAACTGGCTAGT eekddddddddddkke 47 2 85 71 1 2031 542003 n/a Intron 6 AACCATTCCTCACCAA eekddddddddddkke 53 2 87181 2032 542004 n/a Intron 6 AACAGTTGAT eekddddddddddkke 37 2 87 895 2033 542005 n/a Intron 6 GGCTCCTATCATACCT eekddddddddddkke 3 8 2 8 8 943 2034 542006 n/a Intron 6 TAGGTCTCACAACCCT eekddddddddddkke 10 2 8963 8 2035 542007 n/a Intron 6 GTGCATTAGTCTTCCA dddddddkke 74 290035 2036 542008 n/a Intron 7 CCAGGTTAGC eekddddddddddkke 13 290503 2037 542009 n/a Intron 7 TTGACTACCT eekddddddddddkke 50 290924 203 8 542010 n/a Intron 7 GTACCTGCCAGCTACT eekddddddddddkke 35 291 807 2039 EX0n 8 - 54201 1 n/a intron 8 CCTACCTTTGCTGTTT eekddddddddddkke 12 29261 1 2040 junction 542012 n/a Intron 8 AGTCACCAGCCTAAGC eekddddddddddkke 47 292 860 2041 542013 n/a Intron 8 AGGCAACCTGGGAGTG eekddddddddddkke 52 293 377 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 85 295 955 2047 542019 I?a Intron 8 CTGGCCTCAGGGCAAT eekddddddddddkke 51 296186 2048 542020 I?a Intron 8 GTAGACTTGGGTAGGT eekddddddddddkke 53 296680 2049 542022 I?a 3TITR TGGTGCTAAGCTCTCC eekddddddddddkke 67 301009 2050 542023 I?a 3TITR CATGCTCAAGCTGGAA dddddddkke 47 301280 2051 542024 206 Exon2 AAGGTCAACAGCAGCT eekddddddddddkke 93 144990 2052 542025 207 Exon2 CAAGGTCAACAGCAGC eekddddddddddkke 85 144991 2053 542026 208 Exon2 CCAAGGTCAACAGCAG dddddddkke 82 144992 2054 542027 209 Exon2 GCCAAGGTCAACAGCA eekddddddddddkke 84 144993 2055 Table 183 Inhibition of GHR mRNA by deoxy, MOE and (S)—cEt gapmers targeting intronic and exonic s of SEQ ID NOs: 1 and 2 ISIS EDD"): Target 0 SEQ Sequence Chmn?uy PM) Region ition NO‘ 2 1 Start IDNO Start 541262 I?a InHon2 TTGGTTTGTCAATCCT eekddddddddddkke 86 156891 1370 542034 870 Exon7 TCTCACACGCACTTCA dddddddkke 49 290368 2056 542035 871 Exon7 ATCTCACACGCACTTC eekddddddddddkke 39 290369 2057 542036 872 Exon7 GATCTCACACGCACTT eekddddddddddkke 50 290370 2058 542049 I?a Inuonl TGAATCAAGC eekddddddddddkke 85 17928 2059 542050 I?a Inuonl TCTTTCATGAATCAAG eekddddddddddkke 54 17929 2060 542051 I?a Inuonl GTCTTTCATGAATCAA eekddddddddddkke 96 17930 2061 542052 I?a Inuonl GGTCTTTCATGAATCA eekddddddddddkke 98 17931 2062 542053 I?a Inuonl ATGGTCTTTCATGAAT eekddddddddddkke 94 17933 2063 542054 I?a Inuonl GATGGTCTTTCATGAA eekddddddddddkke 73 17934 2064 542055 I?a Inuonl TGATGGTCTTTCATGA eekddddddddddkke 83 17935 2065 542056 I?a Inuonl TATATCAATATTCTCC eekddddddddddkke 75 21821 2066 542057 I?a Inuonl TTATATCAATATTCTC eekddddddddddkke 23 21822 2067 542058 I?a Inuonl GTTATATCAATATTCT eekddddddddddkke 87 21823 2068 542059 I?a Inuonl TTTCTTTAGCAATAGT eekddddddddddkke 85 22519 2069 542060 I?a Inuonl CTTTCTTTAGCAATAG eekddddddddddkke 81 22520 2070 542061 I?a Inuonl GCTTTCTTTAGCAATA eekddddddddddkke 68 22521 2071 542062 I?a Inuonl CTCCATTAGGGTTCTG eekddddddddddkke 91 50948 2072 542063 I?a Inuonl TCTCCATTAGGGTTCT eekddddddddddkke 88 50949 2073 542064 I?a Inuonl TTCTCCATTAGGGTTC dddddddkke 85 50950 2074 542065 I?a Inuonl GTTCTCCATTAGGGTT eekddddddddddkke 84 50951 2075 542066 I?a Inuonl AGGTTGGCAGACAGAC eekddddddddddkke 92 53467 2076 542067 I?a Inuonl CAGGTTGGCAGACAGA eekddddddddddkke 93 53468 2077 542068 I?a Inuonl GCAGGTTGGCAGACAG eekddddddddddkke 91 53469 2078 542069 I?a Inuonl CTTCTTGTGAGCTGGC eekddddddddddkke 95 64885 2079 542070 I?a Inuonl TCTTCTTGTGAGCTGG eekddddddddddkke 89 64886 2080 542071 n/a Intron 1 GTCTTCTTGTGAGCTG eekddddddddddkke 96 648 87 2081 542072 n/a Intron 1 AGTCTTCTTGTGAGCT eekddddddddddkke 81 648 88 2082 542073 n/a Intron 1 TCTTCCACTCACATCC eekddddddddddkke 89 65991 2083 542074 n/a Intron 1 CACTCACATC 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 TTTGACTTCC dddddddkke 86 72108 2087 54207 8 n/a Intron 1 CATAGATTTTGACTTC eekddddddddddkke 42 721 09 208 8 542079 n/a Intron 1 GCATAGATTTTGACTT eekddddddddddkke 66 721 10 2089 542080 n/a Intron 1 AAATGTCAACAGTGCA eekddddddddddkke 97 80639 2090 542081 n/a Intron 1 CATGACTATGTTCTGG eekddddddddddkke 68 1255 95 2091 542082 n/a Intron 1 ACATGACTATGTTCTG eekddddddddddkke 66 1255 96 2092 542083 n/a Intron 1 ACTATGTTCT eekddddddddddkke 74 1255 97 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 TCTGAGCTCT eekddddddddddkke 94 145432 2096 542087 n/a Intron 2 CCTGAATTCTGAGCTC eekddddddddddkke 93 145433 2097 54208 8 n/a Intron 2 GCCTGAATTCTGAGCT eekddddddddddkke 87 145434 209 8 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 2 102 542093 n/a Intron 2 TTGTAATTCT eekddddddddddkke 5 8 148063 2103 542094 n/a Intron 2 CCTGATATTGTAATTC eekddddddddddkke 95 148064 2104 542095 n/a Intron 2 GCCTGATATTGTAATT eekddddddddddkke 85 148065 2 105 542096 n/a Intron 2 TGCCTGATATTGTAAT eekddddddddddkke 86 148066 2106 542097 n/a Intron 2 ATTATGTGCTTTGCCT eekddddddddddkke 86 148907 2107 54209 8 n/a Intron 2 AATTATGTGCTTTGCC eekddddddddddkke 75 148908 2108 542099 n/a Intron 2 CAATTATGTGCTTTGC eekddddddddddkke 8 8 148909 2109 542100 n/a Intron 2 TCAATTATGTGCTTTG dddddddkke 7 8 148910 21 10 542101 n/a Intron 2 GTCAATTATGTGCTTT eekddddddddddkke 97 14891 1 21 1 1 542102 n/a Intron 2 ACCAAACACC eekddddddddddkke 97 15 0973 21 12 542103 n/a Intron 2 TGCCATCACCAAACAC eekddddddddddkke 90 15 0974 21 13 542104 n/a Intron 2 TTGCCATCACCAAACA eekddddddddddkke 89 15 0975 21 14 542105 n/a Intron 2 TGGTGACTCTGCCTGA eekddddddddddkke 9 8 15 13 88 21 15 542106 n/a Intron 2 CTGGTGACTCTGCCTG eekddddddddddkke 96 15 13 89 21 16 542107 n/a Intron 2 GCTGGTGACTCTGCCT eekddddddddddkke 9 8 15 13 90 21 17 542108 n/a Intron 2 TGCTGGTGACTCTGCC eekddddddddddkke 97 15 13 91 21 1 8 542109 n/a Intron 2 CTGCTGGTGACTCTGC eekddddddddddkke 93 15 13 92 21 19 Table 184 Inhibition of GHR mRNA by deoxy, VIOE and (S)-cEt gapmers targeting introns 2 and 3 of SEQ ID NO: 2 ISIS 8158121) Target % SEQ NO 1:21;? sequence Chem‘my Stop Region inhibition IDNO 541262 156891 156906 Intron2 TTGGTTTGTCAATCCT eekddddddddddkke 95 1370 542110 153002 153017 Intr0n2 AGTAGTCAATATTATT eekddddddddddkke 74 2120 542111 153003 153018 Intr0n2 TCAATATTAT eekddddddddddkke 55 2121 542112 153004 153019 Intr0n2 CCAGTAGTCAATATTA eekddddddddddkke 97 2122 542113 153922 153937 Intr0n2 CCTTTGGGTGAATAGC eekddddddddddkke 90 2123 542114 153923 153938 Intr0n2 ACCTTTGGGTGAATAG eekddddddddddkke 71 2124 542115 153924 153939 Intr0n2 CACCTTTGGGTGAATA eekddddddddddkke 78 2125 542116 155595 155610 Intron2 CAACTTGAGGACAATA eekddddddddddkke 89 2126 542118 155597 155612 Intron2 CTCAACTTGAGGACAA eekddddddddddkke 98 2127 542119 156395 156410 Intron2 CAGGAAGAAAGGAACC eekddddddddddkke 95 2128 542120 156396 156411 Intr0n2 CCAGGAAGAAAGGAAC eekddddddddddkke 83 2129 542121 156397 156412 Intr0n2 ACCAGGAAGAAAGGAA eekddddddddddkke 90 2130 542122 156595 156610 Intr0n2 TGCAGTCATGTACACA dddddddkke 97 2131 542123 156596 156611 Intr0n2 CTGCAGTCATGTACAC eekddddddddddkke 90 2132 542124 156597 156612 Intr0n2 TCTGCAGTCATGTACA eekddddddddddkke 81 2133 542125 156890 156905 Intron2 TGGTTTGTCAATCCTT dddddddkke 97 2134 542126 156892 156907 Intron2 CTTGGTTTGTCAATCC eekddddddddddkke 99 2135 542127 157204 157219 Intr0n2 GCTACAATGCACAGGA eekddddddddddkke 98 2136 542128 157205 157220 Intr0n2 TGCTACAATGCACAGG eekddddddddddkke 98 2137 542129 158008 158023 Intr0n2 GATATTTATTGCTGTA eekddddddddddkke 61 2138 542130 158009 158024 Intr0n2 TGATATTTATTGCTGT eekddddddddddkke 41 2139 542131 158010 158025 Intr0n2 CTGATATTTATTGCTG eekddddddddddkke 86 2140 542132 162752 162767 2 AGGGTCTTTACAAAGT dddddddkke 69 2141 542133 162753 162768 Intron2 CAGGGTCTTTACAAAG dddddddkke 71 2142 542134 162754 162769 Intr0n2 TCTTTACAAA eekddddddddddkke 93 2143 542135 166353 166368 Intron2 TTCTGCAGTATCCTAG eekddddddddddkke 84 2144 542136 166354 166369 Intron2 TTTCTGCAGTATCCTA eekddddddddddkke 88 2145 542137 166355 166370 Intron2 GTTTCTGCAGTATCCT eekddddddddddkke 95 2146 542138 166356 166371 Intron2 AGTTTCTGCAGTATCC eekddddddddddkke 92 2147 542139 166357 166372 Intron2 CAGTTTCTGCAGTATC eekddddddddddkke 93 2148 542140 172747 172762 2 CAAATTCCAGTCCTAG eekddddddddddkke 73 2149 542141 172748 172763 2 CCAAATTCCAGTCCTA eekddddddddddkke 91 2150 542142 172749 172764 Intr0n2 TCCAAATTCCAGTCCT eekddddddddddkke 90 2151 542143 175372 175387 Intr0n2 ACCCATTTCATCCATT eekddddddddddkke 94 2152 542144 175373 175388 Intr0n2 AACCCATTTCATCCAT eekddddddddddkke 93 2153 542145 175374 175389 2 GAACCCATTTCATCCA eekddddddddddkke 97 2154 542146 175 375 175 390 Intron 2 GGAACCCATTTCATCC eekddddddddddkke 96 2155 542147 175 376 175 391 Intron 2 AGGAACCCATTTCATC eekddddddddddkke 68 2156 542148 189120 18913 5 Intron 2 GCTTCATGTCTTTCTA dddddddkke 90 2157 542149 189121 18913 6 Intron 2 TGCTTCATGTCTTTCT eekddddddddddkke 96 215 8 542150 189122 18913 7 Intron 2 GTGCTTCATGTCTTTC eekddddddddddkke 97 2159 542151 189485 189500 Intron 2 TAGCAGTCAC eekddddddddddkke 92 2160 542152 189486 189501 Intron 2 ATGAGCTTAGCAGTCA eekddddddddddkke 95 2161 542 153 1 89487 1 89 502 Intron 2 CATGAGCTTAGCAGTC eekddddddddddkke 95 21 62 542154 191 143 191 15 8 Intron 2 TACAGACATAGCTCTA eekddddddddddkke 91 2163 542 155 191 144 191 15 9 Intron 2 ATACAGACATAGCTCT eekddddddddddkke 74 21 64 542 156 191 145 191 160 Intron 2 GATACAGACATAGCTC eekddddddddddkke 91 21 65 542 157 191 146 191 161 Intron 2 GGATACAGACATAGCT eekddddddddddkke 94 21 66 54215 8 198149 198164 Intron 2 TTTAATTCAC eekddddddddddkke 71 2167 542159 19815 0 198165 Intron 2 CTTTAATTCA eekddddddddddkke 81 2168 542160 19815 1 198166 Intron 2 TATGTGGCTTTAATTC eekddddddddddkke 78 2169 542161 199 817 199 832 Intron 2 TGTTCAGTTGCATCAC eekddddddddddkke 91 2170 542162 199 81 8 199 83 3 Intron 2 GTGTTCAGTTGCATCA eekddddddddddkke 89 2171 542163 199 819 199 834 Intron 2 TGTGTTCAGTTGCATC dddddddkke 90 2172 542164 210562 210577 Intron 3 CATCTGGATGTGAGGC eekddddddddddkke 90 2173 542165 210563 21057 8 Intron 3 GGATGTGAGG eekddddddddddkke 78 2174 542166 210564 210579 Intron 3 CACATCTGGATGTGAG eekddddddddddkke 55 2175 542167 219020 21903 5 Intron 3 TCAGGTAATTTCTGGA eekddddddddddkke 82 2176 542168 219021 21903 6 Intron 3 CTCAGGTAATTTCTGG eekddddddddddkke 73 2177 542169 219022 21903 7 Intron 3 TCTCAGGTAATTTCTG eekddddddddddkke 40 2178 542170 225 568 225 5 8 3 Intron 3 TGCTTATTTACCTGGG eekddddddddddkke 90 2179 542171 225 569 225 5 84 Intron 3 TTGCTTATTTACCTGG eekddddddddddkke 90 21 80 542172 225 570 225 5 8 5 Intron 3 TTTGCTTATTTACCTG eekddddddddddkke 79 21 81 542173 225 571 225 5 86 Intron 3 TTTTGCTTATTTACCT eekddddddddddkke 32 21 82 542174 229619 229634 Intron 3 ATGATGTTACTACTAC eekddddddddddkke 63 21 83 542175 229620 22963 5 Intron 3 AATGATGTTACTACTA eekddddddddddkke 53 21 84 542176 229621 22963 6 Intron 3 CAATGATGTTACTACT dddddddkke 12 21 85 542177 232 827 232842 Intron 3 CCCCTAGAGCAATGGT eekddddddddddkke 76 21 86 542178 232 82 8 232843 Intron 3 CCCCCTAGAGCAATGG eekddddddddddkke 83 21 87 542179 232829 232844 Intron 3 TCCCCCTAGAGCAATG eekddddddddddkke 49 2188 542180 237676 237691 Intron 3 GCAGATGCTC eekddddddddddkke 88 21 89 542181 237677 237692 Intron 3 CTCAATTGCAGATGCT eekddddddddddkke 90 2190 542182 23767 8 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 24823 3 248248 Intron 3 AGTATATTCAGTCCAA eekddddddddddkke 94 2194 542186 248234 248249 Intron 3 CAGTATATTCAGTCCA eekddddddddddkke 97 2195 Table 185 Inhibition of GHR mRNA by deoxy, MOE and (S)—cEt gapmers targeting intronic and exonic regions of SEQ ID NOs: 1 and 2 0 SEQ ID If}? NO: 1 Target Region Sequence Chemistry inhilfition NO: 2 SEIEOID Start Start Site 541262 n/a Intron 2 TGTCAATCCT eekddddddddddkke 93 156891 1370 545316 168 "0311;113:330" GAGCTTCGCC eekddddddddddkke 80 3044 2196 545317 173 GEEK?" GTAGGACCTCCGAGCT eekddddddddddkke 74 n/a 2197 545318 177 GEEK?" ACCTGTAGGACCTCCG eekddddddddddkke 70 n/a 2198 545321 213 Exon 2 CAGTGCCAAGGTCAAC eekddddddddddkke 77 144997 2199 545322 225 Exon 2 TCCTGCCAGT eekddddddddddkke 36 145009 2200 545332 361 Exon 4/ Intron 3 CTCGCTCAGGTGAACG eekddddddddddkke 57 268024 2201 545333 366 Exon 4/ Intron 3 AGTCTCTCGCTCAGGT eekddddddddddkke 88 268029 2202 545337 444 Ex‘gi?cggon 4 CCTTCTGGTATAGAAC eekddddddddddkke 21 268107 2203 545340 570 Exon 5 GCTAGTTAGCTTGATA eekddddddddddkke 39 274130 2204 545343 626 "(3111133312)" 4 TCTGGTTGCACTATTT eekddddddddddkke 34 n/a 2205 545344 629 "(3131:;£312" 4 GGATCTGGTTGCACTA eekddddddddddkke 30 n/a 2206 545345 632 Exon 6 GGTGGATCTGGTTGCA dddddddkke 18 278926 2207 545346 638 Exon 6 GCAATGGGTGGATCTG eekddddddddddkke 50 278932 2208 545347 647 Exon 6 CAGTTGAGGGCAATGG dddddddkke 71 278941 2209 545348 651 Exon 6 AGTCCAGTTGAGGGCA eekddddddddddkke 58 278945 2210 545349 655 Exon 6 GTAAAGTCCAGTTGAG eekddddddddddkke 34 278949 2211 545350 660 Exon 6 GTTCAGTAAAGTCCAG dddddddkke 52 278954 2212 545351 685 Exon 6 CTGCATGAATCCCAGT eekddddddddddkke 77 278979 2213 545355 923 Exon 7 ACATAGAGCACCTCAC eekddddddddddkke 38 290421 2214 545356 926 Exon 7 GTTACATAGAGCACCT eekddddddddddkke 79 290424 2215 545357 929 Exon 7 AGTGTTACATAGAGCA eekddddddddddkke 70 290427 2216 545362 1124 EXQEIZé?eg‘lfn 8 TCCTTGAGGAGATCTG eekddddddddddkke 3 n/a 2217 545363 1170 Exon 10 GCTATCATGAATGGCT eekddddddddddkke 69 297587 2218 545364 1180 Exon 10 CGGGTTTATAGCTATC eekddddddddddkke 58 297597 2219 545369 1320 Exon 10 ATCCTTCACCCCTAGG eekddddddddddkke 46 297737 2220 545370 1328 Exon 10 GAGTCGCCATCCTTCA dddddddkke 60 297745 2221 545371 1332 Exon 10 TCCAGAGTCGCCATCC eekddddddddddkke 51 297749 2222 545373 1418 Exon 10 GGCTGAGCAACCTCTG eekddddddddddkke 80 297835 2223 545374 1422 Exon 10 CTGTGGCTGAGCAACC eekddddddddddkke 63 297839 2224 545380 1524 GATAACACTGGGCTGC eekddddddddddkke" 297941 2225 545381 1530 TGCTTGGATAACACTG eekddddddddddkke 297947 2226 545382 1533 CTCTGCTTGGATAACA eekddddddddddkke" 297950 2227 Table 186 tion of GHR mRNA by deoxy, MOE and (S)—cEt gapmers targeting intronic and exonic regions of SEQ ID NOs: 1 and 2 SE? SE)Q If}? N? '. Eggs; Sequence Chemistry inhilfition0 1:31:12 SEQ ID NO St?rt Site 541262 n/a Intron 2 TTGGTTTGTCAATCCT eekddddddddddkke 89 156891 1370 545393 183 8 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 2983 85 2236 545 39 8 19 86 Exon 10 GGACTGTACTATATGA eekddddddddddkke 48 298403 2237 545401 2077 Exon 10 TCAGTTGGTCTGTGCT eekddddddddddkke 60 298494 223 8 545402 2095 Exon 10 GCTAAGGCATGATTTT eekddddddddddkke 5 3 2985 12 2239 545406 2665 Exon 10 CTTGAAGTCT eekddddddddddkke 87 299082 2240 545407 2668 Exon 10 ATAGCCATGCTTGAAG eekddddddddddkke 70 299085 2241 545408 2692 Exon 10 ACACAGTGTGTAGTGT dddddddkke 60 299109 2242 545409 2699 Exon 10 TACACAGTGT eekddddddddddkke 3 1 2991 16 2243 545410 2704 Exon 10 TGCAGTACAC eekddddddddddkke 5 7 299121 2244 54541 1 2739 Exon 10 TAGACTGTAGTTGCTA eekddddddddddkke 5 3 299156 2245 545412 2747 Exon 10 ACCAGCTTTAGACTGT eekddddddddddkke 5 6 299164 2246 545413 2945 Exon 10 GTAAGTTGATCTGTGC eekddddddddddkke 79 299362 2247 545414 2963 Exon 10 TACTTCTTTTGGTGCC eekddddddddddkke 82 2993 80 2248 545416 3212 Exon 10 TCTTGTACCTTATTCC dddddddkke 73 299629 2249 545417 33 06 Exon 10 TGGTTATAGGCTGTGA eekddddddddddkke 90 299723 2250 54541 8 33 09 Exon 10 TTATAGGCTG eekddddddddddkke 8 8 299726 2251 545419 33 13 Exon 10 ATGTGTCTGGTTATAG eekddddddddddkke 68 299730 2252 545420 33 17 Exon 10 GAGTATGTGTCTGGTT eekddddddddddkke 84 299734 2253 545421 4049 Exon 10 GGTCTGCGATAAATGG eekddddddddddkke 69 3 00466 2254 545429 4424 Exon 10 GCCAGACACAACTAGT eekddddddddddkke 5 9 3 00841 2255 54543 0 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 54543 3 143 Exon 1 AGAGCGCGGGTTCGCG eekddddddddddkke 61 3019 2259 545434 n/a "12:2: 1 TACTGACCCCAGTTCC eekddddddddddkke 68 3654 2260 54543 5 n/a "12:5: 1 ACTCTACTGACCCCAG eekddddddddddkke 70 365 8 2261 54543 6 n/a "12:2: 1 GTCACTCTACTGACCC eekddddddddddkke 83 3661 2262 54543 7 n/a "12:5: 1 TTCATGCGGACTGGTG dddddddkke 68 3680 2263 54543 8 n/a 3/11E1:(())rr11 3 GTGAGCATGGACCCCA dddddddkke 94 225436 2264 54543 9 n/a 3/11E1:(())rr11 3 TGATATGTGAGCATGG eekddddddddddkke 8 8 225442 2265 545440 n/a 3/11E1:(())rr113 AAGTTGGTGAGCTTCT eekddddddddddkke 85 2267 85 2266 545441 n/a 3/11E1:(())rr113 CCTTCAAGTTGGTGAG eekddddddddddkke 8 8 226790 2267 545442 n/a 3/11E1:(())rr113 GTAAGATCCTTTTGCC eekddddddddddkke 70 2268 83 2268 545443 n/a CAGCTGTGCAACTTGC eekddddddddddkke 5 0 23 8345 2269 3$36): 3 545444 n/a 3/11E1:(())rr113 GTAGGTAGGG eekddddddddddkke 68 23 8422 2270 545445 n/a 3/11E1:(())rr113 AGAGCCTTGGTAGGTA eekddddddddddkke 85 23 8425 2271 545446 n/a "12:2: 1 CCCGCACAAACGCGCA eekddddddddddkke 10 3614 2272 545447 n/a "12:2: 1 GTCTTCAAGGTCAGTT eekddddddddddkke 92 93208 2273 545448 n/a "12:2: 1 GCCCAGTGAATTCAGC eekddddddddddkke 76 93246 2274 545449 n/a "12:5: 1 AGATGCGCCCAGTGAA dddddddkke 60 93252 2275 54545 0 n/a "12:2: 1 GTAAGATGCGCCCAGT eekddddddddddkke 7 8 93255 2276 54545 1 n/a "12:2: 1 CCAGAAGGCACTTGTA eekddddddddddkke 42 93 301 2277 545452 n/a "12:2: 1 TTTGCAGAAC eekddddddddddkke 1 5 93 340 2278 54545 3 n/a "12:2: 1 CCTTGGTCATGGAAGA eekddddddddddkke 3 5 93 3 5 0 2279 545454 n/a "12:2: 1 TGGTCATGGA eekddddddddddkke 5 5 93 3 5 3 2280 54545 5 n/a "12:5: 1 GAGGTGACCTTGGTCA eekddddddddddkke 70 93 3 5 7 2281 54545 6 n/a "12:2: 1 ATCCAAAGAGGTGACC eekddddddddddkke 41 93 364 2282 54545 7 n/a "12:5: 1 GCCAATCCAAAGAGGT eekddddddddddkke 5 6 93 368 2283 54545 8 n/a "12:5: 1 GGTCTGCCAATCCAAA eekddddddddddkke 79 93 373 2284 54545 9 n/a "12:5: 1 CCCTGGGTCTGCCAAT eekddddddddddkke 68 93 3 7 8 2285 545460 n/a "12:5: 1 GAGATCTCAACAAGGG eekddddddddddkke 52 93427 2286 545461 n/a "12:5: 1 CGCCCATCACTCTTCC eekddddddddddkke 68 93 988 2287 545462 n/a "12:5: 1 CACCTGTCGCCCATCA eekddddddddddkke 67 93 995 2288 545463 n/a "12:5: 1 CATCACCTGTCGCCCA eekddddddddddkke 7 8 93 998 2289 545464 n/a "12:5: 1 CACCTGTCGC eekddddddddddkke 74 94001 2290 545465 n/a "115:5: 1 AATAGTTGTCACCATC eekddddddddddkke 76 94010 2291 545466 n/a "12:5: 1 GCCACCTTTCATGAGA eekddddddddddkke 5 8 94048 2292 545467 n/a "12:5:2 CTCTTGGAAGTAGGTA eekddddddddddkke 89 198762 2293 545468 n/a "1153532 GTTCTCTTGGAAGTAG eekddddddddddkke 80 198765 2294 545469 n/a "1153232 TAAACAGGTTGGTCTG eekddddddddddkke 68 198854 2295 Example 121: Dose-dependent antisense inhibition of human GHR in Hep3B cells by dequ, MOE and (S)-cEt gapmers Gapmers from studies described above exhibiting signi?cant in vitro inhibition of GHR mRNA were selected and tested at various doses in Hep3B cells. The antisense ucleotides were tested in a series of ments that had similar culture ions. 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 "M, 1.25 "M, 2.50 "M, 5.00 "M and 10.00 uM 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 ed according to total RNA content, as measured by RIBOGREEN®. Results are presented as t inhibition of GHR, relative to untreated control cells.

The half maximal tory concentration (IC50) of each ucleotide is also presented. GHR mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells. wo 68618 Table 187 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 541396 30 51 68 74 67 1.4 541262 55 87 90 94 97 0.2 541393 30 38 52 66 81 2.1 541375 41 45 54 64 79 1.6 541438 44 49 75 80 91 0.9 541428 35 32 56 78 88 1.8 541491 13 46 67 55 95 2.0 541435 21 46 55 72 94 1.9 541471 11 49 50 77 89 2.0 541430 24 44 56 57 79 2.2 541492 32 40 65 80 85 1.5 541431 22 46 73 84 92 1.5 Table 188 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 541487 36 46 66 85 92 1.3 541423 33 55 64 80 93 1.2 541452 37 60 79 87 94 0.9 541505 51 75 86 92 97 0.4 541522 54 76 81 90 95 0.3 541539 65 76 85 94 98 0.2 541503 54 65 80 93 97 0.5 541520 43 61 86 94 96 0.7 541515 57 72 85 92 94 0.3 541564 57 72 88 90 97 0.3 541554 43 65 81 89 93 0.7 541509 11 8 19 6 8 >10 541584 59 65 84 91 96 0.3 541585 70 80 93 92 98 0.1 Table 189 0.625 1.250 2.50 5.00 10.00 1c50 ISIS N wo 68618 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 <06 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 190 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM 11M 11M 11M HM (11M) 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 191 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM 11M 11M 11M HM (11M) 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 <06 4 "m- ————m Table 192 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (HM) 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 193 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (HM) 541895 47 72 85 93 94 <0.6 541882 60 67 89 93 97 <0.6 541889 63 80 87 94 97 <0.6 541904 26 78 23 89 93 1.4 545418 0 81 91 94 95 1.7 541930 58 71 82 88 92 <0.6 545439 67 87 93 96 98 <0.6 542024 15 58 78 87 90 1.4 541985 59 81 88 93 97 <0.6 541972 47 58 83 90 92 0.6 541991 57 64 88 92 83 <0.6 541980 33 50 76 72 93 1.2 wo 68618 PCT/U82015/028887 Table 194 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (HM) 541264 26 44 64 79 89 1.6 541265 29 32 62 79 91 1.8 541263 25 40 62 78 93 1.7 541268 57 73 85 90 95 0.3 541266 15 33 46 66 90 2.5 542107 93 97 98 98 98 <0.6 542052 93 96 97 96 98 <0.6 542105 80 92 96 98 97 <0.6 542102 94 96 96 97 98 <0.6 542108 90 92 94 97 99 <0.6 542080 87 93 95 95 97 <0.6 Table 195 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 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 196 0.625 1.250 2.50 5.00 10.00 IC50 ISIS No "M "M HM 11M "M (HM) wo 2015/168618 PCT/U82015/028887 542122 90 94 98 98 99 <0.6 542125 88 97 98 98 99 <0.6 542145 90 96 98 99 99 <0.6 542112 86 94 99 99 99 <0.6 542149 88 93 99 98 99 <0.6 542146 79 93 96 97 98 <06 542153 87 94 97 98 99 <0.6 542119 64 84 93 97 98 <0.6 542137 76 91 97 97 98 <0.6 542152 84 94 96 96 97 <0.6 542157 83 95 98 99 98 <0.6 Table 197 0.625 1.250 2.50 5.00 10.00 1C50 ISIS N0 HM HM HM HM HM (11M) 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 122: Dose-dependent antisense inhibition of human GHR in Hep3B cells by deoxy, MOE and (S)-cEt gapmers Gapmers from s described above exhibiting signi?cant 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 y of 20,000 cells per well and transfected using electroporation with 0.04 "M, 0.11 "M, 0.33 "M, 1.00 "M, and 3.00 uM 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 . GHR mRNA levels were adjusted according to total RNA content, as measured by EEN®. 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 198 0.04 0.11 0.33 1.00 3.00 1c50 ISIS N0 HM HM HM HM HM (11M) 539380 11 16 57 93 98 0.2 541724 0 27 71 66 83 0.3 541748 28 40 71 90 97 0.1 541767 19 38 54 87 98 0.2 541797 23 46 70 88 97 0.1 541766 15 26 49 82 96 0.3 541742 17 28 41 80 95 0.3 541750 33 27 60 89 98 0.2 541749 27 16 62 84 82 0.2 541793 0 14 44 77 96 0.4 541785 4 11 39 75 95 0.4 541752 14 6 45 70 94 0.4 541826 8 34 74 94 99 0.2 541811 6 4 45 79 97 0.4 541822 9 29 67 89 97 0.2 Table 199 0.04 1.00 3.00 1c ISIS No 0.11 "M 0.33 "M "M "M "M (0154)) 539380 0 16 47 82 98 0.4 541819 3 12 50 76 94 0.3 541841 0 19 47 80 95 0.3 541825 0 6 40 74 96 0.4 541827 5 26 48 76 95 0.3 541835 7 11 33 74 93 0.4 541838 21 26 61 90 97 0.2 541833 0 9 41 63 89 0.5 541813 0 17 28 65 92 0.5 541842 5 15 30 72 90 0.4 541804 0 12 3 49 79 1.1 542024 0 0 26 54 76 1.0 ——————m Table 200 0.04 0.11 0.33 1.00 3.00 1C50 ISIS N0 HM HM HM HM HM (11M) 539380 4 18 50 86 95 0.3 542108 15 13 65 86 97 0.2 542101 17 40 68 92 98 0.2 542106 4 23 56 88 98 0.3 542094 0 30 51 86 96 0.3 542086 13 38 50 84 97 0.2 542085 0 27 57 90 98 0.3 542087 7 3 49 80 92 0.4 542109 17 10 56 88 98 0.3 542126 40 63 91 96 99 <0.03 542127 27 47 69 93 97 0.1 542128 11 30 66 90 98 0.2 542118 14 42 77 95 98 0.1 542150 31 46 72 94 98 0.1 542122 13 14 59 90 97 0.3 Table 201 1mg 0g;. 0;;. 0;;. 1.g; 333. (Ch?)I 539380 0 2 50 86 97 0.4 542125 31 32 69 89 96 0.1 542145 15 29 64 91 97 0.2 542112 14 38 61 87 96 0.2 542149 9 37 63 90 97 0.2 542146 13 33 59 82 95 0.2 542153 22 26 63 86 96 0.2 542119 10 20 34 70 87 0.4 542137 3 19 47 77 95 0.3 542152 0 9 47 82 96 0.4 542157 0 26 56 84 96 0.3 542143 8 12 44 81 95 0.3 542144 0 21 42 75 95 0.4 542139 0 14 46 82 97 0.4 542134 3 23 43 72 92 0.4 wo 68618 Table 202 ISIS N0 010/411 0.1V} 013/3 1.10/11) 3.10/11) (1C1?) 539380 0 9 64 85 97 0.3 541870 7 15 48 80 92 0.3 541262 0 29 63 90 98 0.2 541863 0 26 40 82 93 0.4 541875 6 30 71 84 91 0.2 541853 0 13 39 67 91 0.5 541877 0 26 41 79 94 0.4 541881 0 30 54 87 94 0.3 541936 20 41 73 93 98 0.1 541909 0 16 34 64 90 0.5 541907 6 31 59 84 96 0.2 541952 0 0 50 72 92 0.5 541953 0 22 50 80 92 0.4 541914 0 0 46 76 93 0.4 541880 0 13 48 79 89 0.4 Table 203 0.04 0.11 0.33 1.00 3.00 1C50 ISIS N0 11M HM 11M 11M HM (11M) 539380 0 5 53 78 94 0.4 541903 12 20 26 62 88 0.5 541895 3 12 29 66 92 0.5 541882 2 0 27 65 86 0.7 541889 12 12 47 68 87 0.4 541930 0 6 40 59 85 0.6 541985 0 16 41 66 93 0.4 542031 1 0 22 55 80 0.8 541972 0 1 23 46 83 0.9 541991 4 35 42 67 89 0.4 542052 5 28 70 92 98 0.2 542080 0 18 54 87 96 0.3 542051 0 18 52 86 97 0.3 542071 5 3 51 74 95 0.4 542069 0 7 56 85 94 0.3 Table 204 ISIS No 0.10/11 0.1V} 0.13/3 1.10/0 3.10/0 (Ic?) 539380 11 20 54 89 92 0.3 542053 6 14 38 69 74 0.6 542186 14 43 70 90 98 0.2 542185 0 26 48 80 96 0.3 545333 0 4 27 65 90 0.6 545336 0 15 24 43 79 0.9 545373 0 2 9 42 86 1.0 545438 0 24 56 81 92 0.3 545431 0 18 50 73 91 0.4 545447 0 15 34 78 93 0.4 545417 0 11 39 66 87 0.5 545467 12 16 37 76 93 0.4 545441 21 15 20 60 87 0.6 545439 17 24 49 82 91 0.3 Example 123: Dose-dependent antisense inhibition of rhesus monkey GHR in LLC-MK2 cells Gapmers from studies described above exhibiting signi?cant 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 "M, 0.37 "M, 1.11 "M, 3.33 "M, and 10.00 "M concentrations of nse oligonucleotide. After a treatment period of approximately 16 hours, RNA was ed 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 ed by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

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

Table 205 ISIS 0.12 0.37 1.11 3.33 10.00 ICso Chemistry No "M "M "M "M "M (uM) msmnnnnnnDeoxy, MOE and msmnnnnnnDeoxy, MOE and Deoxy, VIOE and 541767 6 10 30 68 88 2.0 (S)-cEt Deoxy, VIOE and 541875 7 19 64 84 96 0.9 (S)-cEt Deoxy, VIOE and 541881 6 24 59 79 91 1.0 (S)-cEt Deoxy’ VICE and 542101 0 5 38 71 81 2.0 {S g-cEt Deoxy, VIOE and 542112 5 17 33 67 76 2.0 (S)-cEt Deoxy, VIOE and 542118 1 6 35 68 86 2.0 (S)-cEt Deoxy, VIOE and 542125 0 12 57 83 93 1.0 (S)-cEt Deoxy, VIOE and 542127 1 0 30 68 84 2.4 (S)-cEt Deoxy, VIOE and 542128 12 0 26 58 83 2.7 (S)-cEt Deoxy, VIOE and 542153 4 0 0 36 59 6.6 (S)-cEt Deoxy, VIOE and 542185 4 0 25 56 87 2.5 (S)-cEt Deoxy, VIOE and 542186 15 23 51 73 90 1.1 (S)-cEt Deoxy, VIOE and 542051 5 19 40 81 94 1.2 (S)-cEt Table 206 . 0.12 0.37 1.11 3.33 10.00 IC50 ISIS N0 Chemlstry M M M M M ( M) 523723 55 VIOE 23 14 31 43 71 3.5 532254 55 VIOE 29 35 42 69 87 0.8 532401 55 VICE 27 28 46 73 88 1.2 533932 55 VICE 10 24 48 70 92 1.2 539376 34 VIOE 21 8 8 35 81 4.3 539399 3'10-4 VICE 2 10 14 18 57 8.3 539404 34 VICE 39 12 25 27 57 7.7 539416 3'10-4 VICE 24 35 44 79 89 1.0 539432 3'10-4 VICE 9 29 42 73 89 1.2 Deoxy, MOE 541262 and (S)-cEt 0 43 63 88 94 0.8 Deoxy, MOE 541742 3 19 35 56 85 1.9 and t Deoxy, MOE 541767 3 24 39 64 86 1.6 and (S)-cEt Deoxy, MOE 545439 19 15 43 74 80 1.7 and (S)—cEt Deoxy, MOEmun" Example 124: Dose-dependent antisense inhibition of GHR in cynomolgus y hepatocytes Gapmers from studies described above exhibiting signi?cant in vitro tion 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 11M, 0.37 11M, 1.11 11M, 3.33 11M, and 10.00 uM 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 tion of GHR, ve to untreated control cells.

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

Table 207 mm Chemistry 3;; (:3; lug; 1;; 133 (?g) 541262 2:338"ng 40 52 75 92 98 0.3 541742 2:338"ng 40 57 51 91 96 0.2 541767 Zihxzs’ngf 36 59 60 78 91 0.4 541875 1:338"ng 54 76 88 95 95 <0.1 541881 2:338"ng 53 75 85 98 98 <0.1 542101 2:338"ng 38 55 78 89 97 0.2 542112 2:338"ng 28 50 74 89 96 0.4 542118 2:338"ng 20 45 69 84 91 0.5 542125 2:338"ng 33 62 77 92 97 0.3 542127 1:338"ng 30 50 65 86 92 0.4 542128 1:338"ng 25 40 52 80 93 0.7 542153 2:338"ng 10 31 51 73 85 1.0 Deoxy, MOE 542185 and (S)—cEt Deoxy, MOE 542186 and (S)—cEt Deoxy, MOE 542051 and (S)—cEt Table 208 0.12 0.37 1.11 3.33 10.00 IC50 ISIS No try.

HM HM HM HM HM (uM) 55 523435 35 47 61 74 85 0.5 55 523723 4 16 40 66 86 1.8 55 532254 14 15 24 16 9 >10 55 532401 37 54 73 88 94 0.3 55 533932 23 40 69 78 86 0.6 34 539376 3 0 44 65 91 2.0 34 539399 0 0 9 42 67 5.0 34 539404 0 0 26 52 71 3.5 34 539416 8 29 62 89 93 0.7 34 539432 0 24 55 85 93 0.9 Deoxy, 541262 VIOE and 23 52 73 92 96 0.4 (S)—cEt Deoxy, 541742 VIOE and 15 51 73 86 97 0.5 (S)—cEt Deoxy, 541767 VIOE and 19 20 39 68 81 1.8 (S)—cEt Deoxy, 545439 VIOE and 0 0 30 61 90 2.4 (S)—cEt Deoxy, 545447 VIOE and 0 17 17 19 27 >10 Example 125: Dose-dependent antisense inhibition of GHR in Hep3B cells Gapmers from studies described above exhibiting signi?cant 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 uM, 0.37 uM, 1.11 uM, 3.33 uM, and 10.00 uM trations 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, ve to untreated control cells.

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

Table 209 0.12 0.37 1.11 3.33 10.00 IC ISIS No M M M M M ( 15;) 541262 25 43 76 85 94 0.5 541742 32 55 76 88 97 0.3 541767 29 56 83 89 97 0.3 541875 38 68 84 93 94 0.1 541881 32 57 81 94 97 0.3 542051 34 66 83 95 98 0.2 542101 25 55 85 95 98 0.3 542112 18 56 83 95 98 0.4 542118 42 61 88 95 97 0.1 542125 30 63 87 95 98 0.2 542127 50 70 91 91 98 0.1 542128 38 63 88 96 98 0.2 542153 37 59 85 94 97 0.2 542185 44 51 76 89 96 0.2 542186 46 59 84 95 97 0.1 Table 210 ISIS No 0.11/2 0.13/7 1.1V} 3:: 10:20 (Ichs/f) 523435 9 26 49 78 93 1.0 523723 7 16 39 72 90 1.4 532254 36 46 69 86 94 0.4 532401 25 54 71 86 91 0.4 533932 8 47 69 80 94 0.7 539376 26 31 54 73 86 0.8 539399 23 43 72 89 94 0.5 539404 30 60 88 95 98 0.2 539416 30 59 84 93 98 0.3 539432 35 62 88 95 98 0.2 541262 43 60 84 89 98 0.2 541742 23 53 73 84 97 0.4 541767 22 49 74 85 92 0.4 545439 41 69 88 95 96 0.1 545447 31 47 63 74 82 0.5 Example 126: Dose-dependent antisense tion of GHR in lgus primary hepatocytes Gapmers from studies bed above exhibiting signi?cant 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 uM, 0.12 uM, 0.37 uM, 1.11 uM, 3.33 uM, and 10.00 uM trations of antisense oligonucleotide. After a ent 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 211 -------10.00 IC50 ISISNO 0.04 M 0.12 M 0.37 M 1.11 M 3.33 M ———————n ———————m ———————m ———————n Example 127: 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 uM, 0.33 uM, 1.00 uM, 1.11 uM, 3.00 uM, and 9.00 uM concentrations of nse oligonucleotide. After a ent 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 t, as measured by RIBOGREEN®. Results are presented as percent inhibition of GHR, relative to untreated control cells.

The half l inhibitory tration (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 212 0.11 0.33 1.00 3.00 9.00 1C50 ISIS N0 HM HM HM HM HM (11M) 227452 11 12 46 73 92 1.4 227488 26 25 39 76 88 1.2 272309 16 14 39 66 91 1.6 272322 13 20 44 70 86 1.4 272328 22 20 24 43 56 5.7 272338 22 24 52 71 85 1.1 532401 34 53 72 87 94 0.3 Example 128: Tolerability of 55 MOE gapmers targeting human GHR in CD1 mice CD1® mice es River, MA) are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense ucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

Trealmenl 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 ted in Table 213. 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 213 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 31 50 0.28 0.15 28 ISIS 533121 176 155 0.19 0.09 27 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 ted in Table 214. ISIS oligonucleotides that caused changes in the levels of any of the logy s outside the expected range for antisense oligonucleotides were excluded in further studies.

Table 214 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC ets (%) (g/dL) (106/11L) 1L) (103/11L) PBS 45 13 8.2 4.1 689 ISIS 523271 42 12 7.9 4.5 1181 ISIS 523324 39 11 7.5 7.9 980 ISIS 523604 33 10 6.9 14.1 507 ISIS 532254 35 10 6.9 7.2 861 ISIS 533121 39 12 7.9 8.4 853 ISIS 533161 49 14 9.3 9.0 607 ISIS 533178-——-m ISIS 533234 1045 Example 129: Tolerability 0f 55 MOE gapmers targeting human GHR in CD1 mice CD1® mice were treated with ISIS nse oligonucleotides selected from s 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 215. 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 ed in further studies.

Table 215 Plasma try s in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 30 59 0.26 0.14 20 ISIS 523715 636 505 0.24 0.14 22 ISIS 523723 57 80 0.20 0.16 23 ISIS 523726 165 167 0.18 0.15 23 ISIS 523736 140 177 0.20 0.15 23 ISIS 523747 96 108 0.17 0.14 23 ISIS 523789 45 74 0.20 0.15 22 ISIS 532395 64 111 0.23 0.12 21 ISIS 532401 47 88 0.21 0.17 22 ISIS 532411 225 426 0.17 0.16 22 ISIS 532420 60 99 0.21 0.12 25 ISIS 532468 319 273 0.15 0.14 21 ISIS 533932 62 81 0.18 0.14 21 Hematology assays Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and analysis, as well as measurements of the various blood cells, such as WB), RBC, and platelets, and total hemoglobin content. The results are presented in Table 216. ISIS oligonucleotides that caused changes in the levels of any of the logy markers outside the expected range for antisense oligonucleotides were excluded in further studies.

Table 216 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC ets (00) (g/dL) (106/11L) 1L) (103/11L) 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 rsrs 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 130: Tolerability 0f 34 MOE gapmers targeting human GHR in CD1 mice CD1® mice were treated with ISIS antisense ucleotides selected from studies bed above and evaluated for changes in the levels of various plasma chemistry markers.

Trealmenl 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 te 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 s AU400e, Melville, NY). The results are presented in Table 217. ISIS oligonucleotides that caused s in the levels of any of the liver or kidney function markers outside the ed range for antisense oligonucleotides were excluded in further s.

Table 217 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 48 63 0.20 0.13 28 ISIS 539302 204 192 0.15 0.15 24 ISIS 539321 726 455 0.17 0.12 27 ISIS 539360 3287 2495 0.58 0.13 22 ISIS 539361 310 226 0.17 0.11 21 ISIS 539376 77 75 0.14 0.12 27 ISIS 539379 134 136 0.16 0.13 24 ISIS 539380 180 188 0.14 0.12 23 ISIS 539383 80 81 0.15 0.12 25 ISIS 539399 119 127 0.13 0.12 24 ISIS 539401 1435 1172 0.24 0.11 24 ISIS 539403 1543 883 0.18 0.12 26 ISIS 539404 75 109 0.16 0.13 23 ISIS 539416 100 107 0.19 0.15 26 ISIS 539432 55 64 0.20 0.14 22 ISIS 539433 86 91 0.12 0.13 22 Hematology assays Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements and is, 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 218. 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 218 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (00) (g/dL) (106/11L) (103/11L) (103/11L) PBS 46 13 8.5 6 954 ISIS 539302 40 11 8.1 13 830 ISIS 539321 39 11 7.8 16 723 ISIS 539360 49 14 9.0 14 671 ISIS 539361 45 13 8.5 9 893 ISIS 539376 42 12 7.7 6 988 ISIS 539379 42 12 8.1 7 795 ISIS 539380 38 10 7.7 8 950 ISIS 539383 45 12 8.4 8 795 ISIS 539399 41 12 8.0 10 895 ISIS 539401 41 11 8.2 9 897 ISIS 539403 33 9 6.2 13 1104 ISIS 539404 42 12 8.4 7 641 ISIS 539416 41 11 7.5 5 686 ISIS 539432 44 12 8.0 6 920 ISIS 539433 40 11 7.4 6 987 Example 131: Tolerability of deoxy, MOE and (S)-cEt gapmers targeting human GHR in CD1 mice CD1® mice were treated with ISIS antisense oligonucleotides ed 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 aneously 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 al chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in Table 219. ISIS oligonucleotides that caused s in the levels of any of the liver or kidney function s outside the ed range for antisense oligonucleotides were excluded in further studies.

Table 219 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN PBS 36 71 0.22 0.18 22 ISIS 541766 2107 1139 0.21 0.21 23 logy 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 t. The results are presented in Table 220. 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 220 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (106/0L) (103/0L) (103/0L) PBS 37 11 7 3 1083 1s1s 541262 38 11 7 6 1010 1s1s 541724 52 16 10 9 940 1s1s 541742 47 14 9 6 1134 1s1s 541748 41 12 8 7 941 1s1s 541749 41 12 8 5 1142 1s1s 541750 42 12 8 4 1409 1s1s 541766 39 11 7 7 989 ISIS 541767 46 14 9 2 994 ISIS 541822 42 12 8 3 1190 ISIS 541826 41 12 8 10 1069 ISIS 541838 44 13 8 6 1005 ISIS 541870 38 11 7 8 1020 ISIS 541875 44 13 8 6 1104 ISIS 541907 40 11 8 9 1271 ISIS 541991 34 10 6 6 1274 Example 132: Tolerability of dequ, MOE and (S)-cEt gapmers targeting human GHR in CD1 mice CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers. The 34 MOE gapmer ISIS 539376 was also included in the study.

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 te the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated al chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in Table 221. 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 ed in further s.

Table 221 Plasma try markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN 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 s are presented in Table 222. ISIS oligonucleotides that caused changes in the levels of any of the hematology markers outside the expected range for antisense ucleotides were excluded in further studies.

Table 222 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC (%) (g/dL) (106/0L) (103/0L) PBS 46 13 8 6 ISIS 541881 53 15 10 7 ISIS 541936 41 11 8 18 ISIS 542051 49 14 9 8 ISIS 542052 46 13 9 9 ISIS 542069 43 13 8 7 ISIS 542085 38 11 7 5 ISIS 542086 49 14 9 9 ISIS 542094 36 10 6 5 ISIS 542101 44 13 9 5 ISIS 542102 27 7 5 25 ISIS 542105 42 12 8 7 ISIS 542106 37 10 7 14 ISIS 542107 41 12 7 17 ISIS 542108 51 14 8 10 ISIS 539376 49 14 10 5 Example 133: Tolerability of dequ, MOE and (S)-cEt gapmers ing human GHR in CD1 mice CD1® mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.

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

Plasma chemistry markers To te the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an ted clinical try analyzer (Hitachi Olympus AU400e, Melville, NY). The s are presented in Table 223. 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 223 Plasma chemistry markers in CD1 mice plasma at week 6 ALT AST Bilirubin Creatinine BUN (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 51 63 0.3 0.14 27 ISIS 542109 3695 2391 0.8 0.19 24 ISIS 542112 119 104 0.3 0.16 28 ISIS 542118 66 86 0.3 0.15 26 ISIS 542122 1112 350 0.3 0.16 27 ISIS 542125 79 92 0.2 0.13 26 ISIS 542126 381 398 0.5 0.14 23 ISIS 542127 54 85 0.3 0.16 26 ISIS 542128 55 89 0.2 0.12 24 ISIS 542145 834 671 0.3 0.11 24 ISIS 542146 163 107 0.2 0.14 30 ISIS 542149 974 752 0.3 0.12 26 ISIS 542150 2840 2126 2.4 0.17 23 ISIS 542153 53 75 0.2 0.14 28 ISIS 542157 137 122 0.3 0.13 25 ISIS 542185 57 72 0.2 0.11 23 ISIS 542186 62 84 0.2 0.12 24 ISIS 545431 2622 1375 3.0 0.15 28 ISIS 545438 1710 1000 0.3 0.14 26 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 224. ISIS ucleotides 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 224 Hematology markers in CD1 mice plasma at week 6 HCT Hemoglobin RBC WBC Platelets (%) (g/dL) (10mm) (103mm (103mm 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 134: bility 0fMOE s targeting human GHR in Sprague-Dawley rats Sprague-Dawley rats are a multipurpose model used for safety and cy 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.

Trealmenl 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 aneously 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 ized and organs and plasma were harvested for ?thher analysis.

Liverfunction To evaluate the effect of ISIS oligonucleotides on c ?mction, plasma levels of transaminases were ed 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 225 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 225 expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver on outside the expected range for antisense oligonucleotides were excluded in ?thher studies.

Table 225 Liver function markers in Sprague-Dawley rats ALT AST Bilirubin (IU/L) (IU/L) (mg/dL) PBS 69 90 0.15 1s1s 523723 79 123 0.12 1s1s 523789 71 105 0.15 1s1s 532254 67 97 0.14 1s1s 532401 61 77 0.12 ISIS 532420 102 127 0.17 1s1s 533178 157 219 0.34 1s1s 533234 71 90 0.11 1s1s 533932 58 81 0.12 1s1s 539376 75 101 0.14 1s1s 539380 86 128 0.16 1s1s 539383 64 94 0.14 1s1s 539399 52 95 0.14 1s1s 539404 88 118 0.13 1s1s 539416 63 104 0.14 1s1s 539432 63 90 0.13 1s1s 539433 69 92 0.13 Kidneyfunction To evaluate the effect of ISIS oligonucleotides on kidney on, 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 226, sed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense ucleotides were excluded in further studies.

Table 226 Kidney ?anction markers (mg/dL) in Sprague-Dawley rats BUN Creatinine ISIS 523789 19 0.37 ISIS 532420 20 0.31 ISIS 533178 20 0.43 ISIS 539376 19 0.36 ISIS 539380 18 0.35 ISIS 539383 19 0.35 ISIS 539404 23 0.39 ISIS 539433 20 0.34 Hematology assays Blood ed 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 227. ISIS ucleotides 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 227 Hematology markers in Sprague-Dawley rats HCT Hemoglobin RBC WBC Platelets (00) (g/dL) (106/HL) (103mm (103mm PBS 46 15 8 11 1078 ISIS 523723 38 12 7 19 626 ISIS 523789 38 12 8 12 702 ISIS 532254 36 12 7 11 547 ISIS 532401 42 14 8 12 858 ISIS 532420 37 12 7 17 542 ISIS 533178 37 12 7 15 1117 ISIS 533234 38 12 7 8 657 ISIS 533932 40 13 7 9 999 ISIS 539376 43 14 9 8 910 ISIS 539380 33 11 5 6 330 ISIS 539383 39 13 7 10 832 ISIS 539399 37 11 7 4 603 ISIS 539404 37 12 7 6 639 ISIS 539416 33 11 6 9 601 ISIS 539432 44 14 9 10 810 ISIS 539433 38 12 7 9 742 Organ weights Liver, heart, spleen and kidney s were ed at the end of the study, and are presented in Table 228. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

Table 228 Organ weights (g) Heart Liver Spleen Kidney PBS 0.35 3.6 0.2 0.8 ISIS 523723 0.31 4.9 0.7 0.8 ISIS 523789 0.34 4.8 0.6 0.8 ISIS 532254 0.32 5.0 0.6 1.0 ISIS 532401 0.32 3.8 0.4 0.8 ISIS 532420 0.29 4.6 0.7 1.0 ISIS 533178 0.34 5.2 0.7 0.9 ISIS 533234 0.30 4.4 0.6 1.0 ISIS 533932 0.31 3.9 0.5 0.9 ISIS 539376 0.29 4.4 0.4 0.8 ————m ——4—9m1-ISIS539404 0.34 ISIS 539416 0.32 Example 135: Tolerability of dequ, MOE, and (S)-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 ted for changes in the levels of various plasma chemistry markers.

Trealmenl 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 e-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.

Liverfunction To te the effect of ISIS ucleotides on hepatic ?mction, 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 229 expressed in IU/L.

Plasma levels of bilirubin were also measured using the same clinical chemistry analyzer and the results are also ted in Table 229 expressed in mg/dL. ISIS oligonucleotides that caused changes in the levels of any markers of liver function e the expected range for antisense oligonucleotides were excluded in further studies.

Table 229 Liver function s in Sprague-Dawley rats ---—(IU/L) (IU/L) (m/dL) ISIS 542112 46 72 0.10 ISIS 542118 42 60 0.08 ISIS 542125 39 67 0.10 ISIS 542127 56 75 0.12 ISIS 542128 45 71 0.12 ISIS 542153 44 69 0.11 ISIS 542185 44 93 0.09 ISIS 542186 51 107 0.12 ISIS 545439 41 73 0.10 ISIS 545447 103 114 0.10 ISIS 541262 106 133 0.12 ISIS 541742 56 102 0.11 ISIS 541767 53 69 0.09 ISIS 541875 70 133 0.08 Kidneyfunction To evaluate the effect of ISIS oligonucleotides on kidney function, plasma levels of blood urea nitrogen (BUN) and creatinine were ed using an automated clinical try analyzer (Hitachi Olympus AU400e, Melville, NY). Results are presented in Table 230, 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 ucleotides were excluded in further studies.

Table 230 Kidney ?Jnction markers (mg/dL) in Sprague-Dawley rats BUN Creatinine PBS 16 0.2 PBS 15 0.2 ISIS 541881 22 0.3 ISIS 542051 18 0.2 ISIS 542101 22 0.3 ISIS 542112 18 0.2 ISIS 542118 18 0.3 ISIS 542125 18 0.3 ISIS 542127 19 0.3 ISIS 542128 18 0.3 ISIS 542153 17 0.3 ISIS 542185 19 0.3 ISIS 542186 19 0.3 ISIS 545439 16 0.2 ISIS 545447 16 0.2 M 541262 logy 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 231. 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 231 Hematology markers in Sprague-Dawley rats HCT Hemoglobin RBC WBC Platelets (0 0) (g/dL) (106/HL) (103/HL) (103/HL) PBS 43 14 7 7 775 PBS 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 232. ISIS oligonucleotides that caused any changes in organ s outside the expected range for antisense oligonucleotides were excluded from further studies.

Table 232 Organ weights (g) Heart Liver Spleen Kidney PBS 0.4 3.7 0.2 0.9 PBS 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 136: Effect of ISIS nse 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 pro?le 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 hnology ation (NCBD 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 gy to the rhesus monkey sequence are ?Jlly 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: 2332). The greater the complementarity between the human oligonucleotide and the rhesus monkey ce, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each ucleotide to SEQ ID NO: 2332 is presented in Table 233. "Start site" indicates the 5’-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence.

Table 233 Antisense oligonucleotides complementary to the rhesus GHR genomic sequence (SEQ ID NO: 2332) Target Target SEQ ID ISIS No Start Stop Chemistry . . NO Site Site 523723 149071 149090 5105 MOE 918 532254 64701 64720 5105 MOE 479 532401 147560 147579 5105 MOE 703 Deoxy’ MOE 541767 152700 152715 1800 and g S g-cEt 541875 210099 210114 Zizxzs’ng 1904 542112 146650 146665 [23%ng 2122 542118 149074 149089 Zizxg’s’ng 2127 542185 245782 245797 Zizxzs’ng 2194 Trealmenl Prior to the study, the monkeys were kept in quarantine during which the s 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 ed male cynomolgus monkeys each were injected aneously 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 s were dosed three times (days 1, 4, and 7) for the ?rst week, and then subsequently once a week for 12 weeks with 40 mg/kg of ISIS oligonucleotide. A l group of 5 cynomolgus monkeys was ed 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 identi?ed for ?thher monitoring and le euthanasia. Scheduled euthanasia of the animals was conducted on day 86 by exsanguination after ketamine/xylazine-induced anesthesia and stration of sodium pentobarbital. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).

Hepatic 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 ofmRNA expression of GHR. s are presented as percent change 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 234, treatment with ISIS antisense oligonucleotides ed in significant reduction of GHR mRNA in comparison to the PBS control. Specifically, treatment with ISIS 532401 resulted in significant reduction ofmRNA expression in all tissues.

Table 234 Percent tion of GHR mRNA in the cynomolgus monkey liver relative to the PBS l ISIS No Liver Kidney WAT 532401 60 47 59 532254 63 65 n.d. 523723 38 0 n.d. 542112 61 60 36 542118 0 22 27 542185 66 53 n.d. 541767 0 14 n.d. 541875 34 77 n.d.

Approximately 1 mL ofblood was ted 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 235. The results indicate that treatment with ISIS oligonucleotides resulted in reduced IGF-l protein levels.

Table 235 Plasma protein levels in the cynomolgus monkey IGF-l (% GH baseline) (ng/mL) PBS 121 19 532401 57 39 532254 51 26 523723 77 16 542112 46 48 542118 97 6 542185 59 32 541767 101 22 541875 45 47 Tolerability studies Body and organ weight ements To evaluate the effect of ISIS ucleotides 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 236. Organ weights were measured on day 86 and the data is also presented in Table 236. The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides. Specifically, ent with ISIS 532401 was well tolerated in terms of the body and organ weights of the monkeys.

Table 236 Final body and organ weights in cynomolgus monkey Sple Kidn Body Wt (kg) en ey r (g) PBS 2.7 2.8 12.3 56.7 532401 2.6 4.0 11.5 58.5 532254 2.6 4.8 15.4 69.5 523723 2.8 3.1 14.8 69.4 542112 2.6 3.5 13.6 60.0 542118 2.7 2.7 11.9 58.6 542185 2.6 5.5 17.2 68.5 541767 2.8 5.1 11.7 65.1 541875 2.8 5.5 13.2 55.0 Liverfunction To evaluate the effect of ISIS oligonucleotides on hepatic ?anction, 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 agulant, which were ?Jged to obtain . Levels of various liver ?mction markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Plasma levels of ALT and AST and bilirubin were measured. The results indicate that antisense ucleotides had no effect on liver function outside the expected range for antisense oligonucleotides. Speci?cally, treatment with ISIS 532401 was well tolerated in terms of the liver function in monkeys.

Kidneyfunction To evaluate the effect of ISIS oligonucleotides on kidney function, blood s 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 ning K2- EDTA anticoagulant, which were centri?Jged to obtain plasma. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).

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

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 KZ-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 er (Bayer, USA).

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

C—reaclz've protein level analysis To evaluate any in?ammatory effect of ISIS oligonucleotides in lgus 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 ature for a minimum of 90 min and then centri?Jged 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 in?ammation, was measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). The s indicate that treatment with ISIS 532401 did not cause in?ammation in monkeys.

Example 137: Measurement of viscosity of ISIS antisense oligonucleotides ing human GHR The viscosity of select nse 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 CF. Oligonucleotides having a viscosity greater than 40 cP would be too viscous to be administered to any subject.

ISIS ucleotides (32-35 mg) were weighed into a glass vial, 120 uL of water was added and the antisense oligonucleotide was dissolved into solution by g the vial at 50°C. Part of (75 uL) the pre- heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometer was set to 250C and the viscosity of the sample was measured. Another part (20 uL) of the ated sample was pipetted into 10 mL of water for UV reading at 260 nM at 850C (Cary UV instrument). The results are presented in Table 237 and indicate that all the antisense oligonucleotides solutions are optimal in their viscosity under the criterion stated above.

Table 237 Viscosity of ISIS antisense oligonucleotides targeting human GHR ISIS . ity Chemistry No. (cP) 523723 55 MOE 8 532254 55 MOE 22 532401 55 MOE 12 Deoxy, MOE 541767 13 and (S)-cEt Deoxy, MOE 541875 33 and (S)-cEt Deoxy, MOE 542112 10 and (S)-cEt Deoxy, MOE 542118 14 and (S)-cEt Deoxy, MOE 542185 17 and (S)-cEt e 138: Effect of ISIS oligonucleotides conjugated with 3-7 vs. unconjugated in a mouse model.

ISIS oligonucleotides targeting murine GHR and that were either unconjugated 0r conjugated with GalNAc3-7 were tested in BALB/c mice for efficacy and tolerability. BALB/c mice are a multipurpose mice model, frequently utilized for safety and efficacy testing.

The oligonucleotides are all 55 MOE s, which are 20 nucleosides in length, wherein the central gap segment comprises often 2’-de0xynucleosides and is ?anked by wing segments on the 5’ direction and the 3 ’ direction comprising five nucleosides each. Each nucleoside in the 5 ’ wing segment and each nucleoside in the 3 ’ wing t has a 2’-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P=S) linkages. All cytosine es throughout each gapmer are 5- methylcytosines. "Start site" indicates the 5 ’-most nucleoside to which the gapmer is targeted in the murine gene sequence. "Stop site" indicates the 3 ’-m0st nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to murine GHR mRNA, designated herein as SEQ ID NO: 2333 (GENBANK Accession No. NM_010284.2). The ucleotides are described in detail in the Table below.

Table 238 ISIS antisense oligonucleotides targeting murine GHR and conjugated with GalNAc3-7 0r unconjugated Target SEQ Sequence Conjugated.

N0. Start ID Site NO 563179 TGCCAACTCACTTGGATGTC N0 772 2334 739949 TGCCAACTCACTTGGATGTC Yes 772 2334 563223 GAGACTTTTCCTTGTACACA N0 3230 2335 706937 GAGACTTTTCCTTGTACACA Yes 3230 2335 Treatment Two groups of seven-week old female BALB/c mice were injected subcutaneously for 4 weeks with 10 mg/kg/week, 25 mg/kg/week, or 50 mg/kg/week 0f ISIS 563223 or ISIS 563179. Two groups of seven- week old female BALB/c mice were injected subcutaneously for 4 weeks with 1 mg/kg/week, 5 mg/kg/week, or 10 mg/kg/week 0f ISIS 706937 or ISIS 739949. One group of female BALB/c mice was ed subcutaneously for 4 weeks with PBS. Mice were ized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

Target reduction To te the efficacy of the ISIS oligonucleotides, plasma IGF-l levels and mRNA expression levels of IGF-1 and GHR in liver, as well as mRNA expression levels of GHR in fat and kidney tissues, were ed. The results are presented in the Tables below.

The results te that the GalNAc3 -7 -conjugated oligonucleotides, ISIS 706937 and ISIS 739949, are 7-8 times more potent than the parent oligonucleotides with the same sequence, ISIS 563223 and ISIS 563179, in reducing GHR liver mRNA levels and were 6- to 8-fold more potent in ng liver and plasma IGF-l levels. Expression of GHR levels in the kidney and fat tissues were not decreased with GalNAc3 -7 - conjugated oligonucleotides, since the 3-7 conjugate group targeted the oligonucleotide speci?cally to the liver. This loss in fat and kidney reduction with GalNAc3conjugated oligonucleotides did not affect reduction of IGF-1.

Table 239 Liver mRNA expression levels (% inhibition) at week 4 mg/kg/wk GHR ED50 IGF-l ED50 62 15 ISIS 563223 25 97 4.2 69 19.4 50 99 77 1 59 24 ISIS 706937 5 97 0.6 63 3.4 98 69 50 22 ISIS 563179 25 67 9.6 31 49.4 50 93 50 1 39 18 ISIS 739949 5 89 1.2 57 6.4 94 45 Table 240 Plasma IGF-l levels (% inhibition) at week 4 mg/kg/wk Week 2 Week 4 PBS - 0 0 13 22 ISIS 563223 25 40 60 50 43 71 1 20 31 ISIS 706937 5 46 64 61 67 19 25 ISIS 563179 25 10 24 50 25 46 1 11 24 ISIS 739949 29 41 Table 241 GHR mR\IA expression levels (% inhibition) in fat and kidney at week 4 mg/kg/wk Fat Kidney 21 45 ISIS 563223 25 30 66 50 62 65 1 0 5 ISIS 706937 5 0 0 0 14 4 38 ISIS 563179 25 14 40 50 20 41 1 4 1 1 ISIS 739949 5 0 1 0 8 Plasma chemistry markers To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, glucose, cholesterol, and cerides were measured using an automated al chemistry analyzer (Beckman Coulter AU480, Brea, CA). The results are presented in the Table below. None of the ISIS oligonucleotides caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense ucleotides. The GalNAc3conjugated oligonucleotides had a ly improved profile over the parent oligonucleotides.

Table 242 Plasma chemistry markers in BALB/c mice plasma at week 4 ALT AST Bilirubin Glucose Cholesterol Triglycerides mg/kg/wk (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) (mg/dL) PBS - 26 58 0.2 165 70 123 23 69 0 .3 157 74 1 86 ISIS 563223 25 39 91 0.3 165 62 160 50 49 118 0.3 159 56 115 1 25 62 0.2 152 64 167 ISIS 706937 5 28 64 0.2 180 53 140 27 65 0 .2 165 5 6 133 28 78 0.4 156 65 131 ISIS 563179 2 8 95 0 .2 152 5 9 1 18 Is1s7s9949——————m The results taken together indicate that oligonucleotides targeting GHR mRNA expression when conjugated with GalNAc3-7 had tenfold r potency and similar or improved tolerability pro?les compared to the parent oligonucleotides. e 139: Tolerability study of an ISIS oligonucleotide conjugated with GalNAc3-7 and ing human GHR in mice.

ISIS 766720 was designed with the same sequence as ISIS 532401, a potent and tolerable oligonucleotide targeting human GHR and described in the s above. ISIS 766720 is a 55 MOE gapmer with mixed backbone chemistry and conjugated with GalNAc3-7. The chemical structure of ISIS 766720 Without the GalNAc3-7 conjugate group is denoted as mCes mCes Aeo mCeo mCes Tds Tds Tds Gds Gds Gds Tds Gds Ads Ads Teo Aeo Ges mCes Ae (SEQ ID NO: 703) and is fully denoted as: Trealmenl Groups of six-week old male CD-1 mice were injected subcutaneously for 6 weeks with 25 mg/kg/week, 50 mg/kg/week, or 100 mg/kg/week of ISIS 766720. One group of mice was injected subcutaneously for 6 weeks (days 1, 5, 15, 22, 29, 36, and 43) with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for ?thher analysis.

Plasma chemistry markers To evaluate the effect of ISIS 766720 on liver and kidney ?mction, plasma levels of transaminases, bilirubin, creatinine and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are ted in the Table below. ISIS 766720 did not cause changes in the levels of any of the liver or kidney ?mction markers outside the ed range for antisense ucleotides and was deemed very tolerable.

Table 243 Plasma chemistry markers in CD-1 mice plasma at week 6 ---—Cream BUN g g (IU/L) (IU/L) (m-/dL) (m/dL) (m/dL) ___-___ 29 47 0 2 0 2 34 ISIS 766720 Body and organ weights Body and organ weights were measured at the end of the study. The results are presented in the Table below. ISIS 766720 did not cause changes in weights outside the expected range for antisense ucleotides and was deemed very tolerable.

Table 244 Weights of CD-1 mice at week 6 - Body Liver Kidney Spleen mg/kg/Wk (%body> (%body> > ___—_— ___"— ISIS 766720 ___—IE- ___—.m-

Claims (8)

Claims:

1. A compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 linked sides having a nucleobase sequence consisting of the sequence 5 recited in SEQ ID NO: 703, wherein the modified oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides; a 5’ wing segment consisting of 5 linked nucleosides; and a 3’ wing segment consisting of 5 linked sides; wherein the gap segment is positioned n the 5’ wing segment and the 3’ wing segment and wherein 10 each nucleoside of each wing t ses a modified sugar, and wherein the conjugate group comprises:

2. The compound according to claim 1, wherein the modified oligonucleotide has the following formula: 15 mCes mCes Aeo mCeo mCes Tds Tds Tds Gds Gds Gds Tds Gds Ads Ads Teo Aeo Ges mCes Ae (SEQ ID NO: 703); wherein A = an adenine nucleobase, mC = a 5-methylcytosine nucleobase, G = a guanine base, 20 T = a thymine nucleobase, e = a 2’-O-methoxyethyl modified sugar moiety, d = a 2’-deoxy sugar moiety, s = a phosphorothioate internucleoside linkage, and o = a phosphodiester internucleoside linkage, wherein the conjugate group comprises a GalNAc3-7 conjugate attached to the 5’ end of the modified ucleotide, wherein the GalNAc3-7 conjugate has the formula: 5 .

3. A compound having the following chemical ure: (SEQ ID NO: 703), or a salt thereof.

4. A compound, wherein the anion form of the nd has the following chemical structure: 5 (SEQ ID NO: 703).

5. A composition comprising the compound of any one of claims 1-4, or a salt f and at least one of a ceutically acceptable carrier or diluent. 5

6. The compound of any one of claims 1-4, or the composition of claim 5, for use in therapy.

7. Use of a therapeutically effective amount of the compound of any one of claims 1-4, or the composition of claim 5, in the manufacture of a medicament for treating a disease associated with excess growth hormone in a human, n (a) the disease associated with excess growth hormone is acromegaly and/or 10 (b) the medicament is formulated to reduce IGF-1 levels.

8. A method of treating a disease associated with excess growth hormone in a non-human, wherein the method comprises administering to the non-human a therapeutically effective amount of the compound of any one of claims 1-4, or the composition of claim 5, wherein (a) the disease associated with excess growth 15 e is acromegaly and/or (b) the treating reduces IGF-1 levels. BIOL0253WOSEQ_ST25