COMPOSITIONS AND METHODS FOR MODULATING COMPLEMENT FACTOR B EXPRESSION

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0251USD1SEQ_ST25.txt created Mar. 18, 2019, which is 204 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

The present embodiments provide methods, compounds, and compositions for treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway by administering a Complement Factor B (CFB) specific inhibitor to a subject.

BACKGROUND

The complement system is part of the host innate immune system involved in lysing foreign cells, enhancing phagocytosis of antigens, clumping antigen-bearing agents, and attracting macrophages and neutrophils. The complement system is divided into three initiation pathways—the classical, lectin, and alternative pathways—that converge at component C3 to generate an enzyme complex known as C3 convertase, which cleaves C3 into C3a and C3b. C3b associates with C3 convertase mediated by CFB and results in generation of C5 convertase, which cleaves C5 into C5a and C5b, which initiates the membrane attack pathway resulting in the formation of the membrane attack complex (MAC) comprising components C5b, C6, C7, C8, and C9. The membrane-attack complex (MAC) forms transmembrane channels and disrupts the phospholipid bilayer of target cells, leading to cell lysis.

In the homeostatic state, the alternative pathway is continuously activated at a low “tickover” level as a result of activation of the alternative pathway by spontaneous hydrolysis of C3 and the production of C3b, which generates C5 convertase.

SUMMARY

The complement system mediates innate immunity and plays an important role in normal inflammatory response to injury, but its dysregulation may cause severe injury. Activation of the alternative complement pathway beyond its constitutive “tickover” level can lead to unrestrained hyperactivity and manifest as diseases of complement dysregulation.

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject by administration of a Complement Factor B (CFB) specific inhibitor. Several embodiments provided herein are drawn to a method of inhibiting expression of CFB in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway by administering a CFB specific inhibitor to the subject. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a CFB specific inhibitor to the subject. In several embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a CFB specific inhibitor to the subject.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21^stedition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2^ndEdition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, 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 nucleoside comprising a sugar comprising fluorine at the 2′ position. Unless otherwise indicated, the fluorine 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 modified sugar moiety.

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

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

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

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

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

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

“Alkyl,” as used herein, means a saturated straight or branched hydrocarbon 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-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, 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. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein, “alkynyl,” means a straight or branched 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 typically 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 radical 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 carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.

As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise 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 substituent groups.

As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing 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 interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.

As used herein, “alkoxy” means a radical formed between 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 limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein, “aminoalkyl” means an amino substituted C₁-C₁₂alkyl 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 further substituent group at the alkyl and/or amino portions.

As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C₁-C₁₂alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without 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 further substituent groups.

“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

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

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

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

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

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

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

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

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

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

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

“Carbohydrate” means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

“Carbohydrate cluster” 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 conjugate clusters).

“Carbohydrate derivative” means any compound which may be synthesized 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 formula: 4′-CH(CH₃)—O-2′.

“Chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence.

“Cleavable bond” means any chemical bond capable of being split. In certain embodiments, 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.

“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 distribution, 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 conjugate 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 3′-hydroxyl group of the 3′ terminal nucleoside of the oligomeric compound. In certain embodiments the point of attachment on the oligomeric 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 embodiments, conjugate groups comprise a cleavable 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 identified by the number and identity of the ligand. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups and is designated “GalNAc₃”. In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups and is designated “GalNAc4”. Specific carbohydrate cluster portions (having specific tether, branching and conjugate 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 embodiments, conjugate compounds may possess or impart one or more properties, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.

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

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

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

“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

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

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

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

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

“Deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).

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

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

“Differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases 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.

“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 relevant conditions.

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

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

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

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

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

“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”

“Halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.

“Heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused 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 sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached 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 further 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 include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

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

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

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

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

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

“Internucleoside neutral 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 nucleosides.

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

“Linkage motif” means a pattern of linkage modifications in an oligonucleotide or region thereof. The nucleosides of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.

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

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

“Locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge.

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

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

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

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

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

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

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

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

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

“Mono or polycyclic ring system” is meant to include all ring systems selected from single or polycyclic radical ring systems wherein the rings are fused or linked and is meant to be inclusive of single and mixed ring systems individually selected from aliphatic, alicyclic, aryl, heteroaryl, aralkyl, arylalkyl, heterocyclic, heteroaryl, hetero¬aromatic and heteroarylalkyl. Such mono and poly cyclic structures can contain rings that each have the same level of saturation or each, independently, have varying degrees of saturation including fully saturated, partially saturated or fully unsaturated. Each ring can comprise ring atoms selected from C, N, O and S to give rise to hetero¬cyclic 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 fused ring has two nitrogen atoms. The mono or polycyclic ring system can be further substituted with substituent groups such as for example phthalimide which has two ═O 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 multiple 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 (2′-OH).

“Naturally occurring internucleoside 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 phospho¬triesters, 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 linking 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 P. D. 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.

“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 linking group links two groups, neither of which is a nucleoside.

“Non-internucleoside phosphorus 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 embodiments, a non-internucleoside phosphorus linking group links two groups, neither of which is a nucleoside.

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

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

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

“Nucleobase 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 nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleoside motif” means a pattern of nucleoside modifications in an oligonucleotide or a region thereof. The linkages of such an oligonucleotide may be modified or unmodified. Unless otherwise indicated, motifs herein describing 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.

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

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

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

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

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

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

“Phosphorus linking group” means a linking group comprising a phosphorus atom. Phosphorus linking groups include without limitation groups having the formula:

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wherein:

R_aand R_dare each, independently, O, S, CH₂, NH, or NJ₁wherein J₁is C₁-C₆alkyl or substituted C₁-C₆alkyl;

R_bis O or S;

R_cis OH, SH, C₁-C₆alkyl, substituted C₁-C₆alkyl, C₁-C₆alkoxy, substituted C₁-C₆alkoxy, amino or substituted amino; and

J₁is R_bis O or S.

Phosphorus linking groups include without limitation, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, phosphoramidate, phosphorothioamidate, thionoalkylphosphonate, phosphotriesters, thionoalkylphosphotriester and boranophosphate.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound

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

“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, compound (e.g., drug).

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

“Protecting group” means any compound 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 0-471-62301-6, John Wiley & Sons, Inc, New York, which is incorporated herein by reference in its entirety.

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

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

“RISC based antisense compound” means an antisense compound wherein at least some of the antisense activity of the antisense compound 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 cleavage of the target nucleic acid by RNase H.

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

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

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

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

“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

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

“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 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unpro¬tected. In certain embodiments, compounds 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 hydro¬carbyl group to a parent compound.

Likewise, as used herein, “substituent” in reference to a chemical functional 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 hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C¬(O)¬Raa), carboxyl (—C(O)O-Raa), aliphatic groups, ali-cyclic groups, alkoxy, substituted oxy (—O—Raa), aryl, aralkyl, heterocyclic radical, hetero¬aryl, hetero-arylalkyl, amino (N(Rbb)¬(Rcc)), imino(=NRbb), amido (C(O)N¬(Rbb)(Rcc) or N(Rbb)C(O)Raa), azido (—N3), nitro (NO2), cyano (—CN), carbamido (OC(O)N(Rbb)(Rcc) or N(Rbb)¬C(O)¬ORaa), ureido (N(Rbb)C(O)¬N(Rbb)(Rcc)), thioureido (N(Rbb)C¬¬¬(S)N(Rbb)¬(Rcc)), guanidinyl (N(Rbb)¬C(═NRbb)-N(Rbb)(Rcc)), amidinyl (C(=NRbb)¬¬N(Rbb)(Rcc) or N(Rbb)C(=NRbb)(Raa)), thiol (—SRbb), sulfinyl (S(O)Rbb), sulfonyl (—S(O)2Rbb) and sulfonamidyl (—S(O)2N(Rbb)(Rcc) or N(Rbb)¬S¬¬(O)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 without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and hetero¬aryl¬alkyl. Selected substituents within the compounds described herein are present to a recursive degree.

“Substituted sugar moiety” means a furanosyl that is not a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, 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 modifications 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 oligomeric 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, sulfur, 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 moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholinos, cyclohexenyls and cyclohexitols.

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

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

“Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

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

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

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“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 internucleoside linkage” means the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.

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

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

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

“Unmodified nucleotide” means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Certain Embodiments

Certain embodiments provide methods, compounds and compositions for inhibiting Complement Factor B (CFB) expression.

Certain embodiments provide antisense compounds targeted to a CFB nucleic acid. In certain embodiments, the CFB nucleic acid has the sequence set forth in GENBANK Accession No. NM_001710.5 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to U.S. Pat. No. 31,861,000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No NW_001116486.1 truncated from nucleotides 536000 to 545000 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. XM_001113553.2 (incorporated herein as SEQ ID NO: 4), or GENBANK Accession No. NM_008198.2 (incorporated herein as SEQ ID NO: 5).

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 6-808.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleobases 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, or 2616-2631 of SEQ ID NO: 1, and wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, or 2616-2631 of SEQ ID NO:1, and wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 1.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleobases 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, or 7846-7862 of SEQ ID NO: 2, and wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2.

Certain embodiments provide a compound comprising a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, and 7846-7862 of SEQ ID NO: 2, and wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2.

In certain embodiments, antisense compounds or oligonucleotides target a region of a CFB nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a CFB 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 nucleobase portion complementary to an equal length portion of a region recited herein. In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting any of the following nucleotide regions of SEQ ID NO: 1: 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, and 2616-2631.

In certain embodiments, antisense compounds or oligonucleotides target a region of a CFB nucleic acid. In certain embodiments, such compounds or oligonucleotides targeted to a region of a CFB 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 nucleobase portion complementary to an equal length portion of a region recited herein. In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting the following nucleotide regions of SEQ ID NO: 2: 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, and 7846-7862.

In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting the 3′UTR of a CFB nucleic acid. In certain aspects, the modified oligonucleotide targets within nucleotides 2574-2626 of a CFB nucleic acid having the nucleobase sequence of SEQ ID NO: 1. In certain aspects, the modified oligonucleotide has at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion within nucleotides 2574-2626 of a CFB nucleic acid having the nucleobase sequence of SEQ ID NO: 1.

In certain embodiments, a compound comprises or consists of a conjugate and a modified oligonucleotide targeting a region of a CFB nucleic acid having the nucleobase sequence of SEQ ID NO: 1 within nucleobases 2457-2631, 2457-2472, 2457-2474, 2457-2476, 2457-2566, 2457-2570, 2457-2571, 2457-2572, 2457-2573, 2457-2574, 2457-2575, 2457-2576, 2457-2577, 2457-2578, 2457-2579, 2457-2580, 2457-2581, 2457-2582, 2457-2583, 2457-2584, 2457-2585, 2457-2586, 2457-2587, 2457-2588, 2457-2589, 2457-2590, 2457-2591, 2457-2592, 2457-2593, 2457-2594, 2457-2595, 2457-2596, 2457-2597, 2457-2598, 2457-2599, 2457-2600, 2457-2601, 2457-2602, 2457-2603, 2457-2604, 2457-2605, 2457-2606, 2457-2607, 2457-2608, 2457-2609, 2457-2610, 2457-2611, 2457-2612, 2457-2613, 2457-2614, 2457-2615, 2457-2616, 2457-2617, 2457-2618, 2457-2619, 2457-2620, 2457-2621, 2457-2622, 2457-2623, 2457-2624, 2457-2625, 2457-2626, 2457-2627, 2457-2628, 2457-2629, 2457-2630, 2457-2631, 2459-2474, 2459-2476, 2459-2566, 2459-2570, 2459-2571, 2459-2572, 2459-2573, 2459-2574, 2459-2575, 2459-2576, 2459-2577, 2459-2578, 2459-2579, 2459-2580, 2459-2581, 2459-2582, 2459-2583, 2459-2584, 2459-2585, 2459-2586, 2459-2587, 2459-2588, 2459-2589, 2459-2590, 2459-2591, 2459-2592, 2459-2593, 2459-2594, 2459-2595, 2459-2596, 2459-2597, 2459-2598, 2459-2599, 2459-2600, 2459-2601, 2459-2602, 2459-2603, 2459-2604, 2459-2605, 2459-2606, 2459-2607, 2459-2608, 2459-2609, 2459-2610, 2459-2611, 2459-2612, 2459-2613, 2459-2614, 2459-2615, 2459-2616, 2459-2617, 2459-2618, 2459-2619, 2459-2620, 2459-2621, 2459-2622, 2459-2623, 2459-2624, 2459-2625, 2459-2626, 2459-2627, 2459-2628, 2459-2629, 2459-2630, 2459-2631, 2461-2476, 2461-2566, 2461-2570, 2461-2571, 2461-2572, 2461-2573, 2461-2574, 2461-2575, 2461-2576, 2461-2577, 2461-2578, 2461-2579, 2461-2580, 2461-2581, 2461-2582, 2461-2583, 2461-2584, 2461-2585, 2461-2586, 2461-2587, 2461-2588, 2461-2589, 2461-2590, 2461-2591, 2461-2592, 2461-2593, 2461-2594, 2461-2595, 2461-2596, 2461-2597, 2461-2598, 2461-2599, 2461-2600, 2461-2601, 2461-2602, 2461-2603, 2461-2604, 2461-2605, 2461-2606, 2461-2607, 2461-2608, 2461-2609, 2461-2610, 2461-2611, 2461-2612, 2461-2613, 2461-2614, 2461-2615, 2461-2616, 2461-2617, 2461-2618, 2461-2619, 2461-2620, 2461-2621, 2461-2622, 2461-2623, 2461-2624, 2461-2625, 2461-2626, 2461-2627, 2461-2628, 2461-2629, 2461-2630, 2461-2631, 2551-2566, 2551-2570, 2551-2571, 2551-2572, 2551-2573, 2551-2574, 2551-2575, 2551-2576, 2551-2577, 2551-2578, 2551-2579, 2551-2580, 2551-2581, 2551-2582, 2551-2583, 2551-2584, 2551-2585, 2551-2586, 2551-2587, 2551-2588, 2551-2589, 2551-2590, 2551-2591, 2551-2592, 2551-2593, 2551-2594, 2551-2595, 2551-2596, 2551-2597, 2551-2598, 2551-2599, 2551-2600, 2551-2601, 2551-2602, 2551-2603, 2551-2604, 2551-2605, 2551-2606, 2551-2607, 2551-2608, 2551-2609, 2551-2610, 2551-2611, 2551-2612, 2551-2613, 2551-2614, 2551-2615, 2551-2616, 2551-2617, 2551-2618, 2551-2619, 2551-2620, 2551-2621, 2551-2622, 2551-2623, 2551-2624, 2551-2625, 2551-2626, 2551-2627, 2551-2628, 2551-2629, 2551-2630, 2551-2631, 2553-2570, 2553-2571, 2553-2572, 2553-2573, 2553-2574, 2553-2575, 2553-2576, 2553-2577, 2553-2578, 2553-2579, 2553-2580, 2553-2581, 2553-2582, 2553-2583, 2553-2584, 2553-2585, 2553-2586, 2553-2587, 2553-2588, 2553-2589, 2553-2590, 2553-2591, 2553-2592, 2553-2593, 2553-2594, 2553-2595, 2553-2596, 2553-2597, 2553-2598, 2553-2599, 2553-2600, 2553-2601, 2553-2602, 2553-2603, 2553-2604, 2553-2605, 2553-2606, 2553-2607, 2553-2608, 2553-2609, 2553-2610, 2553-2611, 2553-2612, 2553-2613, 2553-2614, 2553-2615, 2553-2616, 2553-2617, 2553-2618, 2553-2619, 2553-2620, 2553-2621, 2553-2622, 2553-2623, 2553-2624, 2553-2625, 2553-2626, 2553-2627, 2553-2628, 2553-2629, 2553-2630, 2553-2631, 2554-2573, 2554-2574, 2554-2575, 2554-2576, 2554-2577, 2554-2578, 2554-2579, 2554-2580, 2554-2581, 2554-2582, 2554-2583, 2554-2584, 2554-2585, 2554-2586, 2554-2587, 2554-2588, 2554-2589, 2554-2590, 2554-2591, 2554-2592, 2554-2593, 2554-2594, 2554-2595, 2554-2596, 2554-2597, 2554-2598, 2554-2599, 2554-2600, 2554-2601, 2554-2602, 2554-2603, 2554-2604, 2554-2605, 2554-2606, 2554-2607, 2554-2608, 2554-2609, 2554-2610, 2554-2611, 2554-2612, 2554-2613, 2554-2614, 2554-2615, 2554-2616, 2554-2617, 2554-2618, 2554-2619, 2554-2620, 2554-2621, 2554-2622, 2554-2623, 2554-2624, 2554-2625, 2554-2626, 2554-2627, 2554-2628, 2554-2629, 2554-2630, 2554-2631, 2555-2572, 2555-2573, 2555-2574, 2555-2575, 2555-2576, 2555-2577, 2555-2578, 2555-2579, 2555-2580, 2555-2581, 2555-2582, 2555-2583, 2555-2584, 2555-2585, 2555-2586, 2555-2587, 2555-2588, 2555-2589, 2555-2590, 2555-2591, 2555-2592, 2555-2593, 2555-2594, 2555-2595, 2555-2596, 2555-2597, 2555-2598, 2555-2599, 2555-2600, 2555-2601, 2555-2602, 2555-2603, 2555-2604, 2555-2605, 2555-2606, 2555-2607, 2555-2608, 2555-2609, 2555-2610, 2555-2611, 2555-2612, 2555-2613, 2555-2614, 2555-2615, 2555-2616, 2555-2617, 2555-2618, 2555-2619, 2555-2620, 2555-2621, 2555-2622, 2555-2623, 2555-2624, 2555-2625, 2555-2626, 2555-2627, 2555-2628, 2555-2629, 2555-2630, 2555-2631, 2556-2573, 2556-2574, 2556-2575, 2556-2576, 2556-2577, 2556-2578, 2556-2579, 2556-2580, 2556-2581, 2556-2582, 2556-2583, 2556-2584, 2556-2585, 2556-2586, 2556-2587, 2556-2588, 2556-2589, 2556-2590, 2556-2591, 2556-2592, 2556-2593, 2556-2594, 2556-2595, 2556-2596, 2556-2597, 2556-2598, 2556-2599, 2556-2600, 2556-2601, 2556-2602, 2556-2603, 2556-2604, 2556-2605, 2556-2606, 2556-2607, 2556-2608, 2556-2609, 2556-2610, 2556-2611, 2556-2612, 2556-2613, 2556-2614, 2556-2615, 2556-2616, 2556-2617, 2556-2618, 2556-2619, 2556-2620, 2556-2621, 2556-2622, 2556-2623, 2556-2624, 2556-2625, 2556-2626, 2556-2627, 2556-2628, 2556-2629, 2556-2630, 2556-2631, 2557-2574, 2557-2575, 2557-2576, 2557-2577, 2557-2578, 2557-2579, 2557-2580, 2557-2581, 2557-2582, 2557-2583, 2557-2584, 2557-2585, 2557-2586, 2557-2587, 2557-2588, 2557-2589, 2557-2590, 2557-2591, 2557-2592, 2557-2593, 2557-2594, 2557-2595, 2557-2596, 2557-2597, 2557-2598, 2557-2599, 2557-2600, 2557-2601, 2557-2602, 2557-2603, 2557-2604, 2557-2605, 2557-2606, 2557-2607, 2557-2608, 2557-2609, 2557-2610, 2557-2611, 2557-2612, 2557-2613, 2557-2614, 2557-2615, 2557-2616, 2557-2617, 2557-2618, 2557-2619, 2557-2620, 2557-2621, 2557-2622, 2557-2623, 2557-2624, 2557-2625, 2557-2626, 2557-2627, 2557-2628, 2557-2629, 2557-2630, 2557-2631, 2558-2575, 2558-2576, 2558-2577, 2558-2578, 2558-2579, 2558-2580, 2558-2581, 2558-2582, 2558-2583, 2558-2584, 2558-2585, 2558-2586, 2558-2587, 2558-2588, 2558-2589, 2558-2590, 2558-2591, 2558-2592, 2558-2593, 2558-2594, 2558-2595, 2558-2596, 2558-2597, 2558-2598, 2558-2599, 2558-2600, 2558-2601, 2558-2602, 2558-2603, 2558-2604, 2558-2605, 2558-2606, 2558-2607, 2558-2608, 2558-2609, 2558-2610, 2558-2611, 2558-2612, 2558-2613, 2558-2614, 2558-2615, 2558-2616, 2558-2617, 2558-2618, 2558-2619, 2558-2620, 2558-2621, 2558-2622, 2558-2623, 2558-2624, 2558-2625, 2558-2626, 2558-2627, 2558-2628, 2558-2629, 2558-2630, 2558-2631, 2559-2576, 2559-2577, 2559-2578, 2559-2579, 2559-2580, 2559-2581, 2559-2582, 2559-2583, 2559-2584, 2559-2585, 2559-2586, 2559-2587, 2559-2588, 2559-2589, 2559-2590, 2559-2591, 2559-2592, 2559-2593, 2559-2594, 2559-2595, 2559-2596, 2559-2597, 2559-2598, 2559-2599, 2559-2600, 2559-2601, 2559-2602, 2559-2603, 2559-2604, 2559-2605, 2559-2606, 2559-2607, 2559-2608, 2559-2609, 2559-2610, 2559-2611, 2559-2612, 2559-2613, 2559-2614, 2559-2615, 2559-2616, 2559-2617, 2559-2618, 2559-2619, 2559-2620, 2559-2621, 2559-2622, 2559-2623, 2559-2624, 2559-2625, 2559-2626, 2559-2627, 2559-2628, 2559-2629, 2559-2630, 2559-2631, 2560-2577, 2560-2578, 2560-2579, 2560-2580, 2560-2581, 2560-2582, 2560-2583, 2560-2584, 2560-2585, 2560-2586, 2560-2587, 2560-2588, 2560-2589, 2560-2590, 2560-2591, 2560-2592, 2560-2593, 2560-2594, 2560-2595, 2560-2596, 2560-2597, 2560-2598, 2560-2599, 2560-2600, 2560-2601, 2560-2602, 2560-2603, 2560-2604, 2560-2605, 2560-2606, 2560-2607, 2560-2608, 2560-2609, 2560-2610, 2560-2611, 2560-2612, 2560-2613, 2560-2614, 2560-2615, 2560-2616, 2560-2617, 2560-2618, 2560-2619, 2560-2620, 2560-2621, 2560-2622, 2560-2623, 2560-2624, 2560-2625, 2560-2626, 2560-2627, 2560-2628, 2560-2629, 2560-2630, 2560-2631, 2561-2578, 2561-2579, 2561-2580, 2561-2581, 2561-2582, 2561-2583, 2561-2584, 2561-2585, 2561-2586, 2561-2587, 2561-2588, 2561-2589, 2561-2590, 2561-2591, 2561-2592, 2561-2593, 2561-2594, 2561-2595, 2561-2596, 2561-2597, 2561-2598, 2561-2599, 2561-2600, 2561-2601, 2561-2602, 2561-2603, 2561-2604, 2561-2605, 2561-2606, 2561-2607, 2561-2608, 2561-2609, 2561-2610, 2561-2611, 2561-2612, 2561-2613, 2561-2614, 2561-2615, 2561-2616, 2561-2617, 2561-2618, 2561-2619, 2561-2620, 2561-2621, 2561-2622, 2561-2623, 2561-2624, 2561-2625, 2561-2626, 2561-2627, 2561-2628, 2561-2629, 2561-2630, 2561-2631, 2562-2577, 2562-2578, 2562-2579, 2562-2580, 2562-2581, 2562-2582, 2562-2583, 2562-2584, 2562-2585, 2562-2586, 2562-2587, 2562-2588, 2562-2589, 2562-2590, 2562-2591, 2562-2592, 2562-2593, 2562-2594, 2562-2595, 2562-2596, 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2591-2631, 2592-2611, 2592-2612, 2592-2613, 2592-2614, 2592-2615, 2592-2616, 2592-2617, 2592-2618, 2592-2619, 2592-2620, 2592-2621, 2592-2622, 2592-2623, 2592-2624, 2592-2625, 2592-2626, 2592-2627, 2592-2628, 2592-2629, 2592-2630, 2592-2631, 2593-2608, 2593-2612, 2593-2613, 2593-2614, 2593-2615, 2593-2616, 2593-2617, 2593-2618, 2593-2619, 2593-2620, 2593-2621, 2593-2622, 2593-2623, 2593-2624, 2593-2625, 2593-2626, 2593-2627, 2593-2628, 2593-2629, 2593-2630, 2593-2631, 2594-2612, 2594-2613, 2594-2614, 2594-2615, 2594-2616, 2594-2617, 2594-2618, 2594-2619, 2594-2620, 2594-2621, 2594-2622, 2594-2623, 2594-2624, 2594-2625, 2594-2626, 2594-2627, 2594-2628, 2594-2629, 2594-2630, 2594-2631, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2595-2615, 2595-2616, 2595-2617, 2595-2618, 2595-2619, 2595-2620, 2595-2621, 2595-2622, 2595-2623, 2595-2624, 2595-2625, 2595-2626, 2595-2627, 2595-2628, 2595-2629, 2595-2630, 2595-2631, 2596-2614, 2596-2615, 2596-2616, 2596-2617, 2596-2618, 2596-2619, 2596-2620, 2596-2621, 2596-2622, 2596-2623, 2596-2624, 2596-2625, 2596-2626, 2596-2627, 2596-2628, 2596-2629, 2596-2630, 2596-2631, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2597-2617, 2597-2618, 2597-2619, 2597-2620, 2597-2621, 2597-2622, 2597-2623, 2597-2624, 2597-2625, 2597-2626, 2597-2627, 2597-2628, 2597-2629, 2597-2630, 2597-2631, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2598-2618, 2598-2619, 2598-2620, 2598-2621, 2598-2622, 2598-2623, 2598-2624, 2598-2625, 2598-2626, 2598-2627, 2598-2628, 2598-2629, 2598-2630, 2598-2631, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2599-2619, 2599-2620, 2599-2621, 2599-2622, 2599-2623, 2599-2624, 2599-2625, 2599-2626, 2599-2627, 2599-2628, 2599-2629, 2599-2630, 2599-2631, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2600-2620, 2600-2621, 2600-2622, 2600-2623, 2600-2624, 2600-2625, 2600-2626, 2600-2627, 2600-2628, 2600-2629, 2600-2630, 2600-2631, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2601-2621, 2601-2622, 2601-2623, 2601-2624, 2601-2625, 2601-2626, 2601-2627, 2601-2628, 2601-2629, 2601-2630, 2601-2631, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2602-2622, 2602-2623, 2602-2624, 2602-2625, 2602-2626, 2602-2627, 2602-2628, 2602-2629, 2602-2630, 2602-2631, 2603-2620, 2603-2621, 2603-2622, 2603-2623, 2603-2624, 2603-2625, 2603-2626, 2603-2627, 2603-2628, 2603-2629, 2603-2630, 2603-2631, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2604-2624, 2604-2625, 2604-2626, 2604-2627, 2604-2628, 2604-2629, 2604-2630, 2604-2631, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2605-2625, 2605-2626, 2605-2627, 2605-2628, 2605-2629, 2605-2630, 2605-2631, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2606-2626, 2606-2627, 2606-2628, 2606-2629, 2606-2630, 2606-2631, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2607-2627, 2607-2628, 2607-2629, 2607-2630, 2607-2631, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2608-2628, 2608-2629, 2608-2630, 2608-2631, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2609-2629, 2609-2630, 2609-2631, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2610-2630, 2610-2631, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2611-2631, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, or 2616-2631. In certain aspects, antisense compounds or oligonucleotides target at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases within the aforementioned nucleobase regions.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 50% inhibition: 30-49, 48-63, 150-169, 151-170, 152-171, 154-169, 154-173, 156-171, 156-175, 157-176, 158-173, 158-177, 480-499, 600-619, 638-657, 644-663, 738-757, 1089-1108, 1135-1154, 1141-1160, 1147-1166, 1150-1169, 1153-1172, 1159-1178, 1162-1181, 1165-1184, 1171-1186, 1171-1190, 1173-1188, 1173-1192, 1175-1190, 1175-1194, 1177-1196, 1183-1202, 1208-1227, 1235-1254, 1298-1317, 1304-1323, 1310-1329, 1316-1335, 1319-1338, 1322-1341, 1328-1347, 1349-1368, 1355-1374, 1393-1412, 1396-1415, 1399-1418, 1405-1424, 1421-1440, 1621-1640, 1646-1665, 1646-1665, 1647-1666, 1689-1708, 1749-1768, 1763-1782, 1912-1931, 2073-2092, 2085-2104, 2166-2185, 2172-2191, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2196-2215, 2197-2212, 2197-2216, 2202-2221, 2223-2238, 2223-2242, 2225-2240, 2226-2245, 2227-2242, 2227-2246, 2238-2257, 2241-2260, 2267-2286, 2361-2380, 2388-2407, 2397-2416, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2532-2551, 2550-2569, 2551-2566, 2551-2570, 2552-2568, 2552-2570, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2570, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2573, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2576, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2585, 2570-2587, 2570-2589, 2571-2586, 2571-2588, 2571-2590, 2572-2589, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2590, 2574-2591, 2574-2593, 2575-2590, 2575-2591, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2601, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2601, 2586-2602, 2586-2604, 2586-2605, 2587-2602, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2605, 2588-2606, 2588-2607, 2589-2604, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2590-2609, 2591-2607, 2591-2608, 2591-2609, 2591-2610, 2592-2607, 2592-2608, 2592-2609, 2592-2610, 2592-2611, 2593-2608, 2593-2609, 2593-2610, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2631, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 50% inhibition: 1608-1627, 1685-1704, 1686-1705, 1751-1770, 1769-1784, 1871-1890, 1872-1891, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1878-1897, 1879-1894, 1879-1898, 2288-2307, 2808-2827, 2846-2865, 2852-2871, 2946-2965, 3773-3792, 3819-3838, 3825-3844, 3831-3850, 3834-3853, 3837-3856, 3843-3862, 4151-4166, 4151-4170, 4153-4172, 4159-4178, 4184-4203, 4211-4230, 4609-4628, 4612-4631, 4615-4634, 4621-4640, 4642-4661, 4648-4667, 4686-4705, 4689-4708, 4692-4711, 4698-4717, 4714-4733, 5270-5289, 5295-5314, 5296-5315, 5830-5849, 5890-5909, 5904-5923, 6406-6425, 6662-6681, 6674-6693, 6954-6973, 6960-6979, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6984-7003, 6985-7000, 6985-7004, 6990-7009, 7122-7141, 7125-7144, 7151-7170, 7353-7372, 7362-7381, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7694-7713, 7696-7711, 7696-7715, 7767-7786, 7785-7804, 7786-7801, 7787-7803, 7787-7805, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7805, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7808, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7811, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7820, 7805-7822, 7805-7824, 7806-7821, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7825, 7809-7826, 7809-7828, 7810-7825, 7810-7826, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7836, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7836, 7821-7837, 7821-7839, 7821-7840, 7822-7837, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7839, 7823-7840, 7823-7841, 7823-7842, 7824-7839, 7824-7840, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7843, 7826-7844, 7826-7845, 7827-7842, 7827-7843, 7827-7844, 7827-7845, 7827-7846, 7828-7843, 7828-7844, 7828-7845, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, and 7846-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 60% inhibition: 48-63, 150-169, 152-171, 154-169, 154-173, 156-171, 156-175, 158-173, 158-177, 600-619, 1135-1154, 1141-1160, 1147-1166, 1153-1172, 1171-1186, 1173-1188, 1175-1190, 1749-1768, 1763-1782, 1763-1782, 1912-1931, 2189-2208, 2191-2210, 2193-2212, 2195-2210, 2195-2214, 2197-2212, 2197-2216, 2223-2238, 2225-2240, 2227-2242, 2238-2257, 2448-2467, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2459-2478, 2461-2476, 2461-2480, 2550-2569, 2551-2566, 2552-2571, 2553-2568, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574, 2557-2575, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2583, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2587, 2570-2589, 2571-2588, 2572-2590, 2572-2591, 2573-2590, 2573-2592, 2574-2591, 2574-2593, 2575-2590, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2595, 2577-2596, 2578-2594, 2578-2597, 2579-2598, 2580-2596, 2580-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2598, 2581-2599, 2581-2600, 2582-2598, 2582-2599, 2582-2600, 2582-2601, 2583-2599, 2583-2600, 2583-2601, 2583-2602, 2584-2600, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2602, 2586-2604, 2586-2605, 2587-2603, 2587-2605, 2587-2606, 2588-2603, 2588-2604, 2588-2606, 2588-2607, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2608, 2590-2609, 2591-2607, 2591-2609, 2591-2610, 2592-2608, 2592-2609, 2592-2611, 2593-2608, 2593-2609, 2593-2612, 2594-2609, 2594-2610, 2594-2611, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2612, 2596-2613, 2596-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2618, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2626, 2611-2627, 2611-2628, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2630, 2615-2631, 2615-2631, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 60% inhibition: 1685-1704, 1686-1705, 1769-1784, 1871-1890, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1879-1894, 1879-1898, 2808-2827, 3819-3838, 3825-3844, 3831-3850, 3837-3856, 4151-4166, 5890-5909, 5904-5923, 5904-5923, 6406-6425, 6977-6996, 6979-6998, 6981-7000, 6983-6998, 6983-7002, 6985-7000, 6985-7004, 7122-7141, 7683-7702, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7696-7711, 7696-7715, 7786-7801, 7787-7806, 7788-7803, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7809, 7792-7810, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7818, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7822, 7805-7824, 7806-7823, 7806-7825, 7807-7824, 7807-7825, 7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7825, 7810-7827, 7810-7829, 7811-7828, 7811-7830, 7812-7829, 7812-7830, 7812-7831, 7813-7829, 7813-7832, 7814-7833, 7815-7831, 7815-7832, 7815-7833, 7815-7834, 7816-7832, 7816-7833, 7816-7834, 7816-7835, 7817-7833, 7817-7834, 7817-7835, 7817-7836, 7818-7834, 7818-7835, 7818-7836, 7818-7837, 7819-7835, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7837, 7821-7839, 7821-7840, 7822-7838, 7822-7840, 7822-7841, 7823-7838, 7823-7839, 7823-7841, 7823-7842, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7843, 7825-7844, 7826-7842, 7826-7844, 7826-7845, 7827-7843, 7827-7844, 7827-7846, 7828-7843, 7828-7844, 7828-7847, 7829-7844, 7829-7845, 7829-7846, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7847, 7831-7848, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7853, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, 7846-7862, and 7847-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 70% inhibition: 48-63, 150-169, 152-171, 154-169, 154-173, 156-171, 156-175, 158-173, 158-177, 1135-1154, 1141-1160, 1147-1166, 1171-1186, 1173-1188, 1175-1190, 1749-1768, 1763-1782, 1912-1931, 2193-2212, 2195-2210, 2195-2214, 2197-2212, 2197-2216, 2223-2238, 2225-2240, 2227-2242, 2453-2472, 2455-2474, 2457-2472, 2457-2476, 2459-2474, 2461-2476, 2461-2480, 2550-2569, 2551-2566, 2552-2571, 2553-2570, 2553-2571, 2553-2572, 2554-2571, 2554-2572, 2554-2573, 2554-2573, 2555-2572, 2555-2574, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574, 2557-2576, 2558-2575, 2558-2576, 2558-2577, 2559-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2578, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2566-2585, 2567-2582, 2567-2584, 2567-2586, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2587, 2570-2589, 2571-2588, 2571-2590, 2572-2589, 2572-2591, 2573-2590, 2573-2592, 2574-2591, 2574-2593, 2575-2592, 2575-2594, 2576-2593, 2576-2595, 2577-2594, 2577-2596, 2578-2597, 2579-2598, 2580-2596, 2580-2598, 2580-2599, 2581-2597, 2581-2600, 2582-2598, 2582-2600, 2582-2601, 2583-2599, 2583-2601, 2583-2602, 2584-2600, 2584-2602, 2584-2603, 2585-2601, 2585-2603, 2585-2604, 2586-2605, 2587-2606, 2588-2604, 2588-2606, 2588-2607, 2589-2605, 2589-2606, 2589-2607, 2589-2608, 2590-2605, 2590-2606, 2590-2607, 2590-2609, 2591-2607, 2591-2610, 2592-2611, 2593-2608, 2593-2612, 2594-2609, 2594-2610, 2594-2612, 2594-2613, 2595-2610, 2595-2611, 2595-2612, 2595-2613, 2595-2614, 2596-2611, 2596-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2615, 2599-2616, 2599-2617, 2599-2618, 2600-2615, 2600-2616, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2618, 2601-2619, 2601-2620, 2602-2617, 2602-2618, 2602-2619, 2602-2620, 2602-2621, 2603-2619, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2626, 2608-2627, 2609-2624, 2609-2625, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2627, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2629, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2630, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, 2615-2630, 2615-2631, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 70% inhibition: 1685-1704, 1686-1705, 1769-1784, 1871-1890, 1873-1892, 1875-1890, 1875-1894, 1877-1892, 1877-1896, 1879-1894, 1879-1898, 3819-3838, 3825-3844, 3831-3850, 4151-4166, 5890-5909, 5904-5923, 5904-5923, 6406-6425, 6983-6998, 6983-7002, 6985-7000, 6985-7004, 7688-7707, 7690-7709, 7692-7707, 7692-7711, 7694-7709, 7696-7711, 7696-7715, 7786-7801, 7787-7806, 7788-7805, 7788-7806, 7788-7807, 7789-7806, 7789-7807, 7789-7808, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7809, 7792-7811, 7793-7810, 7793-7811, 7793-7812, 7794-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7813, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7801-7820, 7802-7817, 7802-7819, 7802-7821, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7822, 7805-7824, 7806-7823, 7806-7825, 7807-7824, 7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7827, 7811-7828, 7811-7830, 7812-7829, 7812-7831, 7813-7832, 7814-7833, 7815-7831, 7815-7833, 7815-7834, 7816-7832, 7816-7835, 7817-7833, 7817-7835, 7817-7836, 7818-7834, 7818-7836, 7818-7837, 7819-7835, 7819-7837, 7819-7838, 7820-7836, 7820-7838, 7820-7839, 7821-7840, 7822-7841, 7823-7839, 7823-7841, 7823-7842, 7824-7840, 7824-7841, 7824-7842, 7824-7843, 7825-7840, 7825-7841, 7825-7842, 7825-7844, 7826-7842, 7826-7845, 7827-7846, 7828-7843, 7828-7847, 7829-7844, 7829-7845, 7829-7847, 7829-7848, 7830-7845, 7830-7846, 7830-7847, 7830-7848, 7830-7849, 7831-7846, 7831-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7850, 7834-7851, 7834-7852, 7834-7853, 7835-7850, 7835-7851, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7853, 7836-7854, 7836-7855, 7837-7852, 7837-7853, 7837-7854, 7837-7855, 7837-7856, 7838-7854, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7861, 7843-7862, 7844-7859, 7844-7860, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7845-7862, 7846-7861, 7846-7862, and 7847-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 80% inhibition: 152-171, 154-169, 156-171, 158-173, 1135-1154, 1171-1186, 1173-1188, 1175-1190, 1763-1782, 1912-1931, 2197-2212, 2223-2238, 2225-2240, 2227-2242, 2457-2472, 2459-2474, 2461-2476, 2551-2566, 2553-2570, 2553-2571, 2553-2572, 2554-2573, 2555-2572, 2555-2574, 2556-2573, 2556-2574, 2556-2575, 2557-2574, 2557-2576, 2558-2575, 2558-2576, 2559-2577, 2559-2578, 2560-2577, 2560-2578, 2560-2579, 2561-2578, 2561-2579, 2561-2580, 2562-2577, 2562-2579, 2562-2581, 2563-2580, 2563-2582, 2564-2581, 2564-2583, 2565-2584, 2566-2583, 2567-2584, 2567-2586, 2568-2585, 2568-2587, 2569-2586, 2569-2588, 2570-2587, 2571-2588, 2571-2590, 2572-2589, 2572-2591, 2573-2590, 2573-2592, 2574-2591, 2574-2593, 2575-2592, 2576-2593, 2576-2595, 2577-2594, 2577-2596, 2578-2597, 2580-2598, 2580-2599, 2581-2597, 2581-2600, 2582-2601, 2583-2602, 2584-2603, 2585-2604, 2586-2605, 2587-2606, 2588-2607, 2589-2608, 2590-2606, 2590-2607, 2590-2609, 2591-2610, 2592-2611, 2593-2608, 2593-2612, 2594-2613, 2595-2611, 2595-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2614, 2597-2615, 2597-2616, 2598-2613, 2598-2613, 2598-2614, 2598-2615, 2598-2616, 2598-2617, 2599-2614, 2599-2617, 2599-2618, 2600-2615, 2600-2617, 2600-2618, 2600-2619, 2601-2616, 2601-2617, 2601-2619, 2601-2620, 2602-2618, 2602-2621, 2603-2620, 2603-2621, 2603-2622, 2604-2619, 2604-2620, 2604-2621, 2604-2622, 2604-2623, 2605-2620, 2605-2621, 2605-2622, 2605-2623, 2605-2624, 2606-2621, 2606-2622, 2606-2623, 2606-2624, 2606-2625, 2607-2622, 2607-2623, 2607-2624, 2607-2625, 2607-2626, 2608-2623, 2608-2624, 2608-2625, 2608-2627, 2609-2624, 2609-2626, 2609-2627, 2609-2628, 2610-2625, 2610-2626, 2610-2628, 2610-2629, 2611-2626, 2611-2627, 2611-2629, 2611-2630, 2612-2627, 2612-2628, 2612-2630, 2612-2631, 2613-2628, 2613-2629, 2613-2631, 2614-2629, 2614-2630, 2614-2631, 2615-2630, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 80% inhibition: 1685-1704, 1686-1705, 1873-1892, 1875-1890, 1877-1892, 1879-1894, 3819-3838, 4151-4166, 5904-5923, 6406-6425, 6985-7000, 7692-7707, 7694-7709, 7696-7711, 7786-7801, 7788-7805, 7788-7806, 7788-7807, 7789-7808, 7790-7807, 7790-7809, 7791-7808, 7791-7809, 7791-7810, 7792-7809, 7792-7811, 7793-7810, 7793-7811, 7794-7812, 7794-7813, 7795-7812, 7795-7813, 7795-7814, 7796-7813, 7796-7814, 7796-7815, 7797-7812, 7797-7814, 7797-7816, 7798-7815, 7798-7817, 7799-7816, 7799-7818, 7800-7819, 7801-7818, 7802-7819, 7802-7821, 7803-7820, 7803-7822, 7804-7821, 7804-7823, 7805-7822, 7806-7823, 7806-7825, 7807-7824, 7807-7826, 7808-7825, 7808-7827, 7809-7826, 7809-7828, 7810-7827, 7811-7828, 7812-7829, 7812-7831, 7813-7832, 7814-7833, 7815-7834, 7816-7832, 7816-7835, 7817-7836, 7818-7837, 7819-7838, 7820-7839, 7821-7840, 7822-7841, 7823-7842, 7824-7843, 7825-7841, 7825-7842, 7825-7844, 7826-7845, 7827-7846, 7828-7843, 7828-7847, 7829-7848, 7830-7846, 7830-7849, 7831-7850, 7832-7847, 7832-7848, 7832-7849, 7832-7850, 7832-7851, 7833-7848, 7833-7849, 7833-7850, 7833-7851, 7833-7852, 7834-7849, 7834-7852, 7834-7853, 7835-7850, 7835-7852, 7835-7853, 7835-7854, 7836-7851, 7836-7852, 7836-7854, 7836-7855, 7837-7853, 7837-7856, 7838-7855, 7838-7856, 7838-7857, 7839-7854, 7839-7855, 7839-7856, 7839-7857, 7839-7858, 7840-7855, 7840-7856, 7840-7857, 7840-7858, 7840-7859, 7841-7856, 7841-7857, 7841-7858, 7841-7859, 7841-7860, 7842-7857, 7842-7858, 7842-7859, 7842-7860, 7842-7861, 7843-7858, 7843-7859, 7843-7860, 7843-7862, 7844-7859, 7844-7861, 7844-7862, 7845-7860, 7845-7861, 7846-7862, and 7847-7862.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 1, when targeted by antisense compounds or oligonucleotides, display at least 90% inhibition: 154-169, 156-171, 158-173, 1135-1154, 1171-1186, 1173-1188, 1763-1782, 1912-1931, 2223-2238, 2227-2242, 2459-2474, 2461-2476, 2554-2573, 2555-2574, 2560-2577, 2561-2578, 2561-2579, 2562-2581, 2563-2580, 2563-2582, 2564-2581, 2566-2583, 2567-2584, 2568-2585, 2568-2587, 2569-2586, 2570-2587, 2576-2593, 2577-2594, 2577-2596, 2578-2597, 2580-2599, 2581-2600, 2582-2601, 2583-2602, 2584-2603, 2586-2605, 2587-2605, 2587-2606, 2588-2607, 2589-2608, 2590-2607, 2590-2609, 2592-2611, 2595-2614, 2596-2615, 2597-2612, 2597-2613, 2597-2615, 2597-2616, 2598-2613, 2598-2613, 2598-2617, 2599-2614, 2599-2618, 2600-2615, 2600-2619, 2601-2617, 2601-2620, 2602-2621, 2603-2622, 2604-2623, 2605-2621, 2605-2622, 2605-2624, 2606-2625, 2607-2626, 2608-2623, 2608-2625, 2609-2628, 2611-2627, 2611-2630, 2612-2628, 2612-2631, 2613-2629, 2614-2629, 2615-2630, and 2616-2631.

In certain embodiments, the following nucleotide regions of SEQ ID NO: 2, when targeted by antisense compounds or oligonucleotides, display at least 90% inhibition: 1685-1704, 1686-1705, 1875-1890, 1877-1892, 1879-1894, 3819-3838, 5904-5923, 6406-6425, 7694-7709, 7696-7711, 7789-7808, 7790-7809, 7795-7812, 7795-7813, 7796-7813, 7796-7814, 7797-7814, 7797-7816, 7798-7815, 7798-7817, 7799-7816, 7801-7818, 7802-7819, 7803-7820, 7803-7822, 7804-7821, 7805-7822, 7811-7828, 7812-7829, 7812-7831, 7813-7832, 7815-7834, 7818-7837, 7819-7838, 7821-7840, 7822-7840, 7822-7841, 7825-7842, 7832-7847, 7832-7848, 7832-7850, 7833-7848, 7833-7852, 7834-7849, 7834-7853, 7835-7850, 7836-7852, 7836-7855, 7837-7856, 7838-7856, 7839-7857, 7839-7858, 7840-7856, 7840-7857, 7840-7859, 7843-7858, 7843-7860, and 7846-7862.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 50% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614, 532632, 532635, 532638, 532639, 532686, 532687, 532688, 532689, 532690, 532691, 532692, 532692, 532693, 532694, 532695, 532696, 532697, 532698, 532699, 532700, 532701, 532702, 532703, 532704, 532705, 532706, 532707, 532770, 532775, 532778, 532780, 532791, 532800, 532809, 532810, 532811, 532917, 532952, 588509, 588510, 588511, 588512, 588513, 588514, 588515, 588516, 588517, 588518, 588519, 588520, 588522, 588523, 588524, 588525, 588527, 588528, 588529, 588530, 588531, 588532, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588566, 588567, 588568, 588569, 588570, 588571, 588572, 588573, 588574, 588575, 588576, 588577, 588580, 588581, 588585, 588586, 588589, 588590, 588599, 588603, 588606, 588608, 588610, 588614, 588616, 588628, 588631, 588632, 588634, 588636, 588638, 588640, 588645, 588646, 588654, 588656, 588658, 588660, 588662, 588664, 588670, 588672, 588676, 588682, 588688, 588696, 588698, 588807, 588808, 588809, 588813, 588814, 588815, 588819, 588820, 588822, 588823, 588838, 588839, 588840, 588841, 588842, 588846, 588847, 588848, 588849, 588850, 588851, 588852, 588853, 588854, 588855, 588856, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588864, 588865, 588866, 588867, 588868, 588870, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 588884, 598999, 599000, 599001, 599002, 599003, 599004, 599005, 599006, 599007, 599008, 599009, 599010, 599011, 599012, 599013, 599014, 599015, 599018, 599019, 599023, 599024, 599025, 599026, 599027, 599028, 599029, 599030, 599031, 599032, 599033, 599034, 599035, 599058, 599062, 599063, 599064, 599065, 599070, 599071, 599072, 599073, 599074, 599076, 599077, 599078, 599079, 599080, 599081, 599082, 599083, 599084, 599085, 599086, 599087, 599088, 599089, 599090, 599091, 599092, 599093, 599094, 599095, 599096, 599097, 599098, 599102, 599119, 599123, 599124, 599125, 599126, 599127, 599128, 599132, 599133, 599134, 599135, 599136, 599137, 599138, 599139, 599140, 599141, 599142, 599143, 599144, 599145, 599147, 599148, 599149, 599150, 599151, 599152, 599153, 599154, 599155, 599156, 599157, 599158, 599159, 599178, 599179, 599180, 599181, 599182, 599186, 599187, 599188, 599189, 599190, 599191, 599192, 599193, 599194, 599195, 599196, 599197, 599198, 599199, 599200, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599208, 599209, 599210, 599211, 599212, 599213, 599214, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599241, 599247, 599248, 599249, 599255, 599256, 599257, 599258, 599260, 599261, 599262, 599263, 599264, 599265, 599266, 599267, 599268, 599269, 599270, 599271, 599272, 599273, 599274, 599275, 599276, 599277, 599278, 599279, 599280, 599297, 599299, 599306, 599307, 599308, 599309, 599311, 599312, 599313, 599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321, 599322, 599323, 599324, 599325, 599326, 599327, 599328, 599329, 599330, 599338, 599349, 599353, 599354, 599355, 599356, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599369, 599371, 599372, 599373, 599376, 599378, 599379, 599382, 599383, 599384, 599385, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599394, 599395, 599396, 599397, 599398, 599399, 599400, 599401, 599402, 599403, 599404, 599405, 599406, 599407, 599408, 599409, 599410, 599412, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599425, 599426, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599442, 599443, 599444, 599445, 599446, 599447, 599448, 599450, 599454, 599455, 599456, 599467, 599468, 599469, 599471, 599472, 599473, 599474, 599475, 599476, 599477, 599478, 599479, 599480, 599481, 599482, 599483, 599484, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599493, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599501, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599509, 599512, 599515, 599518, 599531, 599541, 599541, 599546, 599547, 599548, 599549, 599550, 599552, 599553, 599554, 599555, 599557, 599558, 599561, 599562, 599563, 599564, 599565, 599566, 599567, 599568, 599569, 599570, 599577, 599578, 599579, 599580, 599581, 599581, 599582, 599584, 599585, 599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593, 599594, 599595, 601321, 601322, 601323, 601325, 601327, 601328, 601329, 601330, 601332, 601333, 601334, 601335, 601336, 601337, 601338, 601339, 601341, 601342, 601343, 601344, 601345, 601346, 601347, 601348, 601349, 601362, 601367, 601368, 601369, 601371, 601372, 601373, 601374, 601375, 601377, 601378, 601380, 601381, 601382, 601383, 601384, 601385, 601386, 601387, and 601388.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 50% inhibition of a CFB mRNA, SEQ ID NOs: 12, 30, 33, 36, 37, 84, 85, 86, 87, 88, 89, 90, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 198, 203, 206, 208, 219, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 468, 472, 473, 475, 478, 479, 488, 492, 494, 495, 498, 499, 500, 502, 503, 509, 510, 511, 512, 513, 514, 515, 517, 518, 522, 523, 524, 525, 529, 530, 531, 534, 535, 537, 540, 541, 542, 543, 544, 545, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 563, 564, 565, 569, 570, 572, 573, 577, 588, 589, 590, 591, 592, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 623, 640, 641, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 700, 704, 705, 706, 707, 708, 709, 711, 712, 713, 714, 715, 716, 717, 718, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 758, 759, 760, 761, 762, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 813, 833, 834, 841, 846, 849, 850, 867, and 873.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 60% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614, 532635, 532686, 532687, 532688, 532689, 532770, 532800, 532809, 532810, 532811, 532917, 532952, 588512, 588513, 588514, 588515, 588516, 588517, 588518, 588519, 588522, 588523, 588524, 588525, 588527, 588528, 588529, 588530, 588531, 588532, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588566, 588567, 588568, 588569, 588570, 588571, 588572, 588573, 588574, 588575, 588576, 588577, 588636, 588638, 588640, 588664, 588676, 588696, 588698, 588807, 588808, 588814, 588815, 588819, 588820, 588840, 588842, 588846, 588847, 588848, 588849, 588850, 588851, 588852, 588853, 588854, 588855, 588856, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588864, 588866, 588867, 588868, 588870, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 588884, 598999, 599000, 599001, 599002, 599003, 599004, 599005, 599006, 599007, 599008, 599009, 599010, 599011, 599012, 599013, 599014, 599015, 599019, 599024, 599025, 599026, 599027, 599028, 599029, 599030, 599031, 599032, 599033, 599034, 599035, 599064, 599065, 599071, 599072, 599077, 599078, 599079, 599080, 599083, 599084, 599085, 599086, 599087, 599088, 599089, 599090, 599091, 599092, 599093, 599094, 599095, 599096, 599097, 599125, 599126, 599127, 599133, 599134, 599135, 599136, 599138, 599139, 599140, 599141, 599142, 599148, 599149, 599150, 599151, 599152, 599154, 599155, 599156, 599157, 599158, 599159, 599178, 599179, 599180, 599181, 599187, 599188, 599190, 599192, 599193, 599194, 599195, 599196, 599197, 599198, 599199, 599200, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599208, 599209, 599210, 599211, 599212, 599213, 599214, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599247, 599255, 599256, 599257, 599263, 599264, 599265, 599266, 599270, 599271, 599272, 599273, 599274, 599275, 599276, 599277, 599278, 599279, 599280, 599306, 599307, 599308, 599311, 599312, 599313, 599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321, 599322, 599323, 599324, 599325, 599327, 599328, 599329, 599330, 599349, 599353, 599355, 599356, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599369, 599371, 599372, 599373, 599376, 599378, 599379, 599382, 599384, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599394, 599395, 599396, 599397, 599398, 599399, 599400, 599401, 599402, 599403, 599404, 599405, 599406, 599407, 599408, 599409, 599410, 599412, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599425, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599442, 599443, 599444, 599445, 599446, 599447, 599448, 599456, 599467, 599468, 599471, 599472, 599473, 599474, 599475, 599476, 599477, 599478, 599479, 599480, 599481, 599482, 599483, 599484, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599493, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599501, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599512, 599531, 599547, 599548, 599549, 599552, 599553, 599554, 599555, 599557, 599558, 599562, 599563, 599564, 599565, 599566, 599567, 599568, 599569, 599570, 599577, 599578, 599579, 599580, 599581, 599582, 599584, 599585, 599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593, 599594, 599595, 601323, 601327, 601329, 601332, 601333, 601333, 601334, 601335, 601336, 601338, 601339, 601341, 601342, 601343, 601344, 601345, 601346, 601347, 601348, 601349, 601368, 601369, 601371, 601372, 601374, 601375, 601377, 601378, 601380, 601381, 601382, 601383, 601384, 601385, 601386, 601387, and 601388.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 60% inhibition of a CFB mRNA, SEQ ID NOs: 12, 33, 84, 85, 86, 87, 198, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 472, 473, 513, 514, 515, 531, 537, 541, 542, 543, 544, 545, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 564, 565, 569, 570, 577, 590, 592, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 682, 683, 684, 685, 686, 687, 688, 689, 700, 704, 706, 707, 708, 709, 711, 712, 713, 714, 715, 716, 717, 720, 721, 722, 723, 724, 725, 726, 727, 727, 728, 729, 730, 731, 732, 733, 734, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 758, 759, 760, 761, 767, 768, 770, 772, 773, 774, 775, 775, 776, 776, 777, 777, 778, 779, 780, 781, 782, 783, 783, 784, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 813, 833, 834, 841, 846, 849, and 850.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 70% inhibition of a CFB mRNA, ISIS NOs: 516350, 532614, 532686, 532687, 532688, 532770, 532800, 532809, 532810, 532811, 532917, 532952, 588512, 588513, 588514, 588515, 588516, 588517, 588518, 588524, 588529, 588530, 588531, 588532, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588541, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588568, 588569, 588570, 588571, 588572, 588573, 588574, 588575, 588577, 588636, 588638, 588640, 588696, 588698, 588807, 588814, 588815, 588819, 588842, 588847, 588848, 588849, 588850, 588851, 588852, 588853, 588856, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588866, 588867, 588870, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 588884, 599000, 599001, 599003, 599004, 599005, 599008, 599009, 599010, 599011, 599014, 599015, 599024, 599025, 599027, 599028, 599029, 599030, 599031, 599032, 599033, 599034, 599072, 599077, 599080, 599085, 599086, 599087, 599088, 599089, 599090, 599091, 599093, 599094, 599095, 599096, 599097, 599125, 599126, 599134, 599138, 599139, 599148, 599149, 599150, 599151, 599152, 599154, 599155, 599156, 599157, 599158, 599187, 599188, 599193, 599195, 599196, 599197, 599198, 599199, 599200, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599208, 599210, 599211, 599212, 599213, 599214, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599266, 599272, 599272, 599273, 599274, 599275, 599277, 599278, 599279, 599280, 599280, 599306, 599311, 599312, 599313, 599314, 599315, 599316, 599317, 599318, 599319, 599320, 599321, 599322, 599323, 599325, 599327, 599328, 599329, 599330, 599355, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599369, 599371, 599372, 599373, 599378, 599379, 599382, 599384, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599394, 599395, 599396, 599397, 599398, 599399, 599400, 599401, 599402, 599403, 599404, 599405, 599406, 599407, 599408, 599409, 599410, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599442, 599443, 599445, 599446, 599447, 599448, 599472, 599473, 599474, 599475, 599476, 599477, 599478, 599479, 599480, 599481, 599482, 599483, 599484, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599493, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599501, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599512, 599547, 599548, 599552, 599553, 599554, 599555, 599558, 599562, 599563, 599564, 599566, 599567, 599568, 599569, 599570, 599577, 599578, 599579, 599580, 599581, 599582, 599585, 599586, 599587, 599588, 599589, 599590, 599591, 599592, 599593, 599594, 599595, 601332, 601335, 601341, 601343, 601344, 601345, 601346, 601347, 601348, 601349, 601371, 601372, 601380, 601382, 601383, 601384, 601385, 601386, and 601387.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 70% inhibition of a CFB mRNA, SEQ ID NOs: 12, 84, 85, 86, 198, 228, 237, 238, 239, 317, 395, 396, 397, 398, 399, 402, 403, 404, 405, 407, 408, 410, 411, 412, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 464, 465, 472, 473, 513, 514, 515, 541, 542, 543, 544, 545, 546, 547, 549, 550, 551, 552, 553, 554, 555, 556, 557, 564, 565, 569, 592, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 645, 646, 647, 648, 649, 650, 653, 654, 655, 656, 659, 660, 662, 663, 664, 665, 666, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 677, 678, 679, 680, 682, 683, 684, 686, 687, 688, 689, 706, 708, 709, 711, 712, 713, 714, 715, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 767, 768, 773, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 793, 794, 795, 797, 798, 799, 813, 833, 834, 841, 846, 849, 867, and 873.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least an 80% inhibition of a CFB mRNA, ISIS NOs: 532686, 532809, 532810, 532811, 532917, 532952, 588512, 588517, 588518, 588533, 588534, 588535, 588536, 588537, 588538, 588539, 588540, 588542, 588543, 588544, 588545, 588546, 588547, 588548, 588549, 588550, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588563, 588564, 588565, 588571, 588638, 588640, 588696, 588698, 588807, 588814, 588849, 588850, 588851, 588853, 588857, 588858, 588859, 588860, 588861, 588862, 588863, 588866, 588867, 588871, 588872, 588873, 588874, 588875, 588876, 588877, 588878, 588879, 588880, 588881, 588882, 588883, 599001, 599024, 599025, 599033, 599086, 599087, 599088, 599089, 599093, 599094, 599095, 599096, 599134, 599139, 599148, 599149, 599151, 599154, 599155, 599156, 599158, 599188, 599195, 599196, 599198, 599201, 599202, 599203, 599204, 599205, 599206, 599207, 599212, 599213, 599215, 599216, 599217, 599218, 599219, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599230, 599231, 599232, 599233, 599234, 599235, 599236, 599272, 599273, 599275, 599277, 599278, 599279, 599280, 599311, 599313, 599314, 599316, 599317, 599318, 599320, 599321, 599322, 599323, 599327, 599328, 599329, 599330, 599355, 599357, 599358, 599359, 599360, 599361, 599362, 599363, 599364, 599371, 599372, 599373, 599378, 599379, 599382, 599384, 599386, 599387, 599388, 599389, 599390, 599391, 599392, 599393, 599397, 599398, 599399, 599400, 599401, 599403, 599404, 599405, 599407, 599408, 599409, 599410, 599413, 599414, 599415, 599416, 599417, 599418, 599419, 599420, 599421, 599422, 599423, 599424, 599433, 599434, 599435, 599436, 599437, 599438, 599439, 599440, 599441, 599445, 599446, 599447, 599448, 599474, 599476, 599477, 599479, 599481, 599482, 599483, 599485, 599486, 599487, 599488, 599489, 599490, 599491, 599492, 599494, 599495, 599496, 599497, 599498, 599499, 599500, 599502, 599503, 599504, 599505, 599506, 599507, 599508, 599547, 599552, 599553, 599554, 599558, 599563, 599567, 599568, 599569, 599570, 599577, 599578, 599581, 599582, 599585, 599587, 599588, 599590, 599591, 599592, 599593, 599594, 601332, 601344, 601345, 601382, 601383, and 601385.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 80% inhibition of a CFB mRNA, SEQ ID NOs: 84, 237, 238, 239, 317, 395, 397, 411, 412, 413, 414, 415, 417, 418, 419, 420, 421, 422, 423, 425, 426, 427, 429, 430, 431, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 472, 473, 514, 515, 542, 543, 544, 545, 546, 547, 550, 551, 552, 553, 554, 555, 556, 557, 564, 595, 599, 600, 601, 602, 603, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 646, 655, 660, 662, 663, 666, 669, 670, 671, 672, 673, 675, 676, 677, 678, 679, 682, 684, 686, 687, 688, 689, 706, 708, 709, 711, 712, 713, 714, 715, 720, 722, 723, 724, 725, 726, 727, 729, 730, 731, 732, 733, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 768, 775, 776, 778, 781, 782, 783, 784, 785, 787, 788, 789, 790, 791, 792, 793, 794, 799, 813, 833, 834, 841, 849, 867, and 873.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 90% inhibition of a CFB mRNA, ISIS NOs: 532686, 532811, 532917, 588536, 588537, 588538, 588539, 588544, 588545, 588546, 588548, 588551, 588552, 588553, 588554, 588555, 588556, 588557, 588558, 588559, 588560, 588561, 588562, 588564, 588638, 588640, 588696, 588698, 588849, 588850, 588851, 588860, 588866, 588867, 588872, 588873, 588874, 588876, 588877, 588878, 588879, 588881, 588883, 599149, 599188, 599203, 599206, 599220, 599221, 599222, 599223, 599224, 599225, 599226, 599227, 599228, 599229, 599235, 599236, 599279, 599280, 599314, 599321, 599362, 599378, 599390, 599391, 599398, 599399, 599404, 599413, 599414, 599416, 599419, 599420, 599422, 599435, 599437, 599438, 599441, 599483, 599494, 599508, 599552, 599553, 599554, 599568, 599570, 599577, 599581, 599591, 599592, and 599593.

In certain embodiments, the following antisense compounds or oligonucleotides target a region of a CFB nucleic acid and effect at least a 90% inhibition of a CFB mRNA, SEQ ID NOs: 84, 238, 239, 317, 412, 413, 420, 421, 426, 434, 436, 437, 438, 439, 440, 442, 443, 444, 445, 446, 448, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 464, 465, 472, 473, 514, 515, 542, 543, 544, 545, 546, 551, 553, 555, 556, 599, 600, 601, 602, 610, 616, 617, 618, 662, 666, 670, 676, 677, 678, 688, 689, 713, 723, 729, 730, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 755, 756, 768, 783, 793, 833, and 867.

In certain embodiments, a compound can comprise or consist of any oligonucleotide targeted to CFB described herein and a conjugate group.

In certain embodiments, a compound comprises a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides complementary within nucleotides 2193-2212, 2195-2210, 2457-2476, 2571-2590, 2584-2603, 2588-2607, 2592-2611, 2594-2613, 2597-2616, 2600-2619, or 2596-2611 of SEQ ID NO: 1.

In certain embodiments, a compound comprises a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598.

In certain embodiments, a compound comprises a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide has a nucleobase sequence consisting of any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598.

In certain embodiments, any of the foregoing compounds or oligonucleotides can comprise at least one modified internucleoside linkage, at least one modified sugar, and/or at least one modified nucleobase.

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

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

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

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

In certain embodiments, a compound comprises a conjugate group and a modified oligonucleotide comprising:

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

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, or 598.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising or consisting of the sequence recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, or 455, wherein the modified oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

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

a 3′ wing segment consisting of five linked nucleosides;

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

In certain embodiments, a compound comprises or consists of a single-stranded modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 20 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 198, 228, 237, 440, 444, 448, 450, 453, or 455, wherein the oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

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

a 3′ wing segment consisting of five linked nucleosides;

In certain embodiments, a compound comprises or consists of ISIS 588540 and a conjugate group. In certain embodiments, ISIS 588540 has the following chemical structure:

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In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising or consisting of the sequence recited in SEQ ID NO: 549, wherein the modified oligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

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

a 3′ wing segment consisting of three 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 cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain aspects, the modified oligonucleotide has a nucleobase sequence comprising or consisting of the sequence recited in SEQ ID NO: 598, wherein the modified oligonucleotide comprises

a gap segment consisting of ten linked deoxynucleosides;

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

a 3′ wing segment consisting of three linked nucleosides;

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

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

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

In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5′ end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3′ end of the modified oligonucleotide. In certain embodiments, the conjugate group comprises at least one N-Acetylgalactosamine (GalNAc), at least two N-Acetylgalactosamines (GalNAcs), or at least three N-Acetylgalactosamines (GalNAcs).

In certain embodiments, a compound having the following chemical structure comprises or consists of ISIS 588540 with a 5′-X, wherein X is a conjugate group comprising GalNAc as described herein:

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In certain embodiments, a compound comprises or consists of SEQ ID NO: 440, 5′-GalNAc, and chemical modifications as represented by the following chemical structure:

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wherein either R¹is —OCH₂CH₂OCH₃(MOE) and R²is H; or R¹and R²together form a bridge, wherein R¹is —O— and R²is —CH₂—, —CH(CH₃)—, or —CH₂CH₂—, and R¹and R²are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And for each pair of R³and R⁴on the same ring, independently for each ring: either R³is selected from H and —OCH₂CH₂OCH₃and R⁴is H; or R³and R⁴together form a bridge, wherein R³is —O—, and R⁴is —CH₂—, —CH(CH₃)—, or —CH₂CH₂— and R³and R⁴are directly connected such that the resulting bridge is selected from: —O—CH₂—, —O—CH(CH₃)—, and —O—CH₂CH₂—;

And R⁵is selected from H and —CH₃;

And Z is selected from S⁻ and O⁻.

In certain embodiments, a compound comprises ISIS 696844. In certain embodiments, a compound consists of ISIS 696844. In certain embodiments, ISIS 696844 has the following chemical structure:

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In certain embodiments, a compound comprises ISIS 696845. In certain embodiments, a compound consists of ISIS 696845. In certain embodiments, ISIS 696845 has the following chemical structure:

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In certain embodiments, a compound comprises ISIS 698969. In certain embodiments, a compound consists of ISIS 698969. In certain embodiments, ISIS 698969 has the following chemical structure:

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In certain embodiments, a compound comprises ISIS 698970. In certain embodiments, a compound consists of ISIS 698970. In certain embodiments, ISIS 698970 has the following chemical structure:

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Certain embodiments provide compositions comprising any of the compounds comprising or consisting of a modified oligonucleotide targeted to CFB or salt thereof and a conjugate group, and at least one of a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the compounds or compositions as described herein are efficacious by virtue of having at least one of an in vitro IC₅₀of less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM, less than 65 nM, less than 60 nM, less than 55 nM, less than 50 nM, less than 45 nM, less than 40 nM, less than 35 nM, less than 30 nM, less than 25 nM, or less than 20 nM.

In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having at least one of an increase an ALT or AST value of no more than 4 fold, 3 fold, or 2 fold over saline treated animals or an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5%, or 2%. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase of ALT or AST over saline treated animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase in liver, spleen, or kidney weight over saline treated animals.

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

In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound or composition described herein, thereby treating, preventing, or ameliorating the disease. In certain aspects, the complement alternative pathway is activated greater than normal. In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970.

In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD) in a subject comprises administering to the subject a compound or composition described herein, thereby treating, preventing, or ameliorating AMD. In certain aspects, the complement alternative pathway is activated greater than normal. In certain aspects, the AMD is wet AMD. In certain aspects, the AMD is dry AMD, such as Geographic Atrophy. In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration in a subject, such as age-related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy comprises administering to the subject a a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy in a subject comprises administering to the subject a comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD), wet AMD, dry AMD, or Geographic Atrophy in a subject comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the compound or composition is administered to the subject parenterally.

In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound or composition described herein, thereby treating, preventing, or ameliorating the kidney disease. In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of treating, preventing, or ameliorating a kidney disease associated with dysregulation of the complement alternative pathway in a subject comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the complement alternative pathway is activated greater than normal. In certain aspects, the kidney disease is lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any combination thereof. In certain aspects, the kidney disease is associated with C3 deposits, such as C3 deposits in the glomerulus. In certain aspects, the kidney disease is associated with lower than normal circulating C3 levels, such as serum or plasma C3 levels. In certain aspects, administering the compound or composition reduces or inhibits accumulation of ocular C3 levels, such as C3 protein levels. In certain aspects, administering the compound or composition reduces the level of ocular C3 deposits or inhibits accumulation of ocular C3 deposits. In certain aspects, the compound or composition is administered to the subject parenterally. In certain aspects, administering the compound or composition reduces or inhibits accumulation of C3 levels in the kidney, such as C3 protein levels. In certain aspects, administering the compound or composition reduces the level of kidney C3 deposits or inhibits accumulation of kidney C3 deposits, such as C3 levels in the glomerulus. In certain aspects, the subject is identified as having or at risk of having a disease associated with dysregulation of the complement alternative pathway, for example by detecting complement levels or membrane-attack complex levels in the subject's blood and/or performing a genetic test for gene mutations of complement factors associated with the disease.

In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a compound or composition described herein to the subject, thereby inhibiting expression of CFB in the subject. In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of inhibiting expression of Complement Factor B (CFB) in a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, administering the compound or composition inhibits expression of CFB in the eye. In certain aspects, the subject has, or is at risk of having, age related macular degeneration (AMD), such as wet AMD and dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. Geographic Atrophy is considered an advanced form of dry AMD involving degeneration of the retina. In certain aspects, administering the compound or composition inhibits expression of CFB in the kidney, such as in the glomerulus. In certain aspects, the subject has, or is at risk of having, lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any combination thereof.

In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a compound or composition described herein to the subject, thereby reducing or inhibiting accumulation of C3 deposits in the eye of the subject. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the eye of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the subject has, or is at risk of having, age related macular degeneration (AMD), such as wet AMD and dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. In certain aspects, the compound or composition is administered to the subject parenterally.

In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering a compound or composition described herein to the subject, thereby reducing or inhibiting accumulation of C3 deposits in the kidney of the subject. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598. In certain embodiments, a method of reducing or inhibiting accumulation of C3 deposits in the kidney of a subject having, or at risk of having, a disease associated with dysregulation of the complement alternative pathway comprises administering to the subject a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970. In certain aspects, the subject has, or is at risk of having, lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any combination thereof. In certain aspects, the compound or composition is administered to the subject parenterally.

Certain embodiments are drawn to use of a compound or composition described herein for treating a disease associated with dysregulation of the complement alternative pathway. Certain embodiments are drawn to use of a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides and has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 6-808, for treating a disease associated with dysregulation of the complement alternative pathway. Certain embodiments are drawn to use of a compound comprising or consisting of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 198, 228, 237, 440, 444, 448, 450, 453, 455, 549, and 598, for treating a disease associated with dysregulation of the complement alternative pathway. Certain embodiments are drawn to use of a compound comprising or consisting of ISIS 696844, ISIS 696845, ISIS 698969, or ISIS 698970 for treating a disease associated with dysregulation of the complement alternative pathway. In certain aspects, the complement alternative pathway is activated greater than normal. In certain aspects, the disease is macular degeneration, such as age related macular degeneration (AMD), which can be wet AMD or dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. In certain aspects, the disease is a kidney disease such as lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any combination thereof. In certain aspects, the compound or composition is administered to the subject parenterally.

In certain embodiments, a compound or composition described herein is administered parenterally. For example, in certain embodiments the compound or composition can be administered through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound is 10 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compound is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 19 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.

In certain embodiments antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to an CFB nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

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

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

RNase H-Mediated Antisense

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

Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X—Y—Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X—Y—Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

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

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

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

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

each D is a 2′-deoxynucleoside;

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

provided that:

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

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

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

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

RNAi Compounds

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

i. ssRNA Compounds

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

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

embedded image

wherein:

T₁is an optionally protected phosphorus moiety;

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

A has one of the formulas:

embedded image

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

Q₃is O, S, N(R₅) or C(R₆)(R₇);

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

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

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

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

Bx₁is a heterocyclic base moiety;

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

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

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

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

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

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

X₁is O, S or N(E₁);

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

E₁, E₂and E₃are each, independently, H, C₁-C₆alkyl or substituted C₁-C₆alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

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

X₂is O, S or NJ₃;

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

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

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

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

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

In certain embodiments, A has one of the formulas:

embedded image

wherein:

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

In certain embodiments, T₁has the formula:

embedded image

wherein:

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

R_bis O or S. In certain embodiments, R₁, is O and R. and R, are each, independently, OCH₃, OCH₂CH₃or CH(CH₃)₂.

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

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

embedded image

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

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

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

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

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA;

or

ABABBAABBABABAA;

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

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

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

-(A)₂-(B)_x-(A)₂-(C)_y-(A)₃-

wherein: A is a first type of modified nucleoside;

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

x and y are from 1 to 15.

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

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(AB)_xA_y-(D)_z

wherein:

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

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

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

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(A)_x-(D)_z

wherein:

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

A is a first type of modified nucleoside;

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

X is 11-30;

Z is 0-4.

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

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

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

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

5′ most linkage
Central region
3′-region

PS
Alternating PO/PS
6 PS

PS
Alternating PO/PS
7 PS

PS
Alternating PO/PS
8 PS

ii. siRNA Compounds

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Occupancy

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

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode Complement Factor B (CFB) include, without limitation, the following: GENBANK Accession No. NM_001710.5 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to U.S. Pat. No. 31,861,000 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No NW_001116486.1 truncated from nucleotides 536000 to 545000 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. XM_001113553.2 (incorporated herein as SEQ ID NO: 4), or GENBANK Accession No. NM_008198.2 (incorporated herein as SEQ ID NO: 5).

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a CFB nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a CFB nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a CFB nucleic acid).

Non-complementary nucleobases between an antisense compound and a CFB nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a CFB nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a CFB nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a CFB nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a CFB nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a CFB nucleic acid, or specified portion thereof.

The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity. Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

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

In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside 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 certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

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

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 certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate internucleoside 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.

Modified Sugar Moieties

Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁and R₂are each independently H, C₁-C₁₂alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).

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

As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2; 4′—(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as constrained ethyl or cEt) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof see published International Application WO 2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see 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 Dec. 8, 2008).

Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U S. A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; 8,530,640; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 61/026,995 and 61/097,787; Published PCT International applications; WO 2009/067647; WO 2011/017521; WO 2010/036698 WO 1999/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R_a)(R_b)]_n—, —C(R_a)═C(R_b)—, —C(R_a)═N—, —C(═O)—, —C(═NR_a)—, —C(═S)—, —O—, —Si(R_a)₂—, —S(═O)_x—, and —N(R_a)—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

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

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

In certain embodiments, the bridge of a bicyclic sugar moiety is —[C(R_a)(R_b)]_n—, —[C(R_a)(R_b)]_n—O—, —C(R_aR_b)—N(R)—O— or —C(R_aR_b)—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′, 4′—(CH₂)₂-2′, 4′—(CH₂)₃-2′, 4′—CH₂—O-2′, 4′—(CH₂)₂—O-2′, 4′—CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently, H, a protecting group or C₁-C₁₂alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

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

embedded image

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

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

embedded image

wherein:

Bx is a heterocyclic base moiety;

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

R_cis C₁-C₁₂alkyl or an amino protecting group; and

T_aand T_bare each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

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

embedded image

wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

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

In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_c, NJ_cJ_d, SJ_c, N₃, OC(═X)J_c, and NJ_eC(═X)NJ_cJ_d, wherein each J_c, J_dand J_eis, independently, H, C₁-C₆alkyl, or substituted C₁-C₆alkyl and X is O or NJ_c.

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

embedded image

wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

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

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

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wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

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

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

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

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wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

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

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

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

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

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

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wherein:

Bx is a heterocyclic base moiety;

T_aand T_bare each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

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

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

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

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

As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH₂)_nO]_mCH₃, O(CH₂)_nNH₂, O(CH₂)_nCH₃, O(CH₂)_nF, O(CH₂)_nONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_nON[(CH₂)_nCH₃]₂, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:

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In certain embodiments, sugar surrogates are selected having Formula VII:

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

Bx is a heterocyclic base moiety;

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

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

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

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

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

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′, 2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

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

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

Bx is a heterocyclic base moiety;

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

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

As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′ substituents, such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀alkyl, —OCF₃, O—(CH₂)₂—O—CH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_m)(R_n), or O—CH₂—C(═O)—N(R_m)(R_n), where each R_mand R_nis, independently, H or substituted or unsubstituted C₁-C₁₀alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

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

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

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

As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

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

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.

Modified Nucleobases

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

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

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

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

Conjugated Antisense compounds

In certain embodiments, the present disclosure provides conjugated antisense compounds. In certain embodiments, the present disclosure provides conjugated antisense compounds comprising an antisense oligonucleotide complementary to a nucleic acid transcript. In certain embodiments, the present disclosure 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 contacting 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 receptors are expressed on liver cells, particularly 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). Accordingly, conjugates comprising such GalNAc clusters have been used to facilitate uptake of certain compounds into liver cells, specifically 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 concern 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. Pat. No. 8,106,022. Certain conjugate groups described herein are more active and/or easier to synthesize than conjugate groups previously described.

In certain embodiments of the present invention, conjugates are attached to single-stranded antisense compounds, including, but not limited to RNase H based antisense compounds 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 (improved 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 conjugate. Given the required balance of properties for these compounds such improved potency is surprising.

In certain embodiments, conjugate groups herein comprise a cleavable moiety. As noted, without wishing to be bound by mechanism, it is logical that the conjugate should remain on the compound long enough to provide enhancement 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 another, to the antisense compound and/or to the cluster via cleavable bonds (such as those of a phosphodiester linkage). Certain conjugates herein do not comprise a cleavable nucleoside and instead comprise a cleavable bond. It is shown that that sufficient cleavage of the conjugate from the oligonucleotide is provided by at least one bond that is vulnerable 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 attached 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 example, Examples 56, 81, 83, and 84). Further, 5′-attachment allows simpler oligonucleotide synthesis. Typically, oligonucleotides are synthesized on a solid support in the 3′ to 5′ direction. To make 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 approach, 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 standard automated techniques and introduce the conjugate with the final (5′-most) nucleoside or after the oligonucleotide has been cleaved 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. Moreover, 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, providing advantages in manufacturing. For example, the synthesis of certain conjugate groups consists of fewer synthetic steps, resulting in increased yield, relative to conjugate groups previously described. Conjugate groups such as GalNAc₃-10 in Example 46 and GalNAc₃-7 in Example 48 are much simpler than previously described conjugates such as those described in U.S. Pat. Nos. 8,106,022 or 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 oligonucleotides and either strand of double-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 compounds, 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 tolerability remains the same (or indeed even if tolerability worsens only slightly compared 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 consequences 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 replacement of one or more phosphorothioate linkages with phosphodiester linkages also results in reduced cellular uptake and/or loss in potency. In certain embodiments, 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 internucleoside linkage actually exhibit increased potency in vivo even relative to a full phosphorothioate counterpart also comprising the same conjugate. Moreover, since conjugation 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 comprise at least one phosphodiester linkage.

In certain embodiments, conjugation of antisense compounds herein results in increased delivery, uptake and activity in hepatocytes. Thus, more compound is delivered to liver tissue. 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 increase in activity. In such embodiments, productive uptake of the conjugated compound is increased. For example, as shown in Example 102, certain embodiments of GalNAc-containing conjugates increase enrichment of antisense oligonucleotides in hepatocytes versus non-parenchymal cells. This enrichment is beneficial for oligonucleotides that target genes that are expressed in hepatocytes.

In certain embodiments, 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 beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly for non-kidney targets, kidney accumulation is undesired.

In certain embodiments, the present disclosure provides conjugated antisense compounds represented by the formula:

A-B-C-DE-F)_q

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 ligand; and

q is an integer between 1 and 5.

In the above diagram and in similar diagrams herein, the branching 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 formula is:

A-B-C-D-E-F

where q=2, the formula is:

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where q=3, the formula is:

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where q=4, the formula is:

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where q=5, the formula is:

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In certain embodiments, conjugated antisense compounds are provided having the structure:

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In certain embodiments, conjugated antisense compounds are provided having the structure:

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In certain embodiments, conjugated antisense compounds are provided having the structure:

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In certain embodiments, conjugated antisense compounds are provided having the structure:

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The present disclosure provides the following non-limiting numbered embodiments:

Embodiment 1

The conjugated antisense compound of any of embodiments 1179 to 1182, wherein the tether has a structure selected from among:

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wherein each n is independently, 0, 1, 2, 3, 4, 5, 6, or 7.

Embodiment 2

The conjugated antisense compound of any of embodiments 1179 to 1182, wherein the tether has the structure:

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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:

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Embodiment 4

The conjugated antisense compound of any of embodiments 1179 to 1182 or 1688 to 1689, wherein the linker has a structure selected from among:

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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:

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In embodiments having more than one of a particular variable (e.g., more than one “m” or “n”), unless otherwise indicated, 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 embodiments, a cleavable moiety is a cleavable bond. In certain 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 embodiments, the nucleoside or nucleoside analog comprises an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside comprising an optionally protected heterocyclic base selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, 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 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 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 oligonucleotide. 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 embodiments, the cleavable moiety is attached to the antisense oligonucleotide by a phosphodiester linkage. In certain embodiments, the cleavable moiety is attached 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 embodiments, the cleavable moiety is cleaved after the complex has been administered to an animal only after being internalized by a targeted cell. Inside the cell the cleavable moiety is cleaved thereby releasing the active antisense oligonucleotide. While not wanting to be bound by theory it is believed 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:

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wherein each of Bx, Bx₁, Bx₂, and Bx₃is independently a heterocyclic base moiety. In certain embodiments, the cleavable moiety has a structure selected from among the following:

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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-targeting moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support. In certain embodiments, the linker further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker further comprises a covalent attachment to a solid support and further comprises a covalent attachment to a protein binding moiety. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands. In certain embodiments, the linker includes multiple positions for attachment of tethered ligands and is not attached to a branching group. In certain embodiments, the linker further 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 groups. 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 includes 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 antisense 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 embodiments, 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 scaffold includes a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the scaffold includes 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 certain 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 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 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 moiety, 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, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), a vitamin (e.g., folate, vitamin A, vitamin E, biotin, pyridoxal), a peptide, a carbohydrate (e.g., monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide), an endosomolytic component, a steroid (e.g., uvaol, hecigenin, diosgenin), a terpene (e.g., triterpene, e.g., sarsasapogenin, friedelin, epifriedelanol derivatized lithocholic acid), or a cationic lipid. In certain embodiments, the protein binding moiety is a C16 to C22 long chain saturated or unsaturated fatty acid, cholesterol, cholic acid, vitamin E, adamantane or 1-pentafluoropropyl.

In certain embodiments, a linker has a structure selected from among:

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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:

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wherein each n is, independently, from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

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wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

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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 structure selected from among:

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In certain embodiments, a linker has a structure selected from among:

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In certain embodiments, a linker has a structure selected from among:

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In certain embodiments, a linker has a structure selected from among:

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wherein n is from 1 to 20.

In certain embodiments, a linker has a structure selected from among:

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In certain embodiments, a linker has a structure selected from among:

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In certain embodiments, a linker has a structure selected from among:

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In certain embodiments, the conjugate linker has the structure:

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In certain embodiments, the conjugate linker has the structure:

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In certain embodiments, a linker has a structure selected from among:

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In certain embodiments, a linker has a structure selected from among:

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wherein each n is independently, 0, 1, 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 comprise a branching group, one or more tether, and one or more ligand. In certain 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 comprise a targeting moiety comprising a branching group and at least two tethered ligands. In certain embodiments, the branching group attaches the conjugate linker. In certain embodiments, the branching group attaches the cleavable moiety. In certain embodiments, the branching group attaches the antisense oligonucleotide. In certain embodiments, the branching group is covalently attached to the linker and each of the tethered ligands. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxylamino groups. In certain embodiments, the branching group comprises groups selected 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:

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wherein each n is, independently, from 1 to 20;

j is from 1 to 3; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

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wherein each n is, independently, from 1 to 20; and

m is from 2 to 6.

In certain embodiments, a branching group has a structure selected from among:

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In certain embodiments, a branching group has a structure selected from among:

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- wherein each A₁is 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 selected from among:

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- wherein each A₁is 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 selected from among:

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- wherein A₁is 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:

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In certain embodiments, a branching group has a structure selected from among:

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In certain embodiments, a branching group has a structure selected from among:

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2. Certain Tethers

In certain embodiments, conjugate groups comprise one or more tethers covalently attached to the branching group. In certain embodiments, conjugate groups comprise one or more tethers covalently attached 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 comprising 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 certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group.

In certain embodiments, the tether includes one or more cleavable bond. In certain embodiments, the tether is attached to the branching group through 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 embodiments, 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, 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 structure selected from among:

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wherein 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:

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In certain embodiments, a tether has a structure selected from among:

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- wherein each n is, independently, from 1 to 20.

In certain embodiments, a tether has a structure selected from among:

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- wherein L is either a phosphorus linking group or a neutral linking group;
- Z₁is C(═O)O—R₂;
- Z₂is H, C₁-C₆alkyl or substituted C₁-C₆alky;
- R₂is H, C₁-C₆alkyl or substituted C₁-C₆alky; and
- each m₁is, independently, from 0 to 20 wherein at least one m₁is greater than O for each tether.

In certain embodiments, a tether has a structure selected from among:

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In certain embodiments, a tether has a structure selected from among:

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- wherein Z₂is H or CH₃; and
- each m₁is, independently, from 0 to 20 wherein at least one m₁is greater than 0 for each tether.

In certain embodiments, a tether has a structure selected from among:

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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 certain embodiments, a tether comprises a phosphorus linking group and does not comprise any amide bonds.

3. Certain Ligands

In certain embodiments, the present disclosure provides ligands wherein 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 affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, ligands are selected that have an affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine, mannose, glucose, glucosamone and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain 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 galactoseamine 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, α-D-galactosamine, N-Acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-Amino-3-O—[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-Deoxy-2-methylamino-L-glucopyranose, 4,6-Dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-Deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-Glycoloyl-α-neuraminic acid. For example, thio sugars may be selected from the group consisting of 5-Thio-β-D-glucopyranose, Methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-Thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. In certain embodiments, “N-acetyl galactosamine” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, “GalNac” or “Gal-NAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, which includes both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose. In certain embodiments, both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose may be used interchangeably. Accordingly, in structures in which one form is depicted, these structures are intended to include the other form as well. For example, where the structure for an α-form: 2-(Acetylamino)-2-deoxy-D-galactopyranose is shown, this structure is intended to include the other form as well. In certain embodiments, In certain preferred embodiments, the β-form 2-(Acetylamino)-2-deoxy-D-galactopyranose is the preferred embodiment.

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In certain embodiments one or more ligand has a structure selected from among:

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wherein each R₁is selected from OH and NHCOOH.

In certain embodiments one or more ligand has a structure selected from among:

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In certain embodiments one or more ligand has a structure selected from among:

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In certain embodiments one or more ligand has a structure selected from among:

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i. Certain Conjugates

In certain embodiments, conjugate groups comprise the structural features above. In certain such embodiments, conjugate groups have the following structure:

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wherein each n is, independently, from 1 to 20.

In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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wherein each n is, independently, from 1 to 20;

Z is H or a linked solid support;

Q is an antisense compound;

X is O or S; and

Bx is a heterocyclic base moiety.

In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain embodiments, conjugates do not comprise a pyrrolidine.

In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain such embodiments, conjugate groups have the following structure:

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In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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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 following structure:

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wherein 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:

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wherein X is a substituted or unsubstituted tether of four to eleven consecutively bonded atoms and wherein the tether comprises exactly one amide bond.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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wherein Y and Z are independently selected from a C₁-C₁₂substituted 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:

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wherein Y and Z are independently selected from a C₁-C₁₂substituted 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 phosphorothioate.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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wherein Y and Z are independently selected from a C₁-C₁₂substituted or unsubstituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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wherein m and n are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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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:

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wherein X is a substituted or unsubstituted tether of four to thirteen consecutively bonded atoms, and wherein X does not comprise an ether group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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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 conjugate group has the following structure:

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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 conjugate group has the following structure:

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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 unsubstituted C₂-C₁₁alkyl group.

In certain embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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wherein Y is selected from a C₁-C₁₂substituted 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:

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wherein Y is selected from a C₁-C₁₂substituted or unsubstituted alkyl group, or a group comprising an ether, an amine, a piperidine, a phosphate, a phosphodiester, or a phosphorothioate.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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wherein Y is selected from a C₁-C₁₂substituted or unsubstituted alkyl group.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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Wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain such embodiments, the cell-targeting moiety of the conjugate group has the following structure:

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wherein n is 4, 5, 6, 7, or 8.

In certain embodiments, conjugates do not comprise a pyrrolidine.

a Certain Conjugated Antisense Compounds

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-C-DE-F)_q

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 ligand; 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)_q

wherein

A is the antisense oligonucleotide;

C is the conjugate linker

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 such embodiments, the branching group comprises at least one cleavable bond.

In certain embodiments each tether comprises 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-CE-F)_q

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.

A-CE-F)_q

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-DE-F)_q

wherein

A is the antisense oligonucleotide;

B is the cleavable moiety

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 embodiments, a conjugated antisense compound has the following structure:

A-DE-F)_q

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 certain embodiments, a conjugated antisense compound has a structure selected from among the following:

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In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

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In certain embodiments, a conjugated antisense compound has a structure selected from among the following:

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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, cleavable moieties as well as other modifications include without limitation, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO 2012/037254, each of which is incorporated by reference herein in its entirety.

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 without limitation, BIESSEN et al., “The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent Cholesterol Lowering Agent” 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 Chemistry (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) 47:5798-5808, SLIEDREGT et al., “Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic 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 Asialoglycoprotein 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 fully modified oligonucleotide) and any conjugate group comprising at least one, two, or three GalNAc groups. In certain embodiments a conjugated antisense compound comprises any conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly 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., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; 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., J Med Chem, 1995, 38, 1846-1852; Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J Med Chem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol, 2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464; Sato et al., J Am 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 Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO1998/013381; WO2011/038356; WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat. Nos. 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; 7,723,509; 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; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.

In Vitro Testing of Antisense Oligonucleotides

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

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

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

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

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

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

Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments. The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Certain Indications

Examples of renal diseases associated with dysregulation of the complement alternative pathway treatable, preventable, and/or ameliorable with the methods provided herein include C3 glomerulopathy, atypical hemolytic uremic syndrome (aHUS), dense deposit disease (DDD; also known as MPGN Type II or C3Neph), and CFHR5 nephropathy.

Additional renal diseases associated with dysregulation of the complement alternative pathway treatable, preventable, and/or ameliorable with the methods provided herein include IgA nephropathy;

mesangiocapillary (membranoproliferative) glomerulonephritis (MPGN); autoimmune disorders including lupus nephritis and systemic lupus erythematosus (SLE); infection-induced glomerulonephritis (also known as Postinfectious glomerulonephritis); and renal ischemia-reperfusion injury, for example post-transplant renal ischemia-reperfusion injury.

Examples of non-renal disorders associated with dysregulation of the complement alternative pathway treatable and/or preventable with the methods provided herein include ocular diseases such as macular degeneration, for example age-related macular degeneration (AMD), including wet AMD and dry AMD, such as Geographic Atrophy; neuromyelitis optica; corneal disease, such as corneal inflammation; autoimmune uveitis; and diabetic retinopathy. It has been reported that complement system is involved in ocular diseases. Jha P, et al., Mol Immunol (2007) 44(16): 3901-3908. Additional examples of non-renal disorders associated with dysregulation of the complement alternative pathway treatable and/or preventable with the methods provided herein include ANCA-assocaited vasculitis, antiphospholipid syndrome (also known as antiphospholipid antibody syndrome (APS)), asthma, rheumatoid arthritis, Myasthenia Gravis, and multiple sclerosis.

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating a renal disease associated with dysregulation of the complement alternative pathway in a subject by administration of a CFB specific inhibitor, such as an antisense compound targeted to CFB. In certain aspects, the renal disease is lupus nephritis, systemic lupus erythematosus (SLE), dense deposit disease (DDD), C3 glomerulonephritis (C3GN), CFHR5 nephropathy, or atypical hemolytic uremic syndrome (aHUS), or any combination thereof.

Certain embodiments provided herein relate to methods of treating, preventing, or ameliorating macular degeneration, such as age-related macular degeneration (AMD), in a subject by administration of a CFB specific inhibitor, such as an antisense compound targeted to CFB. In certain aspects, the AMD is wet AMD or dry AMD. In certain aspects, dry AMD can be Geographic Atrophy. Studies have demonstrated the association of complement alternative pathway dysregulation and AMD. Complement components are common constituents of ocular drusen, the extracellular material that accumulates in the macula of AMD patients. Furthermore, it has been reported that CFH and CFB variants account for nearly 75% of AMD cases in northern Europe and North America. It has also been found that a specific CFB polymorphism confers protection against AMD. Patel, N. et al., Eye (2008) 22(6):768-76. Additionally, CFB homozygous null mice have lower complement pathway activity, exhibit smaller ocular lesions, and choroidal neovascularization (CNV) after laser photocoagulation. Rohrer, B. et al., Invest Ophthalmol Vis Sci. (2009) 50(7):3056-64. Furthermore, CFB siRNA treatment protects mice from laser induced CNV. Bora, N S et al., J Immunol. (2006) 177(3):1872-8. Studies have also shown that the kidney and eye share developmental pathways and structural features including basement membrane collagen IV protomer composition and vascularity. Savige et al., J Am Soc Nephrol. (2011) 22(8):1403-15. There is evidence that the complement pathway is involved in renal and ocular diseases. For instance, inherited complement regulatory protein deficiency causes predisposition to atypical hemolytic uremic syndrome and AMD. Richards A et al., Adv Immunol. (2007) 96:141-77. Additionally, chronic kidney disease has been associated with AMD. Nitsch, D. et al., Ophthalmic Epidemiol. (2009) 16(3):181-6; Choi, J. et al, Ophthalmic Epidemiol. (2011) 18(6):259-63. Dense deposit disease (DDD), a kidney disease associated with dysregulated complement alternative pathway, is characterized by acute nephritic syndrome and ocular drusen. Cruz and Smith, GeneReviews (2007) July 20. Moreover, mice harboring genetic deletion of a component of the complement alternative pathway have coexisting renal and ocular disease phenotypes. It has been reported that CFH homozygous null mice develop DDD and present retinal abnormalities and visual dysfunction. Pickering et al., Nat Genet. (2002) 31(4):424-8. Mouse models of renal diseases associated with dysregulation of the complement alternative pathway are also accepted as models of AMD. Pennesi M E et al., Mol Aspects Med (2012) 33:487-509. CFH null mice, for example, are an accepted model for renal diseases, such as DDD, and AMD. Furthermore, it has been reported that AMD is associated with the systemic source of complement factors, which accumulate locally in the eye to drive alternative pathway complement activation. Loyet et al., Invest Ophthalmol Vis Sci. (2012) 53(10):6628-37.

EXAMPLES

The following examples illustrate certain embodiments of the present 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 particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.

Example 1: General Method for the Preparation of Phosphoramidites, Compounds 1, 1a and 2

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Bx is a heterocyclic base;

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 Series, 2008, 52(1), 553-554); and also see published PCT International Applications (WO 2011/115818, WO 2010/077578, WO2010/036698, WO2009/143369, WO 2009/006478, and WO 2007/090071), and U.S. Pat. No. 7,569,686).

Example 2: Preparation of Compound 7

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Compounds 3 (2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-β-Dgalactopyranose or galactosamine pentaacetate) is commercially available. Compound 5 was prepared according to published procedures (Weber et al., J. Med. Chem., 1991, 34, 2692).

Example 3: Preparation of Compound 11

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Compounds 8 and 9 are commercially available.

Example 4: Preparation of Compound 18

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Compound 11 was prepared as per the procedures illustrated in Example 3. Compound 14 is commercially available. Compound 17 was prepared using similar procedures reported by Rensen et al., J. Med. Chem., 2004, 47, 5798-5808.

Example 5: Preparation of Compound 23

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Compounds 19 and 21 are commercially available.

Example 6: Preparation of Compound 24

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Compounds 18 and 23 were prepared as per the procedures illustrated in Examples 4 and 5.

Example 7: Preparation of Compound 25

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Compound 24 was prepared as per the procedures illustrated in Example 6.

Example 8: Preparation of Compound 26

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Compound 24 is prepared as per the procedures illustrated in Example 6.

Example 9: General Preparation of Conjugated ASOs Comprising GalNAc₃-1 at the 3′ Terminus, Compound 29

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Wherein the protected GalNAc₃-1 has the structure:

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The GalNAc₃cluster portion of the conjugate group GalNAc₃-1 (GalNAc₃-1_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-1_ahas the formula:

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The solid support bound protected GalNAc₃-1, Compound 25, was prepared as per the procedures illustrated 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 et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite 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 oligomeric compounds having a predetermined sequence and composition. The order and quantity of phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 10: General Preparation Conjugated ASOs Comprising GalNAc₃-1 at the 5′ Terminus, Compound 34

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The Unylinker™ 30 is commercially available. Oligomeric Compound 34 comprising a GalNAc₃-1 cluster at the 5′ terminus is prepared using standard procedures in automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627). Phosphoramidite 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 phosphoramidites added to the solid support can be adjusted to prepare gapped oligomeric compounds as described herein. Such gapped oligomeric compounds can have predetermined composition and base sequence as dictated by any given target.

Example 11: Preparation of Compound 39

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Compounds 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 et al., Eur. J. Org. Chem., 2011, 12, 2346-2353.

Example 12: Preparation of Compound 40

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Compound 38 is prepared as per the procedures illustrated in Example 11.

Example 13: Preparation of Compound 44

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Compounds 23 and 36 are prepared as per the procedures illustrated in Examples 5 and 11. Compound 41 is prepared using similar procedures published in WO 2009082607.

Example 14: Preparation of Compound 45

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Compound 43 is prepared as per the procedures illustrated in Example 13.

Example 15: Preparation of Compound 47

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Compound 46 is commercially available.

Example 16: Preparation of Compound 53

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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 of Compound 54

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Compound 53 is prepared as per the procedures illustrated in Example 16.

Example 18: Preparation of Compound 55

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Compound 53 is prepared as per the procedures illustrated in Example 16.

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 oligomeric 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 β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on an GalNAc₃-1 loaded VIMAD solid support (110 μmol/g, Guzaev et al., 2003) packed in the column. For the coupling step, the phosphoramidites were delivered 4 fold excess over the loading on the solid support and phosphoramidite condensation was carried out for 10 min. All other steps followed standard 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-Dicyanoimidazole (0.7 M) in anhydrous CH₃CN was used as activator during coupling step. Phosphorothioate linkages were introduced by sulfurization with 0.1 M solution of xanthane 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 internucleoside 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) mixture of triethylamine and acetonitrile with a contact time of 45 minutes. The solid-support bound ASOs were suspended in aqueous ammonia (28-30 wt %) and heated at 55° C. 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 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min−1, λ=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 1100 MSD system.

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

Using these methods, 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 5-10-5 MOE gapmer having all phosphorothioate linkages; 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 internucleoside linkages of that compound are phosphodiester linkages. As further summarized in Table 17, two separate antisense compounds targeting SRB-1 were synthesized. ISIS 440762 was a 2-10-2 cEt gapmer with all phosphorothioate internucleoside linkages; ISIS 651900 is the same as ISIS 440762, except that it included a GalNAc₃-1 at its 3′-end.

TABLE 17

Modified ASO targeting ApoC III and SRB-1

SEQ

CalCd
Observed
ID

ASO
Sequence (5′ to 3′)
Target
Mass
Mass
No.

ISIS
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_esT_e
ApoC
7165.4
7164.4
821

304801

III

ISIS
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_es custom-character

ApoC
9239.5
9237.8
822

647535

custom-character

III

ISIS
A_esG_eo^mC_eoT_eoT_eo^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_dsA_dsG_ds^mC_dsT_eoT_eoT_esA_es custom-character

ApoC
9142.9
9140.8
822

647536

custom-character

III

ISIS
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^mC_k
SRB-
4647.0
4646.4
823

440762

1

ISIS
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^m custom-character

SRB-
6721.1
6719.4
824

651900

1

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” 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 designated “GalNAc₃-1a.” This nomenclature is used in the above table to show the full 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 “GalNAc₃-1” 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 referred to herein as a “cluster” or “conjugate cluster” or “GalNAc₃cluster.” In certain instances it is convenient to describe 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.

Treatment

Human ApoCIII transgenic mice were maintained on a 12-hour light/dark cycle and fed ad libitum Teklad lab chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. ASOs were prepared in PBS and sterilized 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 a week for two weeks with ISIS 304801 or 647535 at 0.08, 0.25. 0.75, 2.25 or 6.75 μmol/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 III mRNA Analysis

ApoC III mRNA levels in the mice's livers were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. ApoC III mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of ApoC III mRNA levels for each treatment group, normalized to PBS-treated control and are denoted as “% PBS”. The half maximal effective dosage (ED₅₀) of each ASO is also presented in Table 18, below.

As illustrated, both antisense compounds reduced ApoC III RNA relative 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)_n

TABLE 18

Effect of ASO treatment on ApoC III mRNA

levels in human ApoC III transgenic mice

Dose
%
ED₅₀

Internucleoside
SEQ

ASO
(μmol/kg)
PBS
(μmol/kg)
3′ Conjugate
linkage/Length
ID No.

PBS
0
100
—
—
—

ISIS
0.08
95
0.77
None
PS/20
821

304801
0.75
42

2.25
32

6.75
19

ISIS
0.08
50
0.074
GalNAc₃-1
PS/20
822

647535
0.75
15

2.25
17

6.75
8

ApoC III Protein Analysis (Turbidometric Assay)

Plasma ApoC III protein analysis was determined using procedures reported by Graham et al, Circulation Research, published online before print Mar. 29, 2013.

Approximately 100 μl of plasma isolated from mice was analyzed without dilution using an Olympus Clinical Analyzer and a commercially available turbidometric ApoC III assay (Kamiya, Cat # KAI-006, Kamiya Biomedical, Seattle, Wash.). 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 GalNAc₃-1 (ISIS 647535) was substantially more potent than the antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801).

TABLE 19

Effect of ASO treatment on ApoC III plasma protein

levels in human ApoC III transgenic mice

Dose
%
ED₅₀

Internucleoside
SEQ

ASO
(μmol/kg)
PBS
(μmol/kg)
3′ Conjugate
Linkage/Length
ID No.

PBS
0
100
—
—
—

ISIS
0.08
86
0.73
None
PS/20
821

304801
0.75
51

2.25
23

6.75
13

ISIS
0.08
72
0.19
GalNAc₃-1
PS/20
822

647535
0.75
14

2.25
12

6.75
11

Plasma triglycerides and cholesterol 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 J Biochem Physiol, 37, 911-917, 1959) (Bligh, E and Dyer, W, Can J Biochem Physiol, 37, 911-917, 1959) and measured by using a Beckmann Coulter clinical analyzer and commercially available reagents.

The triglyceride levels were measured relative to PBS injected mice and are denoted as “% PBS”. 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
%
ED₅₀

Internucleoside
SEQ

ASO
(μmol/kg)
PBS
(μmol/kg)
3′ Conjugate
Linkage/Length
ID No.

PBS
0
100
—
—
—

ISIS
0.08
87
0.63
None
PS/20
821

304801
0.75
46

2.25
21

6.75
12

ISIS
0.08
65
0.13
GalNAc₃-1
PS/20
822

647535
0.75
9

2.25
8

6.75
9

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 lowered 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 antisense compound lacking the GalNAc₃-1 conjugate (ISIS 304801). An increase in HDL and a decrease in LDL levels is a cardiovascular beneficial effect of antisense inhibition of ApoC III.

TABLE 21

Effect of ASO treatment on total cholesterol

levels in transgenic mice

Inter-

Total

nucleoside

Dose
Cholesterol

Linkage/
SEQ

ASO
(μmol/kg)
(mg/dL)
3′ Conjugate
Length
ID No.

PBS
0
257
—
—

ISIS
0.08
226
None
PS/20
821

304801
0.75
164

2.25
110

6.75
82

ISIS
0.08
230
GalNAc₃-1
PS/20
822

647535
0.75
82

2.25
86

6.75
99

TABLE 22

Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice

Dose
HDL
LDL

Internucleoside
SEQ

ASO
(μmol/kg)
(mg/dL)
(mg/dL)
3′ Conjugate
Linkage/Length
ID No.

PBS
0
17
28
—
—

ISIS
0.08
17
23
None
PS/20
821

304801
0.75
27
12

2.25
50
4

6.75
45
2

ISIS
0.08
21
21
GalNAc₃-1
PS/20
822

647535
0.75
44
2

2.25
50
2

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 IP-HPLC-MS. The tissue level (μg/g) of full-length ISIS 304801 and 647535 was measured and the results are provided in Table 23. As illustrated, liver concentrations of total full-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 demonstrated by the RNA and protein data above), it is not present at substantially higher concentration in the liver. Indeed, the calculated EC₅₀(provided in Table 23) confirms that the observed increase in potency of the conjugated compound cannot be entirely attributed to increased accumulation. This result suggests that the conjugate improved 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 GalNAc₃-1 conjugated antisense compound in the kidney is lower than that of antisense compound lacking the GalNAc conjugate. This has several beneficial therapeutic implications. For therapeutic indications where activity in the kidney is not sought, exposure to kidney risks kidney toxicity without corresponding benefit. Moreover, high concentration in kidney typically results in loss of compound to the urine resulting in faster clearance. Accordingly, for non-kidney targets, kidney accumulation is undesired. These data suggest that GalNAc₃-1 conjugation reduces kidney accumulation.

TABLE 23

PK analysis of ASO treatment in transgenic mice

Dose
Liver
Kidney
Liver EC₅₀

Internucleoside
SEQ

ASO
(μmol/kg)
(μg/g)
(μg/g)
(μg/g)
3′ Conjugate
Linkage/Length
ID No.

ISIS
0.1
5.2
2.1
53
None
PS/20
821

304801
0.8
62.8
119.6

2.3
142.3
191.5

6.8
202.3
337.7

ISIS
0.1
3.8
0.7
3.8
GalNAc₃-1
PS/20
822

647535
0.8
72.7
34.3

2.3
106.8
111.4

6.8
237.2
179.3

Metabolites of ISIS 647535 were also identified and their masses were confirmed by high resolution mass spectrometry analysis. The cleavage sites and structures of the observed metabolites are shown below. The relative % of full length ASO was calculated using standard procedures and the results are presented in Table 23a. The major metabolite of ISIS 647535 was full-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 cleabable bonds such as esters, peptides, disulfides, phosphoramidates or acyl-hydrazones between the GalNAc₃-1 sugar and the ASO, which can be cleaved 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 lyzosomes, can also be useful.

TABLE 23a

Observed full length metabolites of ISIS 647535

Cleavage
Relative

Metabolite
ASO
site
%

1
ISIS 304801
A
36.1

2
ISIS 304801 + dA
B
10.5

3
ISIS 647535 minus [3 GalNAc]
C
16.1

4
ISIS 647535 minus
D
17.6

[3 GalNAc + 1 5-hydroxy-pentanoic

acid tether]

5
ISIS 647535 minus
D
9.9

[2 GalNAc + 2 5-hydroxy-pentanoic

acid tether]

6
ISIS 647535 minus
D
9.8

[3 GalNAc + 3 5-hydroxy-pentanoic

acid tether]

embedded image

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.

Treatment

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 control. 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 cholesterol, including HDL and LDL fractions were assessed as described above (Example 20). Data from those analyses are presented in Tables 24-28, below. Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. The ALT and AST levels showed that the antisense compounds were well tolerated at all administered 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 GalNAc₃-1 conjugate (ISIS 304801). Further, ISIS 647536, which comprises a GalNAc₃-1 conjugate and some phosphodiester linkages was as potent as ISIS 647535, which comprises the same conjugate and all internucleoside linkages within the ASO are phosphorothioate.

TABLE 24

Effect of ASO treatment on ApoC III mRNA

levels in human ApoC III transgenic mice

Dose
%
ED₅₀

Internucleoside
SEQ

ASO
(mg/kg)
PBS
(mg/kg)
3′ Conjugate
linkage/Length
ID No.

PBS
0
99
—
—
—

ISIS
1
104
13.2
None
PS/20
821

304801
3
92

10
71

30
40

ISIS
0.3
98
1.9
GalNAc₃-1
PS/20
822

647535
1
70

3
33

10
20

ISIS
0.3
103
1.7
GalNAc₃-1
PS/PO/20
822

647536
1
60

3
31

10
21

TABLE 25

Effect of ASO treatment on ApoC III plasma protein

levels in human ApoC III transgenic mice

Dose
%
ED₅₀

Internucleoside
SEQ

ASO
(mg/kg)
PBS
(mg/kg)
3′ Conjugate
Linkage/Length
ID No.

PBS
0
99
—
—
—

ISIS
1
104
23.2
None
PS/20
821

304801
3
92

10
71

30
40

ISIS
0.3
98
2.1
GalNAc₃-1
PS/20
822

647535
1
70

3
33

10
20

ISIS
0.3
103
1.8
GalNAc₃-1
PS/PO/20
822

647536
1
60

3
31

10
21

TABLE 26

Effect of ASO treatment on triglyceride levels in transgenic mice

Dose
%
ED₅₀
3′
Internucleoside
SEQ

ASO
(mg/kg)
PBS
(mg/kg)
Conjugate
Linkage/Length
ID No.

PBS
0
98
—
—
—

ISIS
1
80
29.1
None
PS/20

304801
3
92

821

10
70

30
47

ISIS
0.3
100
2.2
GalNAc₃-1
PS/20
822

647535
1
70

3
34

10
23

ISIS
0.3
95
1.9
GalNAc₃-1
PS/PO/20
822

647536
1
66

3
31

10
23

TABLE 27

Effect of ASO treatment on total cholesterol

levels in transgenic mice

Dose
%

Internucleoside
SEQ

ASO
(mg/kg)
PBS
3′ Conjugate
Linkage/Length
ID No.

PBS
0
96
—
—

ISIS
1
104
None
PS/20
821

304801
3
96

10
86

30
72

ISIS
0.3
93
GalNAc₃-1
PS/20
822

647535
1
85

3
61

10
53

ISIS
0.3
115
GalNAc₃-1
PS/PO/20
822

647536
1
79

3
51

10
54

TABLE 28

Effect of ASO treatment on HDL and LDL cholesterol levels in transgenic mice

Dose
HDL
LDL

Internucleoside
SEQ

ASO
(mg/kg)
% PBS
% PBS
3′ Conjugate
Linkage/Length
ID No.

PBS
0
131
90
—
—

ISIS
1
130
72
None
PS/20
821

304801
3
186
79

10
226
63

30
240
46

ISIS
0.3
98
86
GalNAc₃-1
PS/20
822

647535
1
214
67

3
212
39

10
218
35

ISIS
0.3
143
89
GalNAc₃-1
PS/PO/20
822

647536
1
187
56

3
213
33

10
221
34

These results confirm that the GalNAc₃-1 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 phosphodiester 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 associated with an inflammatory response. Accordingly, reducing the number of phosphorothioate linkages in a compound is expected to reduce the risk of inflammation, 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 present results show that a GalNAc₃-1 conjugated antisense compound where certain 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 compounds are expected to be less proinflammatory (See Example 24 describing an experiment showing reduction of PS results in reduced inflammatory effect).

Example 22: Effect of GalNAc₃-1 Conjugated Modified ASO Targeting SRB-1 In Vivo

ISIS 440762 and 651900, each targeting SRB-1 and described in Table 17, were evaluated in a dose-dependent study for their ability to inhibit SRB-1 in Balb/c mice.

Treatment

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 sacrificed 48 hours following the final administration to determine the SRB-1 mRNA levels in liver using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”.

As illustrated in Table 29, both antisense compounds lowered SRB-1 mRNA levels. Further, the antisense compound comprising the GalNAc₃-1 conjugate (ISIS 651900) was substantially more potent than the antisense compound lacking the GalNAc₃-1 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 nucleosides, in this instance modified nucleosides comprise constrained ethyl sugar moieties (a bicyclic sugar moiety).

TABLE 29

Effect of ASO treatment on SRB-1 mRNA levels in Balb/c mice

Dose
Liver
ED₅₀

Internucleoside
SEQ

ASO
(mg/kg)
% PBS
(mg/kg)
3′ Conjugate
linkage/Length
ID No.

PBS
0
100

—
—

ISIS
0.7
85
2.2
None
PS/14
823

440762
2
55

7
12

20
3

ISIS
0.07
98
0.3
GalNAc₃-1
PS/14
824

651900
0.2
63

0.7
20

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)_nThe 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 centrifuged at rt (18-25° C.) in a horizontal (swing-out) rotor for 30 min. at 1500-1800 RCF with brake off (2700 RPM Beckman Allegra 6R). The cells were retrieved from the buffy coat interface (between Ficoll and polymer gel layers); transferred to a sterile 50 ml conical tube and pooled up to 5 CPT tubes/50 ml conical tube/donor. 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 centrifuged at 330×g for 15 minutes at rt (1215 RPM in Beckman Allegra 6R) and aspirated as much supernatant as possible without disturbing pellet. The cell pellet was dislodged by gently swirling tube and resuspended cells in RPMI+10% FBS+pen/strep (˜1 ml/10 ml starting whole blood volume). A 60 μl sample was pipette into a sample vial (Beckman Coulter) with 600 μl VersaLyse reagent (Beckman Coulter 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 suspension was counted on Vicell XR cell viability analyzer (Beckman Coulter) using PBMC cell type (dilution factor of 1:11 was stored with other parameters). The live cell/ml and viability were recorded. The cell suspension was diluted to 1×10⁷live PBMC/ml in RPMI+10% FBS+pen/strep.

The cells were plated at 5×10⁵in 50 μl/well of 96-well tissue culture plate (Falcon Microtest). 50 μl/well of 2× concentration oligos/controls diluted in RPMI+10% FBS+pen/strep. was added according to experiment template (100₁1.1/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% CO₂, the plates were centrifuged at 400×g for 10 minutes before removing the supernatant for MSD cytokine assay (i.e. human IL-6, IL-10, IL-8 and MCP-1).

Example 24: Evaluation of Proinflammatory Effects in hPBMC Assay for GalNAc₃-1 Conjugated ASOs

The antisense oligonucleotides (ASOs) listed in Table 30 were evaluated for proinflammatory 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, 0.0128, 0.064, 0.32, 1.6, 8, 40 and 200 μM concentrations. After a 24 hr treatment, the cytokine levels were measured.

The levels of IL-6 were used as the primary readout. The EC₅₀and E_maxwas calculated using standard procedures. Results are expressed as the average ratio of E_max/EC₅₀from two donors and is denoted as “E_max/EC₅₀.” The lower ratio indicates a relative decrease in the proinflammatory response and the higher ratio indicates a relative increase in the proinflammatory response.

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

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 full PS antisense compound lacking a GalNAc₃-1 conjugate. Since half-life is not expected to be substantially 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 20-22) 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

Modified ASOs

SEQ ID

ASO
Sequence (5′ to 3′)
Target
No.

ISIS
G_es^mC_esT_esG_esA_esT_dsT_dsA_dsG_dsA_dsG_ds
TNFα
825

104838
A_dsG_dsA_dsG_dsG_esT_es^mC_es^mC_es^mC_e

ISIS
T_es^mC_es^mC_es^mC_dsA_dsT_dsT_dsT_ds^mC_dsA_dsG_ds
CRP
826

353512
G_dsA_dsG_dsA_ds^mC_ds^mC_dsT_esG_esG_e

ISIS
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds
ApoC
821

304801

^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_esT_e
III

ISIS
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds

647535

^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_es custom-character

ApoC
822

custom-character

III

ISIS
A_esG_eo^mC_eoT_eoT_eo^mC_dsT_dsT_dsG_dsT_ds
ApoC
821

616468

^mC_ds^mC_dsA_dsG_ds^mC_dsT_eoT_eoT_esA_esT_e
III

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. “A_do′-GalNAc₃-1a” indicates a conjugate having the structure GalNAc₃-1 shown in Example 9 attached to the 3′-end of the antisense oligonucleotide, as indicated.

TABLE 31

Proinflammatory Effect of ASOs targeting ApoC III in hPBMC assay

EC₅₀
E_max

Internucleoside
SEQ

ASO
(μM)
(μM)
E_max/EC₅₀
3′ Conjugate
Linkage/Length
ID No.

ISIS 353512
0.01
265.9
26,590
None
PS/20
826

(high responder)

ISIS 304801
0.07
106.55
1,522
None
PS/20
821

ISIS 647535
0.12
138
1,150
GalNAc₃-1
PS/20
822

ISIS 616468
0.32
71.52
224
None
PS/PO/20
821

Example 25: Effect of GalNAc₃-1 Conjugated Modified 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 density of 25,000 cells per well were treated with 0.03, 0.08, 0.24, 0.74, 2.22, 6.67 and 20 μM concentrations of modified 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 RIBOGREEN.

The IC₅₀was 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

Modified ASO targeting human ApoC III in primary hepatocytes

IC₅₀

Internucleoside
SEQ

ASO
(μM)
3′ Conjugate
linkage/Length
ID No.

ISIS
0.44
None
PS/20
821

304801

ISIS
0.31
GalNAc₃-1
PS/20
822

647535

In this experiment, the large potency benefits of GalNAc₃-1 conjugation that are observed in vivo were not observed in vitro. Subsequent free uptake experiments in primary hepatocytes in vitro did show increased potency of oligonucleotides comprising various GalNAc conjugates relative to oligonucleotides that lacking the GalNAc conjugate. (see Examples 60, 82, and 92)

Example 26: Effect of PO/PS Linkages on ApoC III ASO Activity

Human ApoC III transgenic mice were injected intraperitoneally once at 25 mg/kg of ISIS 304801, 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 antisense compounds with PO/PS (ISIS 616468) in the wings relative to full PS (ISIS 304801).

TABLE 33

Effect of ASO treatment on ApoC III protein

levels in human ApoC III transgenic mice

Dose
%

Internucleoside
SEQ

ASO
(mg/kg)
PBS
3′ Conjugate
linkage/Length
ID No.

PBS
0
99
—
—

ISIS
25
24
None
Full PS
821

304801
mg/kg/wk

for 2 wks

ISIS
25
40
None
14 PS/6 PO
821

616468
mg/kg/wk

for 2 wks

Example 27: Compound 56

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Compound 56 is commercially available from Glen Research or may be prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 28: Preparation of Compound 60

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Compound 4 was prepared as per the procedures illustrated in Example 2. Compound 57 is commercially available. Compound 60 was confirmed by structural analysis.

Compound 57 is meant to be representative and not intended to be limiting as other mono-protected substituted or unsubstituted alkyl diols including but not limited to those presented in the specification herein can be used to prepare phosphoramidites having a predetermined composition.

Example 29: Preparation of Compound 63

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Compounds 61 and 62 are prepared using procedures similar to those reported by Tober et al., Eur. J. Org. Chem., 2013, 3, 566-577; and Jiang et al., Tetrahedron, 2007, 63(19), 3982-3988.

Alternatively, Compound 63 is prepared using procedures similar to those reported in scientific and patent literature by Kim et al., Synlett, 2003, 12, 1838-1840; and Kim et al., published PCT International Application, WO 2004063208.Example 30: Preparation of Compound 63b

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Compound 63a is prepared using procedures similar to those reported by Hanessian et al., Canadian Journal of Chemistry, 1996, 74(9), 1731-1737.

Example 31: Preparation of Compound 63d

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Compound 63c is prepared using procedures similar to those reported by Chen et al., Chinese Chemical Letters, 1998, 9(5), 451-453.

Example 32: Preparation of Compound 67

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Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 65 is prepared using procedures similar to those reported by Or et al., published PCT International Application, WO 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 specification herein can be used.

Example 33: Preparation of Compound 70

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Compound 64 was prepared as per the procedures illustrated in Example 2. Compound 68 is commercially available. The protecting group used for Compound 68 is meant to be representative and not intended to be limiting as other protecting groups including but not limited to those presented in the specification herein can be used.

Example 34: Preparation of Compound 75a

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Compound 75 is prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 35: Preparation of Compound 79

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Compound 76 was prepared according to published procedures reported by Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454.

Example 36: Preparation of Compound 79a

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Compound 77 is prepared as per the procedures illustrated in Example 35.

Example 37: General Method for the Preparation of Conjugated Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus Via Solid Support (Method I)

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wherein GalNAc₃-2 has the structure:

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The GalNAc₃cluster portion of the conjugate group GalNAc₃-2 (GalNAc₃-2_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-2_ahas the formula:

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The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627)_nThe phosphoramidite Compounds 56 and 60 were prepared as per the procedures illustrated in Examples 27 and 28, respectively. The phosphoramidites illustrated are meant to be representative and not intended to be limiting as other phosphoramidite building blocks including but not limited those presented in the specification 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 predetermined sequence and composition.

Example 38: Alternative Method for the Preparation of Oligomeric Compound 82 Comprising a Phosphodiester Linked GalNAc₃-2 Conjugate at 5′ Terminus (Method II)

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The VIMAD-bound oligomeric compound 79b was prepared using standard procedures for automated DNA/RNA synthesis (see Dupouy et al., Angew. Chem. Int. Ed., 2006, 45, 3623-3627)_nThe GalNAc₃-2 cluster phosphoramidite, Compound 79 was prepared as per the procedures illustrated in Example 35. This alternative method allows a one-step installation of the phosphodiester linked GalNAc₃-2 conjugate to the oligomeric compound at the final 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 specification 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: General Method for the Preparation of Oligomeric Compound 83h Comprising a GalNAc₃-3 Conjugate at the 5′ Terminus (GalNAc₃-1 Modified for 5′ End Attachment) Via Solid Support

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Compound 18 was prepared as per the procedures illustrated in Example 4. Compounds 83a and 83b are commercially available. Oligomeric Compound 83e comprising a phosphodiester linked hexylamine was prepared using standard oligonucleotide synthesis procedures. Treatment of the protected oligomeric compound with aqueous ammonia provided the 5′-GalNAc₃-3 conjugated oligomeric compound (83h).

Wherein GalNAc₃-3 has the structure:

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The GalNAc₃cluster portion of the conjugate group GalNAc₃-3 (GalNAc₃-3_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-3_ahas the formula:

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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

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Wherein GalNAc₃-4 has the structure:

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Wherein CM is a cleavable moiety. In certain embodiments, cleavable moiety is:

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The GalNAc₃cluster portion of the conjugate group GalNAc₃-4 (GalNAc₃-4_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. Wherein GalNAc₃-4_ahas the formula:

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The protected Unylinker functionalized solid support Compound 30 is commercially available. Compound 84 is prepared using procedures similar to those reported in the literature (see Shchepinov et al., Nucleic Acids Research, 1997, 25(22), 4447-4454; Shchepinov et al., Nucleic Acids Research, 1999, 27, 3035-3041; and Hornet et al., Nucleic Acids Research, 1997, 25, 4842-4849).

The phosphoramidite building blocks, Compounds 60 and 79a are prepared as per the procedures illustrated in Examples 28 and 36. 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 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 described 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 synthesis of oligomeric 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. Phosphoramidite compounds 56 and 60 were used to synthesize the phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus. A 0.1 M solution of phosphoramidite in anhydrous acetonitrile was used for β-D-2′-deoxyribonucleoside and 2′-MOE.

The ASO syntheses were performed on ABI 394 synthesizer (1-2 μmol scale) or on GE Healthcare Bioscience ÄKTA oligopilot synthesizer (40-200 μmol scale) by the phosphoramidite coupling method on VIMAD solid support (110 μmol/g, Guzaev et 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 manufacturer. A solution of 6% dichloroacetic acid in toluene was used for removing the dimethoxytrityl (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 sulfurization with 0.1 M solution of xanthane 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 internucleoside linkages with a contact time of 12 minutes.

After the desired sequence was assembled, the cyanoethyl phosphate 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 ammonia (28-30 wt %) and heated at 55° C. 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 μm, 2.54×8 cm, A=100 mM ammonium acetate in 30% aqueous CH₃CN, B=1.5 M NaBr in A, 0-40% of B in 60 min, flow 14 mL min-1, =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 1100 MSD system.

TABLE 34

ASO comprising a phosphodiester linked GalNAc₃-2

conjugate at the 5′ position targeting SRB-1

SEQ

ISIS

CalCd
Observed
ID

No.
Sequence (5′ to 3′)
Mass
Mass
No.

661134

custom-character

6482.2
6481.6
827

custom-character

T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds

A_ds^mC_dsT_dsT_ks^mC_k

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH3 bicyclic nucleoside (e.g. cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of GalNAc₃-2_ais shown in Example 37.

Example 42: General Method for the Preparation of ASOs Comprising a GalNAc₃-3 Conjugate at the 5′ Position Via Solid Phase Techniques (Preparation of ISIS 661166)

The synthesis for ISIS 661166 was performed using similar procedures as illustrated in Examples 39 and 41.

ISIS 661166 is a 5-10-5 MOE gapmer, wherein the 5′ position comprises a GalNAc₃-3 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

hexylamino phosphodiester linkage targeting Malat-1

SEQ

ISIS

Calcd
Observed
ID

No.
Sequence (5′ to 3′)
Conjugate
Mass
Mass
No.

661166
5′- custom-character

′^mC_esG_esG_esT_esG_es

custom-character

8992.16
8990.51
828

^mC_dsA_dsA_dsG_dsG_ds^mC_dsT_dsT_dsA_dsG_ds

G_esA_esA_esT_esT_e

Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO); and “o′” indicates —O—P(═O)(OH)—. Superscript “m” indicates 5-methylcytosines. The structure of “5′-GalNAc₃-3a” is shown in 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-1 In Vivo

ISIS 661134 (see Example 41) comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus was tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 440762 and 651900 (GalNAc₃-1 conjugate at 3′ terminus, see Example 9) were included in the study for comparison and are described previously in Table 17.

Treatment

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 control. Each treatment group consisted of 4 animals. The mice were sacrificed 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, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

As illustrated in Table 35, treatment with antisense oligonucleotides lowered SRB-1 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 conjugate linked at the 3′ terminus (ISIS 651900) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 440762)_nFurther, 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

ASOs containing GalNAc₃-1 or GalNAc₃-2 targeting SRB-1

SRB-1

ISIS
Dosage
mRNA levels
ED₅₀

SEQ

No.
(mg/kg)
(% PBS)
(mg/kg)
Conjugate
ID No.

PBS
0
100
—
—

440762
0.2
116
2.58
No conjugate
823

0.7
91

2
69

7
22

20
5

651900
0.07
95
0.26
3′ GalNAc₃-1
824

0.2
77

0.7
28

2
11

7
8

661134
0.07
107
0.25
5′ GalNAc₃-2
827

0.2
86

0.7
28

2
10

7
6

Structures for 3′ GalNAc₃-1 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 identified and their masses were confirmed by high resolution mass spectrometry analysis. The results showed that the major metabolite detected for the ASO comprising a phosphodiester linked GalNAc₃-2 conjugate at the 5′ terminus (ISIS 661134) was ISIS 440762 (data not shown). No additional metabolites, at a detectable level, were observed. Unlike its counterpart, additional metabolites similar to those reported previously in Table 23a were observed for the ASO having the GalNAc₃-1 conjugate at the 3′ terminus (ISIS 651900)_nThese results suggest that having the phosphodiester linked GalNAc₃-1 or GalNAc₃-2 conjugate may improve the PK profile of ASOs without compromising their potency.

Example 44: Effect of PO/PS Linkages on Antisense Inhibition of ASOs Comprising GalNAc₃-1 Conjugate (See Example 9) at the 3′ Terminus Targeting SRB-1

ISIS 655861 and 655862 comprising a GalNAc₃-1 conjugate at the 3′ terminus each targeting SRB-1 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 comparison.

The ASOs are 5-10-5 MOE gapmers, wherein the gap region comprises ten 2′-deoxyribonucleosides and each wing region comprises five 2′-MOE modified nucleosides. The ASOs were prepared using similar methods as illustrated previously in Example 19 and are described Table 36, below.

TABLE 36

Modified ASOs comprising GalNAc₃-1 conjugate at

the 3′ terminus targeting SRB-1

SEQ

ISIS

ID

No.
Sequence (5′ to 3′)
Chemistry
No.

353382
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
Full PS no
829

(parent)
G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e
conjugate

655861
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
Full PS
830

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_es custom-character

with

conjugate

655862
G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_ds
Mixed

G_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_es custom-character

PS/PO
830

with

custom-character

conjugate

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 655861, 655862 or with PBS treated control. Each treatment 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 final administration to determine the liver SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. SRB-1 mRNA levels were determined relative to total RNA (using Ribogreen), prior to normalization to PBS-treated control. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s 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 potency relative to full PS (ISIS 655861).

TABLE 37

Effect of PO/PS linkages on antisense inhibition of ASOs comprising

GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1

SRB-1

ISIS
Dosage
mRNA levels
ED₅₀

SEQ

No.
(mg/kg)
(% PBS)
(mg/kg)
Chemistry
ID No.

PBS
0
100
—
—

353382
3
76.65
10.4
Full PS without
829

(parent)
10
52.40

conjugate

30
24.95

655861
0.5
81.22
2.2
Full PS with
830

1.5
63.51

GalNAc₃-1

5
24.61

conjugate

15
14.80

655862
0.5
69.57
1.3
Mixed PS/PO
830

1.5
45.78

with GalNAc₃-1

5
19.70

conjugate

15
12.90

Liver transaminase levels, 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 transaminase levels compared to full PS (ISIS 655861).

TABLE 38

Effect of PO/PS linkages on transaminase levels of ASOs comprising

GalNAc₃-1 conjugate at 3′ terminus targeting SRB-1

ISIS
Dosage
ALT
AST

SEQ

No.
(mg/kg)
(U/L)
(U/L)
Chemistry
ID No.

PBS
0
28.5
65
—

353382
3
50.25
89
Full PS without
829

(parent)
10
27.5
79.3
conjugate

30
27.3
97

655861
0.5
28
55.7
Full PS with
830

1.5
30
78
GalNAc₃-1

5
29
63.5

15
28.8
67.8

655862
0.5
50
75.5
Mixed PS/PO
830

1.5
21.7
58.5
with

5
29.3
69
GalNAc₃-1

15
22
61

Example 45: Preparation of PFP Ester, Compound 110a

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Compound 4 (9.5 g, 28.8 mmoles) was treated with compound 103a or 103b (38 mmoles), 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, filtered and reduced under reduced pressure. The resultant oil was purified by silica gel chromatography (2%->10% methanol/dichloromethane) to give compounds 104a and 104b in >80% yield. LCMS and proton NMR was consistent with the structure.

Compounds 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.

Compounds 105a and 105b were treated, 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 structure.

Compounds 108a (60%) and 108b (40%) were treated to the same conditions as for compounds 100a-d (Example 47), to give compounds 109a and 109b 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 structure. Alternatively, Compound 110b can be prepared in a similar manner starting with Compound 109b.

Example 46: General Procedure for Conjugation with PFP Esters (Oligonucleotide 111); Preparation of ISIS 666881 (GalNAc₃-10)

A 5′-hexylamino modified oligonucleotide was synthesized and purified using standard solid-phase oligonucleotide procedures. The 5′-hexylamino modified oligonucleotide was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents of a selected PFP esterified GalNAc₃cluster dissolved in DMSO (50 μL) was added. If the PFP ester precipitated upon addition to the ASO solution DMSO was added until all PFP ester was in solution. The reaction was complete after about 16 h of mixing at room temperature. The resulting solution was diluted with water to 12 mL and then spun down at 3000 rpm in a spin filter with a mass cut off of 3000 Da. This process was repeated twice to remove small molecule impurities. The solution was then lyophilized to dryness and redissolved in concentrated aqueous ammonia and mixed 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 provide the GalNAc₃conjugated oligonucleotide.

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Oligonucleotide 111 is conjugated with GalNAc₃-10. The GalNAc₃cluster portion of the conjugate group GalNAc₃-10 (GalNAc₃-10_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)— as shown in the oligonucleotide (ISIS 666881) synthesized with GalNAc₃-10 below. The structure of GalNAc₃-10 (GalNAc₃-10_a-CM-) is shown below:

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Following this general procedure ISIS 666881 was prepared. 5′-hexylamino modified oligonucleotide, ISIS 660254, was synthesized and purified using standard solid-phase oligonucleotide procedures. ISIS 660254 (40 mg, 5.2 μmol) was dissolved in 0.1 M sodium tetraborate, pH 8.5 (200 μL) and 3 equivalents PFP ester (Compound 110a) dissolved in DMSO (50 μL) was added. The PFP ester precipitated upon addition to the ASO solution requiring additional DMSO (600 μL) to fully 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 filter 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 ammonia 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 μmol).

GalNAc₃-10 conjugated oligonucleotide

SEQ

5′
ID

ASO
Sequence (5′ to 3′)
group
No.

ISIS
NH₂(CH₂)₆-
Hexylamine
831

660254

_oA_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

831

666881

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

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 β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

Example 47: Preparation of Oligonucleotide 102 Comprising GalNAc₃-8

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The triacid 90 (4 g, 14.43 mmol) was dissolved in DMF (120 mL) and N,N-Diisopropylethylamine (12.35 mL, 72 mmoles). Pentafluorophenyl trifluoroacetate (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 mmoles), 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 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%->10% methanol/dichloromethane) to give compounds 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 dichloromethane and 20 mL of trifluoroacetic 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 reduced 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 (3.7 g, 9.6 mmoles) and N,N-Diisopropylethylamine (5 mL) in DMF (20 mL) for 15 minutes. To this was added either compounds 93a or 93b (3 mmoles), 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, filtered and reduced to an oil under reduced pressure. The resultant oil was purified 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.

Compounds 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 filtration thru celite, and the ethanol removed under reduced pressure to give compounds 97a-d in 80-90% yield. LCMS and proton NMR were consistent with the structure.

Compound 23 (0.32 g, 0.53 mmoles) was treated with HBTU (0.2 g, 0.53 mmoles) and N,N-Diisopropylethylamine (0.19 mL, 1.14 mmoles) in DMF (30 mL) 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 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, filtered and reduced to an oil under reduced pressure. The resultant oil was purified 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 (0.17 g, 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 (50 mL) for 15 minutes. To this was added compounds 97a-d (0.51 mmoles), individually, 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, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (5%->20% methanol/dichloromethane) to give compounds 100a-d in 40-60% yield. LCMS and proton NMR was consistent with the structure.

Compounds 100a-d (0.16 mmoles), individually, 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 filtration thru celite, and the organics removed under reduced 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). Pentafluorophenyl trifluoroacetate (0.034 mL, 0.2 mmoles) was added dropwise, under argon, and the reaction was allowed to stir at room temperature for 30 minutes. 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, filtered and reduced to an oil under reduced pressure. The resultant oil was purified by silica gel chromatography (2%->5% methanol/dichloromethane) to give compounds 102a-d in an approximate 80% yield. LCMS and proton NMR were consistent with the structure.

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Oligomeric Compound 102, comprising a GalNAc₃-8 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-8 (GalNAc₃-8_a) 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)-A_d-P(═O)(OH)—.

The structure of GalNAc₃-8 (GalNAc₃-8_a-CM-) is shown below:

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Example 48: Preparation of Oligonucleotide 119 Comprising GalNAc₃-7

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Compound 112 was synthesized following the procedure described 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 filtered 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 confirmed 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 dissolved 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 CH₂Cl₂(100 mL) and washed with aqueous saturated NaHCO₃solution (100 mL) and brine (100 mL). The organic phase was separated, dried (Na₂SO₄), 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 confirmed 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 reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield Compound 115 (quantitative). The structure was confirmed 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 CH₂Cl₂. The organic layer was washed aqueous saturated NaHCO₃solution and brine and dried over anhydrous Na₂SO₄and filtered. 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 dichloromethane to yield Compound 116 (0.84 g, 61%). The structure was confirmed by LC MS and ¹H NMR analysis.

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Compound 116 (0.74 g, 0.4 mmol) was dissolved in 1:1 methanol/ethyl acetate (5 mL/5 mL). Palladium on carbon (wet, 0.074 g) was added. The reaction mixture was flushed with hydrogen and stirred at room temperature under hydrogen for 12 h. The reaction mixture was filtered through a pad of celite. The celite pad was washed with methanol/ethyl acetate (1:1). The filtrate and the washings were combined together and evaporated under reduced pressure to yield compound 117 (0.73 g, 98%). The structure was confirmed 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 μL, 0.4 mmol) and pentafluorophenyl trifluoroacetate (72 μL, 0.42 mmol) were added. The reaction mixture was stirred at room temperature for 12 h and poured into a aqueous saturated NaHCO₃solution. The mixture was extracted with dichloromethane, washed with brine and dried over anhydrous Na₂SO₄. 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 confirmed by LCMS and ¹H and ¹H and ¹⁹F NMR.

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Oligomeric Compound 119, comprising a GalNAc₃-7 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-7 (GalNAc₃-7_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—.

The structure of GalNAc₃-7 (GalNAc₃-7_a-CM-) is shown below:

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Example 49: Preparation of Oligonucleotide 132 Comprising GalNAc₃-5

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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 stirred for 18 h under an argon atmosphere. The reaction was monitored by TLC (ethyl acetate:hexane; 1:1; Rf=0.47)_nThe solvent was removed under reduced pressure. The residue was taken up in EtOAc (300 mL) and washed with 1M NaHSO₄(3×150 mL), aqueous saturated NaHCO₃solution (3×150 mL) and brine (2×100 mL). Organic layer was dried with Na₂SO₄. Drying agent was removed by filtration and organic layer was concentrated by rotary evaporation. Crude mixture was purified 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 confirmed 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 solution of Compound 122 (7.75 g, 13.16 mmol) dissolved in methanol (15 mL). The reaction mixture was stirred at room temperature for 45 min. and monitored by TLC (EtOAc:hexane; 1:1). The reaction mixture was concentrated to half the volume under reduced pressure. The remaining 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 (Na₂SO₄), 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 further purification. M.W.cal:574.36; M.W.fd:575.3 [M+H]⁺.

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Compound 126 was synthesized following the procedure described in the literature (J. Am. Chem. Soc. 2011, 133, 958-963).

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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 N,N-Diisopropylethylamine (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(3×100 mL), aqueous saturated NaHCO₃(3×100 mL) and brine (2×100 mL). The organic phase separated dried (Na₂SO₄), filtered and concentrated. The residue was purified 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]⁺.

Trifluoroacetic 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 temperature for 1 h. The reaction mixture was co-evaporated 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 trifluoro acetate salt and used for next step without further purification. LCMS and ¹H NMR were consistent with structure. Mass m/z 478.2 [M+H]⁺.

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 P₂O₅under reduced pressure in a round bottom flask 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% MeOH/DCM). The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM (30 mL) and washed with 1 M NaHSO₄(3×20 mL), aqueous saturated NaHCO₃(3×20 mL) and brine (3×20 mL). The organic phase was separated, dried over Na₂SO₄, filtered and concentrated. The residue was purified 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 dissolved 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 reaction vessel was flushed with H₂gas. The reaction mixture was stirred at room temperature under H₂atmosphere 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 flask were added compound 130 (75.8 mg, 0.046 mmol), 0.37 M pyridine/DMF (200 μL) and a stir bar. To this solution was added 0.7 M pentafluorophenyl trifluoroacetate/DMF (100 μL) drop wise with stirring. The reaction was completed after 1 h as determined by LC MS. The solvent was removed under reduced pressure and the residue was dissolved in CHCl₃(˜10 mL). The organic layer was partitioned against NaHSO₄(1 M, 10 mL), aqueous saturated NaHCO₃(10 mL) and brine (10 mL) three times each. The organic phase separated and dried over Na₂SO₄, filtered and concentrated to yield Compound 131 (77.7 mg). LCMS is consistent with structure. Used without further purification. Mass m/z 921.3 [M+2H]⁺.

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Oligomeric Compound 132, comprising a GalNAc₃-5 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-5 (GalNAc₃-5_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—.

The structure of GalNAc₃-5 (GalNAc₃-5_a-CM-) is shown below:

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Example 50: Preparation of Oligonucleotide 144 Comprising GalNAc₄-11

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Synthesis of Compound 134. To a Merrifield flask was added aminomethyl VIMAD resin (2.5 g, 450 μmol/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 flask 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 solution was allowed to stir for 5 min and was then added to the Merrifield flask with shaking. The suspension was allowed to shake for 3 h. The reaction 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 coefficient=76000) in DCM and determined to be 238 μmol/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 synthesis 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 observed 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.

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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° C. for 16 h. The solution was cooled and the solid support was filtered. The filtrate was concentrated and the residue dissolved in water and purified by HPLC on a strong anion exchange column. The fractions containing full length compound 144 were pooled together and desalted. The resulting GalNAc₄-11 conjugated oligomeric compound was analyzed by LC-MS and the observed mass was consistent with structure.

The GalNAc₄cluster portion of the conjugate group GalNAc₄-11 (GalNAc₄-11_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—.

The structure of GalNAc₄-11 (GalNAc₄-11_a-CM) is shown below:

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Example 51: Preparation of Oligonucleotide 155 Comprising GalNAc₃-6

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Compound 146 was synthesized as described in the literature (Analytical Biochemistry 1995, 229, 54-60).

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Compound 4 (15 g, 45.55 mmol) and compound 35b (14.3 grams, 57 mmol) were dissolved in CH₂Cl₂(200 ml). Activated molecular sieves (4 Å. 2 g, powdered) were added, and the reaction was allowed to stir for 30 minutes under nitrogen atmosphere. TMS-OTf was 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 NaHCO₃(500 ml) and crushed ice (˜150 g). The organic layer was separated, washed with brine, dried over MgSO₄, filtered, and was concentrated to an orange oil under reduced pressure. The crude material was purified by silica gel column chromatography and eluted with 2-10% MeOH in CH₂Cl₂to 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 bubbling a stream of argon through the solution for 15 minutes. Pearlman's catalyst (palladium hydroxide on carbon, 400 mg) was added, and hydrogen gas was bubbled through the solution for 30 minutes. Upon completion (TLC 10% MeOH in CH₂Cl₂, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly 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-Diisopropylethylamine (DIEA, 3.9 mL, 22 mmol) was added, followed by HBTU (4 g, 10.5 mmol). The reaction mixture was allowed to stir for ˜15 minutes under nitrogen. 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 NaHCO₃and brine. The organics phase was separated, dried (MgSO₄), filtered, and concentrated to an orange syrup. The crude material was purified by column chromatography 2-5% MeOH in CH₂Cl₂to 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 MeOH/EtOAc (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 30 minutes. Upon completion (TLC 10% MeOH in DCM, and LCMS), the catalyst was removed by filtration through a pad of celite. The filtrate was concentrated by rotary evaporation, and was dried briefly 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.

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Compound 146 (0.68 g, 1.73 mmol) was dissolved in dry DMF (20 ml). To this DIEA (450 μL, 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 compound 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 NaHCO₃, 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 CH₂Cl₂to yield Compound 150 (0.62 g, 20%). LCMS and ¹H NMR were consistent with the desired product.

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 filtration (syringe-tip Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly under high vacuum to yield Compound 151 (0.57 g). The LCMS was consistent with the desired product. The product was dissolved in 4 mL dry DMF and was used immediately in the next step.

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Compound 83a (0.11 g, 0.33 mmol) was dissolved in anhydrous DMF (5 mL) and NN-Diisopropylethylamine (75 μL, 1 mmol) and PFP-TFA (90 μL, 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 compound 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 N,N-Diisopropylethylamine (if necessary). The reaction mixture was stirred under nitrogen for ˜30 min. Upon completion, the majority of the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂and washed with aqueous saturated NaHCO₃, followed by brine. The organic phase separated, dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by silica gel column chromatography (2-10% MeOH in CH₂Cl₂) 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 (palladium hydroxide on carbon) 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 Teflon filter, 0.45 μm). The filtrate was concentrated by rotary evaporation, and was dried briefly 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 μL, 0.37 mmol) and PFP-TFA (35 μL, 0.28 mmol) were added. The reaction mixture was stirred under nitrogen for ˜30 min. The reaction mixture turned magenta upon contact, 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 CH₂Cl₂(50 mL), and washed with saturated aqueous NaHCO₃, followed by brine. The organic layer was dried over MgSO₄, filtered, and concentrated to an orange syrup. The residue was purified by column chromatography and eluted with 2-10% MeOH in CH₂Cl₂to yield Compound 154 (0.29 g, 79%). LCMS and ¹H NMR were consistent with the desired product.

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Oligomeric Compound 155, comprising a GalNAc₃-6 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-6 (GalNAc₃-6_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—.

The structure of GalNAc₃-6 (GalNAc₃-6_a-CM-) is shown below:

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Example 52: Preparation of Oligonucleotide 160 Comprising GalNAc₃-9

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Compound 156 was synthesized following the procedure described in the literature (J. Med. Chem. 2004, 47, 5798-5808).

Compound 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 stirred at room temperature under hydrogen for 18 h. The reaction mixture was filtered 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 purified by silica gel column chromatography and eluted with 5-10% methanol 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 reaction mixture was poured into a saturated NaHCO₃aqueous solution. The mixture was extracted with ethyl acetate and the organic layer was washed with brine and dried (Na₂SO₄), filtered and evaporated. The residue obtained was purified by silica gel column chromatography and eluted with 50% ethyl acetate in hexane to yield compound 158 (8.25 g, 73.3%). The structure was confirmed by MS and ¹H NMR analysis.

Compound 158 (7.2 g, 7.61 mmol) was dried over P₂O₅under reduced pressure. The dried compound was dissolved in anhydrous DMF (50 mL). To this 1H-tetrazole (0.43 g, 6.09 mmol) and N-methylimidazole (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 NaHCO₃and brine. The organic phase was separated, dried (Na₂SO₄), 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 confirmed by LCMS and ³¹P NMR analysis.

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Oligomeric Compound 160, comprising a GalNAc₃-9 conjugate group, was prepared using standard oligonucleotide synthesis procedures. Three units of compound 159 were coupled to the solid support, followed by nucleotide phosphoramidites. Treatment of the protected oligomeric compound with aqueous ammonia yielded compound 160. The GalNAc₃cluster portion of the conjugate group GalNAc₃-9 (GalNAc₃-9_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-9 (GalNAc₃-9_a-CM) is shown below:

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Example 53: Alternate Procedure for Preparation of Compound 18 (GalNAc₃-1a and GalNAc₃-3a)

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Lactone 161 was reacted with diamino propane (3-5 eq) or Mono-Boc protected diamino 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 CbzCl to provide 162b as a white solid after purification by column chromatography. Alcohol 162b was further reacted with compound 4 in the presence of TMSOTf to provide 163a which was converted to 163b by removal of the Cbz group using catalytic hydrogenation. The pentafluorophenyl (PFP) ester 164 was prepared by reacting triacid 113 (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 minimizes the formation of byproducts which are formed using the procedure described in Example 4.

Example 54: Alternate Procedure for Preparation of Compound 18 (GalNAc₃-1a and GalNAc₃-3a)

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The triPFP ester 164 was prepared from acid 113 using the procedure 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 trifluoroacetic acid to provide the triamine which was reacted with the PFP activated acid 166 in the presence of a suitable base such as DIPEA to provide Compound 18.

The PFP protected Gal-NAc acid 166 was prepared from the corresponding 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.

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-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃conjugate groups was attached at either the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 39

Modified ASO targeting SRB-1

SEQ

ASO
Sequence (5′ to 3′)
Motif
Conjugate
ID No.

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5
none
829

353382

^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

(parent)

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

custom-character

830

655861

^mC_dsT_dsT_es^mC_es^mC_esT_es custom-character

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

custom-character

830

664078

^mC_dsT_dsT_es^mC_es^mC_esT_es custom-character

ISIS

831

661161
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

665001
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

custom-character

831

^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

The structure of GalNAc₃-1_awas 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.

Treatment

Six week old male Balb/c mice (Jackson 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-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 40, treatment with antisense oligonucleotides lowered SRB-1 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 GalNAc₃-3 and GalNAc₃-8 conjugates linked at the 5′ terminus (ISIS 661161 and ISIS 665001) showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382)_nFurthermore, ISIS 664078, comprising a GalNAc₃-9 conjugate at the 3′ terminus was essentially equipotent compared to ISIS 655861, which comprises a GalNAc₃-1 conjugate at the 3′ terminus. The 5′ conjugated 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 655861 and ISIS 664078)_n

TABLE 40

ASOs containing GalNAc₃-1, 3, 8 or 9 targeting SRB-1

ISIS
Dosage
SRB-1 mRNA

No.
(mg/kg)
(% Saline)
Conjugate

Saline
n/a
100

353382
3
88
none

10
68

30
36

655861
0.5
98
GalNac₃-1 (3′)

1.5
76

5
31

15
20

664078
0.5
88
GalNac₃-9 (3′)

1.5
85

5
46

15
20

661161
0.5
92
GalNac₃-3 (5′)

1.5
59

5
19

15
11

665001
0.5
100
GalNac₃-8 (5′)

1.5
73

5
29

15
13

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 significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 41

ISIS
Dosage

Total

No.
mg/kg
ALT
AST
Bilirubin
BUN
Conjugate

Saline

24
59
0.1
37.52

353382
3
21
66
0.2
34.65
none

10
22
54
0.2
34.2

30
22
49
0.2
33.72

655861
0.5
25
62
0.2
30.65
GalNac₃-1 (3′)

1.5
23
48
0.2
30.97

5
28
49
0.1
32.92

15
40
97
0.1
31.62

664078
0.5
40
74
0.1
35.3
GalNac₃-9 (3′)

1.5
47
104
0.1
32.75

5
20
43
0.1
30.62

15
38
92
0.1
26.2

661161
0.5
101
162
0.1
34.17
GalNac₃-3 (5′)

1.5
g
42
100
0.1
33.37

5
g
23
99
0.1
34.97

15
53
83
0.1
34.8

665001
0.5
28
54
0.1
31.32
GalNac₃-8 (5′)

1.5
42
75
0.1
32.32

5
24
42
0.1
31.85

15
32
67
0.1
31.

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-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the various GalNAc₃conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety) except for ISIS 655861 which had the GalNAc₃conjugate group attached at the 3′ terminus.

TABLE 42

Modified ASO targeting SRB-1

SEQ

ASO
Sequence (5′ to 3′)
Motif
Conjugate
ID No.

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5
no conjugate
829

353382

^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

(parent)

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

custom-character

830

655861

^mC_dsT_dsT_es^mC_es^mC_esT_es custom-character

ISIS

5/10/5

831

664507

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

661161
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

831

^mC_dsT_dsT_es^m_Ces^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

666224

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

666961

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

666981

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

666881

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

The structure of GalNAc₃-1_awas shown previously in Example 9. The structure of GalNAc₃-2_awas shown previously in Example 37. The structure of GalNAc₃-3_awas shown previously in Example 39. The structure of GalNAc₃-5_awas shown previously in Example 49. The structure of GalNAc₃-6_awas shown previously in Example 51. The structure of GalNAc₃-7_awas shown previously in Example 48. The structure of GalNAc₃-10_awas shown previously in Example 46.

Treatment

Six week old male Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 353382, 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-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 43, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. Indeed, the conjugated antisense oligonucleotides showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 353382). The 5′ conjugated antisense oligonucleotides showed a slight increase in potency compared to the 3′ conjugated antisense oligonucleotide.

TABLE 43

ISIS
Dosage
SRB-1 mRNA

No.
(mg/kg)
(% Saline)
Conjugate

Saline
n/a
100.0

353382
3
96.0
none

10
73.1

30
36.1

655861
0.5
99.4
GalNac₃-1 (3′)

1.5
81.2

5
33.9

15
15.2

664507
0.5
102.0
GalNac₃-2 (5′)

1.5
73.2

5
31.3

15
10.8

661161
0.5
90.7
GalNac₃-3 (5′)

1.5
67.6

5
24.3

15
11.5

666224
0.5
96.1
GalNac₃-5 (5′)

1.5
61.6

5
25.6

15
11.7

666961
0.5
85.5
GalNAc₃-6 (5′)

1.5
56.3

5
34.2

15
13.1

666981
0.5
84.7
GalNAc₃-7 (5′)

1.5
59.9

5
24.9

15
8.5

666881
0.5
100.0
GalNAc₃-10 (5′)

1.5
65.8

5
26.0

15
13.0

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 change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 44 below.

TABLE 44

ISIS
Dosage

Total

No.
mg/kg
ALT
AST
Bilirubin
BUN
Conjugate

Saline

26
57
0.2
27

353382
3
25
92
0.2
27
none

10
23
40
0.2
25

30
29
54
0.1
28

655861
0.5
25
71
0.2
34
GalNac₃-1 (3′)

1.5
28
60
0.2
26

5
26
63
0.2
28

15
25
61
0.2
28

664507
0.5
25
62
0.2
25
GalNac₃-2 (5′)

1.5
24
49
0.2
26

5
21
50
0.2
26

15
59
84
0.1
22

661161
0.5
20
42
0.2
29
GalNac₃-3 (5′)

1.5
g
37
74
0.2
25

5
g
28
61
0.2
29

15
21
41
0.2
25

666224
0.5
34
48
0.2
21
GalNac₃-5 (5′)

1.5
23
46
0.2
26

5
24
47
0.2
23

15
32
49
0.1
26

666961
0.5
17
63
0.2
26
GalNAc₃-6 (5′)

1.5
23
68
0.2
26

5
25
66
0.2
26

15
29
107
0.2
28

666981
0.5
24
48
0.2
26
GalNAc₃-7 (5′)

1.5
30
55
0.2
24

5
46
74
0.1
24

15
29
58
0.1
26

666881
0.5
20
65
0.2
27
GalNAc₃-10 (5′)

1.5
23
59
0.2
24

5
45
70
0.2
26

15
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) levels. The study was performed using 3 transgenic mice that express human APOC-III in each group.

TABLE 45

Modified ASO targeting ApoC III

SEQ

Link-
ID

ASO
Sequence (5′ to 3′)
ages
No.

ISIS
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds
PS
821

304801

^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_esT_e

ISIS
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_ds
PS
822

647535
A_dsG_ds^mC_dsT_esT_esT_esA_es custom-character

ISIS
A_esG_eo^mC_eoT_eoT_eo^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_ds
PO/PS
822

647536
A_dsG_ds^mC_dsT_eoT_eoT_esA_es custom-character

The structure of GalNAc₃-1_awas shown previously 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
116

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

ISIS 304801
30
mg/kg
Plasma TG
34
37
50
69
69

ISIS 647535
10
mg/kg
Plasma TG
18
14
24
18
71

ISIS 647536
10
mg/kg
Plasma TG
21
19
15
32
35

As can be seen in the table above the duration of action increased with addition of the 3′-conjugate group compared 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′-Conjugate Group (Comparison of GalNAc₃-1 and GalNAc4-11) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated 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 GalNAc₃-1_awas shown previously in Example 9. The structure of GalNAc₃-11_awas shown previously in Example 50.

Treatment 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 saline. Each treatment group consisted of 4 animals. The mice were sacrificed 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, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment 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 conjugated oligonucleotides, GalNAc₃-1 and GalNAc4-11, were equipotent.

TABLE 47

Modified ASO targeting SRB-1

Dose
% Saline
SEQ ID

ASO
Sequence (5′ to 3′)
mg/kg
control
No.

Saline

100

ISIS
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
0.6
73.45
823

440762

^mC_dsT_dsT_ks^mC_k
2
59.66

6
23.50

ISIS
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
0.2
62.75
824

651900

^mC_dsT_dsT_ks^m custom-character

0.6
29.14

2
8.61

6
5.62

ISIS
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
0.2
63.99
824

663748

^mC_dsT_dsT_ks^m custom-character

0.6
33.53

2
7.58

6
5.52

Capital letters indicate the nucleobase for each nucleoside and ^mC indicates a 5-methyl cytosine. Subscripts: “e” indicates a 2′-MOE modified nucleoside; “k” indicates 6′-(S)—CH₃bicyclic nucleoside; “d” indicates a β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate 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 significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in Table 48 below.

TABLE 48

ISIS
Dosage

Total

No.
mg/kg
ALT
AST
Bilirubin
BUN
Conjugate

Saline

30
76
0.2
40

440762
0.60
32
70
0.1
35
none

2
26
57
0.1
35

6
31
48
0.1
39

651900
0.2
32
115
0.2
39
GalNac₃-1 (3′)

0.6
33
61
0.1
35

2
30
50
0.1
37

6
34
52
0.1
36

663748
0.2
28
56
0.2
36
GalNac₄-11 (3′)

0.6
34
60
0.1
35

2
44
62
0.1
36

6
38
71
0.1
33

Example 59: Effects of GalNAc₃-1 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

Modified ASOs targeting FXI

SEQ

Link-
ID

ASO
Sequence (5′ to 3′)
ages
No.

ISIS
T_esG_esG_esT_esA_esA_dsT_ds^mC_ds^mC_dsA_ds^mC_ds
PS
832

404071
T_dsT_dsT_ds^mC_dsA_esG_esA_esG_esG_e

ISIS
T_esG_esG_esT_esA_esA_dsT_ds^mC_ds^mC_dsA_ds^mC_ds
PS
833

656172
T_dsT_dsT_ds^mC_dsA_esG_esA_esG_es custom-character

ISIS
T_esG_eoG_eoT_eoA_eoA_dsT_ds^mC_ds^mC_dsA_ds^mC_ds
PO/PS
833

656173
T_dsT_dsT_ds^mC_dsA_eoG_eoA_esG_es custom-character

The structure of GalNAc₃-1_awas shown previously in Example 9.

Treatment

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, Oreg.) according to standard protocols. Plasma FXI protein levels were also measured using ELISA. FXI mRNA levels were determined relative to total RNA (using RIBOGREEN®), prior to normalization to PBS-treated control. The results below are presented as the average percent of FXI mRNA levels for each treatment group. The data was normalized to PBS-treated control and is denoted as “% PBS”. The ED₅₀s were measured using similar methods as described previously and are presented below.

TABLE 50

Factor XI mRNA (% Saline)

Dose
%

ASO
mg/kg
Control
Conjugate
Linkages

Saline

100
none

ISIS
3
92
none
PS

404071
10
40

30
15

ISIS
0.7
74
GalNAc₃-1
PS

656172
2
33

6
9

ISIS
0.7
49
GalNAc₃-1
PO/PS

656173
2
22

6
1

As illustrated in Table 50, treatment with antisense oligonucleotides lowered FXI mRNA levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

As illustrated in Table 50a, treatment with antisense oligonucleotides lowered FXI protein levels in a dose-dependent manner. The oligonucleotides comprising a 3′-GalNAc₃-1 conjugate group showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 404071). Between the two conjugated oligonucleotides an improvement in potency was further provided by substituting some of the PS linkages with PO (ISIS 656173).

TABLE 50a

Factor XI protein (% Saline)

Dose
Protein

ASO
mg/kg
(% Control)
Conjugate
Linkages

Saline

100
none

ISIS
3
127
none
PS

404071
10
32

30
3

ISIS
0.7
70
GalNAc₃-1
PS

656172
2
23

6
1

ISIS
0.7
45
GalNAc₃-1
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 standard protocols. Total bilirubin, total albumin, CRE and BUN were also evaluated. The change in body weights was evaluated with no significant change from the saline group. ALTs, ASTs, total bilirubin and BUN values are shown in the table below.

TABLE 51

ISIS
Dosage

Total
Total

No.
mg/kg
ALT
AST
Albumin
Bilirubin
CRE
BUN
Conjugate

Saline

71.8
84.0
3.1
0.2
0.2
22.9

404071
3
152.8
176.0
3.1
0.3
0.2
23.0
none

10
73.3
121.5
3.0
0.2
0.2
21.4

30
82.5
92.3
3.0
0.2
0.2
23.0

656172
0.7
62.5
111.5
3.1
0.2
0.2
23.8
GalNac₃-1 (3′)

2
33.0
51.8
2.9
0.2
0.2
22.0

6
65.0
71.5
3.2
0.2
0.2
23.9

656173
0.7
54.8
90.5
3.0
0.2
0.2
24.9
GalNac₃-1 (3′)

2
85.8
71.5
3.2
0.2
0.2
21.0

6
114.0
101.8
3.3
0.2
0.2
22.7

Example 60: Effects of Conjugated ASOs Targeting SRB-1 In Vitro

The oligonucleotides listed below were tested in a multiple dose study for antisense inhibition of SRB-1 in primary mouse hepatocytes. ISIS 353382 was included as an unconjugated standard. Each of the conjugate groups were attached at the 3′ or 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside cleavable moiety.

TABLE 52

Modified ASO targeting SRB-1

SEQ ID

ASO
Sequence (5′ to 3′)
Motif
Conjugate
No.

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5
none
829

353382

^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

custom-character

830

655861

^mC_dsT_dsT_es^mC_es^mC_esT_es custom-character

ISIS
G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
5/10/5

custom-character

830

655862

^mC_dsT_dsT_eo^mC_eo^mC_esT_es custom-character

ISIS

G_es^mC_esT_esT_es^mC_esA_dsG_ds
5/10/5

custom-character

831

661161
T_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_ds
5/10/5

custom-character

831

665001
T_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_dsmC_dsA_dsT_dsG_dsA_ds
5/10/5

custom-character

830

664078

^mC_dsT_dsT_es^mC_es^mC_esT_es custom-character

ISIS

G_es^mC_esT_esT_es^mC_esA_dsG_ds
5/10/5

custom-character

831

666961
T_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

664507

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

666881

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

666224

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

ISIS

custom-character

5/10/5

831

666981

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

The structure of GalNAc₃-1_awas shown previously in Example 9. The structure of GalNAc₃-3a was shown previously in Example 39. The structure of GalNAc₃-8a was shown previously in Example 47. The structure of GalNAc₃-9a was shown previously in Example 52. The structure of GalNAc₃-6a was shown previously in Example 51. The structure of GalNAc₃-2a was shown previously in Example 37. The structure of GalNAc₃-10a was shown previously in Example 46. The structure of GalNAc₃-5a was shown previously in Example 49. The structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The oligonucleotides 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 modified 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-1 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.

The IC₅₀was calculated using standard methods and the results are presented in Table 53. The results show that, under free uptake conditions in which no reagents or electroporation techniques are used to artificially promote entry of the oligonucleotides into cells, the oligonucleotides comprising a GalNAc conjugate were significantly more potent in hepatocytes than the parent oligonucleotide (ISIS 353382) that does not comprise a GalNAc conjugate.

TABLE 53

IC₅₀
Internucleoside

SEQ

ASO
(nM)
linkages
Conjugate
ID No.

ISIS 353382
190^a
PS
none
829

ISIS 655861

11^a
PS
GalNAc₃-1
830

ISIS 655862
3
PO/PS
GalNAc₃-1
830

ISIS 661161

15^a
PS
GalNAc₃-3
831

ISIS 665001
20
PS
GalNAc₃-8
831

ISIS 664078
55
PS
GalNAc₃-9
830

ISIS 666961

22^a
PS
GalNAc₃-6
831

ISIS 664507
30
PS
GalNAc₃-2
831

ISIS 666881
30
PS
GalNAc₃-10
831

ISIS 666224

30^a
PS
GalNAc₃-5
831

ISIS 666981
40
PS
GalNAc₃-7
831

^aAverage of multiple runs.

Example 61: Preparation of Oligomeric Compound 175 Comprising GalNAc₃-12

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Compound 169 is commercially available. Compound 172 was prepared by addition of benzyl (perfluorophenyl) glutarate to compound 171. The benzyl (perfluorophenyl) 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 prepared from compound 174 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-12 (GalNAc₃-12_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-12 (GalNAc₃-12_a-CM-) is shown below:

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Example 62: Preparation of Oligomeric Compound 180 Comprising GalNAc₃-13

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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 GalNAc₃cluster portion of the conjugate group GalNAc₃-13 (GalNAc₃-13_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In a certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-13 (GalNAc₃-13_a-CM-) is shown below:

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Example 63: Preparation of Oligomeric Compound 188 Comprising GalNAc₃-14

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Compounds 181 and 185 are commercially available. Oligomeric compound 188, comprising a GalNAc₃-14 conjugate group, was prepared from compound 187 using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-14 (GalNAc₃-14_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-14 (GalNAc₃-14_a-CM-) is shown below:

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Example 64: Preparation of Oligomeric Compound 197 Comprising GalNAc₃-15

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Compound 189 is commercially available. Compound 195 was prepared using the general 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 procedures. The GalNAc₃cluster portion of the conjugate group GalNAc₃-15 (GalNAc₃-15_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-15 (GalNAc₃-15_a-CM-) is shown below:

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Example 65: Dose-Dependent Study of Oligonucleotides Comprising a 5′-Conjugate Group (Comparison of GalNAc₃-3, 12, 13, 14, and 15) Targeting SRB-1 In Vivo

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Unconjugated ISIS 353382 was included as a standard. Each of the GalNAc₃conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked 2′-deoxyadenosine nucleoside (cleavable moiety).

TABLE 54

Modified ASOs targeting SRB-1

ISIS

SEQ

No.
Sequence (5′ to 3′)
Conjugate
ID No.

353382
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es
none
829

^mC_es^mC_esT_esT_e

661161

custom-character

GalNAc₃-3
831

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_ds

T_es^m_Ces^mC_esT_esT_e

671144

custom-character

GalNAc₃-12
831

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_ds

T_es^mC_es^mC_esT_esT_e

670061

custom-character

GalNAc₃-13
831

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_ds

T_es^mC_es^mC_esT_esT_e

671261

custom-character

GalNAc₃-14
831

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_ds

T_es^mC_es^mC_esT_esT_e

671262

custom-character

GalNAc₃-15
831

custom-character

G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_ds

T_es^mC_es^mC_esT_esT_e

The structure of GalNAc₃-3_awas shown previously in Example 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 previously in Example 63. The structure of GalNAc₃-15a was shown previously in Example 64.

Treatment

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 353382, 661161, 671144, 670061, 671261, 671262, 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 sacrificed 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, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 55, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. No significant 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 2×15 mg/kg; and ISIS 661161 dosages 5 and 2×2.5 mg/kg). The antisense oligonucleotides comprising the phosphodiester linked GalNAc₃-3, 12, 13, 14, and 15 conjugates showed substantial improvement in potency compared to the unconjugated antisense oligonucleotide (ISIS 335382).

TABLE 55

SRB-1 mRNA (% Saline)

ISIS
Dosage
SRB-1 mRNA
ED₅₀

No.
(mg/kg)
(% Saline)
(mg/kg)
Conjugate

Saline
n/a
100.0
n/a
n/a

353382
3
85.0
22.4
none

10
69.2

30
34.2

2 × 15
36.0

661161
0.5
87.4
2.2
GalNAc₃-3

1.5
59.0

5
25.6

2 × 2.5
27.5

15
17.4

671144
0.5
101.2
3.4
GalNAc₃-12

1.5
76.1

5
32.0

15
17.6

670061
0.5
94.8
2.1
GalNAc₃-13

1.5
57.8

5
20.7

15
13.3

671261
0.5
110.7
4.1
GalNAc₃-14

1.5
81.9

5
39.8

15
14.1

671262
0.5
109.4
9.8
GalNAc₃-15

1.5
99.5

5
69.2

15
36.1

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 significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 56

Total

ISIS
Dosage
ALT
AST
Bilirubin
BUN

No.
(mg/kg)
(U/L)
(U/L)
(mg/dL)
(mg/dL)
Conjugate

Saline
n/a
28
60
0.1
39
n/a

353382
3
30
77
0.2
36
none

10
25
78
0.2
36

30
28
62
0.2
35

2 × 15
22
59
0.2
33

661161
0.5
39
72
0.2
34
GalNAc₃-3

1.5
26
50
0.2
33

5
41
80
0.2
32

2 × 2.5
24
72
0.2
28

15
32
69
0.2
36

671144
0.5
25
39
0.2
34
GalNAc₃-12

1.5
26
55
0.2
28

5
48
82
0.2
34

15
23
46
0.2
32

670061
0.5
27
53
0.2
33
GalNAc₃-13

1.5
24
45
0.2
35

5
23
58
0.1
34

15
24
72
0.1
31

671261
0.5
69
99
0.1
33
GalNAc₃-14

1.5
34
62
0.1
33

5
43
73
0.1
32

15
32
53
0.2
30

671262
0.5
24
51
0.2
29
GalNAc₃-15

1.5
32
62
0.1
31

5
30
76
0.2
32

15
31
64
0.1
32

Example 66: Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Cluster

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice. Each of the GalNAc₃conjugate groups was attached at the 5′ terminus of the respective oligonucleotide by a phosphodiester linked nucleoside (cleavable moiety (CM)).

TABLE 57

Modified ASOs targeting SRB-1

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

661161

GalNAc
₃
-3
_a
-
_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
A_d
831

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

670699

GalNAc
₃
-3
_a
-
_o'
T
_doG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
T_d
834

G_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

670700

GalNAc
₃
-3
_a
-
_o'
A
_eoG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
A_e
831

G_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

670701

GalNAc
₃
-3
_a
-
o'
T
_eoG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
T_e
834

G_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

671165

GalNAc
₃
-13
_a
-
o'
A
_doG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-13a
A_d
831

G_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

“s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_awas shown previously in Example 39. The structure of GalNAc₃-13a was shown previously in Example 62.

Treatment

Six to eight week old C₅₇bl6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once at the dosage shown below with ISIS 661161, 670699, 670700, 670701, 671165, 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-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

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

TABLE 58

SRB-1 mRNA (% Saline)

ISIS
Dosage
SRB-1 mRNA
GalNAc₃

No.
(mg/kg)
(% Saline)
Cluster
CM

Saline
n/a
100.0
n/a
n/a

661161
0.5
87.8
GalNAc₃-3a
A_d

1.5
61.3

5
33.8

15
14.0

670699
0.5
89.4
GalNAc₃-3a
T_d

1.5
59.4

5
31.3

15
17.1

670700
0.5
79.0
GalNAc₃-3a
A_e

1.5
63.3

5
32.8

15
17.9

670701
0.5
79.1
GalNAc₃-3a
T_e

1.5
59.2

5
35.8

15
17.7

671165
0.5
76.4
GalNAc₃-13a
A_d

1.5
43.2

5
22.6

15
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 significant differences from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 56 below.

TABLE 59

Total

ISIS
Dosage
ALT
AST
Bilirubin
BUN
GalNAc₃

No.
(mg/kg)
(U/L)
(U/L)
(mg/dL)
(mg/dL)
Cluster
CM

Saline
n/a
24
64
0.2
31
n/a
n/a

661161
0.5
25
64
0.2
31
GalNAc₃-3a
A_d

1.5
24
50
0.2
32

5
26
55
0.2
28

15
27
52
0.2
31

670699
0.5
42
83
0.2
31
GalNAc₃-3a
T_d

1.5
33
58
0.2
32

5
26
70
0.2
29

15
25
67
0.2
29

670700
0.5
40
74
0.2
27
GalNAc₃-3a
A_e

1.5
23
62
0.2
27

5
24
49
0.2
29

15
25
87
0.1
25

670701
0.5
30
77
0.2
27
GalNAc₃-3a
T_e

1.5
22
55
0.2
30

5
81
101
0.2
25

15
31
82
0.2
24

671165
0.5
44
84
0.2
26
GalNAc₃-13a
A_d

1.5
47
71
0.1
24

5
33
91
0.2
26

15
33
56
0.2
29

Example 67: Preparation of Oligomeric Compound 199 Comprising GalNAc₃-16

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Oligomeric compound 199, comprising a GalNAc₃-16 conjugate group, is prepared using the general procedures illustrated in Examples 7 and 9. The GalNAc₃cluster portion of the conjugate group GalNAc₃-16 (GalNAc₃-16_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-16 (GalNAc₃-16_a-CM-) is shown below:

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Example 68: Preparation of Oligomeric Compound 200 Comprising GalNAc₃-17

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Oligomeric compound 200, comprising a GalNAc₃-17 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-17 (GalNAc₃-17_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-17 (GalNAc₃-17_a-CM-) is shown below:

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Example 69: Preparation of Oligomeric Compound 201 Comprising GalNAc₃-18

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Oligomeric compound 201, comprising a GalNAc₃-18 conjugate group, was prepared using the general procedures illustrated in Example 46. The GalNAc₃cluster portion of the conjugate group GalNAc₃-18 (GalNAc₃-18_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-18 (GalNAc₃-18_a-CM-) is shown below:

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Example 70: Preparation of Oligomeric Compound 204 Comprising GalNAc₃-19

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Oligomeric compound 204, comprising a GalNAc₃-19 conjugate group, was prepared from compound 64 using the general procedures illustrated in Example 52. The GalNAc₃cluster portion of the conjugate group GalNAc₃-19 (GalNAc₃-19_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-19 (GalNAc₃-19_a-CM-) is shown below:

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Example 71: Preparation of Oligomeric Compound 210 Comprising GalNAc₃-20

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Compound 205 was prepared by adding PFP-TFA and DIEA to 6-(2,2,2-trifluoroacetamido)hexanoic acid in acetonitrile, which was prepared by adding triflic anhydride to 6-aminohexanoic acid. The reaction mixture was heated to 80° C., then lowered to rt. Oligomeric compound 210, comprising a GalNAc₃-20 conjugate group, was prepared from compound 208 using the general procedures illustrated in Example 52. The GalNAc₃cluster portion of the conjugate group GalNAc₃-20 (GalNAc₃-20_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-20 (GalNAc₃-20_a-CM-) is shown below:

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Example 72: Preparation of Oligomeric Compound 215 Comprising GalNAc₃-21

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Compound 211 is commercially available. Oligomeric compound 215, comprising a GalNAc₃-21 conjugate group, was prepared from compound 213 using the general procedures illustrated in Example 52. The GalNAc₃cluster portion of the conjugate group GalNAc₃-21 (GalNAc₃-21_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-21 (GalNAc₃-21_a-CM-) is shown below:

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Example 73: Preparation of Oligomeric Compound 221 Comprising GalNAc₃-22

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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 GalNAc₃cluster portion of the conjugate group GalNAc₃-22 (GalNAc₃-22_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, the cleavable moiety is —P(═O)(OH)-A_d-P(═O)(OH)—. The structure of GalNAc₃-22 (GalNAc₃-22_a-CM-) is shown below:

embedded image

Example 74: Effect of Various Cleavable Moieties on Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5′-GalNAc₃Conjugate

TABLE 60

Modified ASOs targeting SRB-1

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

353382
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es
n/a
n/a
829

^mC_es^mC_esT_esT_e

661161

GalNAc
₃
-3
_a
-
_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
A_d
831

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

666904

GalNAc
₃
-3
_a
-
_o'G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
PO
829

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

675441

GalNAc
₃
-17
_a
-
_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-17a
A_d
831

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

675442

GalNAc
₃
-18
_a
-
_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-18a
A_d
831

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

In all tables, 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 β-D-2′-deoxyribonucleoside; “s” indicates a phosphorothioate internucleoside linkage (PS); “o” indicates a phosphodiester internucleoside linkage (PO); and “o′” indicates —O—P(═O)(OH)—. Conjugate groups are in bold.

The structure of GalNAc₃-3_awas 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.

Treatment

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 sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 61, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner. The antisense oligonucleotides comprising a GalNAc conjugate showed similar potencies and were significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 61

SRB-1 mRNA (% Saline)

ISIS
Dosage
SRB-1 mRNA
GalNAc₃

No.
(mg/kg)
(% Saline)
Cluster
CM

Saline
n/a
100.0
n/a
n/a

353382
3
79.38
n/a
n/a

10
68.67

30
40.70

661161
0.5
79.18
GalNAc₃-3a
A_d

1.5
75.96

5
30.53

15
12.52

666904
0.5
91.30
GalNAc₃-3a
PO

1.5
57.88

5
21.22

15
16.49

675441
0.5
76.71
GalNAc₃-17a
A_d

1.5
63.63

5
29.57

15
13.49

675442
0.5
95.03
GalNAc₃-18a
A_d

1.5
60.06

5
31.04

15
19.40

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 significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 62 below.

TABLE 62

Total

ISIS
Dosage
ALT
AST
Bilirubin
BUN
GalNAc₃

No.
(mg/kg)
(U/L)
(U/L)
(mg/dL)
(mg/dL)
Cluster
CM

Saline
n/a
26
59
0.16
42
n/a
n/a

353382
3
23
58
0.18
39
n/a
n/a

10
28
58
0.16
43

30
20
48
0.12
34

661161
0.5
30
47
0.13
35
GalNAc₃-3a
A_d

1.5
23
53
0.14
37

5
26
48
0.15
39

15
32
57
0.15
42

666904
0.5
24
73
0.13
36
GalNAc₃-3a
PO

1.5
21
48
0.12
32

5
19
49
0.14
33

15
20
52
0.15
26

675441
0.5
42
148
0.21
36
GalNAc₃-17a
A_d

1.5
60
95
0.16
34

5
27
75
0.14
37

15
24
61
0.14
36

675442
0.5
26
65
0.15
37
GalNAc₃-18a
A_d

1.5
25
64
0.15
43

5
27
69
0.15
37

15
30
84
0.14
37

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 following the treatment procedures described in Examples 65, 66, and 74. The liver samples were minced and extracted using standard protocols and analyzed by IP-HPLC-MS alongside an internal standard. The combined tissue level (μg/g) of all metabolites was measured by integrating the appropriate UV peaks, and the tissue level of the full-length ASO missing the conjugate (“parent,” which is Isis No. 353382 in this case) was measured using the appropriate extracted ion chromatograms (EIC).

TABLE 63

PK Analysis in Liver

Total
Parent ASO

Tissue Level
Tissue Level

ISIS
Dosage
by UV
by EIC
GalNAc₃

No.
(mg/kg)
(μg/g)
(μg/g)
Cluster
CM

353382
3
8.9
8.6
n/a
n/a

10
22.4
21.0

30
54.2
44.2

661161
5
32.4
20.7
GalNAc₃-3a
A_d

15
63.2
44.1

671144
5
20.5
19.2
GalNAc₃-12a
A_d

15
48.6
41.5

670061
5
31.6
28.0
GalNAc₃-13a
A_d

15
67.6
55.5

671261
5
19.8
16.8
GalNAc₃-14a
A_d

15
64.7
49.1

671262
5
18.5
7.4
GalNAc₃-15a
A_d

15
52.3
24.2

670699
5
16.4
10.4
GalNAc₃-3a
T_d

15
31.5
22.5

670700
5
19.3
10.9
GalNAc₃-3a
A_e

15
38.1
20.0

670701
5
21.8
8.8
GalNAc₃-3a
T_e

15
35.2
16.1

671165
5
27.1
26.5
GalNAc₃-13a
A_d

15
48.3
44.3

666904
5
30.8
24.0
GalNAc₃-3a
PO

15
52.6
37.6

675441
5
25.4
19.0
GalNAc₃-17a
A_d

15
54.2
42.1

675442
5
22.2
20.7
GalNAc₃-18a
A_d

15
39.6
29.0

The results in Table 63 above show that there were greater liver tissue levels of the oligonucleotides comprising a GalNAc₃conjugate group than of the parent oligonucleotide that does not comprise a GalNAc₃conjugate group (ISIS 353382) 72 hours following oligonucleotide administration, particularly when taking into consideration the differences in dosing between the oligonucleotides with and without a GalNAc₃conjugate group. Furthermore, by 72 hours, 40-98% of each oligonucleotide comprising a GalNAc₃conjugate group was metabolized to the parent compound, indicating that the GalNAc₃conjugate groups were cleaved from the oligonucleotides.

Example 76: Preparation of Oligomeric Compound 230 Comprising GalNAc₃-23

embedded image

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 (500 mL) for 16 hours. The reaction was then evaporated to an oil, dissolved in EtOAc and washed with water, sat. NaHCO₃, brine, and dried over Na₂SO₄. The ethyl acetate was concentrated to dryness and purified by column chromatography, eluted with EtOAc/hexanes (1:1) followed by 10% methanol in CH₂Cl₂to give compound 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 (100 mL) 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 Na₂SO₄. The organic layer was concentrated to dryness to give 5.3 g of compound 224 (92%). LCMS and NMR were consistent with the structure. 1-Azidotriethylene glycol (compound 224, 5.53 g, 23.69 mmol) and compound 4 (6 g, 18.22 mmol) were treated with 4 A molecular sieves (5 g), and TMSOTf (1.65 ml, 9.11 mmol) in dichloromethane (100 mL) under an inert atmosphere. After 14 hours, the reaction was filtered to remove the sieves, and the organic layer was washed with sat. NaHCO₃, water, brine, and dried over Na₂SO₄. The organic 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 consistent with the structure. Compound 225 (11.9 g, 23.59 mmol) was hydrogenated in EtOAc/Methanol (4:1, 250 mL) 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 (100 mL) were treated dropwise with pentaflourotrifluoro 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 Na₂SO₄. The organic layer was concentrated to dryness and then recrystallized from heptane to give compound 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 to 10% methanol in dichloromethane to give compound 228. LCMS and NMR were consistent with the structure. Compound 228 (1.7 g, 1.02 mmol) was treated with Raney Nickel (about 2 g wet) in ethanol (100 mL) 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 structure. This solid (0.87 g, 0.53 mmol) was treated with benzylglutaric acid (0.18 g, 0.8 mmol), HBTU (0.3 g, 0.8 mmol) and DIEA (273.7 μl, 1.6 mmol) in DMF (5 mL). 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. NaHCO₃, brine, and dried over Na₂SO₄. 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 filtration 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 μl, 0.66 mmol) was added and the reaction was purged with argon. Pentaflourotriflouro acetate (46.39 μl, 0.4 mmol) was slowly added to the reaction mixture. The color of the reaction changed from pale yellow to burgundy, and gave off a light smoke which was blown away with a stream of argon. The reaction was allowed to stir at room temperature for one hour (completion of reaction was confirmed by LCMS). The solvent was removed under reduced pressure (rotovap) at 70° C. The residue was diluted with DCM and washed with 1N NaHSO₄, brine, saturated sodium bicarbonate and brine again. The organics were dried over Na₂SO₄, 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 illustrated in Example 46. The GalNAc₃cluster portion of the GalNAc₃-23 conjugate group (GalNAc₃-23_a) can be combined with any cleavable moiety to provide a variety of conjugate groups. The structure of GalNAc₃-23 (GalNAc₃-23_a-CM) is shown below:

embedded image

Example 77: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 64

Modified ASOs targeting SRB-1

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

661161

GalNAc
₃
-3
_a
-
_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
A_d
831

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

666904

GalNAc
₃
-3
_a
-
_o'G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-3a
PO
829

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

673502

GalNAc
₃
-10
_a
-
_o'
A
doG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsAdT_ds
GalNAc₃-10a
A_d
831

G_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

677844

GalNAc
₃
-9
_a
-
_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-9a
A_d
831

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

677843

GalNAc
₃
-23
_a
-
_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_ds
GalNAc₃-23a
A_d
831

G_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

655861
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es
GalNAc₃-1a
A_d
830

^mC_esT_esT_eoA_do'-GalNAc₃-1_a

677841
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es
GalNAc₃-19a
A_d
830

^mC_esT_esT_eoA_do'-GalNAc₃-19_a

677842
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es
GalNAc₃-20a
A_d
830

^mC_esT_esT_eoA_do'-GalNAc₃-20_a

The structure of GalNAc₃-1_awas shown previously in Example 9, GalNAc₃-3_awas shown in Example 39, GalNAc₃-9a was shown in Example 52, GalNAc₃-10a was shown in Example 46, GalNAc₃-19_awas shown in Example 70, GalNAc₃-20_awas shown in Example 71, and GalNAc₃-23_awas shown in Example 76.

Treatment

Six to eight week old C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were each injected 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 sacrificed 72 hours following the final administration to determine the SRB-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Table 65, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 65

SRB-1 mRNA (% Saline)

ISIS
Dosage
SRB-1 mRNA
GalNAc₃

No.
(mg/kg)
(% Saline)
Cluster
CM

Saline
n/a
100.0
n/a
n/a

661161
0.5
89.18
GalNAc₃-3a
A_d

1.5
77.02

5
29.10

15
12.64

666904
0.5
93.11
GalNAc₃-3a
PO

1.5
55.85

5
21.29

15
13.43

673502
0.5
77.75
GalNAc₃-10a
A_d

1.5
41.05

5
19.27

15
14.41

677844
0.5
87.65
GalNAc₃-9a
A_d

1.5
93.04

5
40.77

15
16.95

677843
0.5
102.28
GalNAc₃-23a
A_d

1.5
70.51

5
30.68

15
13.26

655861
0.5
79.72
GalNAc₃-1a
A_d

1.5
55.48

5
26.99

15
17.58

677841
0.5
67.43
GalNAc₃-19a
A_d

1.5
45.13

5
27.02

15
12.41

677842
0.5
64.13
GalNAc₃-20a
A_d

1.5
53.56

5
20.47

15
10.23

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were also measured using standard protocols. Total bilirubin and BUN were also evaluated. Changes in body weights were evaluated, with no significant change from the saline group (data not shown). ALTs, ASTs, total bilirubin and BUN values are shown in Table 66 below.

TABLE 66

Total

ISIS
Dosage
ALT
AST
Bilirubin
BUN
GalNAc₃

No.
(mg/kg)
(U/L)
(U/L)
(mg/dL)
(mg/dL)
Cluster
CM

Saline
n/a
21
45
0.13
34
n/a
n/a

661161
0.5
28
51
0.14
39
GalNAc₃-3a
A_d

1.5
23
42
0.13
39

5
22
59
0.13
37

15
21
56
0.15
35

666904
0.5
24
56
0.14
37
GalNAc₃-3a
PO

1.5
26
68
0.15
35

5
23
77
0.14
34

15
24
60
0.13
35

673502
0.5
24
59
0.16
34
GalNAc₃-10a
A_d

1.5
20
46
0.17
32

5
24
45
0.12
31

15
24
47
0.13
34

677844
0.5
25
61
0.14
37
GalNAc₃-9a
A_d

1.5
23
64
0.17
33

5
25
58
0.13
35

15
22
65
0.14
34

677843
0.5
53
53
0.13
35
GalNAc₃-23a
A_d

1.5
25
54
0.13
34

5
21
60
0.15
34

15
22
43
0.12
38

655861
0.5
21
48
0.15
33
GalNAc₃-1a
A_d

1.5
28
54
0.12
35

5
22
60
0.13
36

15
21
55
0.17
30

677841
0.5
32
54
0.13
34
GalNAc₃-19a
A_d

1.5
24
56
0.14
34

5
23
92
0.18
31

15
24
58
0.15
31

677842
0.5
23
61
0.15
35
GalNAc₃-20a
A_d

1.5
24
57
0.14
34

5
41
62
0.15
35

15
24
37
0.14
32

Example 78: Antisense Inhibition In Vivo by Oligonucleotides Targeting Angiotensinogen Comprising a GalNAc₃Conjugate

The oligonucleotides listed below were tested in a dose-dependent study for antisense inhibition of Angiotensinogen (AGT) in normotensive Sprague Dawley rats.

TABLE 67

Modified ASOs targeting AGT

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

552668

^mC_esA_es^mC_esT_esG_esA_dsT_dsT_dsT_dsT_dsT_dsG_ds^mC_ds^mC_ds^mC_dsA_esG_es
n/a
n/a
835

G_esA_esT_e

669509

^mC_esA_es^mC_esT_esG_esA_dsT_dsT_dsT_dsT_dsT_dsG_ds^mC_ds^mC_ds^mC_dsA_esG_es
GalNAc₃-1_a
A_d
836

G_esA_esT_eoA_do'-GalNAC₃-1_a

The structure of GalNAc₃-1_awas shown previously in Example 9.

Treatment

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 sacrificed 72 hours following the final dose. AGT liver mRNA levels were measured using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. AGT plasma protein levels were measured using the Total Angiotensinogen ELISA (Catalog # JP27412, IBL International, Toronto, ON) with plasma diluted 1:20,000. The results below are presented as the average percent of AGT 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 protein levels in a dose-dependent manner, and the oligonucleotide comprising a GalNAc conjugate was significantly more potent than the parent oligonucleotide lacking a GalNAc conjugate.

TABLE 68

AGT liver mRNA and plasma protein levels

AGT liver
AGT plasma

ISIS
Dosage
mRNA
protein
GalNAc₃

No.
(mg/kg)
(% PBS)
(% PBS)
Cluster
CM

PBS
n/a
100
100
n/a
n/a

552668
3
95
122
n/a
n/a

10
85
97

30
46
79

90
8
11

669509
0.3
95
70
GalNAc₃-1a
A_d

1
95
129

3
62
97

10
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

Body Weight

ISIS
Dosage
ALT
AST
(% of
GalNAc₃

No.
(mg/kg)
(U/L)
(U/L)
baseline)
Cluster
CM

PBS
n/a
51
81
186
n/a
n/a

552668
3
54
93
183
n/a
n/a

10
51
93
194

30
59
99
182

90
56
78
170

669509
0.3
53
90
190
GalNAc₃-1a
A_d

1
51
93
192

3
48
85
189

10
56
95
189

Example 79: Duration of Action In Vivo of Oligonucleotides Targeting APOC-III Comprising a GalNAc₃Conjugate

The oligonucleotides listed in Table 70 below were tested in a single dose study for duration of action in mice.

TABLE 70

Modified ASOs targeting APOC-III

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

304801
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_es
n/a
n/a
821

T_esA_esT_e

647535
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_es
GalNAc₃-1a
A_d
822

T_esA_esT_eoA_do'-GalNAc₃-1_a

663083

GalNAc
3
-3
_a
-
_o'
A
_doA_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds
GalNAc₃-3a
A_d
837

^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_esT_e

674449

GalNAC
₃
-7
_a
-
_o′
A
_doA_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds
GalNAc₃-7a
A_d
837

^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_esT_e

674450

GalNAc
₃
-10
_a
-
_o'
A
_doA_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds
GalNAc₃-10a
A_d
837

^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_esT_e

674451

GalNAc
₃
-13
_a
-
_o′
A
_doA_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds
GalNAc₃-13a
A_d
837

^mC_dsA_dsG_ds^mC_dsT_esT_esT_esA_esT_e

The structure of GalNAc₃-1_awas shown previously in Example 9, GalNAc₃-3_awas shown in Example 39, GalNAc₃-7_awas shown in Example 48, GalNAc₃-10_awas shown in Example 46, and GalNAc₃-13_awas shown in Example 62.

Treatment

Six to eight week old transgenic mice that express human APOC-III 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 measured as described in Example 20. The results 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 without 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

Time point

ISIS
Dosage
(days post-
Triglycerides
APOC-III protein
GalNAc₃

No.
(mg/kg)
dose)
(% baseline)
(% baseline)
Cluster
CM

PBS
n/a
3
97
102
n/a
n/a

7
101
98

14
108
98

21
107
107

28
94
91

35
88
90

42
91
105

304801
30
3
40
34
n/a
n/a

7
41
37

14
50
57

21
50
50

28
57
73

35
68
70

42
75
93

647535
10
3
36
37
GalNAc₃-1a
A_d

7
39
47

14
40
45

21
41
41

28
42
62

35
69
69

42
85
102

663083
10
3
24
18
GalNAc₃-3a
A_d

7
28
23

14
25
27

21
28
28

28
37
44

35
55
57

42
60
78

674449
10
3
29
26
GalNAc₃-7a
A_d

7
32
31

14
38
41

21
44
44

28
53
63

35
69
77

42
78
99

674450
10
3
33
30
GalNAc₃-10a
A_d

7
35
34

14
31
34

21
44
44

28
56
61

35
68
70

42
83
95

674451
10
3
35
33
GalNAc₃-13a
A_d

7
24
32

14
40
34

21
48
48

28
54
67

35
65
75

42
74
97

Example 80: Antisense Inhibition In Vivo by Oligonucleotides Targeting Alpha-1 Antitrypsin (A1AT) Comprising a GalNAc₃Conjugate

The oligonucleotides listed in Table 72 below were tested in a study for dose-dependent inhibition of A1AT in mice.

TABLE 72

Modified ASOs targeting A1AT

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

476366
A_es^mC_es^mC_es^mC_esA_esA_dsT_dsT_ds^mC_dsA_dsG_dsA_dsA_dsG_dsG_dsA_esA_es
n/a
n/a
838

G_esG_esA_e

656326
A_es^mC_es^mC_es^mC_esA_esA_dsT_dsT_ds^mC_dsA_dsG_dsA_dsA_dsG_dsG_dsA_esA_es
GalNAc₃-1a
A_d
839

G_esG_esA_eoA_do'-GalNAc₃-1_a

678381

GalNAc
₃
-3
_a
-
_o′
A
_doA_es^mC_es^mC_es^mC_esA_esA_dsT_dsT_ds^mC_dsA_dsG_dsA_ds
GalNAc₃-3a
A_d
840

A_dsG_dsG_dsA_esA_esG_esG_esA_e

678382

GalNAC
₃
-7
_a
-
_o′
A
_doA_es^mC_es^mC_es^mC_esA_esA_dsT_dsT_ds^mC_dsA_dsG_dsA_ds
GalNAc₃-7a
A_d
840

A_dsG_dsG_dsA_esA_esG_esG_esA_e

678383

GalNAc
₃
-10
_a
-
_o′
A
_doA_es^mC_es^mC_es^mC_esA_esA_dsT_dsT_ds^mC_dsA_dsG_ds
GalNAc₃-10a
A_d
840

A_dsA_dsG_dsG_dsA_esA_esG_esG_esA_e

678384

GalNAc
₃
-13
_a
-
_o′
A
_doA_es^mC_es^mC_es^mC_esA_esA_dsT_dsT_ds^mC_dsA_dsG_ds
GalNAc₃-13a
A_d
840

A_dsA_dsG_dsG_dsA_esA_esG_esG_esA_e

Treatment

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 consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. A1AT liver mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. A1AT plasma protein levels were determined using the Mouse Alpha 1-Antitrypsin ELISA (catalog #41-A1AMS-E01, Alpco, Salem, N.H.). The results below are presented as the average percent of A1AT liver mRNA and plasma protein levels for each treatment group, normalized to the PBS control.

As illustrated in Table 73, treatment 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)_n

TABLE 73

A1AT liver mRNA and plasma protein levels

A1AT liver
A1AT plasma

ISIS
Dosage
mRNA
protein
GalNAc₃

No.
(mg/kg)
(% PBS)
(% PBS)
Cluster
CM

PBS
n/a
100
100
n/a
n/a

476366
5
86
78
n/a
n/a

15
73
61

45
30
38

656326
0.6
99
90
GalNAc₃-1a
A_d

2
61
70

6
15
30

18
6
10

678381
0.6
105
90
GalNAc₃-3a
A_d

2
53
60

6
16
20

18
7
13

678382
0.6
90
79
GalNAc₃-7a
A_d

2
49
57

6
21
27

18
8
11

678383
0.6
94
84
GalNAc₃-10a
A_d

2
44
53

6
13
24

18
6
10

678384
0.6
106
91
GalNAc₃-13a
A_d

2
65
59

6
26
31

18
11
15

Liver transaminase and BUN levels in plasma were measured at time of sacrifice using standard protocols. Body weights 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 weight
Liver weight
Kidney weight
Spleen weight

No.
(mg/kg)
(U/L)
(U/L)
(mg/dL)
(% baseline)
(Rel % BW)
(Rel % BW)
(Rel % BW)

PBS
n/a
25
51
37
119
100
100
100

476366
5
34
68
35
116
91
98
106

15
37
74
30
122
92
101
128

45
30
47
31
118
99
108
123

656326
0.6
29
57
40
123
100
103
119

2
36
75
39
114
98
111
106

6
32
67
39
125
99
97
122

18
46
77
36
116
102
109
101

678381
0.6
26
57
32
117
93
109
110

2
26
52
33
121
96
106
125

6
40
78
32
124
92
106
126

18
31
54
28
118
94
103
120

678382
0.6
26
42
35
114
100
103
103

2
25
50
31
117
91
104
117

6
30
79
29
117
89
102
107

18
65
112
31
120
89
104
113

678383
0.6
30
67
38
121
91
100
123

2
33
53
33
118
98
102
121

6
32
63
32
117
97
105
105

18
36
68
31
118
99
103
108

678384
0.6
36
63
31
118
98
103
98

2
32
61
32
119
93
102
114

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 A1AT Comprising a GalNAc₃Cluster

The oligonucleotides listed in Table 72 were tested in a single dose study for duration of action in mice.

Treatment

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 A1AT protein levels were measured via ELISA (see Example 80). The results below are presented as the average percent of plasma A1AT 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 lacking a GalNAc conjugate (ISIS 476366). Furthermore, the oligonucleotides comprising a 5′-GalNAc conjugate (ISIS 678381, 678382, 678383, 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 A1AT protein levels in mice

Time point
A1AT

ISIS
Dosage
(days post-
(% base-
GalNAc₃

No.
(mg/kg)
dose)
line)
Cluster
CM

PBS
n/a
5
93
n/a
n/a

12
93

19
90

25
97

476366
100
5
38
n/a
n/a

12
46

19
62

25
77

656326
18
5
33
GalNAc₃-1a
A_d

12
36

19
51

25
72

678381
18
5
21
GalNAc₃-3a
A_d

12
21

19
35

25
48

678382
18
5
21
GalNAc₃-7a
A_d

12
21

19
39

25
60

678383
18
5
24
GalNAc₃-10a
A_d

12
21

19
45

25
73

678384
18
5
29
GalNAc₃-13a
A_d

12
34

19
57

25
76

Example 82: Antisense Inhibition In Vitro by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃Conjugate

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 Williams E medium and cells were incubated overnight at 37° C. in 5% CO₂. Cells were lysed 16 hours following oligonucleotide addition, and total RNA was purified using RNease 3000 BioRobot (Qiagen). SRB-1 mRNA levels were determined using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. IC₅₀values were determined 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-1 expression in vitro

ISIS

GalNAc

IC₅₀
SEQ

No.
Sequences (5′ to 3′)
Linkages
Cluster
CM
(nM)
ID No.

353382
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds
PS
n/a
n/a
250
829

A_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

655861
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds
PS
GalNAc₃-
A_d
40
830

A_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_eoA_do′-

1_a

GalNAc
₃
-1
_a

661161

GalNAc
₃
-3
_a
-

PS
GalNAc₃-
A_d
40
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

3_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

661162

GalNAc
₃
-3
_a
-

PO/PS
GalNAc₃-
A_d
8
831

_o′
A
_doG_es^mC_eoT_eoT_eo^mT_eoA_dsG_dsT_ds

3_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

664078
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds
PS
GalNAc₃-
A_d
20
830

A_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_eoA_do'-

9_a

GalNAc
₃
-9
_a

665001

GalNAc
₃
-8
_a
-

PS
GalNAc₃-
A_d
70
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_ds-

8_a

A_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

666224

GalNAc
₃
-5
_a
-

PS
GalNAc₃-
A_d
80
831

_o′
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

5_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mc_es^mc_esT_esT_e

666841
G_es^mC_eoT_eoT_eo^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds
PO/PS
n/a
n/a
>250
829

A_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

666881

GalNAc
₃
-10
_a
-

PS
GalNAc₃-
A_d
30
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

10_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

666904

GalNAc
₃
-3
_a
-

PS
GalNAc₃-
PO
9
829

_o′G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_ds

3_a

A_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

666924

GalNAc
₃
-3
_a
-

PS
GalNAc₃-
T_d
15
834

_o′
T
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

3_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

666961

GalNAc
₃
-6
_a
-

PS
GalNAc₃-
A_d
150
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

6_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

666981

GalNAc
₃
-7
_a
-

PS
GalNAc₃-
A_d
20
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

7_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

670061

GalNAc
₃
-13
_a
-

PS
GalNAc₃-
A_d
30
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

13_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

670699

GalNAc
₃
-3
_a
-

PO/PS
GalNAc₃-
T_d
15
834

_o′
T
_doG_es^mC_eoT_eoT_eo^mT_eoA_dsG_dsT_ds

3_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

670700

GalNAc
3
-3
_a
-

PO/PS
GalNAc₃-
A_e
30
831

_o′
A
_eoG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds

3_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT

670701

GalNAc
₃
-3
_a
-

PO/PS
GalNAc₃-
T_e
25
834

_o′
T
_eoG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds

3_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

671144

GalNAc
₃
-12
_a
-

PS
GalNAc₃-
A_d
40
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

12_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

671165

GalNAc
₃
-13
_a
-

PO/PS
GalNAc₃-
A_d
8
831

_o'
A
_doG_es^mC_eoT_eoT_eo^mT_eoA_dsG_dsT_ds

13_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT

671261

GalNAc
₃
-14
_a
-

PS
GalNAc₃-
A_d
>250
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

14_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

671262

GalNAc
₃
-15
_a
-

PS
GalNAc₃-
A_d
>250
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

15_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

673501

GalNAc
₃
-7
_a
-

PO/PS
GalNAc₃-
A_d
30
831

_o'
A
_doG_es^mC_eoT_eoT_eo^mT_eoA_dsG_dsT_ds

7_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

673502

GalNAc
₃
-10
_a
-

PO/PS
GalNAc₃-
A_d
8
831

_o'
A
_doG_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds

10_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

675441

GalNAc
₃
-17
_a
-

PS
GalNAc₃-
A_d
30
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

17_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

675442

GalNAc
₃
-18
_a
-

PS
GalNAc₃-
A_d
20
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

18_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

677841
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds
PS
GalNAc₃-
A_d
40
830

A_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_eoA_do′-

19_a

GalNAc
₃
-19
_a

677842
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds
PS
GalNAc₃-
A_d
30
830

A_ds ^mCasTasT_es^mC_es^mC_esT_esT_eoA_do′-

20_a

GalNAc
₃
-20
_a

677843

GalNAc
₃
-23
_a
-

PS
GalNAc₃-
A_d
40
831

_o'
A
_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds

23_a

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

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

Example 83: Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor XI Comprising a GalNAc₃Cluster

The oligonucleotides listed in Table 77 below were tested in a study for dose-dependent inhibition of Factor XI in mice.

TABLE 77

Modified oligonucleotides targeting Factor XI

ISIS

GalNAc

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

404071
T_esG_esG_esT_esA_esA_dsT_ds^mC_ds^mC_dsA_ds^mC_dsT_dsT_dsT_ds^mC_dsA_es
n/a
n/a
832

G_esA_esG_esGe

656173
T_esG_esG_eoT_eoA_eoA_dsT_ds^mC_ds^mC_dsA_ds^mC_dsT_dsT_dsT_ds^mC_dsA_eo
GalNAc₃-
A_d
833

G_eoA_esG_esG_eoA_do'-GalNAc₃-1_a
1_a

663086

GalNAc
₃
-3
_a
-

GalNAc₃-
A_d
841

_o'
A
_doT_esG_eoG_eoT_eoA_eoA_dsT_ds^mC_ds^mC_dsA_ds^mC_dsT_ds
3_a

T_dsT_ds^mC_dsA_eoG_eoA_esG_esG_e

678347

GalNAc
₃
-7
_a
-

GalNAc₃-
A_d
841

_o'
A
_doT_esG_eoG_eoT_eoA_eoA_dsT_ds^mC_ds^mC_dsA_ds^mC_dsT_ds
7_a

T_dsT_ds^mC_dsA_eoG_eoA_esG_esG_e

678348

GalNAc
₃
-10
_a
-

GalNAc₃-
A_d
841

_o'
A
_doT_esG_eoG_eoT_eoA_eoA_dsT_ds^mC_ds^mC_dsA_ds^mC_ds
10_a

T_dsT_dsT_ds^mC_dsA_eoG_eoA_esG_esG_e

678349

GalNAc
₃
-13
_a
-

GalNAc₃-
A_d
841

_o'
A
_doT_esG_eoG_eoT_eoA_eoA_dsT_ds^mC_ds^mC_dsA_ds^mC_ds
13_a

T_dsT_dsT_ds^mC_dsA_eoG_eoA_esG_esG_e

Treatment

Six to eight week old mice were each injected 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 sacrificed 72 hours following the final dose. Factor XI liver mRNA levels were measured using real-time PCR and normalized to cyclophilin according to standard 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 comprising 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 comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 78

Factor XI liver mRNA, liver transaminase, BUN, and bilirubin levels

Factor XI

ISIS
Dosage
mRNA
ALT
AST
BUN
Bilirubin
GalNAc₃
SEQ

No.
(mg/kg)
(% 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

404071
3
65
41
58
21
0.15
n/a
832

10
33
49
53
23
0.15

30
17
43
57
22
0.14

656173
0.7
43
90
89
21
0.16
GalNAc₃-1a
833

2
9
36
58
26
0.17

6
3
50
63
25
0.15

663086
0.7
33
91
169
25
0.16
GalNAc₃-3a
841

2
7
38
55
21
0.16

6
1
34
40
23
0.14

678347
0.7
35
28
49
20
0.14
GalNAc₃-7a
841

2
10
180
149
21
0.18

6
1
44
76
19
0.15

678348
0.7
39
43
54
21
0.16
GalNAc₃-10a
841

2
5
38
55
22
0.17

6
2
25
38
20
0.14

678349
0.7
34
39
46
20
0.16
GalNAc₃-13a
841

2
8
43
63
21
0.14

6
2
28
41
20
0.14

Example 84: Duration of Action In Vivo of Oligonucleotides Targeting Factor XI Comprising a GalNAc₃Conjugate

The oligonucleotides listed in Table 77 were tested in a single dose study for duration of action in mice.

Treatment

Six to eight week old mice were each injected subcutaneously 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, Minn. (catalog # AF2460 and # BAF2460, respectively) and the OptEIA Reagent Set B (Catalog #550534, BD Biosciences, San Jose, Calif.). The results below are presented as the average percent of plasma Factor XI protein levels for each treatment group, normalized to baseline levels. The results show that the oligonucleotides comprising a GalNAc conjugate were more potent with longer duration of action 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 with an even longer duration of action than the oligonucleotide comprising a 3′-GalNAc conjugate (ISIS 656173).

TABLE 79

Plasma Factor XI protein levels in mice

Time point

ISIS
Dosage
(days post-
Factor XI
GalNAc₃

SEQ

No.
(mg/kg)
dose)
(% baseline)
Cluster
CM
ID No.

PBS
n/a
3
123
n/a
n/a
n/a

10
56

17
100

404071
30
3
11
n/a
n/a
832

10
47

17
52

656173
6
3
1
GalNAc₃-1a
A_d
833

10
3

17
21

663086
6
3
1
GalNAc₃-3a
A_d
841

10
2

17
9

678347
6
3
1
GalNAc₃-7a
A_d
841

10
1

17
8

678348
6
3
1
GalNAc₃-10a
A_d
841

10
1

17
6

678349
6
3
1
GalNAc₃-13a
A_d
841

10
1

17
5

Example 85: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a GalNAc₃Conjugate

Oligonucleotides listed in Table 76 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

Treatment

Six to eight week old C57BL/6 mice were each injected subcutaneously once per week at a dosage shown below, for a total of three doses, with an oligonucleotide 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-1 mRNA levels using real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. The results below are presented as the average percent of liver SRB-1 mRNA levels for each treatment group, normalized to the saline control.

As illustrated in Tables 80 and 81, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner.

TABLE 80

SRB-1 mRNA in liver

ISIS
Dosage
SRB-1 mRNA
GalNAc₃

No.
(mg/kg)
(% Saline)
Cluster
CM

Saline
n/a
100
n/a
n/a

655861
0.1
94
GalNAc₃-1a
A_d

0.3
119

1
68

3
32

661161
0.1
120
GalNAc₃-3a
A_d

0.3
107

1
68

3
26

666881
0.1
107
GalNAc₃-10a
A_d

0.3
107

1
69

3
27

666981
0.1
120
GalNAc₃-7a
A_d

0.3
103

1
54

3
21

670061
0.1
118
GalNAc₃-13a
A_d

0.3
89

1
52

3
18

677842
0.1
119
GalNAc₃-20a
A_d

0.3
96

1
65

3
23

TABLE 81

SRB-1 mRNA in liver

ISIS
Dosage
SRB-1 mRNA
GalNAc₃

No.
(mg/kg)
(% Saline)
Cluster
CM

661161
0.1
107
GalNAc₃-3a
A_d

0.3
95

1
53

3
18

677841
0.1
110
GalNAc₃-19a
A_d

0.3
88

1
52

3
25

Liver transaminase levels, total bilirubin, BUN, and body weights were also measured using standard protocols. Average values for each treatment group are shown in Table 82 below.

TABLE 82

ISIS
Dosage
ALT
AST
Bilirubin
BUN
Body Weight
GalNAc₃

No.
(mg/kg)
(U/L)
(U/L)
(mg/dL)
(mg/dL)
(% baseline)
Cluster
CM

Saline
n/a
19
39
0.17
26
118
n/a
n/a

655861
0.1
25
47
0.17
27
114
GalNAc₃-1a
A_d

0.3
29
56
0.15
27
118

1
20
32
0.14
24
112

3
27
54
0.14
24
115

661161
0.1
35
83
0.13
24
113
GalNAc₃-3a
A_d

0.3
42
61
0.15
23
117

1
34
60
0.18
22
116

3
29
52
0.13
25
117

666881
0.1
30
51
0.15
23
118
GalNAc₃-10a
A_d

0.3
49
82
0.16
25
119

1
23
45
0.14
24
117

3
20
38
0.15
21
112

666981
0.1
21
41
0.14
22
113
GalNAc₃-7a
A_d

0.3
29
49
0.16
24
112

1
19
34
0.15
22
111

3
77
78
0.18
25
115

670061
0.1
20
63
0.18
24
111
GalNAc₃-13a
A_d

0.3
20
57
0.15
21
115

1
20
35
0.14
20
115

3
27
42
0.12
20
116

677842
0.1
20
38
0.17
24
114
GalNAc₃-20a
A_d

0.3
31
46
0.17
21
117

1
22
34
0.15
21
119

3
41
57
0.14
23
118

Example 86: Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃Cluster

Oligonucleotides listed in Table 83 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

Eight week old 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 the tables below or with PBS. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final 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 85-87. After the animals were sacrificed, 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 (AU480, Beckman Coulter, Calif.). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) were used according to standard protocols to determine liver human TTR mRNA levels. The results presented in Tables 84-87 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. Body weights are the average percent weight change from baseline until sacrifice 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 first dose. As illustrated in Tables 84 and 85, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915). Furthermore, the oligonucleotides comprising a GalNAc conjugate and mixed PS/PO internucleoside linkages were even more potent than the oligonucleotide comprising a GalNAc conjugate and full PS linkages.

TABLE 83

Oligonucleotides targeting human TTR

ISIS

GalNAc

SEQ

No.
Sequences (5′ to 3′)
Linkages
Cluster
CM
ID No.

420915
T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_dsA_dsT_dsG_dsA_ds
PS
n/a
n/a
842

A_dsA_esT_es^mC_es^mC_es^mC_e

660261
T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_dsA_dsT_dsG_dsA_ds
PS
GalNAc₃-1a
A_d
843

A_dsA_esT_es^mC_es^mC_es^mC_eoA_do′-GalNAC₃-1_a

682883

GalNAc
₃
-3
_a
-

PS/PO
GalNAc₃-3a
PO
842

_o′T_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds^mC_dsA_ds

T_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

682884

GalNAc
₃
-7
_a
-

PS/PO
GalNAc₃-7a
PO
842

_o′T_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds^mC_dsA_ds

T_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

682885

GalNAc
₃
-10
_a
-

PS/PO
GalNAc₃-
PO
842

_o′T_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds^mC_ds

10a

A_dsT_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

682886

GalNAc
₃
-13
_a
-

PS/PO
GalNAc₃-
PO
842

_o′T_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds^mC_ds

13a

A_dsT_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

684057
T_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds^mC_dsA_dsT_dsG_dsA_ds
PS/PO
GalNAc₃-19a
A_d
843

A_dsA_eoT_eo^mC_es^mC_es^mC_eoA_do'-GalNAc₃-19_a

The legend for Table 85 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_awas shown in Example 39. The structure of GalNAc₃-7_awas shown in Example 48. The structure of GalNAc₃-10_awas shown in Example 46. The structure of GalNAc₃-13_awas shown in Example 62. The structure of GalNAc₃-19_awas shown in Example 70.

TABLE 84

Antisense inhibition of human TTR in vivo

Plasma TTR

Isis
Dosage
TTR mRNA
protein
GalNAc

SEQ

No.
(mg/kg)
(% PBS)
(% PBS)
cluster
CM
ID No.

PBS
n/a
100
100
n/a
n/a

420915
6
99
95
n/a
n/a
842

20
48
65

60
18
28

660261
0.6
113
87
GalNAc₃-1a
A_d
843

2
40
56

6
20
27

20
9
11

TABLE 85

Antisense inhibition of human TTR in vivo

TTR
Plasma TTR protein (% PBS at BL)

Isis
Dosage
mRNA

Day 17
GalNAc

SEQ

No.
(mg/kg)
(% PBS)
BL
Day 3
Day 10
(After sac)
cluster
CM
ID No.

PBS
n/a
100
100
96
90
114
n/a
n/a

420915
6
74
106
86
76
83
n/a
n/a
842

20
43
102
66
61
58

60
24
92
43
29
32

682883
0.6
60
88
73
63
68
GalNAc₃-3a
PO
842

2
18
75
38
23
23

6
10
80
35
11
9

682884
0.6
56
88
78
63
67
GalNAc₃-7a
PO
842

2
19
76
44
25
23

6
15
82
35
21
24

682885
0.6
60
92
77
68
76
GalNAc₃-10a
PO
842

2
22
93
58
32
32

6
17
85
37
25
20

682886
0.6
57
91
70
64
69
GalNAc₃-13a
PO
842

2
21
89
50
31
30

6
18
102
41
24
27

684057
0.6
53
80
69
56
62
GalNAc₃-19a
A_d
843

2
21
92
55
34
30

6
11
82
50
18
13

TABLE 86

Transaminase levels, body weight changes, and relative organ weights

ALT (U/L)
AST (U/L)

Isis
Dosage

Day
Day
Day

Day
Day
Day
Body
Liver
Spleen
Kidney
SEQ

No.
(mg/kg)
BL
3
10
17
BL
3
10
17
(% BL)
(% PBS)
(% PBS)
(% PBS)
ID No.

PBS
n/a
33
34
33
24
58
62
67
52
105
100
100
100
n/a

420915
6
34
33
27
21
64
59
73
47
115
99
89
91
842

20
34
30
28
19
64
54
56
42
111
97
83
89

60
34
35
31
24
61
58
71
58
113
102
98
95

660261
0.6
33
38
28
26
70
71
63
59
111
96
99
92
843

2
29
32
31
34
61
60
68
61
118
100
92
90

6
29
29
28
34
58
59
70
90
114
99
97
95

20
33
32
28
33
64
54
68
95
114
101
106
92

TABLE 87

Transaminase levels, body weight changes, and relative organ weights

ALT (U/L)
AST (U/L)

Isis
Dosage

Day
Day
Day

Day
Day
Day
Body
Liver
Spleen
Kidney
SEQ

No.
(mg/kg)
BL
3
10
17
BL
3
10
17
(% BL)
(% PBS)
(% PBS)
(% PBS)
ID No.

PBS
n/a
32
34
37
41
62
78
76
77
104
100
100
100
n/a

420915
6
32
30
34
34
61
71
72
66
102
103
102
105
842

20
41
34
37
33
80
76
63
54
106
107
135
101

60
36
30
32
34
58
81
57
60
106
105
104
99

682883
0.6
32
35
38
40
53
81
74
76
104
101
112
95
842

2
38
39
42
43
71
84
70
77
107
98
116
99

6
35
35
41
38
62
79
103
65
105
103
143
97

682884
0.6
33
32
35
34
70
74
75
67
101
100
130
99
842

2
31
32
38
38
63
77
66
55
104
103
122
100

6
38
32
36
34
65
85
80
62
99
105
129
95

682885
0.6
39
26
37
35
63
63
77
59
100
109
109
112
842

2
30
26
38
40
54
56
71
72
102
98
111
102

6
27
27
34
35
46
52
56
64
102
98
113
96

682886
0.6
30
40
34
36
58
87
54
61
104
99
120
101
842

2
27
26
34
36
51
55
55
69
103
91
105
92

6
40
28
34
37
107
54
61
69
109
100
102
99

684057
0.6
35
26
33
39
56
51
51
69
104
99
110
102
843

2
33
32
31
40
54
57
56
87
103
100
112
97

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 Comprising a GalNAc₃Cluster

ISIS numbers 420915 and 660261 (see Table 83) were tested in a single dose study for duration of action in mice. ISIS numbers 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 results 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

Time point

ISIS
Dosage
(days post-
TTR
GalNAc₃

SEQ

No.
(mg/kg)
dose)
(% baseline)
Cluster
CM
ID No.

420915
100
3
30
n/a
n/a
842

7
23

10
35

17
53

24
75

39
100

660261
13.5
3
27
GalNAc₃-1a
A_d
843

7
21

10
22

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 percent of plasma TTR levels for each treatment group, normalized to baseline levels.

TABLE 89

Plasma TTR protein levels

Time point

ISIS
Dosage
(days post-
TTR
GalNAc₃

SEQ

No.
(mg/kg)
dose)
(% baseline)
Cluster
CM
ID No.

420915
100
3
48
n/a
n/a
842

7
48

10
48

17
66

31
80

682883
10.0
3
45
GalNAc₃-3a
PO
842

7
37

10
38

17
42

31
65

682885
10.0
3
40

PO
842

7
33
GalNAc₃-10a

10
34

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 GalNAc₃Conjugate

The oligonucleotides listed in Table 90 were tested for splicing modulation of human survival of motor neuron (SMN) in mice.

TABLE 90

Modified ASOs targeting SMN

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

387954
A_esT_esT_es^mC_esA_es^mC_esT_esT_esT_es^mC_esA_esT_esA_esA_esT_esG_es^m
n/a
n/a
844

C_esT_esG_esG_e

699819

GalNAc
₃
-7
_a
-
o′A_esT_esT_es^mC_esA_es^mC_esT_esT_esT_es^mC_esA_esT_esA_esA_es
GalNAc₃-
PO
844

T_esG_es^mC_esT_esG_esG_e
7a

699821

GalNAc
₃
-7
_a
-
_o′A_esT_eoT_eo^mC_eoA_eo^mC_eoT_eoT_eoT_eo^mC_eoA_eoT_eoA_eo
GalNAc₃-
PO
844

A_eoT_eoG_eo^mC_eoT_esG_esG_e
7a

700000
A_esT_esT_es^mC_esA_es^mC_esT_esT_esT_es^mC_esA_esT_esA_esA_esT_esG_es^mC_esT_es
GalNAc₃-
A_d
845

G_esG_eoA_do'-GalNAc₃-1_a
1a

703421
X-ATT^mCA^mCTTT^mCATAATG^mCTGG
n/a
n/a
844

703422

GalNAc
₃
-7
_b-X-ATT^mCA^mCTTT^mCATAATG^mCTGG
GalNAc₃-
n/a
844

7b

The structure of GalNAc₃-7_awas shown previously in Example 48. “X” indicates a 5′ primary amine generated by Gene Tools (Philomath, Oreg.), and GalNAc₃-7_bindicates the structure of GalNAc₃-7_alacking the —NH—C₆—O portion of the linker as shown below:

embedded image

ISIS numbers 703421 and 703422 are morphlino oligonucleotides, wherein each nucleotide of the two oligonucleotides is a morpholino nucleotide.

Treatment

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 according to standard protocols. Total RNA was measured using Ribogreen reagent. The SMN mRNA levels were normalized to total mRNA, and further normalized to the averages for the saline treatment group. The resulting average 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 maintained for multiple modification chemistries, including 2′-MOE and morpholino modified oligonucleotides.

TABLE 91

Effect of oligonucleotides targeting human SMN in vivo

ISIS
Dose

GalNAc₃

SEQ

No.
(mg/kg)
+Exon 7/−Exon 7
Cluster
CM
ID No.

Saline
n/a
1.00
n/a
n/a
n/a

387954
32
1.65
n/a
n/a
844

387954
288
5.00
n/a
n/a
844

699819
32
7.84
GalNAc₃-7a
PO
844

699821
32
7.22
GalNAc₃-7a
PO
844

700000
32
6.91
GalNAc₃-1a
A_d
845

703421
32
1.27
n/a
n/a
844

703422
32
4.12
GalNAc₃-7b
n/a
844

Example 89: Antisense Inhibition In Vivo by Oligonucleotides Targeting Apolipoprotein A (Apo(a)) Comprising a GalNAc₃Conjugate

The oligonucleotides listed in Table 92 below were tested in a study for dose-dependent inhibition of Apo(a) in transgenic mice.

TABLE 92

Modified ASOs targeting Apo(a)

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

494372
T_esG_es^mC_esT_es^mC_es^mC_dsG_dsT_dsT_dsG_dsG_dsT_dsG_ds^mC_ds
n/a
n/a
847

T_dsT_esG_esT_esT_es^mC_e

681257

GalNAc
₃
-7
_a
-
_o'T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-7a
PO
847

T_dsG_ds^mC_dsT_dsT_eoG_eoT_esT_es^mC_e

The structure of GalNAc₃-7_awas shown in Example 48.

Treatment

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 animals. 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 RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) according to standard protocols. Apo(a) plasma protein levels were determined using ELISA, and liver transaminase levels were determined. The mRNA and plasma protein results in Table 93 are presented as the treatment group average percent relative to the PBS treated group. Plasma protein levels were further 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 oligonucleotides 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 oligonucleotide lacking a GalNAc conjugate. 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

Apo(a)
Apo(a) plasma protein (% PBS)

ISIS
Dosage
mRNA

Week
Week
Week
Week
Week
Week

No.
(mg/kg)
(% PBS)
BL
1
2
3
4
5
6

PBS
n/a
100
100
120
119
113
88
121
97

494372
3
80
84
89
91
98
87
87
79

10
30
87
72
76
71
57
59
46

30
5
92
54
28
10
7
9
7

681257
0.3
75
79
76
89
98
71
94
78

1
19
79
88
66
60
54
32
24

3
2
82
52
17
7
4
6
5

10
2
79
17
6
3
2
4
5

TABLE 94

ISIS
Dosage
ALT
AST
Body weight

No.
(mg/kg)
(U/L)
(U/L)
(% baseline)

PBS
n/a
37
54
103

494372
3
28
68
106

10
22
55
102

30
19
48
103

681257
0.3
30
80
104

1
26
47
105

3
29
62
102

10
21
52
107

Example 90: Antisense Inhibition In Vivo by Oligonucleotides Targeting TTR Comprising a GalNAc₃Cluster

Oligonucleotides listed in Table 95 below were tested in a dose-dependent study for antisense inhibition of human transthyretin (TTR) in transgenic mice that express the human TTR gene.

Treatment

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 first 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, Calif.). Real-time PCR and RIBOGREEN® RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.) 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 first dose. As illustrated in Table 96, treatment with antisense oligonucleotides lowered TTR expression levels in a dose-dependent manner. The oligonucleotides comprising a GalNAc conjugate were more potent than the parent lacking a GalNAc conjugate (ISIS 420915), and oligonucleotides comprising a phosphodiester or deoxyadenosine cleavable moiety showed significant improvements in potency compared to the parent lacking a conjugate (see ISIS numbers 682883 and 666943 vs 420915 and see Examples 86 and 87).

TABLE 95

Oligonucleotides targeting human TTR

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Linkages
Cluster
CM
ID No.

420915
T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_dsA_dsT_dsG_dsA_ds
PS
n/a
n/a
842

A_dsA_esT_es^mC_es^mC_es^mC_e

682883

GalNAc
₃
-3
_a
-

PS/PO
GalNAc₃-
PO
842

_o′T_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds^mC_dsA_ds

3a

T_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

666943

GalNAc
₃
-3
_a
-

PS/PO
GalNAc₃-
A_d
846

_o′
A
_doT_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds

3a

^mC_dsA_dsT_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

682887

GalNAc
₃
-7
_a
-

PS/PO
GalNAc₃-
A_d
846

_o′
A
_doT_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds

7a

^mC_dsA_dsT_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

682888

GalNAc
₃
-10
_a
-

PS/PO
GalNAc₃-
A_d
846

_o'
A
_doT_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds

10a

^mC_dsA_dsT_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

682889

GalNAc
₃-13_a-
PS/PO
GalNAc₃-
A_d
846

_o'
A
_doT_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds

13a

^mC_dsA_dsT_dsG_dsA_dsA_dsA_eoT_eo^mC_es^mC_es^mC_e

The legend for Table 95 can be found in Example 74. The structure of GalNAc₃-3_awas shown in Example 39. The structure of GalNAc₃-7_awas shown in Example 48. The structure of GalNAc₃-10_awas shown in Example 46. The structure of GalNAc₃-13_awas shown in Example 62.

TABLE 96

Antisense inhibition of human TTR in vivo

Isis
Dosage
TTR mRNA
TTR protein
GalNAc

No.
(mg/kg)
(% PBS)
(% BL)
cluster
CM

PBS
n/a
100
124
n/a
n/a

420915
6
69
114
n/a
n/a

20
71
86

60
21
36

682883
0.6
61
73
GalNAc₃-3a
PO

2
23
36

6
18
23

666943
0.6
74
93
GalNAc₃-3a
A_d

2
33
57

6
17
22

682887
0.6
60
97
GalNAc₃-7a
A_d

2
36
49

6
12
19

682888
0.6
65
92
GalNAc₃-10a
A_d

2
32
46

6
17
22

682889
0.6
72
74
GalNAc₃-13a
A_d

2
38
45

6
16
18

Example 91: Antisense Inhibition In Vivo by Oligonucleotides Targeting Factor VII Comprising a GalNAc₃Conjugate 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 monkeys.

Treatment

Non-naïve monkeys were each injected subcutaneously 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 treatment group relative to the average value for the PBS group at baseline (BL), the measurements 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 oligonucleotide lacking a GalNAc conjugate.

TABLE 97

Oligonucleotides targeting Factor VII

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Linkages
Cluster
CM
ID No.

407935
A_esT_esG_es^mC_esA_esT_dsG_dsG_dsT_dsG_dsA_dsT_dsG_ds^m
PS
n/a
n/a
848

C_dsT_dsT_es^mC_esT_esG_esA_e

686892

GalNAc
₃
-10
_a
-

PS
GalNAc₃-
PO
848

o′A_esT_esG_es^mC_esA_esT_dsG_dsG_dsT_dsG_ds

10a

A_dsT_dsG_ds^mC_dsT_dsT_es^mC_esT_esG_esA_e

The legend for Table 97 can be found in Example 74. The structure of GalNAc₃-10, was shown in Example 46.

TABLE 98

Factor VII plasma protein levels

ISIS

Dose
Factor VII

No.
Day
(mg/kg)
(% BL)

407935
0
n/a
100

15
10
87

22
n/a
92

29
30
77

36
n/a
46

43
n/a
43

686892
0
3
100

15
10
56

22
n/a
29

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 GalNAc₃Conjugate

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 μM. After incubation 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 quantification reagent (Molecular Probes, Inc.) according to standard protocols. IC₅₀values were determined using Prism 4 software (GraphPad). The results show that regardless of whether the cleavable moiety was a phosphodiester or a phosphodiester-linked deoxyadensoine, the oligonucleotides comprising a GalNAc conjugate were significantly more potent than the parent oligonucleotide lacking a conjugate.

TABLE 99

Inhibition of mouse APOC-III expression in mouse primary hepatocytes

ISIS

IC₅₀
SEQ

No.
Sequences (5′ to 3′)
CM
(nM)
ID No.

440670

^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_esA_esG_es^m
n/a
13.20
849

C_esA_e

661180

^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_es
A_d
1.40
850

A_esG_es^mC_esA_eoA_do′-GalNAC₃-1_a

680771

GalNAc
₃
-3
_a
-

PO
0.70
849

o′
^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_es

A_esG_es^mC_esA_e

680772

GalNAc
₃
-7
_a
-

PO
1.70
849

o′
^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_es

A_esG_es^mC_esA_e

680773

GalNAc
₃
-10
_a
-

PO
2.00
849

o′
^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_es

A_esG_es^mC_esA_e

680774

GalNAc
₃
-13
_a
-

PO
1.50
849

o′
^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_es

A_esG_es^mC_esA_e

681272

GalNAc
₃
-3
_a
-

PO
<0.46
849

o′
^mC_esA_eoG_eo^mC_eoT_eoT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_eo

A_eoG_es^mC_esA_e

681273

GalNAc
₃
-3
_a
-

A_d
1.10
851

o′
A
do
^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds

^mC_esA_esG_es^mC_esA_e

683733

^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_es
A_d
2.50
850

A_esG_es^mC_esA_eoA_do'-GalNAc₃-19_a

The structure of GalNAc₃-1_awas shown previously in Example 9, GalNAc₃-3_awas shown in Example 39, GalNAc₃-7_awas shown in Example 48, GalNAc₃-10_awas shown in Example 46, GalNAc₃-13_awas shown in Example 62, and GalNAc₃-19_awas shown in Example 70.

Example 93: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Mixed Wings and a 5′-GalNAc₃Conjugate

The oligonucleotides listed in Table 100 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 100

Modified ASOs targeting SRB-1

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

449093
T_ksT_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^mC_ks^mC_k
n/a
n/a
852

699806
GalNAc₃-3_a-_o'T_ksT_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_ds
GalNAc₃-
PO
852

T_dsT_ks^mC_ks^mC_k
3a

699807
GalNAc₃-7_a-_o'T_ksT_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_ds
GalNAc₃-
PO
852

T_dsT_ks^mC_ks^mC_k
7a

699809
GalNAc₃-7_a-_o'T_ksT_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_ds
GalNAc₃-
PO
852

T_dsT_es^mC_es^mC_e
7a

699811
GalNAc₃-7_a-_o'T_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_ds
GalNAc₃-
PO
852

T_dsT_ks^mC_ks^mC_k
7a

699813
GalNAc₃-7_a-_o'T_ksT_ds^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_ds
GalNAc₃-
PO
852

T_dsT_ks^mC_ds^mC_k
7a

699815
GalNAc₃-7_a-_o'T_esT_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_ds
GalNAc₃-
PO
852

T_dsT_ks^mC_ks^mC_e
7a

The structure of GalNAc₃-3_awas shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48. Subscripts: “e” indicates 2′-MOE modified nucleoside; “d” indicates β-D-2′-deoxyribonucleoside; “k” indicates 6′-(S)—CH₃bicyclic nucleoside (cEt); “s” indicates phosphorothioate internucleoside linkages (PS); “o” indicates phosphodiester internucleoside linkages (PO). Supersript “m” indicates 5-methylcytosines.

Treatment

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 100 or with saline. Each treatment group consisted of 4 animals. The mice were sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented as the average percent of SRB-1 mRNA levels for each treatment group relative to the saline control group. As illustrated in Table 101, treatment with antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the gapmer oligonucleotides comprising a GalNAc conjugate and having wings that were either full cEt or mixed sugar modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising full cEt modified wings.

Body weights, liver transaminases, total bilirubin, 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-1 mRNA, ALT, AST, BUN, and total

bilirubin levels and body weights

SRB-1

ISIS
Dosage
mRNA
ALT
AST

Body weight

No.
(mg/kg)
(% PBS)
(U/L)
(U/L)
Bil
BUN
(% BL)

PBS
n/a
100
31
84
0.15
28
102

449093
1
111
18
48
0.17
31
104

3
94
20
43
0.15
26
103

10
36
19
50
0.12
29
104

699806
0.1
114
23
58
0.13
26
107

0.3
59
21
45
0.12
27
108

1
25
30
61
0.12
30
104

699807
0.1
121
19
41
0.14
25
100

0.3
73
23
56
0.13
26
105

1
24
22
69
0.14
25
102

699809
0.1
125
23
57
0.14
26
104

0.3
70
20
49
0.10
25
105

1
33
34
62
0.17
25
107

699811
0.1
123
48
77
0.14
24
106

0.3
94
20
45
0.13
25
101

1
66
57
104
0.14
24
107

699813
0.1
95
20
58
0.13
28
104

0.3
98
22
61
0.17
28
105

1
49
19
47
0.11
27
106

699815
0.1
93
30
79
0.17
25
105

0.3
64
30
61
0.12
26
105

1
24
18
41
0.14
25
106

Example 94: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising 2′-Sugar Modifications and a 5′-GalNAc₃Conjugate

The oligonucleotides listed in Table 102 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 102

Modified ASOs targeting SRB-1

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

353382
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_es
n/a
n/a
829

T_esT_e

700989
G_msC_msU_msU_msC_msA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsU_msC_msC_ms
n/a
n/a
853

U_msU_m

666904
GalNAc₃-3_a-_o'G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₃-
PO
829

^mC_dsT_dsT_es^mC_es^mC_esT_esT_e
3a

700991
GalNAc₃-7_a-_o'G_msC_msU_msU_msC_msA_dsG_dsT_ds^mC_dsA_dsT_dsG_ds
Ga1NAc₃-
PO
853

A_ds^mC_dsT_dsU_msC_msC_msU_msU_m
7a

Subscript “m” indicates a 2′-O-methyl modified nucleoside. See Example 74 for complete table legend. The structure of GalNAc₃-3_awas shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

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 modified oligonucleotides comprising a GalNAc conjugate were significantly more potent than the respective parent oligonucleotides lacking a conjugate. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 103

SRB-1 mRNA

ISIS No.
Dosage (mg/kg)
SRB-1 mRNA (% PBS)

PBS
n/a
100

353382
5
116

15
58

45
27

700989
5
120

15
92

45
46

666904
1
98

3
45

10
17

700991
1
118

3
63

10
14

Example 95: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising Bicyclic Nucleosides and a 5′-GalNAc₃Conjugate

The oligonucleotides listed in Table 104 were tested in a dose-dependent study for antisense inhibition of SRB-1 in mice.

TABLE 104

Modified ASOs targeting SRB-1

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No

440762
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^mC_k
n/a
n/a
823

666905
GalNAc₃-3_a-_o'T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^mC_k
GalNAc₃-3_a
PO
823

699782
GalNAc₃-7_a-_o'T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^mC_k
GalNAc₃-7_a
PO
823

699783
GalNAc₃-3_a-_o'T_ls^mC_lsA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ls^mC_l
GalNAc₃-3_a
PO
823

653621
T_ls^mC_lsA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_lsmC_loA_do'-GalNAC₃-1_a
GalNAc₃-1_a
A_d
824

439879
T_gs^mC_gsA_dsG_dsT_ds^mC_dsA_dsT_dG_dsA_ds^mC_dsT_dsT_gs^mC_g
n/a
n/a
823

699789
GalNAc₃-3_a-_o'T_gs^mC_gsA_dsG_dsT_ds^mC_dsA_dsT_dG_dsA_ds^mC_dsT_dsT_gs^mC_g
GalNAc₃-3_a
PO
823

Subscript “g” indicates a fluoro-HNA nucleoside, subscript “1” indicates a locked nucleoside comprising a 2′-O—CH₂-4′ bridge. See the Example 74 table legend for other abbreviations. The structure of GalNAc₃-1_awas shown previously in Example 9, the structure of GalNAc₃-3_awas shown previously in Example 39, and the structure of GalNAc₃-7a was shown previously in Example 48.

Treatment

The study was completed using the protocol described in Example 93. Results are shown in Table 105 below and show that oligonucleotides comprising a GalNAc conjugate and various bicyclic nucleoside modifications were significantly more potent than the parent oligonucleotide lacking a conjugate and comprising bicyclic nucleoside modifications. Furthermore, the oligonucleotide comprising a GalNAc conjugate and fluoro-HNA modifications was significantly more potent than the parent lacking a conjugate and comprising fluoro-HNA modifications. The results of the body weights, liver transaminases, total bilirubin, and BUN measurements indicated that the compounds were all well tolerated.

TABLE 105

SRB-1 mRNA, ALT, AST, BUN, and total

bilirubin levels and body weights

ISIS No.
Dosage (mg/kg)
SRB-1 mRNA (% PBS)

PBS
n/a
100

440762
1
104

3
65

10
35

666905
0.1
105

0.3
56

1
18

699782
0.1
93

0.3
63

1
15

699783
0.1
105

0.3
53

1
12

653621
0.1
109

0.3
82

1
27

439879
1
96

3
77

10
37

699789
0.1
82

0.3
69

1
26

Example 96: Plasma Protein Binding of Antisense Oligonucleotides Comprising a GalNAc₃Conjugate Group

Oligonucleotides listed in Table 70 targeting ApoC-III and oligonucleotides in Table 106 targeting Apo(a) were tested in an ultra-filtration assay in order to assess plasma protein binding.

TABLE 106

Modified oligonucleotides targeting Apo(a)

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No

494372
T_esG_es^mC_esT_es^mC_es^mC_dsG_dsT_dsT_dsG_dsG_dsT_dsG_ds^mC_dsT_dsT_esG_esT_es
n/a
n/a
847

T_es^mC_e

693401
T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_dsT_dsG_ds^mC_dsT_dsT_eoG_eoT_es
n/a
n/a
847

T_es^mC_e

681251
GalNAc₃-7_a-_o'T_esG_es^mC_esT_es^mC_es^mC_dsG_dsT_dsT_dsG_dsG_dsT_dsG_ds^mC_ds
GalNAc₃-
PO
847

T_dsT_esG_esT_esT_es^mC_e
7_a

681257
GalNAC₃-7_a-_o'T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_dsT_dsG_ds^mC_ds
GalNAc₃-
PO
847

T_dsT_eoG_eoT_esT_es^mC_e
7_a

See the Example 74 for table legend. The structure of GalNAc₃-7a was shown previously in Example 48.

Ultrafree-MC ultrafiltration units (30,000 NMWL, low-binding regenerated cellulose membrane, Millipore, Bedford, Mass.) were pre-conditioned with 300 μL of 0.5% Tween 80 and centrifuged at 2000 g for 10 minutes, then with 3004, of a 300 μg/mL solution of a control oligonucleotide in H₂O and centrifuged at 2000 g for 16 minutes. In order to assess non-specific binding to the filters of each test oligonucleotide from Tables 70 and 106 to be used in the studies, 300 μL of a 250 ng/mL solution of oligonucleotide in H₂O at pH 7.4 was placed in the pre-conditioned filters 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 volunteers, cynomolgus monkeys, and CD-1 mice, were purchased from Bioreclamation LLC (Westbury, N.Y.). The test oligonucleotides were added to 1.2 mL aliquots of plasma at two concentrations (5 and 150 μg/mL). An aliquot (300 μL) 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. Aliquots 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 of bound and unbound oligonucleotide in each sample. The average concentration of the filtered sample relative to the concentration of the unfiltered sample is used to determine the percent of oligonucleotide in the plasma that is not bound to plasma proteins (% unbound). The final unbound oligonucleotide values are corrected for non-specific binding by dividing the % unbound by the % recovery 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 μg/mL) in each species of plasma. The results show that GalNAc conjugate groups do not have a significant impact on plasma protein binding. Furthermore, oligonucleotides with full PS internucleoside linkages and mixed PO/PS linkages 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 modified oligonucleotide bound to plasma proteins

Human plasma
Monkey plasma
Mouse plasma

ISIS
5
150
5
150
5
150

No.
μg/mL
μg/mL
μg/mL
μg/mL
μg/mL
μg/mL

304801
99.2
98.0
99.8
99.5
98.1
97.2

663083
97.8
90.9
99.3
99.3
96.5
93.0

674450
96.2
97.0
98.6
94.4
94.6
89.3

494372
94.1
89.3
98.9
97.5
97.2
93.6

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

681257
93.4
90.5
97.6
93.7
95.6
92.7

Example 97: Modified Oligonucleotides Targeting TTR Comprising a GalNAc₃Conjugate Group

The oligonucleotides shown in Table 108 comprising a GalNAc conjugate were designed to target TTR.

TABLE 108

Modified oligonucleotides targeting TTR

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No

666941
GalNAc₃-3_a-_o'A_doT_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds
GalNAc₃-3
A_d
846

^mC_dsA_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

666942
T_es^mC_eoT_eoT_eoG_eoG_dsT_dsT_dsA_ds^mC_dsA_dsT_dsG_ds
GalNAc₃-1
A_d
843

A_dsA_dsA_eoT_eo^mC_es^mC_es^mC_eoA_do'-GalNAc₃-3_a

682876
GalNAc₃-3_a-_o'T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_ds
GalNAc₃-3
PO
842

A_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

682877
GalNAC₃-7_a-_o'T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_ds
GalNAc₃-7
PO
842

A_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

682878
GalNAC₃-10_a-_o'T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_ds
GalNAc₃-10
PO
842

A_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

682879
GalNAC₃-13_a-_o'T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_ds
GalNAc₃-13
PO
842

A_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

682880
GalNAC₃-7_a-_o'A_doT_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds
GalNAc₃-7
A_d
846

^mC_dsA_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

682881
GalNAC₃-10_a-_o'A_doT_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds
GalNAc₃-10
A_d
846

^mC_dsA_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

682882
GalNAC₃-13_a-_o'A_doT_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds
GalNAc₃-13
A_d
846

^mC_dsA_dsT_dsG_dsA_dsA_dsA_esT_es^mC_es^mC_es^mC_e

684056
T_es^mC_esT_esT_esG_esG_dsT_dsT_dsA_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₃-19
A_d
846

A_dsA_esT_es^mC_es^mC_es^mC_eoA_do'-GalNAc₃-19_a

The legend for Table 108 can be found in Example 74. The structure of GalNAc₃-1 was shown in Example 9. The structure of GalNAc₃-3_awas shown in Example 39. The structure of GalNAc₃-7_awas shown in Example 48. The structure of GalNAc₃-10_awas shown in Example 46. The structure of GalNAc₃-13_awas shown in Example 62. The structure of GalNAc₃-19_awas shown in Example 70.

Example 98: Evaluation of Pro-Inflammatory Effects of Oligonucleotides Comprising a GalNAc Conjugate in hPMBC Assay

The oligonucleotides listed in Table 109 and were tested for pro-inflammatory 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 control, and the other oligonucleotides are described in Tables 83, 95, and 108. The results 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 significantly lower pro-inflammatory responses compared to the same oligonucleotides having full PS linkages. Furthermore, the GalNAc conjugate group did not have a significant effect in this assay.

TABLE 109

ISIS No.
E_max/EC₅₀
GalNAc₃cluster
Linkages
CM

353512
3630
n/a
PS
n/a

420915
802
n/a
PS
n/a

682881
1311
GalNAc₃-10
PS
A_d

682888
0.26
GalNAc₃-10
PO/PS
A_d

684057
1.03
GalNAc₃-19
PO/PS
A_d

Example 99: Binding Affinities of Oligonucleotides Comprising a GalNAc Conjugate for the Asialoglycoprotein Receptor

The binding affinities of the oligonucleotides 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, α1-acid glycoprotein (AGP), was incubated in 50 mM sodium acetate buffer (pH 5) with 1 U neuraminidase-agarose for 16 hours at 3TC, 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 January; 32(1):173-81.) In this method, desialylated α1-acid glycoprotein (de-AGP) was added to 10 mM iodine chloride, Na¹²⁵I, and 1 M glycine in 0.25 M NaOH. After incubation for 10 minutes at room temperature, ¹²⁵I-labeled de-AGP was separated from free ¹²⁵I 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 (7.8×300 mm) and a ß-RAM counter. Competition experiments utilizing ¹²⁵I-labeled de-AGP and various GalNAc-cluster containing ASOs were performed as follows. Human HepG2 cells (10⁶cells/ml) were plated on 6-well plates in 2 ml of appropriate growth media. MEM media supplemented with 10% fetal bovine serum (FBS), 2 mM L-Glutamine and 10 mM HEPES was used. Cells were incubated 16-20 hours @ 3TC with 5% and 10% CO₂respectively. Cells were washed with media without FBS prior to the experiment. Cells were incubated for 30 min @37° C. with 1 ml competition mix containing appropriate growth media with 2% FBS, 10⁻⁸M ¹²⁵I-labeled de-AGP and GalNAc-cluster containing ASOs at concentrations ranging from 10⁻¹¹to 10⁻⁵M. Non-specific binding was determined in the presence of 10⁻²M GalNAc sugar. Cells were washed twice with media without FBS to remove unbound ¹²⁵I-labeled de-AGP and competitor GalNAc ASO. Cells were lysed using Qiagen's RLT buffer containing 1% ß-mercaptoethanol. Lysates were transferred to round bottom assay tubes after a brief 10 min freeze/thaw cycle and assayed on a γ-counter. Non-specific binding was subtracted before dividing ¹²⁵I protein counts by the value of the lowest GalNAc-ASO concentration counts. The inhibition curves were fitted according to a single site competition binding equation using a nonlinear regression algorithm to calculate the binding affinities (K_D's).

The results in Table 110 were obtained from experiments performed on five different days. Results for oligonucleotides marked with superscript “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

ISIS

to which GalNAc
K_D

No.
GalNAc conjugate
conjugate is attached
(nM)

661161^a
GalNAc₃-3
5′
3.7

666881^a
GalNAc₃-10
5′
7.6

666981
GalNAc₃-7
5′
6.0

670061
GalNAc₃-13
5′
7.4

655861^a
GalNAc₃-1
3′
11.6

677841^a
GalNAc₃-19
3′
60.8

Example 100: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 111a below were tested in a single dose study for duration of action in mice.

TABLE 111a

Modified ASOs targeting APO(a)

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

681251
GalNAC₃-7_a-_o'T_esG_es^mC_esT_es^mC_es^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-7a
PO
847

T_dsG_ds^mC_dsT_dsT_esG_esT_esT_es^mC_e

681257
GalNAC₃-7_a-_o'T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-7a
PO
847

T_dsG_ds^mC_dsT_dsT_eoG_eoT_esT_es^mC_e

The structure of GalNAc₃-7_awas shown in Example 48.

Treatment

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 presented as the average percent of plasma Apo(a) protein levels for each treatment 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 full PS internucleoside linkages and the oligonucleotide that comprises mixed PO and PS linkages.

TABLE 111b

Apo(a) plasma protein levels

Apo(a) at
Apo(a) at
Apo(a) at

ISIS
Dosage
72 hours
1 week
3 weeks

No.
(mg/kg)
(% BL)
(% BL)
(% BL)

PBS
n/a
116
104
107

681251
0.3
97
108
93

1.0
85
77
57

3.0
54
49
11

10.0
23
15
4

681257
0.3
114
138
104

1.0
91
98
54

3.0
69
40
6

10.0
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 vivo. 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 treated 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 readily cleavable phosphodiester containing linkage. The animals were sacrificed 72 hours after the dose. Liver APOC-III mRNA levels were measured using real-time PCR. APOC-III mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The results are presented in Table 112 as the average percent of APOC-III mRNA levels for each treatment group relative 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 oligonucleotide 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

Modified oligonucleotides targeting mouse APOC-III

APOC-III
SEQ

ISIS

Dosage
mRNA
ID

No.
Sequences (5′ to 3′)
CM
(mg/kg)
(% PBS)
No.

440670

^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_dsT_dsA_ds
n/a
2
92
849

G_dsG_dsG_dsA_ds^mC_esA_esG_es^mC_esA_e

6
86

20
59

60
37

680772
GalNAc₃-7_a-_o'^mC_esA_esG_es^mC_esT_esT_dsT_dsA_ds
PO
0.6
79
849

T_dsT_dsA_dsG_dsG_dsG_dsA_ds^mC_esA_esG_es^mC_esA_e

2
58

6
31

20
13

696847
GalNAc₃-7_a-
n/a
0.6
83
849

_s'
^mC_esA_esG_es^mC_esT_esT_dsT_dsA_dsT_ds
(PS)
2
73

T_dsA_dsG_dsG_dsG_dsA_ds^mC_esA_esG_es^mC_esA_e

6
40

20
28

The structure of GalNAc₃-7_awas shown in Example 48.

Example 102: Distribution in Liver of Antisense Oligonucleotides Comprising a GalNAc Conjugate

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 655861 at a dosage listed in Table 113. Each treatment group consisted of 3 animals except for the 18 mg/kg group for ISIS 655861, which consisted of 2 animals. The animals were sacrificed 48 hours following the dose to determine the liver distribution of the oligonucleotides. In order to measure the number of antisense oligonucleotide molecules per cell, a Ruthenium (II) tris-bipyridine tag (MSD TAG, Meso Scale Discovery) was conjugated to an oligonucleotide probe used to detect the antisense oligonucleotides. The results 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 comprising 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 non-parenchymal 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 majority of the ISIS 655861 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

Concentration

Concentration
Concentration
in non-parenchymal

in whole liver
in hepatocytes
liver cells

(mole-
(mole-
(mole-

ISIS
Dosage
cules*10{circumflex over ( )}6
cules*10{circumflex over ( )}6
cules*10{circumflex over ( )}6

No.
(mg/kg)
per cell)
per cell)
per cell)

353382
3
9.7
1.2
37.2

10
17.3
4.5
34.0

20
23.6
6.6
65.6

30
29.1
11.7
80.0

60
73.4
14.8
98.0

90
89.6
18.5
119.9

655861
0.5
2.6
2.9
3.2

1
6.2
7.0
8.8

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 GalNAc₃Conjugate

The oligonucleotides listed in Table 114 below were tested in a single dose study for duration of action in mice.

TABLE 114

Modified ASOs targeting APOC-III

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

304801
A_esG_es^mC_esT_esT_es^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_dsA_dsG_ds^mC_dsT_esT_es
n/a
n/a
821

T_esA_esT_e

663084
GalNAC₃-3_a-_o'A_doA_esG_eo^mC_eoT_eoT_eo^mC_dsT_dsT_dsG_dsT_ds^mC_ds
GalNAc₃-3a
A_d
837

^mC_dsA_dsG_ds^mC_dsT_eoT_eoT_esA_esT_e

679241
A_esG_eo^mC_eoT_eoT_eo^mC_dsT_dsT_dsG_dsT_ds^mC_ds^mC_dsA_dsG_ds^mC_dsT_eoT_eo
GalNAc₃-19a
A_d
822

T_esA_esT_eoA_do'-GalNAc₃-19_a

The structure of GalNAc₃-3_awas shown in Example 39, and GalNAc₃-19_awas shown in Example 70.

Treatment

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 determine baseline and at 3, 7, 14, 21, 28, 35, and 42 days following the dose. Plasma triglyceride and APOC-III protein levels were measured as described in Example 20. The results in Table 115 are presented as the average percent of plasma triglyceride and APOC-III levels for each treatment group, normalized to baseline levels. 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 exhibited increased duration of action than equivalent oligonucleotides comprising only phosphorothioate internucleoside linkages.

TABLE 115

Plasma triglyceride and APOC-III protein levels in transgenic mice

Time point

APOC-III

ISIS
Dosage
(days post-
Triglycerides
protein
GalNAc₃

No.
(mg/kg)
dose)
(% baseline)
(% baseline)
Cluster
CM

PBS
n/a
3
96
101
n/a
n/a

7
88
98

14
91
103

21
69
92

28
83
81

35
65
86

42
72
88

304801
30
3
42
46
n/a
n/a

7
42
51

14
59
69

21
67
81

28
79
76

35
72
95

42
82
92

663084
10
3
35
28
GalNAc₃-3a
A_d

7
23
24

14
23
26

21
23
29

28
30
22

35
32
36

42
37
47

679241
10
3
38
30
GalNAc₃-19a
A_d

7
31
28

14
30
22

21
36
34

28
48
34

35
50
45

42
72
64

Example 104: Synthesis of Oligonucleotides Comprising a 5′-GalNAc2 Conjugate

embedded image

Compound 120 is commercially 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 mmol) 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 3 h, the reaction mixture was poured into 100 mL of 1 M NaHSO4 and extracted with 2×50 mL ethyl acetate. Organic layers were combined and washed with 3×40 mL sat NaHCO₃and 2× brine, dried with Na₂SO₄, filtered 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 trifluoracetic acid (10 mL) was added. After stirring at room temperature for 2 h, the reaction mixture was concentrated under reduced pressure and co-evaporated with toluene (3×10 mL). The residue was dried under reduced pressure to yield compound 232 as the trifuloracetate 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 (2×100 mL). The organic phase was separated and washed with sat. aqueous NaHCO₃(3×80 mL), 1 M NaHSO₄(3×80 mL) and brine (2×80 mL), then dried (Na₂SO₄), filtered, and concentrated. The residue was purified 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 acetate (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 filtered 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 temperature the reaction mixture was poured into water (40 mL) and extracted with EtOAc (2×50 mL). A standard work-up was completed as described above to yield compound 234. LCMS and NMR were consistent with the structure. Oligonucleotide 235 was prepared using the general procedure described in Example 46. The GalNAc2 cluster portion (GalNAc2-24_a) of the conjugate group GalNAc2-24 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc2-24 (GalNAc2-24_a-CM) is shown below:

embedded image

Example 105: Synthesis of Oligonucleotides Comprising a GalNAc₁-25 Conjugate

embedded image

The synthesis of compound 166 is described in Example 54. Oligonucleotide 236 was prepared using the general procedure described in Example 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.

embedded image

The GalNAc₁cluster portion (GalNAc₁-25_a) 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 (GalNAc₁-25_a-CM) is shown below:

embedded image

Example 106: Antisense Inhibition In Vivo by Oligonucleotides Targeting SRB-1 Comprising a 5% GalNAc2 or a 5′-GalNAc₃Conjugate

Oligonucleotides listed in Tables 116 and 117 were tested in dose-dependent studies for antisense inhibition of SRB-1 in mice.

Treatment

Six to week old, male C57BL/6 mice (Jackson Laboratory, Bar Harbor, Me.) were injected 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 sacrificed 72 hours following the final administration. Liver SRB-1 mRNA levels were measured using real-time PCR. SRB-1 mRNA levels were normalized to cyclophilin mRNA levels according to standard protocols. The antisense oligonucleotides lowered SRB-1 mRNA levels in a dose-dependent manner, and the ED50 results are presented in Tables 116 and 117. Although previous studies showed that trivalent GalNAc-conjugated oligonucleotides were significantly more potent than divalent GalNAc-conjugated oligonucleotides, which were in turn significantly more potent than monovalent 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-1 mRNA levels with similar potencies as shown in Tables 116 and 117.

TABLE 116

Modified oligonucleotides targeting SRB-1

ISIS

GalNAc
ED₅₀
SEQ

No.
Sequences (5′ to 3′)
Cluster
(mg/kg)
ID No

440762
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^mC_k
n/a
4.7
823

686221
GalNAc₂-24_a-_o'A_doT_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₂-24_a
0.39
827

^mC_dsT_dsT_ks^mC_k

686222
GalNAc₃-13_a-_o'A_doT_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₃-13_a
0.41
827

^mC_dsT_dsT_ks^mC_k

See Example 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

Modified oligonucleotides targeting SRB-1

ISIS

GalNAc
ED₅₀
SEQ

No.
Sequences (5′ to 3′)
Cluster
(mg/kg)
ID No

440762
T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_ks^mC_k
n/a
5
823

708561
GalNAc₁-25_a-_o'T_ks^mC_ksA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-25_a
0.4
823

^mC_dsT_dsT_ks^mC_k

See Example 93 for table legend. The structure of GalNAc₁-25a was shown in Example 105.

The concentrations 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 antisense oligonucleotide tissues levels for each treatment group, as measured by UV in units of μg oligonucleotide per gram of liver tissue. The results show that the oligonucleotides comprising a GalNAc conjugate group accumulated in the liver at significantly higher levels than the same dose of the oligonucleotide lacking a GalNAc conjugate group. Furthermore, the antisense oligonucleotides comprising one, two, or three GalNAc ligands in their respective conjugate groups all accumulated in the liver at similar levels. This result is surprising in view of the Khorev et al. literature 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 GalNAc₂or GalNAc₃conjugate group

ISIS
Dosage
[Antisense oligonucleotide]

No.
(mg/kg)
(μg/g)
GalNAc cluster
CM

440762
2
2.1
n/a
n/a

7
13.1

20
31.1

686221
0.2
0.9
GalNAc₂-24a
A_d

0.6
2.7

2
12.0

6
26.5

686222
0.2
0.5
GalNAc₃-13a
A_d

0.6
1.6

2
11.6

6
19.8

TABLE 117b

Liver concentrations of oligonucleotides

comprising a GalNAc₁conjugate group

ISIS
Dosage
[Antisense oligonucleotide]

No.
(mg/kg)
(μg/g)
GalNAc cluster
CM

440762
2
2.3
n/a
n/a

7
8.9

20
23.7

708561
0.2
0.4
GalNAc₁-25_a
PO

0.6
1.1

2
5.9

6
23.7

20
53.9

Example 107: Synthesis of Oligonucleotides Comprising a GalNAc₁-26 or GalNAc₁-27 Conjugate

embedded image

Oligonucleotide 239 is synthesized via coupling of compound 47 (see Example 15) to acid 64 (see Example 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 GalNAc₁cluster portion (GalNAc₁-26_a) of the conjugate group GalNAc₁-26 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-26 (GalNAc₁-26_a-CM) is shown below:

embedded image

In order to add the GalNAc₁conjugate 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 Example 7. The oligonucleotide synthesis is then completed using procedures described in Example 9 in order to form oligonucleotide 240.

embedded image

The GalNAc₁cluster portion (GalNAc₁-27_a) 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 (GalNAc₁-27_a-CM) is shown below:

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Example 108: Antisense Inhibition In Vivo by Oligonucleotides Comprising a GalNAc Conjugate Group Targeting Apo(a) In Vivo

The oligonucleotides listed in Table 118 below were tested in a single dose study in mice.

TABLE 118

Modified ASOs targeting APO(a)

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

494372
T_esG_es^mC_esT_es^mC_es^mC_dsG_dsT_dsT_dsG_dsG_dsT_dsG_ds^mC_ds
n/a
n/a
847

T_dsT_esG_esT_esT_es^mC_e

681251
GalNAc₃-7_a-_o'T_esG_es^mC_esT_es^mC_es^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-7a
PO
847

T_dsG_ds^mC_dsT_dsT_esG_esT_esT_es^mC_e

681255
GalNAc₃-3_a-_o'T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-3a
PO
847

T_dsG_ds^mC_dsT_dsT_eoG_eoT_esT_es^mC_e

681256
GalNAc₃-10_a-_o'T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-10a
PO
847

T_dsG_ds^mC_dsT_dsT_eoG_eoT_esT_es^mC_e

681257
GalNAc₃-7_a-_o'T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-7a
PO
847

T_dsG_ds^mC_dsT_dsT_eoG_eoT_esT_es^mC_e

681258
GalNAc₃-13_a-_o'T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_ds
GalNAc₃-13a
PO
847

T_dsG_ds^mC_dsT_dsT_eoG_eoT_esT_es^mC_e

681260
T_esG_eo^mC_eoT_eo^mC_eo^mC_dsG_dsT_dsT_dsG_dsG_dsT_dsG_ds^mC_dsT_dsT_eoG_eo
GalNAc₃-19a
A_d
854

T_esT_es^mC_eoA_do'-GalNAc₃-19

The structure of GalNAc₃-7_awas shown in Example 48.

Treatment

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 animals. 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 119 are presented as the average percent 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 exhibited 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(a) at 1 week (% BL)

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

embedded image

Oligonucleotide 241 is synthesized using procedures similar to those described in Example 71 to form the phosphoramidite intermediate, followed by procedures described in Example 10 to synthesize the oligonucleotide. The GalNAc₁cluster portion (GalNAc₁-28_a) of the conjugate group GalNAc₁-28 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-28 (GalNAc₁-28_a-CM) is shown below:

embedded image

In order to add the GalNAc₁conjugate group to the 3′-end of an oligonucleotide, procedures similar 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.

embedded image

The GalNAc₁cluster portion (GalNAc₁-29_a) of the conjugate group GalNAc₁-29 can be combined with any cleavable moiety present on the oligonucleotide to provide a variety of conjugate groups. The structure of GalNAc₁-29 (GalNAc₁-29_a-CM) is shown below:

embedded image

Example 110: Synthesis of Oligonucleotides Comprising a GalNAc₁-30 Conjugate

embedded image

Oligonucleotide 246 comprising a GalNAc₁-30 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₁cluster portion (GalNAc₁-30_a) of the conjugate group GalNAc₁-30 can be combined with any cleavable moiety to provide a variety of conjugate groups. In certain embodiments, Y is part of the cleavable moiety. In certain embodiments, Y is part of a stable moiety, and the cleavable moiety is present on the oligonucleotide. The structure of GalNAc₁-30_ais shown below:

embedded image

Example 111: Synthesis of Oligonucleotides Comprising a GalNAc2-31 or GalNAc2-32 Conjugate

embedded image

Oligonucleotide 250 comprising a GalNAc2-31 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc2 cluster portion (GalNAc₂-31_a) 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 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 GalNAc₂-31_ais shown below:

embedded image

The synthesis of an oligonucleotide comprising a GalNAc₂-32 conjugate is shown below.

embedded image

Oligonucleotide 252 comprising a GalNAc₂-32 conjugate group, wherein Y is selected from O, S, a substituted or unsubstituted C₁-C₁₀alkyl, amino, substituted amino, azido, alkenyl or alkynyl, is synthesized as shown above. The GalNAc₂cluster portion (GalNAc₂-32_a) 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 GalNAc₂-32_ais shown below:

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Example 112: Modified Oligonucleotides Comprising a GalNAc₁Conjugate

The oligonucleotides in Table 120 targeting SRB-1 were synthesized with a GalNAc₁conjugate group in order to further test the potency of oligonucleotides comprising conjugate groups that contain one GalNAc ligand.

TABLE 120

ISIS

GalNAc

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

711461
GalNAc₁-25_a-_o'A_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds
GalNAc₁-25_a
A_d
831

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

711462
GalNAc₁-25_a-_o'G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_ds
GalNAc₁-25_a
PO
829

A_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

711463
GalNAc₁-25_a-_o'G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_ds
GalNAc₁-25_a
PO
829

A_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

711465
GalNAc₁-26_a-_o'A_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds
GalNAc₁-26_a
A_d
831

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

711466
GalNAc₁-26_a-_o'G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_ds
GalNAc₁-26_a
PO
829

A_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

711467
GalNAc₁-26_a-_o'G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_ds
GalNAc₁-26_a
PO
829

A_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

711468
GalNAc₁-28_a-_o'A_doG_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds
GalNAc₁-28_a
A_d
831

^mC_dsA_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

711469
GalNAc₁-28_a-_o'G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_ds
GalNAc₁-28_a
PO
829

A_dsT_dsG_dsA_ds^mC_dsT_dsT_es^mC_es^mC_esT_esT_e

711470
GalNAc₁-28_a-_o'G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_ds
GalNAc₁-28_a
PO
829

A_dsT_dsG_dsA_ds^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_e

713844
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-27_a
PO
829

^mC_dsT_dsT_es^mC_es^mC_esT_esT_eo'-GalNAc₁-27_a

713845
G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-27_a
PO
829

^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_eo'-GalNAc₁-27_a

713846
G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-27_a
A_d
830

^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_eoA_do'-GalNAc₁-27_a

713847
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-29_a
PO
829

^mC_dsT_dsT_es^mC_es^mC_esT_esT_eo'-GalNAc₁-29_a

713848
G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-29_a
PO
829

^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_eo'-GalNAc₁-29_a

713849
G_es^mC_esT_esT_es^mC_esA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-29_a
A_d
830

^mC_dsT_dsT_es^mC_es^mC_esT_esT_eoA_do'-GalNAc₁-29_a

713850
G_es^mC_eoT_eoT_eo^mC_eoA_dsG_dsT_ds^mC_dsA_dsT_dsG_dsA_ds
GalNAc₁-29_a
A_d
830

^mC_dsT_dsT_eo^mC_eo^mC_esT_esT_eoA_do'-GalNAc₁-29_a

Example 113: Antisense Inhibition In Vivo by Oligonucleotides Targeting CFB

The oligonucleotides listed in Table 121 were tested in a dose-dependent study for antisense inhibition of human Complement Factor B (CFB) in mice.

TABLE 121

Modified ASOs targeting CFB

ISIS

GalNAc₃

SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
ID No.

588540
A_esT_es^mC_es^mC_es^mC_esA_ds^mC_dsG_ds^mC_ds^mC_ds^mC_ds
n/a
n/a
440

^mC_dsT_dsG_dsT_ds^mC_es^mC_esA_esG_es^mCe

687301
GalNAc₃-3_a-_o'A_esT_es^mC_es^mC_es^mC_esA_ds^mC_dsG_ds
GalNAc₃-3a
PO
440

^mC_ds^mC_ds^mC_ds^mC_dsT_dsG_dsT_ds^mC_es^mC_esA_esG_es^mC_e

The structure of GalNAc₃-3_awas shown previously in Example 39.

Treatment

Transgenic mice that express human CFB (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once per week for 3 weeks (a total of 4 doses) with an oligonucleotide listed in Table 122 or with saline. The four treatment groups that received ISIS No. 588540 were given 6, 12, 25, or 50 mg/kg per dose. The four treatment groups that received ISIS No. 687301 were given 0.25, 0.5, 2, or 6 mg/kg per dose. Each treatment group consisted of 4 animals. The mice were sacrificed 2 days following the final administration to determine the liver and kidney human CFB and cyclophilin mRNA levels using real-time PCR according to standard protocols. The CFB mRNA levels were normalized to the cyclophilin levels, and the averages for each treatment group were used to determine the dose that achieved 50% inhibition of the human CFB transcript expression (ED₅₀). The results are the averages of four experiments completed with two different primer probe sets and are shown in Table 122.

TABLE 122

Potencies of oligonucleotides targeting human CFB in vivo

ISIS
ED₅₀in liver
ED₅₀in kidney
GalNAc₃

No.
(mg/kg)
(mg/kg)
Cluster
CM

588540
7.9
11.7
n/a
n/a

687301
0.49
0.35
GalNAc₃-3a
PO

Liver transaminase levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), in serum were measured relative to saline injected mice using standard protocols. Total bilirubin, BUN, and body weights were also evaluated. The results show that there were no significant changes in any of the treatment groups relative to the saline treated group (data not shown), indicating that both oligonucleotides were very well tolerated.

Example 114: Antisense Inhibition In Vivo by Oligonucleotides Targeting CFB

The oligonucleotides listed in Table 123 were tested in a dose-dependent study for antisense inhibition of human CFB in mice.

Treatment

Transgenic mice that express human CFB (Jackson Laboratory, Bar Harbor, Me.) were injected subcutaneously once with 0.6, 1, 6, or 18 mg/kg of an oligonucleotide listed in Table 123 or with saline. Each treatment group consisted of 4 or 5 animals. The mice were sacrificed 72 hours following the dose to determine the liver human CFB and cyclophilin mRNA levels using real-time PCR according to standard protocols. The CFB mRNA levels were normalized to the cyclophilin levels, and the averages for each treatment group were used to determine the dose that achieved 50% inhibition of the human CFB transcript expression (ED₅₀)_nThe results are shown in Table 123.

TABLE 123

Modified ASOs targeting CFB

ED₅₀ in

ISIS

GalNAc₃

liver
SEQ

No.
Sequences (5′ to 3′)
Cluster
CM
(mg/kg)
ID No.

696844
GalNAc₃-7_a-_o′A_esT_es^mC_es^mC_es^mC_esA_ds^mC_dsG_ds
GalNAc₃-7a
PO
0.86
440

^mC_ds^mC_ds^mC_ds^mC_dsT_dsG_dsT_ds^mC_es^mC_esA_esG_es^mC_e

696845
GalNAc₃-7_a-_o′A_esT_eo^mC_eo^mC_eo^mC_eoA_ds^mC_dsG_ds
GalNAc₃-7a
PO
0.71
440

^mC_ds^mC_ds^mC_ds^mC_dsT_dsG_dsT_ds^mC_eo^mC_eoA_esG_es^mC_e

698969
GalNAc₃-7_a-_o′A_esT_eo^mC_eo^mC_eo^mC_esA_ds^mC_dsG_ds
GalNAc₃-7a
PO
0.51
440

^mC_ds^mC_ds^mC_ds^mC_dsT_dsG_dsT_ds^mC_eo^mC_eoA_esG_es^mC_e

698970
GalNAc₃-7_a-_o′A_esT_es^mC_eo^mC_eo^mC_eoA_ds^mC_dsG_ds
GalNAc₃-7a
PO
0.55
440

^mC_ds^mC_ds^mC_ds^mC_dsT_dsG_dsT_ds^mC_eo^mC_eoA_esG_es^mC_e

The structure of GalNAc₃-7_awas shown previously in Example 48.

Example 115: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 (forward sequence AGTCTCTGTGGCATGGTTTGG, designated herein as SEQ ID NO: 810; reverse sequence GGGCGAATGACTGAGATCTTG, designated herein as SEQ ID NO: 811; probe sequence TACCGATTACCACAAGCAACCATGGCA, designated herein as SEQ ID NO: 812) was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 124

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

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
Region
Sequence
bition
site
site
NO:

532608
20
39
Exon 1
GCTGAGCTGCCAGTCAAGGA
36
1741
1760
6

532609
26
45
Exon 1
GGCCCCGCTGAGCTGCCAGT
16
1747
1766
7

532610
45
64
Exon 1
CGGAACATCCAAGCGGGAGG
11
1766
1785
8

532611
51
70
Exon 1
CTTTCCCGGAACATCCAAGC
26
1772
1791
9

532612
100
119
Exon 1
ATCTGTGTTCTGGCACCTGC
25
1821
1840
10

532613
148
167
Exon 1
GTCACATTCCCTTCCCCTGC
39
1869
1888
11

532614
154
173
Exon 1
GACCTGGTCACATTCCCTTC
71
1875
1894
12

532615
160
179
Exon 1
GACCTAGACCTGGTCACATT
35
1881
1900
13

532616
166
185
Exon 1
ACTCCAGACCTAGACCTGGT
39
1887
1906
14

532617
172
191
Exon 1
GCTGAAACTCCAGACCTAGA
27
1893
1912
15

532618
178
197
Exon 1
GTCCAAGCTGAAACTCCAGA
29
1899
1918
16

532619
184
203
Exon 1
CTCAGTGTCCAAGCTGAAAC
21
1905
1924
17

532620
246
265
Exon 1
AGGAGAGAAGCTGGGCCTGG
31
1967
1986
18

532621
252
271
Exon 1
GAAGGCAGGAGAGAAGCTGG
25
1973
1992
19

532622
336
355
Exon 1-2
GTGGTGGTCACACCTCCAGA
28
n/a
n/a
20

Junction

532623
381
400
Exon 2
CCCTCCAGAGAGCAGGATCC
22
2189
2208
21

532624
387
406
Exon 2
TCTACCCCCTCCAGAGAGCA
37
2195
2214
22

532625
393
412
Exon 2
TTGATCTCTACCCCCTCCAG
30
2201
2220
23

532626
417
436
Exon 2
TGGAGAAGTCGGAAGGAGCC
35
2225
2244
24

532627
423
442
Exon 2
CCCTCTTGGAGAAGTCGGAA
37
2231
2250
25

532628
429
448
Exon 2
GCCTGGCCCTCTTGGAGAAG
0
2237
2256
26

532629
435
454
Exon 2
TCCAGTGCCTGGCCCTCTTG
26
2243
2262
27

532630
458
477
Exon 2
AGAAGCCAGAAGGACACACG
30
2266
2285
28

532631
464
483
Exon 2
ACGGGTAGAAGCCAGAAGGA
43
2272
2291
29

532632
480
499
Exon 2
CGTGTCTGCACAGGGTACGG
57
2288
2307
30

532633
513
532
Exon 2
AGGGTGCTCCAGGACCCCGT
27
2321
2340
31

532634
560
579
Exon 2-3
TTGCTCTGCACTCTGCCTTC
41
n/a
n/a
32

Junction

532635
600
619
Exon 3
TATTCCCCGTTCTCGAAGTC
67
2808
2827
33

532636
626
645
Exon 3
CATTGTAGTAGGGAGACCGG
24
2834
2853
34

532637
632
651
Exon 3
CACTCACATTGTAGTAGGGA
49
2840
2859
35

532638
638
657
Exon 3
TCTCATCACTCACATTGTAG
50
2846
2865
36

532639
644
663
Exon 3
AAGAGATCTCATCACTCACA
52
2852
2871
37

532640
650
669
Exon 3
AGTGGAAAGAGATCTCATCA
34
2858
2877
38

532641
656
675
Exon 3
CATAGCAGTGGAAAGAGATC
32
2864
2883
39

532642
662
681
Exon 3
AACCGTCATAGCAGTGGAAA
45
2870
2889
40

532643
668
687
Exon 3
GAGTGTAACCGTCATAGCAG
36
2876
2895
41

532644
674
693
Exon 3
CCCGGAGAGTGTAACCGTCA
30
2882
2901
42

532645
680
699
Exon 3
CAGAGCCCCGGAGAGTGTAA
27
2888
2907
43

532646
686
705
Exon 3
GATTGGCAGAGCCCCGGAGA
20
2894
2913
44

532647
692
711
Exon 3
AGGTGCGATTGGCAGAGCCC
28
2900
2919
45

532648
698
717
Exon 3
CTTGGCAGGTGCGATTGGCA
24
2906
2925
46

532649
704
723
Exon 3
CATTCACTTGGCAGGTGCGA
28
2912
2931
47

532650
729
748
Exon 3
ATCGCTGTCTGCCCACTCCA
44
2937
2956
48

532651
735
754
Exon 3
TCACAGATCGCTGTCTGCCC
44
2943
2962
49

532652
741
760
Exon 3
CCGTTGTCACAGATCGCTGT
27
2949
2968
50

532653
747
766
Exon 3-4
CCCGCTCCGTTGTCACAGAT
28
n/a
n/a
51

Junction

532654
753
772
Exon 3-4
CAGTACCCCGCTCCGTTGTC
13
n/a
n/a
52

Junction

532655
759
778
Exon 3-4
TTGGAGCAGTACCCCGCTCC
8
n/a
n/a
53

Junction

532656
789
808
Exon 4
ACCTTCCTTGTGCCAATGGG
40
3152
3171
54

532657
795
814
Exon 4
CTGCCCACCTTCCTTGTGCC
41
3158
3177
55

532658
818
837
Exon 4
CGCTGTCTTCAAGGCGGTAC
33
3181
3200
56

532659
835
854
Exon 4
GCTGCAGTGGTAGGTGACGC
32
3198
3217
57

532660
841
860
Exon 4
CCCCCGGCTGCAGTGGTAGG
17
3204
3223
58

532661
847
866
Exon 4
GGTAAGCCCCCGGCTGCAGT
28
3210
3229
59

532662
853
872
Exon 4
ACGCAGGGTAAGCCCCCGGC
13
3216
3235
60

532663
859
878
Exon 4
GGAGCCACGCAGGGTAAGCC
33
3222
3241
61

532664
866
885
Exon 4
GCCGCTGGGAGCCACGCAGG
10
3229
3248
62

532665
891
910
Exon 4
CAAGAGCCACCTTCCTGACA
17
3254
3273
63

532666
897
916
Exon 4
CCGCTCCAAGAGCCACCTTC
25
3260
3279
64

532667
903
922
Exon 4
TCCGTCCCGCTCCAAGAGCC
29
3266
3285
65

532668
909
928
Exon 4
GAAGGCTCCGTCCCGCTCCA
14
3272
3291
66

532669
915
934
Exon 4
TGGCAGGAAGGCTCCGTCCC
18
3278
3297
67

532670
921
940
Exon 4-5
GAGTCTTGGCAGGAAGGCTC
20
n/a
n/a
68

Junction

532671
927
946
Exon 4-5
ATGAAGGAGTCTTGGCAGGA
14
n/a
n/a
69

Junction

532672
956
975
Exon 5
CTTCGGCCACCTCTTGAGGG
45
3539
3558
70

532673
962
981
Exon 5
GGAAAGCTTCGGCCACCTCT
37
3545
3564
71

532674
968
987
Exon 5
AAGACAGGAAAGCTTCGGCC
28
3551
3570
72

532675
974
993
Exon 5
TCAGGGAAGACAGGAAAGCT
16
3557
3576
73

532676
996
1015
Exon 5
TCGACTCCTTCTATGGTCTC
31
3579
3598
74

532677
1033
1052
Exon 5-6
CTTCTGTTGTTCCCCTGGGC
36
n/a
n/a
75

Junction

532678
1068
1087
Exon 6
TTCATGGAGCCTGAAGGGTC
19
3752
3771
76

532679
1074
1093
Exon 6
TAGATGTTCATGGAGCCTGA
24
3758
3777
77

532680
1080
1099
Exon 6
ACCAGGTAGATGTTCATGGA
13
3764
3783
78

532681
1086
1105
Exon 6
TCTAGCACCAGGTAGATGTT
20
3770
3789
79

532682
1092
1111
Exon 6
GATCCATCTAGCACCAGGTA
33
3776
3795
80

532683
1098
1117
Exon 6
CTGTCTGATCCATCTAGCAC
44
3782
3801
81

532684
1104
1123
Exon 6
CCAATGCTGTCTGATCCATC
29
3788
3807
82

532685
1129
1148
Exon 6
TTTGGCTCCTGTGAAGTTGC
40
3813
3832
83

TABLE 125

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

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
Region
Sequence
bition
site
site
NO:

532686
1135
1154
Exon 6
ACACTTTTTGGCTCCTGTGA
91
3819
3838
84

532687
1141
1160
Exon 6
GACTAGACACTTTTTGGCTC
77
3825
3844
85

532688
1147
1166
Exon 6
TAAGTTGACTAGACACTTTT
70
3831
3850
86

532689
1153
1172
Exon 6
CTCAATTAAGTTGACTAGAC
61
3837
3856
87

532690
1159
1178
Exon 6-7
CACCTTCTCAATTAAGTTGA
57
3843
3862
88

Junction

532691
1165
1184
Exon 6-7
ACTTGCCACCTTCTCAATTA
56
n/a
n/a
89

Junction

532692
1171
1190
Exon 6-7
ACCATAACTTGCCACCTTCT
56
n/a
n/a
90

Junction

532693
1177
1196
Exon 7
CTTCACACCATAACTTGCCA
56
4153
4172
91

532694
1183
1202
Exon 7
TCTTGGCTTCACACCATAAC
55
4159
4178
92

532695
1208
1227
Exon 7
ATGTGGCATATGTCACTAGA
55
4184
4203
93

532696
1235
1254
Exon 7
CAGACACTTTGACCCAAATT
55
4211
4230
94

532697
1298
1317
Exon 7-8
GGTCTTCATAATTGATTTCA
53
n/a
n/a
95

Junction

532698
1304
1323
Exon 7-8
ACTTGTGGTCTTCATAATTG
53
n/a
n/a
96

Junction

532699
1310
1329
Exon 7-8
ACTTCAACTTGTGGTCTTCA
52
n/a
n/a
97

Junction

532700
1316
1335
Exon 8
TCCCTGACTTCAACTTGTGG
52
4609
4628
98

532701
1322
1341
Exon 8
TGTTAGTCCCTGACTTCAAC
52
4615
4634
99

532702
1328
1347
Exon 8
TCTTGGTGTTAGTCCCTGAC
51
4621
4640
100

532703
1349
1368
Exon 8
TGTACACTGCCTGGAGGGCC
51
4642
4661
101

532704
1355
1374
Exon 8
TCATGCTGTACACTGCCTGG
51
4648
4667
102

532705
1393
1412
Exon 8
GTTCCAGCCTTCAGGAGGGA
50
4686
4705
103

532706
1399
1418
Exon 8
GGTGCGGTTCCAGCCTTCAG
50
4692
4711
104

532707
1405
1424
Exon 8
ATGGCGGGTGCGGTTCCAGC
50
4698
4717
105

532708
1411
1430
Exon 8
GATGACATGGCGGGTGCGGT
49
4704
4723
106

532709
1417
1436
Exon 8
GAGGATGATGACATGGCGGG
49
4710
4729
107

532710
1443
1462
Exon 8-9
CCCATGTTGTGCAATCCATC
48
n/a
n/a
108

Junction

532711
1449
1468
Exon 9
TCCCCGCCCATGTTGTGCAA
48
5023
5042
109

532712
1455
1474
Exon 9
ATTGGGTCCCCGCCCATGTT
48
5029
5048
110

532713
1461
1480
Exon 9
ACAGTAATTGGGTCCCCGCC
48
5035
5054
111

532714
1467
1486
Exon 9
TCAATGACAGTAATTGGGTC
47
5041
5060
112

532715
1473
1492
Exon 9
ATCTCATCAATGACAGTAAT
47
5047
5066
113

532716
1479
1498
Exon 9
TCCCGGATCTCATCAATGAC
46
5053
5072
114

532717
1533
1552
Exon 9-10
ACATCCAGATAATCCTCCCT
46
n/a
n/a
115

Junction

532718
1539
1558
Exon 9-10
ACATAGACATCCAGATAATC
46
n/a
n/a
116

Junction

532719
1545
1564
Exon 9-10
CCAAACACATAGACATCCAG
46
n/a
n/a
117

Junction

532720
1582
1601
Exon 10
AGCATTGATGTTCACTTGGT
46
5231
5250
118

532721
1588
1607
Exon 10
AGCCAAAGCATTGATGTTCA
45
5237
5256
119

532722
1594
1613
Exon 10
CTTGGAAGCCAAAGCATTGA
45
5243
5262
120

532723
1600
1619
Exon 10
GTCTTTCTTGGAAGCCAAAG
45
5249
5268
121

532724
1606
1625
Exon 10
CTCATTGTCTTTCTTGGAAG
44
5255
5274
122

532725
1612
1631
Exon 10
ATGTTGCTCATTGTCTTTCT
44
5261
5280
123

532726
1618
1637
Exon 10
GAACACATGTTGCTCATTGT
44
5267
5286
124

532727
1624
1643
Exon 10
GACTTTGAACACATGTTGCT
43
5273
5292
125

532728
1630
1649
Exon 10
ATCCTTGACTTTGAACACAT
43
5279
5298
126

532729
1636
1655
Exon 10
TTCCATATCCTTGACTTTGA
43
5285
5304
127

532730
1642
1661
Exon 10
CAGGTTTTCCATATCCTTGA
42
5291
5310
128

532731
1686
1705
Exon 11
CTCAGAGACTGGCTTTCATC
42
5827
5846
129

532732
1692
1711
Exon 11
CAGAGACTCAGAGACTGGCT
42
5833
5852
130

516252
1698
1717
Exon 11
ATGCCACAGAGACTCAGAGA
42
5839
5858
131

532733
1704
1723
Exon 11
CAAACCATGCCACAGAGACT
41
5845
5864
132

532734
1710
1729
Exon 11
TGTTCCCAAACCATGCCACA
41
5851
5870
133

532735
1734
1753
Exon 11
TTGTGGTAATCGGTACCCTT
41
5875
5894
134

532736
1740
1759
Exon 11
GGTTGCTTGTGGTAATCGGT
40
5881
5900
135

532737
1746
1765
Exon 11
TGCCATGGTTGCTTGTGGTA
40
5887
5906
136

532738
1752
1771
Exon 11
TTGGCCTGCCATGGTTGCTT
40
5893
5912
137

532739
1758
1777
Exon 11
GAGATCTTGGCCTGCCATGG
38
5899
5918
138

532740
1803
1822
Exon 12
ACAGCCCCCATACAGCTCTC
38
6082
6101
139

532741
1809
1828
Exon 12
GACACCACAGCCCCCATACA
38
6088
6107
140

532742
1815
1834
Exon 12
TACTCAGACACCACAGCCCC
38
6094
6113
141

532743
1821
1840
Exon 12
ACAAAGTACTCAGACACCAC
37
6100
6119
142

532744
1827
1846
Exon 12
GTCAGCACAAAGTACTCAGA
37
6106
6125
143

532745
1872
1891
Exon 12
TTGATTGAGTGTTCCTTGTC
36
6151
6170
144

532746
1878
1897
Exon 12
CTGACCTTGATTGAGTGTTC
35
6157
6176
145

532747
1909
1928
Exon 13
TATCTCCAGGTCCCGCTTCT
35
6403
6422
146

532748
1967
1986
Exon 13
GAATTCCTGCTTCTTTTTTC
32
6461
6480
147

532749
1973
1992
Exon 13
ATTCAGGAATTCCTGCTTCT
32
6467
6486
148

532750
1979
1998
Exon 13
CATAAAATTCAGGAATTCCT
32
6473
6492
149

532751
1985
2004
Exon 13
CATAGTCATAAAATTCAGGA
31
6479
6498
150

532752
2006
2025
Exon 13
TGAGCTTGATCAGGGCAACG
30
6500
6519
151

532753
2012
2031
Exon 13
TATTCTTGAGCTTGATCAGG
30
6506
6525
152

532754
2048
2067
Exon 13-14
GACAAATGGGCCTGATAGTC
30
n/a
n/a
153

Junction

532755
2070
2089
Exon 14
GTTGTTCCCTCGGTGCAGGG
29
6659
6678
154

532756
2076
2095
Exon 14
GCTCGAGTTGTTCCCTCGGT
28
6665
6684
155

532757
2082
2101
Exon 14
CTCAAAGCTCGAGTTGTTCC
28
6671
6690
156

532758
2088
2107
Exon 14
GGAAGCCTCAAAGCTCGAGT
25
6677
6696
157

532759
2094
2113
Exon 14
GTTGGAGGAAGCCTCAAAGC
23
6683
6702
158

532760
2100
2119
Exon 14
GTGGTAGTTGGAGGAAGCCT
23
6689
6708
159

532761
2106
2125
Exon 14
TGGCAAGTGGTAGTTGGAGG
18
6695
6714
160

532762
2112
2131
Exon 14
TGTTGCTGGCAAGTGGTAGT
14
6701
6720
161

TABLE 126

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

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
Region
Sequence
bition
site
site
NO:

532812
n/a
n/a
Exon 1
TCCAGCTCACTCCCCTGTTG
19
1593
1612
162

532813
n/a
n/a
Exon 1
TAAGGATCCAGCTCACTCCC
40
1599
1618
163

532814
n/a
n/a
Exon 1
CAGAAATAAGGATCCAGCTC
39
1605
1624
164

532815
n/a
n/a
Exon 1
AGGGACCAGAAATAAGGATC
0
1611
1630
165

532816
n/a
n/a
Exon 1
CCACTTAGGGACCAGAAATA
27
1617
1636
166

532817
n/a
n/a
Exon 1
TCCAGGACTCTCCCCTTCAG
39
1682
1701
167

532818
n/a
n/a
Exon 1
AAGTCCCACCCTTTGCTGCC
15
1707
1726
168

532819
n/a
n/a
Exon 1
CTGCAGAAGTCCCACCCTTT
26
1713
1732
169

532820
n/a
n/a
Exon 1
CAGAAACTGCAGAAGTCCCA
8
1719
1738
170

532821
n/a
n/a
Exon 2-
AACCTCTGCACTCTGCCTTC
39
2368
2387
171

Intron

2

532822
n/a
n/a
Exon 2-
CCCTCAAACCTCTGCACTCT
3
2374
2393
172

Intron

2

532823
n/a
n/a
Exon 2-
TCATTGCCCTCAAACCTCTG
19
2380
2399
173

Intron

2

532824
n/a
n/a
Intron 2
CCACACTCATTGCCCTCAAA
37
2386
2405
174

532825
n/a
n/a
Intron 2
CACTGCCCACACTCATTGCC
23
2392
2411
175

532826
n/a
n/a
Intron 2
TTAGGCCACTGCCCACACTC
15
2398
2417
176

532827
n/a
n/a
Intron 2
CTAGTCCTGACCTTGCTGCC
28
2436
2455
177

532828
n/a
n/a
Intron 2
CTCATCCTAGTCCTGACCTT
25
2442
2461
178

532829
n/a
n/a
Intron 2
CCTAGTCTCATCCTAGTCCT
23
2448
2467
179

532830
n/a
n/a
Intron 2
ACCCTGCCTAGTCTCATCCT
30
2454
2473
180

532831
n/a
n/a
Intron 2
CTTGTCACCCTGCCTAGTCT
34
2460
2479
181

532832
n/a
n/a
Intron 2
GCCCACCTTGTCACCCTGCC
36
2466
2485
182

532833
n/a
n/a
Intron 2
CCTAAAACTGCTCCTACTCC
9
2492
2511
183

532834
n/a
n/a
Intron 4
GAGTCAGAAATGAGGTCAAA
19
3494
3513
184

532835
n/a
n/a
Intron
CCCTACTCCCATTTCACCTT
16
5971
5990
185

11

532836
n/a
n/a
Intron 8-
TGTTGTGCAATCCTGCAGAA
25
5013
5032
186

Exon

9

532837
n/a
n/a
Intron 1
AAAGGCTGATGAAGCCTGGC
18
2123
2142
187

532838
n/a
n/a
Intron 7
CCTTTGACCACAAAGTGGCC
21
4461
4480
188

532839
n/a
n/a
Intron
AGGTACCACCTCTTTGTGGG
29
6362
6381
189

12

532840
n/a
n/a
Intron 1-
TGGTGGTCACACCTGAAGAG
34
2143
2162
190

Exon

2

532763
2133
2152
Exon
GCAGGGAGCAGCTCTTCCTT
40
n/a
n/a
191

14-15

Junction

532764
2139
2158
Exon 15
TCCTGTGCAGGGAGCAGCTC
28
6927
6946
192

532765
2145
2164
Exon 15
TTGATATCCTGTGCAGGGAG
41
6933
6952
193

532766
2151
2170
Exon 15
AGAGCTTTGATATCCTGTGC
36
6939
6958
194

532767
2157
2176
Exon 15
ACAAACAGAGCTTTGATATC
33
6945
6964
195

532768
2163
2182
Exon 15
TCAGACACAAACAGAGCTTT
41
6951
6970
196

532769
2169
2188
Exon 15
TCCTCCTCAGACACAAACAG
49
6957
6976
197

532770
2193
2212
Exon 15
ACCTCCTTCCGAGTCAGCTT
61
6981
7000
198

532771
2199
2218
Exon 15
ATGTAGACCTCCTTCCGAGT
39
6987
7006
199

532772
2205
2224
Exon 15
TTCTTGATGTAGACCTCCTT
30
6993
7012
200

532773
2211
2230
Exon 15
TCCCCATTCTTGATGTAGAC
31
6999
7018
201

532774
2217
2236
Exon
TTCTTATCCCCATTCTTGAT
36
n/a
n/a
202

15-16

Junction

532775
2223
2242
Exon
CTGCCTTTCTTATCCCCATT
56
n/a
n/a
203

15-16

Junction

532776
2229
2248
Exon
TCACAGCTGCCTTTCTTATC
33
n/a
n/a
204

15-16

Junction

532777
2235
2254
Exon 16
TCTCTCTCACAGCTGCCTTT
38
7119
7138
205

532778
2241
2260
Exon 16
TGAGCATCTCTCTCACAGCT
51
7125
7144
206

532779
2247
2266
Exon 16
GCATATTGAGCATCTCTCTC
39
7131
7150
207

532780
2267
2286
Exon 16
TGACTTTGTCATAGCCTGGG
56
7151
7170
208

532781
2273
2292
Exon 16
TGTCCTTGACTTTGTCATAG
36
7157
7176
209

532782
2309
2328
Exon 16
CAGTACAAAGGAACCGAGGG
30
7193
7212
210

532783
2315
2334
Exon 16
CTCCTCCAGTACAAAGGAAC
21
7199
7218
211

532784
2321
2340
Exon 16
GACTCACTCCTCCAGTACAA
31
7205
7224
212

532785
2327
2346
Exon 16
CATAGGGACTCACTCCTCCA
30
7211
7230
213

532786
2333
2352
Exon 16
GGTCAGCATAGGGACTCACT
31
7217
7236
214

532787
2352
2371
Exon
TCACCTCTGCAAGTATTGGG
42
7236
7255
215

16-17

Junction

532788
2358
2377
Exon
CCAGAATCACCTCTGCAAGT
32
n/a
n/a
216

16-17

Junction

532789
2364
2383
Exon
GGGCCGCCAGAATCACCTCT
35
n/a
n/a
217

16-17

Junction

532790
2382
2401
Exon 17
CTCTTGTGAACTATCAAGGG
33
7347
7366
218

532791
2388
2407
Exon 17
CGACTTCTCTTGTGAACTAT
52
7353
7372
219

532792
2394
2413
Exon 17
ATGAAACGACTTCTCTTGTG
16
7359
7378
220

532793
2400
2419
Exon
ACTTGAATGAAACGACTTCT
45
7365
7384
221

17-18

Junction

532794
2406
2425
Exon
ACACCAACTTGAATGAAACG
18
n/a
n/a
222

17-18

Junction

532795
2427
2446
Exon 18
TCCACTACTCCCCAGCTGAT
30
7662
7681
223

532796
2433
2452
Exon 18
CAGACATCCACTACTCCCCA
38
7668
7687
224

532797
2439
2458
Exon 18
TTTTTGCAGACATCCACTAC
35
7674
7693
225

532798
2445
2464
Exon 18
TTCTGGTTTTTGCAGACATC
45
7680
7699
226

532799
2451
2470
Exon 18
TGCCGCTTCTGGTTTTTGCA
47
7686
7705
227

532800
2457
2476
Exon 18
TGCTTTTGCCGCTTCTGGTT
61
7692
7711
228

532801
2463
2482
Exon 18
GGTACCTGCTTTTGCCGCTT
47
7698
7717
229

532802
2469
2488
Exon 18
TGAGCAGGTACCTGCTTTTG
31
7704
7723
230

532803
2517
2536
Exon 18
TTCAGCCAGGGCAGCACTTG
41
7752
7771
231

532804
2523
2542
Exon 18
TTCTCCTTCAGCCAGGGCAG
44
7758
7777
232

532805
2529
2548
Exon 18
TGGAGTTTCTCCTTCAGCCA
46
7764
7783
233

532806
2535
2554
Exon 18
TCATCTTGGAGTTTCTCCTT
49
7770
7789
234

532807
2541
2560
Exon 18
AAATCCTCATCTTGGAGTTT
30
7776
7795
235

532808
2547
2566
Exon 18
AAACCCAAATCCTCATCTTG
20
7782
7801
236

532809
2571
2590
Exon 18
GTCCAGCAGGAAACCCCTTA
65
7806
7825
237

532810
2577
2596
Exon 18
GCCCCTGTCCAGCAGGAAAC
74
7812
7831
238

532811
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
96
7834
7853
239

TABLE 127

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

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
Region
Sequence
bition
site
site
NO:

532841
n/a
n/a
Intron 6-
AACTTGCCACCTGTGGGTGA
4142
4161
11
240

Exon 7

532842
n/a
n/a
Exon 15-
TCACCTTATCCCCATTCTTG
7007
7026
16
241

Intron 15

532843
n/a
n/a
Intron 11
TCAACTTTCACAAACCACCA
6015
6034
19
242

532844
n/a
n/a
Intron 16-
CCGCCAGAATCACCTGCAAG
7326
7345
33
243

Exon 17

532845
n/a
n/a
Intron 10
AGGAGGAATGAAGAAGGCTT
5431
5450
29
244

532846
n/a
n/a
Intron 13
GCCTTTCCTCAGGGATCTGG
6561
6580
26
245

532847
n/a
n/a
Intron 4
AAATGTCTGGGAGTGTCAGG
3477
3496
18
246

532848
n/a
n/a
Intron 15
GCCTAGAGTGCCTCCTTAGG
7038
7057
20
247

532849
n/a
n/a
Intron 17
GGCATCTCCCCAGATAGGAA
7396
7415
16
248

532850
n/a
n/a
Intron 6
AGGGAGCTAGTCCTGGAAGA
3906
3925
14
249

532851
n/a
n/a
Intron 1-
ACACCTGAAGAGAAAGGCTG
2135
2154
6
250

Exon 2

532852
n/a
n/a
Intron 7
CCCTTTGACCACAAAGTGGC
4462
4481
25
251

532853
n/a
n/a
Intron 7
GCCCTCAAGGTAGTCTCATG
4354
4373
26
252

532854
n/a
n/a
Intron 6
AAGGGAAGGAGGACAGAATA
3977
3996
18
253

532855
n/a
n/a
Intron 1
AAAGGCCAAGGAGGGATGCT
2099
2118
9
254

532856
n/a
n/a
Exon 8-
AGAGGTCCCTTCTGACCATC
4736
4755
4
255

Intron 8

532857
n/a
n/a
Intron 8
GCTGGGACAGGAGAGAGGTC
4749
4768
0
256

532858
n/a
n/a
Intron 4
TCAAATGTCTGGGAGTGTCA
3479
3498
13
257

532859
n/a
n/a
Intron 10
AGAAGGAGAATGTGCTGAAA
5801
5820
20
258

532860
n/a
n/a
Intron 17
TGCTGACCACTTGGCATCTC
7408
7427
20
259

532861
n/a
n/a
Intron 11
CAACTTTCACAAACCACCAT
6014
6033
18
260

532862
n/a
n/a
Intron 10
AGCTCTGTGATTCTAAGGTT
5497
5516
15
261

532863
n/a
n/a
Intron 6-
CCACCTGTGGGTGAGGAGAA
4136
4155
16
262

Exon 7

532864
n/a
n/a
Exon 17-
GAGGACTCACTTGAATGAAA
7373
7392
21
263

Intron 17

532865
n/a
n/a
Intron 6
TGGAATGATCAGGGAGCTAG
3916
3935
30
264

532866
n/a
n/a
Intron 5
GTCCCTTCTCCATTTTCCCC
3659
3678
26
265

532867
n/a
n/a
Intron 7
TCAACTTTTTAAGTTAATCA
4497
4516
14
266

532868
n/a
n/a
Intron 6
GGGTGAGGAGAACAAGGCGC
4128
4147
21
267

532869
n/a
n/a
Intron 7
CTTCCAAGCCATCTTTTAAC
4553
4572
5
268

532870
n/a
n/a
Exon 17-
AGGACTCACTTGAATGAAAC
7372
7391
18
269

Intron 17

532871
n/a
n/a
Intron 10
TTCCAGGCAACTAGAGCTTC
5412
5431
15
270

532872
n/a
n/a
Exon 1
CAGAGTCCAGCCACTGTTTG
1557
1576
13
271

532873
n/a
n/a
Intron 17-
CCAACCTGCAGAGGCAGTGG
7638
7657
23
272

Exon 18

532874
n/a
n/a
Intron 16
TGCAAGGAGAGGAGAAGCTG
7312
7331
10
273

532875
n/a
n/a
Exon 9-
CTAGGCAGGTTACTCACCCA
5120
5139
21
274

Intron 9

532876
n/a
n/a
Intron 6-
CACCATAACTTGCCACCTGT
4148
4167
41
275

Exon 7

532877
n/a
n/a
Intron 12
TAGGTACCACCTCTTTGTGG
6363
6382
27
276

532878
n/a
n/a
Intron 11
CTTGACCTCACCTCCCCCAA
5954
5973
13
277

532879
n/a
n/a
Intron 12
CCACCTCTTTGTGGGCAGCT
6357
6376
33
278

532880
n/a
n/a
Intron 11
TTCACAAACCACCATCTCTT
6009
6028
8
279

532881
n/a
n/a
Exon 3-
TTCTCACCTCCGTTGTCACA
2958
2977
17
280

Intron 3

532882
n/a
n/a
Intron 12
GAAAGTGGGAGGTGTTGCCT
6225
6244
19
281

532883
n/a
n/a
Intron 1
ACAGCAGGAAGGGAAGGTTA
2075
2094
34
282

532884
n/a
n/a
Intron 17
CATGCTGACCACTTGGCATC
7410
7429
18
283

532885
n/a
n/a
Exon 4-
GGTCACCTTGGCAGGAAGGC
3286
3305
0
284

Intron 4

532886
n/a
n/a
Intron 8
GTATAGTGTTACAAGTGGAC
4804
4823
13
285

532887
n/a
n/a
Intron 7
GGACTTCCCTTTGACCACAA
4468
4487
18
286

532888
n/a
n/a
Intron 11
TCACCTTGACCTCACCTCCC
5958
5977
20
287

532889
n/a
n/a
Intron 15
TAGAGTGCCTCCTTAGGATG
7035
7054
27
288

532890
n/a
n/a
Intron 7
TGACTTCAACTTGTGGTCTG
4605
4624
16
289

532891
n/a
n/a
Intron 10
CAGAGAAGGAGAATGTGCTG
5804
5823
25
290

532892
n/a
n/a
Intron 14-
AGGGAGCAGCTCTTCCTCTG
6919
6938
47
291

Exon 15

532893
n/a
n/a
Intron 5-
TGTTCCCCTGGGTGCCAGGA
3710
3729
24
292

Exon 6

532894
n/a
n/a
Intron 10
GGCCTGGCTGTTTTCAAGCC
5612
5631
15
293

532895
n/a
n/a
Intron 10-
GACTGGCTTTCATCTGGCAG
5821
5840
25
294

Exon 11

532896
n/a
n/a
Intron 10
GAAGGCTTTCCAGGCAACTA
5419
5438
19
295

532897
n/a
n/a
Exon 17-
TCACTTGAATGAAACGACTT
7367
7386
11
296

Intron 17

532898
n/a
n/a
Intron 1
GGCCCCAAAAGGCCAAGGAG
2106
2125
5
297

532899
n/a
n/a
Intron 16-
AATCACCTGCAAGGAGAGGA
7319
7338
19
298

Exon 17

532900
n/a
n/a
Intron 12
GACCTTCAGTTGCATCCTTA
6183
6202
25
299

532901
n/a
n/a
Intron 1
TGATGAAGCCTGGCCCCAAA
2117
2136
0
300

532902
n/a
n/a
Intron 12
TAGAAAGTGGGAGGTGTTGC
6227
6246
0
301

532903
n/a
n/a
Intron 12
CCCATCCCTGACTGGTCTGG
6295
6314
14
302

532904
n/a
n/a
Intron 8
CCATGGGTATAGTGTTACAA
4810
4829
13
303

532905
n/a
n/a
Intron 2
GTGTTCTCTTGACTTCCAGG
2586
2605
23
304

532906
n/a
n/a
Intron 13
GGCCTGCTCCTCACCCCAGT
6597
6616
27
305

532907
n/a
n/a
Intron 10
GAGGCCTGGCTGTTTTCAAG
5614
5633
32
306

532908
n/a
n/a
Exon 1
GACTCTCCCCTTCAGTACCT
1677
1696
16
307

532909
n/a
n/a
Intron 8
CATGGGTATAGTGTTACAAG
4809
4828
10
308

532910
n/a
n/a
Intron 10
GAAGGAGAATGTGCTGAAAA
5800
5819
0
309

532911
n/a
n/a
Intron 7
TCACCTGGTCTTCCAAGCCA
4562
4581
0
310

532912
n/a
n/a
Intron 17
CTCCCCAGATAGGAAAGGGA
7391
7410
0
311

532913
n/a
n/a
Exon 17-
GGACTCACTTGAATGAAACG
7371
7390
0
312

Intron 17

532914
n/a
n/a
Intron 16-
GGCCGCCAGAATCACCTGCA
7328
7347
30
313

Exon 17

532915
n/a
n/a
Exon 17-
CTCACTTGAATGAAACGACT
7368
7387
22
314

Intron 17

532916
n/a
n/a
Intron 13
CTTTCCCAGCCTTTCCTCAG
6569
6588
28
315

532918
n/a
n/a
Intron 12
AGAAAGTGGGAGGTGTTGCC
6226
6245
3
316

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
7839
7858
90
317

TABLE 128

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

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
Region
Sequence
bition
site
site
NO:

532919
n/a
n/a
Exon 1
CCAGGACTCTCCCCTTCAGT
1681
1700
4
318

532920
n/a
n/a
Intron 6
AGGGAAGGAGGACAGAATAG
3976
3995
25
319

532921
n/a
n/a
Intron 4
GAAATGAGGTCAAATGTCTG
3488
3507
30
320

532922
n/a
n/a
Intron 4
GGAGAGTCAGAAATGAGGTC
3497
3516
25
321

532923
n/a
n/a
Intron 12
GTAGAAAGTGGGAGGTGTTG
6228
6247
26
322

532924
n/a
n/a
Intron 10
TAGAAAGATCTCTGAAGTGC
5521
5540
24
323

532925
n/a
n/a
Intron 13
CTGCTCCTCACCCCAGTCCT
6594
6613
26
324

532926
n/a
n/a
Intron 11
CTACTGGGATTCTGTGCTTA
5927
5946
30
325

532927
n/a
n/a
Intron 1
CCCAAAAGGCCAAGGAGGGA
2103
2122
13
326

532928
n/a
n/a
Intron 17
TGACCACTTGGCATCTCCCC
7405
7424
27
327

532929
n/a
n/a
Intron 16-
CCTGCAAGGAGAGGAGAAGC
7314
7333
29
328

Exon 17

532930
n/a
n/a
Exon 16-
CTCTCACCTCTGCAAGTATT
7239
7258
44
329

Intron 16

532931
n/a
n/a
Intron 1
CCCCAAAAGGCCAAGGAGGG
2104
2123
21
330

532932
n/a
n/a
Intron 7
GTCTTCCAAGCCATCTTTTA
4555
4574
20
331

532933
n/a
n/a
Intron 8
GTTACAAGTGGACTTAAGGG
4797
4816
30
332

532934
n/a
n/a
Intron 8-
CCCATGTTGTGCAATCCTGC
5017
5036
30
333

Exon 9

532935
n/a
n/a
Intron 15
GAGGTGGGAAGCATGGAGAA
7091
7110
17
334

532936
n/a
n/a
Intron 14
TGCTCCCACCACTGTCATCT
6874
6893
21
335

532937
n/a
n/a
Exon 9-
AGGCAGGTTACTCACCCAGA
5118
5137
18
336

Intron 9

532938
n/a
n/a
Intron 11
TACTGGGATTCTGTGCTTAC
5926
5945
15
337

532939
n/a
n/a
Intron 13
GCCTTTCCCAGCCTTTCCTC
6571
6590
27
338

532940
n/a
n/a
Intron 8-
GTGCAATCCTGCAGAAGAGA
5009
5028
21
339

Exon 9

532941
n/a
n/a
Intron 8
ACAGGAGAGAGGTCCCTTCT
4743
4762
20
340

532942
n/a
n/a
Intron 10
CCCAAAAGGAGAAAGGGAAA
5717
5736
14
341

532943
n/a
n/a
Intron 2
AAGCCCAGGGTAAATGCTTA
2557
2576
32
342

532944
n/a
n/a
Intron 1
GATGAAGCCTGGCCCCAAAA
2116
2135
22
343

532945
n/a
n/a
Intron 10
TGGCAGAGAAGGAGAATGTG
5807
5826
22
344

532946
n/a
n/a
Intron 13
TTCCCAGCCTTTCCTCAGGG
6567
6586
35
345

532947
n/a
n/a
Intron 10
GGCAGAGAAGGAGAATGTGC
5806
5825
30
346

532948
n/a
n/a
Intron 10
ACAGTGCCAGGAAACAAGAA
5471
5490
25
347

532949
n/a
n/a
Exon 9-
TAGGCAGGTTACTCACCCAG
5119
5138
22
348

Intron 9

532950
n/a
n/a
Intron 2
TTCTCTTGACTTCCAGGGCT
2583
2602
22
349

532951
n/a
n/a
Intron 13
CCTGCTCCTCACCCCAGTCC
6595
6614
16
350

532953
n/a
n/a
Intron 7
TCCCACTAACCTCCATTGCC
4422
4441
14
351

532954
n/a
n/a
Intron 7
TTCCCTTTGACCACAAAGTG
4464
4483
16
352

532955
n/a
n/a
Intron 9
CTGGGTCCTAGGCAGGTTAC
5127
5146
30
353

532956
n/a
n/a
Intron 10
TCCAGGCAACTAGAGCTTCA
5411
5430
20
354

532957
n/a
n/a
Intron 8-
GCCCATGTTGTGCAATCCTG
5018
5037
45
355

Exon 9

532958
n/a
n/a
Intron 7
GGTTCCCACTAACCTCCATT
4425
4444
18
356

532959
n/a
n/a
Intron 3
AGGTAGAGAGCAAGAGTTAC
3052
3071
26
357

532960
n/a
n/a
Intron 7
CCACTAACCTCCATTGCCCA
4420
4439
10
358

532961
n/a
n/a
Intron 11
TCACAAACCACCATCTCTTA
6008
6027
40
359

532962
n/a
n/a
Exon 9-
TACTCACCCAGATAATCCTC
5110
5129
27
360

Intron 9

532963
n/a
n/a
Intron 13
TGCTCCTCACCCCAGTCCTC
6593
6612
24
361

532964
n/a
n/a
Intron 15-
TCTCACAGCTGCCTTTCTGT
7115
7134
25
362

Exon 16

532965
n/a
n/a
Exon 17-
GAAAGGGAGGACTCACTTGA
7379
7398
11
363

Intron 17

532966
n/a
n/a
Intron 7
CCATCTTTTAACCCCAGAGA
4545
4564
18
364

532967
n/a
n/a
Intron 13
TCCTCACCCCAGTCCTCCAG
6590
6609
27
365

532968
n/a
n/a
Intron 10
CTGGCAGAGAAGGAGAATGT
5808
5827
15
366

532969
n/a
n/a
Intron 17
TCTCCCCAGATAGGAAAGGG
7392
7411
23
367

532970
n/a
n/a
Intron 14
ACTTCAGCTGCTCCCACCAC
6882
6901
18
368

532971
n/a
n/a
Intron 1
GACAGCAGGAAGGGAAGGTT
2076
2095
13
369

532972
n/a
n/a
Intron 13-
GGAGACAAATGGGCCTATAA
6640
6659
33
370

Exon 14

532973
n/a
n/a
Intron 14
CTGCTCCCACCACTGTCATC
6875
6894
11
371

532974
n/a
n/a
Intron 10
AGGAATGAAGAAGGCTTTCC
5428
5447
21
372

532975
n/a
n/a
Intron 14
GGGATCTCATCCTTATCCTC
6741
6760
31
373

532976
n/a
n/a
Intron 9
GTGCTGGGTCCTAGGCAGGT
5130
5149
16
374

532977
n/a
n/a
Intron 1
CAAAAGGCCAAGGAGGGATG
2101
2120
14
375

532978
n/a
n/a
Intron 17
CCATGCTGACCACTTGGCAT
7411
7430
20
376

532979
n/a
n/a
Intron 8
GGAGGCTGGGACAGGAGAGA
4753
4772
25
377

532980
n/a
n/a
Intron 14-
GGAGCAGCTCTTCCTCTGGA
6917
6936
36
378

Exon 15

532981
n/a
n/a
Exon 3-
TCTCACCTCCGTTGTCACAG
2957
2976
20
379

Intron 3

532982
n/a
n/a
Intron 13
CAGTCCTCCAGCCTTTCCCA
6581
6600
21
380

532983
n/a
n/a
Intron 13
AGTCCTCCAGCCTTTCCCAG
6580
6599
22
381

532984
n/a
n/a
Intron 4-
TGAAGGAGTCTGGGAGAGTC
3509
3528
12
382

Exon 5

532985
n/a
n/a
Intron 16-
CAGAATCACCTGCAAGGAGA
7322
7341
20
383

Exon 17

532986
n/a
n/a
Exon 17-
TAGGAAAGGGAGGACTCACT
7382
7401
3
384

Intron 17

532987
n/a
n/a
Exon 4-
ACCTTGGCAGGAAGGCTCCG
3282
3301
12
385

Intron 4

532988
n/a
n/a
Intron 13-
GAGACAAATGGGCCTATAAA
6639
6658
15
386

Exon 14

532989
n/a
n/a
Intron 1
CTGAAGAGAAAGGCTGATGA
2131
2150
17
387

532990
n/a
n/a
Intron 6
AATGATCAGGGAGCTAGTCC
3913
3932
30
388

532991
n/a
n/a
Intron 17
CTTAGCTGACCTAAAGGAAT
7557
7576
22
389

532992
n/a
n/a
Intron 8
TGGGTATAGTGTTACAAGTG
4807
4826
17
390

532993
n/a
n/a
Intron 1
TGAAGAGAAAGGCTGATGAA
2130
2149
19
391

532994
n/a
n/a
Intron 8
GTGTTACAAGTGGACTTAAG
4799
4818
25
392

532995
n/a
n/a
Intron 6
ACCTGTGGGTGAGGAGAACA
4134
4153
24
393

532996
n/a
n/a
Exon 9-
TCACCCAGATAATCCTCCCT
5107
5126
36
394

Intron 9

532952
2608
2627
Exon 18
TGTTGTCGCAGCTGTTTTAA
7843
7862
90
395

Example 116: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 4,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3460_MGB (forward sequence CGAAGCAGCTCAATGAAATCAA, designated herein as SEQ ID NO: 813; reverse sequence TGCCTGGAGGGCCTTCTT, designated herein as SEQ ID NO: 814; probe sequence AGACCACAAGTTGAAGTC, designated herein as SEQ ID NO: 815) was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

TABLE 129

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

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
Region
Sequence
bition
site
site
NO:

532686
1135
1154
Exon 6
ACACTTTTTGGCTCCTGTGA
48
3819
3838
84

532687
1141
1160
Exon 6
GACTAGACACTTTTTGGCTC
63
3825
3844
85

532688
1147
1166
Exon 6
TAAGTTGACTAGACACTTTT
47
3831
3850
86

532689
1153
1172
Exon 6
CTCAATTAAGTTGACTAGAC
57
3837
3856
87

532690
1159
1178
Exon 6-7
CACCTTCTCAATTAAGTTGA
49
3843
3862
88

Junction

532691
1165
1184
Exon 6-7
ACTTGCCACCTTCTCAATTA
33
n/a
n/a
89

Junction

532692
1171
1190
Exon 6-7
ACCATAACTTGCCACCTTCT
67
n/a
n/a
90

Junction

532693
1177
1196
Exon 7
CTTCACACCATAACTTGCCA
56
4153
4172
91

532694
1183
1202
Exon 7
TCTTGGCTTCACACCATAAC
50
4159
4178
92

532695
1208
1227
Exon 7
ATGTGGCATATGTCACTAGA
53
4184
4203
93

532696
1235
1254
Exon 7
CAGACACTTTGACCCAAATT
52
4211
4230
94

532697
1298
1317
Exon 7-8
GGTCTTCATAATTGATTTCA
59
n/a
n/a
95

Juncion

532698
1304
1323
Exon 7-8
ACTTGTGGTCTTCATAATTG
52
n/a
n/a
96

Juncion

532699
1310
1329
Exon 7-8
ACTTCAACTTGTGGTCTTCA
85
n/a
n/a
97

Juncion

532700
1316
1335
Exon 8
TCCCTGACTTCAACTTGTGG
96
4609
4628
98

532701
1322
1341
Exon 8
TGTTAGTCCCTGACTTCAAC
56
4615
4634
99

532702
1328
1347
Exon 8
TCTTGGTGTTAGTCCCTGAC
86
4621
4640
100

532703
1349
1368
Exon 8
TGTACACTGCCTGGAGGGCC
35
4642
4661
101

532704
1355
1374
Exon 8
TCATGCTGTACACTGCCTGG
12
4648
4667
102

532705
1393
1412
Exon 8
GTTCCAGCCTTCAGGAGGGA
27
4686
4705
103

532706
1399
1418
Exon 8
GGTGCGGTTCCAGCCTTCAG
67
4692
4711
104

532707
1405
1424
Exon 8
ATGGCGGGTGCGGTTCCAGC
26
4698
4717
105

532708
1411
1430
Exon 8
GATGACATGGCGGGTGCGGT
28
4704
4723
106

532709
1417
1436
Exon 8
GAGGATGATGACATGGCGGG
6
4710
4729
107

532710
1443
1462
Exon 8-9
CCCATGTTGTGCAATCCATC
35
n/a
n/a
108

Junction

532711
1449
1468
Exon 9
TCCCCGCCCATGTTGTGCAA
28
5023
5042
109

532712
1455
1474
Exon 9
ATTGGGTCCCCGCCCATGTT
19
5029
5048
110

532713
1461
1480
Exon 9
ACAGTAATTGGGTCCCCGCC
29
5035
5054
111

532714
1467
1486
Exon 9
TCAATGACAGTAATTGGGTC
49
5041
5060
112

532715
1473
1492
Exon 9
ATCTCATCAATGACAGTAAT
45
5047
5066
113

532716
1479
1498
Exon 9
TCCCGGATCTCATCAATGAC
54
5053
5072
114

532717
1533
1552
Exon 9-
ACATCCAGATAATCCTCCCT
22
n/a
n/a
115

10

Junction

532718
1539
1558
Exon 9-
ACATAGACATCCAGATAATC
8
n/a
n/a
116

10

Junction

532719
1545
1564
Exon 9-
CCAAACACATAGACATCCAG
30
n/a
n/a
117

10

Junction

532720
1582
1601
Exon 10
AGCATTGATGTTCACTTGGT
62
5231
5250
118

532721
1588
1607
Exon 10
AGCCAAAGCATTGATGTTCA
46
5237
5256
119

532722
1594
1613
Exon 10
CTTGGAAGCCAAAGCATTGA
35
5243
5262
120

532723
1600
1619
Exon 10
GTCTTTCTTGGAAGCCAAAG
43
5249
5268
121

532724
1606
1625
Exon 10
CTCATTGTCTTTCTTGGAAG
40
5255
5274
122

532725
1612
1631
Exon 10
ATGTTGCTCATTGTCTTTCT
49
5261
5280
123

532726
1618
1637
Exon 10
GAACACATGTTGCTCATTGT
68
5267
5286
124

532727
1624
1643
Exon 10
GACTTTGAACACATGTTGCT
54
5273
5292
125

532728
1630
1649
Exon 10
ATCCTTGACTTTGAACACAT
61
5279
5298
126

532729
1636
1655
Exon 10
TTCCATATCCTTGACTTTGA
55
5285
5304
127

532730
1642
1661
Exon 10
CAGGTTTTCCATATCCTTGA
51
5291
5310
440

532731
1686
1705
Exon 10-
CTCAGAGACTGGCTTTCATC
41
5827
5846
129

11

Junction

532732
1692
1711
Exon 11
CAGAGACTCAGAGACTGGCT
59
5833
5852
130

516252
1698
1717
Exon 11
ATGCCACAGAGACTCAGAGA
57
5839
5858
131

532733
1704
1723
Exon 11
CAAACCATGCCACAGAGACT
34
5845
5864
132

532734
1710
1729
Exon 11
TGTTCCCAAACCATGCCACA
51
5851
5870
133

532735
1734
1753
Exon 11
TTGTGGTAATCGGTACCCTT
50
5875
5894
134

532736
1740
1759
Exon 11
GGTTGCTTGTGGTAATCGGT
64
5881
5900
135

532737
1746
1765
Exon 11
TGCCATGGTTGCTTGTGGTA
40
5887
5906
136

532738
1752
1771
Exon 11
TTGGCCTGCCATGGTTGCTT
49
5893
5912
137

532739
1758
1777
Exon 11
GAGATCTTGGCCTGCCATGG
47
5899
5918
138

532740
1803
1822
Exon 12
ACAGCCCCCATACAGCTCTC
48
6082
6101
139

532741
1809
1828
Exon 12
GACACCACAGCCCCCATACA
40
6088
6107
140

532742
1815
1834
Exon 12
TACTCAGACACCACAGCCCC
33
6094
6113
141

532743
1821
1840
Exon 12
ACAAAGTACTCAGACACCAC
39
6100
6119
142

532744
1827
1846
Exon 12
GTCAGCACAAAGTACTCAGA
45
6106
6125
143

532745
1872
1891
Exon 12
TTGATTGAGTGTTCCTTGTC
42
6151
6170
144

532746
1878
1897
Exon 12
CTGACCTTGATTGAGTGTTC
53
6157
6176
145

532747
1909
1928
Exon 13
TATCTCCAGGTCCCGCTTCT
31
6403
6422
146

532748
1967
1986
Exon 13
GAATTCCTGCTTCTTTTTTC
30
6461
6480
147

532749
1973
1992
Exon 13
ATTCAGGAATTCCTGCTTCT
40
6467
6486
148

532750
1979
1998
Exon 13
CATAAAATTCAGGAATTCCT
45
6473
6492
149

532751
1985
2004
Exon 13
CATAGTCATAAAATTCAGGA
43
6479
6498
150

532752
2006
2025
Exon 13
TGAGCTTGATCAGGGCAACG
61
6500
6519
151

532753
2012
2031
Exon 13
TATTCTTGAGCTTGATCAGG
47
6506
6525
152

532754
2048
2067
Exon 13-
GACAAATGGGCCTGATAGTC
35
n/a
n/a
153

14

Junction

532755
2070
2089
Exon 14
GTTGTTCCCTCGGTGCAGGG
43
6659
6678
154

532756
2076
2095
Exon 14
GCTCGAGTTGTTCCCTCGGT
51
6665
6684
155

532757
2082
2101
Exon 14
CTCAAAGCTCGAGTTGTTCC
36
6671
6690
156

532758
2088
2107
Exon 14
GGAAGCCTCAAAGCTCGAGT
54
6677
6696
157

532759
2094
2113
Exon 14
GTTGGAGGAAGCCTCAAAGC
52
6683
6702
158

532760
2100
2119
Exon 14
GTGGTAGTTGGAGGAAGCCT
22
6689
6708
159

532761
2106
2125
Exon 14
TGGCAAGTGGTAGTTGGAGG
34
6695
6714
160

532762
2112
2131
Exon 14
TGTTGCTGGCAAGTGGTAGT
52
6701
6720
161

Example 117: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 5,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘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 130

Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1

SEQ ID
SEQ ID

NO: 1
NO: 1

%
SEQ

ISIS
start
stop
Target

inhi-
ID

NO
site
site
Region
Sequence
bition
NO:

588570
150
169
Exon 1
TGGTCACATTCCCTTCCCCT
54
396

588571
152
171
Exon 1
CCTGGTCACATTCCCTTCCC
63
397

532614
154
173
Exon 1
GACCTGGTCACATTCCCTTC
64
12

588572
156
175
Exon 1
TAGACCTGGTCACATTCCCT
62
398

588573
158
177
Exon 1
CCTAGACCTGGTCACATTCC
53
399

588566
2189
2208
Exon 15
CCTTCCGAGTCAGCTTTTTC
60
400

588567
2191
2210
Exon 15
CTCCTTCCGAGTCAGCTTTT
61
401

532770
2193
2212
Exon 15
ACCTCCTTCCGAGTCAGCTT
77
198

588568
2195
2214
Exon 15
AGACCTCCTTCCGAGTCAGC
72
402

588569
2197
2216
Exon 15
GTAGACCTCCTTCCGAGTCA
46
403

588574
2453
2472
Exon 18
TTTGCCGCTTCTGGTTTTTG
46
404

588575
2455
2474
Exon 18
CTTTTGCCGCTTCTGGTTTT
41
405

532800
2457
2476
Exon 18
TGCTTTTGCCGCTTCTGGTT
69
228

588576
2459
2478
Exon 18
CCTGCTTTTGCCGCTTCTGG
61
406

588577
2461
2480
Exon 18
TACCTGCTTTTGCCGCTTCT
51
407

516350
2550
2569
Exon 18
AGAAAACCCAAATCCTCATC
71
408

588509
2551
2570
Exon 18
TAGAAAACCCAAATCCTCAT
58
409

588510
2552
2571
Exon 18
ATAGAAAACCCAAATCCTCA
57
410

588511
2553
2572
Exon 18
TATAGAAAACCCAAATCCTC
57
411

588512
2554
2573
Exon 18
TTATAGAAAACCCAAATCCT
44
412

588513
2555
2574
Exon 18
CTTATAGAAAACCCAAATCC
37
413

588514
2556
2575
Exon 18
CCTTATAGAAAACCCAAATC
50
414

588515
2557
2576
Exon 18
CCCTTATAGAAAACCCAAAT
45
415

588516
2558
2577
Exon 18
CCCCTTATAGAAAACCCAAA
60
416

588517
2559
2578
Exon 18
ACCCCTTATAGAAAACCCAA
67
417

588518
2560
2579
Exon 18
AACCCCTTATAGAAAACCCA
57
418

588519
2561
2580
Exon 18
AAACCCCTTATAGAAAACCC
61
419

588520
2562
2581
Exon 18
GAAACCCCTTATAGAAAACC
27
420

588521
2563
2582
Exon 18
GGAAACCCCTTATAGAAAAC
25
421

588522
2564
2583
Exon 18
AGGAAACCCCTTATAGAAAA
36
422

588523
2565
2584
Exon 18
CAGGAAACCCCTTATAGAAA
36
423

588524
2566
2585
Exon 18
GCAGGAAACCCCTTATAGAA
46
424

588525
2567
2586
Exon 18
AGCAGGAAACCCCTTATAGA
38
425

588526
2568
2587
Exon 18
CAGCAGGAAACCCCTTATAG
47
426

588527
2569
2588
Exon 18
CCAGCAGGAAACCCCTTATA
68
427

588528
2570
2589
Exon 18
TCCAGCAGGAAACCCCTTAT
63
428

532809
2571
2590
Exon 18
GTCCAGCAGGAAACCCCTTA
85
237

588529
2572
2591
Exon 18
TGTCCAGCAGGAAACCCCTT
76
429

588530
2573
2592
Exon 18
CTGTCCAGCAGGAAACCCCT
74
430

588531
2574
2593
Exon 18
CCTGTCCAGCAGGAAACCCC
75
431

588532
2575
2594
Exon 18
CCCTGTCCAGCAGGAAACCC
73
432

588533
2576
2595
Exon 18
CCCCTGTCCAGCAGGAAACC
82
433

532810
2577
2596
Exon 18
GCCCCTGTCCAGCAGGAAAC
88
238

588534
2578
2597
Exon 18
CGCCCCTGTCCAGCAGGAAA
86
434

588535
2579
2598
Exon 18
ACGCCCCTGTCCAGCAGGAA
86
435

588536
2580
2599
Exon 18
CACGCCCCTGTCCAGCAGGA
93
436

588537
2581
2600
Exon 18
CCACGCCCCTGTCCAGCAGG
92
437

588538
2582
2601
Exon 18
CCCACGCCCCTGTCCAGCAG
94
438

588539
2583
2602
Exon 18
TCCCACGCCCCTGTCCAGCA
96
439

588540
2584
2603
Exon 18
ATCCCACGCCCCTGTCCAGC
88
440

588541
2585
2604
Exon 18
AATCCCACGCCCCTGTCCAG
79
441

588542
2586
2605
Exon 18
CAATCCCACGCCCCTGTCCA
83
442

588543
2587
2606
Exon 18
TCAATCCCACGCCCCTGTCC
86
443

588544
2588
2607
Exon 18
TTCAATCCCACGCCCCTGTC
90
444

588545
2589
2608
Exon 18
ATTCAATCCCACGCCCCTGT
92
445

588546
2590
2609
Exon 18
AATTCAATCCCACGCCCCTG
92
446

588547
2591
2610
Exon 18
TAATTCAATCCCACGCCCCT
88
447

588548
2592
2611
Exon 18
TTAATTCAATCCCACGCCCC
93
448

588549
2593
2612
Exon 18
TTTAATTCAATCCCACGCCC
88
449

588550
2594
2613
Exon 18
TTTTAATTCAATCCCACGCC
89
450

588551
2595
2614
Exon 18
GTTTTAATTCAATCCCACGC
94
451

588552
2596
2615
Exon 18
TGTTTTAATTCAATCCCACG
93
452

588553
2597
2616
Exon 18
CTGTTTTAATTCAATCCCAC
96
453

588554
2598
2617
Exon 18
GCTGTTTTAATTCAATCCCA
98
454

532811
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
97
239

532811
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
95
239

588555
2600
2619
Exon 18
CAGCTGTTTTAATTCAATCC
93
455

588556
2601
2620
Exon 18
GCAGCTGTTTTAATTCAATC
96
456

588557
2602
2621
Exon 18
CGCAGCTGTTTTAATTCAAT
98
457

588558
2603
2622
Exon 18
TCGCAGCTGTTTTAATTCAA
95
458

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
97
317

588559
2605
2624
Exon 18
TGTCGCAGCTGTTTTAATTC
95
459

588560
2606
2625
Exon 18
TTGTCGCAGCTGTTTTAATT
92
460

588561
2607
2626
Exon 18
GTTGTCGCAGCTGTTTTAAT
93
461

532952
2608
2627
Exon 18
TGTTGTCGCAGCTGTTTTAA
88
395

588562
2609
2628
Exon 18/
TTGTTGTCGCAGCTGTTTTA
90
462

Repeat

588563
2610
2629
Exon 18/
TTTGTTGTCGCAGCTGTTTT
89
463

Repeat

588564
2611
2630
Exon 18/
TTTTGTTGTCGCAGCTGTTT
92
464

Repeat

588565
2612
2631
Exon 18/
TTTTTGTTGTCGCAGCTGTT
88
465

Repeat

TABLE 131

Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
Region
Sequence
bition
site
site
NO:

588685
n/a
n/a
Exon 1
GGATCCAGCTCACTCCCCTG
48
1596
1615
466

588686
n/a
n/a
Exon 1
AAATAAGGATCCAGCTCACT
29
1602
n/a
467

588688
n/a
n/a
Exon 1
GACCAGAAATAAGGATCCAG
58
1608
1627
468

588690
n/a
n/a
Exon 1
CTTAGGGACCAGAAATAAGG
45
1614
1633
469

588692
n/a
n/a
Exon 1
CACCCACTTAGGGACCAGAA
36
1620
1639
470

588694
n/a
n/a
Exon 1
ACCACCCACTTAGGGACCAG
47
1622
1641
471

588696
n/a
n/a
Exon 1
AGGTCCAGGACTCTCCCCTT
96
1685
1704
472

588698
n/a
n/a
Exon 1
AAGGTCCAGGACTCTCCCCT
96
1686
1705
473

588700
n/a
n/a
Exon 1
AAACTGCAGAAGTCCCACCC
2
1716
1735
474

588586
30
49
Exon 1
GGAGGGCCCCGCTGAGCTGC
59
1751
1770
475

588587
48
67
Exon 1
TCCCGGAACATCCAAGCGGG
45
1769
1788
476

588588
56
75
Exon 1
CATCACTTTCCCGGAACATC
39
1777
n/a
477

588589
151
170
Exon 1
CTGGTCACATTCCCTTCCCC
29
1872
1891
478

588590
157
176
Exon 1
CTAGACCTGGTCACATTCCC
47
1878
1897
479

588591
339
358
Exon 1-2
GGAGTGGTGGTCACACCTCC
44
n/a
n/a
480

Junction

588592
384
403
Exon 2
ACCCCCTCCAGAGAGCAGGA
43
2192
2211
481

588593
390
409
Exon 2
ATCTCTACCCCCTCCAGAGA
34
2198
2217
482

588594
467
486
Exon 2
GGTACGGGTAGAAGCCAGAA
17
2275
2294
483

588595
671
690
Exon 3
GGAGAGTGTAACCGTCATAG
37
2879
2898
484

588596
689
708
Exon 3
TGCGATTGGCAGAGCCCCGG
18
2897
2916
485

588597
695
714
Exon 3
GGCAGGTGCGATTGGCAGAG
32
2903
2922
486

588598
707
726
Exon 3
GGCCATTCACTTGGCAGGTG
45
2915
2934
487

588599
738
757
Exon 3
TTGTCACAGATCGCTGTCTG
52
2946
2965
488

588600
924
943
Exon 4-5
AAGGAGTCTTGGCAGGAAGG
39
n/a
n/a
489

Junction

588601
931
950
Exon 4-5
GTACATGAAGGAGTCTTGGC
37
n/a
n/a
490

Junction

588602
959
978
Exon 5
AAGCTTCGGCCACCTCTTGA
21
3542
3561
491

588603
1089
1108
Exon 6
CCATCTAGCACCAGGTAGAT
22
3773
3792
492

588604
1108
1127
Exon 6
GGCCCCAATGCTGTCTGATC
21
3792
3811
493

588606
1150
1169
Exon 6
AATTAAGTTGACTAGACACT
56
3834
3853
494

588608
1162
1181
Exon 6-7
TGCCACCTTCTCAATTAAGT
50

19
495

Junction

588578
1167
1186
Exon 6-7
TAACTTGCCACCTTCTCAAT
23
n/a
n/a
496

Junction

588579
1169
1188
Exon 6-7
CATAACTTGCCACCTTCTCA
23
n/a
n/a
497

Junction

532692
1171
1190
Exon 6-7
ACCATAACTTGCCACCTTCT
15
n/a
n/a
90

Junction

588580
1173
1192
Exon 6-7
ACACCATAACTTGCCACCTT
16
n/a
n/a
498

Junction

588581
1175
1194
Exon 6-7
TCACACCATAACTTGCCACC
14
4151
4170
499

Junction

588610
1319
1338
Exon 8
TAGTCCCTGACTTCAACTTG
50
4612
4631
500

588612
1325
1344
Exon 8
TGGTGTTAGTCCCTGACTTC
47
4618
4637
501

588614
1396
1415
Exon 8
GCGGTTCCAGCCTTCAGGAG
47
4689
4708
502

588616
1421
1440
Exon 8
TCATGAGGATGATGACATGG
51
4714
4733
503

588618
1446
1465
Exon 9
CCGCCCATGTTGTGCAATCC
18
5020
5039
504

588620
1458
1477
Exon 9
GTAATTGGGTCCCCGCCCAT
40
5032
5051
505

588623
1482
1501
Exon 9
AAGTCCCGGATCTCATCAAT
40
5056
5075
506

588624
1542
1561
Exon 9-10
AACACATAGACATCCAGATA
45
n/a
n/a
507

Junction

588626
1585
1604
Exon 10
CAAAGCATTGATGTTCACTT
43
5234
5253
508

588628
1621
1640
Exon 10
TTTGAACACATGTTGCTCAT
45
5270
5289
509

588631
1646
1665
Exon 10
CTTCCAGGTTTTCCATATCC
53
5295
5314
510

588632
1647
1666
Exon 10
TCTTCCAGGTTTTCCATATC
56
5296
5315
511

588634
1689
1708
Exon 11
AGACTCAGAGACTGGCTTTC
35
5830
5849
512

588636
1749
1768
Exon 11
GCCTGCCATGGTTGCTTGTG
55
5890
5909
513

588638
1763
1782
Exon 11
TGACTGAGATCTTGGCCTGC
78
5904
5923
514

588640
1912
1931
Exon 13
TTCTATCTCCAGGTCCCGCT
95
6406
6425
515

588642
1982
2001
Exon 13
AGTCATAAAATTCAGGAATT
44
6476
6495
516

588645
2073
2092
Exon 14
CGAGTTGTTCCCTCGGTGCA
40
6662
6681
517

588646
2085
2104
Exon 14
AGCCTCAAAGCTCGAGTTGT
57
6674
6693
518

588648
2091
2110
Exon 14
GGAGGAAGCCTCAAAGCTCG
48
6680
6699
519

588651
2097
2116
Exon 14
GTAGTTGGAGGAAGCCTCAA
40
6686
6705
520

588652
2103
2122
Exon 14
CAAGTGGTAGTTGGAGGAAG
43
6692
6711
521

588654
2166
2185
Exon 15
TCCTCAGACACAAACAGAGC
13
6954
6973
522

588656
2172
2191
Exon 15
TTCTCCTCCTCAGACACAAA
55
6960
6979
523

588658
2196
2215
Exon 15
TAGACCTCCTTCCGAGTCAG
44
6984
7003
524

588660
2202
2221
Exon 15
TTGATGTAGACCTCCTTCCG
50
6990
7009
525

588582
2219
2238
Exon 15-16
CTTTCTTATCCCCATTCTTG
19
n/a
n/a
526

Junction

588583
2221
2240
Exon 15-16
GCCTTTCTTATCCCCATTCT
14
n/a
n/a
527

Junction

532775
2223
2242
Exon 15-16
CTGCCTTTCTTATCCCCATT
3
n/a
n/a
203

Junction

588584
2225
2244
Exon 15-16
AGCTGCCTTTCTTATCCCCA
18
n/a
n/a
528

Junction

588662
2226
2245
Exon 15-16
CAGCTGCCTTTCTTATCCCC
27
n/a
n/a
529

Junction

588585
2227
2246
Exon 15-16
ACAGCTGCCTTTCTTATCCC
59
n/a
n/a
530

Junction

588664
2238
2257
Exon 16
GCATCTCTCTCACAGCTGCC
49
7122
7141
531

588666
2276
2295
Exon 16
AGATGTCCTTGACTTTGTCA
41
7160
7179
532

588668
2330
2349
Exon 16
CAGCATAGGGACTCACTCCT
41
7214
7233
533

588670
2361
2380
Exon 16-17
CCGCCAGAATCACCTCTGCA
43
n/a
n/a
534

Junction

588672
2397
2416
Exon 17
TGAATGAAACGACTTCTCTT
52
7362
7381
535

588674
2430
2449
Exon 18
ACATCCACTACTCCCCAGCT
39
7665
7684
536

588676
2448
2467
Exon 18
CGCTTCTGGTTTTTGCAGAC
69
7683
7702
537

588678
2454
2473
Exon 18
TTTTGCCGCTTCTGGTTTTT
46
7689
7708
538

588680
2466
2485
Exon 18
GCAGGTACCTGCTTTTGCCG
47
7701
7720
539

588682
2532
2551
Exon 18
TCTTGGAGTTTCTCCTTCAG
58
7767
7786
540

532811
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
10
7834
7853
239

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
11
7839
7858
317

Example 118: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 3,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 4-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE, 3-10-4 MOE, 3-10-7 MOE, 6-7-6-MOE, 6-8-6 MOE, or 5-7-5 MOE gapmers, or as deoxy, MOE, and (S)-cEt oligonucleotides.

The 4-8-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four and five nucleosides respectively. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-7-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-4 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and four nucleosides respectively. The 3-10-7 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and seven nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

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 modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification.

“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 CFB mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_001710.5) or the human CFB genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NT_007592.15 truncated from nucleotides 31852000 to 31861000), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 132

Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

532811
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
10
7834
7853
eeeeeddddddddddeeeee
239

588884
48
63
Exon 1
GGAACATCCAAGCGGG
79
1769
1784
eekdddddddddddke
541

588872
154
169
Exon 1
TGGTCACATTCCCTTC
91
1875
1890
eekdddddddddddke
542

588873
156
171
Exon 1
CCTGGTCACATTCCCT
91
1877
1892
eekdddddddddddke
543

588874
158
173
Exon 1
GACCTGGTCACATTCC
91
1879
1894
eekdddddddddddke
544

588878
1171
1186
Exon 6-7
TAACTTGCCACCTTCT
92
n/a
n/a
eekdddddddddddke
545

Junction

588879
1173
1188
Exon 6-7
CATAACTTGCCACCTT
94
n/a
n/a
eekdddddddddddke
546

Junction

588880
1175
1190
Exon 6-7
ACCATAACTTGCCACC
89
4151
4166
eekdddddddddddke
547

Junction

588869
2193
2208
Exon 15
CCTTCCGAGTCAGCTT
17
6981
6996
eekdddddddddddke
548

588870
2195
2210
Exon 15
CTCCTTCCGAGTCAGC
78
6983
6998
eekdddddddddddke
549

588871
2197
2212
Exon 15
ACCTCCTTCCGAGTCA
80
6985
7000
eekdddddddddddke
550

588881
2223
2238
Exon 15-
CTTTCTTATCCCCATT
93
n/a
n/a
eekdddddddddddke
551

16

Junction

588882
2225
2240
Exon 15-
GCCTTTCTTATCCCCA
88
n/a
n/a
eekdddddddddddke
552

16

Junction

588883
2227
2242
Exon 15-
CTGCCTTTCTTATCCC
90
n/a
n/a
eekdddddddddddke
553

16

Junction

588875
2457
2472
Exon 18
TTTGCCGCTTCTGGTT
81
7692
7707
eekdddddddddddke
554

588876
2459
2474
Exon 18
CTTTTGCCGCTTCTGG
95
7694
7709
eekdddddddddddke
555

588877
2461
2476
Exon 18
TGCTTTTGCCGCTTCT
91
7696
7711
eekdddddddddddke
556

588807
2551
2566
Exon 18
AAACCCAAATCCTCAT
82
7786
7801
eekdddddddddddke
557

588808
2553
2568
Exon 18
GAAAACCCAAATCCTC
69
7788
7803
eekdddddddddddke
558

588809
2555
2570
Exon 18
TAGAAAACCCAAATCC
51
7790
7805
eekdddddddddddke
559

588810
2556
2571
Exon 18
ATAGAAAACCCAAATC
23
7791
7806
eekdddddddddddke
560

588811
2559
2574
Exon 18
CTTATAGAAAACCCAA
13
7794
7809
eekdddddddddddke
561

588812
2560
2575
Exon 18
CCTTATAGAAAACCCA
29
7795
7810
eekdddddddddddke
562

588813
2561
2576
Exon 18
CCCTTATAGAAAACCC
53
7796
7811
eekdddddddddddke
563

588814
2562
2577
Exon 18
CCCCTTATAGAAAACC
86
7797
7812
eekdddddddddddke
564

588815
2563
2578
Exon 18
ACCCCTTATAGAAAAC
76
7798
7813
eekdddddddddddke
565

588816
2564
2579
Exon 18
AACCCCTTATAGAAAA
33
7799
7814
eekdddddddddddke
566

588817
2565
2580
Exon 18
AAACCCCTTATAGAAA
48
7800
7815
eekdddddddddddke
567

588818
2566
2581
Exon 18
GAAACCCCTTATAGAA
44
7801
7816
eekdddddddddddke
568

588819
2567
2582
Exon 18
GGAAACCCCTTATAGA
74
7802
7817
eekdddddddddddke
569

588820
2568
2583
Exon 18
AGGAAACCCCTTATAG
68
7803
7818
eekdddddddddddke
570

588821
2569
2584
Exon 18
CAGGAAACCCCTTATA
45
7804
7819
eekdddddddddddke
571

588822
2570
2585
Exon 18
GCAGGAAACCCCTTAT
50
7805
7820
eekdddddddddddke
572

588823
2571
2586
Exon 18
AGCAGGAAACCCCTTA
54
7806
7821
eekdddddddddddke
573

588824
2572
2587
Exon 18
CAGCAGGAAACCCCTT
35
7807
7822
eekdddddddddddke
574

588825
2573
2588
Exon 18
CCAGCAGGAAACCCCT
11
7808
7823
eekdddddddddddke
575

588826
2574
2589
Exon 18
TCCAGCAGGAAACCCC
19
7809
7824
eekdddddddddddke
576

588827
2575
2590
Exon 18
GTCCAGCAGGAAACCC
42
7810
7825
eekdddddddddddke
577

588828
2576
2591
Exon 18
TGTCCAGCAGGAAACC
0
7811
7826
eekdddddddddddke
578

588829
2577
2592
Exon 18
CTGTCCAGCAGGAAAC
49
7812
7827
eekdddddddddddke
579

588830
2578
2593
Exon 18
CCTGTCCAGCAGGAAA
11
7813
7828
eekdddddddddddke
580

588831
2579
2594
Exon 18
CCCTGTCCAGCAGGAA
20
7814
7829
eekdddddddddddke
581

588832
2580
2595
Exon 18
CCCCTGTCCAGCAGGA
19
7815
7830
eekdddddddddddke
582

588833
2581
2596
Exon 18
GCCCCTGTCCAGCAGG
12
7816
7831
eekdddddddddddke
583

588834
2582
2597
Exon 18
CGCCCCTGTCCAGCAG
10
7817
7832
eekdddddddddddke
584

588835
2583
2598
Exon 18
ACGCCCCTGTCCAGCA
13
7818
7833
eekdddddddddddke
585

588836
2584
2599
Exon 18
CACGCCCCTGTCCAGC
13
7819
7834
eekdddddddddddke
586

588837
2585
2600
Exon 18
CCACGCCCCTGTCCAG
39
7820
7835
eekdddddddddddke
587

588838
2586
2601
Exon 18
CCCACGCCCCTGTCCA
54
7821
7836
eekdddddddddddke
588

588839
2587
2602
Exon 18
TCCCACGCCCCTGTCC
51
7822
7837
eekdddddddddddke
589

588840
2588
2603
Exon 18
ATCCCACGCCCCTGTC
65
7823
7838
eekdddddddddddke
590

588841
2589
2604
Exon 18
AATCCCACGCCCCTGT
59
7824
7839
eekdddddddddddke
591

588842
2590
2605
Exon 18
CAATCCCACGCCCCTG
70
7825
7840
eekdddddddddddke
592

588843
2591
2606
Exon 18
TCAATCCCACGCCCCT
0
7826
7841
eekdddddddddddke
593

588844
2592
2607
Exon 18
TTCAATCCCACGCCCC
48
7827
7842
eekdddddddddddke
594

588845
2593
2608
Exon 18
ATTCAATCCCACGCCC
46
7828
7843
eekdddddddddddke
595

588846
2594
2609
Exon 18
AATTCAATCCCACGCC
67
7829
7844
eekdddddddddddke
596

588847
2595
2610
Exon 18
TAATTCAATCCCACGC
75
7830
7845
eekdddddddddddke
597

588848
2596
2611
Exon 18
TTAATTCAATCCCACG
76
7831
7846
eekdddddddddddke
598

588849
2597
2612
Exon 18
TTTAATTCAATCCCAC
94
7832
7847
eekdddddddddddke
599

588850
2598
2613
Exon 18
TTTTAATTCAATCCCA
91
7833
7848
eekdddddddddddke
600

588851
2599
2614
Exon 18
GTTTTAATTCAATCCC
91
7834
7849
eekdddddddddddke
601

588852
2600
2615
Exon 18
TGTTTTAATTCAATCC
78
7835
7850
eekdddddddddddke
602

588853
2601
2616
Exon 18
CTGTTTTAATTCAATC
81
7836
7851
eekdddddddddddke
603

588854
2602
2617
Exon 18
GCTGTTTTAATTCAAT
63
7837
7852
eekdddddddddddke
604

588855
2603
2618
Exon 18
AGCTGTTTTAATTCAA
65
7838
7853
eekdddddddddddke
605

588856
2604
2619
Exon 18
CAGCTGTTTTAATTCA
76
7839
7854
eekdddddddddddke
606

588857
2605
2620
Exon 18
GCAGCTGTTTTAATTC
89
7840
7855
eekdddddddddddke
607

588858
2606
2621
Exon 18
CGCAGCTGTTTTAATT
89
7841
7856
eekdddddddddddke
608

588859
2607
2622
Exon 18
TCGCAGCTGTTTTAAT
89
7842
7857
eekdddddddddddke
609

588860
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
76
7843
7858
eekdddddddddddke
610

588861
2609
2624
Exon 18
TGTCGCAGCTGTTTTA
87
7844
7859
eekdddddddddddke
611

588862
2610
2625
Exon 18
TTGTCGCAGCTGTTTT
85
7845
7860
eekdddddddddddke
612

588863
2611
2626
Exon 18
GTTGTCGCAGCTGTTT
87
7846
7861
eekdddddddddddke
613

588864
2612
2627
Exon 18
TGTTGTCGCAGCTGTT
67
7847
7862
eekdddddddddddke
614

588865
2613
2628
Exon 18
TTGTTGTCGCAGCTGT
51
n/a
n/a
eekdddddddddddke
615

588866
2614
2629
Exon 18
TTTGTTGTCGCAGCTG
95
n/a
n/a
eekdddddddddddke
616

588867
2615
2630
Exon 18
TTTTGTTGTCGCAGCT
92
n/a
n/a
eekdddddddddddke
617

588868
2616
2631
Exon 18
TTTTTGTTGTCGCAGC
66
n/a
n/a
eekdddddddddddke
618

TABLE 133

Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 or SEQ

ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop
ID

NO
site
site
region
Sequence
bition
site
site
NO:

588685
n/a
n/a
Exon 1
GGATCCAGCTCACTCCCCTG
14
1596
1615
466

588686
n/a
n/a
Exon 1
AAATAAGGATCCAGCTCACT
2
1602
1621
467

588688
n/a
n/a
Exon 1
GACCAGAAATAAGGATCCAG
3
1608
1627
468

588690
n/a
n/a
Exon 1
CTTAGGGACCAGAAATAAGG
10
1614
1633
469

588692
n/a
n/a
Exon 1
CACCCACTTAGGGACCAGAA
23
1620
1639
470

588694
n/a
n/a
Exon 1
ACCACCCACTTAGGGACCAG
23
1622
1641
471

588696
n/a
n/a
Exon 1
AGGTCCAGGACTCTCCCCTT
15
1685
1704
472

588698
n/a
n/a
Exon 1
AAGGTCCAGGACTCTCCCCT
19
1686
1705
473

588700
n/a
n/a
Exon 1
AAACTGCAGAAGTCCCACCC
16
1716
1735
474

588586
30
49
Exon 1
GGAGGGCCCCGCTGAGCTGC
11
1751
1770
475

588587
48
67
Exon 1
TCCCGGAACATCCAAGCGGG
14
1769
1788
476

588588
56
75
Exon 1
CATCACTTTCCCGGAACATC
18
1777
1796
477

588589
151
170
Exon 1
CTGGTCACATTCCCTTCCCC
59
1872
1891
478

588590
157
176
Exon 1
CTAGACCTGGTCACATTCCC
59
1878
1897
479

588591
339
358
Exon 1-2
GGAGTGGTGGTCACACCTCC
45
n/a
n/a
480

Junction

588592
384
403
Exon 2
ACCCCCTCCAGAGAGCAGGA
39
2192
2211
481

588593
390
409
Exon 2
ATCTCTACCCCCTCCAGAGA
29
2198
2217
482

588594
467
486
Exon 2
GGTACGGGTAGAAGCCAGAA
47
2275
2294
483

588595
671
690
Exon 3
GGAGAGTGTAACCGTCATAG
44
2879
2898
484

588596
689
708
Exon 3
TGCGATTGGCAGAGCCCCGG
43
2897
2916
638

588597
695
714
Exon 3
GGCAGGTGCGATTGGCAGAG
34
2903
2922
486

588598
707
726
Exon 3
GGCCATTCACTTGGCAGGTG
17
2915
2934
487

588599
738
757
Exon 3
TTGTCACAGATCGCTGTCTG
37
2946
2965
488

588600
924
943
Exon 3-4
AAGGAGTCTTGGCAGGAAGG
18
n/a
n/a
489

Junction

588601
931
950
Exon 3-4
GTACATGAAGGAGTCTTGGC
32
n/a
n/a
490

Junction

588602
959
978
Exon 5
AAGCTTCGGCCACCTCTTGA
45
3542
3561
491

588603
1089
1108
Exon 6
CCATCTAGCACCAGGTAGAT
52
3773
3792
492

588604
1108
1127
Exon 6
GGCCCCAATGCTGTCTGATC
39
3792
3811
493

588606
1150
1169
Exon 6
AATTAAGTTGACTAGACACT
37
3834
3853
494

588608
1162
1181
Exon 6-7
TGCCACCTTCTCAATTAAGT
21
n/a
n/a
648

Junction

588578
1167
1186
Exon 6-7
TAACTTGCCACCTTCTCAAT
22
n/a
n/a
496

Junction

588579
1169
1188
Exon 6-7
CATAACTTGCCACCTTCTCA
21
n/a
n/a
497

Junction

532692
1171
1190
Exon 6-7
ACCATAACTTGCCACCTTCT
56
n/a
n/a
90

Junction

588580
1173
1192
Exon 6-7
ACACCATAACTTGCCACCTT
50
n/a
n/a
498

Junction

588581
1175
1194
Exon 7
TCACACCATAACTTGCCACC
50
4151
4170
499

588610
1319
1338
Exon 8
TAGTCCCTGACTTCAACTTG
47
4612
4631
500

588612
1325
1344
Exon 8
TGGTGTTAGTCCCTGACTTC
47
4618
4637
501

588614
1396
1415
Exon 8
GCGGTTCCAGCCTTCAGGAG
51
4689
4708
502

588616
1421
1440
Exon 8
TCATGAGGATGATGACATGG
18
4714
4733
503

588618
1446
1465
Exon 9
CCGCCCATGTTGTGCAATCC
40
5020
5039
504

588620
1458
1477
Exon 9
GTAATTGGGTCCCCGCCCAT
40
5032
5051
505

588623
1482
1501
Exon 9
AAGTCCCGGATCTCATCAAT
45
5056
5075
506

588624
1542
1561
Exon 9-10
AACACATAGACATCCAGATA
43
n/a
n/a
507

Junction

588626
1585
1604
Exon 10
CAAAGCATTGATGTTCACTT
45
5234
5253
508

588628
1621
1640
Exon 10
TTTGAACACATGTTGCTCAT
53
5270
5289
509

588631
1646
1665
Exon 10
CTTCCAGGTTTTCCATATCC
56
5295
5314
510

588632
1647
1666
Exon 10
TCTTCCAGGTTTTCCATATC
35
5296
5315
511

588634
1689
1708
Exon 11
AGACTCAGAGACTGGCTTTC
55
5830
5849
512

588636
1749
1768
Exon 11
GCCTGCCATGGTTGCTTGTG
78
5890
5909
513

588638
1763
1782
Exon 11
TGACTGAGATCTTGGCCTGC
95
5904
5923
514

588640
1912
1931
Exon 13
TTCTATCTCCAGGTCCCGCT
44
6406
6425
515

588642
1982
2001
Exon 13
AGTCATAAAATTCAGGAATT
40
6476
6495
516

588645
2073
2092
Exon 14
CGAGTTGTTCCCTCGGTGCA
57
6662
6681
517

588646
2085
2104
Exon 14
AGCCTCAAAGCTCGAGTTGT
48
6674
6693
518

588648
2091
2110
Exon 14
GGAGGAAGCCTCAAAGCTCG
40
6680
6699
519

588651
2097
2116
Exon 14
GTAGTTGGAGGAAGCCTCAA
43
6686
6705
520

588652
2103
2122
Exon 14
CAAGTGGTAGTTGGAGGAAG
13
6692
6711
521

588654
2166
2185
Exon 15
TCCTCAGACACAAACAGAGC
55
6954
6973
522

588656
2172
2191
Exon 15
TTCTCCTCCTCAGACACAAA
44
6960
6979
523

588658
2196
2215
Exon 15
TAGACCTCCTTCCGAGTCAG
50
6984
7003
524

588660
2202
2221
Exon 15
TTGATGTAGACCTCCTTCCG
27
6990
7009
525

588582
2219
2238
Exon 15-
CTTTCTTATCCCCATTCTTG
49
n/a
n/a
526

16

Junction

588583
2221
2240
Exon 15-
GCCTTTCTTATCCCCATTCT
41
n/a
n/a
527

16

Junction

532775
2223
2242
Exon 15-
CTGCCTTTCTTATCCCCATT
41
n/a
n/a
203

16

Junction

588584
2225
2244
Exon 15-
AGCTGCCTTTCTTATCCCCA
43
n/a
n/a
528

16

Junction

588662
2226
2245
Exon 15-
CAGCTGCCTTTCTTATCCCC
52
n/a
n/a
529

16

Junction

588585
2227
2246
Exon 15-
ACAGCTGCCTTTCTTATCCC
39
n/a
n/a
530

16

Junction

588664
2238
2257
Exon 16
GCATCTCTCTCACAGCTGCC
69
7122
7141
531

588666
2276
2295
Exon 16
AGATGTCCTTGACTTTGTCA
46
7160
7179
532

588668
2330
2349
Exon 16
CAGCATAGGGACTCACTCCT
47
7214
7233
533

588670
2361
2380
Exon 16-
CCGCCAGAATCACCTCTGCA
58
n/a
n/a
534

17

Junction

588672
2397
2416
Exon 17
TGAATGAAACGACTTCTCTT
48
7362
7381
535

588674
2430
2449
Exon 18
ACATCCACTACTCCCCAGCT
29
7665
7684
536

588676
2448
2467
Exon 18
CGCTTCTGGTTTTTGCAGAC
58
7683
7702
537

588678
2454
2473
Exon 18
TTTTGCCGCTTCTGGTTTTT
45
7689
7708
538

588680
2466
2485
Exon 18
GCAGGTACCTGCTTTTGCCG
36
7701
7720
539

588682
2532
2551
Exon 18
TCTTGGAGTTTCTCCTTCAG
47
7767
7786
540

532811
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
96
7834
7853
239

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
96
7839
7858
317

TABLE 134

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

598973
2552
2568
Exon 18
GAAAACCCAAATCCTCA
40
7787
7803
3-10-4
619

599036
2552
2568
Exon 18
GAAAACCCAAATCCTCA
18
7787
7803
5-7-5
619

598974
2553
2569
Exon 18
AGAAAACCCAAATCCTC
28
7788
7804
3-10-4
620

599037
2553
2569
Exon 18
AGAAAACCCAAATCCTC
19
7788
7804
5-7-5
620

598975
2554
2570
Exon 18
TAGAAAACCCAAATCCT
15
7789
7805
3-10-4
621

599038
2554
2570
Exon 18
TAGAAAACCCAAATCCT
32
7789
7805
5-7-5
621

598976
2555
2571
Exon 18
ATAGAAAACCCAAATCC
12
7790
7806
3-10-4
622

599039
2555
2571
Exon 18
ATAGAAAACCCAAATCC
7
7790
7806
5-7-5
622

598977
2557
2573
Exon 18
TTATAGAAAACCCAAAT
13
7792
7808
3-10-4
623

599040
2557
2573
Exon 18
TTATAGAAAACCCAAAT
13
7792
7808
5-7-5
623

598978
2558
2574
Exon 18
CTTATAGAAAACCCAAA
0
7793
7809
3-10-4
624

599041
2558
2574
Exon 18
CTTATAGAAAACCCAAA
0
7793
7809
5-7-5
624

598979
2559
2575
Exon 18
CCTTATAGAAAACCCAA
8
7794
7810
3-10-4
625

599042
2559
2575
Exon 18
CCTTATAGAAAACCCAA
19
7794
7810
5-7-5
625

598980
2560
2576
Exon 18
CCCTTATAGAAAACCCA
42
7795
7811
3-10-4
626

599043
2560
2576
Exon 18
CCCTTATAGAAAACCCA
10
7795
7811
5-7-5
626

598981
2561
2577
Exon 18
CCCCTTATAGAAAACCC
20
7796
7812
3-10-4
627

599044
2561
2577
Exon 18
CCCCTTATAGAAAACCC
12
7796
7812
5-7-5
627

598982
2562
2578
Exon 18
ACCCCTTATAGAAAACC
10
7797
7813
3-10-4
628

599045
2562
2578
Exon 18
ACCCCTTATAGAAAACC
3
7797
7813
5-7-5
628

598983
2563
2579
Exon 18
AACCCCTTATAGAAAAC
0
7798
7814
3-10-4
629

599046
2563
2579
Exon 18
AACCCCTTATAGAAAAC
18
7798
7814
5-7-5
629

598984
2564
2580
Exon 18
AAACCCCTTATAGAAAA
0
7799
7815
3-10-4
630

599047
2564
2580
Exon 18
AAACCCCTTATAGAAAA
7
7799
7815
5-7-5
630

598985
2565
2581
Exon 18
GAAACCCCTTATAGAAA
0
7800
7816
3-10-4
631

599048
2565
2581
Exon 18
GAAACCCCTTATAGAAA
9
7800
7816
5-7-5
631

598986
2566
2582
Exon 18
GGAAACCCCTTATAGAA
0
7801
7817
3-10-4
632

599049
2566
2582
Exon 18
GGAAACCCCTTATAGAA
18
7801
7817
5-7-5
632

598988
2567
2583
Exon 18
AGGAAACCCCTTATAGA
0
7802
7818
3-10-4
633

599050
2567
2583
Exon 18
AGGAAACCCCTTATAGA
8
7802
7818
5-7-5
633

598989
2568
2584
Exon 18
CAGGAAACCCCTTATAG
0
7803
7819
3-10-4
634

598990
2569
2585
Exon 18
GCAGGAAACCCCTTATA
8
7804
7820
3-10-4
635

598991
2570
2586
Exon 18
AGCAGGAAACCCCTTAT
25
7805
7821
3-10-4
636

598992
2571
2587
Exon 18
CAGCAGGAAACCCCTTA
12
7806
7822
3-10-4
637

598993
2572
2588
Exon 18
CCAGCAGGAAACCCCTT
37
7807
7823
3-10-4
638

598994
2573
2589
Exon 18
TCCAGCAGGAAACCCCT
29
7808
7824
3-10-4
639

598995
2574
2590
Exon 18
GTCCAGCAGGAAACCCC
42
7809
7825
3-10-4
640

598996
2575
2591
Exon 18
TGTCCAGCAGGAAACCC
36
7810
7826
3-10-4
641

598997
2576
2592
Exon 18
CTGTCCAGCAGGAAACC
18
7811
7827
3-10-4
642

598998
2577
2593
Exon 18
CCTGTCCAGCAGGAAAC
27
7812
7828
3-10-4
643

598999
2578
2594
Exon 18
CCCTGTCCAGCAGGAAA
61
7813
7829
3-10-4
644

599000
2580
2596
Exon 18
GCCCCTGTCCAGCAGGA
71
7815
7831
3-10-4
645

599001
2581
2597
Exon 18
CGCCCCTGTCCAGCAGG
80
7816
7832
3-10-4
646

599002
2582
2598
Exon 18
ACGCCCCTGTCCAGCAG
68
7817
7833
3-10-4
647

599003
2583
2599
Exon 18
CACGCCCCTGTCCAGCA
71
7818
7834
3-10-4
648

599004
2584
2600
Exon 18
CCACGCCCCTGTCCAGC
76
7819
7835
3-10-4
649

599005
2585
2601
Exon 18
CCCACGCCCCTGTCCAG
70
7820
7836
3-10-4
650

599006
2586
2602
Exon 18
TCCCACGCCCCTGTCCA
65
7821
7837
3-10-4
651

599007
2587
2603
Exon 18
ATCCCACGCCCCTGTCC
60
7822
7838
3-10-4
652

599008
2588
2604
Exon 18
AATCCCACGCCCCTGTC
72
7823
7839
3-10-4
653

599009
2589
2605
Exon 18
CAATCCCACGCCCCTGT
79
7824
7840
3-10-4
654

599010
2590
2606
Exon 18
TCAATCCCACGCCCCTG
73
7825
7841
3-10-4
655

599011
2591
2607
Exon 18
TTCAATCCCACGCCCCT
79
7826
7842
3-10-4
656

599012
2592
2608
Exon 18
ATTCAATCCCACGCCCC
67
7827
7843
3-10-4
657

599013
2593
2609
Exon 18
AATTCAATCCCACGCCC
65
7828
7844
3-10-4
658

599014
2594
2610
Exon 18
TAATTCAATCCCACGCC
74
7829
7845
3-10-4
659

599015
2595
2611
Exon 18
TTAATTCAATCCCACGC
71
7830
7846
3-10-4
660

599016
2596
2612
Exon 18
TTTAATTCAATCCCACG
48
7831
7847
3-10-4
661

599017
2597
2613
Exon 18
TTTTAATTCAATCCCAC
34
7832
7848
3-10-4
662

599018
2598
2614
Exon 18
GTTTTAATTCAATCCCA
56
7833
7849
3-10-4
663

599019
2599
2615
Exon 18
TGTTTTAATTCAATCCC
60
7834
7850
3-10-4
664

599020
2600
2616
Exon 18
CTGTTTTAATTCAATCC
0
7835
7851
3-10-4
665

599021
2601
2617
Exon 18
GCTGTTTTAATTCAATC
33
7836
7852
3-10-4
666

599022
2602
2618
Exon 18
AGCTGTTTTAATTCAAT
17
7837
7853
3-10-4
667

599023
2603
2619
Exon 18
CAGCTGTTTTAATTCAA
52
7838
7854
3-10-4
668

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
86
7839
7858
5-10-5
317

599024
2604
2620
Exon 18
GCAGCTGTTTTAATTCA
88
7839
7855
3-10-4
669

599025
2605
2621
Exon 18
CGCAGCTGTTTTAATTC
85
7840
7856
3-10-4
670

599026
2606
2622
Exon 18
TCGCAGCTGTTTTAATT
69
7841
7857
3-10-4
671

599027
2607
2623
Exon 18
GTCGCAGCTGTTTTAAT
77
7842
7858
3-10-4
672

599028
2608
2624
Exon 18
TGTCGCAGCTGTTTTAA
73
7843
7859
3-10-4
673

599029
2609
2625
Exon 18
TTGTCGCAGCTGTTTTA
78
7844
7860
3-10-4
674

599030
2610
2626
Exon 18
GTTGTCGCAGCTGTTTT
75
7845
7861
3-10-4
675

599031
2611
2627
Exon 18
TGTTGTCGCAGCTGTTT
77
7846
7862
3-10-4
676

599032
2612
2628
Exon 18/
TTGTTGTCGCAGCTGTT
79
n/a
n/a
3-10-4
677

Repeat

599033
2613
2629
Exon 18/
TTTGTTGTCGCAGCTGT
80
n/a
n/a
3-10-4
678

Repeat

599034
2614
2630
Exon 18/
TTTTGTTGTCGCAGCTG
78
n/a
n/a
3-10-4
679

Repeat

599035
2615
2631
Exon 18/
TTTTTGTTGTCGCAGCT
63
n/a
n/a
3-10-4
680

Repeat

TABLE 135

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599098
2552
2568
Exon 18
GAAAACCCAAATCCTCA
57
7787
7803
4-8-5
619

599099
2553
2569
Exon 18
AGAAAACCCAAATCCTC
33
7788
7804
4-8-5
620

599100
2554
2570
Exon 18
TAGAAAACCCAAATCCT
32
7789
7805
4-8-5
621

599101
2555
2571
Exon 18
ATAGAAAACCCAAATCC
47
7790
7806
4-8-5
622

599102
2557
2573
Exon 18
TTATAGAAAACCCAAAT
59
7792
7808
4-8-5
623

599103
2558
2574
Exon 18
CTTATAGAAAACCCAAA
10
7793
7809
4-8-5
624

599104
2559
2575
Exon 18
CCTTATAGAAAACCCAA
3
7794
7810
4-8-5
625

599105
2560
2576
Exon 18
CCCTTATAGAAAACCCA
45
7795
7811
4-8-5
626

599106
2561
2577
Exon 18
CCCCTTATAGAAAACCC
49
7796
7812
4-8-5
627

599107
2562
2578
Exon 18
ACCCCTTATAGAAAACC
35
7797
7813
4-8-5
628

599108
2563
2579
Exon 18
AACCCCTTATAGAAAAC
17
7798
7814
4-8-5
629

599109
2564
2580
Exon 18
AAACCCCTTATAGAAAA
36
7799
7815
4-8-5
630

599110
2565
2581
Exon 18
GAAACCCCTTATAGAAA
20
7800
7816
4-8-5
631

599111
2566
2582
Exon 18
GGAAACCCCTTATAGAA
20
7801
7817
4-8-5
632

599112
2567
2583
Exon 18
AGGAAACCCCTTATAGA
15
7802
7818
4-8-5
633

599113
2568
2584
Exon 18
CAGGAAACCCCTTATAG
19
7803
7819
4-8-5
634

599051
2568
2584
Exon 18
CAGGAAACCCCTTATAG
26
7803
7819
5-7-5
634

599114
2569
2585
Exon 18
GCAGGAAACCCCTTATA
18
7804
7820
4-8-5
635

599052
2569
2585
Exon 18
GCAGGAAACCCCTTATA
21
7804
7820
5-7-5
635

599115
2570
2586
Exon 18
AGCAGGAAACCCCTTAT
31
7805
7821
4-8-5
636

599053
2570
2586
Exon 18
AGCAGGAAACCCCTTAT
25
7805
7821
5-7-5
636

599116
2571
2587
Exon 18
CAGCAGGAAACCCCTTA
39
7806
7822
4-8-5
637

599054
2571
2587
Exon 18
CAGCAGGAAACCCCTTA
36
7806
7822
5-7-5
637

599117
2572
2588
Exon 18
CCAGCAGGAAACCCCTT
46
7807
7823
4-8-5
638

599055
2572
2588
Exon 18
CCAGCAGGAAACCCCTT
22
7807
7823
5-7-5
638

599118
2573
2589
Exon 18
TCCAGCAGGAAACCCCT
40
7808
7824
4-8-5
639

599056
2573
2589
Exon 18
TCCAGCAGGAAACCCCT
32
7808
7824
5-7-5
639

599119
2574
2590
Exon 18
GTCCAGCAGGAAACCCC
50
7809
7825
4-8-5
640

599057
2574
2590
Exon 18
GTCCAGCAGGAAACCCC
46
7809
7825
5-7-5
640

599120
2575
2591
Exon 18
TGTCCAGCAGGAAACCC
30
7810
7826
4-8-5
641

599058
2575
2591
Exon 18
TGTCCAGCAGGAAACCC
52
7810
7826
5-7-5
641

599121
2576
2592
Exon 18
CTGTCCAGCAGGAAACC
31
7811
7827
4-8-5
642

599059
2576
2592
Exon 18
CTGTCCAGCAGGAAACC
24
7811
7827
5-7-5
642

599122
2577
2593
Exon 18
CCTGTCCAGCAGGAAAC
23
7812
7828
4-8-5
643

599060
2577
2593
Exon 18
CCTGTCCAGCAGGAAAC
37
7812
7828
5-7-5
643

599123
2578
2594
Exon 18
CCCTGTCCAGCAGGAAA
51
7813
7829
4-8-5
644

599061
2578
2594
Exon 18
CCCTGTCCAGCAGGAAA
34
7813
7829
5-7-5
644

599124
2580
2596
Exon 18
GCCCCTGTCCAGCAGGA
56
7815
7831
4-8-5
645

599062
2580
2596
Exon 18
GCCCCTGTCCAGCAGGA
51
7815
7831
5-7-5
645

599125
2581
2597
Exon 18
CGCCCCTGTCCAGCAGG
70
7816
7832
4-8-5
646

599063
2581
2597
Exon 18
CGCCCCTGTCCAGCAGG
56
7816
7832
5-7-5
646

599126
2582
2598
Exon 18
ACGCCCCTGTCCAGCAG
76
7817
7833
4-8-5
647

599064
2582
2598
Exon 18
ACGCCCCTGTCCAGCAG
61
7817
7833
5-7-5
647

599127
2583
2599
Exon 18
CACGCCCCTGTCCAGCA
67
7818
7834
4-8-5
648

599065
2583
2599
Exon 18
CACGCCCCTGTCCAGCA
64
7818
7834
5-7-5
648

599066
2584
2600
Exon 18
CCACGCCCCTGTCCAGC
40
7819
7835
5-7-5
649

599067
2585
2601
Exon 18
CCCACGCCCCTGTCCAG
37
7820
7836
5-7-5
650

599068
2586
2602
Exon 18
TCCCACGCCCCTGTCCA
31
7821
7837
5-7-5
651

599069
2587
2603
Exon 18
ATCCCACGCCCCTGTCC
39
7822
7838
5-7-5
652

599070
2588
2604
Exon 18
AATCCCACGCCCCTGTC
59
7823
7839
5-7-5
653

599071
2589
2605
Exon 18
CAATCCCACGCCCCTGT
63
7824
7840
5-7-5
657

599072
2590
2606
Exon 18
TCAATCCCACGCCCCTG
74
7825
7841
5-7-5
655

599073
2591
2607
Exon 18
TTCAATCCCACGCCCCT
53
7826
7842
5-7-5
656

599074
2592
2608
Exon 18
ATTCAATCCCACGCCCC
56
7827
7843
5-7-5
657

599075
2593
2609
Exon 18
AATTCAATCCCACGCCC
49
7828
7844
5-7-5
658

599076
2594
2610
Exon 18
TAATTCAATCCCACGCC
54
7829
7845
5-7-5
659

599077
2595
2611
Exon 18
TTAATTCAATCCCACGC
79
7830
7846
5-7-5
660

599078
2596
2612
Exon 18
TTTAATTCAATCCCACG
67
7831
7847
5-7-5
661

599079
2597
2613
Exon 18
TTTTAATTCAATCCCAC
69
7832
7848
5-7-5
662

599080
2598
2614
Exon 18
GTTTTAATTCAATCCCA
79
7833
7849
5-7-5
663

599081
2599
2615
Exon 18
TGTTTTAATTCAATCCC
57
7834
7850
5-7-5
664

599082
2600
2616
Exon 18
CTGTTTTAATTCAATCC
50
7835
7851
5-7-5
665

599083
2601
2617
Exon 18
GCTGTTTTAATTCAATC
67
7836
7852
5-7-5
666

599084
2602
2618
Exon 18
AGCTGTTTTAATTCAAT
60
7837
7853
5-7-5
667

599085
2603
2619
Exon 18
CAGCTGTTTTAATTCAA
71
7838
7854
5-7-5
668

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
82
7839
7858
5-10-5
317

599086
2604
2620
Exon 18
GCAGCTGTTTTAATTCA
81
7839
7855
5-7-5
669

599087
2605
2621
Exon 18
CGCAGCTGTTTTAATTC
88
7840
7856
5-7-5
670

599088
2606
2622
Exon 18
TCGCAGCTGTTTTAATT
84
7841
7857
5-7-5
671

599089
2607
2623
Exon 18
GTCGCAGCTGTTTTAAT
81
7842
7858
5-7-5
672

599090
2608
2624
Exon 18
TGTCGCAGCTGTTTTAA
77
7843
7859
5-7-5
673

599091
2609
2625
Exon 18
TTGTCGCAGCTGTTTTA
74
7844
7860
5-7-5
674

599092
2610
2626
Exon 18
GTTGTCGCAGCTGTTTT
66
7845
7861
5-7-5
675

599093
2611
2627
Exon 18
TGTTGTCGCAGCTGTTT
89
7846
7862
5-7-5
676

599094
2612
2628
Exon 18/
TTGTTGTCGCAGCTGTT
82
n/a
n/a
5-7-5
677

Repeat

599095
2613
2629
Exon 18/
TTTGTTGTCGCAGCTGT
87
n/a
n/a
5-7-5
678

Repeat

599096
2614
2630
Exon 18/
TTTTGTTGTCGCAGCTG
85
n/a
n/a
5-7-5
679

Repeat

599097
2615
2631
Exon 18/
TTTTTGTTGTCGCAGCT
78
n/a
n/a
5-7-5
680

Repeat

TABLE 136

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599510
2552
2570
Exon 18
TAGAAAACCCAAATCCTCA
45
7787
7805
5-9-5
681

599331
2553
2571
Exon 18
ATAGAAAACCCAAATCCTC
46
7788
7806
5-9-5
682

599332
2554
2572
Exon 18
TATAGAAAACCCAAATCCT
38
7789
7807
5-9-5
683

599333
2556
2574
Exon 18
CTTATAGAAAACCCAAATC
1
7791
7809
5-9-5
684

599334
2557
2575
Exon 18
CCTTATAGAAAACCCAAAT
5
7792
7810
5-9-5
685

599335
2558
2576
Exon 18
CCCTTATAGAAAACCCAAA
34
7793
7811
5-9-5
686

599336
2559
2577
Exon 18
CCCCTTATAGAAAACCCAA
40
7794
7812
5-9-5
687

599337
2560
2578
Exon 18
ACCCCTTATAGAAAACCCA
39
7795
7813
5-9-5
688

599338
2561
2579
Exon 18
AACCCCTTATAGAAAACCC
57
7796
7814
5-9-5
689

599339
2562
2580
Exon 18
AAACCCCTTATAGAAAACC
26
7797
7815
5-9-5
690

599281
2562
2580
Exon 18
AAACCCCTTATAGAAAACC
15
7797
7815
6-7-6
690

599340
2563
2581
Exon 18
GAAACCCCTTATAGAAAAC
17
7798
7816
5-9-5
691

599282
2563
2581
Exon 18
GAAACCCCTTATAGAAAAC
12
7798
7816
6-7-6
691

599341
2564
2582
Exon 18
GGAAACCCCTTATAGAAAA
23
7799
7817
5-9-5
692

599283
2564
2582
Exon 18
GGAAACCCCTTATAGAAAA
18
7799
7817
6-7-6
692

599342
2565
2583
Exon 18
AGGAAACCCCTTATAGAAA
10
7800
7818
5-9-5
693

599284
2565
2583
Exon 18
AGGAAACCCCTTATAGAAA
14
7800
7818
6-7-6
693

599343
2566
2584
Exon 18
CAGGAAACCCCTTATAGAA
10
7801
7819
5-9-5
694

599285
2566
2584
Exon 18
CAGGAAACCCCTTATAGAA
13
7801
7819
6-7-6
694

599344
2567
2585
Exon 18
GCAGGAAACCCCTTATAGA
22
7802
7820
5-9-5
695

599286
2567
2585
Exon 18
GCAGGAAACCCCTTATAGA
31
7802
7820
6-7-6
695

599345
2568
2586
Exon 18
AGCAGGAAACCCCTTATAG
19
7803
7821
5-9-5
696

599287
2568
2586
Exon 18
AGCAGGAAACCCCTTATAG
12
7803
7821
6-7-6
696

599346
2569
2587
Exon 18
CAGCAGGAAACCCCTTATA
30
7804
7822
5-9-5
697

599288
2569
2587
Exon 18
CAGCAGGAAACCCCTTATA
28
7804
7822
6-7-6
697

599347
2570
2588
Exon 18
CCAGCAGGAAACCCCTTAT
46
7805
7823
5-9-5
698

599289
2570
2588
Exon 18
CCAGCAGGAAACCCCTTAT
32
7805
7823
6-7-6
698

599348
2571
2589
Exon 18
TCCAGCAGGAAACCCCTTA
44
7806
7824
5-9-5
699

599290
2571
2589
Exon 18
TCCAGCAGGAAACCCCTTA
24
7806
7824
6-7-6
699

599349
2572
2590
Exon 18
GTCCAGCAGGAAACCCCTT
60
7807
7825
5-9-5
700

599291
2572
2590
Exon 18
GTCCAGCAGGAAACCCCTT
38
7807
7825
6-7-6
700

599350
2573
2591
Exon 18
TGTCCAGCAGGAAACCCCT
49
7808
7826
5-9-5
701

599292
2573
2591
Exon 18
TGTCCAGCAGGAAACCCCT
35
7808
7826
6-7-6
701

599351
2575
2593
Exon 18
CCTGTCCAGCAGGAAACCC
46
7810
7828
5-9-5
702

599293
2575
2593
Exon 18
CCTGTCCAGCAGGAAACCC
12
7810
7828
6-7-6
702

599352
2576
2594
Exon 18
CCCTGTCCAGCAGGAAACC
49
7811
7829
5-9-5
703

599294
2576
2594
Exon 18
CCCTGTCCAGCAGGAAACC
38
7811
7829
6-7-6
703

599353
2577
2595
Exon 18
CCCCTGTCCAGCAGGAAAC
64
7812
7830
5-9-5
704

599295
2577
2595
Exon 18
CCCCTGTCCAGCAGGAAAC
33
7812
7830
6-7-6
704

599354
2578
2596
Exon 18
GCCCCTGTCCAGCAGGAAA
56
7813
7831
5-9-5
705

599296
2578
2596
Exon 18
GCCCCTGTCCAGCAGGAAA
13
7813
7831
6-7-6
705

599355
2580
2598
Exon 18
ACGCCCCTGTCCAGCAGGA
81
7815
7833
5-9-5
706

599297
2580
2598
Exon 18
ACGCCCCTGTCCAGCAGGA
57
7815
7833
6-7-6
706

599356
2581
2599
Exon 18
CACGCCCCTGTCCAGCAGG
64
7816
7834
5-9-5
707

599298
2581
2599
Exon 18
CACGCCCCTGTCCAGCAGG
39
7816
7834
6-7-6
707

599299
2582
2600
Exon 18
CCACGCCCCTGTCCAGCAG
55
7817
7835
6-7-6
708

599300
2583
2601
Exon 18
CCCACGCCCCTGTCCAGCA
45
7818
7836
6-7-6
709

599301
2584
2602
Exon 18
TCCCACGCCCCTGTCCAGC
39
7819
7837
6-7-6
710

599302
2585
2603
Exon 18
ATCCCACGCCCCTGTCCAG
27
7820
7838
6-7-6
711

599303
2586
2604
Exon 18
AATCCCACGCCCCTGTCCA
35
7821
7839
6-7-6
712

599304
2587
2605
Exon 18
CAATCCCACGCCCCTGTCC
16
7822
7840
6-7-6
713

599305
2588
2606
Exon 18
TCAATCCCACGCCCCTGTC
41
7823
7841
6-7-6
714

599306
2589
2607
Exon 18
TTCAATCCCACGCCCCTGT
70
7824
7842
6-7-6
715

599307
2590
2608
Exon 18
ATTCAATCCCACGCCCCTG
66
7825
7843
6-7-6
716

599308
2591
2609
Exon 18
AATTCAATCCCACGCCCCT
68
7826
7844
6-7-6
717

599309
2592
2610
Exon 18
TAATTCAATCCCACGCCCC
52
7827
7845
6-7-6
718

599310
2593
2611
Exon 18
TTAATTCAATCCCACGCCC
39
7828
7846
6-7-6
719

599311
2594
2612
Exon 18
TTTAATTCAATCCCACGCC
83
7829
7847
6-7-6
720

599312
2595
2613
Exon 18
TTTTAATTCAATCCCACGC
72
7830
7848
6-7-6
721

599313
2596
2614
Exon 18
GTTTTAATTCAATCCCACG
86
7831
7849
6-7-6
722

599314
2597
2615
Exon 18
TGTTTTAATTCAATCCCAC
91
7832
7850
6-7-6
723

599315
2598
2616
Exon 18
CTGTTTTAATTCAATCCCA
71
7833
7851
6-7-6
724

599316
2599
2617
Exon 18
GCTGTTTTAATTCAATCCC
89
7834
7852
6-7-6
725

599317
2600
2618
Exon 18
AGCTGTTTTAATTCAATCC
87
7835
7853
6-7-6
726

599318
2601
2619
Exon 18
CAGCTGTTTTAATTCAATC
81
7836
7854
6-7-6
727

599319
2602
2620
Exon 18
GCAGCTGTTTTAATTCAAT
75
7837
7855
6-7-6
728

599320
2603
2621
Exon 18
CGCAGCTGTTTTAATTCAA
84
7838
7856
6-7-6
729

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
92
7839
7858
5-10-5
317

599321
2604
2622
Exon 18
TCGCAGCTGTTTTAATTCA
90
7839
7857
6-7-6
730

599322
2605
2623
Exon 18
GTCGCAGCTGTTTTAATTC
89
7840
7858
6-7-6
731

599323
2606
2624
Exon 18
TGTCGCAGCTGTTTTAATT
81
7841
7859
6-7-6
732

599324
2607
2625
Exon 18
TTGTCGCAGCTGTTTTAAT
68
7842
7860
6-7-6
733

599325
2608
2626
Exon 18
GTTGTCGCAGCTGTTTTAA
71
7843
7861
6-7-6
734

599326
2609
2627
Exon 18
TGTTGTCGCAGCTGTTTTA
52
7844
7862
6-7-6
735

599327
2610
2628
Exon 18/
TTGTTGTCGCAGCTGTTTT
88
n/a
n/a
6-7-6
736

Repeat

599328
2611
2629
Exon 18/
TTTGTTGTCGCAGCTGTTT
87
n/a
n/a
6-7-6
737

Repeat

599329
2612
2630
Exon 18/
TTTTGTTGTCGCAGCTGTT
84
n/a
n/a
6-7-6
738

Repeat

599330
2613
2631
Exon 18/
TTTTTGTTGTCGCAGCTGT
87
n/a
n/a
6-7-6
739

Repeat

TABLE 137

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599512
2552
2571
Exon 18
ATAGAAAACCCAAATCCTCA
74
7787
7806
3-10-7
410

599449
2553
2572
Exon 18
TATAGAAAACCCAAATCCTC
43
7788
7807
3-10-7
411

599450
2554
2573
Exon 18
TTATAGAAAACCCAAATCCT
51
7789
7808
3-10-7
412

599451
2555
2574
Exon 18
CTTATAGAAAACCCAAATCC
35
7790
7809
3-10-7
413

599452
2556
2575
Exon 18
CCTTATAGAAAACCCAAATC
34
7791
7810
3-10-7
414

599453
2557
2576
Exon 18
CCCTTATAGAAAACCCAAAT
44
7792
7811
3-10-7
415

599454
2558
2577
Exon 18
CCCCTTATAGAAAACCCAAA
54
7793
7812
3-10-7
416

599455
2559
2578
Exon 18
ACCCCTTATAGAAAACCCAA
53
7794
7813
3-10-7
417

599456
2560
2579
Exon 18
AACCCCTTATAGAAAACCCA
69
7795
7814
3-10-7
418

599457
2561
2580
Exon 18
AAACCCCTTATAGAAAACCC
46
7796
7815
3-10-7
419

599458
2562
2581
Exon 18
GAAACCCCTTATAGAAAACC
0
7797
7816
3-10-7
420

599459
2563
2582
Exon 18
GGAAACCCCTTATAGAAAAC
12
7798
7817
3-10-7
421

599460
2564
2583
Exon 18
AGGAAACCCCTTATAGAAAA
17
7799
7818
3-10-7
422

599461
2565
2584
Exon 18
CAGGAAACCCCTTATAGAAA
24
7800
7819
3-10-7
423

599462
2566
2585
Exon 18
GCAGGAAACCCCTTATAGAA
33
7801
7820
3-10-7
424

599463
2567
2586
Exon 18
AGCAGGAAACCCCTTATAGA
38
7802
7821
3-10-7
425

599464
2568
2587
Exon 18
CAGCAGGAAACCCCTTATAG
33
7803
7822
3-10-7
426

599465
2569
2588
Exon 18
CCAGCAGGAAACCCCTTATA
49
7804
7823
3-10-7
427

599466
2570
2589
Exon 18
TCCAGCAGGAAACCCCTTAT
45
7805
7824
3-10-7
428

599467
2571
2590
Exon 18
GTCCAGCAGGAAACCCCTTA
60
7806
7825
3-10-7
237

599468
2572
2591
Exon 18
TGTCCAGCAGGAAACCCCTT
61
7807
7826
3-10-7
429

599469
2573
2592
Exon 18
CTGTCCAGCAGGAAACCCCT
52
7808
7827
3-10-7
430

599470
2574
2593
Exon 18
CCTGTCCAGCAGGAAACCCC
45
7809
7828
3-10-7
431

599471
2575
2594
Exon 18
CCCTGTCCAGCAGGAAACCC
67
7810
7829
3-10-7
432

599472
2576
2595
Exon 18
CCCCTGTCCAGCAGGAAACC
79
7811
7830
3-10-7
433

599473
2577
2596
Exon 18
GCCCCTGTCCAGCAGGAAAC
72
7812
7831
3-10-7
238

599474
2578
2597
Exon 18
CGCCCCTGTCCAGCAGGAAA
87
7813
7832
3-10-7
434

599475
2579
2598
Exon 18
ACGCCCCTGTCCAGCAGGAA
76
7814
7833
3-10-7
435

599476
2580
2599
Exon 18
CACGCCCCTGTCCAGCAGGA
81
7815
7834
3-10-7
436

599477
2581
2600
Exon 18
CCACGCCCCTGTCCAGCAGG
83
7816
7835
3-10-7
437

599478
2582
2601
Exon 18
CCCACGCCCCTGTCCAGCAG
72
7817
7836
3-10-7
438

599479
2583
2602
Exon 18
TCCCACGCCCCTGTCCAGCA
81
7818
7837
3-10-7
439

599480
2584
2603
Exon 18
ATCCCACGCCCCTGTCCAGC
77
7819
7838
3-10-7
440

599481
2585
2604
Exon 18
AATCCCACGCCCCTGTCCAG
83
7820
7839
3-10-7
441

599482
2586
2605
Exon 18
CAATCCCACGCCCCTGTCCA
87
7821
7840
3-10-7
442

599483
2587
2606
Exon 18
TCAATCCCACGCCCCTGTCC
90
7822
7841
3-10-7
443

599484
2588
2607
Exon 18
TTCAATCCCACGCCCCTGTC
72
7823
7842
3-10-7
444

599485
2589
2608
Exon 18
ATTCAATCCCACGCCCCTGT
82
7824
7843
3-10-7
445

599486
2590
2609
Exon 18
AATTCAATCCCACGCCCCTG
84
7825
7844
3-10-7
446

599487
2591
2610
Exon 18
TAATTCAATCCCACGCCCCT
84
7826
7845
3-10-7
447

599488
2592
2611
Exon 18
TTAATTCAATCCCACGCCCC
87
7827
7846
3-10-7
448

599489
2593
2612
Exon 18
TTTAATTCAATCCCACGCCC
87
7828
7847
3-10-7
449

599490
2594
2613
Exon 18
TTTTAATTCAATCCCACGCC
86
7829
7848
3-10-7
450

599491
2595
2614
Exon 18
GTTTTAATTCAATCCCACGC
87
7830
7849
3-10-7
451

599492
2596
2615
Exon 18
TGTTTTAATTCAATCCCACG
88
7831
7850
3-10-7
452

599493
2597
2616
Exon 18
CTGTTTTAATTCAATCCCAC
75
7832
7851
3-10-7
453

599433
2597
2616
Exon 18
CTGTTTTAATTCAATCCCAC
89
7832
7851
6-8-6
453

599494
2598
2617
Exon 18
GCTGTTTTAATTCAATCCCA
90
7833
7852
3-10-7
454

599434
2598
2617
Exon 18
GCTGTTTTAATTCAATCCCA
89
7833
7852
6-8-6
454

599495
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
88
7834
7853
3-10-7
239

599435
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
91
7834
7853
6-8-6
239

599496
2600
2619
Exon 18
CAGCTGTTTTAATTCAATCC
89
7835
7854
3-10-7
455

599436
2600
2619
Exon 18
CAGCTGTTTTAATTCAATCC
89
7835
7854
6-8-6
455

599497
2601
2620
Exon 18
GCAGCTGTTTTAATTCAATC
89
7836
7855
3-10-7
456

599437
2601
2620
Exon 18
GCAGCTGTTTTAATTCAATC
91
7836
7855
6-8-6
456

599498
2602
2621
Exon 18
CGCAGCTGTTTTAATTCAAT
88
7837
7856
3-10-7
457

599438
2602
2621
Exon 18
CGCAGCTGTTTTAATTCAAT
90
7837
7856
6-8-6
457

599499
2603
2622
Exon 18
TCGCAGCTGTTTTAATTCAA
81
7838
7857
3-10-7
458

599439
2603
2622
Exon 18
TCGCAGCTGTTTTAATTCAA
88
7838
7857
6-8-6
458

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
90
7839
7858
5-10-5
317

599500
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
88
7839
7858
3-10-7
317

599440
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
88
7839
7858
6-8-6
317

599501
2605
2624
Exon 18
TGTCGCAGCTGTTTTAATTC
78
7840
7859
3-10-7
459

599441
2605
2624
Exon 18
TGTCGCAGCTGTTTTAATTC
90
7840
7859
6-8-6
459

599502
2606
2625
Exon 18
TTGTCGCAGCTGTTTTAATT
87
7841
7860
3-10-7
460

599442
2606
2625
Exon 18
TTGTCGCAGCTGTTTTAATT
76
7841
7860
6-8-6
460

599503
2607
2626
Exon 18
GTTGTCGCAGCTGTTTTAAT
83
7842
7861
3-10-7
461

599443
2607
2626
Exon 18
GTTGTCGCAGCTGTTTTAAT
77
7842
7861
6-8-6
461

599504
2608
2627
Exon 18
TGTTGTCGCAGCTGTTTTAA
89
7843
7862
3-10-7
395

599444
2608
2627
Exon 18
TGTTGTCGCAGCTGTTTTAA
69
7843
7862
6-8-6
395

599505
2609
2628
Exon 19/
TTGTTGTCGCAGCTGTTTTA
83
n/a
n/a
3-10-7
462

Repeat

599445
2609
2628
Exon 19/
TTGTTGTCGCAGCTGTTTTA
85
n/a
n/a
6-8-6
462

Repeat

599506
2610
2629
Exon 19/
TTTGTTGTCGCAGCTGTTTT
89
n/a
n/a
3-10-7
463

Repeat

599446
2610
2629
Exon 19/
TTTGTTGTCGCAGCTGTTTT
85
n/a
n/a
6-8-6
463

Repeat

599507
2611
2630
Exon 19/
TTTTGTTGTCGCAGCTGTTT
82
n/a
n/a
3-10-7
464

Repeat

599447
2611
2630
Exon 19/
TTTTGTTGTCGCAGCTGTTT
83
n/a
n/a
6-8-6
464

Repeat

599508
2612
2631
Exon 19/
TTTTTGTTGTCGCAGCTGTT
90
n/a
n/a
3-10-7
465

Repeat

599448
2612
2631
Exon 19/
TTTTTGTTGTCGCAGCTGTT
87
n/a
n/a
6-8-6
465

Repeat

Example 119: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 2,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 4-8-5 MOE, 5-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE, 6-7-6-MOE, 3-10-5 MOE, or 6-8-6 MOE gapmers.

The 4-8-5 MOE gapmers are 17 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four and five nucleosides respectively. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises often 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and five nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

TABLE 138

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599160
2560
2577
Exon 18
CCCCTTATAGAAAACCCA
26
7795
7812
5-8-5
740

599161
2561
2578
Exon 18
ACCCCTTATAGAAAACCC
20
7796
7813
5-8-5
741

599162
2562
2579
Exon 18
AACCCCTTATAGAAAACC
12
7797
7814
5-8-5
742

599163
2563
2580
Exon 18
AAACCCCTTATAGAAAAC
11
7798
7815
5-8-5
743

599164
2564
2581
Exon 18
GAAACCCCTTATAGAAAA
11
7799
7816
5-8-5
744

599165
2566
2583
Exon 18
AGGAAACCCCTTATAGAA
0
7801
7818
5-8-5
745

599166
2567
2584
Exon 18
CAGGAAACCCCTTATAGA
12
7802
7819
5-8-5
746

599167
2568
2585
Exon 18
GCAGGAAACCCCTTATAG
14
7803
7820
5-8-5
747

599168
2569
2586
Exon 18
AGCAGGAAACCCCTTATA
16
7804
7821
5-8-5
748

599169
2570
2587
Exon 18
CAGCAGGAAACCCCTTAT
24
7805
7822
5-8-5
749

599170
2571
2588
Exon 18
CCAGCAGGAAACCCCTTA
37
7806
7823
5-8-5
750

599171
2572
2589
Exon 18
TCCAGCAGGAAACCCCTT
30
7807
7824
5-8-5
751

599172
2573
2590
Exon 18
GTCCAGCAGGAAACCCCT
43
7808
7825
5-8-5
752

599173
2574
2591
Exon 18
TGTCCAGCAGGAAACCCC
47
7809
7826
5-8-5
753

599174
2575
2592
Exon 18
CTGTCCAGCAGGAAACCC
27
7810
7827
5-8-5
754

599175
2576
2593
Exon 18
CCTGTCCAGCAGGAAACC
30
7811
7828
5-8-5
755

599176
2577
2594
Exon 18
CCCTGTCCAGCAGGAAAC
34
7812
7829
5-8-5
756

599177
2578
2595
Exon 18
CCCCTGTCCAGCAGGAAA
41
7813
7830
5-8-5
757

599178
2580
2597
Exon 18
CGCCCCTGTCCAGCAGGA
67
7815
7832
5-8-5
758

599179
2581
2598
Exon 18
ACGCCCCTGTCCAGCAGG
61
7816
7833
5-8-5
759

599180
2582
2599
Exon 18
CACGCCCCTGTCCAGCAG
62
7817
7834
5-8-5
760

599181
2583
2600
Exon 18
CCACGCCCCTGTCCAGCA
63
7818
7835
5-8-5
761

599128
2584
2600
Exon 18
CCACGCCCCTGTCCAGC
55
7819
7835
4-8-5
649

599182
2584
2601
Exon 18
CCCACGCCCCTGTCCAGC
58
7819
7836
5-8-5
762

599129
2585
2601
Exon 18
CCCACGCCCCTGTCCAG
41
7820
7836
4-8-5
650

599183
2585
2602
Exon 18
TCCCACGCCCCTGTCCAG
43
7820
7837
5-8-5
763

599130
2586
2602
Exon 18
TCCCACGCCCCTGTCCA
46
7821
7837
4-8-5
651

599184
2586
2603
Exon 18
ATCCCACGCCCCTGTCCA
32
7821
7838
5-8-5
764

599131
2587
2603
Exon 18
ATCCCACGCCCCTGTCC
30
7822
7838
4-8-5
652

599185
2587
2604
Exon 18
AATCCCACGCCCCTGTCC
35
7822
7839
5-8-5
765

599132
2588
2604
Exon 18
AATCCCACGCCCCTGTC
52
7823
7839
4-8-5
653

599186
2588
2605
Exon 18
CAATCCCACGCCCCTGTC
55
7823
7840
5-8-5
766

599133
2589
2605
Exon 18
CAATCCCACGCCCCTGT
66
7824
7840
4-8-5
654

599187
2589
2606
Exon 18
TCAATCCCACGCCCCTGT
72
7824
7841
5-8-5
767

599134
2590
2606
Exon 18
TCAATCCCACGCCCCTG
80
7825
7841
4-8-5
655

599188
2590
2607
Exon 18
TTCAATCCCACGCCCCTG
92
7825
7842
5-8-5
768

599135
2591
2607
Exon 18
TTCAATCCCACGCCCCT
61
7826
7842
4-8-5
656

599189
2591
2608
Exon 18
ATTCAATCCCACGCCCCT
52
7826
7843
5-8-5
769

599136
2592
2608
Exon 18
ATTCAATCCCACGCCCC
68
7827
7843
4-8-5
657

599190
2592
2609
Exon 18
AATTCAATCCCACGCCCC
62
7827
7844
5-8-5
770

599137
2593
2609
Exon 18
AATTCAATCCCACGCCC
51
7828
7844
4-8-5
658

599191
2593
2610
Exon 18
TAATTCAATCCCACGCCC
54
7828
7845
5-8-5
771

599138
2594
2610
Exon 18
TAATTCAATCCCACGCC
71
7829
7845
4-8-5
659

599192
2594
2611
Exon 18
TTAATTCAATCCCACGCC
66
7829
7846
5-8-5
772

599139
2595
2611
Exon 18
TTAATTCAATCCCACGC
80
7830
7846
4-8-5
660

599193
2595
2612
Exon 18
TTTAATTCAATCCCACGC
74
7830
7847
5-8-5
773

599140
2596
2612
Exon 18
TTTAATTCAATCCCACG
66
7831
7847
4-8-5
786

599194
2596
2613
Exon 18
TTTTAATTCAATCCCACG
66
7831
7848
5-8-5
774

599141
2597
2613
Exon 18
TTTTAATTCAATCCCAC
63
7832
7848
4-8-5
662

599195
2597
2614
Exon 18
GTTTTAATTCAATCCCAC
86
7832
7849
5-8-5
775

599142
2598
2614
Exon 18
GTTTTAATTCAATCCCA
69
7833
7849
4-8-5
663

599196
2598
2615
Exon 18
TGTTTTAATTCAATCCCA
82
7833
7850
5-8-5
776

599143
2599
2615
Exon 18
TGTTTTAATTCAATCCC
59
7834
7850
4-8-5
664

599197
2599
2616
Exon 18
CTGTTTTAATTCAATCCC
79
7834
7851
5-8-5
777

599144
2600
2616
Exon 18
CTGTTTTAATTCAATCC
52
7835
7851
4-8-5
665

599198
2600
2617
Exon 18
GCTGTTTTAATTCAATCC
86
7835
7852
5-8-5
778

599145
2601
2617
Exon 18
GCTGTTTTAATTCAATC
53
7836
7852
4-8-5
666

599199
2601
2618
Exon 18
AGCTGTTTTAATTCAATC
72
7836
7853
5-8-5
779

599146
2602
2618
Exon 18
AGCTGTTTTAATTCAAT
42
7837
7853
4-8-5
667

599200
2602
2619
Exon 18
CAGCTGTTTTAATTCAAT
76
7837
7854
5-8-5
780

599147
2603
2619
Exon 18
CAGCTGTTTTAATTCAA
55
7838
7854
4-8-5
668

599201
2603
2620
Exon 18
GCAGCTGTTTTAATTCAA
87
7838
7855
5-8-5
781

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
93
7839
7858
5-10-5
317

599148
2604
2620
Exon 18
GCAGCTGTTTTAATTCA
84
7839
7855
4-8-5
669

599202
2604
2621
Exon 18
CGCAGCTGTTTTAATTCA
89
7839
7856
5-8-5
782

599149
2605
2621
Exon 18
CGCAGCTGTTTTAATTC
92
7840
7856
4-8-5
670

599203
2605
2622
Exon 18
TCGCAGCTGTTTTAATTC
90
7840
7857
5-8-5
783

599150
2606
2622
Exon 18
TCGCAGCTGTTTTAATT
75
7841
7857
4-8-5
671

599151
2607
2623
Exon 18
GTCGCAGCTGTTTTAAT
80
7842
7858
4-8-5
672

599152
2608
2624
Exon 18
TGTCGCAGCTGTTTTAA
76
7843
7859
4-8-5
673

599153
2609
2625
Exon 18
TTGTCGCAGCTGTTTTA
56
7844
7860
4-8-5
674

599154
2610
2626
Exon 18
GTTGTCGCAGCTGTTTT
85
7845
7861
4-8-5
675

599155
2611
2627
Exon 18
TGTTGTCGCAGCTGTTT
89
7846
7862
4-8-5
676

599156
2612
2628
Exon 18/
TTGTTGTCGCAGCTGTT
83
n/a
n/a
4-8-5
813

Repeat

599157
2613
2629
Exon 18/
TTTGTTGTCGCAGCTGT
78
n/a
n/a
4-8-5
678

Repeat

599158
2614
2630
Exon 18/
TTTTGTTGTCGCAGCTG
83
n/a
n/a
4-8-5
679

Repeat

599159
2615
2631
Exon 18/
TTTTTGTTGTCGCAGCT
65
n/a
n/a
4-8-5
680

Repeat

599204
2606
2623
Exon 18
GTCGCAGCTGTTTTAATT
83
7841
7858
5-8-5
784

TABLE 139

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599509
2552
2570
Exon 18
TAGAAAACCCAAATCCTCA
45
7787
7805
6-7-6
681

599213
2553
2570
Exon 18
TAGAAAACCCAAATCCTC
89
7788
7805
3-10-5
785

599273
2553
2571
Exon 18
ATAGAAAACCCAAATCCTC
85
7788
7806
6-7-6
682

599214
2554
2571
Exon 18
ATAGAAAACCCAAATCCT
79
7789
7806
3-10-5
786

599274
2554
2572
Exon 18
TATAGAAAACCCAAATCCT
75
7789
7807
6-7-6
683

599215
2555
2572
Exon 18
TATAGAAAACCCAAATCC
81
7790
7807
3-10-5
787

599216
2556
2573
Exon 18
TTATAGAAAACCCAAATC
87
7791
7808
3-10-5
788

599275
2556
2574
Exon 18
CTTATAGAAAACCCAAATC
84
7791
7809
6-7-6
684

599217
2557
2574
Exon 18
CTTATAGAAAACCCAAAT
84
7792
7809
3-10-5
789

599276
2557
2575
Exon 18
CCTTATAGAAAACCCAAAT
68
7792
7810
6-7-6
685

599218
2558
2575
Exon 18
CCTTATAGAAAACCCAAA
82
7793
7810
3-10-5
790

599277
2558
2576
Exon 18
CCCTTATAGAAAACCCAAA
82
7793
7811
6-7-6
686

599219
2559
2576
Exon 18
CCCTTATAGAAAACCCAA
81
7794
7811
3-10-5
791

599278
2559
2577
Exon 18
CCCCTTATAGAAAACCCAA
84
7794
7812
6-7-6
687

599220
2560
2577
Exon 18
CCCCTTATAGAAAACCCA
92
7795
7812
3-10-5
740

599279
2560
2578
Exon 18
ACCCCTTATAGAAAACCCA
92
7795
7813
6-7-6
688

599221
2561
2578
Exon 18
ACCCCTTATAGAAAACCC
93
7796
7813
3-10-5
741

599280
2561
2579
Exon 18
AACCCCTTATAGAAAACCC
90
7796
7814
6-7-6
689

599222
2562
2579
Exon 18
AACCCCTTATAGAAAACC
95
7797
7814
3-10-5
742

599223
2563
2580
Exon 18
AAACCCCTTATAGAAAAC
93
7798
7815
3-10-5
743

599224
2564
2581
Exon 18
GAAACCCCTTATAGAAAA
90
7799
7816
3-10-5
744

599225
2566
2583
Exon 18
AGGAAACCCCTTATAGAA
93
7801
7818
3-10-5
745

599226
2567
2584
Exon 18
CAGGAAACCCCTTATAGA
95
7802
7819
3-10-5
746

599227
2568
2585
Exon 18
GCAGGAAACCCCTTATAG
94
7803
7820
3-10-5
747

599228
2569
2586
Exon 18
AGCAGGAAACCCCTTATA
96
7804
7821
3-10-5
748

599229
2570
2587
Exon 18
CAGCAGGAAACCCCTTAT
92
7805
7822
3-10-5
749

599230
2571
2588
Exon 18
CCAGCAGGAAACCCCTTA
88
7806
7823
3-10-5
750

599231
2572
2589
Exon 18
TCCAGCAGGAAACCCCTT
83
7807
7824
3-10-5
751

599232
2573
2590
Exon 18
GTCCAGCAGGAAACCCCT
89
7808
7825
3-10-5
752

599233
2574
2591
Exon 18
TGTCCAGCAGGAAACCCC
83
7809
7826
3-10-5
753

599234
2575
2592
Exon 18
CTGTCCAGCAGGAAACCC
88
7810
7827
3-10-5
754

599235
2576
2593
Exon 18
CCTGTCCAGCAGGAAACC
91
7811
7828
3-10-5
755

599236
2577
2594
Exon 18
CCCTGTCCAGCAGGAAAC
90
7812
7829
3-10-5
756

599237
2578
2595
Exon 18
CCCCTGTCCAGCAGGAAA
34
7813
7830
3-10-5
757

599238
2580
2597
Exon 18
CGCCCCTGTCCAGCAGGA
14
7815
7832
3-10-5
758

599239
2581
2598
Exon 18
ACGCCCCTGTCCAGCAGG
10
7816
7833
3-10-5
759

599240
2582
2599
Exon 18
CACGCCCCTGTCCAGCAG
26
7817
7834
3-10-5
760

599241
2583
2600
Exon 18
CCACGCCCCTGTCCAGCA
11
7818
7835
3-10-5
761

599242
2584
2601
Exon 18
CCCACGCCCCTGTCCAGC
24
7819
7836
3-10-5
762

599243
2585
2602
Exon 18
TCCCACGCCCCTGTCCAG
23
7820
7837
3-10-5
763

599244
2586
2603
Exon 18
ATCCCACGCCCCTGTCCA
29
7821
7838
3-10-5
764

599245
2587
2604
Exon 18
AATCCCACGCCCCTGTCC
11
7822
7839
3-10-5
765

599246
2588
2605
Exon 18
CAATCCCACGCCCCTGTC
0
7823
7840
3-10-5
766

599247
2589
2606
Exon 18
TCAATCCCACGCCCCTGT
21
7824
7841
3-10-5
767

599248
2590
2607
Exon 18
TTCAATCCCACGCCCCTG
0
7825
7842
3-10-5
768

599249
2591
2608
Exon 18
ATTCAATCCCACGCCCCT
9
7826
7843
3-10-5
769

599250
2592
2609
Exon 18
AATTCAATCCCACGCCCC
4
7827
7844
3-10-5
770

599251
2593
2610
Exon 18
TAATTCAATCCCACGCCC
12
7828
7845
3-10-5
771

599252
2594
2611
Exon 18
TTAATTCAATCCCACGCC
2
7829
7846
3-10-5
772

599253
2595
2612
Exon 18
TTTAATTCAATCCCACGC
28
7830
7847
3-10-5
773

599254
2596
2613
Exon 18
TTTTAATTCAATCCCACG
27
7831
7848
3-10-5
774

599255
2597
2614
Exon 18
GTTTTAATTCAATCCCAC
38
7832
7849
3-10-5
775

599256
2598
2615
Exon 18
TGTTTTAATTCAATCCCA
36
7833
7850
3-10-5
776

599257
2599
2616
Exon 18
CTGTTTTAATTCAATCCC
48
7834
7851
3-10-5
777

599258
2600
2617
Exon 18
GCTGTTTTAATTCAATCC
19
7835
7852
3-10-5
778

599259
2601
2618
Exon 18
AGCTGTTTTAATTCAATC
36
7836
7853
3-10-5
779

599260
2602
2619
Exon 18
CAGCTGTTTTAATTCAAT
58
7837
7854
3-10-5
780

599261
2603
2620
Exon 18
GCAGCTGTTTTAATTCAA
35
7838
7855
3-10-5
781

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
96
7839
7858
5-10-5
317

599262
2604
2621
Exon 18
CGCAGCTGTTTTAATTCA
52
7839
7856
3-10-5
782

599263
2605
2622
Exon 18
TCGCAGCTGTTTTAATTC
66
7840
7857
3-10-5
783

599264
2606
2623
Exon 18
GTCGCAGCTGTTTTAATT
48
7841
7858
3-10-5
784

599265
2607
2624
Exon 18
TGTCGCAGCTGTTTTAAT
46
7842
7859
3-10-5
792

599205
2607
2624
Exon 18
TGTCGCAGCTGTTTTAAT
83
7842
7859
5-8-5
792

599266
2608
2625
Exon 18
TTGTCGCAGCTGTTTTAA
76
7843
7860
3-10-5
793

599206
2608
2625
Exon 18
TTGTCGCAGCTGTTTTAA
90
7843
7860
5-8-5
793

599267
2609
2626
Exon 18
GTTGTCGCAGCTGTTTTA
53
7844
7861
3-10-5
794

599207
2609
2626
Exon 18
GTTGTCGCAGCTGTTTTA
82
7844
7861
5-8-5
794

599268
2610
2627
Exon 18
TGTTGTCGCAGCTGTTTT
58
7845
7862
3-10-5
795

599208
2610
2627
Exon 18
TGTTGTCGCAGCTGTTTT
70
7845
7862
5-8-5
795

599269
2611
2628
Exon 18/
TTGTTGTCGCAGCTGTTT
38
n/a
n/a
3-10-5
796

Repeat

599209
2611
2628
Exon 18/
TTGTTGTCGCAGCTGTTT
50
n/a
n/a
5-8-5
796

Repeat

599270
2612
2629
Exon 18/
TTTGTTGTCGCAGCTGTT
46
n/a
n/a
3-10-5
797

Repeat

599210
2612
2629
Exon 18/
TTTGTTGTCGCAGCTGTT
76
n/a
n/a
5-8-5
797

Repeat

599271
2613
2630
Exon 18/
TTTTGTTGTCGCAGCTGT
64
n/a
n/a
3-10-5
798

Repeat

599211
2613
2630
Exon 18/
TTTTGTTGTCGCAGCTGT
78
n/a
n/a
5-8-5
798

Repeat

599272
2614
2631
Exon 18/
TTTTTGTTGTCGCAGCTG
89
n/a
n/a
3-10-5
799

Repeat

599212
2614
2631
Exon 18/
TTTTTGTTGTCGCAGCTG
84
n/a
n/a
5-8-5
799

Repeat

TABLE 140

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
Start
stop
Target

inhi-
Start
Stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599511
2552
2571
Exon 18
ATAGAAAACCCAAATCCTCA
38
7787
7806
6-8-6
410

599389
2553
2572
Exon 18
TATAGAAAACCCAAATCCTC
80
7788
7807
6-8-6
411

599390
2554
2573
Exon 18
TTATAGAAAACCCAAATCCT
92
7789
7808
6-8-6
412

599391
2555
2574
Exon 18
CTTATAGAAAACCCAAATCC
90
7790
7809
6-8-6
413

599392
2556
2575
Exon 18
CCTTATAGAAAACCCAAATC
87
7791
7810
6-8-6
414

599393
2557
2576
Exon 18
CCCTTATAGAAAACCCAAAT
87
7792
7811
6-8-6
415

599394
2558
2577
Exon 18
CCCCTTATAGAAAACCCAAA
74
7793
7812
6-8-6
416

599395
2559
2578
Exon 18
ACCCCTTATAGAAAACCCAA
78
7794
7813
6-8-6
417

599396
2560
2579
Exon 18
AACCCCTTATAGAAAACCCA
77
7795
7814
6-8-6
418

599397
2561
2580
Exon 18
AAACCCCTTATAGAAAACCC
89
7796
7815
6-8-6
419

599398
2562
2581
Exon 18
GAAACCCCTTATAGAAAACC
90
7797
7816
6-8-6
420

599399
2563
2582
Exon 18
GGAAACCCCTTATAGAAAAC
91
7798
7817
6-8-6
421

599400
2564
2583
Exon 18
AGGAAACCCCTTATAGAAAA
88
7799
7818
6-8-6
422

599401
2565
2584
Exon 18
CAGGAAACCCCTTATAGAAA
85
7800
7819
6-8-6
423

599402
2566
2585
Exon 18
GCAGGAAACCCCTTATAGAA
77
7801
7820
6-8-6
424

599403
2567
2586
Exon 18
AGCAGGAAACCCCTTATAGA
85
7802
7821
6-8-6
425

599404
2568
2587
Exon 18
CAGCAGGAAACCCCTTATAG
90
7803
7822
6-8-6
426

599405
2569
2588
Exon 18
CCAGCAGGAAACCCCTTATA
89
7804
7823
6-8-6
427

599406
2570
2589
Exon 18
TCCAGCAGGAAACCCCTTAT
72
7805
7824
6-8-6
428

599407
2571
2590
Exon 18
GTCCAGCAGGAAACCCCTTA
87
7806
7825
6-8-6
237

599408
2572
2591
Exon 18
TGTCCAGCAGGAAACCCCTT
87
7807
7826
6-8-6
429

599409
2573
2592
Exon 18
CTGTCCAGCAGGAAACCCCT
83
7808
7827
6-8-6
430

599410
2574
2593
Exon 18
CCTGTCCAGCAGGAAACCCC
88
7809
7828
6-8-6
431

599411
2575
2594
Exon 18
CCCTGTCCAGCAGGAAACCC
45
7810
7829
6-8-6
432

599412
2576
2595
Exon 18
CCCCTGTCCAGCAGGAAACC
66
7811
7830
6-8-6
433

599413
2577
2596
Exon 18
GCCCCTGTCCAGCAGGAAAC
92
7812
7831
6-8-6
238

599414
2578
2597
Exon 18
CGCCCCTGTCCAGCAGGAAA
92
7813
7832
6-8-6
434

599415
2579
2598
Exon 18
ACGCCCCTGTCCAGCAGGAA
87
7814
7833
6-8-6
435

599416
2580
2599
Exon 18
CACGCCCCTGTCCAGCAGGA
91
7815
7834
6-8-6
436

599417
2581
2600
Exon 18
CCACGCCCCTGTCCAGCAGG
84
7816
7835
6-8-6
437

599357
2582
2600
Exon 18
CCACGCCCCTGTCCAGCAG
88
7817
7835
5-9-5
708

599418
2582
2601
Exon 18
CCCACGCCCCTGTCCAGCAG
85
7817
7836
6-8-6
438

599358
2583
2601
Exon 18
CCCACGCCCCTGTCCAGCA
86
7818
7836
5-9-5
709

599419
2583
2602
Exon 18
TCCCACGCCCCTGTCCAGCA
91
7818
7837
6-8-6
833

599359
2584
2602
Exon 18
TCCCACGCCCCTGTCCAGC
85
7819
7837
5-9-5
834

599420
2584
2603
Exon 18
ATCCCACGCCCCTGTCCAGC
91
7819
7838
6-8-6
440

599360
2585
2603
Exon 18
ATCCCACGCCCCTGTCCAG
89
7820
7838
5-9-5
711

599421
2585
2604
Exon 18
AATCCCACGCCCCTGTCCAG
87
7820
7839
6-8-6
441

599361
2586
2604
Exon 18
AATCCCACGCCCCTGTCCA
89
7821
7839
5-9-5
712

599422
2586
2605
Exon 18
CAATCCCACGCCCCTGTCCA
90
7821
7840
6-8-6
442

599362
2587
2605
Exon 18
CAATCCCACGCCCCTGTCC
94
7822
7840
5-9-5
713

599423
2587
2606
Exon 18
TCAATCCCACGCCCCTGTCC
85
7822
7841
6-8-6
841

599363
2588
2606
Exon 18
TCAATCCCACGCCCCTGTC
88
7823
7841
5-9-5
714

599424
2588
2607
Exon 18
TTCAATCCCACGCCCCTGTC
88
7823
7842
6-8-6
444

599364
2589
2607
Exon 18
TTCAATCCCACGCCCCTGT
88
7824
7842
5-9-5
715

599425
2589
2608
Exon 18
ATTCAATCCCACGCCCCTGT
68
7824
7843
6-8-6
445

599365
2590
2608
Exon 18
ATTCAATCCCACGCCCCTG
48
7825
7843
5-9-5
716

599426
2590
2609
Exon 18
AATTCAATCCCACGCCCCTG
55
7825
7844
6-8-6
446

599366
2591
2609
Exon 18
AATTCAATCCCACGCCCCT
28
7826
7844
5-9-5
717

599427
2591
2610
Exon 18
TAATTCAATCCCACGCCCCT
13
7826
7845
6-8-6
849

599367
2592
2610
Exon 18
TAATTCAATCCCACGCCCC
21
7827
7845
5-9-5
718

599428
2592
2611
Exon 18
TTAATTCAATCCCACGCCCC
39
7827
7846
6-8-6
448

599368
2593
2611
Exon 18
TTAATTCAATCCCACGCCC
20
7828
7846
5-9-5
719

599429
2593
2612
Exon 18
TTTAATTCAATCCCACGCCC
18
7828
7847
6-8-6
449

599369
2594
2612
Exon 18
TTTAATTCAATCCCACGCC
78
7829
7847
5-9-5
720

599430
2594
2613
Exon 18
TTTTAATTCAATCCCACGCC
24
7829
7848
6-8-6
450

599370
2595
2613
Exon 18
TTTTAATTCAATCCCACGC
25
7830
7848
5-9-5
721

599431
2595
2614
Exon 18
GTTTTAATTCAATCCCACGC
30
7830
7849
6-8-6
451

599371
2596
2614
Exon 18
GTTTTAATTCAATCCCACG
84
7831
7849
5-9-5
722

599432
2596
2615
Exon 18
TGTTTTAATTCAATCCCACG
29
7831
7850
6-8-6
452

599372
2597
2615
Exon 18
TGTTTTAATTCAATCCCAC
83
7832
7850
5-9-5
723

599373
2598
2616
Exon 18
CTGTTTTAATTCAATCCCA
81
7833
7851
5-9-5
724

599374
2599
2617
Exon 18
GCTGTTTTAATTCAATCCC
26
7834
7852
5-9-5
725

599375
2600
2618
Exon 18
AGCTGTTTTAATTCAATCC
26
7835
7853
5-9-5
726

599376
2601
2619
Exon 18
CAGCTGTTTTAATTCAATC
62
7836
7854
5-9-5
727

599377
2602
2620
Exon 18
GCAGCTGTTTTAATTCAAT
21
7837
7855
5-9-5
728

599378
2603
2621
Exon 18
CGCAGCTGTTTTAATTCAA
90
7838
7856
5-9-5
729

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
95
7839
7858
5-10-5
867

599379
2604
2622
Exon 18
TCGCAGCTGTTTTAATTCA
88
7839
7857
5-9-5
730

599380
2605
2623
Exon 18
GTCGCAGCTGTTTTAATTC
37
7840
7858
5-9-5
869

599381
2606
2624
Exon 18
TGTCGCAGCTGTTTTAATT
33
7841
7859
5-9-5
732

599382
2607
2625
Exon 18
TTGTCGCAGCTGTTTTAAT
81
7842
7860
5-9-5
733

599383
2608
2626
Exon 18
GTTGTCGCAGCTGTTTTAA
54
7843
7861
5-9-5
734

599384
2609
2627
Exon 18
TGTTGTCGCAGCTGTTTTA
85
7844
7862
5-9-5
873

599385
2610
2628
Exon 18/
TTGTTGTCGCAGCTGTTTT
59
n/a
n/a
5-9-5
736

Repeat

599386
2611
2629
Exon 18/
TTTGTTGTCGCAGCTGTTT
81
n/a
n/a
5-9-5
737

Repeat

599387
2612
2630
Exon 18/
TTTTGTTGTCGCAGCTGTT
80
n/a
n/a
5-9-5
738

Repeat

599388
2613
2631
Exon 18/
TTTTTGTTGTCGCAGCTGT
84
n/a
n/a
5-9-5
739

Repeat

Example 120: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed deoxy, MOE and (S)-cEt oligonucleotides. 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 modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; and ‘e’ indicates a MOE modification.

TABLE 141

Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1

or SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
start
stop
Target

inhi-
start
stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599513
2551
2566
Exon 18
AAACCCAAATCCTCAT
11
7786
7801
ekkeekkddddddddk
557

599514
2553
2568
Exon 18
GAAAACCCAAATCCTC
13
7788
7803
ekkeekkddddddddk
801

599515
2555
2570
Exon 18
TAGAAAACCCAAATCC
54
7790
7805
ekkeekkddddddddk
559

599516
2559
2574
Exon 18
CTTATAGAAAACCCAA
16
7794
7809
ekkeekkddddddddk
561

599517
2560
2575
Exon 18
CCTTATAGAAAACCCA
29
7795
7810
ekkeekkddddddddk
562

599518
2561
2576
Exon 18
CCCTTATAGAAAACCC
55
7796
7811
ekkeekkddddddddk
563

599519
2562
2577
Exon 18
CCCCTTATAGAAAACC
31
7797
7812
ekkeekkddddddddk
564

599520
2563
2578
Exon 18
ACCCCTTATAGAAAAC
14
7798
7813
ekkeekkddddddddk
565

599521
2564
2579
Exon 18
AACCCCTTATAGAAAA
9
7799
7814
ekkeekkddddddddk
566

599522
2565
2580
Exon 18
AAACCCCTTATAGAAA
8
7800
7815
ekkeekkddddddddk
567

599523
2566
2581
Exon 18
GAAACCCCTTATAGAA
6
7801
7816
ekkeekkddddddddk
568

599524
2567
2582
Exon 18
GGAAACCCCTTATAGA
14
7802
7817
ekkeekkddddddddk
569

599525
2568
2583
Exon 18
AGGAAACCCCTTATAG
6
7803
7818
ekkeekkddddddddk
570

599526
2569
2584
Exon 18
CAGGAAACCCCTTATA
16
7804
7819
ekkeekkddddddddk
571

599527
2570
2585
Exon 18
GCAGGAAACCCCTTAT
0
7805
7820
ekkeekkddddddddk
572

599528
2571
2586
Exon 18
AGCAGGAAACCCCTTA
6
7806
7821
ekkeekkddddddddk
573

599529
2572
2587
Exon 18
CAGCAGGAAACCCCTT
6
7807
7822
ekkeekkddddddddk
574

599530
2574
2589
Exon 18
TCCAGCAGGAAACCCC
29
7809
7824
ekkeekkddddddddk
576

599531
2575
2590
Exon 18
GTCCAGCAGGAAACCC
64
7810
7825
ekkeekkddddddddk
577

599532
2576
2591
Exon 18
TGTCCAGCAGGAAACC
43
7811
7826
ekkeekkddddddddk
578

599533
2577
2592
Exon 18
CTGTCCAGCAGGAAAC
25
7812
7827
ekkeekkddddddddk
820

599534
2578
2593
Exon 18
CCTGTCCAGCAGGAAA
12
7813
7828
ekkeekkddddddddk
580

599535
2580
2595
Exon 18
CCCCTGTCCAGCAGGA
16
7815
7830
ekkeekkddddddddk
582

599536
2582
2597
Exon 18
CGCCCCTGTCCAGCAG
27
7817
7832
ekkeekkddddddddk
584

599537
2583
2598
Exon 18
ACGCCCCTGTCCAGCA
35
7818
7833
ekkeekkddddddddk
585

599538
2584
2599
Exon 18
CACGCCCCTGTCCAGC
26
7819
7834
ekkeekkddddddddk
586

599539
2585
2600
Exon 18
CCACGCCCCTGTCCAG
33
7820
7835
ekkeekkddddddddk
587

599540
2586
2601
Exon 18
CCCACGCCCCTGTCCA
27
7821
7836
ekkeekkddddddddk
588

599541
2587
2602
Exon 18
TCCCACGCCCCTGTCC
52
7822
7837
ekkeekkddddddddk
589

599542
2588
2603
Exon 18
ATCCCACGCCCCTGTC
16
7823
7838
ekkeekkddddddddk
590

599543
2589
2604
Exon 18
AATCCCACGCCCCTGT
19
7824
7839
ekkeekkddddddddk
591

599544
2590
2605
Exon 18
CAATCCCACGCCCCTG
33
7825
7840
ekkeekkddddddddk
831

599545
2591
2606
Exon 18
TCAATCCCACGCCCCT
24
7826
7841
ekkeekkddddddddk
593

599546
2592
2607
Exon 18
TTCAATCCCACGCCCC
54
7827
7842
ekkeekkddddddddk
594

599547
2593
2608
Exon 18
ATTCAATCCCACGCCC
87
7828
7843
ekkeekkddddddddk
595

599548
2594
2609
Exon 18
AATTCAATCCCACGCC
79
7829
7844
ekkeekkddddddddk
596

599549
2595
2610
Exon 18
TAATTCAATCCCACGC
62
7830
7845
ekkeekkddddddddk
597

599550
2596
2611
Exon 18
TTAATTCAATCCCACG
52
7831
7846
ekkeekkddddddddk
598

599551
2597
2612
Exon 18
TTTAATTCAATCCCAC
27
7832
7847
ekkeekkddddddddk
599

599577
2597
2613
Exon 18
TTTTAATTCAATCCCAC
90
7832
7848
eeekkddddddddkeee
662

599552
2598
2613
Exon 18
TTTTAATTCAATCCCA
92
7833
7848
ekkeekkddddddddk
600

599578
2598
2614
Exon 18
GTTTTAATTCAATCCCA
88
7833
7849
eeekkddddddddkeee
663

599553
2599
2614
Exon 18
GTTTTAATTCAATCCC
91
7834
7849
ekkeekkddddddddk
601

599579
2599
2615
Exon 18
TGTTTTAATTCAATCCC
79
7834
7850
eeekkddddddddkeee
664

599554
2600
2615
Exon 18
TGTTTTAATTCAATCC
90
7835
7850
ekkeekkddddddddk
602

599580
2600
2616
Exon 18
CTGTTTTAATTCAATCC
79
7835
7851
eeekkddddddddkeee
665

599555
2601
2616
Exon 18
CTGTTTTAATTCAATC
79
7836
7851
ekkeekkddddddddk
846

599581
2601
2617
Exon 18
GCTGTTTTAATTCAATC
90
7836
7852
eeekkddddddddkeee
666

599556
2602
2617
Exon 18
GCTGTTTTAATTCAAT
47
7837
7852
ekkeekkddddddddk
604

599582
2602
2618
Exon 18
AGCTGTTTTAATTCAAT
89
7837
7853
eeekkddddddddkeee
849

599557
2603
2618
Exon 18
AGCTGTTTTAATTCAA
67
7838
7853
ekkeekkddddddddk
850

599583
2603
2619
Exon 18
CAGCTGTTTTAATTCAA
49
7838
7854
eeekkddddddddkeee
668

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAAT
78
7839
7858
eeeeeddddddddddeee
317

TCA

ee

599558
2604
2619
Exon 18
CAGCTGTTTTAATTCA
80
7839
7854
ekkeekkddddddddk
606

599584
2604
2620
Exon 18
GCAGCTGTTTTAATTCA
66
7839
7855
eeekkddddddddkeee
669

599559
2605
2620
Exon 18
GCAGCTGTTTTAATTC
38
7840
7855
ekkeekkddddddddk
607

599585
2605
2621
Exon 18
CGCAGCTGTTTTAATTC
80
7840
7856
eeekkddddddddkeee
670

599560
2606
2621
Exon 18
CGCAGCTGTTTTAATT
16
7841
7856
ekkeekkddddddddk
608

599586
2606
2622
Exon 18
TCGCAGCTGTTTTAATT
78
7841
7857
eeekkddddddddkeee
671

599561
2607
2622
Exon 18
TCGCAGCTGTTTTAAT
58
7842
7857
ekkeekkddddddddk
609

599587
2607
2623
Exon 18
GTCGCAGCTGTTTTAAT
81
7842
7858
eeekkddddddddkeee
672

588860
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
92
7843
7858
eekdddddddddddke
610

599562
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
78
7843
7858
ekkeekkddddddddk
610

599588
2608
2624
Exon 18
TGTCGCAGCTGTTTTAA
81
7843
7859
eeekkddddddddkeee
673

599563
2609
2624
Exon 18
TGTCGCAGCTGTTTTA
86
7844
7859
ekkeekkddddddddk
611

599589
2609
2625
Exon 18
TTGTCGCAGCTGTTTTA
75
7844
7860
eeekkddddddddkeee
674

599564
2610
2625
Exon 18
TTGTCGCAGCTGTTTT
75
7845
7860
ekkeekkddddddddk
612

599590
2610
2626
Exon 18
GTTGTCGCAGCTGTTTT
88
7845
7861
eeekkddddddddkeee
675

599565
2611
2626
Exon 18
GTTGTCGCAGCTGTTT
65
7846
7861
ekkeekkddddddddk
613

599591
2611
2627
Exon 18
TGTTGTCGCAGCTGTTT
94
7846
7862
eeekkddddddddkeee
676

599566
2612
2627
Exon 18
TGTTGTCGCAGCTGTT
72
7847
7862
ekkeekkddddddddk
614

599592
2612
2628
Exon 18/
TTGTTGTCGCAGCTGTT
90
n/a
n/a
eeekkddddddddkeee
677

Repeat

599567
2613
2628
Exon 18/
TTGTTGTCGCAGCTGT
82
n/a
n/a
ekkeekkddddddddk
615

Repeat

599593
2613
2629
Exon 18/
TTTGTTGTCGCAGCTGT
95
n/a
n/a
eeekkddddddddkeee
678

Repeat

599568
2614
2629
Exon 18/
TTTGTTGTCGCAGCTG
92
n/a
n/a
ekkeekkddddddddk
616

Repeat

599594
2614
2630
Exon 18/
TTTTGTTGTCGCAGCTG
86
n/a
n/a
eeekkddddddddkeee
679

Repeat

599569
2615
2630
Exon 18/
TTTTGTTGTCGCAGCT
89
n/a
n/a
ekkeekkddddddddk
617

Repeat

599595
2615
2631
Exon 18/
TTTTTGTTGTCGCAGCT
76
n/a
n/a
eeekkddddddddkeee
680

Repeat

599570
2616
2631
Exon 18/
TTTTTGTTGTCGCAGC
95
n/a
n/a
ekkeekkddddddddk
618

Repeat

Example 121: Antisense Inhibition of Human Complement Factor B (CFB) in HepG2 Cells by MOE Gapmers

Additional antisense oligonucleotides were designed targeting human Complement Factor B (CFB) nucleic acid and were tested for their effects on CFB mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as deoxy, MOE and (S)-cEt oligonucleotides, or as 5-8-5 MOE, 5-9-5 MOE, 5-10-5 MOE, 6-7-6-MOE, 3-10-5 MOE, or 6-8-6 MOE gapmers.

The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of nine 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. The 3-10-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three and five nucleosides respectively. The 6-7-6 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises of eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

TABLE 142

Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1

or SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
start
stop
Target

inhi-
start
stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

601152
2551
2566
Exon 18
AAACCCAAATCCTCAT
22
7786
7801
eekkdddddddddkee
557

601218
2551
2566
Exon 18
AAACCCAAATCCTCAT
21
7786
7801
edkkdddddddddeee
557

601153
2552
2567
Exon 18
AAAACCCAAATCCTCA
27
7787
7802
eekkdddddddddkee
800

601219
2552
2567
Exon 18
AAAACCCAAATCCTCA
19
7787
7802
edkkdddddddddeee
800

601154
2553
2568
Exon 18
GAAAACCCAAATCCTC
23
7788
7803
eekkdddddddddkee
558

601220
2553
2568
Exon 18
GAAAACCCAAATCCTC
24
7788
7803
edkkdddddddddeee
558

601155
2554
2569
Exon 18
AGAAAACCCAAATCCT
20
7789
7804
eekkdddddddddkee
801

601221
2554
2569
Exon 18
AGAAAACCCAAATCCT
0
7789
7804
edkkdddddddddeee
801

601156
2555
2570
Exon 18
TAGAAAACCCAAATCC
11
7790
7805
eekkdddddddddkee
559

601222
2555
2570
Exon 18
TAGAAAACCCAAATCC
23
7790
7805
edkkdddddddddeee
559

601157
2556
2571
Exon 18
ATAGAAAACCCAAATC
9
7791
7806
eekkdddddddddkee
560

601223
2556
2571
Exon 18
ATAGAAAACCCAAATC
0
7791
7806
edkkdddddddddeee
560

601158
2557
2572
Exon 18
TATAGAAAACCCAAAT
0
7792
7807
eekkdddddddddkee
802

601224
2557
2572
Exon 18
TATAGAAAACCCAAAT
0
7792
7807
edkkdddddddddeee
802

601159
2558
2573
Exon 18
TTATAGAAAACCCAAA
2
7793
7808
eekkdddddddddkee
803

601225
2558
2573
Exon 18
TTATAGAAAACCCAAA
0
7793
7808
edkkdddddddddeee
803

601160
2559
2574
Exon 18
CTTATAGAAAACCCAA
0
7794
7809
eekkdddddddddkee
561

601226
2559
2574
Exon 18
CTTATAGAAAACCCAA
0
7794
7809
edkkdddddddddeee
561

601161
2560
2575
Exon 18
CCTTATAGAAAACCCA
1
7795
7810
eekkdddddddddkee
562

601227
2560
2575
Exon 18
CCTTATAGAAAACCCA
14
7795
7810
edkkdddddddddeee
562

601162
2561
2576
Exon 18
CCCTTATAGAAAACCC
9
7796
7811
eekkdddddddddkee
563

601228
2561
2576
Exon 18
CCCTTATAGAAAACCC
9
7796
7811
edkkdddddddddeee
563

601163
2562
2577
Exon 18
CCCCTTATAGAAAACC
0
7797
7812
eekkdddddddddkee
564

601164
2563
2578
Exon 18
ACCCCTTATAGAAAAC
3
7798
7813
eekkdddddddddkee
565

601165
2564
2579
Exon 18
AACCCCTTATAGAAAA
0
7799
7814
eekkdddddddddkee
566

601166
2565
2580
Exon 18
AAACCCCTTATAGAAA
0
7800
7815
eekkdddddddddkee
567

601167
2566
2581
Exon 18
GAAACCCCTTATAGAA
0
7801
7816
eekkdddddddddkee
568

601168
2567
2582
Exon 18
GGAAACCCCTTATAGA
0
7802
7817
eekkdddddddddkee
569

601169
2568
2583
Exon 18
AGGAAACCCCTTATAG
0
7803
7818
eekkdddddddddkee
570

601170
2569
2584
Exon 18
CAGGAAACCCCTTATA
10
7804
7819
eekkdddddddddkee
571

601171
2570
2585
Exon 18
GCAGGAAACCCCTTAT
9
7805
7820
eekkdddddddddkee
572

601172
2571
2586
Exon 18
AGCAGGAAACCCCTTA
15
7806
7821
eekkdddddddddkee
573

601173
2572
2587
Exon 18
CAGCAGGAAACCCCTT
29
7807
7822
eekkdddddddddkee
574

601174
2573
2588
Exon 18
CCAGCAGGAAACCCCT
25
7808
7823
eekkdddddddddkee
575

601175
2574
2589
Exon 18
TCCAGCAGGAAACCCC
15
7809
7824
eekkdddddddddkee
576

601176
2575
2590
Exon 18
GTCCAGCAGGAAACCC
18
7810
7825
eekkdddddddddkee
577

601177
2576
2591
Exon 18
TGTCCAGCAGGAAACC
10
7811
7826
eekkdddddddddkee
578

601178
2577
2592
Exon 18
CTGTCCAGCAGGAAAC
11
7812
7827
eekkdddddddddkee
579

601179
2578
2593
Exon 18
CCTGTCCAGCAGGAAA
19
7813
7828
eekkdddddddddkee
580

601180
2579
2594
Exon 18
CCCTGTCCAGCAGGAA
7
7814
7829
eekkdddddddddkee
581

601181
2580
2595
Exon 18
CCCCTGTCCAGCAGGA
3
7815
7830
eekkdddddddddkee
582

601182
2581
2596
Exon 18
GCCCCTGTCCAGCAGG
0
7816
7831
eekkdddddddddkee
583

601183
2582
2597
Exon 18
CGCCCCTGTCCAGCAG
4
7817
7832
eekkdddddddddkee
584

601184
2583
2598
Exon 18
ACGCCCCTGTCCAGCA
14
7818
7833
eekkdddddddddkee
585

601185
2584
2599
Exon 18
CACGCCCCTGTCCAGC
26
7819
7834
eekkdddddddddkee
586

601186
2585
2600
Exon 18
CCACGCCCCTGTCCAG
8
7820
7835
eekkdddddddddkee
587

601187
2586
2601
Exon 18
CCCACGCCCCTGTCCA
18
7821
7836
eekkdddddddddkee
588

601188
2587
2602
Exon 18
TCCCACGCCCCTGTCC
20
7822
7837
eekkdddddddddkee
589

601189
2588
2603
Exon 18
ATCCCACGCCCCTGTC
12
7823
7838
eekkdddddddddkee
590

601190
2589
2604
Exon 18
AATCCCACGCCCCTGT
33
7824
7839
eekkdddddddddkee
591

601191
2590
2605
Exon 18
CAATCCCACGCCCCTG
52
7825
7840
eekkdddddddddkee
592

601192
2591
2606
Exon 18
TCAATCCCACGCCCCT
46
7826
7841
eekkdddddddddkee
593

601193
2592
2607
Exon 18
TTCAATCCCACGCCCC
30
7827
7842
eekkdddddddddkee
594

601194
2593
2608
Exon 18
ATTCAATCCCACGCCC
41
7828
7843
eekkdddddddddkee
595

601195
2594
2609
Exon 18
AATTCAATCCCACGCC
40
7829
7844
eekkdddddddddkee
596

601196
2595
2610
Exon 18
TAATTCAATCCCACGC
71
7830
7845
eekkdddddddddkee
597

601197
2596
2611
Exon 18
TTAATTCAATCCCACG
42
7831
7846
eekkdddddddddkee
598

601198
2597
2612
Exon 18
TTTAATTCAATCCCAC
63
7832
7847
eekkdddddddddkee
599

601199
2598
2613
Exon 18
TTTTAATTCAATCCCA
51
7833
7848
eekkdddddddddkee
600

601200
2599
2614
Exon 18
GTTTTAATTCAATCCC
65
7834
7849
eekkdddddddddkee
601

601201
2600
2615
Exon 18
TGTTTTAATTCAATCC
49
7835
7850
eekkdddddddddkee
602

601202
2601
2616
Exon 18
CTGTTTTAATTCAATC
33
7836
7851
eekkdddddddddkee
603

601203
2602
2617
Exon 18
GCTGTTTTAATTCAAT
63
7837
7852
eekkdddddddddkee
604

601204
2603
2618
Exon 18
AGCTGTTTTAATTCAA
69
7838
7853
eekkdddddddddkee
605

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATT
73
7839
7858
eeeeeddddddddddeeee
317

CA

e

601205
2604
2619
Exon 18
CAGCTGTTTTAATTCA
51
7839
7854
eekkdddddddddkee
606

601206
2605
2620
Exon 18
GCAGCTGTTTTAATTC
43
7840
7855
eekkdddddddddkee
607

601207
2606
2621
Exon 18
CGCAGCTGTTTTAATT
52
7841
7856
eekkdddddddddkee
608

601208
2607
2622
Exon 18
TCGCAGCTGTTTTAAT
61
7842
7857
eekkdddddddddkee
609

588860
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
75
7843
7858
eekdddddddddddke
610

601209
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
73
7843
7858
eekkdddddddddkee
610

601210
2609
2624
Exon 18
TGTCGCAGCTGTTTTA
80
7844
7859
eekkdddddddddkee
611

601211
2610
2625
Exon 18
TTGTCGCAGCTGTTTT
64
7845
7860
eekkdddddddddkee
612

601212
2611
2626
Exon 18
GTTGTCGCAGCTGTTT
86
7846
7861
eekkdddddddddkee
613

601213
2612
2627
Exon 18
TGTTGTCGCAGCTGTT
87
7847
7862
eekkdddddddddkee
614

601214
2613
2628
Exon 18/
TTGTTGTCGCAGCTGT
84
n/a
n/a
eekkdddddddddkee
615

Repeat

601215
2614
2629
Exon 18/
TTTGTTGTCGCAGCTG
78
n/a
n/a
eekkdddddddddkee
616

Repeat

601216
2615
2630
Exon 18/
TTTTGTTGTCGCAGCT
73
n/a
n/a
eekkdddddddddkee
617

Repeat

601217
2616
2631
Exon 18/
TTTTTGTTGTCGCAGC
66
n/a
n/a
eekkdddddddddkee
618

Repeat

TABLE 143

Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1

or SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
start
stop
Target

inhi-
start
stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

601284
2551
2566
Exon 18
AAACCCAAATCCTCAT
8
7786
7801
ekkdddddddddkeee
557

601285
2552
2567
Exon 18
AAAACCCAAATCCTCA
15
7787
7802
ekkdddddddddkeee
800

601286
2553
2568
Exon 18
GAAAACCCAAATCCTC
21
7788
7803
ekkdddddddddkeee
558

601287
2554
2569
Exon 18
AGAAAACCCAAATCCT
9
7789
7804
ekkdddddddddkeee
801

601288
2555
2570
Exon 18
TAGAAAACCCAAATCC
0
7790
7805
ekkdddddddddkeee
559

601289
2556
2571
Exon 18
ATAGAAAACCCAAATC
40
7791
7806
ekkdddddddddkeee
560

601290
2557
2572
Exon 18
TATAGAAAACCCAAAT
16
7792
7807
ekkdddddddddkeee
802

601291
2558
2573
Exon 18
TTATAGAAAACCCAAA
15
7793
7808
ekkdddddddddkeee
803

601292
2559
2574
Exon 18
CTTATAGAAAACCCAA
5
7794
7809
ekkdddddddddkeee
561

601293
2560
2575
Exon 18
CCTTATAGAAAACCCA
15
7795
7810
ekkdddddddddkeee
562

601294
2561
2576
Exon 18
CCCTTATAGAAAACCC
3
7796
7811
ekkdddddddddkeee
563

601229
2562
2577
Exon 18
CCCCTTATAGAAAACC
15
7797
7812
edkddddddddddeee
564

601295
2562
2577
Exon 18
CCCCTTATAGAAAACC
5
7797
7812
ekkdddddddddkeee
564

601230
2563
2578
Exon 18
ACCCCTTATAGAAAAC
14
7798
7813
edkkdddddddddeee
565

601296
2563
2578
Exon 18
ACCCCTTATAGAAAAC
0
7798
7813
ekkdddddddddkeee
565

601231
2564
2579
Exon 18
AACCCCTTATAGAAAA
14
7799
7814
edkkdddddddddeee
566

601297
2564
2579
Exon 18
AACCCCTTATAGAAAA
14
7799
7814
ekkdddddddddkeee
566

601232
2565
2580
Exon 18
AAACCCCTTATAGAAA
15
7800
7815
edkddddddddddeee
567

601298
2565
2580
Exon 18
AAACCCCTTATAGAAA
7
7800
7815
ekkdddddddddkeee
567

601233
2566
2581
Exon 18
GAAACCCCTTATAGAA
0
7801
7816
edkddddddddddeee
568

601299
2566
2581
Exon 18
GAAACCCCTTATAGAA
0
7801
7816
ekkdddddddddkeee
568

601234
2567
2582
Exon 18
GGAAACCCCTTATAGA
0
7802
7817
edkddddddddddeee
569

601300
2567
2582
Exon 18
GGAAACCCCTTATAGA
9
7802
7817
ekkdddddddddkeee
569

601235
2568
2583
Exon 18
AGGAAACCCCTTATAG
3
7803
7818
edkddddddddddeee
570

601301
2568
2583
Exon 18
AGGAAACCCCTTATAG
14
7803
7818
ekkdddddddddkeee
570

601236
2569
2584
Exon 18
CAGGAAACCCCTTATA
0
7804
7819
edkkdddddddddeee
571

601302
2569
2584
Exon 18
CAGGAAACCCCTTATA
0
7804
7819
ekkdddddddddkeee
571

601237
2570
2585
Exon 18
GCAGGAAACCCCTTAT
16
7805
7820
edkkdddddddddeee
572

601303
2570
2585
Exon 18
GCAGGAAACCCCTTAT
16
7805
7820
ekkdddddddddkeee
572

601238
2571
2586
Exon 18
AGCAGGAAACCCCTTA
11
7806
7821
edkkdddddddddeee
573

601304
2571
2586
Exon 18
AGCAGGAAACCCCTTA
10
7806
7821
ekkdddddddddkeee
573

601239
2572
2587
Exon 18
CAGCAGGAAACCCCTT
21
7807
7822
edkkdddddddddeee
574

601305
2572
2587
Exon 18
CAGCAGGAAACCCCTT
7
7807
7822
ekkdddddddddkeee
574

601240
2573
2588
Exon 18
CCAGCAGGAAACCCCT
6
7808
7823
edkkdddddddddeee
575

601241
2574
2589
Exon 18
TCCAGCAGGAAACCCC
10
7809
7824
edkkdddddddddeee
576

601242
2575
2590
Exon 18
GTCCAGCAGGAAACCC
19
7810
7825
edkkdddddddddeee
577

601243
2576
2591
Exon 18
TGTCCAGCAGGAAACC
10
7811
7826
edkkdddddddddeee
578

601244
2577
2592
Exon 18
CTGTCCAGCAGGAAAC
28
7812
7827
edkkdddddddddeee
579

601245
2578
2593
Exon 18
CCTGTCCAGCAGGAAA
5
7813
7828
edkkdddddddddeee
580

601246
2579
2594
Exon 18
CCCTGTCCAGCAGGAA
18
7814
7829
edkkdddddddddeee
581

601247
2580
2595
Exon 18
CCCCTGTCCAGCAGGA
4
7815
7830
edkkdddddddddeee
582

601248
2581
2596
Exon 18
GCCCCTGTCCAGCAGG
6
7816
7831
edkkdddddddddeee
583

601249
2582
2597
Exon 18
CGCCCCTGTCCAGCAG
18
7817
7832
edkkdddddddddeee
584

601250
2583
2598
Exon 18
ACGCCCCTGTCCAGCA
26
7818
7833
edkkdddddddddeee
585

601251
2584
2599
Exon 18
CACGCCCCTGTCCAGC
27
7819
7834
edkkdddddddddeee
586

601252
2585
2600
Exon 18
CCACGCCCCTGTCCAG
21
7820
7835
edkkdddddddddeee
587

601253
2586
2601
Exon 18
CCCACGCCCCTGTCCA
0
7821
7836
edkkdddddddddeee
588

601254
2587
2602
Exon 18
TCCCACGCCCCTGTCC
31
7822
7837
edkkdddddddddeee
589

601255
2588
2603
Exon 18
ATCCCACGCCCCTGTC
3
7823
7838
edkkdddddddddeee
590

601256
2589
2604
Exon 18
AATCCCACGCCCCTGT
21
7824
7839
edkkdddddddddeee
591

601257
2590
2605
Exon 18
CAATCCCACGCCCCTG
47
7825
7840
edkkdddddddddeee
592

601258
2591
2606
Exon 18
TCAATCCCACGCCCCT
48
7826
7841
edkkdddddddddeee
593

601259
2592
2607
Exon 18
TTCAATCCCACGCCCC
38
7827
7842
edkkdddddddddeee
594

601260
2593
2608
Exon 18
ATTCAATCCCACGCCC
33
7828
7843
edkkdddddddddeee
595

601261
2594
2609
Exon 18
AATTCAATCCCACGCC
17
7829
7844
edkkdddddddddeee
596

601262
2595
2610
Exon 18
TAATTCAATCCCACGC
40
7830
7845
edkkdddddddddeee
597

601263
2596
2611
Exon 18
TTAATTCAATCCCACG
31
7831
7846
edkkdddddddddeee
598

601264
2597
2612
Exon 18
TTTAATTCAATCCCAC
72
7832
7847
edkkdddddddddeee
599

601265
2598
2613
Exon 18
TTTTAATTCAATCCCA
48
7833
7848
edkkdddddddddeee
600

601266
2599
2614
Exon 18
GTTTTAATTCAATCCC
64
7834
7849
edkkdddddddddeee
601

601267
2600
2615
Exon 18
TGTTTTAATTCAATCC
43
7835
7850
edkkdddddddddeee
602

601268
2601
2616
Exon 18
CTGTTTTAATTCAATC
44
7836
7851
edkkdddddddddeee
603

601269
2602
2617
Exon 18
GCTGTTTTAATTCAAT
66
7837
7852
edkkdddddddddeee
604

601270
2603
2618
Exon 18
AGCTGTTTTAATTCAA
47
7838
7853
edkkdddddddddeee
605

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATT
3
7839
7858
eeeeeddddddddddeeee
317

CA

e

601271
2604
2619
Exon 18
CAGCTGTTTTAATTCA
26
7839
7854
edkkdddddddddeee
606

601272
2605
2620
Exon 18
GCAGCTGTTTTAATTC
33
7840
7855
edkkdddddddddeee
607

601273
2606
2621
Exon 18
CGCAGCTGTTTTAATT
34
7841
7856
edkkdddddddddeee
608

601274
2607
2622
Exon 18
TCGCAGCTGTTTTAAT
39
7842
7857
edkkdddddddddeee
609

588860
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
72
7843
7858
eekdddddddddddke
610

601275
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
65
7843
7858
edkkdddddddddeee
610

601276
2609
2624
Exon 18
TGTCGCAGCTGTTTTA
65
7844
7859
edkkdddddddddeee
611

601277
2610
2625
Exon 18
TTGTCGCAGCTGTTTT
51
7845
7860
edkkdddddddddeee
612

601278
2611
2626
Exon 18
GTTGTCGCAGCTGTTT
78
7846
7861
edkkdddddddddeee
613

601279
2612
2627
Exon 18
TGTTGTCGCAGCTGTT
79
7847
7862
edkkdddddddddeee
614

601280
2613
2628
Exon 18/
TTGTTGTCGCAGCTGT
70
n/a
n/a
edkkdddddddddeee
615

Repeat

601281
2614
2629
Exon 18/
TTTGTTGTCGCAGCTG
78
n/a
n/a
edkkdddddddddeee
616

Repeat

601282
2615
2630
Exon 18/
TTTTGTTGTCGCAGCT
68
n/a
n/a
edkkdddddddddeee
617

Repeat

601283
2616
2631
Exon 18/
TTTTTGTTGTCGCAGC
61
n/a
n/a
edkkdddddddddeee
618

Repeat

TABLE 144

Inhibition of CFB mRNA by deoxy, MOE and (S)-cEt oligonucleotides targeting SEQ ID NO: 1

or SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
start
stop
Target

inhi-
start
stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

601306
2573
2588
Exon 18
CCAGCAGGAAACCCCT
22
7808
7823
ekkdddddddddkeee
575

601307
2574
2589
Exon 18
TCCAGCAGGAAACCCC
22
7809
7824
ekkdddddddddkeee
576

601308
2575
2590
Exon 18
GTCCAGCAGGAAACCC
33
7810
7825
ekkdddddddddkeee
577

601309
2576
2591
Exon 18
TGTCCAGCAGGAAACC
33
7811
7826
ekkdddddddddkeee
578

601310
2577
2592
Exon 18
CTGTCCAGCAGGAAAC
28
7812
7827
ekkdddddddddkeee
579

601311
2578
2593
Exon 18
CCTGTCCAGCAGGAAA
33
7813
7828
ekkdddddddddkeee
580

601312
2579
2594
Exon 18
CCCTGTCCAGCAGGAA
13
7814
7829
ekkdddddddddkeee
581

601313
2580
2595
Exon 18
CCCCTGTCCAGCAGGA
32
7815
7830
ekkdddddddddkeee
582

601314
2581
2596
Exon 18
GCCCCTGTCCAGCAGG
0
7816
7831
ekkdddddddddkeee
583

601315
2582
2597
Exon 18
CGCCCCTGTCCAGCAG
36
7817
7832
ekkdddddddddkeee
584

601316
2583
2598
Exon 18
ACGCCCCTGTCCAGCA
39
7818
7833
ekkdddddddddkeee
585

601317
2584
2599
Exon 18
CACGCCCCTGTCCAGC
33
7819
7834
ekkdddddddddkeee
586

601356
2584
2599
Exon 18
CACGCCCCTGTCCAGC
27
7819
7834
kdkdddddddddeeee
586

601318
2585
2600
Exon 18
CCACGCCCCTGTCCAG
35
7820
7835
ekkdddddddddkeee
587

601357
2585
2600
Exon 18
CCACGCCCCTGTCCAG
26
7820
7835
kdkdddddddddeeee
587

601319
2586
2601
Exon 18
CCCACGCCCCTGTCCA
33
7821
7836
ekkdddddddddkeee
588

601358
2586
2601
Exon 18
CCCACGCCCCTGTCCA
26
7821
7836
kdkdddddddddeeee
588

601320
2587
2602
Exon 18
TCCCACGCCCCTGTCC
25
7822
7837
ekkdddddddddkeee
589

601359
2587
2602
Exon 18
TCCCACGCCCCTGTCC
23
7822
7837
kdkdddddddddeeee
589

601321
2588
2603
Exon 18
ATCCCACGCCCCTGTC
50
7823
7838
ekkdddddddddkeee
590

601360
2588
2603
Exon 18
ATCCCACGCCCCTGTC
33
7823
7838
kdkdddddddddeeee
590

601322
2589
2604
Exon 18
AATCCCACGCCCCTGT
52
7824
7839
ekkdddddddddkeee
591

601361
2589
2604
Exon 18
AATCCCACGCCCCTGT
48
7824
7839
kdkdddddddddeeee
591

601323
2590
2605
Exon 18
CAATCCCACGCCCCTG
67
7825
7840
ekkdddddddddkeee
592

601362
2590
2605
Exon 18
CAATCCCACGCCCCTG
51
7825
7840
kdkdddddddddeeee
592

601324
2591
2606
Exon 18
TCAATCCCACGCCCCT
42
7826
7841
ekkdddddddddkeee
593

601363
2591
2606
Exon 18
TCAATCCCACGCCCCT
42
7826
7841
kdkdddddddddeeee
593

601325
2592
2607
Exon 18
TTCAATCCCACGCCCC
52
7827
7842
ekkdddddddddkeee
594

601364
2592
2607
Exon 18
TTCAATCCCACGCCCC
48
7827
7842
kdkdddddddddeeee
594

601326
2593
2608
Exon 18
ATTCAATCCCACGCCC
27
7828
7843
ekkdddddddddkeee
595

601365
2593
2608
Exon 18
ATTCAATCCCACGCCC
36
7828
7843
kdkdddddddddeeee
595

601327
2594
2609
Exon 18
AATTCAATCCCACGCC
66
7829
7844
ekkdddddddddkeee
596

601366
2594
2609
Exon 18
AATTCAATCCCACGCC
49
7829
7844
kdkdddddddddeeee
596

601328
2595
2610
Exon 18
TAATTCAATCCCACGC
55
7830
7845
ekkdddddddddkeee
597

601367
2595
2610
Exon 18
TAATTCAATCCCACGC
57
7830
7845
kdkdddddddddeeee
597

601329
2596
2611
Exon 18
TTAATTCAATCCCACG
69
7831
7846
ekkdddddddddkeee
598

601368
2596
2611
Exon 18
TTAATTCAATCCCACG
68
7831
7846
kdkdddddddddeeee
598

601330
2597
2612
Exon 18
TTTAATTCAATCCCAC
58
7832
7847
ekkdddddddddkeee
599

601369
2597
2612
Exon 18
TTTAATTCAATCCCAC
65
7832
7847
kdkdddddddddeeee
599

601331
2598
2613
Exon 18
TTTTAATTCAATCCCA
45
7833
7848
ekkdddddddddkeee
600

601370
2598
2613
Exon 18
TTTTAATTCAATCCCA
42
7833
7848
kdkdddddddddeeee
600

601332
2599
2614
Exon 18
GTTTTAATTCAATCCC
84
7834
7849
ekkdddddddddkeee
601

601371
2599
2614
Exon 18
GTTTTAATTCAATCCC
79
7834
7849
kdkdddddddddeeee
601

601333
2600
2615
Exon 18
TGTTTTAATTCAATCC
61
7835
7850
ekkdddddddddkeee
602

601372
2600
2615
Exon 18
TGTTTTAATTCAATCC
71
7835
7850
kdkdddddddddeeee
602

601334
2601
2616
Exon 18
CTGTTTTAATTCAATC
61
7836
7851
ekkdddddddddkeee
603

601373
2601
2616
Exon 18
CTGTTTTAATTCAATC
57
7836
7851
kdkdddddddddeeee
603

601335
2602
2617
Exon 18
GCTGTTTTAATTCAAT
73
7837
7852
ekkdddddddddkeee
604

601374
2602
2617
Exon 18
GCTGTTTTAATTCAAT
66
7837
7852
kdkdddddddddeeee
604

601336
2603
2618
Exon 18
AGCTGTTTTAATTCAA
64
7838
7853
ekkdddddddddkeee
605

601375
2603
2618
Exon 18
AGCTGTTTTAATTCAA
61
7838
7853
kdkdddddddddeeee
605

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATT
66
7839
7858
eeeeedddddddddde
317

CA

eeee

601337
2604
2619
Exon 18
CAGCTGTTTTAATTCA
53
7839
7854
ekkdddddddddkeee
606

601376
2604
2619
Exon 18
CAGCTGTTTTAATTCA
39
7839
7854
kdkdddddddddeeee
606

601338
2605
2620
Exon 18
GCAGCTGTTTTAATTC
67
7840
7855
ekkdddddddddkeee
607

601377
2605
2620
Exon 18
GCAGCTGTTTTAATTC
67
7840
7855
kdkdddddddddeeee
607

601339
2606
2621
Exon 18
CGCAGCTGTTTTAATT
63
7841
7856
ekkdddddddddkeee
608

601378
2606
2621
Exon 18
CGCAGCTGTTTTAATT
60
7841
7856
kdkdddddddddeeee
608

601340
2607
2622
Exon 18
TCGCAGCTGTTTTAAT
40
7842
7857
ekkdddddddddkeee
609

601379
2607
2622
Exon 18
TCGCAGCTGTTTTAAT
36
7842
7857
kdkdddddddddeeee
609

588860
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
84
7843
7858
eekdddddddddddke
610

601341
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
74
7843
7858
ekkdddddddddkeee
610

601380
2608
2623
Exon 18
GTCGCAGCTGTTTTAA
78
7843
7858
kdkdddddddddeeee
610

601342
2609
2624
Exon 18
TGTCGCAGCTGTTTTA
68
7844
7859
ekkdddddddddkeee
611

601381
2609
2624
Exon 18
TGTCGCAGCTGTTTTA
66
7844
7859
kdkdddddddddeeee
611

601343
2610
2625
Exon 18
TTGTCGCAGCTGTTTT
71
7845
7860
ekkdddddddddkeee
612

601382
2610
2625
Exon 18
TTGTCGCAGCTGTTTT
84
7845
7860
kdkdddddddddeeee
612

601344
2611
2626
Exon 18
GTTGTCGCAGCTGTTT
87
7846
7861
ekkdddddddddkeee
613

601383
2611
2626
Exon 18
GTTGTCGCAGCTGTTT
85
7846
7861
kdkdddddddddeeee
613

601345
2612
2627
Exon 18
TGTTGTCGCAGCTGTT
82
7847
7862
ekkdddddddddkeee
614

601384
2612
2627
Exon 18
TGTTGTCGCAGCTGTT
79
7847
7862
kdkdddddddddeeee
614

601346
2613
2628
Exon 18/
TTGTTGTCGCAGCTGT
73
n/a
n/a
ekkdddddddddkeee
615

Repeat

601385
2613
2628
Exon 18/
TTGTTGTCGCAGCTGT
84
n/a
n/a
kdkdddddddddeeee
615

Repeat

601347
2614
2629
Exon 18/
TTTGTTGTCGCAGCTG
70
n/a
n/a
ekkdddddddddkeee
616

Repeat

601386
2614
2629
Exon 18/
TTTGTTGTCGCAGCTG
71
n/a
n/a
kdkdddddddddeeee
616

Repeat

601348
2615
2630
Exon 18/
TTTTGTTGTCGCAGCT
71
n/a
n/a
ekkdddddddddkeee
617

Repeat

601387
2615
2630
Exon 18/
TTTTGTTGTCGCAGCT
76
n/a
n/a
kdkdddddddddeeee
617

Repeat

601349
2616
2631
Exon 18/
TTTTTGTTGTCGCAGC
71
n/a
n/a
ekkdddddddddkeee
618

Repeat

601388
2616
2631
Exon 18/
TTTTTGTTGTCGCAGC
67
n/a
n/a
kdkdddddddddeeee
618

Repeat

TABLE 145

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
start
stop
Target

inhi-
start
stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599357
2582
2600
Exon 18
CCACGCCCCTGTCCAGCAG
26
7817
7835
5-9-5
708

599358
2583
2601
Exon 18
CCCACGCCCCTGTCCAGCA
22
7818
7836
5-9-5
709

599359
2584
2602
Exon 18
TCCCACGCCCCTGTCCAGC
13
7819
7837
5-9-5
710

599360
2585
2603
Exon 18
ATCCCACGCCCCTGTCCAG
7
7820
7838
5-9-5
711

599361
2586
2604
Exon 18
AATCCCACGCCCCTGTCCA
11
7821
7839
5-9-5
712

599362
2587
2605
Exon 18
CAATCCCACGCCCCTGTCC
14
7822
7840
5-9-5
713

599363
2588
2606
Exon 18
TCAATCCCACGCCCCTGTC
17
7823
7841
5-9-5
714

599364
2589
2607
Exon 18
TTCAATCCCACGCCCCTGT
20
7824
7842
5-9-5
715

599365
2590
2608
Exon 18
ATTCAATCCCACGCCCCTG
22
7825
7843
5-9-5
716

599366
2591
2609
Exon 18
AATTCAATCCCACGCCCCT
13
7826
7844
5-9-5
717

599367
2592
2610
Exon 18
TAATTCAATCCCACGCCCC
11
7827
7845
5-9-5
718

599368
2593
2611
Exon 18
TTAATTCAATCCCACGCCC
10
7828
7846
5-9-5
719

599369
2594
2612
Exon 18
TTTAATTCAATCCCACGCC
19
7829
7847
5-9-5
720

599370
2595
2613
Exon 18
TTTTAATTCAATCCCACGC
23
7830
7848
5-9-5
721

599371
2596
2614
Exon 18
GTTTTAATTCAATCCCACG
4
7831
7849
5-9-5
722

599372
2597
2615
Exon 18
TGTTTTAATTCAATCCCAC
16
7832
7850
5-9-5
723

599373
2598
2616
Exon 18
CTGTTTTAATTCAATCCCA
3
7833
7851
5-9-5
724

599374
2599
2617
Exon 18
GCTGTTTTAATTCAATCCC
10
7834
7852
5-9-5
725

599375
2600
2618
Exon 18
AGCTGTTTTAATTCAATCC
17
7835
7853
5-9-5
726

599376
2601
2619
Exon 18
CAGCTGTTTTAATTCAATC
18
7836
7854
5-9-5
727

599377
2602
2620
Exon 18
GCAGCTGTTTTAATTCAAT
22
7837
7855
5-9-5
728

599378
2603
2621
Exon 18
CGCAGCTGTTTTAATTCAA
11
7838
7856
5-9-5
729

599511
2552
2571
Exon 18
ATAGAAAACCCAAATCCTCA
7
7787
7806
6-8-6
410

599389
2553
2572
Exon 18
TATAGAAAACCCAAATCCTC
22
7788
7807
6-8-6
411

599390
2554
2573
Exon 18
TTATAGAAAACCCAAATCCT
21
7789
7808
6-8-6
412

599391
2555
2574
Exon 18
CTTATAGAAAACCCAAATCC
27
7790
7809
6-8-6
413

599392
2556
2575
Exon 18
CCTTATAGAAAACCCAAATC
30
7791
7810
6-8-6
414

599393
2557
2576
Exon 18
CCCTTATAGAAAACCCAAAT
30
7792
7811
6-8-6
415

599394
2558
2577
Exon 18
CCCCTTATAGAAAACCCAAA
28
7793
7812
6-8-6
416

599395
2559
2578
Exon 18
ACCCCTTATAGAAAACCCAA
23
7794
7813
6-8-6
417

599396
2560
2579
Exon 18
AACCCCTTATAGAAAACCCA
53
7795
7814
6-8-6
418

599397
2561
2580
Exon 18
AAACCCCTTATAGAAAACCC
33
7796
7815
6-8-6
419

599398
2562
2581
Exon 18
GAAACCCCTTATAGAAAACC
58
7797
7816
6-8-6
420

599399
2563
2582
Exon 18
GGAAACCCCTTATAGAAAAC
23
7798
7817
6-8-6
421

599400
2564
2583
Exon 18
AGGAAACCCCTTATAGAAAA
54
7799
7818
6-8-6
422

599401
2565
2584
Exon 18
CAGGAAACCCCTTATAGAAA
30
7800
7819
6-8-6
423

599402
2566
2585
Exon 18
GCAGGAAACCCCTTATAGAA
25
7801
7820
6-8-6
424

599403
2567
2586
Exon 18
AGCAGGAAACCCCTTATAGA
17
7802
7821
6-8-6
425

599404
2568
2587
Exon 18
CAGCAGGAAACCCCTTATAG
20
7803
7822
6-8-6
426

599405
2569
2588
Exon 18
CCAGCAGGAAACCCCTTATA
12
7804
7823
6-8-6
427

599406
2570
2589
Exon 18
TCCAGCAGGAAACCCCTTAT
51
7805
7824
6-8-6
428

599407
2571
2590
Exon 18
GTCCAGCAGGAAACCCCTTA
39
7806
7825
6-8-6
237

599408
2572
2591
Exon 18
TGTCCAGCAGGAAACCCCTT
53
7807
7826
6-8-6
429

599409
2573
2592
Exon 18
CTGTCCAGCAGGAAACCCCT
65
7808
7827
6-8-6
430

599410
2574
2593
Exon 18
CCTGTCCAGCAGGAAACCCC
56
7809
7828
6-8-6
431

599411
2575
2594
Exon 18
CCCTGTCCAGCAGGAAACCC
60
7810
7829
6-8-6
432

599412
2576
2595
Exon 18
CCCCTGTCCAGCAGGAAACC
61
7811
7830
6-8-6
433

599413
2577
2596
Exon 18
GCCCCTGTCCAGCAGGAAAC
40
7812
7831
6-8-6
238

599414
2578
2597
Exon 18
CGCCCCTGTCCAGCAGGAAA
41
7813
7832
6-8-6
434

599415
2579
2598
Exon 18
ACGCCCCTGTCCAGCAGGAA
37
7814
7833
6-8-6
435

599416
2580
2599
Exon 18
CACGCCCCTGTCCAGCAGGA
54
7815
7834
6-8-6
436

599417
2581
2600
Exon 18
CCACGCCCCTGTCCAGCAGG
36
7816
7835
6-8-6
437

599418
2582
2601
Exon 18
CCCACGCCCCTGTCCAGCAG
53
7817
7836
6-8-6
438

599419
2583
2602
Exon 18
TCCCACGCCCCTGTCCAGCA
54
7818
7837
6-8-6
439

599420
2584
2603
Exon 18
ATCCCACGCCCCTGTCCAGC
50
7819
7838
6-8-6
440

599421
2585
2604
Exon 18
AATCCCACGCCCCTGTCCAG
48
7820
7839
6-8-6
441

599422
2586
2605
Exon 18
CAATCCCACGCCCCTGTCCA
55
7821
7840
6-8-6
442

599423
2587
2606
Exon 18
TCAATCCCACGCCCCTGTCC
75
7822
7841
6-8-6
443

599424
2588
2607
Exon 18
TTCAATCCCACGCCCCTGTC
69
7823
7842
6-8-6
444

599425
2589
2608
Exon 18
ATTCAATCCCACGCCCCTGT
77
7824
7843
6-8-6
445

599426
2590
2609
Exon 18
AATTCAATCCCACGCCCCTG
60
7825
7844
6-8-6
446

599427
2591
2610
Exon 18
TAATTCAATCCCACGCCCCT
72
7826
7845
6-8-6
447

599428
2592
2611
Exon 18
TTAATTCAATCCCACGCCCC
81
7827
7846
6-8-6
448

599429
2593
2612
Exon 18
TTTAATTCAATCCCACGCCC
68
7828
7847
6-8-6
449

599430
2594
2613
Exon 18
TTTTAATTCAATCCCACGCC
58
7829
7848
6-8-6
450

599431
2595
2614
Exon 18
GTTTTAATTCAATCCCACGC
70
7830
7849
6-8-6
451

599432
2596
2615
Exon 18
TGTTTTAATTCAATCCCACG
85
7831
7850
6-8-6
452

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
85
7839
7858
5-10-5
317

599379
2604
2622
Exon 18
TCGCAGCTGTTTTAATTCA
73
7839
7857
5-9-5
730

599380
2605
2623
Exon 18
GTCGCAGCTGTTTTAATTC
77
7840
7858
5-9-5
731

599381
2606
2624
Exon 18
TGTCGCAGCTGTTTTAATT
69
7841
7859
5-9-5
732

599382
2607
2625
Exon 18
TTGTCGCAGCTGTTTTAAT
58
7842
7860
5-9-5
733

599383
2608
2626
Exon 18
GTTGTCGCAGCTGTTTTAA
52
7843
7861
5-9-5
734

599384
2609
2627
Exon 18
TGTTGTCGCAGCTGTTTTA
63
7844
7862
5-9-5
735

599385
2610
2628
Exon 18/
TTGTTGTCGCAGCTGTTTT
53
n/a
n/a
5-9-5
736

Repeat

599386
2611
2629
Exon 18/
TTTGTTGTCGCAGCTGTTT
63
n/a
n/a
5-9-5
737

Repeat

599387
2612
2630
Exon 18/
TTTTGTTGTCGCAGCTGTT
64
n/a
n/a
5-9-5
438

Repeat

599388
2613
2631
Exon 18/
TTTTTGTTGTCGCAGCTGT
66
n/a
n/a
5-9-5
739

Repeat

TABLE 146

Inhibition of CFB mRNA by MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2

SEQ

ISIS
start
stop
Target

inhi-
start
stop

ID

NO
site
site
region
Sequence
bition
site
site
Motif
NO:

599213
2553
2570
Exon 18
TAGAAAACCCAAATCCTC
0
7788
7805
3-10-5
785

599214
2554
2571
Exon 18
ATAGAAAACCCAAATCCT
0
7789
7806
3-10-5
786

599215
2555
2572
Exon 18
TATAGAAAACCCAAATCC
36
7790
7807
3-10-5
787

599216
2556
2573
Exon 18
TTATAGAAAACCCAAATC
8
7791
7808
3-10-5
788

599217
2557
2574
Exon 18
CTTATAGAAAACCCAAAT
5
7792
7809
3-10-5
789

599218
2558
2575
Exon 18
CCTTATAGAAAACCCAAA
0
7793
7810
3-10-5
790

599219
2559
2576
Exon 18
CCCTTATAGAAAACCCAA
8
7794
7811
3-10-5
791

599220
2560
2577
Exon 18
CCCCTTATAGAAAACCCA
0
7795
7812
3-10-5
740

599221
2561
2578
Exon 18
ACCCCTTATAGAAAACCC
54
7796
7813
3-10-5
741

599222
2562
2579
Exon 18
AACCCCTTATAGAAAACC
3
7797
7814
3-10-5
742

599223
2563
2580
Exon 18
AAACCCCTTATAGAAAAC
0
7798
7815
3-10-5
743

599224
2564
2581
Exon 18
GAAACCCCTTATAGAAAA
0
7799
7816
3-10-5
744

599225
2566
2583
Exon 18
AGGAAACCCCTTATAGAA
60
7801
7818
3-10-5
745

599226
2567
2584
Exon 18
CAGGAAACCCCTTATAGA
0
7802
7819
3-10-5
746

599227
2568
2585
Exon 18
GCAGGAAACCCCTTATAG
37
7803
7820
3-10-5
747

599228
2569
2586
Exon 18
AGCAGGAAACCCCTTATA
0
7804
7821
3-10-5
748

599229
2570
2587
Exon 18
CAGCAGGAAACCCCTTAT
39
7805
7822
3-10-5
749

599230
2571
2588
Exon 18
CCAGCAGGAAACCCCTTA
10
7806
7823
3-10-5
750

599231
2572
2589
Exon 18
TCCAGCAGGAAACCCCTT
16
7807
7824
3-10-5
751

599232
2573
2590
Exon 18
GTCCAGCAGGAAACCCCT
9
7808
7825
3-10-5
752

599233
2574
2591
Exon 18
TGTCCAGCAGGAAACCCC
44
7809
7826
3-10-5
753

599234
2575
2592
Exon 18
CTGTCCAGCAGGAAACCC
14
7810
7827
3-10-5
754

599235
2576
2593
Exon 18
CCTGTCCAGCAGGAAACC
0
7811
7828
3-10-5
755

599236
2577
2594
Exon 18
CCCTGTCCAGCAGGAAAC
43
7812
7829
3-10-5
756

599237
2578
2595
Exon 18
CCCCTGTCCAGCAGGAAA
0
7813
7830
3-10-5
757

599238
2580
2597
Exon 18
CGCCCCTGTCCAGCAGGA
9
7815
7832
3-10-5
758

599239
2581
2598
Exon 18
ACGCCCCTGTCCAGCAGG
36
7816
7833
3-10-5
759

599240
2582
2599
Exon 18
CACGCCCCTGTCCAGCAG
11
7817
7834
3-10-5
760

599241
2583
2600
Exon 18
CCACGCCCCTGTCCAGCA
51
7818
7835
3-10-5
761

599242
2584
2601
Exon 18
CCCACGCCCCTGTCCAGC
7
7819
7836
3-10-5
762

599243
2585
2602
Exon 18
TCCCACGCCCCTGTCCAG
47
7820
7837
3-10-5
763

599244
2586
2603
Exon 18
ATCCCACGCCCCTGTCCA
37
7821
7838
3-10-5
764

599245
2587
2604
Exon 18
AATCCCACGCCCCTGTCC
35
7822
7839
3-10-5
765

599246
2588
2605
Exon 18
CAATCCCACGCCCCTGTC
21
7823
7840
3-10-5
766

599247
2589
2606
Exon 18
TCAATCCCACGCCCCTGT
61
7824
7841
3-10-5
767

599248
2590
2607
Exon 18
TTCAATCCCACGCCCCTG
51
7825
7842
3-10-5
768

599249
2591
2608
Exon 18
ATTCAATCCCACGCCCCT
58
7826
7843
3-10-5
769

599250
2592
2609
Exon 18
AATTCAATCCCACGCCCC
49
7827
7844
3-10-5
770

599251
2593
2610
Exon 18
TAATTCAATCCCACGCCC
46
7828
7845
3-10-5
771

599252
2594
2611
Exon 18
TTAATTCAATCCCACGCC
32
7829
7846
3-10-5
772

599253
2595
2612
Exon 18
TTTAATTCAATCCCACGC
23
7830
7847
3-10-5
773

599254
2596
2613
Exon 18
TTTTAATTCAATCCCACG
0
7831
7848
3-10-5
774

599255
2597
2614
Exon 18
GTTTTAATTCAATCCCAC
61
7832
7849
3-10-5
775

599256
2598
2615
Exon 18
TGTTTTAATTCAATCCCA
64
7833
7850
3-10-5
776

599257
2599
2616
Exon 18
CTGTTTTAATTCAATCCC
66
7834
7851
3-10-5
777

599258
2600
2617
Exon 18
GCTGTTTTAATTCAATCC
59
7835
7852
3-10-5
778

599259
2601
2618
Exon 18
AGCTGTTTTAATTCAATC
40
7836
7853
3-10-5
779

599260
2602
2619
Exon 18
CAGCTGTTTTAATTCAAT
38
7837
7854
3-10-5
780

599261
2603
2620
Exon 18
GCAGCTGTTTTAATTCAA
54
7838
7855
3-10-5
781

599509
2552
2570
Exon 18
TAGAAAACCCAAATCCTCA
54
7787
7805
6-7-6
681

599273
2553
2571
Exon 18
ATAGAAAACCCAAATCCTC
0
7788
7806
6-7-6
682

599274
2554
2572
Exon 18
TATAGAAAACCCAAATCCT
57
7789
7807
6-7-6
683

599275
2556
2574
Exon 18
CTTATAGAAAACCCAAATC
0
7791
7809
6-7-6
684

599276
2557
2575
Exon 18
CCTTATAGAAAACCCAAAT
44
7792
7810
6-7-6
685

599277
2558
2576
Exon 18
CCCTTATAGAAAACCCAAA
0
7793
7811
6-7-6
686

599278
2559
2577
Exon 18
CCCCTTATAGAAAACCCAA
0
7794
7812
6-7-6
687

599279
2560
2578
Exon 18
ACCCCTTATAGAAAACCCA
20
7795
7813
6-7-6
688

599280
2561
2579
Exon 18
AACCCCTTATAGAAAACCC
70
7796
7814
6-7-6
689

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
85
7839
7858
5-10-5
317

599262
2604
2621
Exon 18
CGCAGCTGTTTTAATTCA
49
7839
7856
3-10-5
782

599263
2605
2622
Exon 18
TCGCAGCTGTTTTAATTC
49
7840
7857
3-10-5
783

599264
2606
2623
Exon 18
GTCGCAGCTGTTTTAATT
62
7841
7858
3-10-5
784

599265
2607
2624
Exon 18
TGTCGCAGCTGTTTTAAT
63
7842
7859
3-10-5
792

599266
2608
2625
Exon 18
TTGTCGCAGCTGTTTTAA
41
7843
7860
3-10-5
793

599267
2609
2626
Exon 18
GTTGTCGCAGCTGTTTTA
52
7844
7861
3-10-5
794

599268
2610
2627
Exon 18
TGTTGTCGCAGCTGTTTT
51
7845
7862
3-10-5
795

599269
2611
2628
Exon 18/
TTGTTGTCGCAGCTGTTT
58
n/a
n/a
3-10-5
796

Repeat

599270
2612
2629
Exon 18/
TTTGTTGTCGCAGCTGTT
69
n/a
n/a
3-10-5
797

Repeat

599271
2613
2630
Exon 18/
TTTTGTTGTCGCAGCTGT
69
n/a
n/a
3-10-5
798

Repeat

599272
2614
2631
Exon 18/
TTTTTGTTGTCGCAGCTG
72
n/a
n/a
3-10-5
799

Repeat

599205
2607
2624
Exon 18
TGTCGCAGCTGTTTTAAT
54
7842
7859
5-8-5
792

599206
2608
2625
Exon 18
TTGTCGCAGCTGTTTTAA
62
7843
7860
5-8-5
793

599207
2609
2626
Exon 18
GTTGTCGCAGCTGTTTTA
62
7844
7861
5-8-5
794

599208
2610
2627
Exon 18
TGTTGTCGCAGCTGTTTT
66
7845
7862
5-8-5
795

599209
2611
2628
Exon 18/
TTGTTGTCGCAGCTGTTT
60
n/a
n/a
5-8-5
796

Repeat

599210
2612
2629
Exon 18/
TTTGTTGTCGCAGCTGTT
62
n/a
n/a
5-8-5
797

Repeat

599211
2613
2630
Exon 18/
TTTTGTTGTCGCAGCTGT
65
n/a
n/a
5-8-5
798

Repeat

599212
2614
2631
Exon 18/
TTTTTGTTGTCGCAGCTG
67
n/a
n/a
5-8-5
799

Repeat

TABLE 147

Inhibition of CFB mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1 or

SEQ ID NO: 2

SEQ ID
SEQ ID

SEQ ID
SEQ ID

NO: 1
NO: 1

%
NO: 2
NO: 2
SEQ

ISIS
start
stop
Target

inhi-
start
stop
ID

NO
site
site
region
Sequence
bition
site
site
NO:

588570
150
169
Exon 1
TGGTCACATTCCCTTCCCCT
72
1871
1890
396

588571
152
171
Exon 1
CCTGGTCACATTCCCTTCCC
80
1873
1892
397

532614
154
173
Exon 1
GACCTGGTCACATTCCCTTC
65
1875
1894
12

588572
156
175
Exon 1
TAGACCTGGTCACATTCCCT
74
1877
1896
398

588573
158
177
Exon 1
CCTAGACCTGGTCACATTCC
72
1879
1898
399

588566
2189
2208
Exon 15
CCTTCCGAGTCAGCTTTTTC
66
6977
6996
400

588567
2191
2210
Exon 15
CTCCTTCCGAGTCAGCTTTT
66
6979
6998
401

532770
2193
2212
Exon 15
ACCTCCTTCCGAGTCAGCTT
64
6981
7000
198

588568
2195
2214
Exon 15
AGACCTCCTTCCGAGTCAGC
78
6983
7002
402

588569
2197
2216
Exon 15
GTAGACCTCCTTCCGAGTCA
74
6985
7004
403

588574
2453
2472
Exon 18
TTTGCCGCTTCTGGTTTTTG
71
7688
7707
404

588575
2455
2474
Exon 18
CTTTTGCCGCTTCTGGTTTT
72
7690
7709
405

532800
2457
2476
Exon 18
TGCTTTTGCCGCTTCTGGTT
71
7692
7711
228

588576
2459
2478
Exon 18
CCTGCTTTTGCCGCTTCTGG
59
7694
7713
406

588577
2461
2480
Exon 18
TACCTGCTTTTGCCGCTTCT
76
7696
7715
407

516350
2550
2569
Exon 18
AGAAAACCCAAATCCTCATC
58
7785
7804
408

588509
2551
2570
Exon 18
TAGAAAACCCAAATCCTCAT
6
7786
7805
409

588510
2552
2571
Exon 18
ATAGAAAACCCAAATCCTCA
10
7787
7806
410

588511
2553
2572
Exon 18
TATAGAAAACCCAAATCCTC
9
7788
7807
411

588512
2554
2573
Exon 18
TTATAGAAAACCCAAATCCT
80
7789
7808
412

588513
2555
2574
Exon 18
CTTATAGAAAACCCAAATCC
70
7790
7809
413

588514
2556
2575
Exon 18
CCTTATAGAAAACCCAAATC
71
7791
7810
414

588515
2557
2576
Exon 18
CCCTTATAGAAAACCCAAAT
78
7792
7811
415

588516
2558
2577
Exon 18
CCCCTTATAGAAAACCCAAA
72
7793
7812
416

588517
2559
2578
Exon 18
ACCCCTTATAGAAAACCCAA
80
7794
7813
417

588518
2560
2579
Exon 18
AACCCCTTATAGAAAACCCA
80
7795
7814
418

588519
2561
2580
Exon 18
AAACCCCTTATAGAAAACCC
62
7796
7815
419

588520
2562
2581
Exon 18
GAAACCCCTTATAGAAAACC
59
7797
7816
420

588521
2563
2582
Exon 18
GGAAACCCCTTATAGAAAAC
40
7798
7817
421

588522
2564
2583
Exon 18
AGGAAACCCCTTATAGAAAA
66
7799
7818
422

588523
2565
2584
Exon 18
CAGGAAACCCCTTATAGAAA
63
7800
7819
423

588524
2566
2585
Exon 18
GCAGGAAACCCCTTATAGAA
70
7801
7820
424

588525
2567
2586
Exon 18
AGCAGGAAACCCCTTATAGA
67
7802
7821
425

588526
2568
2587
Exon 18
CAGCAGGAAACCCCTTATAG
0
7803
7822
426

588527
2569
2588
Exon 18
CCAGCAGGAAACCCCTTATA
11
7804
7823
427

588528
2570
2589
Exon 18
TCCAGCAGGAAACCCCTTAT
15
7805
7824
428

532809
2571
2590
Exon 18
GTCCAGCAGGAAACCCCTTA
75
7806
7825
237

588529
2572
2591
Exon 18
TGTCCAGCAGGAAACCCCTT
16
7807
7826
429

588530
2573
2592
Exon 18
CTGTCCAGCAGGAAACCCCT
16
7808
7827
430

588531
2574
2593
Exon 18
CCTGTCCAGCAGGAAACCCC
19
7809
7828
431

588532
2575
2594
Exon 18
CCCTGTCCAGCAGGAAACCC
15
7810
7829
432

588533
2576
2595
Exon 18
CCCCTGTCCAGCAGGAAACC
29
7811
7830
433

532810
2577
2596
Exon 18
GCCCCTGTCCAGCAGGAAAC
74
7812
7831
238

588534
2578
2597
Exon 18
CGCCCCTGTCCAGCAGGAAA
21
7813
7832
434

588535
2579
2598
Exon 18
ACGCCCCTGTCCAGCAGGAA
16
7814
7833
435

588536
2580
2599
Exon 18
CACGCCCCTGTCCAGCAGGA
0
7815
7834
436

588537
2581
2600
Exon 18
CCACGCCCCTGTCCAGCAGG
8
7816
7835
437

588538
2582
2601
Exon 18
CCCACGCCCCTGTCCAGCAG
10
7817
7836
438

588539
2583
2602
Exon 18
TCCCACGCCCCTGTCCAGCA
23
7818
7837
439

588540
2584
2603
Exon 18
ATCCCACGCCCCTGTCCAGC
16
7819
7838
440

588541
2585
2604
Exon 18
AATCCCACGCCCCTGTCCAG
16
7820
7839
441

588542
2586
2605
Exon 18
CAATCCCACGCCCCTGTCCA
12
7821
7840
442

588543
2587
2606
Exon 18
TCAATCCCACGCCCCTGTCC
26
7822
7841
443

588544
2588
2607
Exon 18
TTCAATCCCACGCCCCTGTC
26
7823
7842
444

588545
2589
2608
Exon 18
ATTCAATCCCACGCCCCTGT
31
7824
7843
445

588546
2590
2609
Exon 18
AATTCAATCCCACGCCCCTG
22
7825
7844
446

588547
2591
2610
Exon 18
TAATTCAATCCCACGCCCCT
12
7826
7845
447

588548
2592
2611
Exon 18
TTAATTCAATCCCACGCCCC
20
7827
7846
448

588549
2593
2612
Exon 18
TTTAATTCAATCCCACGCCC
26
7828
7847
449

588550
2594
2613
Exon 18
TTTTAATTCAATCCCACGCC
32
7829
7848
450

588551
2595
2614
Exon 18
GTTTTAATTCAATCCCACGC
48
7830
7849
451

588552
2596
2615
Exon 18
TGTTTTAATTCAATCCCACG
57
7831
7850
452

588553
2597
2616
Exon 18
CTGTTTTAATTCAATCCCAC
49
7832
7851
453

588554
2598
2617
Exon 18
GCTGTTTTAATTCAATCCCA
64
7833
7852
454

532811
2599
2618
Exon 18
AGCTGTTTTAATTCAATCCC
78
7834
7853
239

588555
2600
2619
Exon 18
CAGCTGTTTTAATTCAATCC
48
7835
7854
455

588556
2601
2620
Exon 18
GCAGCTGTTTTAATTCAATC
55
7836
7855
456

588557
2602
2621
Exon 18
CGCAGCTGTTTTAATTCAAT
51
7837
7856
457

588558
2603
2622
Exon 18
TCGCAGCTGTTTTAATTCAA
51
7838
7857
458

532917
2604
2623
Exon 18
GTCGCAGCTGTTTTAATTCA
82
7839
7858
317

588559
2605
2624
Exon 18
TGTCGCAGCTGTTTTAATTC
58
7840
7859
459

588560
2606
2625
Exon 18
TTGTCGCAGCTGTTTTAATT
72
7841
7860
460

588561
2607
2626
Exon 18
GTTGTCGCAGCTGTTTTAAT
75
7842
7861
461

532952
2608
2627
Exon 18
TGTTGTCGCAGCTGTTTTAA
39
7843
7862
395

588562
2609
2628
Exon 18/
TTGTTGTCGCAGCTGTTTTA
53
n/a
n/a
462

Repeat

588563
2610
2629
Exon 18/
TTTGTTGTCGCAGCTGTTTT
62
n/a
n/a
463

Repeat

588564
2611
2630
Exon 18/
TTTTGTTGTCGCAGCTGTTT
63
n/a
n/a
464

Repeat

588565
2612
2631
Exon 18/
TTTTTGTTGTCGCAGCTGTT
64
n/a
n/a
465

Repeat

Example 122: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells by 5-10-5 MOE Gapmers

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.313 μM, 0.625 μM, 1.25 μM, 2.50 μM, 5.00 μM, or 10.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

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

TABLE 148

ISIS
0.313
0.625
1.25
2.50
5.00
10.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
(μM)

532614
7
13
43
72
65
71
2.2

532635
12
0
3
28
0
0
>10

532692
26
0
12
52
55
74
3.7

532770
21
18
32
73
64
88
1.8

532775
8
0
26
35
47
59
6.2

532800
0
5
30
65
50
75
3.1

532809
12
30
28
40
46
66
4.6

532810
28
44
32
69
84
95
1.2

532811
66
83
90
94
97
99
<0.3

532917
64
85
88
96
97
99
<0.3

532952
50
53
68
80
91
94
0.4

Example 123: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. The antisense oligonucleotides were tested in a number of experiments with 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 0.08 μM, 0.25 μM, 0.74 μM, 2.22 μM, 6.67 μM, and 20.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

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

TABLE 149

ISIS
0.08
0.25
0.74
2.22
6.67
20.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
(μM)

532811
19
53
81
87
96
97
0.2

588834
7
42
64
92
98
98
0.5

588835
11
30
66
89
97
97
0.5

588836
14
40
61
91
97
97
0.5

588837
6
39
67
89
96
97
0.5

588838
0
27
41
81
87
97
1.0

588842
17
51
68
86
93
95
0.3

588843
21
38
72
90
95
96
0.4

588870
9
31
56
88
95
97
0.6

588871
14
25
47
79
93
97
0.7

588872
18
28
59
84
92
97
0.6

TABLE 150

ISIS
0.08
0.25
0.74
2.22
6.67
20.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
(μM)

532811
31
70
89
94
97
97
0.1

588844
31
60
77
91
95
96
0.1

588846
32
52
78
89
95
97
0.2

588847
22
52
77
91
95
97
0.2

588848
20
40
73
91
96
98
0.3

588851
40
52
82
94
97
97
0.1

588854
17
55
59
84
94
96
0.4

588855
10
32
56
82
93
96
0.6

588856
13
46
75
90
96
97
0.3

588857
11
52
73
94
96
97
0.3

588858
19
48
75
94
97
98
0.3

TABLE 151

ISIS
0.08
0.25
0.74
2.22
6.67
20.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
(μM)

532811
42
66
88
96
97
98
0.1

588859
18
46
66
90
96
97
0.4

588860
55
80
94
97
97
97
<0.1

588861
24
61
86
93
96
97
0.2

588862
25
64
85
94
96
98
0.1

588863
50
73
89
96
96
98
<0.1

588864
52
80
92
96
98
98
<0.1

588865
46
72
91
96
96
99
<0.1

588866
47
76
88
96
97
98
<0.1

588867
43
69
83
92
96
99
0.1

588868
43
56
65
84
93
97
0.1

TABLE 152

ISIS
0.08
0.25
0.74
2.22
6.67
20.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
(μM)

532810
0
14
38
72
89
96
1.2

532811
18
54
79
93
96
97
0.3

532952
19
34
73
87
94
96
0.4

588534
17
13
44
77
93
97
0.9

588544
12
43
69
86
89
93
0.4

588545
17
55
67
86
91
93
0.3

588546
10
32
67
85
91
93
0.6

588552
27
54
76
90
94
97
0.2

588553
32
68
87
93
95
97
0.1

588560
16
54
76
90
94
96
0.3

588561
18
45
68
85
93
96
0.4

TABLE 153

ISIS
0.08
0.25
0.74
2.22
6.67
20.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
(μM)

532811
22
60
82
94
97
98
0.2

588536
2
38
65
90
96
97
0.6

588537
12
38
63
87
94
97
0.5

588547
19
35
61
86
93
97
0.5

588548
19
36
75
88
95
96
0.4

588554
0
76
92
95
97
97
<0.1

588555
31
61
89
96
97
98
0.1

588556
33
56
82
95
94
97
0.1

588562
12
39
71
87
94
97
0.4

588563
25
48
72
86
94
96
0.3

588564
15
33
63
89
91
97
0.5

TABLE 154

ISIS
0.08
0.25
0.74
2.22
6.67
20.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
(μM)

532811
39
68
86
96
98
98
0.1

588538
0
40
82
94
97
98
0.3

588539
34
65
88
95
98
98
0.1

588540
30
51
81
91
97
98
0.2

588549
31
57
82
95
96
98
0.1

588550
34
65
88
96
98
98
0.1

588551
47
66
87
96
98
99
<0.1

588557
40
84
95
98
98
98
<0.1

588558
45
73
93
97
98
99
<0.1

588559
51
69
83
96
98
99
<0.1

588565
19
56
81
92
96
98
0.2

Example 124: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. The antisense oligonucleotides were tested in a number of experiments with 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 0.06 μM, 0.25 μM, 1.00 μM, and 4.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

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

TABLE 155

ISIS
0.06
0.25
1.00
4.00
IC₅₀

No
μM
μM
μM
μM
(μM)

532917
31
58
87
92
0.2

588860
18
50
79
93
0.3

599001
16
28
69
90
0.5

599024
14
32
74
90
0.4

599025
0
31
56
92
0.7

599032
28
44
62
88
0.3

599033
28
46
80
92
0.2

599077
8
20
59
80
0.8

599080
9
33
48
76
0.9

599086
7
22
53
83
0.8

599087
21
31
74
87
0.4

599088
13
37
69
82
0.5

599089
3
36
55
79
0.7

599093
25
59
79
88
0.2

599094
19
29
75
89
0.4

599095
29
43
67
87
0.3

599096
23
51
70
88
0.3

599149
20
53
82
92
0.3

599188
0
21
62
85
0.8

TABLE 156

ISIS
0.06
0.25
1.00
4.00
IC₅₀

No
μM
μM
μM
μM
(μM)

532917
0
42
81
91
0.4

588860
17
49
74
92
0.3

599155
29
52
67
87
0.3

599198
3
25
64
89
0.6

599201
13
26
67
91
0.5

599202
0
44
72
87
0.5

599203
22
41
75
88
0.3

599314
12
34
71
84
0.5

599316
7
37
66
88
0.5

599317
8
1
54
83
1.0

599321
8
33
70
85
0.5

599322
24
38
66
87
0.4

599327
22
32
66
89
0.4

599328
0
31
59
88
0.7

599330
5
43
67
84
0.5

599374
23
42
80
91
0.3

599378
21
57
80
93
0.2

599380
23
56
82
93
0.2

599432
17
37
73
93
0.4

TABLE 157

ISIS
0.06
0.25
1.00
4.00
IC₅₀

No
μM
μM
μM
μM
(μM)

532917
23
65
76
93
0.2

588860
17
60
76
90
0.3

601282
48
68
81
88
0.1

601269
18
59
80
94
0.2

601276
34
64
81
91
0.1

601275
14
39
78
90
0.4

601344
52
84
92
94
<0.06

601383
53
81
86
94
<0.06

601382
41
76
88
94
0.1

601385
52
74
89
91
<0.06

601332
41
69
86
94
0.1

601345
36
75
86
95
0.1

601371
34
72
91
93
0.1

601384
50
78
91
95
<0.06

601380
28
57
83
92
0.2

601387
48
61
82
88
0.1

601341
28
65
83
91
0.2

601346
31
69
82
93
0.1

601335
24
56
85
92
0.2

TABLE 158

ISIS
0.06
0.25
1.00
4.00
IC₅₀

No
μM
μM
μM
μM
(μM)

532917
31
66
86
93
0.1

588860
28
62
85
94
0.2

599208
24
50
71
89
0.3

599261
31
49
81
94
0.2

599267
41
48
80
88
0.2

599268
28
56
75
92
0.2

599313
14
24
71
92
0.5

599441
24
57
80
87
0.2

599494
13
55
86
94
0.3

599552
30
69
93
95
0.1

599553
34
71
93
96
0.1

599554
30
74
93
96
0.1

599568
40
77
90
97
0.1

599570
61
82
93
96
<0.06

599577
18
62
81
93
0.2

599581
27
60
80
94
0.2

599591
49
74
93
96
<0.06

599592
46
76
90
94
0.1

599593
44
72
91
95
0.1

TABLE 159

ISIS
0.06
0.25
1.00
4.00
IC₅₀

No
μM
μM
μM
μM
(μM)

532917
25
56
84
92
0.2

588860
11
51
80
92
0.3

599547
23
60
82
90
0.2

599569
42
73
85
88
0.1

599578
29
49
82
89
0.2

599582
21
56
78
91
0.2

599590
24
62
80
90
0.2

601209
21
49
85
88
0.3

601210
34
64
86
92
0.1

601212
46
68
88
90
0.1

601213
54
80
90
92
<0.06

601214
38
77
88
95
0.1

601215
42
64
85
92
0.1

601216
45
57
76
89
0.1

601264
29
58
86
95
0.2

601278
51
82
83
93
<0.06

601279
44
80
92
96
0.1

601280
44
73
87
94
0.1

601281
51
80
91
94
<0.06

Example 125: Dose-Dependent Antisense Inhibition of Human CFB in HepG2 Cells

Gapmers from studies described above exhibiting in vitro inhibition of CFB mRNA were selected and tested at various doses in HepG2 cells. Additionally, a deoxy, MOE and (S)-cEt oligonucleotide, ISIS 594430, was designed with the same sequence (CTCCTTCCGAGTCAGC, SEQ ID NO: 549) and target region (target start site 2195 of SEQ ID NO: 1 and target start site 6983 of SED ID NO: 2) as ISIS 588870, another deoxy, MOE, and (S)-cEt oligonucleotide. ISIS 594430 is a 3-10-3 (S)-cEt gapmer.

Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.01 μM, 0.04 μM, 0.12 μM, 0.37 μM, 1.11 μM, 3.33 μM, and 10.00 μM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and CFB mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3459 was used to measure mRNA levels. CFB mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of CFB, relative to untreated control cells.

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

TABLE 160

ISIS
0.01
0.04
0.12
0.37
1.11
3.33
10.00
IC₅₀

No
μM
μM
μM
μM
μM
μM
μM
(μM)

588536
0
0
0
5
45
73
94
1.4

588548
0
0
0
19
52
78
90
1.2

588553
0
0
9
42
76
85
94
0.6

588555
0
52
23
58
78
83
95
0.3

588847
4
1
18
45
67
84
96
0.5

588848
0
3
13
38
67
83
95
0.6

594430
0
0
10
34
50
55
84
1.4

Example 126: Tolerability of MOE Gapmers Targeting Human CFB in CD1 Mice

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

Study 1 (with 5-10-5 MOE Gapmers)

Groups of seven-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotide. A group of male CD1 mice was injected subcutaneously once a week for 6 weeks with PBS. One group of mice was injected with subcutaneously once a week for 6 weeks with 100 mg/kg of control oligonucleotide ISIS 141923 (CCTTCCCTGAAGGTTCCTCC, designated herein as SEQ ID NO: 809, 5-10-5 MOE gapmer with no known murine target). 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, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. 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 161

Plasma chemistry markers in CD1 mice plasma on day 40

ALT (IU/L)
AST (IU/L)
BUN (mg/dL)

PBS
25
46
20

ISIS 532614
513
407
22

ISIS 532692
131
130
24

ISIS 532770
36
53
25

ISIS 532775
193
158
23

ISIS 532800
127
110
25

ISIS 532809
36
42
22

ISIS 532810
229
286
26

ISIS 532811
197
183
21

ISIS 532917
207
204
27

ISIS 532952
246
207
25

ISIS 141923
39
67
23

Weights Body weights of the mice were measured on day 40 before sacrificing the mice. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 162

Weights (g) of CD1 mice on day 40

Body
Kidney
Liver
Spleen

PBS
44
0.8
2.0
0.1

ISIS 532614
43
0.7
4.3
0.2

ISIS 532692
42
0.7
2.6
0.2

ISIS 532770
42
0.6
2.3
0.2

ISIS 532775
42
0.7
2.5
0.2

ISIS 532800
43
0.6
2.8
0.3

ISIS 532809
42
0.6
2.2
0.1

ISIS 532810
43
0.6
2.3
0.2

ISIS 532811
41
0.7
2.4
0.2

ISIS 532917
42
0.7
3.0
0.2

ISIS 532952
44
0.8
2.5
0.3

ISIS 141923
41
0.6
2.0
0.1

Study 2 (with 5-10-5 MOE Gapmers)

Groups of six- to eight-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotide. Two groups of male CD1 mice were injected subcutaneously once a week for 6 weeks with PBS. One group of mice was injected with subcutaneously once a week for 6 weeks with 100 mg/kg of control oligonucleotide ISIS 141923. 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, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. 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 163

Plasma chemistry markers in CD1 mice plasma on day 45

ALT
AST
Albumin
BUN

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

PBS
39
53
2.9
29

PBS
50
97
2.9
30

ISIS 141923
163
174
4.1
25

ISIS 532810
321
297
2.5
26

ISIS 532952
182
199
2.7
27

ISIS 588534
276
248
2.6
29

ISIS 588536
48
60
2.9
31

ISIS 588537
72
79
4.0
25

ISIS 588538
63
67
4.5
29

ISIS 588539
238
177
3.9
28

ISIS 588545
496
256
4.4
24

ISIS 588547
323
210
4.4
25

ISIS 588548
61
63
4.2
27

ISIS 588549
127
132
4.1
23

ISIS 588551
302
282
4.2
22

ISIS 588552
76
98
4.0
30

ISIS 588558
1066
521
3.9
27

ISIS 588559
76
94
4.1
26

ISIS 588561
502
500
4.4
26

ISIS 588563
50
99
4.4
28

Weights

Body weights of the mice were measured on day 42. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 45. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 164

Weights (g) of CD1 mice on day 40

Body
Kidney
Liver
Spleen

PBS
44
0.7
2.4
0.1

PBS
43
0.7
2.4
0.2

ISIS 141923
43
0.6
2.4
0.2

ISIS 532810
41
0.6
1.9
0.1

ISIS 532952
43
0.6
2.4
0.2

ISIS 588534
44
0.7
2.8
0.2

ISIS 588536
43
0.7
2.7
0.2

ISIS 588537
43
0.7
2.4
0.2

ISIS 588538
44
0.7
2.8
0.2

ISIS 588539
44
0.6
2.7
0.2

ISIS 588545
44
0.8
3.3
0.3

ISIS 588547
42
0.6
3.3
0.3

ISIS 588548
43
0.6
2.8
0.2

ISIS 588549
42
0.6
2.8
0.3

ISIS 588551
39
0.6
2.2
0.2

ISIS 588552
41
0.6
2.2
0.2

ISIS 588558
44
0.7
3.3
0.3

ISIS 588559
43
0.6
2.7
0.3

ISIS 588561
40
0.7
2.4
0.3

ISIS 588563
41
0.7
2.4
0.2

Study 3 (with 5-10-5 MOE Gapmers)

Groups of six- to eight-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotide. Two groups of male CD1 mice were injected subcutaneously once 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

TABLE 165

Plasma chemistry markers in CD1 mice plasma on day 42

ALT
AST
Albumin
BUN

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

PBS
37
108
3.1
30

PBS
45
51
3.0
27

ISIS 588544
209
168
2.9
26

ISIS 588546
526
279
3.0
22

ISIS 588550
82
136
2.7
25

ISIS 588553
79
105
3.0
24

ISIS 588554
112
220
3.2
19

ISIS 588555
95
162
2.8
25

ISIS 588556
345
236
3.0
26

ISIS 588557
393
420
2.8
24

ISIS 588560
109
148
2.7
27

ISIS 588562
279
284
2.8
22

ISIS 588564
152
188
3.0
23

ISIS 588565
247
271
2.8
28

Weights Body weights of the mice were measured on day 42. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 42. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 166

Weights (g) of CD1 mice on day 40

Body
Kidney
Liver
Spleen

PBS
42
0.7
2.4
0.1

PBS
41
0.7
2.4
0.2

ISIS 588544
44
0.6
1.9
0.1

ISIS 588546
43
0.6
2.4
0.2

ISIS 588550
41
0.7
2.8
0.2

ISIS 588553
44
0.7
2.7
0.2

ISIS 588554
40
0.7
2.4
0.2

ISIS 588555
44
0.7
2.8
0.2

ISIS 588556
39
0.6
2.7
0.2

ISIS 588557
41
0.8
3.3
0.3

ISIS 588560
38
0.6
3.2
0.3

ISIS 588562
41
0.6
2.8
0.2

ISIS 588564
40
0.6
2.8
0.3

ISIS 588565
39
0.6
2.2
0.2

Study 4 (with (S) cEt Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups of ten-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide from the studies described above. In addition, two oligonucleotides, ISIS 594431 and ISIS 594432, were designed as 3-10-3 (S)-cEt gapmers and were also tested in this study. ISIS 594431 (ACCTCCTTCCGAGTCA, SEQ ID NO: 550) targets the same region as ISIS 588871, a deoxy, MOE and (S)-cEt gapmer (target start site 2197 of SEQ ID NO: 1 and target start site 6985 of SEQ ID NO: 2). ISIS 594432 (TGGTCACATTCCCTTC, SEQ ID NO: 542) targets the same region as ISIS 588872 a deoxy, MOE and (S)-cEt gapmer (target start site 154 of SEQ ID NO: 1 and target start site 1875 of SEQ ID NO: 2).

Two groups of male CD1 mice were injected subcutaneously once 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, albumin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. 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 167

Plasma chemistry markers in CD1 mice plasma on day 42

ALT
AST
Albumin
Creatinine
BUN

Chemistry
(IU/L)
(IU/L)
(g/dL)
(mg/dL)
(mg/dL)

PBS
—
71
77
2.7
0.2
29

PBS
—
30
36
2.7
0.2
26

ISIS 588834
Deoxy, MOE and (S)-cEt
436
510
2.8
0.2
25

ISIS 588835
Deoxy, MOE and (S)-cEt
70
98
3.0
0.2
27

ISIS 588836
Deoxy, MOE and (S)-cEt
442
312
2.7
0.2
27

ISIS 588846
Deoxy, MOE and (S)-cEt
50
75
2.5
0.1
28

ISIS 588847
Deoxy, MOE and (S)-cEt
44
71
2.6
0.1
24

ISIS 588848
Deoxy, MOE and (S)-cEt
47
70
2.4
0.1
27

ISIS 588857
Deoxy, MOE and (S)-cEt
1287
655
2.7
0.2
26

ISIS 588858
Deoxy, MOE and (S)-cEt
1169
676
2.5
0.2
26

ISIS 588859
Deoxy, MOE and (S)-cEt
1036
1300
3.2
0.2
25

ISIS 588861
Deoxy, MOE and (S)-cEt
749
466
3.1
0.1
24

ISIS 588862
Deoxy, MOE and (S)-cEt
1564
1283
2.9
0.2
22

ISIS 588863
Deoxy, MOE and (S)-cEt
477
362
2.8
0.1
23

ISIS 588864
Deoxy, MOE and (S)-cEt
118
165
2.9
0.2
27

ISIS 588866
Deoxy, MOE and (S)-cEt
843
784
3.2
0.2
25

ISIS 594430
3-10-3 (S)-cEt
89
99
2.4
0.1
28

ISIS 594431
3-10-3 (S)-cEt
590
433
3.0
0.2
24

ISIS 594432
3-10-3 (S)-cEt
2595
2865
2.4
0.1
25

Weights

Body weights of the mice were measured on day 39. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 42. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 168

Weights (g) of CD1 mice

Chemistry
Body
Kidney
Liver
Spleen

PBS
—
37
0.6
2.1
0.1

PBS
—
45
0.7
2.5
0.2

ISIS 588834
Deoxy, MOE and (S)-cEt
40
0.6
3.2
0.2

ISIS 588835
Deoxy, MOE and (S)-cEt
38
0.7
2.8
0.3

ISIS 588836
Deoxy, MOE and (S)-cEt
41
0.7
2.3
0.2

ISIS 588837
Deoxy, MOE and (S)-cEt
38
0.6
2.4
0.3

ISIS 588846
Deoxy, MOE and (S)-cEt
39
0.6
2.3
0.2

ISIS 588847
Deoxy, MOE and (S)-cEt
40
0.7
2.5
0.2

ISIS 588848
Deoxy, MOE and (S)-cEt
43
0.7
2.6
0.3

ISIS 588857
Deoxy, MOE and (S)-cEt
39
0.6
3.3
0.2

ISIS 588858
Deoxy, MOE and (S)-cEt
37
0.6
3.4
0.2

ISIS 588859
Deoxy, MOE and (S)-cEt
41
0.7
2.5
0.3

ISIS 588861
Deoxy, MOE and (S)-cEt
39
0.6
2.6
0.4

ISIS 588862
Deoxy, MOE and (S)-cEt
34
0.6
2.5
0.4

ISIS 588863
Deoxy, MOE and (S)-cEt
40
0.6
2.7
0.3

ISIS 588864
Deoxy, MOE and (S)-cEt
40
0.7
2.3
0.2

ISIS 588866
Deoxy, MOE and (S)-cEt
45
0.7
3.0
0.2

ISIS 594430
3-10-3 (S)-cEt
39
0.6
2.2
0.2

ISIS 594431
3-10-3 (S)-cEt
36
0.6
3.2
0.2

ISIS 594432
3-10-3 (S)-cEt
31
0.4
1.9
0.1

Study 5 (with MOE Gapmers, (S) cEt Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 50 mg/kg of ISIS oligonucleotide. Two groups of male CD1 mice were injected subcutaneously once 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

TABLE 169

Plasma chemistry markers in CD1 mice plasma on day 42

ALT
AST
Albumin
Creatinine
BUN

Chemistry
(IU/L)
(IU/L)
(g/dL)
(mg/dL)
(mg/dL)

PBS
—
33
84
2.9
0.2
28

PBS
—
32
65
2.5
0.1
27

ISIS 532692
5-10-5 MOE
363
281
3.0
0.2
30

ISIS 532770
5-10-5 MOE
69
100
2.9
0.1
28

ISIS 532775
5-10-5 MOE
371
333
2.6
0.1
29

ISIS 532800
5-10-5 MOE
104
106
2.7
0.1
31

ISIS 532809
5-10-5 MOE
69
127
2.8
0.1
26

ISIS 588540
5-10-5 MOE
66
110
2.8
0.1
26

ISIS 588838
3-10-3 (S)-cEt
391
330
2.9
0.1
25

ISIS 588842
Deoxy, MOE and (S)-cEt
224
264
2.6
0.1
26

ISIS 588843
3-10-3 (S)-cEt
185
160
2.8
0.1
24

ISIS 588844
Deoxy, MOE and (S)-cEt
304
204
2.7
0.1
25

ISIS 588851
Deoxy, MOE and (S)-cEt
186
123
2.7
0.1
31

ISIS 588854
Deoxy, MOE and (S)-cEt
1232
925
2.7
0.1
25

ISIS 588855
Deoxy, MOE and (S)-cEt
425
321
2.7
0.1
28

ISIS 588856
Deoxy, MOE and (S)-cEt
78
101
2.4
0.1
31

ISIS 588865
Deoxy, MOE and (S)-cEt
126
145
2.5
0.1
23

ISIS 588867
Deoxy, MOE and (S)-cEt
108
112
2.5
0.1
32

ISIS 588868
Deoxy, MOE and (S)-cEt
61
124
2.5
0.1
28

ISIS 588870
Deoxy, MOE and (S)-cEt
48
69
2.4
0.1
31

ISIS 588871
Deoxy, MOE and (S)-cEt
723
881
2.5
0.1
24

ISIS 588872
Deoxy, MOE and (S)-cEt
649
654
2.7
0.1
26

Weights

Body weights of the mice were measured on day 40. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 42. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 170

Weights (g) of CD1 mice

Chemistry
Body
Kidney
Liver
Spleen

PBS
—
46
0.7
2.3
0.2

PBS
—
44
0.7
2.3
0.2

ISIS 532692
5-10-5 MOE
44
0.6
2.8
0.2

ISIS 532770
5-10-5 MOE
43
0.6
2.2
0.2

ISIS 532775
5-10-5 MOE
43
0.6
2.8
0.2

ISIS 532800
5-10-5 MOE
47
0.7
2.9
0.2

ISIS 532809
5-10-5 MOE
44
0.7
2.6
0.2

ISIS 588540
5-10-5 MOE
44
0.7
2.5
0.2

ISIS 588838
3-10-3 (S)-cEt
45
0.7
3.1
0.2

ISIS 588842
Deoxy, MOE and (S)-cEt
41
0.6
2.6
0.2

ISIS 588843
3-10-3 (S)-cEt
43
0.7
2.9
0.2

ISIS 588844
Deoxy, MOE and (S)-cEt
43
0.7
2.8
0.2

ISIS 588851
Deoxy, MOE and (S)-cEt
46
0.6
2.6
0.2

ISIS 588854
Deoxy, MOE and (S)-cEt
45
0.7
4.1
0.2

ISIS 588855
Deoxy, MOE and (S)-cEt
44
0.7
2.9
0.3

ISIS 588856
Deoxy, MOE and (S)-cEt
44
0.7
3.2
0.2

ISIS 588865
Deoxy, MOE and (S)-cEt
45
0.7
2.6
0.3

ISIS 588867
Deoxy, MOE and (S)-cEt
46
0.7
3.2
0.3

ISIS 588868
Deoxy, MOE and (S)-cEt
42
0.7
2.9
0.3

ISIS 588870
Deoxy, MOE and (S)-cEt
43
0.6
2.2
0.2

ISIS 588871
Deoxy, MOE and (S)-cEt
41
0.7
3.1
0.2

ISIS 588872
Deoxy, MOE and (S)-cEt
39
0.6
3.2
0.3

Study 6 (with Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 50 mg/kg of deoxy, MOE, and (S)-cEt oligonucleotides. Two groups of male CD1 mice were injected subcutaneously once 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, albumin, creatinine, bilirubin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in the Table below. 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 171

Plasma chemistry markers in CD1 mice plasma on day 45

ALT
AST
Albumin
Creatinine
Bilirubin
BUN

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

PBS
39
78
3.4
0.2
0.2
31

PBS
37
59
2.9
0.1
0.2
27

ISIS 599552
167
208
3.0
0.1
0.2
32

ISIS 599553
43
86
2.9
0.1
0.2
28

ISIS 599554
57
101
2.2
0.2
0.2
31

ISIS 599569
469
530
3.5
0.2
0.3
27

ISIS 599577
37
84
2.9
0.1
0.1
31

ISIS 599578
45
104
2.8
0.1
0.2
30

ISIS 599581
54
88
3.1
0.1
0.2
31

ISIS 599590
1741
1466
3.1
0.1
0.3
25

ISIS 599591
2230
1183
3.2
0.1
0.3
27

ISIS 601209
68
104
2.9
0.1
0.2
30

ISIS 601212
1795
968
3.2
0.1
0.3
22

ISIS 601215
424
385
3.1
0.1
0.4
25

ISIS 601216
90
125
2.9
0.1
0.2
29

ISIS 601276
946
366
2.9
0.1
0.5
31

ISIS 601282
831
540
3.3
0.2
0.2
32

Weights

Body weights of the mice were measured on day 40. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 45. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 172

Weights (g) of CD1 mice

Body
Kidney
Liver
Spleen

PBS
40
0.7
2.1
0.2

PBS
42
0.8
2.3
0.2

ISIS 599552
38
0.6
2.3
0.2

ISIS 599553
39
0.7
2.2
0.2

ISIS 599554
39
0.7
2.4
0.2

ISIS 599569
39
0.7
2.2
0.2

ISIS 599577
41
0.7
2.5
0.2

ISIS 599578
37
0.6
2.0
0.2

ISIS 599581
40
0.6
2.5
0.2

ISIS 599590
34
0.6
3.5
0.2

ISIS 599591
38
0.8
2.7
0.2

ISIS 601209
42
0.7
2.6
0.3

ISIS 601212
38
0.6
2.9
0.2

ISIS 601215
36
0.7
2.6
0.2

ISIS 601216
42
0.6
2.7
0.2

ISIS 601276
42
0.7
3.2
0.2

ISIS 601282
38
0.7
3.2
0.2

Study 7 (with MOE Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of ISIS oligonucleotides. One group of male CD1 mice was injected subcutaneously once 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

TABLE 173

Plasma chemistry markers in CD1 mice plasma on day 45

ALT
AST
Albumin
Creatinine
BUN

Chemistry
(IU/L)
(IU/L)
(g/dL)
(mg/dL)
(mg/dL)

PBS
—
120
102
2.7
0.2
26

ISIS 588842
Deoxy, MOE and (S)-cEt
177
164
2.7
0.1
23

ISIS 588843
Deoxy, MOE and (S)-cEt
98
194
2.7
0.1
24

ISIS 588851
Deoxy, MOE and (S)-cEt
91
142
2.6
0.1
23

ISIS 588856
Deoxy, MOE and (S)-cEt
78
110
2.7
0.1
23

ISIS 599024
3-10-4 MOE
91
108
2.7
0.1
23

ISIS 599087
5-7-5 MOE
198
183
2.6
0.2
28

ISIS 599093
5-7-5 MOE
3285
2518
2.6
0.2
24

ISIS 599149
4-8-5 MOE
30
64
2.9
0.2
25

ISIS 599155
4-8-5 MOE
145
189
2.6
0.2
25

ISIS 599202
5-8-5 MOE
150
128
2.8
0.2
23

ISIS 599203
5-8-5 MOE
111
127
2.8
0.2
24

ISIS 599208
5-8-5 MOE
146
178
2.9
0.2
22

ISIS 599261
3-10-5 MOE
144
165
2.8
0.2
26

ISIS 599267
3-10-5 MOE
96
132
2.6
0.2
27

ISIS 599268
3-10-5 MOE
87
115
2.6
0.1
23

ISIS 599322
6-7-6 MOE
115
138
2.7
0.1
22

ISIS 599374
5-9-5 MOE
375
271
2.6
0.1
21

ISIS 599378
5-9-5 MOE
77
99
2.7
0.1
23

ISIS 599441
6-8-6 MOE
150
250
2.9
0.1
23

Weights

Body weights of the mice were measured on day 44. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 49. The results are presented in the Table below. ISIS oligonucleotides that caused changes in the weights outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 174

Weights (g) of CD1 mice

Chemistry
Body
Kidney
Liver
Spleen

PBS
—
39
0.6
1.9
0.1

ISIS 588842
Deoxy, MOE and (S)-cEt
38
0.5
2.1
0.1

ISIS 588843
Deoxy, MOE and (S)-cEt
41
0.6
2.4
0.2

ISIS 588851
Deoxy, MOE and (S)-cEt
42
0.6
2.2
0.2

ISIS 588856
Deoxy, MOE and (S)-cEt
42
0.7
2.6
0.2

ISIS 599024
3-10-4 MOE
41
0.6
4.0
0.2

ISIS 599087
5-7-5 MOE
44
0.8
2.6
0.3

ISIS 599093
5-7-5 MOE
39
0.6
2.3
0.2

ISIS 599149
4-8-5 MOE
42
0.7
2.8
0.2

ISIS 599155
4-8-5 MOE
41
0.7
2.1
0.2

ISIS 599202
5-8-5 MOE
43
0.6
2.6
0.2

ISIS 599203
5-8-5 MOE
42
0.6
2.6
0.2

ISIS 599208
5-8-5 MOE
40
0.6
2.1
0.2

ISIS 599261
3-10-5 MOE
39
0.7
3.4
0.3

ISIS 599267
3-10-5 MOE
42
0.8
2.5
0.3

ISIS 599268
3-10-5 MOE
41
0.7
2.1
0.2

ISIS 599322
6-7-6 MOE
43
0.6
2.2
0.2

ISIS 599374
5-9-5 MOE
37
0.6
2.2
0.2

ISIS 599378
5-9-5 MOE
43
0.7
2.7
0.2

ISIS 599441
6-8-6 MOE
42
0.6
2.5
0.3

Study 8 (with MOE Gapmers, Deoxy, MOE and (S)-cEt Oligonucleotides, and (S)-cEt Gapmers)

Groups of eight- to nine-week old male CD1 mice were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers, or 50 mg/kg of deoxy, MOE and (S)-cEt oligonucleotides or (S)-cEt gapmers. One group of male CD1 mice was injected subcutaneously once 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

TABLE 175

Plasma chemistry markers in CD1 mice plasma on day 43

Dose
ALT
AST
Albumin
Creatinine
BUN

Chemistry
(mg/kg/wk)
(IU/L)
(IU/L)
(g/dL)
(mg/dL)
(mg/dL)

PBS
—
—
37
57
2.5
0.08
26

ISIS 532770
5-10-5 MOE
100
57
73
2.5
0.07
24

ISIS 532800
5-10-5 MOE
100
74
126
2.8
0.10
26

ISIS 532809
5-10-5 MOE
100
83
73
2.5
0.07
23

ISIS 588540
5-10-5 MOE
100
106
102
2.7
0.09
27

ISIS 588544
5-10-5 MOE
100
66
62
2.6
0.10
24

ISIS 588548
5-10-5 MOE
100
48
67
2.6
0.08
23

ISIS 588550
5-10-5 MOE
100
65
106
2.5
0.10
25

ISIS 588553
5-10-5 MOE
100
78
90
2.6
0.09
25

ISIS 588555
5-10-5 MOE
100
94
89
2.5
0.08
23

ISIS 588848
Deoxy, MOE
50
38
54
2.3
0.07
25

and (S)-cEt

ISIS 594430
3-10-3 (S)-cEt
50
63
72
2.5
0.10
27

Weights

Body weights of the mice were measured on day 36. Weights of organs, liver, kidney, and spleen were also measured after the mice were sacrificed on day 43. The results for the organ weights were expressed as a ratio to the body weights and normalized to the PBS control ratio.

TABLE 176

Organ Weights/Body weight (BW) of CD1 mice

Dose
Kidney/
Liver/
Spleen/

Chemistry
(mg/kg/wk)
BW
BW
BW

PBS
—
—
1.0
1.0
1.0

ISIS 532770
5-10-5 MOE
100
1.4
1.1
1.0

ISIS 532800
5-10-5 MOE
100
1.5
1.1
0.9

ISIS 532809
5-10-5 MOE
100
1.3
1.2
0.9

ISIS 588540
5-10-5 MOE
100
1.3
1.2
1.0

ISIS 588544
5-10-5 MOE
100
1.6
1.1
1.0

ISIS 588548
5-10-5 MOE
100
1.7
1.2
1.0

ISIS 588550
5-10-5 MOE
100
1.5
1.2
1.0

ISIS 588553
5-10-5 MOE
100
1.5
1.0
0.8

ISIS 588555
5-10-5 MOE
100
1.8
1.2
1.0

ISIS 588848
Deoxy, MOE
50
1.3
1.0
0.9

and (S)-cEt

ISIS 594430
3-10-3 (S)-cEt
50
1.4
1.1
0.9

Cytokine Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for measurements of the various cytokine levels, such as IL-6, MDC, MIP1β, IP-10, MCP1, MIP-1α, and RANTES. The results are presented in Table 54.

TABLE 177

Cytokine levels (pg/mL) in CD1 mice plasma

Chemistry
IL-6
MDC
MIP1β
IP-10
MCP1
MIP-1α
RANTES

PBS
—
70
16
23
20
17
6
2

ISIS 532770
5-10-5 MOE
101
18
146
116
101
24
6

ISIS 532800
5-10-5 MOE
78
17
83
53
105
1
3

ISIS 532809
5-10-5 MOE
66
19
60
32
55
20
4

ISIS 588540
5-10-5 MOE
51
18
126
70
75
4
3

ISIS 588544
5-10-5 MOE
157
14
94
34
102
1
3

ISIS 588548
5-10-5 MOE
164
12
90
66
84
10
4

ISIS 588550
5-10-5 MOE
58
21
222
124
157
3
5

ISIS 588553
5-10-5 MOE
62
14
183
60
103
9
4

ISIS 588555
5-10-5 MOE
70
19
172
171
178
16
9

ISIS 588848
Deoxy, MOE
59
13
61
27
63
12
4

and (S)-cEt

ISIS 594430
3-10-3 (S)-cEt
48
14
56
38
85
10
3

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for measurements of hematocrit (HCT), as well as of the various blood cells, such as WBC, RBC, and platelets, and total hemoglobin (Hb) content. The results are presented in Table 55.

TABLE 178

Hematology markers in CD1 mice plasma

HCT
Hb
WBC
RBC
Platelets

Chemistry
(%)
(g/dL)
(10³/μL)
(10⁶/μL)
(10³/μL)

PBS
—
46
15
7
9
960

ISIS 532770
5-10-5 MOE
45
14
5
9
879

ISIS 532800
5-10-5 MOE
45
14
5
9
690

ISIS 532809
5-10-5 MOE
46
14
6
9
1005

ISIS 588540
5-10-5 MOE
49
15
6
10
790

ISIS 588544
5-10-5 MOE
36
11
7
7
899

ISIS 588548
5-10-5 MOE
46
14
6
9
883

ISIS 588550
5-10-5 MOE
42
13
8
8
721

ISIS 588553
5-10-5 MOE
45
14
6
9
719

ISIS 588555
5-10-5 MOE
43
13
8
9
838

ISIS 588848
Deoxy, MOE
40
15
8
10
840

and (S)-cEt

ISIS 594430
3-10-3 (S)-cEt
45
14
8
9
993

Example 127: Tolerability of Antisense Oligonucleotides Targeting Human CFB in Sprague-Dawley Rats

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

Study 1 (with 5-10-5 MOE Gapmers)

Male Sprague-Dawley rats, seven- to eight-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of 5-10-5 MOE gapmers. One control group of 6 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

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

TABLE 179

Liver function markers in Sprague-Dawley rats

ALT (IU/L)
AST (IU/L)

PBS
66
134

ISIS 588544
101
329

ISIS 588550
69
157

ISIS 588553
88
304

ISIS 588554
202
243

ISIS 588555
94
113

ISIS 588556
102
117

ISIS 588560
206
317

ISIS 588564
292
594

Kidney Function

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

TABLE 180

Kidney function markers (mg/dL) in Sprague-Dawley rats

BUN
Creatinine

PBS
18
3.5

ISIS 588544
21
3.1

ISIS 588550
21
3.0

ISIS 588553
22
2.8

ISIS 588554
23
3.0

ISIS 588555
22
3.5

ISIS 588556
21
3.2

ISIS 588560
26
2.4

ISIS 588564
24
2.7

Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.

TABLE 181

Weights (g)

Body
Liver
Spleen
Kidney

PBS
422
16
1.2
3.9

ISIS 588544
353
15
1.7
2.9

ISIS 588550
321
14
2.1
3.2

ISIS 588553
313
15
2.3
3.2

ISIS 588554
265
11
1.6
2.7

ISIS 588555
345
14
1.4
3.3

ISIS 588556
328
13
1.7
3.1

ISIS 588560
270
13
2.4
3.0

ISIS 588564
253
12
2.9
3.0

Study 2 (with Deoxy, MOE and (S)-cEt Oligonucleotides)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of deoxy, MOE, and (S)-cEt oligonucleotides. Two control groups of 3 rats each were injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 42 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase), and albumin were measured and the results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 182

Liver function markers in Sprague-Dawley rats

ALT (IU/L)
AST (IU/L)
Albumin (g/dL)

PBS
55
150
3.4

PBS
64
91
3.5

ISIS 588554
52
92
3.2

ISIS 588835
971
844
4.1

ISIS 588842
317
359
3.8

ISIS 588843
327
753
2.9

ISIS 588846
70
111
3.2

ISIS 588847
65
100
3.0

ISIS 588864
91
109
3.0

ISIS 594430
85
106
3.7

Kidney Function

TABLE 183

Kidney function markers (mg/dL) in Sprague-Dawley rats

BUN
Creatinine

PBS
17
0.4

PBS
21
0.4

ISIS 588554
20
0.4

ISIS 588835
23
0.5

ISIS 588842
22
0.4

ISIS 588843
51
0.4

ISIS 588846
25
0.5

ISIS 588847
23
0.5

ISIS 588864
23
0.4

ISIS 594430
22
0.5

Weights

TABLE 184

Weights (g)

Body
Liver
Spleen
Kidney

PBS
466
16
0.9
3.8

PBS
485
16
0.9
3.6

ISIS 588554
393
15
2.3
2.6

ISIS 588835
387
16
1.0
3.3

ISIS 588842
414
22
1.5
3.7

ISIS 588843
427
20
2.5
4.2

ISIS 588846
366
16
2.1
3.3

ISIS 588847
402
15
1.6
3.1

ISIS 588864
364
15
2.1
3.8

ISIS 594430
420
16
1.2
3.6

Study 3 (with MOE Gapmers)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers. One control group of 6 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 43 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 185

Liver function markers in Sprague-Dawley rats

ALT
AST
Albumin

Chemistry
(IU/L)
(IU/L)
(g/dL)

PBS
—
52
110
3.7

ISIS 588563
5-10-5 MOE
175
291
2.9

ISIS 599024
3-10-4 MOE
139
173
1.4

ISIS 599093
5-7-5 MOE
116
238
2.6

ISIS 599149
4-8-5 MOE
232
190
3.4

ISIS 599155
4-8-5 MOE
108
215
2.5

ISIS 599202
5-8-5 MOE
65
86
3.5

ISIS 599203
5-8-5 MOE
71
97
3.1

ISIS 599208
5-8-5 MOE
257
467
1.9

ISIS 599261
3-10-5 MOE
387
475
1.5

ISIS 599267
3-10-5 MOE
201
337
2.7

Kidney Function

TABLE 186

Kidney function markers (mg/dL) in Sprague-Dawley rats

Chemistry
BUN
Creatinine

PBS
—
16
0.3

ISIS 588563
5-10-5 MOE
26
0.4

ISIS 599024
3-10-4 MOE
135
1.2

ISIS 599093
5-7-5 MOE
29
0.4

ISIS 599149
4-8-5 MOE
23
0.4

ISIS 599155
4-8-5 MOE
29
0.4

ISIS 599202
5-8-5 MOE
19
0.4

ISIS 599203
5-8-5 MOE
22
0.4

ISIS 599208
5-8-5 MOE
26
0.3

ISIS 599261
3-10-5 MOE
228
1.6

ISIS 599267
3-10-5 MOE
24
0.4

Weights

TABLE 187

Weights (g)

Chemistry
Body
Liver
Spleen
Kidney

PBS
—
471
16
1.0
4.1

ISIS 588563
5-10-5 MOE
311
16
3.4
4.1

ISIS 599024
3-10-4 MOE
297
11
1.0
3.5

ISIS 599093
5-7-5 MOE
332
18
4.1
5.0

ISIS 599149
4-8-5 MOE
388
16
2.3
3.7

ISIS 599155
4-8-5 MOE
290
15
2.9
4.5

ISIS 599202
5-8-5 MOE
359
13
1.3
3.2

ISIS 599203
5-8-5 MOE
334
14
1.8
3.3

ISIS 599208
5-8-5 MOE
353
29
4.7
4.6

ISIS 599261
3-10-5 MOE
277
10
0.9
3.2

ISIS 599267
3-10-5 MOE
344
21
3.9
4.7

Study 4 (with MOE Gapmers)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers. One control group of 6 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 42 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 188

Liver function markers in Sprague-Dawley rats

ALT
AST
Albumin

Chemistry
(IU/L)
(IU/L)
(g/dL)

PBS
—
48
77
3.9

ISIS 532800
5-10-5 MOE
72
111
3.4

ISIS 532809
5-10-5 MOE
59
89
3.8

ISIS 588540
5-10-5 MOE
146
259
3.8

ISIS 599268
3-10-5 MOE
175
206
2.7

ISIS 599322
6-7-6 MOE
523
567
3.3

ISIS 599374
5-9-5 MOE
114
176
3.0

ISIS 599378
5-9-5 MOE
124
116
3.2

ISIS 599380
5-9-5 MOE
148
210
3.4

ISIS 599441
6-8-6 MOE
51
91
2.6

Kidney Function

TABLE 189

Kidney function markers (mg/dL) in Sprague-Dawley rats

Chemistry
BUN
Creatinine

PBS
—
15
0.4

ISIS 532800
5-10-5 MOE
26
0.5

ISIS 532809
5-10-5 MOE
18
0.5

ISIS 588540
5-10-5 MOE
22
0.5

ISIS 599268
3-10-5 MOE
28
0.5

ISIS 599322
6-7-6 MOE
24
0.5

ISIS 599374
5-9-5 MOE
29
0.5

ISIS 599378
5-9-5 MOE
22
0.4

ISIS 599380
5-9-5 MOE
26
0.5

ISIS 599441
6-8-6 MOE
24
0.4

Weights

TABLE 190

Weights (g)

Chemistry
Body
Liver
Spleen
Kidney

PBS
—
502
16
0.9
3.7

ISIS 532800
5-10-5 MOE
376
16
2.0
3.4

ISIS 532809
5-10-5 MOE
430
16
1.4
3.4

ISIS 588540
5-10-5 MOE
391
16
1.8
3.5

ISIS 599268
3-10-5 MOE
332
16
3.6
3.6

ISIS 599322
6-7-6 MOE
348
13
2.1
3.4

ISIS 599374
5-9-5 MOE
302
12
2.0
3.3

ISIS 599378
5-9-5 MOE
332
11
1.1
2.8

ISIS 599380
5-9-5 MOE
350
11
1.5
3.3

ISIS 599441
6-8-6 MOE
368
16
2.5
3.3

Study 5 (with MOE Gapmers and Deoxy, MOE and (S)-cEt Oligonucleotides)

Male Sprague-Dawley rats, nine- to ten-week old, were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmer or with 50 mg/kg of deoxy, MOE and (S)-cEt oligonucleotides. One control group of 4 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma levels of transaminases were measured on day 42 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. ISIS oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 191

Liver function markers in Sprague-Dawley rats

ALT
AST
Albumin

Chemistry
(IU/L)
(IU/L)
(g/dL)

PBS
—
49
74
3.3

ISIS 532770
5-10-5 MOE
95
132
3.3

ISIS 588851
Deoxy, MOE, and (S)-cEt
47
72
3.1

ISIS 588856
Deoxy, MOE, and (S)-cEt
56
75
3.0

ISIS 588865
Deoxy, MOE, and (S)-cEt
62
84
2.9

ISIS 588867
Deoxy, MOE, and (S)-cEt
73
214
2.9

ISIS 588868
Deoxy, MOE, and (S)-cEt
59
83
3.1

ISIS 588870
Deoxy, MOE, and (S)-cEt
144
144
3.4

Kidney Function

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

TABLE 192

Kidney function markers (mg/dL) in

the plasma of Sprague-Dawley rats

Chemistry
BUN
Creatinine

PBS
—
18
0.3

ISIS 532770
5-10-5 MOE
20
0.4

ISIS 588851
Deoxy, MOE, and (S)-cEt
20
0.4

ISIS 588856
Deoxy, MOE, and (S)-cEt
22
0.4

ISIS 588865
Deoxy, MOE, and (S)-cEt
24
0.5

ISIS 588867
Deoxy, MOE, and (S)-cEt
22
0.4

ISIS 588868
Deoxy, MOE, and (S)-cEt
19
0.4

ISIS 588870
Deoxy, MOE, and (S)-cEt
20
0.5

TABLE 193

Kidney function markers (mg/dL) in

the urine of Sprague-Dawley rats

Chemistry
Total protein
Creatinine

PBS
—
80
92

ISIS 532770
5-10-5 MOE
466
69

ISIS 588851
Deoxy, MOE, and (S)-cEt
273
64

ISIS 588856
Deoxy, MOE, and (S)-cEt
259
68

ISIS 588865
Deoxy, MOE, and (S)-cEt
277
67

ISIS 588867
Deoxy, MOE, and (S)-cEt
337
68

ISIS 588868
Deoxy, MOE, and (S)-cEt
326
75

ISIS 588870
Deoxy, MOE, and (S)-cEt
388
82

Weights

TABLE 194

Weights (g)

Chemistry
Body
Liver
Spleen
Kidney

PBS
—
489
16
0.9
3.5

ISIS 532770
5-10-5 MOE
372
15
1.7
3.1

ISIS 588851
Deoxy, MOE, and
285
14
1.4
3.2

(S)-cEt

ISIS 588856
Deoxy, MOE, and
415
15
1.1
3.3

(S)-cEt

ISIS 588865
Deoxy, MOE, and
362
14
2.0
3.3

(S)-cEt

ISIS 588867
Deoxy, MOE, and
406
15
2.4
3.4

(S)-cEt

ISIS 588868
Deoxy, MOE, and
399
15
1.5
3.4

(S)-cEt

ISIS 588870
Deoxy, MOE, and
446
14
1.4
3.3

(S)-cEt

Study 6 (with MOE Gapmers, Deoxy, MOE and (S)-cEt Oligonucleotides, and (S)-cEt Gapmers)

Male rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 rats each were injected subcutaneously once a week for 6 weeks with 100 mg/kg of MOE gapmers or with 50 mg/kg of deoxy, MOE and (S)-cEt oligonucleotide or (S)-cEt gapmer. One control group of 4 rats was injected subcutaneously once a week for 6 weeks with PBS. Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.

Liver Function

TABLE 195

Liver function markers

Albu-

Dose
ALT
AST
min

Chemistry
(mg/kg/wk)
(IU/L)
(IU/L)
(g/dL)

PBS
—
—
54
73
4.3

ISIS 532770
5-10-5 MOE
100
57
114
4.4

ISIS 532800
5-10-5 MOE
100
176
180
4.3

ISIS 532809
5-10-5 MOE
100
71
132
4.1

ISIS 588540
5-10-5 MOE
100
89
202
4.4

ISIS 588544
5-10-5 MOE
100
75
152
3.9

ISIS 588548
5-10-5 MOE
100
50
71
4.1

ISIS 588550
5-10-5 MOE
100
80
133
3.6

ISIS 588553
5-10-5 MOE
100
59
112
3.9

ISIS 588555
5-10-5 MOE
100
97
142
3.8

ISIS 588848
Deoxy, MOE
50
53
82
3.9

and (S)-cEt

ISIS 594430
3-10-3 (S)-cEt
50
198
172
4.4

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, urine levels of total protein and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in the Table below. ISIS oligonucleotides that caused changes in the levels of any of the kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.

TABLE 196

Total protein/creatinine ratio in the urine of rats

Chemistry
Dose (mg/kg/wk)
P/C ratio

PBS
—
—
1.1

ISIS 532770
5-10-5 MOE
100
8.3

ISIS 532800
5-10-5 MOE
100
6.5

ISIS 532809
5-10-5 MOE
100
6.1

ISIS 588540
5-10-5 MOE
100
10.1

ISIS 588544
5-10-5 MOE
100
7.9

ISIS 588548
5-10-5 MOE
100
6.6

ISIS 588550
5-10-5 MOE
100
7.6

ISIS 588553
5-10-5 MOE
100
7.0

ISIS 588555
5-10-5 MOE
100
6.2

ISIS 588848
Deoxy, MOE
50
5.2

and (S)-cEt

ISIS 594430
3-10-3 (S)-cEt
50
5.3

Weights

Body weight measurements were taken on day 39. Liver, heart, spleen and kidney weights were measured at the end of the study on day 42, and are presented in the Table below. The results for the organ weights were expressed as a ratio to the body weights and normalized to the PBS control ratio.

TABLE 197

Organ weights/Body weight (BW) ratios

Dose
Spleen/
Liver/
Kidney/

Chemistry
(mg/kg/wk)
BW
BW
BW

PBS
—
—
1.0
1.0
1.0

ISIS 532770
5-10-5 MOE
100
2.0
1.2
1.0

ISIS 532800
5-10-5 MOE
100
2.8
1.3
1.0

ISIS 532809
5-10-5 MOE
100
2.2
1.1
1.0

ISIS 588540
5-10-5 MOE
100
2.2
1.4
1.0

ISIS 588544
5-10-5 MOE
100
2.5
1.3
1.1

ISIS 588548
5-10-5 MOE
100
2.1
1.3
1.1

ISIS 588550
5-10-5 MOE
100
3.9
1.4
1.1

ISIS 588553
5-10-5 MOE
100
4.1
1.4
1.4

ISIS 588555
5-10-5 MOE
100
1.8
1.3
1.0

ISIS 588848
Deoxy, MOE
50
3.1
1.3
1.1

and (S)-cEt

ISIS 594430
3-10-3 (S)-cEt
50
1.7
1.0
1.1

Example 128: Efficacy of Antisense Oligonucleotides Against CFB mRNA in hCFB Mice

Selected compounds were tested for efficacy in human CFB transgenic mice, founder line #6 The human CFB gene is located on chromosome 6: position 31913721-31919861. A Fosmid (ABC14-50933200C23) containing the CFB sequence was selected to make transgenic mice expressing the human CFB gene. Cla I (31926612) and Age I (31926815) restriction enzymes were used to generate a 22,127 bp fragment containing the CFB gene for pronuclear injection. DNA was confirmed by restriction enzyme analysis using Pvu I. The 22,127 bp DNA fragment was injected into C57BL/6NTac embryos. 6 positive founders were bred. Founder #6 expressed the liver human CFB mRNA and was crossbreed to the 3^rdgeneration. Progeny from 3′ generation mice were used to evaluate human CFB ASOs for human CFB mRNA reduction.

Treatment

Groups of 3 mice each were injected subcutaneously twice a week for the first week with 50 mg/kg of ISIS oligonucleotides, followed by once a week dosing with 50 mg/kg of ISIS oligonucleotides for an additional three weeks. One control group of 4 mice was injected subcutaneously twice a week for 2 weeks for the first week with PBS for the first week for an additional three weeks. Forty eight hours after the last dose, mice were euthanized and organs and plasma were harvested for further analysis.

RNA Analysis

At the end of the dosing period, RNA was extracted from the liver and kidney for real-time PCR analysis of CFB mRNA levels. Human CFB mRNA levels were measured using the human primer probe set RTS3459. CFB mRNA levels were normalized to RIBOGREEN®, and also to the housekeeping gene, Cyclophilin. Results were calculated as percent inhibition of CFB mRNA expression compared to the control. All the antisense oligonucleotides effected inhibition of human CFB mRNA levels in the liver.

TABLE 198

Percent reduction of CFB mRNA levels in hCFB mice

Normalized to
Normalized to

ISIS No
RIBOGREEN
Cyclophilin

532770
86
87

532800
88
87

532809
69
69

588540
95
94

588544
91
91

588548
78
77

588550
89
88

588553
94
94

588555
94
94

588848
83
82

594430
78
76

Example 129: In Vivo Antisense Inhibition of Murine CFB

Several antisense oligonucleotides were designed that were targeted to murine CFB mRNA (GENBANK Accession No. NM_008198.2, incorporated herein as SEQ ID NO: 5). The target start sites and sequences of each oligonucleotide are described in the table below. The chimeric antisense oligonucleotides in the table below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleosides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

TABLE 199

Gapmers targeting murine CFB

Target Start

ISIS

Site on SEQ
SEQ ID

No
Sequence
ID NO: 5
NO

516269
GCATAAGAGGGTACCAGCTG
2593
804

516272
GTCCTTTAGCCAGGGCAGCA
2642
805

516323
TCCACCCATGTTGTGCAAGC
1568
806

516330
CCACACCATGCCACAGAGAC
1826
807

516341
TTCCGAGTCAGGCTCTTCCC
2308
808

Treatment

Groups of four C57BL/6 mice each were injected with 50 mg/kg of ISIS 516269, ISIS 516272, ISIS 516323, ISIS 516330, or ISIS 516341 administered weekly for 3 weeks. A control group of mice was injected with phosphate buffered saline (PBS) administered weekly for 3 weeks.

CFB RNA Analysis

At the end of the study, RNA was extracted from liver tissue for real-time PCR analysis of CFB, using primer probe set RTS3430 (forward sequence GGGCAAACAGCAATTTGTGA, designated herein as SEQ ID NO: 816; reverse sequence TGGCTACCCACCTTCCTTGT, designated herein as SEQ ID NO: 817; probe sequence CTGGATACTGTCCCAATCCCGGTATTCCX, designated herein as SEQ ID NO: 818). The mRNA levels were normalized using RIBOGREEN®. As shown in the Table below, some of the antisense oligonucleotides achieved reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 200

Percent inhibition of murine CFB mRNA in C57BL/6 mice

ISIS No
%

516269
29

516272
72

516323
77

516330
62

516341
72

Protein Analysis

CFB protein levels were measured in the kidney, liver, plasma, and in the eye by western Blot using goat anti-CFB antibody (Sigma Aldrich). Results are presented as percent inhibition of CFB, relative to PBS control. ‘n/a’ indicates that measurements were not taken for that sample. As shown in the Table below, antisense inhibition of CFB by ISIS oligonucleotides resulted in a reduction of CFB protein in various tissues. As shown in the Table below, systemic administration of ISIS oligonucleotides was effective in reducing CFB levels in the eye.

TABLE 201

Percent inhibition of murine CFB protein in C57BL/6 mice

ISIS No
Kidney
Liver
Plasma
Eye

516269
20
58
n/a
70

516272
48
74
n/a
99

516323
73
85
90
93

516330
77
80
n/a
n/a

516341
80
88
68
n/a

Example 130: Dose-Dependent Antisense Inhibition of Murine CFB

Groups of four C57BL/6 mice each were injected with 25 mg/kg, 50 mg/kg, or 100 mg/kg of ISIS 516272, and ISIS 516323 administered weekly for 6 weeks. Another two groups of mice were injected with 100 mg/kg of ISIS 516330 or ISIS 516341 administered weekly for 6 weeks. Two control groups of mice were injected with phosphate buffered saline (PBS) administered weekly for 6 weeks.

CFB RNA Analysis

RNA was extracted from liver and kidney tissues for real-time PCR analysis of CFB, using primer probe set RTS3430. The mRNA levels were normalized using RIBOGREEN®. As shown in the Table below, the antisense oligonucleotides achieved dose-dependent reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 202

Percent inhibition of murine CFB mRNA in C57BL/6 mice

ISIS No
Dose (mg/kg/wk)
Liver
Kidney

516272
25
39
32

50
73
36

100
87
42

516323
25
36
41

50
65
47

100
79
71

516330
100
85
45

516341
200
89
65

Protein Analysis

CFB protein levels were measured in the plasma by western Blot using goat anti-CFB antibody (Sigma Aldrich). As shown in the table below, antisense inhibition of CFB by the ISIS oligonucleotides resulted in a reduction of CFB protein. Results are presented as percent inhibition of CFB, relative to PBS control. ‘n/a’ indicates that measurements were not taken for that sample.

CFB protein levels were also measured in the eye by Western Blot. All treatment groups demonstrated an inhibition of CFB by 95%, with some sample measurements being below detection levels of the assay.

TABLE 203

Percent inhibition of murine CFB protein in C57BL/6 mice

ISIS No
Dose (mg/kg/wk)
Liver

516272
25
32

50
70

100
83

516323
25
43

50
80

100
90

516330
100
n/a

516341
200
n/a

Example 131: Effect of Antisense Inhibition of CFB in the NZB/W F1 Mouse Model

The NZB/W F1 is the oldest classical model of lupus, where the mice develop severe lupus-like phenotypes comparable to that of lupus patients (Theofilopoulos, A. N. and Dixon, F. J. Advances in Immunology, vol. 37, pp. 269-390, 1985)_nThese lupus-like phenotypes include lymphadenopathy, splenomegaly, elevated serum antinuclear autoantibodies (ANA) including anti-dsDNA IgG, a majority of which are IgG2a and IgG3, and immune complex-mediated glomerulonephritis (GN) that becomes apparent at 5-6 months of age, leading to kidney failure and death at 10-12 months of age.

Study 1

A study was conducted to demonstrate that treatment with antisense oligonucleotides targeting CFB would improve renal pathology in the mouse model. Female NZB/W F1 mice, 17 weeks old, were purchased from Jackson Laboratories. Groups of 16 mice each received doses of 100 μg/kg/week of ISIS 516272 or ISIS 516323 for 20 weeks. Another group of 16 mice received doses of 100 μg/kg/week of control oligonucleotide ISIS 141923 for 20 weeks. Another group of 10 mice received doses of PBS for 20 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. The mRNA levels were normalized using RIBOGREEN®. As shown in the Table below, some of the antisense oligonucleotides achieved reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 204

Percent inhibition of murine CFB mRNA in NZB/W F1 mice

ISIS No
Liver
Kidney

516272
55
25

516323
63
43

141923
0
0

Proteinuria

Proteinuria is expected in 60% of animals in this mouse model. The cumulative incidence of severe proteinuria was measured by calculating the total protein to creatinine ratio using a clinical analyzer. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB achieved reduction of proteinuria in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 205

Percent cumulative incidence of severe

proteinuria in NZB/W F1 mice

%

PBS
40

ISIS 516272
6

ISIS 516323
0

ISIS 141923
25

Survival

Survival of the mice was monitored by keeping count of the mice at the start of treatment and then again at week 20. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB increased survival in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 206

Number of surviving mice and % survival

Week 1
Week 20
% survival at week 20

PBS
10
6
60

ISIS 516272
16
15
94

ISIS 516323
16
16
100

ISIS 141923
16
12
75

Glomerular Deposition

The amount of C3 deposition, as well as IgG deposition, in the glomeruli of the kidneys was measured by immunohistochemistry with an anti-C3 antibody. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB achieved reduction of both C3 and IgG depositions in the kidney glomeruli compared to the PBS control and the control oligonucleotide treated mice.

TABLE 207

Percent inhibition of glomerula deposition in NZB/W F1 mice

ISIS No
C3
IgG

516272
45
20

516323
48
2

141923
0
0

Study 2

Female NZB/W F1 mice, 16 weeks old, were purchased from Jackson Laboratories. A group of 10 mice received doses of 100 μg/kg/week of ISIS 516323 for 12 weeks. Another group of 10 mice received doses of 100 μg/kg/week of control oligonucleotide ISIS 141923 for 12 weeks. Another group of 10 mice received doses of PBS for 12 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the table below, treatment with ISIS 516323 achieved reduction of murine CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 208

Percent inhibition of murine CFB mRNA in NZB/W F1 mice

ISIS No
Liver
Kidney

516323
75
46

141923
0
6

Proteinuria

The cumulative incidence of severe proteinuria was assessed by measuring urine total protein to creatinine ratio, as well as by measuring total microalbumin levels. The results are presented in the tables below and demonstrate that treatment with antisense oligonucleotides targeting CFB reduced proteinuria in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 209

Proteinuria in NZB/W F1 mice measured

as urine microalbumin levels (mg/dl)

ISIS No
Week 0
Week 6
Week 8
Week 10

516323
0
0
5.4
0.4

141923
0
8.28
8.6
5.6

TABLE 210

Proteinuria in NZB/W F1 mice measured

as total protein to creatinine ratio

ISIS No
Week 0
Week 6
Week 8
Week 10

516323
5.5
7.8
8.6
7.2

141923
6.9
10.0
13.5
7.2

Survival

Survival of the mice was monitored by keeping count of the mice at the start of treatment and then again at week 12. The results are presented in the table below and demonstrate that treatment with antisense oligonucleotides targeting CFB increased survival in the mice compared to the PBS control and the control oligonucleotide treated mice.

TABLE 211

Number of surviving mice

Week 1
Week 12

PBS
10
9

ISIS 516323
10
10

ISIS 141923
10
9

Example 132: Effect of Antisense Inhibition of CFB in the MRL Mouse Model

The MRL/lpr lupus nephritis mouse model develops an SLE-like phenotype characterized by lymphadenopathy due to an accumulation of double negative (CD4⁻ CD8⁻) and B220+ T-cells. These mice display an accelerated mortality rate. In addition, the mice have high concentrations of circulating immunoglobulins, which included elevated levels of autoantibodies such as ANA, anti-ssDNA, anti-dsDNA, anti-Sm, and rheumatoid factors, resulting in large amounts of immune complexes (Andrews, B. et al., J. Exp. Med. 148: 1198-1215, 1978).

Treatment

A study was conducted to investigate whether treatment with antisense oligonucleotides targeting CFB would reverse renal pathology in the mouse model. Female MRL/lpr mice, 14 weeks old, were purchased from Jackson Laboratories. A group of 10 mice received doses of 50 μg/kg/week of ISIS 516323 for 7 weeks. Another group of 10 mice received doses of 50 μg/kg/week of control oligonucleotide ISIS 141923 for 7 weeks. Another group of 10 mice received doses of PBS for 7 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the Table below, ISIS 516323 reduced CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 212

Percent inhibition of murine CFB mRNA in MRL/lpr mice

ISIS No
%

516323
68

141923
4

Renal Pathology

Renal pathology was evaluated by two methods. Histological sections of the kidney were stained with Haematoxylin &Eosin. The PBS control demonstrated presence of multiglomerular crescents tubular casts, which is a symptom of glomerulosclerosis. In contrast, the sections from mice treated with ISIS 516323 showed absent crescents tubular casts with minimal bowman capsule fibrotic changes, moderate to severe segmental mesangial cell expansion and glomerular basement membrane thickening.

Accumulation of C3 in the kidney was also assessed by immunohistochemistry with anti-C3 antibodies. The whole kidney C3 immunohistochemistry intensity score was calculated by intensity scoring system, which was computed by capturing 10 glomeruli per kidney and calculation the intensity of positive C3 staining. The results are presented in the table below and demonstrate that treatment with ISIS 516323 reduced renal C3 accumulation compared to the control groups.

TABLE 213

Renal C3 accumulation in MRL/lpr mice

Whole kidney C3
C3 quantification (area/

intensity score
total area) % of average PBS

PBS
2.5
100

ISIS 516323
1.6
68

ISIS 141923
2.2
99

Plasma C3 Levels

Reduction of CFB inhibits activation of the alternative complement pathway, preventing C3 consumption and leading to an apparent elevation of plasma C3 levels. Plasma C3 levels from terminal bleed were measured by clinical analyzer. The results are presented in the table below and demonstrate that treatment with ISIS 516323 increased C3 levels (p<0.001) in the plasma compared to the control groups.

TABLE 214

Plasma C3 levels (mg/dL) in MRL/lpr mice

ISIS No
C3

516323
28

141923
16

The results indicate that treatment with antisense oligonucleotides targeting CFB reverses renal pathology in the lupus mouse model.

Example 133: Effect of Antisense Inhibition of CFB in the CFH Het Mouse Model

CFH heterozygous (CFH Het, CFH^+/−) mouse model express a mutant Factor H protein in combination with the full-length mouse protein (Pickering, M. C. et al., J. Exp. Med. 2007. 204: 1249-56)_nRenal histology remains normal in these mice up to six months old.

Study 1

Groups of 8 CFH^+/− mice, 6 weeks old, each received doses of 75 mg/kg/week of ISIS 516323 or ISIS 516341 for 6 weeks. Another group of 8 mice received doses of 75 mg/kg/week of control oligonucleotide ISIS 141923 for 6 weeks. Another group of 8 mice received doses of PBS for 6 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the Table below, the antisense oligonucleotides reduced CFB over the PBS control. Results are presented as percent inhibition of CFB, relative to control.

TABLE 215

Percent inhibition of murine CFB mRNA in CFH^+/− mice

ISIS No
Liver
Kidney

516323
80
38

516341
90
44

141923
0
17

Plasma C3 levels

Reduction of CFB inhibits activation of the alternative complement pathway, preventing C3 consumption and leading to an apparent elevation of plasma C3 levels. Plasma C3 levels from terminal plasma collection were measured by clinical analyzer. The results are presented in the table below and demonstrate that treatment with ISIS 516323 increased C3 to normal levels in the plasma.

TABLE 216

Plasma C3 levels (mg/dL) in CFH^+/− mice

ISIS No
C3

516323
15

516341
17

141923
8

Study 2

Groups of 5 CFH^+/− mice each received doses of 12.5 mg/kg/week, 25 mg/kg/week, 50 mg/kg/week, 75 mg/kg/week, or 100 mg/kg/week of ISIS 516323 or ISIS 516341 for 6 weeks. Another group of 5 mice received doses of 75 μg/kg/week of control oligonucleotide ISIS 141923 for 6 weeks. Another group of 5 mice received doses of PBS for 6 weeks and served as the control group to which all the other groups were compared. Terminal endpoints were collected 48 hours after the last dose was injected.

CFB RNA Analysis

RNA was extracted from liver and kidney tissue for real-time PCR analysis of CFB, using primer probe set RTS3430. As shown in the Table below, the antisense oligonucleotides reduced CFB over the PBS control in a dose dependent manner. Results are presented as percent inhibition of CFB, relative to control.

TABLE 217

Percent inhibition of murine CFB mRNA in

the liver of CFH^+/− mice

ISIS No
Dose (mg/kg/week)
%

516323
12.5
34

25
51

50
72

75
79

100
92

516341
12.5
38

25
57

50
89

75
92

100
90

141923
75
13

Plasma C3 levels

Reduction of CFB inhibits activation of the alternative complement pathway, preventing C3 consumption and leading to an apparent elevation of plasma C3 levels. Plasma C3 levels from terminal plasma collection were measured by clinical analyzer. The results are presented in the table below and demonstrate that treatment with ISIS oligonucleotides targeting CFB increased C3 levels in the plasma.

TABLE 218

Plasma C3 levels (mg/dL) in CFH^+/− mice

Dose (mg/kg/week)
C3

PBS
—
10.1

516323
12.5
11.4

25
15.5

50
17.0

75
18.3

100
18.8

516341
12.5
12.1

25
16.3

50
18.6

75
22.1

100
19.1

141923
75
8.9

Example 134: Effect of ISIS Antisense Oligonucleotides Targeting Human CFB in Cynomolgus Monkeys

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

At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus monkeys was compared to a rhesus monkey sequence for homology. It is expected that ISIS oligonucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well. The human antisense oligonucleotides tested are cross-reactive with the rhesus genomic sequence (GENBANK Accession No. NW_001116486.1 truncated from nucleotides 536000 to 545000, designated herein as SEQ ID NO: 3). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the rhesus monkey sequence. The start and stop sites of each oligonucleotide targeted to SEQ ID NO: 3 is presented in the Table below. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence. ‘Mismatches’ indicates the number of nucleobases in the human oligonucleotide that are mismatched with the rhesus genomic sequence.

TABLE 219

Antisense oligonucleotides complementary to the

rhesus CFB genomic sequence (SEQ ID NO: 3)

ISIS
Target

SEQ

No
Start Site
Mismatches
Chemistry
ID NO

532770
6788
0
5-10-5 MOE
198

532800
7500
0
5-10-5 MOE
228

532809
7614
0
5-10-5 MOE
237

588540
7627
1
5-10-5 MOE
440

588544
7631
1
5-10-5 MOE
444

588548
7635
1
5-10-5 MOE
448

588550
7637
1
5-10-5 MOE
450

588553
7640
1
5-10-5 MOE
453

588555
7643
0
5-10-5 MOE
455

588848
7639
1
Deoxy, MOE and cEt
598

594430
6790
0
3-10-3 cEt
549

Treatment

Prior to the study, the monkeys were kept in quarantine for at least a 30 day period, during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed between 2 and 4 kg. Eleven groups of 4-6 randomly assigned male cynomolgus monkeys each were injected subcutaneously with ISIS oligonucleotide or PBS at four sites on the back in a clockwise rotation (i.e. left, top, right, and bottom), one site per dose. The monkeys were given four loading doses of PBS or 40 mg/kg of ISIS 532800, ISIS 532809, ISIS 588540, ISIS 588544, ISIS 588548, ISIS 588550, ISIS 588553, ISIS 588555, ISIS 588848, or ISIS 594430 for the first week (days 1, 3, 5, and 7), and were subsequently dosed once a week for 12 weeks (days 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84) with PBS or 40 mg/kg of ISIS oligonucleotide. ISIS 532770 was tested in a separate study with similar conditions with two male and two female cynomolgus monkeys in the group.

Hepatic Target Reduction
RNA Analysis

On day 86, liver and kidney samples were collected in duplicate (approximately 250 mg each) for CFB mRNA analysis. The samples were flash frozen in liquid nitrogen at necropsy within approximately 10 minutes of sacrifice.

RNA was extracted from liver and kidney for real-time PCR analysis of measurement of mRNA expression of CFB. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. RNA levels were also normalized with the house-keeping gene, Cyclophilin A. RNA levels were measured with the primer probe sets RTS3459, described above, or RTS4445_MGB (forward sequence CGAAGAAGCTCAGTGAAATCAA, designated herein as SEQ ID NO: 819; reverse sequence TGCCTGGAGGGCCCTCTT, designated herein as SEQ ID NO: 820; probe sequence AGACCACAAGTTGAAGTC, designated herein as SEQ ID NO: 815).

As shown in the Tables below, treatment with ISIS antisense oligonucleotides resulted in reduction of CFB mRNA in comparison to the PBS control. Analysis of CFB mRNA levels revealed that several of the ISIS oligonucleotides reduced CFB levels in liver and/or kidney. Here ‘0’ indicates that the expression levels were not inhibited. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions.

TABLE 220

Percent inhibition of CFB mRNA in the cynomolgus

monkey liver relative to the PBS control

ISIS
RTS3459/
RTS3459/
RTS445_MGB/
RTS445_MGB/

No
Cyclophilin A
RIBOGREEN
Cyclophilin A
RIBOGREEN

532770*
12
37
24
45

532800
54
45
56
46

588540
31
27
28
24

588548
68
67
68
67

588550
53
39
51
37

588553
74
59
74
59

588555
73
71
71
69

588848
9
0
6
0

594430
24
26
23
25

TABLE 221

Percent inhibition of CFB mRNA in the cynomolgus

monkey kidney relative to the PBS control

ISIS
RTS3459/
RTS3459/
RTS445_MGB/
RTS445_MGB/

No
Cyclophilin A
RIBOGREEN
Cyclophilin A
RIBOGREEN

532770*
34
56
2
31

532800
36
30
43
37

588540
70
71
67
69

588548
83
84
82
83

588550
81
77
78
74

588553
86
84
86
85

588555
32
34
48
50

588848
89
91
87
90

594430
33
37
19
23

Protein Analysis

Approximately 1 mL of blood was collected from all available animals at day 85 and placed in tubes containing the potassium salt of EDTA. The blood samples were placed in wet-ice or Kryorack immediately, and centrifuged (3000 rpm for 10 min at 4° C.) to obtain plasma (approximately 0.4 mL) within 60 minutes of collection. Plasma levels of CFB were measured in the plasma by radial immunodiffusion (RID), using a polyclonal anti-Factor B antibody. The results are presented in the Table below. ISIS 532770 was tested in a separate study and plasma protein levels were measured on day 91 or 92 in that group.

Analysis of plasma CFB revealed that several ISIS oligonucleotides reduced protein levels in a sustained manner. ISIS 532770, which was tested in a separate study, reduced CFB protein levels on day 91/92 by 50% compared to baseline values. The reduction in plasma CFB protein levels correlates well with liver CFB mRNA level reduction in the corresponding groups of animals.

TABLE 222

Plasma protein levels (% baseline

values) in the cynomolgus monkey

Day 1
Day 30
Day 58
Day 72
Day 86

PBS
113
115
95
83
86

ISIS 532800
117
68
52
39
34

ISIS 532809
104
121
100
80
71

ISIS 588540
108
72
61
40
38

ISIS 588544
118
74
53
33
29

ISIS 588548
110
41
28
20
16

ISIS 588550
104
64
54
38
37

ISIS 588553
97
42
35
18
16

ISIS 588555
107
35
37
18
18

ISIS 588848
116
95
92
69
71

ISIS 594430
104
64
59
45
46

Tolerability Studies
Body Weight Measurements

To evaluate the effect of ISIS oligonucleotides on the overall health of the animals, body and organ weights were measured and are presented in the Table below. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys. The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides.

TABLE 223

Final body weights (g) in cynomolgus monkey

Day
Day
Day
Day
Day
Day
Day

1
14
28
42
56
70
84

PBS
2887
2953
3028
3094
3125
3143
3193

ISIS 532770*
2963
2947
2966
3050
3097
3138
3160

ISIS 532800
2886
2976
3072
3149
3220
3269
3265

ISIS 532809
2755
2836
2927
2983
3019
3071
3098

ISIS 588540
2779
2834
2907
2934
2981
3034
3057

ISIS 588544
2837
2896
3009
3064
3132
3163
3199

ISIS 588548
2694
2816
2882
2990
3073
3149
3161

ISIS 588550
2855
2988
3062
3188
3219
3282
3323

ISIS 588553
3033
3156
3256
3335
3379
3372
3442

ISIS 588555
2757
2863
2965
3022
3075
3088
3158

ISIS 588848
2850
3018
3032
3187
3230
3212
3291

ISIS 594430
2884
2963
2953
3149
3187
3204
3256

TABLE 224

Final organ weights (g) in cynomolgus monkey

Spleen
Heart
Kidney
Liver

PBS
2.8
11.6
11.9
55.8

ISIS 532770*
5.0
11.3
20.6
77.9

ISIS 532800
6.2
11.9
18.6
94.4

ISIS 588540
4.0
11.4
13.5
67.1

ISIS 588548
4.1
11.7
17.3
72.0

ISIS 588550
5.8
10.9
18.5
81.8

ISIS 588553
5.0
12.7
17.2
85.9

ISIS 588555
4.7
11.8
15.9
88.3

ISIS 588848
5.0
12.7
14.4
75.7

ISIS 594430
3.9
11.9
14.8
69.9

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, blood samples were collected from all the study groups. The blood samples were collected from the cephalic, saphenous, or femoral veins, 48 hours post-dosing. The monkeys were fasted overnight prior to blood collection. Blood (1.5 mL) was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged (approximately 3,000 rpm for 10 min) to obtain serum. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).

Plasma levels of ALT and AST were measured and the results are presented in the Table below, expressed in IU/L. Bilirubin, a liver function marker, was similarly measured and is presented in the Table below expressed in mg/dL. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys. The results indicate that most of the antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides.

TABLE 225

Liver chemistry marker levels in cynomolgus

monkey plasma on day 86

ALT (IU/L)
AST (IU/L)
Bilirubin (mg/dL)

PBS
71
57
0.3

ISIS 532770*
59
58
0.1

ISIS 532800
65
86
0.1

ISIS 532809
35
58
0.1

ISIS 588540
70
88
0.2

ISIS 588544
55
97
0.2

ISIS 588548
61
85
0.2

ISIS 588550
94
84
0.2

ISIS 588553
44
65
0.2

ISIS 588555
63
84
0.2

ISIS 588848
69
65
0.2

ISIS 594430
86
53
0.2

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, blood samples were collected from all the study groups. The blood samples were collected from the cephalic, saphenous, or femoral veins, 48 hours post-dosing. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged (approximately 3,000 rpm for 10 min) to obtain serum. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Results are presented in the Table below, expressed in mg/dL. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys.

For urinalysis, fresh urine from all the animals was collected in the morning using a clean cage pan on wet ice. Food was removed overnight the day before urine collection but water was supplied. Urine samples (approximately 1 mL) were analyzed for protein to creatinine (P/C) ratio using a Toshiba 200FR NEO automated chemistry analyzer (Toshiba Co., Japan). ‘n.d.’ indicates that the urine protein level was under the detection limit of the analyzer.

The plasma and urine chemistry data indicate that most of the ISIS oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides.

TABLE 226

Renal chemistry marker levels (mg/dL)

in cynomolgus monkey plasma on day 86

BUN
Creatinine
Total protein

PBS
28
0.9
8.0

ISIS 532770*
20
0.9
6.9

ISIS 532800
25
0.9
7.5

ISIS 532809
23
0.8
7.4

ISIS 588540
30
0.8
7.5

ISIS 588544
26
0.9
7.4

ISIS 588548
25
0.9
7.6

ISIS 588550
24
0.9
7.2

ISIS 588553
25
0.8
7.2

ISIS 588555
25
0.8
7.6

ISIS 588848
24
0.9
7.5

ISIS 594430
25
0.8
7.2

TABLE 227

Renal chemistry marker levels in cynomolgus

monkey urine on day 44 and day 86

Day 44
Day 86

PBS
0.03
n.d.

ISIS 532800
0.01
n.d.

ISIS 532809
0.01
n.d.

ISIS 588540
0.03
n.d.

ISIS 588544
0.01
0.09

ISIS 588548
0.01
0.01

ISIS 588550
0.04
0.01

ISIS 588553
0.05
n.d.

ISIS 588555
0.03
0.03

ISIS 588848
0.09
n.d.

ISIS 594430
0.03
n.d.

Hematology

To evaluate any effect of ISIS oligonucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 0.5 mL of blood was collected from each of the available study animals in tubes containing K₂-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA120 hematology analyzer (Bayer, USA). The data is presented in the Tables below. ‘*’ indicates that the oligonucleotide was tested in a separate study with similar conditions and is the average of the measurements from male and female monkeys.

The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose.

TABLE 228

Blood cell counts in cynomolgus monkeys

RBC
Platelets
WBC
Neutrophils
Lymphocytes
Monocytes

(×10⁶/μL)
(×10³/μL)
(×10³/μL)
(% WBC)
(% total)
(% total)

PBS
5.8
347
9.4
42.7
53.1
3.0

ISIS 532770*
5.4
386
10.8
22.3
71.7
3.3

ISIS 532800
5.6
360
13.1
29.5
61.1
6.5

ISIS 532809
5.2
400
11.5
56.6
38.2
2.5

ISIS 588540
5.5
367
11.7
50.9
42.7
2.1

ISIS 588544
5.2
373
14.3
56.6
37.6
4.3

ISIS 588548
5.1
373
9.7
40.4
54.3
3.9

ISIS 588550
6.1
343
9.9
32.1
61.7
4.6

ISIS 588553
5.2
424
9.3
41.7
53.2
3.6

ISIS 588555
5.1
411
9.6
45.1
49.7
3.5

ISIS 588848
5.7
370
10.0
39.8
55.8
3.1

ISIS 594430
5.7
477
10.6
47.3
47.8
3.6

TABLE 229

Hematologic parameters in cynomolgus monkeys

Hemoglobin (g/dL)
HCT (%)

PBS
14.1
46.6

ISIS 532770*
12.4
40.9

ISIS 532800
12.3
40.5

ISIS 532809
12.2
40.4

ISIS 588540
12.5
41.5

ISIS 588544
11.9
38.1

ISIS 588548
12.3
39.6

ISIS 588550
13.4
45.0

ISIS 588553
12.6
39.8

ISIS 588555
11.6
38.1

ISIS 588848
13.2
42.7

ISIS 594430
13.4
43.1

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide was measured in the kidney and liver tissues. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in the Table below, expressed as μgig liver or kidney tissue.

TABLE 230

Antisense oligonucleotide distribution

Kidney (μg/g)
Liver (μg/g)
Kidney/Liver ratio

ISIS 532800
3881
1633
2.4

ISIS 588540
3074
1410
2.2

ISIS 588548
3703
1233
3.0

ISIS 588550
4242
860
4.9

ISIS 588553
3096
736
4.2

ISIS 588555
4147
1860
2.2

ISIS 588848
2235
738
3.0

ISIS 594430
1548
752
2.1

Example 135: 6 Week Efficacy Study of Unconjugated and 5′-THA-GalNAc3 Conjugated Antisense Oligonucleotides Targeted to Human CFB in Transgenic Mice

Two antisense oligonucleotides having the same nucleobase sequence: uncongugated antisense oligonucleotide ISIS 588540 and 5′-THA-GalNAc₃-conjugated antisense oligonucleotide ISIS 696844, were tested in human CFB transgenic mice (hCFB-Tg mice).

The mice were administered subcutaneously with ISIS 696844 at doses of 0.1, 1.25, 0.5, 2.0, 6.0, or 12.0 mg/kg/week or with ISIS 588540 at doses of 2, 6, 12, 25, or 50 mg/kg/week for 6 weeks. A control group of mice were administered subcutaneously with PBS for 6 weeks. Mice were sacrificed 48 hours after the last dose. Hepatic mRNA levels were analyzed by qRT-PCR.

Study 1

The results are presented in the Table below and demonstrate that the 5′-THA-GalNAc₃-conjugated antisense oligonucleotide targeting CFB is more potent than the unconjugated antisense oligonucleotide with the same sequence.

TABLE 231

Efficacy of antisense oligonucleotides targeting CFB

ED₅₀(mg/kg)
ED₇₅(mg/kg)

ISIS 588540
4.52
9.26

ISIS 696844
0.52
1.12

Study 2

Liver mRNA levels were measured with two different primer probe sets targeting different regions of the mRNA and normalized to either RIBOGREEN® (RGB) or Cyclophilin. The primer probe sets were RTS3459, described above, and RTS3460 (forward sequence CGAAGCAGCTCAATGAAATCAA, designated herein as SEQ ID NO: 813; reverse sequence TGCCTGGAGGGCCTTCTT, designated herein as SEQ ID NO: 814; probe sequence AGACCACAAGTTGAAGTC, designated herein as SEQ ID NO: 815). The results are presented in the Table below and demonstrate that the 5′-THA-GalNAc₃-conjugated antisense oligonucleotide targeting CFB is more potent than the unconjugated antisense oligonucleotide with the same sequence, irrespective of the primer probe set used.

TABLE 231

Efficacy of antisense oligonucleotides targeting CFB

ED₅₀
ED₅₀
ED₅₀
ED₅₀
ED₇₅
ED₇₅
ED₇₅
ED₇₅

RTS3459
RTS3460
RTS3459
RTS3460
RTS3459
RTS3460
RTS3459
RTS3460

(RGB)
(RGB)
(Cyclophilin)
(Cyclophilin)
(RGB)
(RGB)
(Cyclophilin)
(Cyclophilin)

ISIS 588540
4.5
4.1
5.2
5.4
9.3
10.0
10.0
9.3

ISIS 696844
0.5
0.5
0.6
0.5
1.1
1.3
1.2
0.9

	Number	Date	Country
	62076273	Nov 2014	US
	61987471	May 2014	US

	Number	Date	Country
Parent	15307526	Oct 2016	US
Child	16357018		US

COMPOSITIONS AND METHODS FOR MODULATING COMPLEMENT FACTOR B EXPRESSION

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