EA042765B1 - EXPRESSION MODULATORS PCSK9 - Google Patents

Description

In one aspect, the present invention provides a compound of the formula (SEQ ID NO: 1016)

O or a pharmaceutically acceptable salt thereof. Preferably the pharmaceutically acceptable salt is the sodium salt or the potassium salt.

In yet another aspect, the present invention provides a compound, or a pharmaceutically acceptable salt thereof, consisting of a modified oligonucleotide of length 16 linked nucleosides having the nucleotide sequence shown in SEQ ID NO: 1016, wherein the modified oligonucleotide contains:

a gap segment consisting of ten linked deoxynucleosides;

5' end flank segment consisting of three linked nucleosides; And

3'-terminal flank segment, consisting of three linked nucleosides;

- 1 042765 where the gap segment is located between the 5'-terminal flank segment and the 3'-terminal flank segment, where each nucleoside of each flank segment contains cEt-caxap; where each internucleoside bond is a phosphothioate bond; and where each cytosine is 5-methylcytosine.

In another aspect, the present invention provides a compound, or a pharmaceutically acceptable salt thereof, comprising a modified oligonucleotide having a length of 16 linked nucleosides, which has a nucleobase sequence consisting of SEQ ID NO: 1016, wherein the modified oligonucleotide comprises:

a gap segment consisting of linked deoxynucleosides;

5'-terminal flank segment, consisting of linked nucleosides; And

3'-terminal flank segment consisting of linked nucleosides;

where the gap segment is located between the 5'-end flank segment and the 3'-end flank segment, where the 5'-end flank segment and the 3'-end flank segment contain cEt-caxapa; where each internucleoside bond is a phosphothioate bond; and where each cytosine is a 5-methyl cytosine.

In another aspect, the present invention provides the use of a compound or a pharmaceutically acceptable salt thereof according to any of the above aspects in therapy, in particular in the treatment, prevention or amelioration of a disease associated with PCSK9, for example where the disease is hypercholesterolemia, dyslipidemia or mixed dyslipidemia.

Certain embodiments contemplated herein are directed to compounds and compositions that are more effective or of greater therapeutic value than publicly disclosed compounds.

Detailed description

It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory only and are not intended to limit the claimed embodiments. In this document, the use of the singular form includes the plural form, unless specifically noted otherwise. In this document, the use of or means and/or, unless otherwise indicated. Moreover, the use of the term including, as well as other forms such as includes and included, is not limiting.

The section headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter being described. All documents or portions of documents cited in this application, including without limitation patents, patent applications, articles, books, scientific papers, and GenBank and NCBI reference sequence records, are hereby expressly incorporated by reference to the portions of the document discussed in this document, and in their entirety.

It is understood that the sequence given under each of the SEQ ID NOs in the examples contained herein is independent of any modification of the sugar component, internucleoside bond, or nucleobase. By virtue of this, the compounds defined under SEQ ID NO may independently contain one or more modifications of the sugar component, internucleoside bond, or nucleobase. For compounds described under the ISIS number, it indicates a combination of nucleobase sequence, chemical modification, and motif.

Definitions

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

2'-deoxynucleoside means a nucleoside containing a 2'-H(H)-furanosyl sugar moiety found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, the 2'-deoxynucleoside may contain a modified nucleobase or may contain an RNA nucleobase (uracil).

2'-O-methoxyethyl (also 2'-MOE and 2'-O(CH ₂ ) ₂ -OCH ₃ ) refers to the O-methoxyethyl modification at the 2'-position of the furanosyl ring. 2'-O-methoxyethyl modified sugar is a modified sugar.

2'-MOE-nucleoside (also 2'-O-methoxyethylnucleoside) means a nucleoside containing a 2'-MOE-modified sugar moiety.

2'-substituted nucleoside or 2'-modified nucleoside means a nucleoside containing a 2'-substituted or 2'-modified sugar moiety. As used herein, 2'-substituted or 2-modified with respect to a sugar moiety means that the sugar moiety contains at least one 2' substituent group other than H or OH.

A 3' target site refers to a target nucleic acid nucleotide that is complementary to the 3'-most nucleotide of a particular compound.

A 5' target site refers to a target nucleic acid nucleotide that is complementary to the 5'-most nucleotide of a particular compound.

5'-methylcytosine means a cytosine with a methyl group attached at the 5' position.

- 2 042765

Approximate means within ± 10% of the value. For example, if compounds are indicated to have at least about 70% inhibition of PCSK9, PCSK9 levels are understood to be suppressed in the range of 60% to 80%.

The introduction or implementation of the introduction refers to the ways of introduction to the individual of the compounds or compositions provided in this document, to perform their intended function. An example route of administration that can be used includes, without limitation, parenteral administration such as subcutaneous, intravenous, or intramuscular injection or infusion.

Reduction in intensity refers to improvement or amelioration of at least one manifestation, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, reducing the intensity includes delaying or slowing the progression or reducing the severity of one or more manifestations of the condition or disease. The progression or severity of manifestations can be determined using subjective or objective indicators that are known to experts in this field.

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, non-human apes and chimpanzees.

Antisense activity refers to any detectable and/or measurable activity associated with hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a reduction in the amount or expression of a target nucleic acid or the protein encoded by that target nucleic acid compared to levels of the target nucleic acid or levels of the target protein in the absence of the target antisense compound.

An antisense compound means a compound containing an oligonucleotide and optionally one or more additional components such as a conjugate group or an end group. Examples of antisense compounds include single-stranded and double-stranded compounds such as oligonucleotides, ribozymes, siRNA, shRNA, ssRNA, and compounds whose activity depends on the degree of active site occupancy.

Antisense suppression refers to the reduction in the levels of a target nucleic acid in the presence of an antisense compound complementary to the target nucleic acid compared to the levels of the target nucleic acid in the absence of the antisense compound.

Antisense mechanisms are all such mechanisms involving the hybridization of a compound with a target nucleic acid, where the result or effect of hybridization is either destruction of the target or occupation of the target with concomitant blocking of a cellular mechanism involving, for example, transcription or splicing.

An antisense oligonucleotide means an oligonucleotide having a nucleic base sequence that is complementary to a target nucleic acid or region or segment thereof. In certain embodiments, the antisense oligonucleotide is capable of specific hybridization with a target nucleic acid, or region or segment thereof.

Bicyclic nucleoside or BNA means a nucleoside containing a bicyclic sugar moiety. Bicyclic sugar or bicyclic sugar component means a modified sugar component containing two rings, where the second ring is formed by a bridge connecting two atoms in the first ring, thereby providing the formation of a bicyclic structure. In certain embodiments, the first ring of the bicyclic sugar moiety is a furanosyl moiety. In certain embodiments, the bicyclic sugar moiety does not contain a furanosyl moiety.

A branching group means a group of atoms with at least 3 positions that can form covalent bonds with at least 3 groups. In certain embodiments, the branching group provides multiple reactive sites for attaching bound ligands to the oligonucleotide via a conjugating linker and/or a cleavable component.

A cell-targeting component means a conjugated group or fragment of a conjugated group that is capable of binding to a specific cell type or specific cell types.

cEt or conformationally restricted to ethyl means a bicyclic furanosyl sugar component containing a bridge connecting the 4'-carbon atom and the 2'-carbon atom, and the bridge has the formula: 4'-CH(CH ₃ )-O-2'.

cEt-nucleoside means a nucleoside containing a cEt-modified sugar moiety.

Chemical modification in a compound describes the substitutions or changes resulting from a chemical reaction of any of the structural units in the compound compared to the initial state of such a structural unit. A modified nucleoside means a nucleoside independently having a modified sugar moiety and/or a modified nucleobase. A modified oligonucleotide means an oligonucleotide containing at least one modified internucleoside bond, a modified sugar and/or a modified nucleobase.

A chemically distinct region refers to a region of a compound that is chemically different in some way from another region of the same compound. For example, a region with 2'-O-methoxyethyl nucleotides is chemically different from a region with nucleotides without 2'-O-methoxyethyl modifications.

Chimeric antisense compounds means antisense compounds that have at least 2 chemically distinct regions, with multiple subunits per position.

A cleavable bond means any chemical bond that can be broken. In certain embodiments, the cleavable bond is selected from an amide, polyamide, ester, ester, one or both esters in a phosphodiester bond, phosphoester, carbamate, disulfide, or peptide.

A cleavable component means a bond or group of atoms that is cleaved under physiological conditions, for example, within a cell, animal or human.

Complementary in relation to an oligonucleotide means that the nucleobase sequence of such an oligonucleotide or one or more of its regions corresponds to the nucleobase sequence of another oligonucleotide or nucleic acid or one or more of their regions when the two nucleobase sequences are aligned in opposite directions. Nucleobase matches or complementary nucleobases described herein are limited to the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine ( ^m C) and guanine (G) unless otherwise noted. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity for each nucleoside and may contain one or more nucleobase mismatches. In contrast, fully complementary or 100% complementary in relation to oligonucleotides means that such oligonucleotides have nucleobase matches for each nucleoside without any nucleobase mismatch.

A conjugated group means a group of atoms that is attached to an oligonucleotide. Conjugated groups contain a conjugated component and a conjugated linker that attaches the conjugated component to the oligonucleotide.

Conjugate linker means a group of atoms containing at least one bond that connects the conjugated component to the oligonucleotide.

A conjugated component means a group of atoms that is attached to an oligonucleotide via a conjugating linker.

Adjacent, in relation to an oligonucleotide, refers to nucleosides, nucleobases, sugar moieties, or internucleoside bonds that are directly adjacent to each other. For example, adjacent nucleobases means nucleobases that are directly adjacent to each other in sequence.

Designed or designed for refers to a method of designing a compound that specifically hybridizes to a selected nucleic acid molecule.

Modified in different ways means chemical modifications or chemical substituents that are different from each other, including no modifications. Thus, for example, the MOE nucleoside and the unmodified DNA nucleoside are modified in different ways, even though the DNA nucleoside is unmodified. Similarly, DNA and RNA are modified in different ways, even though both are naturally occurring unmodified nucleosides. Nucleosides that are the same but contain different nucleobases are not modified in different ways. For example, a nucleoside containing a 2'-OMe-modified sugar and an unmodified adenine nucleobase and a nucleoside containing a 2'-OMe-modified sugar and an unmodified thymine nucleobase are not modified in different ways.

Double-stranded antisense compound means an antisense compound containing two oligomeric compounds that are complementary to each other and form a duplex, and where one of the two said oligomeric compounds contains an oligonucleotide.

An effective amount means an amount of a compound sufficient to achieve the desired physiological result in an individual in need of the compound. The effective amount may vary for individuals depending on the health and physical condition of the individual being treated, the taxonomic group of individuals being treated, the composition of the composition, the assessment of the medical condition of the individual, as well as other factors taken into account.

Efficiency means the ability to deliver the desired effect.

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Expression includes all the functions by which the information encoded in the gene is converted into structures present and functioning in the cell. Such structures include, without limitation, transcription and translation products.

Gapmer means an oligonucleotide containing an internal region having several nucleosides that promote cleavage by RNase H, located between external regions having one or more nucleosides, where the nucleosides forming the internal region are chemically different from the nucleoside or nucleosides forming the external regions. The inner area may be called a gap and the outer areas may be called flanks.

Hybridization means annealing of oligonucleotides and/or nucleic acids. Without being limited to a particular mechanism, the most common hybridization mechanism involves hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding between complementary nucleobases. In certain embodiments, complementary nucleic acid molecules include, without limitation, an antisense compound and a target nucleic acid. In certain embodiments, complementary nucleic acid molecules include, without limitation, an oligonucleotide and a target nucleic acid.

Directly adjacent means that there are no intermediate elements between immediately adjacent elements of the same type (eg, there are no intermediate nucleobases between immediately adjacent nucleobases).

Subject means a human or non-human animal chosen for treatment or therapy.

Suppression of expression or activity refers to a reduction or blocking of expression or activity compared to expression or activity in an untreated or control sample and does not necessarily indicate complete elimination of expression or activity.

An internucleoside bond means a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. Modified internucleoside bond means any internucleoside bond other than a naturally occurring phosphate internucleoside bond. Non-phosphate bonds are referred to herein as modified internucleoside bonds.

Extended oligonucleotides are oligonucleotides that contain one or more additional nucleosides compared to the oligonucleotide disclosed herein, for example, the original oligonucleotide.

Linked nucleosides means adjacent nucleosides linked to each other by an internucleoside bond.

Linker nucleoside means a nucleoside that links an oligonucleotide to a conjugated moiety. Linker nucleosides are located in the conjugating linker of the compound. Linker nucleosides are not considered part of the oligonucleotide portion of the compound, even if they are adjacent to the oligonucleotide.

Mismatched or non-complementary means a nucleobase of the first oligonucleotide that is not complementary to the corresponding nucleotide base of the second oligonucleotide or target nucleic acid in the alignment of the first and second oligonucleotides. For example, nucleobases, including but not limited to the universal nucleobases inosine and hypoxanthine, are capable of hybridizing to at least one nucleobase, but are nonetheless mismatched or non-complementary to the nucleobase to which they hybridize. As another example, the nucleobase of the first oligonucleotide that is unable to hybridize with the corresponding nucleobase of the second oligonucleotide or target nucleic acid, when the first and second oligonucleotides are aligned, is a mismatched or non-complementary nucleobase.

Modulation refers to the change or adjustment of a trait in a cell, tissue, organ, or organism. For example, modulating PCSK9 RNA can mean an increase or decrease in the level of PCSK9 RNA and/or PCSK9 protein in a cell, tissue, organ, or organism. A modulator brings about a change in a cell, tissue, organ, or organism. For example, a PCSK9 compound may be a modulator that reduces the amount of PCSK9 RNA and/or PCSK9 protein in a cell, tissue, organ, or organism.

MOE means methoxyethyl.

A monomer refers to one structural unit of an oligomer. Monomers include, without limitation, nucleosides and nucleotides.

A motif means a characteristic region of unmodified and/or modified sugar components, nucleobases and/or internucleoside bonds in an oligonucleotide.

Natural or naturally occurring remedies are found in nature.

Non-bicyclic modified sugar or non-bicyclic modified sugar moiety means a modified sugar moiety that contains a modification, such as a substituent, that does not bridge between two sugar atoms to form a second ring.

Nucleic acid refers to molecules composed of monomeric nucleotides. Nucleic acid includes, without limitation, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

Nucleic base means a heterocyclic component capable of pairing with the base of another nucleic acid. As used herein, the naturally occurring nucleobase is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). A modified nucleobase is a naturally occurring nucleobase that has been chemically modified. A universal base or universal nucleobase is a nucleobase other than a naturally occurring nucleobase and a modified nucleobase and capable of pairing with any nucleobase.

The sequence of nucleic bases means the order in which adjacent nucleic bases are arranged in a nucleic acid or oligonucleotide, regardless of any sugar or internucleoside bond.

Nucleoside means a compound containing a nucleic base and a sugar component. The nucleic base and the sugar component are independently unmodified or modified.

Modified nucleoside means a nucleoside containing a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include nucleosides with nitrogenous bases removed that lack a nucleobase.

Oligomeric compound means a compound containing one oligonucleotide and optionally one or more additional components such as a conjugate group or an end group.

Oligonucleotide means a polymer of linked nucleosides, each of which may be independently modified or unmodified. Unless otherwise indicated, oligonucleotides consist of 8-80 linked nucleosides. Modified oligonucleotide means an oligonucleotide where at least one sugar, nucleobase or internucleoside bond is modified. An unmodified oligonucleotide means an oligonucleotide that does not contain any modification of a sugar, nucleobase or internucleoside bond.

Parent oligonucleotide means an oligonucleotide whose sequence is used as a basis for constructing more oligonucleotides with a similar sequence but different lengths, motifs and/or chemical structures. Newly designed oligonucleotides may have the same or overlapping sequence as compared to the original oligonucleotide.

Parenteral administration means administration by injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intracerebroventricular administration.

A pharmaceutically acceptable carrier or diluent means any substance suitable for use when administered to an individual. For example, a pharmaceutically acceptable carrier may be a sterile aqueous solution such as PBS or water for injection.

Pharmaceutically acceptable salts means the physiologically and pharmaceutically acceptable salts of compounds such as oligomeric compounds or oligonucleotides, i.e. salts that retain the necessary biological activity of the parent compound and do not impart undesirable toxicological properties to it.

Pharmaceutical agent means a compound that provides a therapeutically beneficial effect when administered to an individual.

Pharmaceutical composition means a mixture of substances suitable for administration to an individual. For example, the pharmaceutical composition may contain one or more compounds, or a salt thereof, and a sterile aqueous solution.

Phosphothioate bond means a modified phosphate bond in which one of the non-bridging oxygen atoms is replaced by a sulfur atom. The phosphothioate internucleoside bond is a modified internucleoside bond.

The phosphorus component means a group of atoms containing a phosphorus atom. In certain embodiments, the implementation of the phosphorus component includes mono-, di - or triphosphate or phosphothioate.

Fragment means a specific number of contiguous (i.e. linked) nucleic bases

- 6 042765 nucleic acid. In certain embodiments, a fragment is a certain number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a fragment is a certain number of contiguous nucleobases of an oligomeric compound.

Prevention refers to delaying or preventing the onset, development, or progression of a disease, disorder, or condition for a period of time ranging from a few minutes to an indefinite period.

Prodrug means a compound in a form outside the body, which, when administered to an individual, is metabolized to another form within his body or cells. In certain embodiments, the metabolized form is the active or more active form of the compound (eg, drug). In general, prodrug conversion within the body is facilitated by the action of the enzyme(s) (eg, endogenous or viral enzyme) or chemical(s) present in cells or tissues and/or physiological conditions.

Decreasing means reducing to a lesser degree, size, amount or number.

A RefSeq No. is a unique combination of letters and numbers assigned to a sequence that indicates that the sequence corresponds to a particular target transcript (eg, target gene). Such sequence and target gene information (collectively, the gene record) can be found in a genetic sequence database. Genetic sequence databases include the NCBI Reference Sequence Database, GenBank, the European Nucleotide Archive and the Japan DNA Data Bank (the latter three form the International Nucleotide Sequence Database Collaboration or INSDC).

A region is defined as a target nucleic acid fragment having at least one identifiable structure, function, or characteristic.

Compound for RNAi means an antisense compound that acts at least in part through RISC or Ago2, but not through RNase H, to modulate the target nucleic acid and/or the protein encoded by the target nucleic acid. RNAi compounds include, but are not limited to, double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimetics.

Segments are defined as smaller fragments or sub-fragments of regions within a nucleic acid.

Side effects mean physiological disease and/or conditions associated with treatment that differ from the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, kidney function abnormalities, hepatotoxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, elevated serum aminotransferase levels may indicate hepatotoxicity or abnormal liver function. For example, elevated levels of bilirubin may indicate hepatotoxicity or abnormal liver function.

Single-stranded in relation to the connection means that the connection has only one oligonucleotide. Self-complementary means an oligonucleotide that hybridizes at least partially to itself. A single oligonucleotide compound, where the compound oligonucleotide is self-complementary, is a single stranded compound. A single stranded compound may be capable of binding to a complementary compound to form a duplex.

Sites are defined as unique positions of nucleic bases within a target nucleic acid.

Specifically hybridizable refers to an oligonucleotide having a sufficient degree of complementarity between the oligonucleotide and the target nucleic acid to induce the desired effect, while exhibiting minimal or no such effects on non-target nucleic acids. In certain embodiments, specific hybridization occurs under physiological conditions.

Specific inhibition in relation to a target nucleic acid means reducing or blocking the expression of a target nucleic acid while exhibiting lesser, minimal or no such effects on non-target nucleic acids. A decrease does not necessarily indicate complete elimination of expression of the target nucleic acid.

Standard cell assay means the assay(s) described in the examples and their acceptable variations.

Standard in vivo experiment means the procedure(s) described in the example(s) and acceptable variations thereof.

Sugar component means an unmodified sugar component or a modified sugar component. The unmodified sugar component or unmodified sugar means the 2'-OH(H)-furanosyl component found in RNA (the unmodified sugar component of RNA) or the 2'-H(H)-component found in DNA (the unmodified sugar component DNA). The unmodified sugar components have one hydrogen atom at each of the 1', 3', and 4' positions, an oxygen atom at the 3' position, and two hydrogen atoms at the 5' position. Modified sugar component or modified sugar means a modified furanosyl sugar component or sugar simulant. A modified furanosyl sugar component means a furanosyl sugar containing a non-hydrogen substituent in place of at least one hydrogen atom of the unmodified sugar component. In certain embodiments, the modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar components include bicyclic sugars and non-bicyclic sugars.

Sugar simulant means a modified sugar moiety, other than a furanosyl moiety, that can link a nucleobase to another group, such as an internucleoside bond, conjugate group, or end group, in an oligonucleotide. Modified nucleosides containing mimic sugars may be incorporated into an oligonucleotide at one or more positions, and such oligonucleotides are capable of hybridizing to complementary compounds or nucleic acids.

PCSK9 means any nucleic acid or PCSK9 protein. Nucleic acid PCSK9 means any nucleic acid encoding PCSK9. For example, in certain embodiments, the PCSK9 nucleic acid includes a DNA sequence encoding PCSK9, an RNA sequence transcribed from DNA encoding PCSK9 (including genomic DNA containing introns and exons), and an mRNA sequence encoding PCSK9. mRNA PCSK9 refers to an mRNA encoding a PCSK9 protein. The target can be specified in upper or lower case.

A specific PCSK9 inhibitor refers to any agent capable of specifically inhibiting the expression or activity of PCSK9 RNA and/or PCSK9 protein at the molecular level. For example, specific inhibitors of PCSK9 include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of suppressing the expression of PCSK9 RNA and/or PCSK9 protein.

A target gene refers to a gene encoding a target.

Targeting means the specific hybridization of a compound with a target nucleic acid in order to induce the desired effect.

All of a target nucleic acid, a target RNA, a target RNA transcript, and a target nucleic acid means a nucleic acid that is capable of being targeted by the compounds described herein.

Target region means a target nucleic acid fragment to which one or more compounds are targeted.

Target segment means the nucleotide sequence of the target nucleic acid to which the compound is targeted. The 5' target site refers to the 5'-terminal nucleotide of the target segment. The 3' target site refers to the 3'-terminal nucleotide of the target segment.

An end group means a chemical group or a group of atoms that is covalently attached to the end of an oligonucleotide.

A therapeutically effective amount means an amount of a compound, pharmaceutical agent, or composition that has a therapeutically beneficial effect on an individual.

Treatment refers to the administration of a compound or pharmaceutical composition to an animal for the purpose of effecting a change or improvement in a disease, disorder, or condition in the animal.

Certain Embodiments

In certain embodiments, methods, compounds, and compositions are provided for suppressing PCSK9 expression.

In certain embodiments, compounds that target a PCSK9 nucleic acid are provided. In certain embodiments, the PCSK9 nucleic acid has the sequence listed under RefSeq or GENBANK Accession No. NM_174936.3 and GENBANK Accession No. NC_000001.11 with nucleotides truncated 55036001-55068000 (incorporated by reference disclosed herein under SEQ ID NO: 1 and SEQ ID NO: 2, respectively). In certain embodiments, the compound is an antisense compound or an oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and having the sequence

- 8,042,765 nucleobases comprising at least 8 contiguous nucleobases from the nucleic acid sequence of SEQ ID NO: 1016. In certain embodiments, the compound is an antisense compound or an oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded. In certain embodiments, the modified oligonucleotide is 16 linked nucleosides in length.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 9 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 11 contiguous nucleobases from the nucleobase sequences of SEQ ID NO: 1016. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound is an antisense compound or an oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided that contains a modified oligonucleotide consisting of the nucleic base sequence of SEQ ID NO: 1016. In certain embodiments, the compound is an antisense compound or an oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and within nucleobases 6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949, and 28105-28136 being complementary to SEQ ID NO: 2, wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95%, or 100% complementary to SEQ ID NO: 2. In certain embodiments, the compound is an antisense compound or an oligomeric compound. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded.

In certain embodiments, a compound is provided comprising a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence containing a fragment of at least 8 contiguous nucleobases complementary to an equal length fragment of nucleobases 6356-6371, 12843-12947, 12905- 12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949 and 28105-28136 in SEQ ID NO: 2, wherein said modified oligonucleotide is at least 85%, at least 90%, at least 95 % or 100% complementary to SEQ ID NO: 2. In certain embodiments, the compound is an antisense compound or an oligomeric

- 9 042765 connection. In certain embodiments, the connection is single stranded. In certain embodiments, the connection is double stranded. In certain embodiments, the modified oligonucleotide is 16 linked nucleosides in length.

In certain embodiments, the compounds target the PCSK9 nucleic acid region. In certain embodiments, such compounds targeting the PCSK9 nucleic acid region have an adjacent nucleobase fragment that is complementary to an equal length fragment of the nucleic bases in that region. For example, a fragment may be a fragment of at least 8, 9, 10, 11, 12, 13, 14, 15, 16 contiguous nucleobases complementary to an equal length fragment in the region referred to herein. In certain embodiments, such compounds target the following nucleotide regions under SEQ ID NO: 2: 6356-6371, 12843-12947, 12905-12948, 17681-17700, 19653-19673, 27626-27669, 27895-27949, and 28105-28136 . In certain embodiments, these compounds are antisense compounds, oligomeric compounds, or oligonucleotides.

In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a fragment of at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases complementary to an equal length fragment within nucleotides 6356 -6371, 12909-12924, 17685-17700, 19658-19673, 27643-27658, 27906-27921, 27916-27931, 27917-27932, and 28107-28122 in SEQ ID NO: 2.

In certain embodiments, the compound contains a modified oligonucleotide having a length of 16 linked nucleosides and within the range of nucleotides 6356-6371, 12909-12924, 17685-17700, 19658-19673, 27643-27658, 27906-27921, 27916-27931, 27917-27 and 28107-28122 being complementary to SEQ ID NO: 2.

In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence containing a fragment of at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobases of the sequence of SEQ ID NO : 1016.

In certain embodiments, the compound contains a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising SEQ ID NOs: 1016.

In certain embodiments, the compound comprises a modified oligonucleotide having a nucleobase sequence consisting of SEQ ID NO: 1016.

In certain embodiments, compounds targeting PCSK9 are ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681. Of the more than 1540 compounds that were screened in as described in the Examples section below, ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 proved to be the best leader connections. In particular, ISIS 863633 showed the best combination of properties in terms of efficacy and tolerability of more than 1540 compounds.

In certain embodiments, any of the above modified oligonucleotides contains at least one modified internucleoside bond, at least one modified sugar, and/or at least one modified nucleobase.

In certain embodiments, any of the above modified oligonucleotides contains at least one modified sugar. In certain embodiments, at least one modified sugar contains a 2'-O-methoxyethyl group. In certain embodiments, at least one modified sugar is a bicyclic sugar, such as containing a 4'-CH(CH ₃ )-O-2' group, a 4'-CH2-O-2' group, or a 4'-(CH2 )2-O-2'.

In certain embodiments, the modified oligonucleotide contains at least one modified internucleoside bond, such as a phosphothioate internucleoside bond.

In certain embodiments, any of the above modified oligonucleotides contains at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the above modified oligonucleotides contains:

a gap segment consisting of linked deoxynucleosides;

5'-terminal flank segment consisting of linked nucleosides; And

3'-terminal flank segment consisting of linked nucleosides;

where the gap segment is located between the 5'-flank segment and the 3'-flank segment, and where each nucleoside of each flank segment contains a modified sugar. In certain embodiments, the modified oligonucleotide is 16 linked long

- 10 042765 nucleosides and has a nucleic base sequence containing the sequence

SEQ ID NO: 1016.

In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleobases, which has or consists of a nucleobase sequence containing the sequence mentioned under SEQ ID NO: 1016, wherein the modified oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

5' end flank segment consisting of three linked nucleosides; And

3'-terminal flank segment, consisting of three linked nucleosides;

where the gap segment is located between the 5'-terminal flank segment and the 3'-terminal flank segment, where each nucleoside of each flank segment contains cEt-caxap; where each internucleoside bond is a phosphothioate bond, and where each cytosine is 5 methylcytosine.

In certain embodiments, the compound comprises or consists of ISIS 863633 or a salt thereof, which have the following chemical structure:

In certain embodiments, the compound comprises or consists of ISIS 863633 or a salt thereof, which have the following chemical structure:

- 11 042765

In certain embodiments, the compound comprises or consists of the sodium salt of ISIS 863633, which has the following chemical structure:

In any of the above embodiments, the compound or oligonucleotide may be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to the nucleic acid encoding PCSK9 .

In any of the above embodiments, the connection may be single stranded.

- 12 042765

In certain embodiments, the compound contains deoxyribonucleotides. In certain embodiments, the connection is double stranded. In certain embodiments, the connection is double-stranded and contains ribonucleotides. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound.

In any of the above embodiments, the connection may be 16 linked nucleosides in length. In certain embodiments, the compound comprises or consists of an oligonucleotide.

In certain embodiments, the implementation of the connection contains a modified oligonucleotide described in this document, and a conjugated group. In certain embodiments, the conjugated group is linked to the modified oligonucleotide at the 5' end of the modified oligonucleotide. In certain embodiments, the conjugated group is linked to the modified oligonucleotide at the 3' end of the modified oligonucleotide. In certain embodiments, the conjugated group contains at least one N-acetylgalactosamine (GalNAc), at least two N-acetylgalactosamine (GalNAc), or at least three N-acetylgalactosamine (GalNAc).

In certain embodiments, the compounds or compositions provided herein comprise a pharmaceutically acceptable salt of the modified oligonucleotide. In certain embodiments, the salt is a sodium salt. In certain embodiments, the salt is a potassium salt.

In certain embodiments, the compounds or compositions described herein are active by virtue of having at least one of an in vitro IC ₅₀ of less than 5 μM, less than 4.5 μM, less than 4 μM, less than 3, 5 μM, less than 3 μM, less than 2.5 μM, less than 2 μM, less than 1.5 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM , less than 0.5 μM, less than 0.4 μM, less than 0.3 μM, less than 0.2 μM, or less than 0.1 μM.

In certain embodiments, the compounds or compositions described herein are well tolerated, as demonstrated by having at least one increase in alanine transaminase (ALT) or aspartate transaminase (AST) levels of no more than 4-fold, 3-fold, or 2-fold , compared with animals treated with saline, or an increase in the mass of the liver, spleen or kidney, constituting no more than 30, 20, 15, 12, 10, 5 or 2%, compared with animals treated with control. In certain embodiments, the compounds or compositions described herein are well tolerated as demonstrated by no increase in ALT or AST levels compared to control treated animals. In certain embodiments, the compounds or compositions described herein are well tolerated, as demonstrated by no increase in liver, spleen, or kidney weight compared to control-treated animals.

In certain embodiments, a composition is provided comprising a compound according to any of the above embodiments, or any pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition has a viscosity of less than about 40 centipoise (cP), less than about 30 cP, less than about 20 cP, less than about 15 cP, or less than about 10 cP. In certain embodiments, a composition having any of the above viscosities contains 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, approximately 225 mg/ml, approximately 250 mg/ml, approximately 275 mg/ml or approximately 300 mg/ml. In certain embodiments, a composition having any of the above values for viscosity and/or compound concentration has a temperature corresponding to room temperature or about 20°C, about 21°C, about 22°C, about 23°C, about 24°C , approximately 25°C, approximately 26°C, approximately 27°C, approximately 28°C, approximately 29°C or approximately 30°C.

Some indications

Certain embodiments provided herein relate to methods for suppressing PCSK9 expression, which may be useful for treating, preventing, or ameliorating a disease associated with PCSK9 in an individual by administering a compound that targets PCSK9. In certain embodiments, the compound may be a specific inhibitor of PCSK9. In certain embodiments, the compound may be an antisense compound, an oligomeric compound, or an oligonucleotide targeting PCSK9.

Examples of PCSK9-associated diseases amenable to treatment, prevention, and/or amelioration using the methods provided herein include cardiovascular disease, dyslipidemia, mixed dyslipidemia, hypercholesterolemia, lowering LDL cholesterol, and lowering atherogenic apolipoprotein (a)

[Lp(a)].

In certain embodiments, a method of treating, preventing, or ameliorating a PCSK9-associated disease in a subject comprises administering to the subject a compound containing a specific PCSK9 inhibitor, thereby providing treatment, prevention, or amelioration of the disease. In certain embodiments, the individual is identified as having a disease associated with PCSK9, or at risk of having it. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides that has a nucleobase sequence comprising SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a nucleobase sequence consisting of SEQ ID NO: 1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound. In certain embodiments, the compound is administered parenterally to an individual. In certain embodiments, administration of the compound reduces LDL cholesterol levels, decreases Lp(a) levels, induces LDL receptor (LDL-R) activity, and regulates LDL cholesterol homeostasis via LDL receptors.

In certain embodiments, a method of treating, preventing, or ameliorating cardiovascular disease comprises administering to an individual a compound containing a specific PCSK9 inhibitor, thereby providing treatment, prevention, or amelioration of cardiovascular disease. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound contains a modified oligonucleotide having a length of 8-80 linked nucleosides and having a nucleobase sequence containing at least 8 contiguous nucleobases of SEQ ID NO: 1016. In certain embodiments, the compound contains a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides , which has a nucleobase sequence comprising SEQ ID Ns: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a nucleobase sequence consisting of SEQ ID NO: 1016. In any of the above embodiments, the modified oligonucleotide may be 16 linked nucleosides in length .

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound. In certain embodiments, the compound is administered parenterally to an individual. In certain embodiments, administration of the compound reduces LDL cholesterol levels. In certain embodiments, Lp(a) levels are reduced as a result of compound administration. In certain embodiments, LDL receptor (LDL-R) activity is induced by administration of the compound. IN

- 14 042765 certain embodiments, administration of the compound regulates LDL cholesterol homeostasis via LDL receptors.

In certain embodiments, a method of suppressing PCSK9 expression in an individual having a disease associated with PCSK9, or at risk of having one, comprises administering to the individual a compound containing a specific inhibitor of PCSK9, thereby causing suppression of PCSK9 expression in the individual. In certain embodiments, administration of the compound suppresses hepatic expression of PCSK9. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the individual has or is at risk for dyslipidaemia. In certain embodiments, the individual has or is at risk of hypercholesterolemia. In certain embodiments, the individual has, or is at risk of, mixed dyslipidaemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides that has a nucleobase sequence comprising SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a nucleobase sequence consisting of SEQ ID NO: 1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound. In certain embodiments, the compound is administered parenterally to an individual. In certain embodiments, administration of the compound reduces LDL cholesterol levels. In certain embodiments, Lp(a) levels are reduced as a result of compound administration. In certain embodiments, LDL receptor (LDL-R) activity is induced by administration of the compound. In certain embodiments, administration of the compound regulates LDL cholesterol homeostasis via LDL receptors.

In certain embodiments, a method for suppressing PCSK9 expression in a cell comprises contacting the cell with a compound containing a specific inhibitor of PCSK9, thereby causing the expression of PCSK9 in the cell to be suppressed. In certain embodiments, the cell is a hepatocyte. In certain embodiments, the cell is located in the liver. In certain embodiments, the cell is located in the liver of an individual who has or is at risk for cardiovascular disease. In certain embodiments, the cell is located in the liver of an individual who has or is at risk for dyslipidemia. In certain embodiments, the cell is located in the liver of an individual who has or is at risk for mixed dyslipidemia. In certain embodiments, the cell is located in the liver of an individual who has or is at risk of hypercholesterolemia. In certain embodiments, administration of the compound suppresses hepatic expression of PCSK9. In certain embodiments, the individual has or is at risk of developing a cardiovascular disease. In certain embodiments, the individual has or is at risk for dyslipidaemia. In certain embodiments, the individual has, or is at risk of, mixed dyslipidaemia. In certain embodiments, the individual has or is at risk of hypercholesterolemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID

NO: 1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound.

In certain embodiments, a method for reducing or suppressing LDL cholesterol levels, reducing or suppressing Lp(a) levels, inducing LDL receptor (LDL-R) activity, or regulating LDL cholesterol homeostasis via LDL receptors in the liver of an individual having a PCSK9 associated disease, or the risk of its presence includes administering to an individual a compound containing a specific inhibitor of PCSK9, thereby reducing or suppressing LDL and Lp(a) cholesterol levels, inducing LDL receptor (LDL-R) activity, and regulating LDL cholesterol homeostasis via LDL receptors in the individual's liver . In certain embodiments, the individual has or is at risk of developing a cardiovascular disease. In certain embodiments, the individual has or is at risk for dyslipidaemia. In certain embodiments, the individual has, or is at risk of, mixed dyslipidaemia. In certain embodiments, the individual has or is at risk of hypercholesterolemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the sequence nucleic bases SEQ ID NO: 1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound. In certain embodiments, the compound is administered parenterally to an individual. In certain embodiments, the individual is identified as having a disease associated with PCSK9, or at risk of having it.

Certain embodiments encompass a compound containing a specific inhibitor of PCSK9 for use in the treatment of a disease associated with PCSK9. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the disease is dyslipidemia. In certain embodiments, the disease is mixed dyslipidemia. In certain embodiments, the disease is hypercholesterolemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases from any of the nucleobase sequences of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide, having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID NO: 1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound. In certain embodiments, the compound is administered parenterally to an individual.

Certain embodiments encompass a compound containing a specific inhibitor of PCSK9 for use in lowering or suppressing LDL cholesterol levels, reducing or suppressing Lp(a) levels, inducing LDL receptor (LDL-R) activity, and regulating LDL cholesterol homeostasis via LDL receptors in an individual, having a cardiovascular disease or the risk of having it. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases from any of the nucleobase sequences of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide, having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID NO: 1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound.

Certain embodiments encompass the use of a compound containing a specific inhibitor of PCSK9 for the manufacture or preparation of a medicament for the treatment of a disease associated with PCSK9. Certain embodiments encompass the use of a compound containing a specific inhibitor of PCSK9 for the preparation of a medicament for the treatment of a disease associated with PCSK9. In certain embodiments, the disease is a cardiovascular disease. In certain embodiments, the disease is dyslipidemia. In certain embodiments, the disease is mixed dyslipidemia. In certain embodiments, the disease is hypercholesterolemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets PCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of the nucleic base sequences of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID NO:1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound.

Certain embodiments encompass the use of a compound containing a specific inhibitor of PCSK9 for the manufacture or preparation of a medicament for lowering or suppressing LDL cholesterol levels, lowering or suppressing Lp(a) levels, inducing LDL receptor (LDL-R) activity, and regulating cholesterol homeostasis. LDL via LDL receptors in an individual having or at risk of having a cardiovascular disease. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. Certain embodiments encompass the use of a compound containing a specific PCSK9 inhibitor for the preparation of a medicament for lowering or suppressing LDL cholesterol levels, reducing or suppressing Lp(a) levels, inducing LDL receptor (LDL-R) activity, and regulating LDL cholesterol homeostasis by LDL receptors in an individual with or at risk of cardiovascular disease. In certain embodiments, the cardiovascular disease is dyslipidemia or hypercholesterolemia. In certain embodiments, the compound contains an antisense compound that targets PCSK9. In certain embodiments, the compound contains an oligonucleotide that targets HaPCSK9. In certain embodiments, the compound comprises a modified oligonucleotide having a length of 8-80 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases from the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound comprises a modified oligonucleotide, having a length of 16 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 1016. B

- 17 042765 In certain embodiments, the compound comprises a modified oligonucleotide consisting of the nucleobase sequence of SEQ ID NO: 1016. In certain embodiments, the compound contains a modified oligonucleotide having a length of 16 linked nucleosides, which has a nucleobase sequence comprising SEQ ID NO:

1016.

In certain embodiments, the connection is ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681.

In any of the above embodiments, the connection may be single strand or double strand. In any of the above embodiments, the compound may be an antisense compound or an oligomeric compound.

In any of the above methods or uses, the compound may be targeted to PCSK9. In certain embodiments, the compound comprises or consists of a modified oligonucleotide, eg, a modified oligonucleotide having a length of 16 linked nucleosides. In certain embodiments, the modified oligonucleotide is at least 80%, 85%, 90%, 95%, or 100% complementary to any of the nucleic base sequences mentioned under SEQ ID NOs: 1, 2, 1545-1550. In certain embodiments, the modified oligonucleotide contains at least one modified internucleoside bond, at least one modified sugar, and/or at least one modified nucleobase. In certain embodiments, the modified internucleoside bond is a phosphothioate internucleoside bond, the modified sugar is a bicyclic sugar or a 2'-O-methoxyethyl-modified sugar, and the modified nucleobase is 5-methylcytosine. In certain embodiments, the modified oligonucleotide contains a gap segment consisting of linked deoxynucleosides; 5'-terminal flank segment consisting of linked nucleosides; and a 3' flank segment consisting of linked nucleosides, wherein the gap segment is located between and immediately adjacent to the 5' flank segment and the 3' flank segment, and wherein each nucleoside of each flank segment contains a modified sugar.

In any of the above embodiments, the modified oligonucleotide is 16 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is at least 80%, 85%, 90%, 95%, or 100% complementary to any of the nucleobase sequences mentioned under SEQ ID NOs: 1, 2, 1545-1550. In certain embodiments, the modified oligonucleotide contains at least one modified internucleoside bond, at least one modified sugar, and/or at least one modified nucleobase. In certain embodiments, the modified internucleoside bond is a phosphothioate internucleoside bond, the modified sugar is a bicyclic sugar or a 2'-O-methoxyethyl modified sugar, and the modified nucleobase is 5methylcytosine. In certain embodiments, the modified oligonucleotide contains a gap segment consisting of linked 2'-deoxynucleosides; 5'-terminal flank segment consisting of linked nucleosides; and a 3' flank segment consisting of linked nucleosides, wherein the gap segment is located between and immediately adjacent to the 5' flank segment and the 3' flank segment, and wherein each nucleoside of each flank segment contains a modified sugar.

In any of the above methods or uses, the compound comprises or consists of a modified oligonucleotide having a length of 16 linked nucleosides and having a nucleobase sequence comprising SEQ ID NO: 1016, wherein the modified oligonucleotide comprises:

a gap segment consisting of linked 2'-deoxynucleosides; 5'-terminal flank segment consisting of linked nucleosides; and a 3'-terminal flank segment consisting of linked nucleosides;

where the gap segment is located between the 5'-flank segment and the 3'-flank segment, and where each nucleoside of each flank segment contains a modified sugar.

In any of the above methods or uses, the compound comprises or consists of a modified oligonucleotide having a length of 16 linked nucleobases, which has or consists of a nucleobase sequence comprising or consisting of the sequence of SEQ ID NO: 1016, wherein the modified oligonucleotide comprises:

a gap segment consisting of ten linked deoxynucleosides;

5' end flank segment consisting of three linked nucleosides; And

3'-terminal flank segment, consisting of three linked nucleosides;

where the gap segment is located between the 5'-terminal flank segment and the 3'-terminal flank segment, where each nucleoside of each flank segment contains cEt-caxap; where each internucleoside bond is a phosphothioate bond; where each cytosine is 5-18 042765 methylcytosine. In certain embodiments, the modified oligonucleotide is 16 linked nucleosides in length.

In any of the above methods or uses, the compound comprises or consists of ISIS 863633 or a salt thereof, which have the following chemical structure:

ABOUT

In any of the above methods or uses, the compound comprises or consists of ISIS 863633 or a salt thereof, which have the following chemical structure:

In any of the above methods or uses, the compound contains or consists of the sodium salt of ISIS 863633, which has the following chemical structure:

- 19 042765

In any of the above methods or routes of administration, the compound may be administered parenterally. For example, in certain embodiments, the compound may be administered by injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intrathecal or intracerebroventricular administration.

Some connections

In certain embodiments, the compounds described herein may be antisense compounds. In certain embodiments, the antisense compound comprises or consists of an oligomeric compound. In certain embodiments, the implementation of the oligomeric compound contains a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleic base sequence that is complementary to the target nucleic acid sequence.

In certain embodiments, a compound described herein comprises or consists of a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleic base sequence that is complementary to the target nucleic acid sequence.

In certain embodiments, the connection or antisense connection is single stranded. Such a single-stranded compound or antisense compound contains or consists of an oligomeric compound. In certain embodiments, such an oligomeric compound contains or consists of an oligonucleotide and optionally a conjugated group. In certain embodiments, the oligonucleotide is an antisense oligonucleotide. In certain embodiments, the implementation of the oligonucleotide is modified. In certain embodiments, the oligonucleotide of the single-stranded antisense compound or oligomeric compound contains a self-complementary nucleobase sequence.

In certain embodiments, the connections are double stranded. Such double stranded compounds comprise a first modified oligonucleotide having a region complementary to the target nucleic acid and a second modified oligonucleotide having a region complementary to the first modified oligonucleotide. In certain embodiments, the modified oligonucleotide is an RNA oligonucleotide. In such embodiments, the thymine nucleobase in the modified oligonucleotide is replaced with a uracil nucleobase. In certain embodiments, the implementation of the connection contains a conjugated group. In certain embodiments, one of the modified oligonucleotides is conjugated. In certain embodiments, both modified oligonucleotides are conjugated. In certain embodiments, the first modified oligonucleotide is conjugated. In certain variants,

- 20 042765 the second modified oligonucleotide is conjugated. In certain embodiments, the first modified oligonucleotide is 12-30 linked nucleosides in length and the second modified oligonucleotide is 12-30 linked nucleosides in length. In certain embodiments, one of the modified oligonucleotides has a nucleobase sequence comprising at least 8 contiguous nucleobases from SEQ ID NO: 1016.

In certain embodiments, the antisense compounds are double stranded. Such double stranded antisense compounds comprise a first oligomeric compound having a region complementary to the target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound. The first oligomeric connection of such double-stranded antisense compounds typically contains or consists of a modified oligonucleotide and optionally a conjugated group. The oligonucleotide of the second oligomeric compound of such a double-stranded antisense compound may be modified or unmodified. Either or both of the oligomeric compounds of the double-stranded antisense compound may contain a conjugated group. Oligomeric compounds of double-stranded antisense compounds may contain non-complementary overhang nucleosides.

Examples of single-stranded and double-stranded compounds include, but are not limited to, oligonucleotides, siRNAs, microRNA-targeting oligonucleotides, and single-stranded compounds for RNAi such as small hairpin RNAs (shRNA), single-stranded siRNAs (ssRNA), and microRNA mimetics.

In certain embodiments, a compound described herein has a nucleic base sequence that, when written in the 5'-3' direction, contains a sequence that is inversely complementary to the target nucleic acid segment of the target nucleic acid to which it targets.

In certain embodiments, a compound described herein contains an oligonucleotide having a length of 10-30 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 12-30 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 12-22 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 14-30 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 14-20 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 15-30 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 15-20 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 16-30 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 16-20 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 17-30 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 17-20 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 18-30 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 18-21 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 18-20 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 20-30 linked subunits. In other words, such oligonucleotides are 12-30 linked subunits, 14-30 linked subunits, 14-20 subunits, 15-30 subunits, 15-20 subunits, 16-30 subunits, 16-20 subunits, 17-30 subunits, 17 -20 subunits, 18-30 subunits, 18-20 subunits, 18-21 subunits, 20-30 subunits, or 12-22 linked subunits, respectively. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 14 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 16 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 17 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 18 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 19 linked subunits. In certain embodiments, a compound described herein contains an oligonucleotide having a length of 20 linked subunits. In other embodiments, a compound described herein contains an oligonucleotide of 8-80, 12-50, 13-30, 13-50, 14-30, 1450, 15-30, 15-50, 16-30, 16- 50, 17-30, 17-50, 18-22, 18-24, 18-30, 18-50, 19-22, 19-30, 19-50 or 20-30

- 21 042765 known subunits. In certain such embodiments, a compound described herein comprises an oligonucleotide having a length of 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, or a length in the range limited by any two of the above values. In certain embodiments, the associated subunits are nucleotides, nucleosides, or nucleobases.

In certain embodiments, the compound may further comprise additional components or elements, such as a conjugate group, that are attached to the oligonucleotide. In certain embodiments, such compounds are antisense compounds. In certain embodiments, such compounds are oligomeric compounds. In embodiments in which the conjugated group contains a nucleoside (ie, a nucleoside that links the conjugated group to the oligonucleotide), the nucleoside of the conjugated group is not counted in the length of the oligonucleotide.

In certain embodiments, the connections may be shortened or truncated. For example, one subunit can be removed from the 5' end (5' end truncation) or, alternatively, from the 3' end (3' end truncation). A truncated or truncated compound targeting the PCSK9 nucleic acid may have two subunits removed from the 5' end, or alternatively two subunits may be removed from the 3' end of the compound. Alternatively, the removed nucleosides may be distributed throughout the compound.

If there is one additional subunit in the extended connection, the additional subunit may be located at the 5' or 3' end of the connection. When there are two or more additional subunits, the added subunits can be adjacent to each other, for example, in a compound having two subunits added at the 5' end (5' end addition) or alternatively at the 3' end (3 '-end addition) of the connection. Alternatively, the added subunits may be distributed throughout the compound.

It is possible to increase or decrease the length of a compound, such as an oligonucleotide, and/or introduce mismatched bases without eliminating activity (Woolf et al. Proc. Natl. Acad. Sci. USA 1992, 89:73057309; Gautschi et al. J. Natl. Cancer Inst March 2001, 93:463-471; Maher and Dolnick Nuc Acid Res. 1998, 16:3341-3358). However, seemingly small changes in the sequence, chemical structures, and motifs of an oligonucleotide can greatly affect one or more of the many properties required for clinical research (Seth et al. J. Med. Chem. 2009, 52, 10; Egli et al J. Am. Chem. Soc. 2011, 133, 16642).

In certain embodiments, the compounds described herein are interfering RNA (for RNAi) compounds, which include double-stranded RNA (also referred to as short interfering RNA or siRNA) compounds and single-stranded RNAi (or ssRNAi) compounds. ). Such compounds perform their function at least in part through the RISC signaling pathway to destroy and/or sequester the target nucleic acid (hence include microRNA/microRNA mimetic compounds). As used herein, the term siRNA is intended to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence-specific RNAi, e.g., short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (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, the term RNAi, as used herein, is intended to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational repression, or epigenetic mechanisms.

In certain embodiments, a compound described herein may contain any of the oligonucleotide sequences described herein that target PCSK9. In certain embodiments, the connection may be double stranded. In certain embodiments, the connection comprises a first strand containing a fragment of 16 contiguous nucleobases from SEQ ID NO: 1016 and a second strand. In certain embodiments, the implementation of the connection contains the first strand containing the sequence of nucleic bases under any of SEQ ID NO: 3-1540, and the second strand. In certain embodiments, the implementation of the connection contains ribonucleotides, while the first strand contains uracil (U) instead of thymine (T) in any of SEQ ID NO: 3-1540. In certain embodiments, the connection comprises (i) a first strand containing a nucleobase sequence complementary to the site and PCSK9 targeted by any of SEQ ID NO: 3-1540, and (ii) a second strand. In certain embodiments, the compound contains one or more modified nucleotides whose 2'

- 22 042765 position in the sugar contains a halogen (such as a fluorine group; 2'-F) or contains an alkoxy group (such as a methoxy group; 2'-OMe). In certain embodiments, the compound contains at least one 2'L sugar modification and at least one 2'OMe sugar modification. In certain embodiments, at least one 2'-P sugar modification and at least one 2'-OMe sugar modification are arranged in alternating order for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases along the strand of a dsRNA-based compound. In certain embodiments, the compound contains one or more bonds between adjacent nucleotides other than a naturally occurring phosphodiester bond. Examples of such bonds include phosphoramide, phosphothioate and phosphodithioate bonds. The compounds may also be chemically modified nucleic acid molecules as disclosed in US Pat.

In certain embodiments, the first strand of the junction is the siRNA guide strand and the second strand of the junction is the siRNA follower strand. In certain embodiments, the second strand of the connection is complementary to the first strand. In certain embodiments, each strand of connection is 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides in length. In certain embodiments, the first or second strand of the connection may contain a conjugated group.

In certain embodiments, a compound described herein may contain any of the oligonucleotide sequences described herein that target PCSK9. In certain embodiments, the connection is single stranded. In certain embodiments, such a connection is a single-stranded connection for RNAi (ssRNAi). In certain embodiments, the compound contains a fragment of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19 or 20 contiguous nucleobases from any of SEQ ID NOs: 3-1540. In certain embodiments, the compound contains the nucleobase sequence of any of SEQ ID NO: 31540. In certain embodiments, the compound contains ribonucleotides, with uracil (U) located in place of thymine (T) in any of SEQ ID NO: 3-1540. In certain embodiments, the compound contains a nucleobase sequence that is complementary to a site in PCSK9 targeted by any of SEQ ID NOs: 3-1540. In certain embodiments, the compound contains one or more modified nucleotides that have a halogen (such as a fluoro group; 2'-F) or an alkoxy group (such as a methoxy group; 2'-OMe) at the 2' position on the sugar. In certain embodiments, the compound contains at least one 2'L sugar modification and at least one 2'OMe sugar modification. In certain embodiments, at least one 2'-P sugar modification and at least one 2'-OMe sugar modification are arranged in alternating order for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleobases along the connection strand. In certain embodiments, the compound contains one or more bonds between adjacent nucleotides other than a naturally occurring phosphodiester bond. Examples of such bonds include phosphoramide, phosphothioate and phosphodithioate bonds. The compounds may also be chemically modified nucleic acid molecules as disclosed in US Pat. No. 6,673,661. consists of 16, 17, 18, 19, 20, 21, 22 or 23 linked nucleosides. In certain embodiments, the implementation of the connection may contain a conjugated group.

In certain embodiments, the compounds described herein contain modified oligonucleotides. Certain modified oligonucleotides have one or more centers of asymmetry and therefore form enantiomers, diastereomers and other stereoisomeric configurations that can be defined in terms of absolute stereochemistry as (R) or (S), as α or β, as, for example, in the case of anomers sugars, or as (D) or (L), such as in the case of amino acids, etc. Modified oligonucleotides provided herein include all such possible isomers, including their racemic and optically pure forms, unless otherwise specified. Likewise, all cis and trans isomers and tautomeric forms are also included.

The compounds described herein include variants in which one or more atoms are replaced by a non-radioactive isotope or a radioactive isotope of the specified element. For example, compounds according to this document, which contain hydrogen atoms, cover all possible substitutions of deuterium for each of the hydrogen atoms 1H. Isotopic substitutions covered by the compounds of this document include, without limitation: ² H or ³ H instead of 1H, ¹³ C or ¹⁴ C instead of ¹² C, ¹⁵ N instead of ¹⁴ N, ¹⁷ O or ¹⁸ O instead of ¹⁶ O, and ³³ S, ³⁴ S, ³⁵ S, or ³⁶ S instead of ³² S. In certain embodiments, substitutions with non-radioactive isotopes can impart novel properties to the compound that are favorable for its use as a therapeutic or research tool. In certain embodiments, radioactive isotope substitutions may make the compound suitable for research or diagnostic purposes, such as imaging analysis.

Some mechanisms

In certain embodiments, the compounds described herein contain or consist of modified oligonucleotides. In certain embodiments, the compounds described herein are antisense compounds. In certain embodiments, the compounds comprise oligomeric compounds. In certain embodiments, the compounds described herein are capable of hybridizing to a target nucleic acid resulting in at least one form of antisense activity. In certain embodiments, the compounds described herein selectively target one or more target nucleic acids. Such compounds contain a nucleic base sequence that hybridizes to one or more target nucleic acids resulting in one or more forms of the desired antisense activity, and does not hybridize to one or more non-target nucleic acids or does not hybridize to one or more non-target nucleic acids in a manner that results in significant unwanted antisense activity.

In certain forms of antisense activity, hybridization of a compound described herein with a target nucleic acid results in the recruitment of a protein that cleaves the target nucleic acid. For example, certain compounds described herein result in RNase H-mediated cleavage of the target nucleic acid. RNase H is a cellular endonuclease that cleaves an RNA strand in an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, the compounds described herein are DNA-like enough to induce RNase H activity. In addition, in certain embodiments, one or more non-DNA-like nucleosides are allowed in the gap segment of the gapmer.

In certain forms of antisense activity, the compounds described herein, or a fragment of the compound, are incorporated into an RNA-inducible silencing complex (RISC), which ultimately leads to cleavage of the target nucleic acid. For example, certain compounds described herein result in cleavage of the target nucleic acid by the Argonaute protein. Compounds that are included in RISC are RNAI compounds. Connections for RNAi can be double-stranded (siRNA) or single-stranded (ssRNA).

In certain embodiments, hybridization of the compounds described herein with a target nucleic acid does not result in the recruitment of a protein that cleaves the target nucleic acid. In certain such embodiments, hybridization of a compound to a target nucleic acid results in a change in the splicing of the target nucleic acid. In certain embodiments, hybridization of a compound to a target nucleic acid results in suppression of the binding interaction between the target nucleic acid and the protein or other nucleic acid. In certain such embodiments, hybridization of a compound to a target nucleic acid results in a change in the translation of the target nucleic acid.

Forms of antisense activity can be observed directly or indirectly. In certain embodiments, observing or detecting antisense activity involves observing or detecting a change in the amount of a target nucleic acid or protein encoded by that target nucleic acid, a change in the ratio of splice variants of the nucleic acid or protein, and/or a phenotypic change in a cell or animal.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide containing a region complementary to the target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid encodes a protein. In certain such embodiments, the target nucleic acid is selected from mRNA and pre-mRNA containing intron, exon, and untranslated regions. In certain embodiments, the target RNA is an mRNA. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain such embodiments, the target region lies entirely within the intron. In certain embodiments, the target region encompasses the exon-intron junction. In certain embodiments, at least 50% of the target region is within an intron.

- 24 042765

The nucleotide sequences that encode PCSK9 include, without limitation, the following: RefSeq # and GENBANK Access # NM_174936.3 (SEQ ID NO: 1), GENBANK Access # NC_000001.11 with nucleotides cut off 55036001-55068000 (SEQ ID NO: 2 ), GENBANK Access No. AK124635.1 (SEQ ID NO: 1545), GENBANK Access No. NT_032977.8 with nucleotide cuts 25475000-25504000 (SEQ ID NO: 1546), GENBANK Access No. DA092236.1 (SEQ ID NO: 1547), GENBANK Access No. DA803830.1 (SEQ ID NO: 1548), GENBANK Access No. DC352135.1 (SEQ ID NO: 1549), and GENBANK Access No. NR_110451.1 (SEQ ID NO: 1550).

Hybridization

In some embodiments, hybridization occurs between a compound disclosed herein and a PCSK9 nucleic acid. The most common hybridization mechanism involves the formation of hydrogen bonds (for example, the formation of hydrogen bonds in the form of Watson-Crick, Hoogsteen or reverse Hoogsteen interaction) between complementary nucleic bases of nucleic acid molecules.

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

Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art. In certain embodiments, the compounds provided herein can specifically hybridize to a PCSK9 nucleic acid.

complementarity

An oligonucleotide is said to be complementary to another nucleic acid if the nucleic base sequence of such an oligonucleotide or one or more of its regions corresponds to the nucleic base sequence of another oligonucleotide or nucleic acid or one or more regions thereof when the two nucleic base sequences are aligned in opposite directions. Nucleobase matches or complementary nucleobases described herein are limited to the following pairs: adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), and 5-methylcytosine (mC ) and guanine (G) unless otherwise noted. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity for each nucleoside and may contain one or more nucleobase mismatches. An oligonucleotide is fully complementary or 100% complementary if such oligonucleotides have nucleobase matches for each nucleoside without any nucleobase mismatch.

In certain embodiments, the compounds described herein contain or consist of modified oligonucleotides. In certain embodiments, the compounds described herein are antisense compounds. In certain embodiments, the compounds comprise oligomeric compounds. Non-complementary nucleobases between the compound and the PCSK9 nucleic acid may be allowed, provided that the compound retains the ability to specifically hybridize to the target nucleic acid. Moreover, the compound may hybridize to one or more segments of the PCSK9 nucleic acid such that intermediate or adjacent segments do not participate in the hybridization event (eg, loop, mismatch, or hairpin).

In certain embodiments, the compounds provided herein, or a specified fragment thereof, are complementary to the PCSK9 nucleic acid, its target region, target segment, or specified fragment by an amount of, at least, or up to 70, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. In certain embodiments, the compounds provided herein, or a specified fragment thereof, are 70-75, 75-80, 80-85, 85-90, 90-95 complementary to the PCSK9 nucleic acid, its target region, target segment, or specified fragment. , 95-100% or any value within these ranges. The percent complementarity of a compound to a target nucleic acid can be determined using standard methods.

For example, a compound in which 18 of the compound's 20 nucleobases are complementary to the target region, and therefore will specifically hybridize, will be 90 percent complementary. In this example, the remaining non-complementary nucleobases may cluster or alternate with the complementary nucleobases and do not need to be adjacent to each other or to the complementary nucleobases. Accordingly, a compound that is 18 nucleobases long, having four non-complementary nucleobases that are flanked by two regions that are fully complementary to the target nucleic acid, will have a total complementarity with the target nucleic acid of 77.8%. Complementarity percentage of a compound with a region

- 25 042765 target nucleic acids can be determined in the usual way using BLAST programs (Basic Local Alignment Finder) 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 sequence homology, identity, or complementarity can be determined, for example, using the Gap program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, Madison, Wisconsin) using the default settings, which uses Smith's algorithm -Waterman (Adv. Appl. Math., 1981, 2, 482-489).

In certain embodiments, the compounds described herein, or certain fragments thereof, are fully complementary (i.e., 100% complementary) to the target nucleic acid or a certain fragment thereof. For example, the compound may be fully complementary to the PCSK9 nucleic acid or its target region or target segment or target sequence. As used herein, fully complementary means that each nucleobase of a compound is complementary to the corresponding nucleobase of the target nucleic acid. For example, a 20 nucleobase compound is fully complementary to a 400 nucleobase target nucleic acid, provided that there is a corresponding 20 nucleobase fragment in the target nucleic acid that is fully complementary to the compound. Fully complementary can also be used in relation to a specific fragment of the first and/or second nucleic acid. For example, a 20 nucleobase fragment in a 30 nucleobase compound may be fully complementary to a 400 nucleobase target nucleic acid. A 20 nucleobase fragment in a 30 nucleobase compound is fully complementary to a target sequence if there is a corresponding 20 nucleobase fragment in the target sequence in which each nucleobase is complementary to a nucleobase in the 20 nucleobase fragment in the compound. At the same time, the entire 30 nucleobase compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases in the compound are also complementary to the target sequence.

In certain embodiments, the compounds described herein contain one or more non-matching nucleic bases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by this mismatch, but activity against the non-target molecule is reduced by an even greater amount. Thus, in certain such embodiments, compound selectivity is improved. In certain embodiments, the mismatch has a specific location within the oligonucleotide having the gapmer motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5' end of the gap region. In certain such embodiments, the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3' end of the gap region. In certain such embodiments, the mismatch is at position 1, 2, 3, or 4 from the 5' end of the flank region. In certain such embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3' end of the flank region. In certain embodiments, the mismatch has a specific location within the oligonucleotide lacking the gapmer motif. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5' end of the oligonucleotide. In certain such embodiments, the mismatch is at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3' end of the oligonucleotide.

The location of the non-complementary nucleobase may be at the 5' end or at the 3' end of the compound. Alternatively, the non-complementary nucleobase or nucleobases may be in an internal junction position. Where there are two or more non-complementary nucleobases, they may be contiguous (ie, linked) or non-contiguous. In one embodiment, the non-complementary nucleobase is located in the flank segment of the gapmer oligonucleotide.

In certain embodiments, compounds described herein that are or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length contain no more than 4, no more than 3, no more 2 or at most 1 non-complementary nucleic base relative to the target nucleic acid, such as PCSK9 nucleic acid, or a specified fragment thereof.

In certain embodiments, compounds described herein that are or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 in length , 28, 29, or 30 nucleobases, contain 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 with respect to the target nucleic acid, such as PCSK9 nucleic acid, or its a specific fragment.

In certain embodiments, the compounds described herein also

- 26 042765 include those compounds that are complementary to the target nucleic acid fragment. As used herein, a fragment refers to a specific number of contiguous (ie linked) nucleic bases within a region or segment of a target nucleic acid. A fragment can also refer to a specific number of contiguous nucleobases in a compound. In certain embodiments, the compounds are complementary to a fragment of at least 8 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 9 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 10 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 11 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 12 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 13 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 14 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 15 nucleobases in the target segment. In certain embodiments, the compounds are complementary to a fragment of at least 16 nucleobases in the target segment. Also contemplated are compounds that are complementary to a fragment of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleobases in the target segment or fragment within a range limited by any two of these values.

Identity

The compounds provided herein may also share a certain percentage of identity with a particular nucleotide sequence, SEQ ID NO, or a compound represented by a particular ION number, or a fragment thereof. In certain embodiments, the compounds described herein are antisense compounds or oligomeric compounds. In certain embodiments, the compounds described herein are modified oligonucleotides. As used herein, a compound is identical to the sequence disclosed herein if it has the same ability to form nucleic base pairs. For example, RNA that contains uracil instead of thymidine in the disclosed DNA sequence would be considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Also contemplated are shortened and extended versions of the compounds described herein, as well as compounds having non-identical bases relative to the compounds provided herein. Non-identical bases may be adjacent to each other or distributed throughout the connection. The percent identity of a compound is calculated by the number of bases that have identical base pairing properties compared to the sequence to which it is being compared.

In certain embodiments, the compounds described herein, or fragments thereof, are identical to one or more compounds or SEQ ID NO or fragment thereof disclosed herein by an amount equal to or equal to at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%. In certain embodiments, the compounds described herein are about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or any percentage in between. with respect to a specific nucleotide sequence, SEQ ID NO, or a compound represented by a specific ION number, or a fragment thereof, wherein the compounds contain an oligonucleotide having one or more non-matching nucleic bases. In certain such embodiments, the mismatch is at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 5' end of the oligonucleotide. In certain such embodiments, the mismatch is at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 from the 3' end of the oligonucleotide.

In certain embodiments, the compounds described herein contain or consist of antisense compounds. In certain embodiments, a fragment of an antisense compound is compared to an equal length fragment in a target nucleic acid. In certain embodiments, a fragment of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases is compared to an equal length fragment in the nucleobase acid targets.

In certain embodiments, the compounds described herein contain or consist of oligonucleotides. In certain embodiments, an oligonucleotide fragment is compared to an equal length fragment in a target nucleic acid. In certain embodiments, a fragment of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobases is compared to an equal length fragment in the nucleobase target acid.

- 27 042765

Some modified compounds

In certain embodiments, the compounds described herein contain or consist of oligonucleotides consisting of linked nucleosides. The oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides contain at least one modification compared to unmodified RNA or DNA (i.e. contain at least one modified nucleoside (containing a modified sugar component and/or a modified nucleobase) and/or at least one modified internucleoside bond) .

A. Modified nucleosides.

Modified nucleosides contain a modified sugar moiety or a modified nucleobase, or both a modified sugar moiety and a modified nucleobase.

1. Modified sugar components.

In certain embodiments, the sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, the modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, the modified sugar components are sugar simulants. Such sugar simulants may contain one or more substitutions corresponding to substitutions in other types of modified sugar components.

In certain embodiments, the modified sugar moieties are non-bicyclic modified sugar moieties containing a furanosyl ring with one or more acyclic substituents, including but not limited to substituents at the 2', 4', and/or 5' positions. In certain embodiments, one or more of the acyclic substituents on the non-bicyclic modified sugar moieties are branched. Examples of 2'-substituent groups suitable for non-bicyclic modified sugar components include, without limitation: 2'-F, 2'-OCH ₃ (OMe or O-methyl) and 2'-O(CH ₂ ) ₂ OCH ₃ (MOE) . In certain embodiments, the 2'-substituent groups are selected from halo, allyl, amino, azido, SH, CN, OCN, CF ₃ , OCF ₃ , OC ₁ -C ₁₀ -alkoxy substituted with OC ₁ -C ₁₀ -alkoxy, OC ₁ -C ₁₀ -alkyl substituted with OC ₁ -C ₁₀ -alkyl, S-alkyl, N(R _m )-alkyl, O-alkenyl, S-alkenyl, N(R _m )-alkenyl, Oalkynyl, S-alkynyl, N (R _m )-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, Oalkaryl, O-aralkyl, O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ ON(R _m )(R _n ) or OCH2C(=O)-N(R _m )(R _n ), where each Rm and R _n is independently H, an amino protecting group, or substituted or unsubstituted C ₁ -C ₁₀ alkyl, and 2'-substituting groups described in Cook et al., US 6531584; Cook et al., US 5859221; and Cook et al., US 6005087. In certain embodiments, such 2' substituent groups may be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol , thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4'-substituent groups suitable for linear non-bicyclic modified sugar moieties include, without limitation, alkoxy (eg, methoxy), alkyl, and the groups described in Manoharan et al., WO 2015/106128. Examples of 5'-substituent groups suitable for non-bicyclic modified sugar moieties include, without limitation: 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy. In certain embodiments, the non-bicyclic modified sugars contain more than one non-bridging substituent on the sugar, such as in the case of 2'-F-5'-methyl modified sugar moieties, as well as the modified sugar moieties and modified nucleosides described in Migawa et al., US2010/190837 and Rajeev et al., US2013/0203836.

In certain embodiments, the 2'-substituted nucleoside or non-bicyclic 2'-modified nucleoside contains a sugar moiety containing a linear 2'-substituent group selected from: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH=CH2, OCH2CH=CH2, OCH ₂ CH ₂ OCH ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ ON(R _m )(R _n ), O(CH ₂ ) ₂ O(CH ₂ ) ₂ N( CH ₃ ) ₂ and N-substituted acetamide (OCH ₂ C(=O)-N(R _m )(R _n )), where each R _m and R _n independently represents H, an amino protecting group, or a substituted or unsubstituted C ₁ -C ₁₀ -alkyl.

In certain embodiments, the 2'-substituted nucleoside or non-bicyclic 2'-modified nucleoside contains a sugar moiety containing a linear 2'-substituent group selected from: F, OCF3, OCH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON( CH3)2, O(CH2)2O(CH2)2N(CH3)2 and OCH2C(=O)-N(H)CH3 (NMA).

In certain embodiments, the 2'-substituted nucleoside or non-bicyclic 2'-modified nucleoside contains a sugar moiety containing a linear 2'-substituent group selected from: F, OCH3, and OCH2CH2OCH3.

Nucleosides containing modified sugar moieties, such as non-bicyclic modified sugar moieties, are designated by the position(s) of substitution(s) in the sugar moiety of the nucleoside. For example, nucleosides containing 2'-substituted

- 28 042765 or 2-modified sugar components are called 2'-substituted nucleosides or 2-modified nucleosides.

Certain modified sugar moieties contain a bridging substituent on the sugar that forms a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety contains a bridge between the 4' and 2' atoms of the furanose ring. Examples of such 4'-2'-bridging substituents on a sugar include, without limitation: 4'-CH2-2', 4'-(CH2>2-2', 4'-(CH2)3-2', 4'-CH2 -O-2' (LNA), 4'-CH2-S-2', 4'(CH ₂ ) ₂ -O-2' (ENA), 4'-CH(CH ₃ )-O-2' (called conformationally limiting ethyl or cEt in S-configuration), 4'-CH ₂ -O-CH ₂ -2', 4'-CH2-N(R)-2', 4'-CH(CH2OCH ₃ )-O-2 ' (conformationally restrictive MOE or cMOE) and its analogues (see e.g. Seth et al., US 7399845, Bhat et al., US 7569686, Swayze et al., US 7741457, and Swayze et al., US 8022193) , 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see, for example, Seth et al., US 8278283), 4'-CH2-N(OCH ₃ )-2' and its analogs (see, for example, Prakash et al., US 8278425), 4'-CH2-ON(CH3)-2' (see, for example, Allerson et al., US 7696345 and Allerson et al., US 8124745 ), 4'-CH2-C(H)(CH3)-2' (see e.g. Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4'-CH2C( =CH2)-2' and its analogs (see, for example, Seth et al., US 8278426), 4'-C(R _a R _b )-N(R)-O-2', 4'-C( RaRb)-ON(R)-2', 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2', where each R, R _a and R _b independently represents is H, a protecting group or C ₁ -C ₁₂ -alkyl (see, for example, Imanishi et al., US 7427672).

In certain embodiments, such 4'-2' bridges independently contain 1 to 4 linked groups independently selected from: -[C(R _a )(R _b )] _n -, -[C(R _a )(R _b )] _n -O-, -C(R _a )=C(R _b )-, -C(R _a )=N-, -C(=NRa)-, -C(=O)-, -C( =S)-, -O-, -Si(Ra)2-, -S(=O) _x - and -N(Ra)-;

where x is 0, 1 or 2;

n is 1, 2, 3 or 4;

each R _a and Rb is independently H, a protecting group, hydroxyl, C ₁ -C ₁₂ alkyl, substituted with C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted with C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ -alkynyl, substituted C ₂ C ₁₂ -alkynyl, C ₅ -C ₂₀ -aryl, substituted C ₅ -C ₂₀ -aryl, heterocyclic radical, substituted heterocyclic radical, heteroaryl, substituted heteroaryl, alicyclic C ₅ -C ₇ -radical, substituted alicyclic C ₅ -C7 radical, halogen, OJ1, NJ1J2, SJ1, N ₃ , COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) ₂ - J1) or sulfoxyl (S(=O)-J1); and each J1 and J2 is independently H, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ - alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₅ -C ₂₀ -aryl, substituted C ₅ -C ₂₀ -aryl, acyl (C(=O)-H), substituted acyl, heterocyclic radical, substituted heterocyclic radical, C ₁ -C ₁₂ -aminoalkyl substituted with C ₁ -C ₁₂ -aminoαalkyl or a protective group.

Additional bicyclic sugar moieties are known in the art, see for example: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443, Albaek et al., J. Org. Chem., 2006, 71, 77317740, 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, 8362-8379; Elayadi et al., Curr. Opinion Inverts. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wengel et al., U.S. 7053207, Imanishi et al., U.S. 6268490, Imanishi et al. U.S. 6770748, Imanishi et al., U.S. RE44779; Wengel et al., U.S. 6794499, Wengel et al., U.S. 6670461; Wengel et al., U.S. 7034133, Wengel et al., U.S. 8080644; Wengel et al., U.S. 8034909; Wengel et al., U.S. 8153365; Wengel et al., U.S. 7572582; and Ramasamy et al., U.S. 6525191, Torsten et al., WO 2004/106356, Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. 7547684; Seth et al., U.S. 7666854; Seth et al., U.S. 8088746; Seth et al., U.S. 7750131; Seth et al., U.S. 8030467; Seth et al., U.S. 8268980; Seth et al., U.S. 8546556; Seth et al., U.S. 8530640; Migawa et al., U.S. 9012421; Seth et al., U.S. 8501805; Allerson et al., US2008/0039618; and Migawa et al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosides comprising such bicyclic sugar moieties are further defined by isomeric configuration. For example, the LNA nucleoside (described herein) may be in the α-L configuration or in the β-D configuration.

LNA (PD-configuration) α-L-LNA (aL-configuration) bridge = 4-CH2-O-2' bridge = 4'-CH ₂ -O-2' aL-methyleneoxy-modified (4'-CH2-O -2') or α-L-LNA bicyclic nucleosides were incorporated into oligonucleotides that exhibited antisense activity (Frieden et al., Nucleic Acids Research, 2003, 27, 6365-6372). In this document, the general description of bicyclic nucleosides includes both isomeric configurations. If positions of specific bicyclic nucleosides (e.g. LNA or cEt) are identified in illustrated

- 29 042765 exemplified herein, they are in the β-D configuration, unless otherwise indicated.

In certain embodiments, the modified sugar components contain one or more non-bridged substituents on the sugar and one or more bridged substituents on the sugar (eg, in the case of 5'-substituted and 4'-2'-bridged sugars).

In certain embodiments, the modified sugar components are sugar simulants. In certain such embodiments, the implementation of the oxygen atom in the sugar component is replaced by, for example, a sulfur, carbon or nitrogen atom. In certain similar embodiments, such modified sugar components also contain bridging and/or non-bridging substituents as described herein. For example, certain sugar mimics contain a 4' sulfur atom and a substitution at the 2' position (see, for example, Bhat et al., U.S. 7,875,733 and Bhat et al., U.S. 7,939,677) and/or the 5' position.

In certain embodiments, the sugar simulants comprise rings with a number of atoms other than 5. For example, in certain embodiments, the sugar simulant comprises six-membered tetrahydropyran (THP). Such tetrahydropyrans may be further modified or substituted. Nucleosides containing such modified tetrahydropyrans include, without limitation, hexite nucleic acid (HNA), annit nucleic acid (ANA), mannitol nucleic acid (MNA) (see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841854), fluoro-HNA:

F

F-HNA (F-HNA, see e.g. Swayze et al., U.S. 8088904; Swayze et al., U.S. 8440803; and Swayze et al., U.S. 9005906, F-HNA may also be referred to as F-THP or 3' -fluorotetrahydropyran) and nucleosides containing additional modified THP compounds of the following formula:

where independently for each specified modified THP nucleoside:

Bx is a nucleobase component;

each of T ₃ and T ₄ is independently an internucleoside linking group linking the modified THP nucleoside to the rest of the oligonucleotide, or one of T ₃ and T ₄ is an internucleoside linking group linking the modified THP nucleoside to the rest of the oligonucleotide and the other is from T ₃ and T ₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5' or 3' end group;

each of q _b q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ independently represents H, C ₁ -C ₆ -alkyl, substituted with C ₁ C ₆ -alkyl, C ₂ -C ₆ -alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl; and each of R1 and R2 is independently selected from hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ _b N ₃ , OC(=X)J _b OC(=X)NJJ ₂ , NJ ₃ C(=X)NJJ ₂ , and CN, where X is O, S, or NJ1 and J1, J2, and J3 are each independently H or C ₁ -C ₆ alkyl.

In certain embodiments, modified THP nucleosides are provided wherein each of q _b q ₂ , q ₃ , q ₄ , q ₅ , q _{6 ,} and q ₇ is H. In certain embodiments, at least one of q _b q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ is other than H. In certain embodiments, at least one of q _b q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ is methyl . In certain embodiments, modified THP nucleosides are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is F and R2 is H, in certain embodiments, R1 is methoxy and R2 is H, and in certain embodiments, R1 is methoxyethoxy and _R2 is H.

In certain embodiments, the sugar mimics comprise rings containing more than 5 atoms and more than one heteroatom. For example, nucleosides containing morpholine sugar components and their use in oligonucleotides have been reported (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. 5698685; Summerton et al. , U.S. 5166315; Summerton et al., U.S. 5185444; and Summerton et al., U.S. 5034506). As used herein, the term morpholine component means a sugar simulant with the following structure:

- 30 042765

In certain embodiments, the implementation of the morpholine components can be modified, for example, by adding or changing various substituent groups in the above structure of the morpholine component. Such sugar simulants are referred to herein as modified morpholine components.

In certain embodiments, the sugar simulants contain acyclic components. Examples of nucleosides and oligonucleotides containing such acyclic sugar mimics include, without limitation, peptidic nucleic acid (PNA), acyclic butyl nucleic acid (see, for example, Kumar et al., Org. Biomol. Chem., 2013, 77, 5853- 5865), as well as the nucleosides and oligonucleotides described in Manoharan et al., US2013/130378.

Many other bicyclic and tricyclic ring systems of sugars and sugar mimics are known in the art and can be used in modified nucleosides.

2. Modified nucleic bases.

Nucleic bases (or bases) with modifications or substitutions are structurally distinct from, but functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleic bases can take part in the formation of hydrogen bonds. Such nucleobase modifications may confer nuclease stability, binding affinity, or some other beneficial biological property to antisense compounds.

In certain embodiments, the compounds described herein contain modified oligonucleotides. In certain embodiments, the modified oligonucleotides comprise one or more nucleosides containing an unmodified nucleobase. In certain embodiments, the modified oligonucleotides comprise one or more nucleosides containing the modified nucleobase. In certain embodiments, the modified oligonucleotides contain one or more nucleosides that do not contain a nucleobase, referred to as base-deleted nucleosides.

In certain embodiments, the modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl- or alkynyl-substituted pyrimidines, alkyl-substituted purines, and N-2-, N-6-, and O-6-substituted purines. In certain embodiments, the modified nucleobases are selected from: 2-aminopropyladenine, 5hydroxymethylcytosine, 5-hydroxymethylcytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2thiocytosine, 5-propynyl(CΞC-CH ₃ )-uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino , 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl, 5-halogenuracil and 5-halogencytosine, 7-methylguanine, 7-methyladenine, 2-P-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-M-benzoyladenine, 2-Nisobutyrylguanine, 4-N-benzoylcytosine, 4-N- benzoyluracil, 5-methyl-4-M-benzoylcytosine, 5-methyl4-N-benzoyluracil, universal bases, hydrophobic bases, non-specific pairing bases, oversized bases and fluorinated bases. Additional modified nucleobases include tricyclic pyrimidines such as 1,3-diazaphenoxazin-2-one, 1,3-diazaphenothiazin-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazin-2-one (G- figurative clip). Modified nucleobases may also include nucleobases in which the purine or pyrimidine base has been replaced by other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2pyridone. Additional nucleobases include those disclosed in Merigan et al., US 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858-859 ; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288; and nucleobases disclosed in chapters 6 and 15 of Antisense Drug Technology, Crooke ST, Ed., CRC Press, 2008, pages 163-166 and 442-443.

Publications that describe the preparation of some of the above modified nucleobases, as well as other modified nucleobases, include, without limitation, Manoharan et al., US2003/0158403, Manoharan et al., US2003/0175906; Dinh et al., U.S. 4845205; Spielvogel et al., U.S. 5130302; Rogers et al., U.S. 5134066; Bischofberger et al., U.S. 5175273; Urdea et al., U.S. 5367066; Benner et al., U.S. 5432272; Matteucci et al., U.S. 5434257; Gmeiner et al., U.S. 5457187; Cook et al., U.S. 5459255; Froehler et al., U.S. 5484908; Matteucci et al., U.S. 5502177; Hawkins et al., U.S.

- 31 042765

5525711; Haralambidis et al., U.S. 5552540; Cook et al., U.S. 5587469; Froehler et al., U.S. 5594121; Switzer et al., U.S. 5596091; Cook et al., U.S. 5614617; Froehler et al., U.S. 5645985; Cook et al., U.S. 5681941; Cook et al., U.S. 5811534; Cook et al., U.S. 5750692; Cook et al., U.S. 5948903; Cook et al., U.S. 5587470; Cook et al.

U.S. 5457191; Matteucci et al., U.S. 5763588; Froehler et al., U.S. 5830653; Cook et al., U.S. 5808027; Cook et al., U.S. 6166199; and Matteucci et al., U.S. 6005096.

In certain embodiments, compounds targeting the PCSK9 nucleic acid comprise one or more modified nucleic bases. In certain embodiments, the modified nucleobase is 5'-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

3. Modified internucleoside bonds.

The naturally occurring internucleoside bond in RNA and DNA is a 3'-5' phosphodiester bond. In certain embodiments, the compounds described herein having one or more modified, i.e. non-naturally occurring, internucleoside bonds are often preferred over antisense compounds with naturally occurring internucleoside bonds due to their desirable properties such as, for example, increased cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

In certain embodiments, compounds targeting the PCSK9 nucleic acid contain one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside bonds are phosphothioate bonds. In certain embodiments, each internucleoside bond of the antisense compound is a phosphothioate internucleoside bond.

In certain embodiments, the compounds described herein contain oligonucleotides. Oligonucleotides with modified internucleoside bonds contain internucleoside bonds that retain a phosphorus atom, as well as internucleoside bonds that do not have a phosphorus atom. Exemplary phosphorus-containing internucleoside bonds include, without limitation, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphothioate bonds. Methods for preparing phosphorus-containing and phosphorus-free bonds are well known.

In certain embodiments, the nucleosides of the modified oligonucleotides may be linked to each other via any internucleoside bond. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Exemplary phosphorus-containing internucleoside bonds include, but are not limited to, phosphate bonds, which include phosphodiester bond (P=O) (also referred to as unmodified or naturally occurring bonds), phosphotriester, methylphosphonate, phosphoramidate, as well as phosphothioate (P=S) and phosphodithioate (HS -P=S) links. Exemplary phosphorus-free internucleoside linking groups include, without limitation, methylenemethylimino (-CH2N(CH ₃ )-O-CH ₂ -), thiodiester, thionocarbamate (-OC(=O)(NH)-S-); siloxane (-O-SiH ₂ -O-) and N,N'-dimethylhydrazine (-CH2-N(CH ₃ )-N(CH ₃ )-) groups. Modified internucleoside bonds, as opposed to naturally occurring phosphate bonds, can be used to modify, typically increase, the resistance of an oligonucleotide to nucleases. In certain embodiments, internucleoside bonds having a chiral atom can be obtained as a racemic mixture or as individual enantiomers. Illustrative chiral internucleoside bonds include, without limitation, alkyl phosphonate and phosphothioate bonds. Methods for preparing phosphorus-containing and phosphorus-free internucleoside bonds are well known to those skilled in the art.

Neutral internucleoside bonds include, but are not limited to, phosphotriester, methylphosphonate bonds, MMI (3'-CH2-N(CH ₃ )-O-5'), 3-amide (3'-CH ₂ -C(=O)-N(H) -5'), 4-amide (3'-CH ₂ -N(H)C(=O)-5'), formacetal (3'-O-CH ₂ -O-5'), methoxypropyl and thioformacetal bonds ( 3'-S-CH ₂ O-5'). Additional neutral internucleoside bonds include non-ionic bonds, including siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfonate ester, and amide bonds (see, for example: Carbohydrate Modifications in Antisense Research; YS Sanghvi and PD Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Additional neutral internucleoside bonds include non-ionic bonds containing a combination of N, O, S and CH ₂ constituents.

In certain embodiments, the implementation of the oligonucleotides contain modified internucleoside linkages located along the oligonucleotide or its region in the form of a specific characteristic site or motif of the modified internucleoside bonds. In certain embodiments, the internucleoside bonds are arranged in a gap-containing motif. In such embodiments, the internucleoside bonds in each of the two flank regions are different from the internucleoside bonds in the gap region. In certain embodiments, the internucleoside bonds in the flanks are phosphodiester and the internucleoside bonds in the gap are phosphothioate. The nucleoside motif is independently chosen so that such oligonucleotides having a motif from

- 32 042765 internucleoside bonds containing a gap may or may not have a nucleoside motif containing a gap, and if they do have a nucleoside motif containing a gap, then the length of the flanks and the gap may or may not be the same.

In certain embodiments, the implementation of the oligonucleotides contain a region having an alternating motif of internucleoside bonds. In certain embodiments, the implementation of the oligonucleotides contain a region with uniformly modified internucleoside bonds. In certain such embodiments, the implementation of the oligonucleotide contains a region having homogeneous bonds, representing phosphothioate internucleoside bonds. In certain embodiments, the oligonucleotide has uniform phosphothioate linkages. In certain embodiments, each internucleoside bond of the oligonucleotide is selected from phosphodiester and phosphothioate. In certain embodiments, each internucleoside bond of the oligonucleotide is selected from phosphodiester and phosphothioate, and at least one internucleoside bond is phosphothioate.

In certain embodiments, the oligonucleotide contains at least 6 phosphothioate internucleoside bonds. In certain embodiments, the oligonucleotide contains at least 8 phosphothioate internucleoside bonds. In certain embodiments, the oligonucleotide contains at least 10 phosphothioate internucleoside bonds. In certain embodiments, the implementation of the oligonucleotide contains at least one block consisting of at least 6 consecutive phosphothioate internucleoside bonds. In certain embodiments, the implementation of the oligonucleotide contains at least one block consisting of at least 8 consecutive phosphothioate internucleoside bonds. In certain embodiments, the implementation of the oligonucleotide contains at least one block consisting of at least 10 consecutive phosphothioate internucleoside bonds. In certain embodiments, the implementation of the oligonucleotide contains at least one block, consisting of at least 12 consecutive phosphothioate internucleoside bonds. 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 at the 3' end of the oligonucleotide.

In certain embodiments, the oligonucleotides contain one or more methylphosphonate linkages. In certain embodiments, oligonucleotides having a gapmeric nucleoside motif comprise a bond motif containing bonds all of which are phosphothioate except for one or two methylphosphonate bonds. In certain embodiments, one methylphosphonate bond is located in the central gap of an oligonucleotide having a gapmeric nucleoside motif.

In certain embodiments, it is desirable to order the number of phosphothioate internucleoside bonds and phosphodiester internucleoside bonds to maintain nuclease resistance. In certain embodiments, it is desirable to order the number and position of phosphothioate internucleoside bonds and the number and position of phosphodiester internucleoside bonds to maintain nuclease resistance. In certain embodiments, the number of phosphothioate internucleoside bonds can be reduced and the number of phosphodiester internucleoside bonds can be increased. In certain embodiments, the number of phosphothioate internucleoside bonds can be reduced and the number of phosphodiester internucleoside bonds can be increased while still maintaining nuclease resistance. In certain embodiments, it is desirable to reduce the number of phosphothioate internucleoside bonds while still maintaining nuclease resistance. In certain embodiments, it is desirable to increase the number of phosphodiester internucleoside bonds while still maintaining nuclease resistance.

4. Some motives.

In certain embodiments, the compounds described herein contain oligonucleotides. Oligonucleotides may have a motif, for example, a characteristic region of unmodified and/or modified sugar components, nucleobases and/or internucleoside bonds. In certain embodiments, the modified oligonucleotides comprise one or more modified nucleosides containing a modified sugar. In certain embodiments, the modified oligonucleotides comprise one or more modified nucleosides containing a modified nucleobase. In certain embodiments, the modified oligonucleotides contain one or more modified internucleoside linkages. In such embodiments, a characteristic site or motif defines modified, unmodified, and variously modified sugar moieties, nucleobases, and/or internucleoside linkages of the modified oligonucleotide. In certain embodiments, each characteristic region of sugar components, nucleobases, and internucleoside bonds is independent of the others. Thus, a modified oligonucleotide can be described using its motif from

- 33 042765 sugars, a nucleobase motif and/or an internucleoside linkage motif (as used herein, a nucleobase motif describes nucleobase modifications regardless of the nucleobase sequence).

a. Some motifs from sugars.

In certain embodiments, the compounds described herein contain oligonucleotides. In certain embodiments, the implementation of the oligonucleotides contain one or more types of modified sugar and/or unmodified sugar components, located along the oligonucleotide or region in the form of a specific characteristic area or motif of sugars. In some instances, such sugar motifs include, without limitation, any sugar modifications discussed herein.

In certain embodiments, the modified oligonucleotides comprise or consist of a region having a gapmer motif, which contains two outer regions, or flanks, and a central or inner region, or gap. The three regions of the gapmer motif (5'flank, gap, and 3'flank) form a contiguous sequence of nucleosides in which at least some of the sugar components of the nucleosides of each of the flanks differ from at least some of the sugar components of the gap nucleosides. In particular, at least the sugar components of the nucleosides of each flank that are closest to the gap (the 3'-terminal nucleoside of the 5'-flank and the 5'-flank-most nucleoside of the 3'-flank) differ from the sugar components of adjacent nucleosides. gap, which thus defines the boundary between the flanks and the gap (i.e., the articulation point of the flank and the gap). In certain embodiments, all sugar components in the gap are the same. In certain embodiments, the gap comprises one or more nucleosides having a sugar moiety that is different from the sugar moiety of one or more other gap nucleosides. In certain embodiments, all sugar motifs of the two flanks are the same (symmetrical gapmer). In certain embodiments, the 5'-flank sugar motif is different from the 3'-flank sugar motif (asymmetric gapmer).

In certain embodiments, the flanks of the gapmer contain 1-5 nucleosides. In certain embodiments, the flanks of the gapmer contain 2-5 nucleosides. In certain embodiments, the flanks of the gapmer contain 3-5 nucleosides. In certain embodiments, all gapmer nucleosides are modified nucleosides.

In certain embodiments, the gap gapmer contains 7-12 nucleosides. In certain embodiments, the gap gapmer contains 7-10 nucleosides. In certain embodiments, the gap gapmer contains 8-10 nucleosides. In certain embodiments, the gap gapmer contains 10 nucleosides. In a specific embodiment, each Gap Gapmer nucleoside is an unmodified 2'-deoxynucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In such embodiments, the nucleosides on the gap side of each flank and gap articulation are unmodified 2'-deoxynucleosides, and the nucleosides on the flank side of each flank and gap articulation are modified nucleosides. In certain such embodiments, each gap nucleoside is an unmodified 2'-deoxynucleoside. In certain such embodiments, each nucleoside of each flank is a modified nucleoside.

In certain embodiments, the modified oligonucleotide has a fully modified sugar motif, wherein each nucleoside of the modified oligonucleotide contains a modified sugar moiety. In certain embodiments, the modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, with each nucleoside of the region containing a modified sugar motif. In certain embodiments, the modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside in the fully modified region contains the same modified sugar moiety, and such region is referred to herein as a uniformly modified sugar motif. In certain embodiments, the fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified oligonucleotide contains the same 2' modification.

In certain embodiments, the modified oligonucleotide may contain the sugar motif described in Swayze et al., US2010/0197762; Freier et al., US2014/0107330; Freier et al., US2015/0184153; and Seth et al., US2015/0267195, each of which is incorporated herein by reference in its entirety.

b. Some motifs from nucleic bases.

In certain embodiments, the compounds described herein contain oligonucleotides. In certain embodiments, the implementation of the oligonucleotides contain modified and/or unmodified nucleic bases located along the oligonucleotide

- 34 042765 or its areas in the form of a certain characteristic area or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is modified. In certain embodiments, each purine or each pyrimidine is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each uracil is modified. In certain embodiments, each cytosine is modified. In certain embodiments, some or all of the cytosine nucleobases in the modified oligonucleotide are 5-methylcytosine.

In certain embodiments, the modified oligonucleotides comprise a block of modified nucleic bases. In certain such embodiments, the block is located at the 3' end of the oligonucleotide. In certain embodiments, the block is located within 3 nucleosides at the 3' end of the oligonucleotide. In certain embodiments, the block is at the 5' end of the oligonucleotide. In certain embodiments, the block is located within 3 nucleosides at the 5' end of the oligonucleotide.

In certain embodiments, oligonucleotides having a gapmer motif comprise a nucleoside containing a modified nucleobase. In certain such embodiments, one nucleoside containing the modified nucleobase is located in the central gap of an oligonucleotide having a gapmeric motif. In certain such embodiments, the implementation of the sugar component of the specified nucleoside is a 2'-deoxyribosyl component. In certain embodiments, the modified nucleobase is selected from 2-thiopyrimidine and 5-propine pyrimidine.

c. Some motifs from internucleoside bonds.

In certain embodiments, the compounds described herein contain oligonucleotides. In certain embodiments, the implementation of the oligonucleotides contain modified and/or unmodified internucleoside bonds located along the oligonucleotide or its region in the form of a specific characteristic site or motif. In certain embodiments, virtually each internucleoside linking group is a phosphate internucleoside bond (P=O). In certain embodiments, each internucleoside linking group of the modified oligonucleotide is phosphothioate (P=S). In certain embodiments, each internucleoside linking group of the modified oligonucleotide is independently selected from phosphothioate and phosphate internucleoside linkages. In certain embodiments, the sugar motif of the modified oligonucleotide is a gapmer and all internucleoside bonds in the gap are modified. In certain such embodiments, some or all of the internucleoside bonds in the flanks are unmodified phosphate bonds. In certain embodiments, terminal internucleoside bonds are modified.

5. Some modified oligonucleotides.

In certain embodiments, the compounds described herein contain modified oligonucleotides. In certain embodiments, the above modifications (sugars, nucleobases, internucleoside bonds) are included in the modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification, motifs, and overall length values. In certain embodiments, each of these parameters is independent of the others. Thus, unless otherwise indicated, each internucleoside bond of an oligonucleotide having a sugar gapmer motif may or may not be modified and may or may not correspond to the sugar gapmer pattern. For example, the internucleoside bonds in the flank regions of the sugar gapmer may be the same or different from each other and may be the same as or different from the internucleoside bonds in the gap region of the sugar motif. Similarly, such gapmeric oligonucleotides may contain one or more modified nucleobases, regardless of the gapmeric nature of the sugar modifications. In addition, in some cases, an oligonucleotide is described by the total length or length range or lengths or length ranges of two or more regions (e.g., regions from nucleosides having said sugar modifications), under such circumstances it may be possible to select for each range such amounts that in the result is an oligonucleotide having an overall length outside the specified range. Under such circumstances, the requirements for both elements must be satisfied. For example, in certain embodiments, the modified oligonucleotide is composed of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, where region A is composed of 2-6 linked nucleosides having the indicated sugar motif, region B consists of 6-10 linked nucleosides having the indicated sugar motif, and region C consists of 2-6 linked nucleosides having the indicated sugar motif. Such options

- 35 042765 implementations do not include modified oligonucleotides in which A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (despite the fact that these numbers of nucleosides are allowed according to the requirements for A, B and C), since the total length of such an oligonucleotide is 22, which exceeds the upper limit of the total length of the modified oligonucleotide (20). Herein, if the description of an oligonucleotide is silent on one or more parameters, then that parameter is not limited. Thus, a modified oligonucleotide described only as having a sugar gapmer motif without further description can be of any length, any internucleoside linkage motif, and any nucleobase motif. Unless otherwise indicated, all modifications are independent of the nucleobase sequence.

Some conjugated compounds

In certain embodiments, the compounds described herein contain or consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugated groups and/or end groups. Conjugated groups consist of one or more conjugated components and a conjugated linker that links the conjugated component to the oligonucleotide. Conjugated groups may be attached to any one or both ends of the oligonucleotide and/or at any internal position. In certain embodiments, the conjugated groups are attached to the nucleoside of the modified oligonucleotide at the 2' position. In certain embodiments, the conjugated groups attached to either one or both ends of the oligonucleotide are end groups. In certain such embodiments, conjugated groups or end groups are attached at the 3' and/or 5' end of the oligonucleotides. In certain such embodiments, conjugated groups (or end groups) are attached at the 3' end of the oligonucleotides. In certain embodiments, the conjugated groups are attached near the 3' end of the oligonucleotides. In certain embodiments, conjugated groups (or end groups) are attached at the 5' end of the oligonucleotides. In certain embodiments, the conjugated groups are attached near the 5' end of the oligonucleotides.

In certain embodiments, the implementation of the oligonucleotide is modified. In certain embodiments, the compound oligonucleotide has a nucleobase sequence that is complementary to the target nucleic acid. In certain embodiments, the oligonucleotides are complementary to messenger RNA (mRNA). In certain embodiments, the oligonucleotides are complementary to the sense transcript.

Examples of end groups include, without limitation, conjugated groups, cap groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.

A. Some conjugated groups.

In certain embodiments, one or more conjugated groups are covalently attached to the oligonucleotides. In certain embodiments, the conjugated groups modify one or more properties of the oligonucleotide to which they are attached, including, without limitation, pharmacodynamic characteristics, pharmacokinetic characteristics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge, and clearance. In certain embodiments, the conjugated groups impart a new property to the oligonucleotide to which they are attached, for example, fluorophores or reporter groups provide for the detection of the oligonucleotide.

Some conjugated groups and conjugated moieties have been described previously, for example: cholesterol component (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g. ., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chain, e.g. residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49 -54), a phospholipid such as dihexadecyl rac glycerol or triethylammonium 1,2-di-O-hexadecyl rac glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantanoacetic acid, a palmityl component (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), -octadecylamine or hexylaminocarbonyloxycholesterol component (Crooke et al., J. Pharmacol. Exp. Ther., 1996, i, 923-937), tocopherol group (Nishina et al., Molecular Therapy Nucleic Acids, 2015, 4, e220; doi:10.1038/mtna.2014.72 and Nishina et al., Molecular Therapy, 2008, 16, 734-740) or a GalNAc cluster (e.g. WO2014/179620).

1. Conjugated components.

Conjugated components include, without limitation, intercalators, reporter molecules,

- 36 042765 polyamines, polyamides, peptides, carbohydrates (e.g. GalNAc), vitamin components, polyethylene glycols, thioethers, polyesters, cholesterols, thiocholesterols, cholic acid components, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores and dyes.

In certain embodiments, the conjugate component contains the active drug, e.g., aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, benzothiadiazide, chlorthiazide, diazepine, indomethicin, barbiturate, cephalosporin, sulfa drug, antidiabetic agent, antibacterial agent or antibiotic.

2. Conjugating linkers.

The conjugated components are attached to the oligonucleotides using conjugating linkers. In some compounds, the conjugated group is a single chemical bond (ie, the conjugated component is attached to the oligonucleotide with a conjugating linker via a single bond). In certain embodiments, the conjugating linker contains a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units, such as ethylene glycol, nucleoside, or amino acid units.

In certain embodiments, the conjugating linker contains one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugating linker contains groups selected from alkyl, amino, oxo, amide, and ether groups. In certain embodiments, the implementation of the conjugating linker contains groups selected from alkyl and amide groups. In certain embodiments, the implementation of the conjugating linker contains groups selected from alkyl and ether groups. In certain embodiments, the implementation of the conjugating linker contains at least one phosphorus-containing component. In certain embodiments, the implementation of the conjugating linker contains at least one phosphate group. In certain embodiments, the conjugating linker contains at least one neutral linking group.

In certain embodiments, conjugation linkers, including the conjugation linkers described above, are bifunctional linkers, such as those known in the art to be useful for attaching conjugated groups to parent compounds, such as the oligonucleotides provided herein. Typically, the bifunctional linking component contains at least two functional groups. One of the functional groups is selected to bind to a particular site in the compound and the other is selected to bind to a conjugated group. Examples of functional groups used in the bifunctional linker include, without limitation, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, the bifunctional linking components contain one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugating linkers include, but are not limited to, pyrrolidine, 8-amino-3,6dioxaoctanoic acid (ADO), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other conjugating linkers include, without limitation, substituted or unsubstituted C ₁ -C ₁ o-alkyl, substituted or unsubstituted C 2 -C 10 alkenyl, or substituted or unsubstituted C ₂ -C ₁₀ alkynyl, where a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy , benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain embodiments, the implementation of the conjugating linkers contain 1-10 linker nucleosides. In certain embodiments, such linker nucleosides are modified nucleosides. In certain embodiments, such linker nucleosides contain a modified sugar moiety. In certain embodiments, the linker nucleosides are unmodified. In certain embodiments, the linker nucleosides contain an optionally protected heterocyclic base selected from purine, substituted purine, pyrimidine, or substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-Nbenzoyladenine, guanine, and 2-N- isobutyrylguanine. Generally, it is desirable that the linker nucleosides are cleaved from the compound after it has reached the target tissue. Accordingly, linker nucleosides are typically linked to each other and to the rest of the compound via cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.

In this document, linker nucleosides are not considered part of the oligonucleotide. Accordingly, in embodiments in which the compound contains an oligonucleotide consisting of linked nucleosides in the specified amount or range of amounts and/or characterized by the specified percentage of complementarity with respect to the reference nucleic acid, and the compound also contains a conjugated group containing a conjugating linker containing linker nucleosides, these linker nucleosides are not taken into account when determining the length of the oligonucleotide and are not used in determining the percentage of complementarity of the oligonucleotide with respect to the reference nucleic acid. For example, the compound may contain (1) a modified oligonucleotide consisting of 8-30 nucleosides, and (2) a conjugated group containing 1-10 linker nucleosides adjacent to the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such a compound is greater than 30. Alternatively, the compound may contain a modified oligonucleotide of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such a compound does not exceed 30. Unless otherwise indicated, conjugating linkers contain no more than 10 linker nucleosides. In certain embodiments, the implementation of the conjugating linkers contain no more than 5 linker nucleosides. In certain embodiments, the implementation of the conjugating linkers contain no more than 3 linker nucleosides. In certain embodiments, the implementation of the conjugating linkers contain no more than 2 linker nucleosides. In certain embodiments, the implementation of the conjugating linkers contain no more than 1 linker nucleoside.

In certain embodiments, it is desirable that the conjugated group be cleaved from the oligonucleotide. For example, under certain circumstances, compounds containing a particular conjugated component are better taken up by cells of a particular type, however, after absorption of the compound, it is desirable that the conjugated group is cleaved off to release the unconjugated or original oligonucleotide. Thus, some conjugates may contain one or more cleavable components, typically within the conjugate linker. In certain embodiments, the cleavable component is a cleavable bond. In certain embodiments, a cleavable component is a group of atoms containing at least one cleavable bond. In certain embodiments, the cleavable component contains a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, the cleavable component is selectively cleaved within a cellular or subcellular compartment, such as a lysosome. In certain embodiments, the cleavable component is selectively cleaved by endogenous enzymes such as nucleases.

In certain embodiments, the cleavable bond is selected from an amide, an ester, an ester, one or both of the esters in a phosphodiester bond, a phosphoester, a carbamate, or a disulfide. In certain embodiments, the cleavable bond is one or both of the esters in the phosphodiester bond. In certain embodiments, the cleavable component contains a phosphate or phosphodiester. In certain embodiments, the cleavable component is a phosphate bond between the oligonucleotide and the conjugated component or conjugated group.

In certain embodiments, the cleavage component comprises or consists of one or more linker nucleosides. In certain such embodiments, one or more linker nucleosides are linked to each other and/or to the remainder of the compound via cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, the cleavable moiety is a 2'-deoxynucleoside that is attached to the 3' or 5' nucleoside of the oligonucleotide via a phosphate internucleoside bond and is covalently attached to the remainder of the conjugate linker or conjugate moiety via a phosphate or phosphothioate bond. In certain such embodiments, the cleavable component is 2'-deoxyadenosine.

3. Certain cell-targeting conjugates In certain embodiments, the conjugate group comprises a cell-targeting conjugate. In certain embodiments, the conjugate group has the general formula:

| Ligand — ]__| Branching,] __ (Conjugating ι__g Cleavable 1__§

V element ^P group m ¹ linker 'j ¹ component ^J k

Cell-targeting component where n is 1 to about 3, m is 0 if n is 1, m is 1 if n is 2 or more, j is 1 or 0, and k is 1 or 0.

In certain embodiments, n is 1, j is 1, and k is 0. In certain embodiments, n is 1, j is 0, and k is 1. In certain embodiments,

- 38 042765 max n is 1, j is 1, and k is 1. In certain embodiments, n is 2, j is 1, and k is 0. In certain embodiments, n is 2, j is 0, and k is 1. In certain embodiments, n is 2, j is 1, and k is 1. In certain embodiments, n is 3, j is 1, and k is 0. In certain embodiments, n is 3, j is 0, and k is 1. In certain embodiments, n is 3, j is 1, and k is 1.

In certain embodiments, the implementation of the conjugated groups contain components that target the cell, which have at least one associated ligand. In certain embodiments, the cell-targeting components comprise two linked ligands covalently attached to a branching group. In certain embodiments, the cell-targeting components comprise three linked ligands covalently attached to a branching group.

In certain embodiments, the cell targeting component contains a branching group containing one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain embodiments, the branching group contains a branched aliphatic group containing groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the branched aliphatic group contains groups selected from alkyl, amino, oxo, amide, and ether groups. In certain such embodiments, the branched aliphatic group contains groups selected from alkyl, amino, and ether groups. In certain similar embodiments, the branched aliphatic group contains groups selected from an alkyl and an ether group. In certain embodiments, the branching group contains a mono- or polycyclic ring system.

In certain embodiments, each cell-targeting component binding element contains one or more groups selected from alkyl, substituted alkyl, ether, thioether, disulfide, amino, oxo, amide, phosphodiester, and polyethylene glycol in any combination. In certain embodiments, each linking element is a linear aliphatic group containing one or more groups selected from alkyl, ether, thioether, disulfide, amino, oxo, amide, and polyethylene glycol in any combination. In certain embodiments, each linking element is a linear aliphatic group containing one or more groups selected from alkyl, phosphodiester, ether, amino, oxo, and amide in any combination. In certain embodiments, each linking element is a linear aliphatic group containing one or more groups selected from alkyl, ether, amino, oxo, and amide in any combination. In certain embodiments, each linking element is a linear aliphatic group containing one or more groups selected from alkyl, amino, and oxo in any combination. In certain embodiments, each linking element is a linear aliphatic group containing one or more groups selected from alkyl and oxo in any combination. In certain embodiments, each linking element is a linear aliphatic group containing one or more groups selected from alkyl and phosphodiester in any combination. In certain embodiments, each linking element contains at least one phosphorus-containing linking group or neutral linking group. In certain embodiments, each linking element contains a chain length of from about 6 to about 20 atoms. In certain embodiments, each linking element contains a chain length of from about 10 to about 18 atoms. In certain embodiments, each linking element contains a chain length of approximately 10 atoms.

In certain embodiments, each ligand of the cell-targeting component has an affinity for at least one type of receptor on the target cell. In certain embodiments, each ligand has an affinity for at least one type of receptor on the surface of a mammalian liver cell. In certain embodiments, each ligand has an affinity for the liver asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is independently selected from galactose, N-acetylgalactosamine (GalNAc), mannose, glucose, glucosamine, and fucose. In certain embodiments, each ligand is N-acetylgalactosamine (GalNAc). In certain embodiments, the cell-targeting component contains 3 GalNAc ligands. In certain embodiments, the cell-targeting component contains 2 GalNAc ligands. In certain embodiments, the cell-targeting component comprises 1 GalNAc ligand.

In certain embodiments, each cell targeting component ligand is a carbohydrate, a carbohydrate derivative, a modified carbohydrate, a polysaccharide, a modified

- 39 042765 cited polysaccharide or polysaccharide derivative. In certain such embodiments, the conjugated group contains a carbohydrate cluster (see, for example, Maier et al., Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting, Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et al ., Design and Synthesis of Novel NAcetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor, J. Med. Chem. 2004, 47, 5798-5808, which are incorporated herein by reference in their entirety). In certain such embodiments, each ligand is an amino sugar or a thio sugar. For example, the amino sugars may be selected from any number of compounds known in the art, such as sialic acid, α-D-galactosamine, β-muramic acid, 2-deoxy-2-methylamino-b-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-glycoloylα-neuraminic acid. For example, thiosugars can be selected from 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-glucoheptopyranoside.

In certain embodiments, the implementation of the conjugated groups contain a component that targets a cell, characterized by the following formula:

but he

AcHN

In certain embodiments, the implementation of the conjugated groups contain a component that targets a cell, characterized by the following formula:

but he

In certain embodiments, the implementation of the conjugated groups contain a component that targets a cell, characterized by the following formula:

AcHN

In certain embodiments, the compounds described herein contain a conjugate group described herein as LICA-1. LICA-1 is shown below without the optional cleavable component at the end of the conjugating linker:

- 40 042765

In certain embodiments, the compounds described herein comprise

LICA-1 and the cleavable component in the conjugating linker and have the following formula:

cell component where oligo is an oligonucleotide.

Illustrative publications that outline the preparation of some of the aforementioned conjugated groups and compounds containing conjugated groups, linking elements, conjugating linkers, branching groups, ligands, cleavable components, as well as other modifications, include, without limitation, US 5994517, US 6300319, US 6660720, US 6906182, US 7262177, US 7491805, US 8106022, US 7723509, US 9127276, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO 2012/037254, J. Med al. Chem. 1995, 38, 1846-1852, Lee et al., Bioorganic & Medicinal Chemistry 2011,19, 2494-2500, Rensen et al., J. Biol. Chem. 2001, 276, 37577-37584, Rensen et al., J. Med Chem. 2004, 47, 5798-5808, Sliedregt et al., J. Med Chem. 1999, 42, 609-618, and Valentijn et al., Tetrahedron, 1997, 53, 759-770, each of which is incorporated herein by reference in its entirety.

In certain embodiments, the compounds described herein contain modified oligonucleotides containing a gapmeric or fully modified motif and a conjugated group containing at least one, two, or three GalNAc ligands. In certain embodiments, the compounds described herein contain a conjugate group, which can be found in any of the following literature: 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, 317328; 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, 17841792; 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, An

- 41 042765 tisense 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 publications 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; US Pat. Nos. 4,751,219; 8552163; 6908903; 7262177; 5994517; 6300319; 8106022; 7491805; 7491805; 7582744; 8137695; 6383812; 6525031; 6660720; 7723509; 8541548; 8344125; 8313772; 8349308; 8450467; 8501930; 8158601; 7262177; 6906182; 6620916; 8435491; 8404862; 7851615; publications of US patent applications 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.

Compositions and methods for formulating pharmaceutical compositions.

The compounds described herein can be mixed with pharmaceutically acceptable active or inert substances to obtain pharmaceutical compositions or formulations. Compositions and methods of formulating pharmaceutical compositions depend on a number of criteria, including, without limitation, the route of administration or the degree of disease.

In certain embodiments, pharmaceutical compositions are provided that contain one or more compounds, or a salt thereof. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compounds comprise or consist of the modified oligonucleotide. In certain such embodiments, the implementation of the pharmaceutical composition contains a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, the implementation of the pharmaceutical composition contains a sterile saline solution and one or more compounds. In certain embodiments, such a pharmaceutical composition consists of a sterile saline solution and one or more compounds. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, the implementation of the pharmaceutical composition contains one or more compounds and sterile water. In certain embodiments, the pharmaceutical composition consists of a single compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, the pharmaceutical composition contains one or more compounds and phosphate buffered saline (PBS). In certain embodiments, the pharmaceutical composition consists of a single compound and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS. Compositions and methods of formulating pharmaceutical compositions depend on a number of criteria, including, without limitation, the route of administration or the degree of disease.

A compound described herein that targets a PCSK9 nucleic acid can be used in pharmaceutical compositions by combining the compound with a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, the pharmaceutically acceptable diluent is water, such as sterile water suitable for injection. Accordingly, in one embodiment, the methods described herein use a pharmaceutical composition comprising a compound that targets a PCSK9 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is water. In certain embodiments, the compound comprises or consists of a modified oligonucleotide as provided herein.

Pharmaceutical compositions containing the compounds provided herein encompass any pharmaceutically acceptable salts, esters or salts of such esters or any other oligonucleotide which, when administered to an animal, including a human, is capable of providing it (directly or indirectly) with its biologically active metabolite or residue. In certain embodiments, the compounds are antisense compounds or oligomeric compounds. In certain embodiments, the compound comprises or consists of a modified oligonucleotide. Accordingly, for example, the present invention also encompasses pharmaceutically acceptable salts of the compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, without limitation, the sodium and potassium salts.

The prodrug may include additional nucleosides at one or both termini of the compound, which are cleaved by endogenous nucleases in the body to form the active compound.

In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.

Some selected compounds.

Approximately 1540 newly designed compounds and several previously disclosed compounds with different lengths, chemical structures and motifs were tested for their effect on human PCSK9 mRNA in vitro in several cell types (examples 1-2). Of the 1540 compounds tested for efficacy in a single dose in vitro, 183 selected compounds were tested for dose-dependent suppression in HepG2 cells (Example 2). Of the 183 compounds tested in dose response assays, 134 oligonucleotides were selected for in vivo single dose tolerability studies in rodents. These 134 oligonucleotides were conjugated at the 3' end to the terminal cap structure THA-C6-GalNAc3-(3R,5S)-5-(hydroxymethyl)pyrrolidin-3-olphosphate (hereinafter referred to as 3'-THA).

In a single dose rodent tolerance model, BALB/c mice were dosed with a high concentration of 3'-THA oligonucleotide and levels of liver function markers such as alanine transaminase and aspartate transaminase were measured (Example 3). ISIS 863413 (SEQ ID NO: 1096), ISIS 863419 (SEQ ID NO: 1419), ISIS 863424 (SEQ ID NO: 1297), ISIS 863425 (SEQ ID NO: 1528), ISIS 863427 (SEQ ID NO: 1145), ISIS 863433 (SEQ ID NO: 1223), ISIS 863434 (SEQ ID NO: 377), ISIS 863436 (SEQ ID NO: 763), ISIS 863437 (SEQ ID NO: 994), ISIS 863438 (SEQ ID NO: 1071), ISIS 863439 (SEQ ID NO: 1147), ISIS 863441 (SEQ ID NO: 1302), ISIS 863444 (SEQ ID NO: 1149), ISIS 863445 (SEQ ID NO: 455), ISIS 863448 (SEQ ID NO: 1380), ISIS 863452 (SEQ ID NO: 691), ISIS 863472 (SEQ ID NO: 926), ISIS 863473 (SEQ ID NO: 1233), ISIS 863474 (SEQ ID NO: 619), ISIS 863475 (SEQ ID NO: 388), ISIS 863477 (SEQ ID NO: 1542), ISIS 863479 (SEQ ID NO: 1158), ISIS 863480 (SEQ ID NO: 1235), ISIS 863481 (SEQ ID NO: 389), ISIS 863482 (SEQ ID NO: 1312), ISIS 863483 (SEQ ID NO: 1240), ISIS 863484 (SEQ ID NO: 549), ISIS 863485 (SEQ ID NO: 550), ISIS 863486 (SEQ ID NO: 781), ISIS 863489 (SEQ ID NO: 939), ISIS 863490 (SEQ ID NO: 1016), ISIS 863491 (SEQ ID NO: 1243), ISIS 863493 (SEQ ID NO: 629), ISIS 863494 (SEQ ID NO: 1017), ISIS 863495 (SEQ ID NO: 1092), ISIS 863496 (SEQ ID NO: 1244), ISIS 863497 (SEQ ID NO: 554), ISIS 863498 (SEQ ID NO: 478), ISIS 863499 (SEQ ID NO: 1094), ISIS 863502 (SEQ ID NO: 563), ISIS 863506 (SEQ ID NO: 410), ISIS 863507 (SEQ ID NO: 1411), ISIS 863509 (SEQ ID NO: 721), ISIS 863510 (SEQ ID NO: 1258), ISIS 863511 (SEQ ID NO: 645), ISIS 863512 (SEQ ID NO: 955), ISIS 863514 (SEQ ID NO: 1413), ISIS 863516 (SEQ ID NO: 881), ISIS 863517 (SEQ ID NO: 648), ISIS 863518 (SEQ ID NO: 725), ISIS 863520 (SEQ ID NO: 1111), ISIS 863522 (SEQ ID NO: 1188), ISIS 863524 (SEQ ID NO: 885), ISIS 863525 (SEQ ID NO: 504), ISIS 863526 (SEQ ID NO: 1043), ISIS 863527 (SEQ ID NO: 1195), ISIS 863531 (SEQ ID NO: 426), ISIS 863533 (SEQ ID NO: 427), ISIS 863536 (SEQ ID NO: 585), ISIS 863537 (SEQ ID NO: 1047), ISIS 863538 (SEQ ID NO: 353), ISIS 863539 (SEQ ID NO: 1352), ISIS 863541 (SEQ ID NO: 1203), ISIS 863545 (SEQ ID NO: 1516), ISIS 863547 (SEQ ID NO: 1213), ISIS 863548 (SEQ ID NO: 367), ISIS 863549 (SEQ ID NO: 1061), ISIS 863550 (SEQ ID NO: 523), ISIS 863552 (SEQ ID NO: 831) and ISIS 863553 (SEQ ID NO: 908) were considered tolerated in this study and were selected for further evaluation in a transgenic mouse model.

In a mouse tolerance model transgenic for PCSK9, mice were dosed with 3'-THA-conjugated oligonucleotides and plasma levels of alanine transaminase, aspartate transaminase, cholesterol, HDL cholesterol, LDL cholesterol and triglycerides were measured (Example 4). Было обнаружено, что ISIS 863433, ISIS 863490, ISIS 863512, ISIS 863527, ISIS 863538, ISIS 863425, ISIS 863438, ISIS 863439, ISIS 863444, ISIS 863413, ISIS 863434, ISIS 863436, ISIS 863494 и ISIS 863539 являются переносимыми. Selected oligonucleotide sequences were conjugated at the 5' end to a trishexylamino-(THA)-C6GalNAC3 terminal cap structure (hereinafter referred to as 5'-THA) and tested in the CD-1 mouse model. Олигонуклеотиды, конъюгированные с 5'-THA, представляют собой ISIS 845219, ISIS 863568, ISIS 863576, ISIS 863577, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863637, ISIS 863655, ISIS 863670, ISIS 863681 и ISIS 863682.

In the CD-1 mouse tolerance model, mice were injected with various doses of the antisense oligonucleotide for six weeks and markers of tolerance were measured, such as markers of liver and kidney function (ALT, AST, plasma albumin, BUN, creatinine, and bilirubin levels), hematological markers (blood cell count for RBC, WBC, platelets, neutrophils, lymphocytes and monocytes, as well as hemoglobin, hematocrit and MCV), as well as body and organ weights (example 5). For further evaluation in the rat model, only those oligonucleotides were selected that, upon treatment with plasma levels of these markers, were within the expected range after treatment with antisense oligonucleotides. ISIS 863568, ISIS 863576, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681 were selected for further study in a rat model.

In the Sprague-Dawley rat model, rats were similarly dosed for 6 weeks with antisense oligonucleotides and markers of tolerability were measured, such as markers of liver function (ALT, AST, albumin, BUN, creatinine, and bilirubin levels in plasma), markers of kidney function (urinary creatinine and total protein levels); and weekly body weights and endpoints of organ weights (Example 6).

These oligonucleotides were also tested for their viscosity (Example 8) and all of them were found to have optimal viscosity according to the testing criteria.

ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 were tested for efficacy in a four-week dose-response study in the PCSK9 transgenic mouse model (Example 7). At the end of 4 weeks, mouse livers were analyzed for PCSK9 mRNA expression levels and ED ₅₀ calculated. In addition, plasma PCSK9 protein levels, plasma LDL cholesterol levels, and liver LDL receptor levels were measured. It was observed that treatment with antisense oligonucleotides resulted in downregulation of PCSK9 mRNA expression levels in the liver, a decrease in plasma levels of LDL cholesterol, and a corresponding increase in liver levels of LDL receptors. In particular, processing with ISIS 863633 was found to be effective.

ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670, and ISIS 863681 were tested for potency, pharmacokinetic profile9, and tolerability in a 12-week craze-eater study. Treatment with compounds that fully cross-reacted with the rhesus monkey gene sequence caused a decrease in PCSK9 mRNA expression in the liver tissue. In particular, treatment with ISIS 863633, ISIS 863670 and ISIS 863681 caused a decrease in PCSK9 mRNA expression in liver tissue compared to PBS control. It was noted that ISIS 863633 caused the greatest decrease in PCSK9 mRNA expression compared to the PBS control. Changes in PCSK9 protein levels, LDL cholesterol levels, total cholesterol levels, and LDL receptor levels in the liver are a consequence of downregulation of PCSK9 mRNA levels. Treatment with ISIS 863633, ISIS 863670 and ISIS 863681 caused a decrease in PCSK9 protein levels, LDL cholesterol levels, total cholesterol levels and induction of liver LDL receptor levels, with ISIS 863633 causing the most significant change. Treatment with antisense oligonucleotides did not cause any changes in HDL cholesterol or triglyceride levels. Therefore, in terms of potency, ISIS 863633 was the most effective in the monkey study. The monkeys tolerated compound treatment well, in particular treatment with ISIS 863633.

The new compounds were compared with previously designed compounds, including ISIS 405879 and ISIS 405995, which were previously identified as among the most effective antisense compounds in vitro (see, for example, US patent 8084437), as well as ISIS 431131 and ISIS 480604, which were previously described in US patent 9127276 (example 10). A head-to-head comparison study demonstrated that ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681 were more effective than any of the previously publicly disclosed oligonucleotides.

Accordingly, compounds with any one or more improved properties are contemplated herein. In certain embodiments, the compounds described herein are effective and tolerable.

Examples

The following examples describe a screening method for identifying leader compounds targeting PCSK9. Of over 1540 antisense oligonucleotides that were screened, ISIS 863568, ISIS 863579, ISIS 863581, ISIS 863582, ISIS 863587, ISIS 863633, ISIS 863655, ISIS 863670 and ISIS 863681 were found to be the best leader compounds. In particular, ISIS 863633 showed the best combination of properties in terms of efficacy and tolerability of over 1540 oligonucleotides.

Non-limiting disclosure and inclusion by reference.

Although the sequence listing that accompanies this filed application identifies each sequence as RNA or DNA as required, in reality these sequences can be modified by any combination of chemical modifications. One skilled in the art will readily appreciate that the designation RNA or DNA to describe modified oligonucleotides is, in some cases, arbitrary. For example, an oligonucleotide containing a nucleoside containing a 2'OH-sugar component and a thymine base can be described as DNA having a modified sugar (2'-OH instead of the natural 2'-H in DNA) or as RNA having a modified base ( thymine (methylated uracil) instead of natural uracil in RNA). It is contemplated herein that the designation O in mRNA suppression assays only indicates that the antisense oligonucleotide does not suppress mRNA expression levels.

Accordingly, the nucleic acid sequences provided herein, including but not limited to those listed, are intended to encompass nucleic acids containing any combination of naturally occurring or modified RNA and/or DNA,

- 44 042765 including but not limited to such nucleic acids with modified nucleic bases. As a further example and without limitation, an oligonucleotide having the nucleobase sequence ATCGATCG encompasses any oligonucleotide having such a nucleobase sequence, whether modified or unmodified, including, without limitation, those compounds that contain RNA bases, such as compounds having the sequence AUCGAUCG and compounds having multiple DNA bases and multiple RNA bases such as AUCGATCG as well as compounds having other modified nucleobases such as ATmCGAUCG where ^m C indicates a cytosine base containing a methyl group in 5-position.

Although some of the compounds described herein, compositions and methods have been specifically described in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to be limiting. Each of the references mentioned in this application is incorporated herein by reference in its entirety.

Example 1 Antisense suppression of human PCSK9 in HepG2 cells with cEt gapmers 3-10-3.

Antisense oligonucleotides were designed to target the PCSK9 nucleic acid and tested for their effects on PCSK9 mRNA in vitro. Chimeric antisense oligonucleotides are referred to in the tables below as cEt gapmers 3-10-3. Gapmers are 16 nucleosides long, with a central gap segment containing ten 2'-deoxynucleosides and flanked by 5' and 3' flank segments, each containing three nucleosides. Each nucleoside in the 5' flank segment and each nucleoside in the 3' flank segment has a cEt sugar modification. All internucleoside bonds in each gapmer are phosphothioate (P=S) bonds. All cytosine residues in each gapmer are 5-methylcytosine.

The start site indicates the outermost 5' nucleoside targeted by the gapmer in the human gene sequence. The stop site indicates the most 3' end nucleoside targeted by the gapmer in the human gene sequence. Each gapmer listed in the tables below targets either the human PCSK9 mRNA referred herein as SEQ ID NO: 1 (GENBANK Accession No. NM_174936.3) or the human PCSK9 genomic sequence referred to herein as SEQ ID NO. : 2 (accession no. in GENBANK NC_000001.11 with cut-off nucleotides 55036001-55068000). n/a indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

Antisense oligonucleotides were tested in a series of experiments in which culture conditions were similar. The results of each experiment are presented in the individual tables shown below.

Research 1.

Cultured HepG2 cells at a density of 20,000 cells/well were transfected by electroporation with 1000 nM antisense oligonucleotide. ISIS 431131 previously disclosed in WO2014179620 was also included in the study as a comparison oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer and probe kit from ABI (ID no. Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels were corrected according to total RNA measured with RIBOGREEN®. Results are presented as percent suppression of PCSK9 compared to untreated control cells. Some of the newly designed oligonucleotides were more effective than the previously disclosed oligonucleotide ISIS 431131.

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Table 1 Suppression of PCSK9 mRNA by cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop website Subsequence % depressed SEQID: 2, start site SEQ ID2: stopsite SE Q ID NO 4311 31 1015 1034 GTCACACTTGCTGG CCTGTC 3 N/A N/A 3 8593 73 N/A N/A TCTGCGTCCTTC CA 36 4562 4577 4 8593 77 N/A N/A AGCATCTGCCGTCC TT 62 4566 4581 5 8593 81 N/A N/A TGTCAAAGCACCAC AGC 31 5010 5025 6 8593 85 N/A N/A TGACTTGTCAAGCA CC 61 5015 5030 7 8593 89 N/A N/A ATAAGTGACTTGTC AA 45 5020 5035 8 8593 93 N/A N/A GCATTGTAAGTTCA CT 84 5138 5153 9 8593 97 N/A N/A GCTTGCATTGTAAG TT 29 5142 5157 10 8594 01 N/A N/A GGTTTTCCCAGCTC TG 85 5897 5912 eleven 8594 05 N/A N/A CTCTGGTTTTCCCA GC 72 5901 5916 12 8594 09 N/A N/A CCACCTCTGGTTTT CC 67 5905 5920 13 8594 13 N/A N/A GTCTGCTCCAACTG CT 43 6714 6729 14 8594 17 N/A N/A CTCTTGTCTGCTCC AA 55 6719 6734 15 8594 21 N/A N/A TTTAGCTCTTGTCT GC 45 6724 6739 16 8594 25 N/A N/A GCAAGTCCCTGGCTA GA 18 11238 11253 17 8594 29 N/A N/A CACCTGCAAGTCCC TG 15 11243 11258 18 8594 33 N/A N/A ATTGCCACCTGCAA GT 4 11248 11263 19

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8594 37 N/A N/A TCATTCTCTCCTCA GG 58 11294 11309 20 8594 41 N/A N/A TGGACCACCACAT TG 34 11383 11398 21 8594 45 N/A N/A TGCATGGACCCACC AC 61 11387 11402 22 8594 49 N/A N/A CATGTTGCATGGAC CC 61 11392 11407 23 8594 53 N/A N/A ACAGTAGCCCCCC AAC 46 11608 11623 24 8594 57 N/A N/A TCACACAGTAGCCC CC 52 11612 11627 25 8594 61 N/A N/A AAAGTCACACAGT AGC 51 11616 11631 26 8594 65 N/A N/A GGTCACACAGTTTG GG 58 11639 11654 27 8594 69 N/A N/A ACATATGCAAGGT CAC 74 11649 11664 28 8594 73 N/A N/A ACTCAGACATATGC AA 32 11655 11670 29 8594 77 N/A N/A AGTGTCCCTCTTGG TC 59 11950 11965 thirty 8594 81 N/A N/A TGCCAGTGTCCCTC TT 37 11954 11969 31 8594 85 N/A N/A TGATCCTCTGCCAG TG 58 11962 11977 32 8594 89 N/A N/A TCTGTGATCCTCTG CC 50 11966 11981 33 8594 93 N/A N/A GGTCTCTGTGATCC TC 67 11970 11985 34 8594 97 N/A N/A AGCTCTAGGGCCC ATG 6 12671 12686 35 8595 01 N/A N/A AGCCAGCTCTAGG GCC 0 12675 12690 36 8595 05 N/A N/A GTGGGTGCTCAAA ACG 28 12924 12939 37 8595 09 N/A N/A AATAATGCCCCCGT AG 26 13176 13191 38 8595 13 N/A N/A CTGGTTAATAATGC CC 47 13182 13197 39 8595 17 N/A N/A CTGGATACATTGGC AG 25 14125 14140 40

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8595 21 N/A N/A CTCTGAGTGTGTTA GG 54 14473 14488 41 8595 25 N/A N/A TGCTCTCTGAGTGT GT 26 14477 14492 42 8595 29 N/A N/A GCATGCTGAGCAG GTC 78 14541 14556 43 8595 33 N/A N/A TGTAGCATGCTGAG CA 73 14545 14560 44 8595 37 N/A N/A ATTCAGTGGCTGCT CT 0 14755 14770 45 8595 41 N/A N/A GAGCAATTCAGTG GCT 24 14760 14775 46 8593 10 979 994 TCCTCGGGCACATT CT 34 16371 16386 47 4684 60 1075 1090 TTGGCCCACGCCGGC AT 8 16705 16720 48 8595 45 N/A N/A ATTTAGCAGCTACG GC 35 17441 17456 49 8595 49 N/A N/A CAACTATTTAGCAG CT 36 17446 17461 50 8595 53 N/A N/A AGGTTACCGCTGTC AG 73 20914 20929 51 8595 57 N/A N/A GACCTAGGTTACCG CT 33 20919 20934 52 8593 14 1498 1513 CCACTCTGTGACAC AA 79 21470 21485 53 8593 18 1503 1518 ATGTCCCACTCTGT GA 49 21475 21490 54 8595 61 N/A N/A CTCGAAGGTAAGC CGC 66 21891 21906 55

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8595 65 N/A N/A CAGCACTGTCCTGC AG 20 22880 22895 56 8595 69 N/A N/A TTTTGCTGGTTTG AG 34 23030 23045 57 8595 73 N/A N/A CACTTTTTTGCTGG TT 58 23034 23049 58 8595 77 N/A N/A AATTCCACTTTTTTT GC 15 23039 23054 59 8595 81 N/A N/A ATCTGTGCACTATC CT 28 23069 23084 60 8595 85 N/A N/A TGGCATCTGTGCAC TA 55 23073 23088 61 8595 89 N/A N/A TCTCACACGAGCAT TA 16 23176 23191 62 8595 93 N/A N/A CTGCCTCTCACACG AG 46 23181 23196 63 8595 97 N/A N/A TCTTATTGGAGGAG GAA 23 23853 23868 64 8596 01 N/A N/A TGTTTTCGACACAG GG 68 24225 24240 65 8596 05 N/A N/A GGCATGTTTTCGAC AC 68 24229 24244 66 8596 09 N/A N/A CATCTTTTCAGTAC TC 43 26516 26531 67 8596 13 N/A N/A ATCGCATCTTTCA GT 31 26520 26535 68 8593 22 2429 2444 CTGTCACTGGAGCT CC 26 27572 27587 69 8593 25 2495 2510 GTCGGAACCATTTTT AA 34 27638 27653 70

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8593 29 2501 2516 GGACAAGTCGGAA CCA 29 27644 27659 71 8593 33 2505 2520 AGAGGGACAAGTC GGA 61 27648 27663 72 8593 37 3461 3476 ATCTCCGCCAGGCC AG 24 28604 28619 73 8593 41 3465 3480 AAGCATCTCCGCCA GG 68 28608 28623 74 8593 45 3470 3485 CTTAGAAGCATCTC CG 61 28613 28628 75 8593 49 3476 3491 CCATGCCTTAGAAG CA 58 28619 28634 76 8593 53 3538 3553 TGTCTGCTTGCTTG GG 62 28681 28696 77 8593 57 3544 3559 GATAAATGTCTGCT TG 37 28687 28702 78 8593 61 3593 3608 GTCTAGAAAAGTT GGC 63 28736 28751 79 8593 65 3597 3612 ACAGGTCTAGAAA AGT 64 28740 28755 80 8593 69 3606 3621 AAAAGCAAAACAG GTC 33 28749 28764 81

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table 2

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop Subsequence % depressed SEQ ID: 2, start SEQ ID 2: stop SEQ ID NO website VY site website 4311 31 1015 1034 GTCACACTTGCTGGC CTGTC 0 N/A N/A 3 8593 74 N/A N/A ATCTGCCGTCCTTCCC 42 4563 4578 82 8593 78 N/A N/A CAGCATCTGCCGTCC T 62 4567 4582 83 8593 82 N/A N/A CTTGTCAAGCACCAC A 61 5012 5027 84 8593 86 N/A N/A GTGACTTGTCAAGCA C 10 5016 5031 85 8593 90 N/A N/A TGTAAGTTCACTCAT G 56 5134 5149 86 8593 94 N/A N/A TGCATTGTAAGTTCA C 71 5139 5154 87 8593 98 N/A N/A GGGCTTGCATTGTAA G 13 5144 5159 88 8594 02 N/A N/A TGGTTTTTCCCAGCTCT 78 5898 5913 89 8594 06 N/A N/A CCTCTGGTTTTCCAG 61 5902 5917 90 8594 10 N/A N/A TCCACCTCTGGTTTTC 41 5906 5921 91 8594 14 N/A N/A TTGTCTGCTTCCAACT G 39 6716 6731 92 8594 18 N/A N/A GCTCTTGTCTGCTCCA 71 6720 6735 93

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8594 22 N/A N/A TATAAATCTCCCATC C 0 7280 7295 94 8594 26 N/A N/A CTGCAAGTCCCTGCT A 12 11240 1125 5 95 8594 thirty N/A N/A CCACCTGCAAGTCCC T 26 11244 1125 9 96 8594 34 N/A N/A CTCTCCTCAGGCCAG G 64 11289 ISO 4 97 8594 38 N/A N/A CCCACCACATTGCAT C 37 11379 1139 4 98 8594 42 N/A N/A ATGGACCACCACAT T 10 11384 1139 9 99 8594 46 N/A N/A TTGCATGGACCCACC A 40 11388 1140 3 100 8594 50 N/A N/A CGAGAACCGGTAAGG G 34 11447 1146 2 101 8594 54 N/A N/A CACAGTAGCCCCCCA A 32 11609 1162 4 102 8594 58 N/A N/A GTCACACAGTAGCCC C 69 11613 1162 8 103 8594 62 N/A N/A GCAAAGTCACACAGT A 64 11618 1163 3 104 8594 66 N/A N/A AGGTCACACAGTTTG G 83 11640 1165 5 105 8594 70 N/A N/A GACATATGCAAGGTC A 40 11650 1166 5 106 8594 74 N/A N/A TCCCTCTTGGTCTCTG 55 11946 1196 1 107 8594 78 N/A N/A CAGTGTCCCTCTTGG T 67 11951 1196 6 108

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8594 82 N/A N/A CTGCCAGTGTCCCTC T 1 11955 1197 0 109 8594 86 N/A N/A GTGATCCTCTGCCAG T 35 11963 1197 8 110 8594 90 N/A N/A CTCTGTGATCCCTGC 68 11967 1198 2 111 8594 94 N/A N/A GGGTCTCTGTGATCC T 33 11971 1198 6 112 8594 98 N/A N/A CAGCTCTAGGGCCCA T 56 12672 1268 7 113 8595 02 N/A N/A CAGCCAGCTCTAGGG C 31 12676 1269 1 114 8595 06 N/A N/A AGTGGGTGCTCAAAAA C 23 12925 1294 0 115 8595 10 N/A N/A TTAATAATGCCCCCG T 32 13178 1319 3 116 8595 14 N/A N/A CCTGGTTAATAATGC C 36 13183 1319 8 117 8595 18 N/A N/A GAGTGTGTTAGGAGC T 83 14469 1448 4 118 8595 22 N/A N/A TCTCTGAGTGTGTTA G 21 14474 1448 9 119 8595 26 N/A N/A CTTGCTCTCTGAGTGT 42 14479 1449 4 120 8595 thirty N/A N/A AGCATGCTGAGCAGG T 54 14542 1455 7 121 8595 34 N/A N/A CTGTAGCATGCTGAG C 71 14546 1456 1 122 8595 38 N/A N/A AATTCAGTGGCTGCT C 27 14756 1477 1 123

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8595 42 N/A N/A AGAGCAATTCAGTGG C 42 14761 1477 6 124 8593 AND 981 996 CCTCCTCGGGCACAT T 14 16373 1638 8 125 4684 60 1075 1090 TTGGCCACGCCGGCA T 0 16705 1672 0 48 8595 46 N/A N/A CTATTTAGCAGCTAC G 57 17443 1745 8 126 8595 50 N/A N/A TCAACTATTTAGCAG C 56 17447 1746 2 127 8595 54 N/A N/A TAGGTTACCGCTGTC A 47 20915 2093 0 128 8595 58 N/A N/A GGACCTAGGTTACCG C 49 20920 2093 5 129 8593 15 1499 1514 CCCACTCTGTGACAC A 94 21471 2148 6 130 8593 19 1505 1520 TGATGTCCCACTCTG T 31 21477 2149 2 131 8595 62 N/A N/A GTCCTGCAGCCTCTA G thirty 22873 2288 8 132 8595 66 N/A N/A CCAGCACTGTCCTGC A 54 22881 2289 6 133 8595 70 N/A N/A _{TTTTTTGCTGGTTTGA} 33 23031 2304 6 134 8595 74 N/A N/A CCACTTTTTTGCTGGT 21 23035 2305 0 135 8595 78 N/A N/A CAATTCCACTTTTTTG 41 23040 2305 5 136 8595 82 N/A N/A CATCTGTGCACTATC C 48 23070 2308 5 137

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8595 86 N/A N/A ATGGCATCTGTGCAC T thirty 23074 2308 9 138 8595 90 N/A N/A CCTCTCACACGAGCA T 48 23178 2319 3 139 8595 94 N/A N/A TCTGCCTCTCACACG A 29 23182 2319 7 140 8595 98 N/A N/A TTCGACACAGGGTAG C 67 24221 2423 6 141 8596 02 N/A N/A ATGTTTTCGACACAG G 66 24226 2424 1 142 8596 06 N/A N/A TGGGCATGTTTTCGA C 0 24231 2424 6 143 8596 10 N/A N/A GCATCTTTTCAGTACT 55 26517 2653 2 144 8596 14 N/A N/A CATCGCATCTTTCA G 40 26521 2653 6 145 4684 84 2430 2445 GCTGTCACTGGAGCT C 46 27573 2758 8 146 8593 26 2497 2512 AAGTCGGAACCATTT T 56 27640 2765 5 147 8593 thirty 2502 2517 GGGACAAGTCGGAAC C 57 27645 2766 0 148 8593 34 3458 3473 TCCGCCAGGCCAGTG A 33 28601 2861 6 149 8593 38 3462 3477 CATCTCCCGCCAGGCC A 57 28605 2862 0 150 8593 42 3466 3481 GAAGCATCTCCGCCA G 56 28609 2862 4 151 8593 46 3472 3487 GCCTTAGAAGCATCT C 67 28615 2863 0 152 8593 50 3477 3492 ACCATGCCTTAGAAG C 41 28620 2863 5 153 8593 54 3540 3555 AATGTCTGCTTGCT G 69 28683 2869 8 154 8593 58 3546 3561 AAGATAAATGTCTGC T 69 28689 2870 4 155 8593 62 3594 3609 GGTCTAGAAAAGTTG G 51 28737 2875 2 156 8593 66 3602 3617 GCAAAACAGGTCTAG A 52 28745 2876 0 157 8593 70 3632 3647 ACCCAGAATAAATAT C 28 28775 2879 0 158

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Table 3

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, start site SEQ ID 2: stop website SEQ ID NO 4311 31 1015 1034 GTCACACTTGCTGGC CTGTC 10 N/A N/A 3 8593 75 N/A N/A CATCTGCCGTCCTTC WITH 28 4564 4579 159 8593 79 N/A N/A CCAGCATCTGCCGTC WITH 55 4568 4583 160 8593 83 N/A N/A ACTTGTCAAGCACC AC 66 5013 5028 161 8593 87 N/A N/A AGTGACTTGTCAAG SA 84 5017 5032 162 8593 91 N/A N/A ATTGTAAGTTCACTC A 70 5136 5151 163 8593 95 N/A N/A TTGCATTGTAAGTTC A 82 5140 5155 164 8593 99 N/A N/A AAATTGAATGAATT GG 26 5768 5783 165 8594 03 N/A N/A CTGGTTTTCCCAGCT WITH 73 5899 5914 166 8594 07 N/A N/A ACCTCTGGTTTTCCC A 77 5903 5918 167 8594 AND N/A N/A TTCCACCTCTGGTTT T 23 5907 5922 168 8594 15 N/A N/A CTTGTCTGCTTCCAAC T 22 6717 6732 169 8594 19 N/A N/A AGCTCTTGTCTGCTC WITH 81 6721 6736 170 8594 23 N/A N/A ATTGAGGGAAAAAAA TS 12 7468 7483 171

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8594 27 N/A N/A CCTGCAAGTCCCGC T 42 11241 1125 6 172 8594 31 N/A N/A GCCACCTGCAAGTC CC 47 11245 1126 0 173 8594 35 N/A N/A ATTCTCTCCTCAGGC C 52 11292 ISO 7 174 8594 39 N/A N/A GACCCACCACATTG CA 48 11381 1139 6 175 8594 43 N/A N/A CATGGACCCACCAC AT 25 11385 1140 0 176 8594 47 N/A N/A GTTGCATGGACCCA CC 65 11389 1140 4 177 8594 51 N/A N/A AGTAGCCCCCCAAC TT 0 11606 1162 1 178 8594 55 N/A N/A ACACAGTAGCCCCC CA 42 11610 1162 5 179 8594 59 N/A N/A AGTCACACAGTAGC CC 83 11614 1162 9 180 8594 63 N/A N/A CACACAGTTTGGGT GT 37 11636 1165 1 181 8594 67 N/A N/A ATATGCAAGGTCAC AC 49 11647 1166 2 182 8594 71 N/A N/A AGACATATGCAAGG TC 46 11651 1166 6 183 8594 75 N/A N/A TGTCCCTCTTGGTCT C 61 11948 1196 3 184 8594 79 N/A N/A CCAGTGTCCCTCTTG G 35 11952 1196 7 185 8594 83 N/A N/A TCTGCCAGTGTCCCCT C 27 11956 1197 1 186 8594 87 N/A N/A TGTGATTCCTCTGCCA G 42 11964 1197 9 187 8594 91 N/A N/A TCTCTGTGATCCCTCT G 73 11968 1198 3 188 8594 95 N/A N/A TCTAGGGCCCATGCT T 21 12668 1268 3 189 8594 99 N/A N/A CCAGCTCTAGGGCC CA 53 12673 1268 8 190 8595 03 N/A N/A GGCAGCCAGCTTCTA GG 42 12678 1269 3 191

-57042765

8595 07 N/A N/A CGCACAGTGGGTGC TC 41 12930 1294 5 192 8595 AND N/A N/A GGTTAATAATGCCCC C 58 13180 1319 5 193 8595 15 N/A N/A GCCTGGTTAATAATG C 0 13184 1319 9 194 8595 19 N/A N/A CTGAGTGTGTTAGG AG 52 14471 1448 6 195 8595 23 N/A N/A CTCTCTGAGTGTGTT A 57 14475 1449 0 196 8595 27 N/A N/A TGCTGAGCAGGTCCT T 47 14538 1455 3 197 8595 31 N/A N/A TAGCATGCTGAGCA GG 79 14543 1455 8 198 8595 35 N/A N/A TTCTGTAGCATGCTG A 50 14548 1456 3 199 8595 39 N/A N/A CAATTCAGTGGCTGC T 62 14757 1477 2 200 8595 43 N/A N/A ACAGAGCAATTCAG TG 49 14763 1477 8 201 4684 60 1075 1090 TTGGCCCACGCCGGC AT 29 16705 1672 0 48 8595 47 N/A N/A ACTATTTAGCAGCTA C 65 17444 1745 9 202 8595 51 N/A N/A GTCAACTATTTAGCA G 73 17448 1746 3 203 8595 55 N/A N/A CTAGGTTACCGCTGT C 60 20916 2093 1 204 8595 59 N/A N/A GGGACCTAGGTTAC CG 31 20921 2093 6 205

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8593 12 1495 1510 CTCTGTGACACAAA GC 69 21467 2148 2 206 8593 16 1501 1516 GTCCCACTCTGTGAC A 66 21473 2148 8 207 8595 63 N/A N/A GCACTGTCCTGCAGC C 44 22878 2289 3 208 8595 67 N/A N/A ATCCAGCACTGTCCT G 47 22883 2289 8 209 8595 71 N/A N/A _{CTTTTTTGCTGGTTT} G 77 23032 2304 7 210 8595 75 N/A N/A TCCACTTTTTTGCTG G 0 23036 2305 1 211 8595 79 N/A N/A TGTGCACTATCCTGT A 39 23066 2308 1 212 8595 83 N/A N/A GCATCTGTGCACTAT C 73 23071 2308 6 213 8595 87 N/A N/A AGATGGCATCTGTG CA 65 23076 2309 1 214 8595 91 N/A N/A GCCTCTCACACGAG CA 57 23179 2319 4 215 8595 95 N/A N/A ACTCTGCCTCTCACA C 27 23184 2319 9 216 8595 99 N/A N/A TTTTCGACACAGGGT A 69 24223 2423 8 217 8596 03 N/A N/A CATGTTTTCGACACA G 69 24227 2424 2 218 8596 07 N/A N/A CTTTTCAGTACTCTA T 28 26513 2652 8 219 8596 AND N/A N/A CGCATCTTTTCAGTA C 70 26518 2653 3 220

- 59 042765

8596 15 N/A N/A TCCATCGCATCTTT C 51 26523 2653 8 221 8593 20 2425 2440 CACTGGAGCTCTG GG 45 27568 2758 3 222 8593 23 2431 2446 GGCTGTCACTGGAG CT 33 27574 2758 9 223 8593 27 2498 2513 CAAGTCGGAACCAT TT 52 27641 2765 6 224 8593 31 2503 2518 AGGGACAAGTCGGA AC 28 27646 2766 1 225 8593 35 3459 3474 CTCCGCCAGGCCAG TG 25 28602 2861 7 226 8593 39 3463 3478 GCATCTCCCGCCAGG CC 56 28606 2862 1 227 8593 43 3467 3482 AGAAGCATCTCCGC CA 63 28610 2862 5 228 8593 47 3474 3489 ATGCCTTAGAAGCA TC 8 28617 2863 2 229 8593 51 3478 3493 GACCATGCCTTAGA AG thirty 28621 2863 6 230 8593 55 3541 3556 AAATGTCTGCTTGCT T 51 28684 2869 9 231 8593 59 3589 3604 AGAAAAGTTGGCTG TA 37 28732 2874 7 232 8593 63 3595 3610 AGGTCTAGAAAAGT TG 74 28738 2875 3 233 8593 67 3604 3619 AAGCAAAACAGGTC TA 37 28747 2876 2 234 8593 71 3633 3648 AACCCAGAATAAAT AT thirty 28776 2879 1 235

- 60 042765

Table 4

Suppression of PCSK9 mRNA by 3-10-3 cEt gapmers targeting

SEQ ID NO: 1 and 2

No. ISIS SEQ ID: 1, starting site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, start site SEQ ID2: stopsite SEQID NO 43113 1 1015 1034 GTCACACTTGCTGGCCT GTC 0 N/A N/A 3 46846 0 1075 1090 TTGGCCACGCCGGCAT 25 16705 16720 48 85931 3 1497 1512 CACTCTGTGACACAAA 59 21469 21484 236 85931 7 1502 1517 TGTCCCACTCTGTGAC 10 21474 21489 237 85932 1 2428 2443 TGTCACTGGAGCTCCT 17 27571 27586 238 85932 4 2432 2447 GGGCTGTCACTGGAGC 46 27575 27590 239 85932 8 2499 2514 ACAAGTCGGAACCATT 60 27642 27657 240 85933 2 2504 2519 GAGGGACAAGTCGGAA 1 27647 27662 241 85933 6 3460 3475 TCTCCGCCAGGCCAGT 20 28603 28618 242 85934 0 3464 3479 AGCATCTCCCGCCAGGC 60 28607 28622 243 85934 4 3468 3483 TAGAAGCATCTCCGCC 59 28611 28626 244

- 61 042765

85934 8 3475 3490 CATGCCTTAGAAGCAT 0 28618 28633 245 85935 2 3480 3495 CCGACCATGCCTTAGA 63 28623 28638 246 85935 6 3542 3557 TAAATGTCTGCTTGCT 67 28685 28700 247 85936 0 3591 3606 CTAGAAAAGTTGGCTG 59 28734 28749 248 85936 4 3596 3611 CAGGTCTAGAAAAGTT 44 28739 28754 249 85936 8 3605 3620 AAAGCAAAACAGGTCT 62 28748 28763 250 85937 2 N/A N/A TGCCGTCCTTCCCACCC 17 4560 4575 251 85937 6 N/A N/A GCATCTGCCGTCCTTC 40 4565 4580 252 85938 0 N/A N/A TCCCAGCATCTGCCGT 49 4570 4585 253 85938 4 N/A N/A GACTTGTCAAGCACCA 83 5014 5029 254 85938 8 N/A N/A AAGTGACTTGTCAAGC 77 5018 5033 255 85939 2 N/A N/A CATTGTAAGTTCACTC 77 5137 5152 256 85939 6 N/A N/A CTTGCATTGTAAGTTC 65 5141 5156 257 85940 0 N/A N/A TTTCCCAGCTCTGTAT 28 5894 5909 258 85940 4 N/A N/A TCTGGTTTCCCAGCT 48 5900 5915 259

- 62 042765

85940 8 N/A N/A CACCTCTGGTTTTCCC 78 5904 5919 260 85941 2 N/A N/A CTTCCACCTCTGGTTT 28 5908 5923 261 85941 6 N/A N/A TCTTGTCTGCTTCCAAC 13 6718 6733 262 85942 0 N/A N/A TAGCTCTTGTCTGCTC 39 6722 6737 263 85942 4 N/A N/A ACCCTAGGTGTACTTT 27 7779 7794 264 85942 8 N/A N/A ACCTGCAAGTCCCGC 31 11242 11257 265 85943 2 N/A N/A TGCCACCTGCAAGTCC 44 11246 11261 266 85943 6 N/A N/A CATTCTCTCTCAGGC 38 11293 11308 267 85944 0 N/A N/A GGACCCACCACATTGC 19 11382 11397 268 85944 4 N/A N/A GCATGGACCCACACACA 56 11386 11401 269 85944 8 N/A N/A TGTTGCATGGACCAC 53 11390 11405 270 85945 2 N/A N/A CAGTAGCCCCCCAACT 19 11607 11622 271 85945 6 N/A N/A CACACAGTAGCCCCCC 56 11611 11626 272 85946 0 N/A N/A AAGTCACACAGTAGCC 32 11615 11630 273 85946 4 N/A N/A GTCACACAGTTTGGGT 69 11638 11653 274

- 63 042765

85946 8 N/A N/A CATATGCAAGGTCACA 59 11648 11663 275 85947 2 N/A N/A TCAGACATATGCAAGG 72 11653 11668 276 85947 6 N/A N/A GTGTCCCTCTTGGTCT 67 11949 11964 277 85948 0 N/A N/A GCCAGTGTCCCTCTTG 46 11953 11968 278 85948 4 N/A N/A CCTCTGCCAGTGTCCC 34 11958 11973 279 85948 8 N/A N/A CTGTGTGATCCCTGCCA 58 11965 11980 280 85949 2 N/A N/A GTCTCTGTGATCCCTCT 71 11969 11984 281 85949 6 N/A N/A GCTCTAGGGCCCATGC 16 12670 12685 282 85950 0 N/A N/A GCCAGCTCTAGGGCCC 16 12674 12689 283 85950 4 N/A N/A AGAGAAATGCATGCTA 68 12841 12856 284 85950 8 N/A N/A GGCGCACAGTGGGTGC 0 12932 12947 285 85951 2 N/A N/A TGGTTAATAATGCCCC 40 13181 13196 286 85951 6 N/A N/A TTGCCTGGTTAATAAT 12 13186 13201 287 85952 0 N/A N/A TCTGAGTGTGTTAGGA 62 14472 14487 288 85952 4 N/A N/A GCTCTCTGAGTGTGTT 56 14476 14491 289

- 64 042765

85952 8 N/A N/A CATGCTGAGCAGGTCC 66 14540 14555 290 85953 2 N/A N/A GTAGCATGCTGAGCAG 79 14544 14559 291 85953 6 N/A N/A TCAGTGGCTGCTCTGA 24 14753 14768 292 85954 0 N/A N/A GCAATTCAGTGGCTGC 33 14758 14773 293 85954 4 N/A N/A TAGTTACACACAGAA 17 16208 16223 294 85954 8 N/A N/A AACTATTTAGCAGCTA 43 17445 17460 295 85955 2 N/A N/A CGTCAACTATTTAGCA 66 17449 17464 296 85955 6 N/A N/A CCTAGGTTACCGCTGT 33 20917 20932 297 85956 0 N/A N/A GCACAGACCCTGACTG 23 21567 21582 298 85956 4 N/A N/A AGCACTGTCCTGCAGC 48 22879 22894 299 85956 8 N/A N/A TTTTGCTGGTTTGAGA 52 23029 23044 300 85957 2 N/A N/A ACTTTTTTGCTGGTTTT 70 23033 23048 301 85957 6 N/A N/A ATTCCACTTTTTTGCT 24 23038 23053 302 85958 0 N/A N/A TCTGTGCACTATCCTG 38 23068 23083 303 85958 4 N/A N/A GGCATCTGTGCACTAT 75 23072 23087 304

- 65 042765

85958 8 N/A N/A TCACACGAGCATTAAG 34 23174 23189 305 85959 2 N/A N/A TGCCTCTCACACGAGC 38 23180 23195 306 85959 6 N/A N/A TAGACAGGATCAACTC 18 23406 23421 307 85960 0 N/A N/A GTTTTCGACACAGGGT 61 24224 24239 308 85960 4 N/A N/A GCATGTTTTCGACACA 63 24228 24243 309 85960 8 N/A N/A ATCTTTTCAGTACTCT 48 26515 26530 310 85961 2 N/A N/A TCGCATCTTTTCAGTA 31 26519 26534 311 85961 6 N/A N/A AGCAGCTAGGCCACAG 20 26841 26856 312

Research 2.

Cultured HepG2 cells at a density of 20,000 cells/well were transfected by electroporation with 3,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer and probe kit from ABI (ID no. Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels were adjusted according to total RNA measured with RIBOGREEN®. Results are presented as percent suppression of PCSK9 compared to untreated control cells.

Table 5

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, starting site SEQ ID 2: stopsite SEQ ID NO 848304 757 772 AACTTCAAGGCCA GCT 66 N/A N/A 313 848215 108 123 CTGAACTGAACGG CGG 68 3583 3598 314 848231 320 335 GGCGAGGAGACCT AGA 87 3795 3810 315 848247 485 500 ACGCAAGGCTAGC ACC 77 3960 3975 316 848788 N/A N/A GGCAAGCCCGCTTT CT 88 4456 4471 317 848804 N/A N/A CCCTTTTAAAGATT AG 67 5053 5068 318 848820 N/A N/A CCAGAGAAGAAAC ATS 62 5524 5539 319

- 66 042765

848836 N/A N/A AAGAATCTGGAGC TGC 84 6430 6445 320 848852 N/A N/A CACTGAGGACCAA ATT 65 6806 6821 321 848868 N/A N/A GCTATATAAAGGA ATA 65 7161 7176 322 848884 N/A N/A CATGAGCAAGTCA CTC 65 7387 7402 323 848900 N/A N/A AGTATAGTAGATG ATA 82 7720 7735 324 848262 614 629 CGAGAGGTGGGTC TCC 54 7887 7902 325 466848 673 688 GTGAGGTATCCCCG GC 88 7946 7961 326 848289 689 704 GACATGCAGGATCT TG 79 7962 7977 327 848916 N/A N/A GGGAATTCATACA GA 72 8193 8208 328 848932 N/A N/A CACCGAAGATGTG ASA 51 8521 8536 329 848948 N/A N/A AAGGGAAAGGCCT GAG 53 9040 9055 330 848964 N/A N/A CCCCACAGGCAGC CTC 61 9610 9625 331 848980 N/A N/A CCGGAGGACAGAC TAG 27 10368 10383 332 848319 862 877 TGGTATTCATCCGC CC 60 10623 10638 333 848996 N/A N/A AGCCAAACGGAGC TGG 51 11093 11108 334 849012 N/A N/A AAAGGAACAGGCT CTT 56 11842 11857 335 849028 N/A N/A CTCTAGGGCCCATG CT 56 12669 12684 336 849044 N/A N/A GGATACACAGGCT CGC 96 13108 13123 337 849060 N/A N/A GATGATGTGACAC TG 86 13864 13879 338 849076 N/A N/A GGCCAGCAAGGTG GGC 35 14277 14292 339 849092 N/A N/A TGCTAGTAGGTCTG GG 72 14675 14690 340

- 67 042765

849108 N/A N/A CCAAAGGAAGACT TCA 70 15400 15415 341 849124 N/A N/A ATTTAAAGACTCAA AG 16 15870 15885 342 849140 N/A N/A ACATTTGTGGGAGA GG 56 16250 16265 343 848334 984 999 CGTCCTCCTCGGGC AC 85 16376 16391 344 848348 1018 1033 TCACACTTGCTGGC CT 52 16648 16663 345 848364 1061 1076 ATCCCGGCCGCTGA CC 57 16691 16706 346 468460 1075 1090 TTGGCCCACGCCGGC AT 48 16705 16720 48 848379 1085 1100 GCTGGCACCCTTGG CC 57 16715 16730 347 849156 N/A N/A AAGACATAAAGAC ATS 89 17083 17098 348 849172 N/A N/A CAGAAGGTTATTGA TT 68 17735 17750 349 849188 N/A N/A TGCTAGTTATTAAG CA 15 18080 18095 350 849204 N/A N/A CAGTAACAGCTTCTT TT 80 19020 19035 351 849220 N/A N/A CCTTATTATCCCTT TC 70 19396 19411 352 849236 N/A N/A CAACATCAAATTCT GC 92 19658 19673 353 848395 1165 1180 TTCCGAATAAACTC CA 73 19996 20011 354

- 68 042765

848411 1345 1360 ACCTCGGGAGCTG AGG 20 20176 20191 355 849252 N/A N/A GATGGAGGTTTCGA GC 87 20381 20396 356 849268 N/A N/A TATCAAGTGGTTCT AA 44 21039 21054 357 848427 1458 1473 CACCAATGATGTCC TC 45 21430 21445 358 849284 N/A N/A TCTTATCGGCCAGG TG 75 21810 21825 359 848443 1608 1623 TGGCAGAGAGTG GAT 21 22101 22116 360 848459 1730 1745 CCATACAGTCCTGC AA 40 22512 22527 361 848475 1807 1822 GAGCAGCTCAGCA GCT 44 22589 22604 362 849300 N/A N/A TACAGAAGAGCTG GAG 61 22767 22782 363 849316 N/A N/A ACACAGAGTGGTTT CA 79 23262 23277 364 848489 1951 1966 CTGCAGTTGGCCTG GG 57 23571 23586 365 848505 2006 2021 GTGGCAGTGGACA CGG 71 23626 23641 366 849332 N/A N/A AGGAGAAGTAAGG TCA 91 23828 23843 367 849348 N/A N/A GGGCATGTTTTTCGA CA 65 24230 24245 368 849364 N/A N/A TTCCACCCAGAGAT GG 34 25105 25120 369

- 69 042765

849380 N/A N/A CGGTATGGTGGTGG CA 71 25630 25645 370 849396 N/A N/A GGGCAAGTGGATC CAA 38 26364 26379 371 849412 N/A N/A CCACACTTCATTTTC TC 35 27298 27313 372 848521 2225 2240 GCAGGCCACGGTC ACC 67 27368 27383 373 848537 2483 2498 TTAAAGCTCAGCCC CA 67 27626 27641 374 848553 2560 2575 CCCCGGAAAAGGCG GAA 39 27703 27718 375 848569 2704 2719 CCGAGCACAGCTC GAC 62 27847 27862 376 848584 2739 2754 ACGGACATCGGCA CAT 92 27882 27897 377 848600 2776 2791 GGCACGGAACAAG AGC 87 27919 27934 378 848616 2888 2903 ACCTTTCACACTCA CC 92 28031 28046 379 468491 2971 2986 TGCCATCCAGAAAA GCT 64 28114 28129 380 848646 3085 3100 CACCTTTGGGTGTT GC 80 28228 28243 381 848662 3132 3147 CCACTGCACACTGC CG 87 28275 28290 382 848678 3220 3235 TGGTTCCAGGTTTC TT 87 28363 28378 383 848693 3253 3268 CCTGCTGTGTGGAGC TT 93 28396 28411 384 848709 3290 3305 CTTCAGAGCCAGCC CA 65 28433 28448 385 848725 3380 3395 CCGAGCTTCCTGGT CT 92 28523 28538 386 848741 3433 3448 GGGTGATAACGGA AAA 62 28576 28591 387 848756 3547 3562 AAAGATAAATGTCT GC 92 28690 28705 388 848772 3631 3646 CCCAGAATAAATAT CT 89 28774 28789 389

- 70 042765

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

Table 6

No. ISIS SEQ ID: 1, starting site SEQ ID: 1, stop site Subsequence PCSK9 (% suppression) SEQ ID: 2, starting site SEQ ID2: stopsite SEQ ID NO 848216 123 138 CTCCAGGCCTCAGACC C 82 3598 3613 390 848232 353 368 GCCCATGAGGGCCAG G 67 3828 3843 391 848248 486 501 AACGCAAGGCTAGCA C 62 3961 3976 392 848789 N/A N/A CTATAATGGCAAGCC C 76 4463 4478 393 848805 N/A N/A CGTAGGGACGATTGT C 65 5073 5088 394 848821 N/A N/A ATGAACTTGAGGAGG C 85 5558 5573 395 848837 N/A N/A TTGGAAGAATCTGGA G 81 6434 6449 396 848853 N/A N/A CAACACTGAGGACCA A 77 6809 6824 397 848869 N/A N/A GGCAACACTTCTTAA A 60 7219 7234 398 848885 N/A N/A GTGCAGCCATGAGCA A 73 7394 7409 399 848901 N/A N/A ACAGAGTATAGTAGA T 73 7724 7739 400 848263 616 631 TGCGAGAGGTGGGTC T 42 7889 7904 401 848275 674 689 GGTGAGGTATCCCCG G 67 7947 7962 402 848290 690 705 AGACATGCAGGATCT T 56 7963 7978 403 848917 N/A N/A TCAAATGAAGATAGA C 68 8237 8252 404 848933 N/A N/A GAATACCCAGTCCCCC T 49 8548 8563 405 848949 N/A N/A CTCAAGGGAAAGGCC T 59 9043 9058 406 848965 N/A N/A CATGGCAGCGGTGAA C 47 9638 9653 407 848981 N/A N/A GATCAAACCTGTCCC C 42 10427 10442 408 848997 N/A N/A TGCAGACCGTTTTCC A 83 11221 11236 409

- 71 042765

849013 N/A N/A GGAAAGGAACAGGCT C 88 11844 11859 410 849029 N/A N/A TTTGAGGGCGGCAGC C 32 12687 12702 411 849045 N/A N/A TGCTGGATACACAGG C 79 13112 13127 412 849061 N/A N/A TGTACCTGGGTTCTG C 86 13885 13900 413 849077 N/A N/A CACTAGATATTGAGC T 55 14329 14344 414 849093 N/A N/A CTATGCTAGTAGGTC T 51 14678 14693 415 849109 N/A N/A GGCCAAAGGAAGACT T 22 15402 15417 416 849125 N/A N/A CTGCAAGAAAGACAA C 73 15889 15904 417 848320 887 902 CACCAGGCTGCCTCC G 49 16279 16294 418 848335 985 1000 CCGTCCTCCTCGGGC A 81 16377 16392 419 849141 N/A N/A CATCAGACGGCCGTG C 40 16416 16431 420 848349 1019 1034 GTCACACTTGCTGGC C 62 16649 16664 421 468459 1063 1078 GCATCCCGGCCCGCTG A 76 16693 16708 422 468460 1075 1090 TTGGCCACGCCGGCA T 46 16705 16720 48 848380 1091 1106 GCGCATGCTGGCACC C 45 16721 16736 423

- 72 042765

849157 N/A N/A GATAACATAACAAAA G 29 17096 17111 424 849173 N/A N/A GCAGAAGGTTATTGA T 64 17736 17751 425 849189 N/A N/A ACAGCTGCTAGTTAT T 96 18085 18398 18100 18413 426 849205 N/A N/A TGCTACTGTCAAACAG T 86 19032 19047 427 849221 N/A N/A CAATAACCTTATTAT C 6 19402 19417 428 849237 N/A N/A AGAGAACAGACTGAG G 83 19727 19742 429 848396 1170 1185 GGCTTTTCCGAATAA A 43 20001 20016 430 849253 N/A N/A GGCAGAGGACGCAG GG 40 20538 20553 431 849269 N/A N/A ACTATTCGGTGTATC A 82 21050 21065 432 848428 1466 1481 GCTGGAGGCACCAAT G 0 21438 21453 433 849285 N/A N/A GTCTTATCGGCCAGG T 86 21811 21826 434 848444 1617 1632 TGACATCTTTGGCAG A 39 22110 22125 435 848460 1731 1746 ACCATACAGTCCTGC A 19 22513 22528 436 848476 1814 1829 GAAACTGGAGCAGCT C 54 22596 22611 437 849301 N/A N/A CATTGAAAATCCATC C 83 22895 22910 438

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849317 N/A N/A CCCAAGGAAGACTGT T 56 23318 23333 439 848490 1977 1992 CCTCAGCTGGTGGAG C 54 23597 23612 440 848506 2007 2022 GGTGGCAGTGGACAC G 46 23627 23642 441 849333 N/A N/A AGGTAATACCTTTTTC 51 23871 23886 442 849349 N/A N/A CACCATCCCTTGATG C 46 24285 24300 443 849365 N/A N/A GAAAACACCATCTTT C 26 25118 25133 444 849381 N/A N/A GGCGGTATGGTGGTG G 71 25632 25647 445 849397 N/A N/A ACCCAGGGCAAGTGG A 56 26369 26384 446 849413 N/A N/A CCCCACCCACACTTC A 53 27304 27319 447 848522 2227 2242 TCGCAGGCCACGGTC A 69 27370 27385 448 848538 2485 2500 TTTTAAAGCTCAGCC C 38 27628 27643 449 848554 2565 2580 AGCAGCCCCGGAAAG G 67 27708 27723 450 848570 2706 2721 ACCCGAGCACAGCTC G 90 27849 27864 451 848585 2741 2756 CCACGGACATCGGCA C 93 27884 27899 452 848601 2777 2792 TGGCACGGAACAAGA G 54 27920 27935 453

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848617 2898 2913 GGCCATCAGCACCTT T 63 28041 28056 454 848632 2976 2991 CTAGATGCCATCCAG A 91 28119 28134 455 848647 3088 3103 GGCCACCTTTGGGTG T 46 28231 28246 456 848663 3141 3156 GTGCATGCACCACTG WITH 78 28284 28299 457 848679 3221 3236 CTGGTTCCAGGTTTC 88 28364 28379 458 848694 3254 3269 TCCTGCTGTGTGTGGC T 91 28397 28412 459 848710 3302 3317 GAAGAGGCTTGGCTT WITH 17 28445 28460 460 848726 3381 3396 ACCGAGCTTCCTGGT WITH 38 28524 28539 461 848742 3438 3453 GGCCTGGGTGATAAC G 26 28581 28596 462 848757 3550 3565 CCAAAAGATAAATGT WITH 46 28693 28708 463 848773 3638 3653 TACAAAAACCCAGAAT A 59 28781 28796 464 848305 758 773 CAACTTCAAGGCCAG WITH 79 N/A N/A 465 848412 1349 1364 GATGACCTCGGGAGC T 38 N/A N/A 466

Research 3.

Cultured HepG2 cells at a density of 20,000 cells per well were transfected by electroporation with 1000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer and probe kit from ABI (ID no. Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels were adjusted according to total RNA measured with RIBOGREEN®. Results are presented as percent suppression of PCSK9 compared to untreated control cells.

- 75 042765

Table 7

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, start site SEQ ID2: stopsite SEQID NO 848205 4 19 CACCAGAGCCCCAT CG 3 3479 3494 467 848221 189 204 CTGGGAGCCGCTGC TG 42 3664 3679 468 848237 389 404 TGGCAGCGGCCACC AG 0 3864 3879 469 848253 506 521 GGCCAGGCCGTCCT CC 51 3981 3996 470 848778 N/A N/A CCGCACCTTGGCGC AG 0 4035 4050 471 848794 N/A N/A GAAACAGATGGAA TAC 28 4626 4641 472 848810 N/A N/A ACCCAGCACACTCA GA 0 5291 5306 473 848826 N/A N/A GTACTCTGTGCAGT GG 80 5748 5763 474 848842 N/A N/A GAGTAGAACAGAG TCC 54 6544 6559 475

- 76 042765

848858 N/A N/A TTAATAATCAGCCT TC 46 6933 6948 476 848874 N/A N/A TACAAATGCAGGCA GA 40 7256 7271 477 848890 N/A N/A CTCGACAACAGGTT TT 65 7577 7592 478 848906 N/A N/A AGGAACATGATGA CAT 0 7811 7826 479 848268 644 659 CTGCAGGCGGCGG GCA 0 7917 7932 480 848279 679 694 ATCTTGGTGAGGTA TC 40 7952 7967 481 848294 695 710 ATGGAAGACATGC AGG 44 7968 7983 482 848922 N/A N/A TAAAATGACTCAGG CT 19 8406 8421 483 848938 N/A N/A CATCAAGTTAGAGG CC 49 8650 8665 484 848954 N/A N/A TATACGGGTACCTT CT 28 9143 9158 485 848970 N/A N/A GCTCAGTGCAAACT GC 0 10094 10109 486 848309 779 794 CTCGATGTAGTCGA CA 62 10540 10555 487 848986 N/A N/A CCCGAGAAGTGGA AAC 12 10729 10744 488 849002 N/A N/A CCAAGATCCCACGA GA 56 11458 11473 489 849018 N/A N/A GAGAAAGTGGTCCT GC 35 12122 12137 490

- 77 042765

849034 N/A N/A CAGCAATAACTGAT TT 42 12825 12840 491 849044 N/A N/A GGATACACAGGCTC GC 83 13108 13123 337 849050 N/A N/A CGCCTGAGAAGCTC AG 44 13353 13368 492 849066 N/A N/A CCACACAACGCACA TC 15 14043 14058 493 849082 N/A N/A GACCAAACAGTGCT CG 28 14360 14375 494 849098 N/A N/A CTACAAAGACCTTT TC 0 14849 14864 495 849114 N/A N/A TAGGAGAAAGTAG GGA 26 15533 15548 496 849130 N/A N/A GAATATCAATATCT AA 19 15983 15998 497 848325 931 946 TCCCGGTGGTCACT CT 2 16323 16338 498 848340 990 1005 GGGTCCCGTCCTCC TC 38 16382 16397 499 849146 N/A N/A CCACATGAGAAAG ACC 0 16614 16629 500 848354 1026 1041 CATGACTGTCACAC TT 23 16656 16671 501 848369 1074 1089 TGGCCACGCCGGCA TC 17 16704 16719 502 468460 1075 1090 TTGGCCCACGCCGGC AT 4 16705 16720 48 848385 1114 1129 TGGCAGTTGAGCAC GC 0 16744 16759 503

- 78 042765

849162 N/A N/A GTCTAGAAAAAGTC CT 65 17257 17272 504 849178 N/A N/A CCGTGCCAGGTCAT GC 78 17957 17972 505 849194 N/A N/A CTGGGATACAGACA CC 42 18254 18269 506 849210 N/A N/A AATTAAAAGACTCC AT 18 19154 19169 507 849226 N/A N/A TATTAGCAATTACA CC 45 19502 19517 508 849242 N/A N/A TTACAGGCACAGAG TG 25 19914 19929 509 848401 1228 1243 CTGTACCACCCGC CA eleven 20059 20074 510 849258 N/A N/A AACAAGATTCCTTC CC thirty 20814 20829 511 849274 N/A N/A GGCAGAACTCTGGC AC 56 21163 21178 512 848417 1402 1417 CCCAAAGTCCCCAG GG 0 21374 21389 513 849290 N/A N/A GGTGAAAGATGGT GAT 10 22003 22018 514 848433 1544 1559 CATCATGGCTGCAA TG 0 22037 22052 515 848449 1636 1651 GGGAACCAGGCCTC AT 25 22129 22144 516 848465 1736 1751 TGCTGACCATACAG TC 0 22518 22533 517 849306 N/A N/A GTGCACTATCCTGT AG 28 23065 23080 518

- 79 042765

849322 N/A N/A CAAAGACGGAAAT GGG 44 23462 23477 519 848481 1925 1940 GCACCTGGCAATGG CG 34 23545 23560 520 848495 1993 2008 CGGGTCCCCATGCT GG 0 23613 23628 521 849338 N/A N/A TGCTTAGCACTCAT CA 0 24102 24117 522 849354 N/A N/A CCTACATGCCAGCC TG 73 24472 24487 523 849370 N/A N/A AAAGAGATGCTGG CCT 28 25228 25243 524 848511 2082 2097 GCACAGGCGGCTTG TG 28 25413 25428 525 849386 N/A N/A CCATATTTATGCAC AT 62 25806 25821 526 849402 N/A N/A TTTCAGTACTCTAT AT 12 26511 26526 527 848527 2372 2387 AACGGCTGTCACGG CC 42 27515 27530 528 848543 2507 2522 AGAGAGGGACAAG TCG 47 27650 27665 529 848559 2603 2618 TTCCAGGCAAGGAG GC 23 27746 27761 530 848575 2712 2727 GGCAGCACCCGAG CAC 72 27855 27870 531 848590 2757 2772 ATAAAAGTCATTCT GC 15 27900 27915 532 848606 2787 2802 ATTGAATGCCTGGC AC 36 27930 27945 533

- 80 042765

848622 2919 2934 CCACAGTTAGCTGG AG 45 28062 28077 534 848637 3036 3051 GCTCAGGAAACCA AGG 12 28179 28194 535 848652 3122 3137 CTGCCGAGTCAGTC CT 66 28265 28280 536 848668 3172 3187 TGCCGGGTAGTGGA GC 49 28315 28330 537 848683 3226 3241 CCCCTCTGGTTCCA GG 43 28369 28384 538 848699 3261 3276 GCTCAGTTCCTGCT GT 18 28404 28419 539 848715 3317 3332 AGCCGGGTGAAGT AAG 26 28460 28475 540 848731 3386 3401 CACTCACCGAGCTT CC 73 28529 28544 541 848747 3471 3486 CCTTAGAAGCATCT CC 52 28614 28629 542 848762 3576 3591 GTAAAAAGGCAAC AGA 36 28719 28734 543

- 81 042765

Table 8

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Sequential th % depressed nya SEQ ID: 2, starting site SEQ ID2: stopsite SEQ ID NO 848206 7 22 CGCCACCAGAGC CCCA thirty 3482 3497 544 848222 207 222 CGGAATCCTGGC TGGG 55 3682 3697 545 848238 476 491 TAGCACCAGCTC CTCG 53 3951 3966 546 848254 520 535 TGCTCGGGTGCTTT CGG 35 3995 4010 547 848779 N/A N/A GGCTGCGGGTTC GCCC 13 4070 4085 548 848795 N/A N/A CGAGAATACCTC CGCC 73 4682 4697 549 848811 N/A N/A GCTGAGTAAGGA CTTG 64 5317 5332 550 848827 N/A N/A TTCCGCTAAATAA AAA 9 5781 5796 551 848843 N/A N/A TCAGAGTAGAAC AGAG 51 6547 6562 552 848859 N/A N/A GAGGGAGGTGCC AAGC 31 6954 6969 553 848875 N/A N/A CTACAAATGCAG GCAG 63 7257 7272 554 848891 N/A N/A AACTACGGGCCA CACT 7 7591 7606 555 848907 N/A N/A ACGGATCCTGGC CCCA 45 7834 7849 556 848269 663 678 CCCGGCGGGCAG CCTG 18 7936 7951 557 848280 680 695 GATCTTGGTGAG GTAT 1 7953 7968 558

- 82 042765

848295 699 714 GGCCATGGAAGA CATG 18 7972 7987 559 848923 N/A N/A CTAGAGTCATGCT TTT 0 8425 8440 560 848939 N/A N/A GTGGAGAATCAG TGTG 40 8683 8698 561 848955 N/A N/A TTGAAGTCCAGCT CTC 16 9258 9273 562 848971 N/A N/A GCAATTCGGTTTG TCC 65 10140 1015 5 563 848310 795 810 AGACAGAGGAGT CCTC 31 10556 1057 1 564 848987 N/A N/A GATCATTTAAGG CAAG 26 10850 1086 5 565 849003 N/A N/A TCCCAAGATCCC ACGA 5 11460 1147 5 566 849019 N/A N/A CAAGAGAAGCTT CTCC 20 12152 1216 7 567 849035 N/A N/A CTACAGCAATAA CTGA 46 12828 1284 3 568 849044 N/A N/A GGATACACAGGC TCGC 78 13108 1312 3 337 849051 N/A N/A GAGCAGGGAGCT CATT 0 13395 1341 0 569 849067 N/A N/A CTAGAAGACAGC ACAG 15 14085 1410 0 570 849083 N/A N/A CCGACCTGAAGA CATC 41 14380 1439 5 571 849099 N/A N/A GCCCACCGCATA ATCC 18 14897 1491 2 572

- 83 042765

849115 N/A N/A GACAAAGGTTAG GGTA 46 15549 1556 4 573 849131 N/A N/A TGAATATCAATAT STA thirty 15984 1599 9 574 848326 939 954 CCTCGATTTCCCG GTG thirty 16331 1634 6 575 848341 992 1007 GCGGGTCCCGTC CTCC 0 16384 1639 9 576 849147 N/A N/A GACCACATGAGA AAGA 0 16616 1663 1 577 848355 1029 1044 TGCCATGACTGTC ASA 35 16659 1667 4 578 468460 1075 1090 TTGGCCCACGCCG GCAT 19 16705 1672 0 48 848370 1076 1091 CTTGGCCACGCC GGCA 0 16706 1672 1 579 848386 1133 1148 GCTAACCGTGCC CTTC 20 16763 1677 8 580 849163 N/A N/A CAGTAAGGGAGA GAAC 3 17363 1737 8 581 849179 N/A N/A CCCGTGCCAGGT CATG 38 17958 1797 3 582 849195 N/A N/A AGCTGCTAGTTAT TTA 44 18396 1841 1 583 849211 N/A N/A GAAACAGGGACA GTTG 25 19189 1920 4 584 849227 N/A N/A GGAAGATATTAG CAAT 78 19508 1952 3 585 849243 N/A N/A CTTACAGGCACA GAGT 27 19915 1993 0 586

- 84 042765

848402 1238 1253 GAGGACGCGGCT GTAC 25 20069 2008 4 587 849259 N/A N/A GTGGAACAGAT TCCT 40 20818 2083 3 588 849275 N/A N/A AGGCAGAACTCT GGCA 56 21164 2117 9 589 848418 1414 1429 CCAAAGTTGGTC CCCA 0 21386 2140 1 590 848434 1546 1561 AGCATCATGGCT GCAA 49 22039 2205 4 591 848450 1641 1656 CCTCAGGGAACC AGGC 28 22134 2214 9 592 849291 N/A N/A TGCCATCCCTGCTT ACC thirty 22209 2222 4 593 848466 1738 1753 TGTGCTGACCATA CAG 20 22520 2253 5 594 849307 N/A N/A CACCAAAATGCT GCAA 39 23094 2310 9 595 849323 N/A N/A GGCCTTAGAGTC AAAG 0 23473 2348 8 596 468479 1926 1941 AGCACCTGGCAA TGGC 47 23546 2356 1 597 848496 1997 2012 GACACGGGTCCC CATG 28 23617 2363 2 598 849339 N/A N/A ACTAAGCTAAGT GCTT 7 24113 2412 8 599 849355 N/A N/A GCAAAATGGTGC TCTT 52 24690 2470 5 600 849371 N/A N/A ACCTAGAAACAA CTCA 23 25251 2526 6 601

- 85 042765

848512 2084 2099 CAGCACAGGCGG CTTG 14 25415 2543 0 602 849387 N/A N/A ATGCATATTGCAT TTC 44 25828 2584 3 603 849403 N/A N/A GGCCAGACCACA CTCC 13 26808 2682 3 604 848528 2375 2390 GGCAACGGCTGT CACG 52 27518 2753 3 605 848544 2509 2524 TGAGAGAGGGAC AAGT 27 27652 2766 7 606 848560 2613 2628 AGTGAGTGAGTT CCAG 65 27756 2777 1 607 848576 2713 2728 TGGCAGCACCCG AGCA 56 27856 2787 1 608 848591 2761 2776 CTCAATAAAAGT CATT 45 27904 2791 9 609 848607 2791 2806 GAGGATTGAATG CCTG 37 27934 2794 9 610 848623 2921 2936 CTCCACAGTTAGC TGG 35 28064 2807 9 611 848638 3045 3060 GTAAAGGTGGCT CAGG 46 28188 2820 3 612 848653 3123 3138 ACTGCCGAGTCA GTCC 29 28266 2828 1 613 848669 3180 3195 GTGTACCCTGCCG GGT 3 28323 2833 8 614 848684 3227 3242 CCCCCTCTGGTTC CAG 54 28370 2838 5 615 848700 3263 3278 TGGCTCAGTTCCT GCT 36 28406 2842 1 616 848716 3321 3336 GCCCAGCCGGGT GAAG 26 28464 2847 9 617 848732 3388 3403 ATCACTCACCGA GCTT 69 28531 2854 6 618 848748 3473 3488 TGCCTTAGAAGC ATCT 68 28616 2863 1 619 848763 3578 3593 CTGTAAAAAAGGC AACA 52 28721 2873 6 620

- 86 042765

Table 9

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Sequential th % depressed AND I SEQID: 2, start site SEQ ID2: stop website SEQ ID NO 8482 07 12 27 GATCACGCCACC AGAG 0 3487 3502 621 8482 23 209 224 CGCGGAATCCTG GCTG 1 3684 3699 622 8482 39 477 492 CTAGCACCAGCT CCTC 38 3952 3967 623 8482 55 533 548 GGCTGTGGTTCC GTGC 29 4008 4023 624 8487 80 N/A N/A AAACAGCACCGC ACCG 0 4090 4105 625 8487 96 N/A N/A CCGCACACGGTC GGCA 35 4821 4836 626

- 87 042765

8488 12 N/A N/A GAACAAGGAAGA GGGC 39 5341 5356 627 8488 28 N/A N/A CAAAGACCCATC TGAA 32 5803 5818 628 8488 44 N/A N/A TCATGAATCAAG TCCA 71 6650 6665 629 8488 60 N/A N/A GCAGAGATCAAT CACA 36 6997 7012 630 8488 76 N/A N/A TCCCATCCAGAT GCTC 34 7272 7287 631 8488 92 N/A N/A ATTCAGGCAAAA ATGG 12 7608 7623 632 8482 70 665 680 TCCCCGGCGGGC AGCC 24 7938 7953 633 8482 81 681 696 GGATCTTGGTGA GGTA 23 7954 7969 634 8482 96 704 719 AAGAAGGCCATG GAAG 38 7977 7992 635 8489 08 N/A N/A TGCCATTCCCAA AAAG 0 8045 8060 636 8489 24 N/A N/A CAAAGGAATTTTT GGAC 0 8442 8457 637 8489 40 N/A N/A TGCCATGTGGAG AATC 24 8689 8704 638 8489 56 N/A N/A TCGAACCCAGGC TGGT 0 9286 9301 639 8489 72 N/A N/A CATGACATAACA GCAC 24 10158 1017 3 640 8483 AND 797 812 AAAGACAGAGGA GTCC 28 10558 1057 3 641

- 88 042765

8489 88 N/A N/A AAAGAGGATCAT TTAA 0 10856 1087 1 642 8490 04 N/A N/A CAAGAACGACAA AGCT 48 11493 1150 8 643 8490 20 N/A N/A GGACACGGAAGA GGCA 63 12166 1218 1 644 8490 36 N/A N/A ACAGAGAAATGC ATGC 66 12843 1285 8 645 8490 44 N/A N/A GGATACACAGGC TCGC 81 13108 1312 3 337 8490 52 N/A N/A CTCTATCTTCCAA ACC 22 13578 1359 3 646 8490 68 N/A N/A ATACAGGCTTAT CTAG 3 14097 1411 2 647 8490 84 N/A N/A CAATAGGCATCT ACCA 59 14444 1445 9 648 8491 00 N/A N/A AGGACTTACAGC CCAC 12 14907 1492 2 649 8491 16 N/A N/A GCCCATGCAGGA CAAA eleven 15559 1557 4 650 8491 32 N/A N/A TATGAATATCAA TATC 0 15986 1600 1 651 4684 53 950 965 CATGACCCTGCC CTCG 45 16342 1635 7 652 8483 42 995 1010 GAAGCGGGTCCC GTCC 17 16387 1640 2 653 8491 48 N/A N/A ACACAAGGACCA CATG 0 16623 1663 8 654 8483 56 1040 1055 TGCCAGGTGGGT GCCA 26 16670 1668 5 655

- 89 042765

4684 60 1075 1090 TTGGCCCACGCCG GCAT 0 16705 1672 0 48 8483 71 1077 1092 CCTTGGCCACGC CGGC 37 16707 1672 2 656 8483 87 1135 1150 CCGCTAACCGTG CCCT 17 16765 1678 0 657 8491 64 N/A N/A CTTGAGCTGTGC GACC 27 17497 1751 2 658 8491 80 N/A N/A CCCCGTGCCAGG TCAT 26 17959 1797 4 659 8491 96 N/A N/A ACAACAGCCACA TTTA 56 18413 1842 8 660 8492 12 N/A N/A TGTGAAACAGGG ACAG 17 19192 1920 7 661 8492 28 N/A N/A TAATATATACATC STA 0 19564 1957 9 662 8492 44 N/A N/A CCTTACAGGCAC AGAG 16 19916 1993 1 663 8484 03 1241 1256 GTTGAGGACGCG GCTG 19 20072 2008 7 664 8492 60 N/A N/A CCAACGCGCGCG CGCG 4 20870 2088 5 665 8492 76 N/A N/A GAAAATCTGACT GCCC 43 21180 2119 5 666 8484 19 1416 1431 GGCCAAAAGTTGG TCCC 0 21388 2140 3 667 8484 35 1550 1565 AGACAGCATCAT GGCT 43 22043 2205 8 668 8484 51 1656 1671 TCAGTACCCGCT GGTC 41 22149 2216 4 669

- 90 042765

8492 92 N/A N/A GGTCACACAGAC CTCC 24 22252 2226 7 670 8484 67 1744 1759 CCCGAGTGTGCT GACC 39 22526 2254 1 671 8493 08 N/A N/A TTCCACCAAAAT GCTG 7 23097 2311 2 672 8484 82 1927 1942 CAGCACCTGGCA ATGG 10 23547 2356 2 673 8484 97 1998 2013 GGACACGGGTCC CCAT 32 23618 2363 3 674 8493 24 N/A N/A GGCAAGCCCAGC CTCC 0 23667 2368 2 675 8493 40 N/A N/A AACGATAGCTAG AATT 12 24143 2415 8 676 8493 56 N/A N/A TGGTAGGGTTGT GGTT 0 24782 2479 7 677 8493 72 N/A N/A GCAAGAGCTAGG AAAC 60 25265 2528 0 678 8485 13 2086 2101 CTCAGCACAGGC GGCT 41 25417 2543 2 679 8493 88 N/A N/A ATGCATGCATATT GCA 0 25832 2584 7 680 8494 04 N/A N/A CTTCAGACTCCA GCCT 0 26944 2695 9 681 8485 29 2378 2393 GATGGCAACGGC TGTC 0 27521 2753 6 682 8485 45 2511 2526 GCTGAGAGAGGG ACAA 54 27654 2766 9 683 8485 61 2617 2632 CCAGAGTGAGTG AGTT thirty 27760 2777 5 684

- 91 042765

8485 77 2714 2729 CTGGCAGCACCC GAGC 52 27857 2787 2 685 8485 92 2762 2777 GCTCAATAAAAG TCAT 44 27905 2792 0 686 8486 08 2795 2810 ACCTGAGGATTG AATG 16 27938 2795 3 687 8486 24 2944 2959 AATCAGGGAGCC CCCA 0 28087 2810 2 688 8486 39 3047 3062 GAGTAAAGGTGG CTCA 19 28190 2820 5 689 8486 54 3124 3139 CACTGCCGAGTC AGTC 51 28267 2828 2 690 8486 70 3186 3201 GCGAATGTGTAC CCTG 81 28329 2834 4 691 8486 85 3229 3244 CGCCCCCTCTGGT TCC 57 28372 2838 7 692 8487 01 3264 3279 CTGGCTCAGTTCC TGC 69 28407 2842 2 693 8487 17 3330 3345 AAATGAGGAGCC CAGC 42 28473 2848 8 694 8487 33 3392 3407 TGCCATCACTCAC CGA 48 28535 2855 0 695 8487 49 3482 3497 CCCCGACCATGC CTTA 43 28625 2864 0 696 8487 64 3580 3595 GGCTGTAAAAAAG GCAA 68 28723 2873 8 697

- 92 042765

Table 10

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % depressed SEQ ID: 2, starting site SEQ ID 2: stopsite SEQ ID NO 8482 08 15 thirty GCAGATCACGCCA CCA 0 3490 3505 698 8482 24 212 227 GCGCGCGGAATCC TGG 21 3687 3702 699 8482 40 478 493 GCTAGCACCAGCT CCT 37 3953 3968 700 4684 41 541 556 TGGAAGGTGGCTG TGG 0 4016 4031 701 8487 81 N/A N/A CCGAGAGGAAACA GCA 52 4098 4113 702 8487 97 N/A N/A AGCAAACTCGCCC CGC 55 4833 4848 703 8488 13 N/A N/A GGCTCAGGGAACA AGG 39 5349 5364 704 8488 29 N/A N/A CACCATCATCGCT GAA 39 5834 5849 705 8488 45 N/A N/A GCAGACATACCTG CTT 24 6693 6708 706 8488 61 N/A N/A CTACATGTGCAGA GAT 52 7005 7020 707 8488 77 N/A N/A ATAAATCCCCATC CA 36 7279 7294 708 8488 93 N/A N/A TTTCAAATGTGCCA TT 22 7621 7636 709

- 93 042765

4684 43 666 681 ATCCCCGGCGGGC AGC 52 7939 7954 710 8482 82 682 697 AGGATCTTGGTGA GGT 47 7955 7970 711 8482 97 732 747 CGCCACTCATCTTC AC 23 8005 8020 712 8489 09 N/A N/A GGCCTACTAAGCA CAG 49 8100 8115 713 8489 25 N/A N/A TTCAAAGGAATTTTT GG 0 8444 8459 714 8489 41 N/A N/A ACCCAGGTCCAGA CTC 44 8793 8808 715 8489 57 N/A N/A GGTTAGACAGCCA ATA 48 9318 9333 716 8489 73 N/A N/A CTAAATCATGACA TAA 14 10164 1017 9 717 8483 12 799 814 GCAAAGACAGAGG AGT 46 10560 1057 5 718 8489 89 N/A N/A TCCAAGGTAAGTG CAG 52 10924 1093 9 719 8490 05 N/A N/A GACAAGAACGACA AAG 46 11495 1151 0 720 8490 21 N/A N/A GACAATGAAGAGG AGA 69 12240 1225 5 721 8490 37 N/A N/A CTCAATACCTGAC AGA 50 12854 1286 9 722 8490 44 N/A N/A GGATACACAGGCT CGC 81 13108 1312 3 337 8490 53 N/A N/A TGGTAAAAGCCCC CAC 39 13677 1369 2 723

- 94 042765

8490 69 N/A N/A ATACATTGGCAGA CAG 56 14121 1413 6 724 8490 85 N/A N/A ACTCATCAATAGG CAT 79 14450 1446 5 725 8491 01 N/A N/A CATCACAGGACTT ASA 0 14913 1492 8 726 8491 17 N/A N/A CAAGAGACTCACT GGG 16 15581 1559 6 727 8491 33 N/A N/A TGCCAAGAAGGAC CCA 61 16020 1603 5 728 8483 27 953 968 GACCATGACCCTG CCC 41 16345 1636 0 729 8483 43 997 1012 TGGAAGCGGGTCC CGT eleven 16389 1640 4 730 8491 49 N/A N/A TGCTCGACGAACA CAA 0 16633 1664 8 731 8483 57 1053 1068 CGCTGACCACCCC TGC 33 16683 1669 8 732 4684 60 1075 1090 TTGGCCCACGCCGG CAT 7 16705 1672 0 48 8483 72 1078 1093 CCCTTGGCCCACGC CGG 49 16708 1672 3 733 8483 88 1137 1152 TGCCGCTAACCGT GCC 10 16767 1678 2 734 8491 65 N/A N/A GTGGAAGTCAAGC TGC 42 17550 1756 5 735 8491 81 N/A N/A GCCCCGTGCCAGG TCA 58 17960 1797 5 736 8491 97 N/A N/A TAGGAGACAGCTA GTG 25 18486 1850 1 737

- 95 042765

8492 13 N/A N/A CACAAACTTGAAC AGA 38 19248 1926 3 738 8492 29 N/A N/A CTTAATATATACAT CC 0 19566 1958 1 739 8492 45 N/A N/A GCCAAGGTCACCC CTT 0 19963 1997 8 740 8484 04 1283 1298 GACCAGCACGACC CCA 13 20114 2012 9 741 8492 61 N/A N/A ACCTAGGTTACCG CTG 33 20918 2093 3 742 8492 77 N/A N/A AGGAAAATCTGAC TGC 26 21182 2119 7 743 8484 20 1423 1438 ACACAGCGGCCAAA AGT 0 21395 2141 0 744 8484 36 1552 1567 GCAGACAGCATCA TGG 76 22045 2206 0 745 8484 52 1658 1673 GGTCAGTACCGCGC TGG 29 22151 2216 6 746 8492 93 N/A N/A CCTATTTAAGGTG GCG 51 22311 2232 6 747 8484 68 1753 1768 CGTGTAGGCCCCG AGT 44 22535 2255 0 748 8493 09 N/A N/A CAGAAATGCCTGC CCC 64 23141 2315 6 749 8484 83 1928 1943 GCAGCACCTGGCA ATG 0 23548 2356 3 750 8484 98 1999 2014 TGGACACGGGTCC CCA 2 23619 2363 4 751 8493 25 N/A N/A CCTCACCCCAGGG CAA 0 23678 2369 3 752

- 96 042765

8493 41 N/A N/A GTAACGATAGCTA GAA 52 24145 2416 0 753 8493 57 N/A N/A TGAACATGGTAGG GTT 62 24788 2480 3 754 8493 73 N/A N/A AGGCAAGAGCTAG GAA 51 25267 2528 2 755 8485 14 2113 2128 ACGCACTGGTTGG GCT 5 25444 2545 9 756 8493 89 N/A N/A GTTAAATAGATCA GAG 15 25989 2600 4 757 8494 05 N/A N/A CCCCATAGCCTGC CCC 0 27058 2707 3 758 8485 thirty 2381 2396 GCAGATGGCAACG GCT 14 27524 2753 9 759 8485 46 2520 2535 CCATGGAGGGCTG AGA 3 27663 2767 8 760 8485 62 2619 2634 ACCCAGAGTGAGT GAG 29 27762 2777 7 761 8485 78 2715 2730 GCTGGCAGCACCC GAG 18 27858 2787 3 762 8485 93 2763 2778 AGCTCAATAAAAG TCA 61 27906 2792 1 763 8486 09 2820 2835 GAAGAATCCTGCC TCC 42 27963 2797 8 764 8486 25 2946 2961 TTAATCAGGGAGC CCC 31 28089 2810 4 765 8486 40 3050 3065 GCAGAGTAAAGGT GGC 38 28193 2820 8 766 8486 55 3125 3140 ACACTGCCGAGTC AGT 32 28268 2828 3 767 8486 71 3188 3203 GTGCGAATGTGTA CCC 68 28331 2834 6 768 8486 86 3237 3252 GGCAGGCACGCCC CCT 17 28380 2839 5 769 8487 02 3265 3280 TCTGGCTCAGTTCC TG 51 28408 2842 3 770 8487 18 3332 3347 AAAAATGAGGAGC CCA 17 28475 2849 0 771 8487 34 3415 3430 TTCCATGCCTGCAG GC 35 28558 2857 772 8487 50 3502 3517 GGACAGTTGTTGG CCC 25 28645 2866 0 773 8487 65 3588 3603 GAAAAGTTGGCTG TAA 22 28731 2874 6 774

- 97 042765

Table 11

mRNA suppression of PCSK9. with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % suppression SEQID: 2, starting site SEQ ID2: stopsite SEQ ID NO 848209 51 66 ACCTTCTAGGGTGT GG 0 3526 3541 775 848225 242 257 CTGAAGTTCAGGA GCA 35 3717 3732 776 848241 479 494 GGCTAGCACCAGCT CC 58 3954 3969 777 848256 549 564 CGCAGCGGTGGAA GGT 15 4024 4039 778 848782 N/A N/A GCGAAGAGCCCTC GGC 33 4158 4173 779 848798 N/A N/A GGGAAGAAGCTTC CCA 0 4882 4897 780 848814 N/A N/A AGAAAGTCAAAGG CTC 74 5360 5375 781 848830 N/A N/A GACACCATCATCGC TG 67 5836 5851 782 848846 N/A N/A GGCAGACATACCT GCT 0 6694 6709 783 848862 N/A N/A GGCCTTAAGCTGCT TT 10 7048 7063 784 848878 N/A N/A ATATAAATCTCCCA TC 9 7281 7296 785 848894 N/A N/A TCACATGGTTATAT AA 37 7640 7655 786 848271 667 682 TATCCCCGGCGGGC AG 35 7940 7955 787 848283 683 698 CAGGATCTTGGTGA GG 49 7956 7971 788 848298 740 755 CAGCAGGTCGCCA CTC 5 8013 8028 789 848910 N/A N/A AGCAATGGGCCTA STA 0 8107 8122 790 848926 N/A N/A AGCAACAGCTTCA AAG AND 8453 8468 791 848942 N/A N/A CTGATTAACCCATG GG 46 8833 8848 792 848958 N/A N/A TTACAGATAGAGG AAT 0 9365 9380 793

- 98 042765

848974 N/A N/A GGACATGGAGTGA AGC 7 10200 10215 794 848313 801 816 GGGCAAAGACAGA GGA eleven 10562 10577 795 848990 N/A N/A GGTAGAGTCACCAT CA 40 10953 10968 796 849006 N/A N/A TCTCACAGCAACCT GT 0 11548 11563 797 849022 N/A N/A TCCTAACCCCCACA AC 0 12373 12388 798 849038 N/A N/A TGCAGGGCTAAAAA TCA 34 12880 12895 799 849044 N/A N/A GGATACACAGGCT CGC 78 13108 13123 337 849054 N/A N/A GGCAGGTGGGCCT GGT 0 13689 13704 800 849070 N/A N/A GGATACATTGGCA GAC 41 14123 14138 801 849086 N/A N/A GTGTTAGGAGCTTT CA 60 14465 14480 802 849102 N/A N/A AGACACATCACAG GAC 27 14918 14933 803 849118 N/A N/A AAGAAGCAGGCAC TGG 32 15608 15623 804 849134 N/A N/A AAGGAAAGGGAGG CCT 0 16081 16096 805 848328 964 979 TCGAAGTCGGTGAC CA 17 16356 16371 806 848344 999 1014 TGTGGAAGCGGGT CCC 24 16391 16406 807

- 99 042765

848358 1055 1070 GCCGCTGACCACCC CT 0 16685 16700 808 468460 1075 1090 TTGGCCCACGCCGGC AT thirty 16705 16720 48 848373 1079 1094 ACCCTTGGCCACGC CG thirty 16709 16724 809 848389 1145 1160 TATGAGGGTGCCGC TA 4 16775 16790 810 849150 N/A N/A ACTTACCTATGAGG GT 0 16782 16797 811 849166 N/A N/A GGCTTACAGTGGA AGT 8 17558 17573 812 849182 N/A N/A TGCCCCGTGCCAGG TC 41 17961 17976 813 849198 N/A N/A CCAGGAGCCGTGG CCA 12 18537 18552 814 849214 N/A N/A ACACAAACTTGAA CAG 10 19249 19264 815 849230 N/A N/A TCTTAATATATACA TC 0 19567 19582 816 849246 N/A N/A GAACAAAGCCAAG GTC 0 19970 19985 817 848405 1286 1301 GGTGACCAGCACG ACC 0 20117 20132 818 849262 N/A N/A CCTTGATAGGCCAG GG 0 20937 20952 819 849278 N/A N/A TCCCACTCAAATGT CC 0 21205 21220 820 848421 1425 1440 CCACACAGCGGCC AAA 0 21397 21412 821

- 100 042765

848437 1554 1569 CGGCAGACAGCAT CAT 72 22047 22062 822 848453 1681 1696 GGCAGGGCGGCCA CCA 0 22174 22189 823 849294 N/A N/A AAAATATTGCACA GCC 0 22353 22368 824 848469 1760 1775 GGCCATCCGTGTAG GC 0 22542 22557 825 849310 N/A N/A AGCATTAAGACCC AT 21 23167 23182 826 848484 1929 1944 GGCAGCACCTGGC AAT 17 23549 23564 827 848499 2000 2015 GTGGACACGGGTC CCC 17 23620 23635 828 849326 N/A N/A AGAAAGAGACCCC TCC 0 23692 23707 829 849342 N/A N/A AAACAATAGTAAC GAT 0 24153 24168 830 849358 N/A N/A TCTGAACATGGTAG GG 58 24790 24805 831 849374 N/A N/A GCCCATCCCCCATCA GA 6 25303 25318 832 848515 2157 2172 GGGCATGGCAGCA GGA 17 25488 25503 833 849390 N/A N/A CTTCAGCATGCAGC TC 6 26093 26108 834 849406 N/A N/A TGAAACTGATGGCC CC 22 27070 27085 835 848531 2383 2398 CAGCAGATGGCAA CGG 25 27526 27541 836

- 101 042765

848547 2522 2537 GGCCATGGAGGGC TGA 0 27665 27680 837 848563 2623 2638 AGGCACCCAGAGT GAG 42 27766 27781 838 848579 2716 2731 AGCTGGCAGCACC CGA 20 27859 27874 839 848594 2770 2785 GAACAAGAGCTCA ATA 13 27913 27928 840 848610 2822 2837 GGGAAAGAATCCTG CCT 25 27965 27980 841 848626 2948 2963 CATTAATCAGGA GCC 21 28091 28106 842 848641 3052 3067 GAGCAGAGTAAAG GTG 23 28195 28210 843 848656 3126 3141 CACACTGCCGAGTC AG 56 28269 28284 844 848672 3213 3228 AGGTTTCTTTCCTCT GT 60 28356 28371 845 848687 3243 3258 GAGCTTGGCAGGC ACG 67 28386 28401 846 848703 3266 3281 TTCTGGCTCAGTTC CT 20 28409 28424 847 848719 3334 3349 GTAAAAATGAGGA GCC 29 28477 28492 848 848735 3421 3436 AAAAAGTTCCATGC CT 9 28564 28579 849 848751 3506 3521 GGAGGGACAGTTG TTG 0 28649 28664 850 848766 3590 3605 TAGAAAAGTTGGCT GT 22 28733 28748 851

- 102042765

Table 12

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % depressed SEQ ID: 2, start site SEQ ID 2: stop website SE Q ID NO 8483 45 1012 1027 TTGCTGGCCTGTCT GT 3 N/A N/A 852 8483 90 1148 1163 GCCTATGAGGGTG CCG 0 N/A N/A 853 8484 54 1708 1723 TGCCAACCTGCCC CAT 8 N/A N/A 854 8482 10 56 71 CGGAAACCTTTCTA GGG 0 3531 3546 855 8482 26 244 259 AGCTGAAGTTCAG GAG 50 3719 3734 856 8482 42 480 495 AGGCTAGCACCAG CTC 37 3955 3970 857 8482 57 552 567 TGGCGCAGCGGTG GAA 0 4027 4042 858 8487 83 N/A N/A CCACTACCCGTCC TCC 0 4211 4226 859

- 103 042765

8487 99 N/A N/A CGGGAAGAAGCTT CCC 0 4883 4898 860 8488 15 N/A N/A CCCCACAACATCC CTC 5 5377 5392 861 8488 31 N/A N/A AAAGAACTGTGGA CAT 43 5935 5950 862 8488 47 N/A N/A AGGCAGACATACC TGC 0 6695 6710 863 8488 63 N/A N/A CTGCAAGCTCTCC AGG 17 7095 7110 864 8488 79 N/A N/A CAAGAGTGACGGT TAT 45 7295 7310 865 8488 95 N/A N/A TATTATTTCACATG GTT 41 7646 7661 866 8482 72 668 683 GTATCCCCGGCGG GCA 37 7941 7956 867 8482 84 684 699 GCAGGATCTTGGT GAG 0 7957 7972 868 8482 99 743 758 CTCCAGCAGGTCG CCA 20 8016 8031 869 8489 AND N/A N/A ATACATACTTGCT GTC 21 8137 8152 870 8489 27 N/A N/A TGGATAGCAACAG CTT 70 8458 8473 871 8489 43 N/A N/A TGCTGAGCTGATT AAC 27 8840 8855 872 8489 59 N/A N/A AATTATTATAACT GGT 0 9381 9396 873 8489 75 N/A N/A GGGCAGGATGGAC ATG 4 10209 1022 4 874

- 104 042765

8483 14 817 832 TTCCACGGGATTGC TCT 3 10578 1059 3 875 8489 91 N/A N/A TTAAAGGATCTGG TCC 31 11011 1102 6 876 8490 07 N/A N/A GTGTATGCAAAAGT CAC 55 11624 1163 9 877 8490 23 N/A N/A AGGGAACAGTGGC TGC 42 12407 1242 2 878 8490 39 N/A N/A TTATTGAATGGTA AGA 37 12905 1292 0 879 8490 44 N/A N/A GGATACACAGGCT CGC 77 13108 1312 3 337 8490 55 N/A N/A CTGCAAGCATGGC CAG 33 13710 1372 5 880 8490 71 N/A N/A ACAACTGGATACA TTG 57 14129 1414 4 881 8490 87 N/A N/A CACTTTGGTTTCTT CT 28 14560 1457 5 882 8491 03 N/A N/A CTTATGGCTTCGGT CA 42 15107 1512 2 883 8491 19 N/A N/A AGATACCAGGAGG GCT 48 15730 1574 5 884 8491 35 N/A N/A ATAGACAAGGAAA GGG 68 16087 1610 2 885 8483 29 971 986 CACATTCTCGAAG TCG 25 16363 1637 8 886 8483 59 1056 1071 GGCCGCTGACCAC CCC 19 16686 1670 1 887 4684 60 1075 1090 TTGGCCCACGCCGG CAT 10 16705 1672 0 48

- 105 042765

8483 74 1080 1095 CACCCTTGGCCAC GCC 37 16710 1672 5 888 8491 51 N/A N/A ATGGAGAGAGACC AGC 20 16811 1682 6 889 8491 67 N/A N/A CACCAGAGCCAGT GTT 0 17590 1760 5 890 8491 83 N/A N/A CTGCCCCGTGCCA GGT 0 17962 1797 7 891 8491 99 N/A N/A TGTCAAATGAGGT GTG 15 18586 1860 1 892 8492 15 N/A N/A ATGAGAAGGGCAC ACT 0 19342 1935 7 893 8492 31 N/A N/A AACTCTTAATATA TAC 0 19570 1958 5 894 8492 47 N/A N/A AGGAACAAAGCCCA AGG 31 19972 1998 7 895 8484 06 1293 1308 CGGCAGCGGTGAC CAG 0 20124 2013 9 896 8492 63 N/A N/A GCCTTGATAGGCC AGG 33 20938 2095 3 897 8492 79 N/A N/A AGAGAAAGGAGCC CAA 0 21290 2130 5 898 8484 22 1427 1442 GTCCACACAGCGG CCA 5 21399 2141 4 899 8484 38 1558 1573 GGCTCGGCAGACA GCA 58 22051 2206 6 900 8492 95 N/A N/A GGCTTAAAGAACA TAC 28 22397 2241 2 901 8484 70 1772 1787 GGCGACGGCTGTG GCC 0 22554 2256 9 902

- 106 042765

8493 AND N/A N/A GAGCATTAAGAC CCA 32 23168 2318 3 903 4684 80 1930 1945 AGGCAGCACCTGG CAA 19 23550 2356 5 904 8485 00 2001 2016 AGTGGACACGGGT CCC 56 23621 2363 6 905 8493 27 N/A N/A TCACACTTGTGAG GAC 0 23737 2375 2 906 8493 43 N/A N/A AAAGCAACGGGTG ATG 0 24170 2418 5 907 8493 59 N/A N/A GTAAGATGGAAAG AGA 55 24877 2489 2 908 8493 75 N/A N/A CTGGGATGCTCCG TCT 0 25324 2533 9 909 8485 16 2172 2187 TGCATTCCAGACC TGG 6 25503 2551 8 910 8493 91 N/A N/A TGCTAACAACCTT CAG 0 26103 2611 8 911 8494 07 N/A N/A TACCATGCCAGTG CCA 29 27098 2711 3 912 8485 32 2386 2401 CGGCAGCAGATGG CAA 27 27529 2754 4 913 8485 48 2527 2542 TGCCAGGCCATGG AGG 0 27670 2768 5 914 8485 64 2628 2643 GGAGGAGGCACCC AGA 20 27771 2778 6 915 4684 87 2720 2735 GAGCAGCTGGCAG CAC 42 27863 2787 8 916 8485 95 2771 2786 GGAACAAGAGCTC AAT 47 27914 2792 9 917

- 107 042765

8486 AND 2828 2843 ATCCATGGGAAGA ATS 15 27971 2798 6 918 8486 27 2951 2966 CTCCATTAATCAG GGA 26 28094 2810 9 919 8486 42 3055 3070 ATAGAGCAGAGTA AAG 0 28198 2821 3 920 8486 57 3127 3142 GCACACTGCCGAG TCA 46 28270 2828 5 921 8486 73 3214 3229 CAGGTTTCTTCCTC TG 34 28357 2837 2 922 8486 88 3245 3260 GTGAGCTTGGCAG GCA 70 28388 2840 3 923 8487 04 3267 3282 TTTCTGGCTCAGTT CC 54 28410 2842 5 924 8487 20 3336 3351 CCGTAAAAATGAG GAG 17 28479 2849 4 925 8487 36 3423 3438 GGAAAAAAGTTCCA TGC 57 28566 2858 1 926 4685 02 3508 3523 AAGGAGGGACAGT TGT 0 28651 2866 6 927 8487 67 3592 3607 TCTAGAAAAGTTG GCT 39 28735 2875 0 928

- 108 042765

Table 13

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % depressed SEQ ID: 2, start SEQ ID2: stopsite SEQ ID NO VY site 8482 58 561 576 ACGGATCCTTGGC GCA 38 N/A N/A 929 8483 00 753 768 TCAAGGCCAGCTC CAG 23 N/A N/A 930 8483 46 1014 1029 ACTTGCTGGCCTG TST 0 N/A N/A 931 8483 91 1154 1169 CTCCAGGCCTATG AGG 0 N/A N/A 932 8482 AND 58 73 TGCGGAAAACCTTC TAG 0 3533 3548 933 8482 27 258 273 GAGGACTGTGCAG GAG 42 3733 3748 934 8482 43 481 496 AAGGCTAGCACCA GCT 23 3956 3971 935 8487 84 N/A N/A GGATATCCTGGCA GTG 0 4350 4365 936 8488 00 N/A N/A GAAAAAGCTAGTG GTC thirty 4905 4920 937 8488 16 N/A N/A CTTGAAATGCCCT TTC 0 5408 5423 938 8488 32 N/A N/A TACATTTCAGACG GTG 61 6279 6294 939 8488 48 N/A N/A ATTATAGCAGCCA STA 13 6761 6776 940 8488 64 N/A N/A ATTTATTTCAGCTC ATG 1 7114 7129 941

- 109 042765

8488 80 N/A N/A TGGGAAAGTCAAG AGT 44 7304 7319 942 8488 96 N/A N/A GCCAACTATTATT ATT 26 7655 7670 943 4668 46 669 684 GGTATCCCCGGCG GGC 45 7942 7957 944 8482 85 685 700 TGCAGGATCTTGG TGA 0 7958 7973 945 8489 12 N/A N/A GATAAGTGCTCAA TAC 33 8149 8164 946 8489 28 N/A N/A CTGGATAGCAACA GCT 71 8459 8474 947 8489 44 N/A N/A CGCCACAGGCCTT GGT thirty 8881 8896 948 8489 60 N/A N/A CCAATTATTATAA CTG 21 9383 9398 949 8489 76 N/A N/A AGCCAGGCTGTGC CAA 0 10231 1024 6 950 8483 15 824 839 CTCCAGGTTCCAC GGG 0 10585 1060 0 951 8489 92 N/A N/A ATTAAAGGATCTG GTC 2 11012 1102 7 952 8490 08 N/A N/A CAGACATATGCAA GGT 68 11652 1166 7 953 8490 24 N/A N/A GCCCAAGGGAGA GGAG 0 12421 1243 6 954 8490 40 N/A N/A GTTATTATTGAAT GGT 80 12909 1292 4 955 8490 44 N/A N/A GGATACACAGGCT CGC 76 13108 1312 3 337

- 110 042765

8490 56 N/A N/A CAGGAGGCTGCA GCA 49 13717 1373 2 956 8490 72 N/A N/A CAGGAAGCAGCCC AAG 0 14175 1419 0 957 8490 88 N/A N/A CTCCACTGATCAG TCC 49 14582 1459 7 958 8491 04 N/A N/A CCAAATATGCTGC AGA 20 15185 1520 0 959 8491 20 N/A N/A AGAGACAGGAAG CTGC 63 15808 1582 3 960 8491 36 N/A N/A CACAGAAACTACA GAG 0 16199 1621 4 961 8483 thirty 974 989 GGGCACATTCTCG AAG 0 16366 1638 1 962 8483 60 1057 1072 CGGCCGCTGACCA CCC 0 16687 1670 2 963 4684 60 1075 1090 TTGGCCCACGCCGG CAT 0 16705 1672 0 48 8483 75 1081 1096 GCACCCTTGGCCA CGC 24 16711 1672 6 964 8491 52 N/A N/A CCAGATGGAGAGA GAC 26 16815 1683 0 965 8491 68 N/A N/A GGCTAGTGGGCTG CCT 0 17613 1762 8 966 8491 84 N/A N/A CCCTGCCCCGTGC CAG 0 17964 1797 9 967 8492 00 N/A N/A GCCTAACGCAGCT CTG 62 18644 1865 9 968 8492 16 N/A N/A GAGGAATGAGAA GGGC 61 19347 1936 2 969

- 111 042765

8492 32 N/A N/A CCGCACGGTCACA TGA 19 19592 1960 7 970 8492 N/A N/A GTGCCACAAGAAG 9 20268 2028 3 971 48 CCC 20286 2030 1 8484 07 1301 1316 GAAGTTGCCGGCA GCG 24 20132 2014 7 972 8492 64 N/A N/A TAAATTCGGCCGC CAG 9 20958 2097 3 973 8484 23 1433 1448 AAAGAGGTCCACA CAG 0 21405 2142 0 974 8492 80 N/A N/A CTGTTAGCATCAC GGT 25 21542 2155 7 975 8484 39 1574 1589 GGCCAGGGTGAGC TCC 0 22067 2208 2 976 8492 96 N/A N/A GAGAGAGGAGGG CTTA 0 22407 2242 2 977 8484 55 1716 1731 AAAACAGCTGCCA ACC 0 22498 2251 3 978 8484 71 1774 1789 CGGGCGACGGCTG TGG 0 22556 2257 1 979 8493 12 N/A N/A ACACGAGCATTAA GAC 45 23172 2318 7 980 8484 85 1932 1947 GCAGGCAGCACCCT GGC 7 23552 2356 7 981 8485 01 2002 2017 CAGTGGACACGGG TCC 37 23622 2363 7 982 8493 28 N/A N/A CTGCAAGTCAGGC TTG 20 23764 2377 9 983

- 112 042765

8493 44 N/A N/A TTTAAAGCAACGG GTG 47 24173 2418 8 984 8493 60 N/A N/A AGCTACACTGAGG CTC 23 24906 2492 1 985 8485 17 2177 2192 GACTTTGCATTCC AGA 41 25508 2552 3 986 8493 76 N/A N/A GGCAGAGGAAAG CCAG 0 25355 2537 0 987 8493 92 N/A N/A TGTCACATTCCAG GGC 43 26171 2618 6 988 8494 08 N/A N/A AGATAGACAGATG CCT 8 27113 2712 8 989 8485 33 2402 2417 CGCCAGGTGCCGG CTC 0 27545 2756 0 990 8485 49 2550 2565 GCGGAAGCATCCC CAT 0 27693 2770 8 991 8485 65 2669 2684 CCCCACAGTGAGG GAG 20 27812 2782 7 992 8485 80 2727 2742 ACATTGGGAGCAG CTG 34 27870 2788 5 993 8485 96 2772 2787 CGGAACAAGAGCT CAA 70 27915 2793 0 994 8486 12 2832 2847 CCCTATCCATGGG AAG 39 27975 2799 0 995 8486 28 2959 2974 AGCTAAGCCTCCA TTA 15 28102 2811 7 996 8486 43 3057 3072 GCATAGAGCAGAG TAA 0 28200 2821 5 997 8486 58 3128 3143 TGCACACTGCCGA GTC 49 28271 2828 6 998 8486 74 3215 3230 CCAGGTTTCTTTCCT CT 58 28358 2837 3 999 8486 89 3246 3261 TGTGAGCTTGGCA GGC 39 28389 2840 4 1000 8487 05 3268 3283 GTTTCTGGCTCAG TTC 3 28411 2842 6 1001 8487 21 3338 3353 ACCCGTAAAAATG AGG 0 28481 2849 6 1002 8487 37 3425 3440 ACGGAAAAAAGTTC CAT 18 28568 2858 1003 8487 52 3511 3526 CTCAAGGAGGGAC AGT 33 28654 2866 9 1004 8487 68 3599 3614 AAACAGGTCTAGA AAA 17 28742 2875 7 1005

- 113 042765

Table 14

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, start site SEQ ID2: stopsite SEQ ID NO 468456 1015 1030 CACTTGCTGGCCT GTC 22 N/A N/A 1006 848259 565 580 CTCCACGGATCCT TGG 0 N/A N/A 1007 848301 754 769 TTCAAGGCCAGC TCCA 42 N/A N/A 1008 848392 1159 1174 ATAAACTCCAGG CCTA 37 N/A N/A 1009 848212 64 79 CGTCGCTGCGGA AACC 41 3539 3554 1010 848228 293 308 GTCCACGCCGGC ggcg 32 3768 3783 1011 848244 482 497 CAAGGCTAGCAC CAGC 45 3957 3972 1012 848785 N/A N/A GAAATCTGGGCA GGAT 39 4362 4377 1013 848801 N/A N/A CACCACAGCTAG TGAG 28 5003 5018 1014 848817 N/A N/A CACTATTTTCCAGA ASA 20 5445 5460 1015 848833 N/A N/A AATAATCTCATGT CAG 77 6356 6371 1016 848849 N/A N/A CAAATTATAGCA GCCA 85 6764 6779 1017 848865 N/A N/A GATTTATTCAGCT CAT 65 7115 7130 1018 848881 N/A N/A CCACAGTACCTAT GAC 0 7330 7345 1019 848897 N/A N/A CAAATGCGGACC AAAA 38 7694 7709 1020 848273 670 685 AGGTATCCCCGG CGGG 26 7943 7958 1021 848286 686 701 ATGCAGGATCTT GGTG 0 7959 7974 1022 848913 N/A N/A CGATAAGTGCTC AATA 25 8150 8165 1023 848929 N/A N/A AAAGAAGGGACT TCAC 29 8476 8491 1024

- 114 042765

848945 N/A N/A ACTTAGACTACG GGTT 25 8931 8946 1025 848961 N/A N/A TCCTTAAATGAAT GTT 41 9399 9414 1026 848977 N/A N/A CTTGATCAGGCTG GGA 0 10332 1034 7 1027 848316 833 848 GGTAATCCCGCTCC AGG 59 10594 1060 9 1028 848993 N/A N/A TCCCATTAAAGG ATCT 23 11016 1103 1 1029 849009 N/A N/A ACACATGACCGA CCAG 0 11773 1178 8 1030 849025 N/A N/A CTCCAGCCAAGC CCTT 40 12570 1258 5 1031 849041 N/A N/A TCTGGATACACTG TTG 27 13008 1302 3 1032 849044 N/A N/A GGATACACAGGC TCGC 85 13108 1312 3 337 849057 N/A N/A GGTCAGGAGGCT GCAA 61 13720 1373 5 1033 849073 N/A N/A TGCTCAGGAAGC AGCC 43 14179 1419 4 1034 849089 N/A N/A CCCTAAGGCCTCC AGT 41 14610 1462 5 1035 849105 N/A N/A CTGAACAGCACC TCTG 37 15220 1523 5 1036 849121 N/A N/A TTAGAGACAGGA AGCT 28 15810 1582 5 1037 849137 N/A N/A CTTATAGTTAACA CAC 0 16212 1622 7 1038

- 115 042765

848331 980 995 CTCCTCGGGCAC ATTC 7 16372 1638 7 1039 848361 1058 1073 CCGGCCGCTGAC CACC thirty 16688 1670 3 1040 468460 1075 1090 TTGGCCCACGCCG GCAT 31 16705 1672 0 48 848376 1082 1097 GGCACCCTTGGC CACG 52 16712 1672 7 1041 849153 N/A N/A GCCCAAGCCACC TCCC 0 16840 1685 5 1042 849169 N/A N/A AGGAAAAGTCTCA GGGC 78 17681 1769 6 1043 849185 N/A N/A AGCCCCTGCCCC GTGC 18 17967 1798 2 1044 849201 N/A N/A GGCCTAACGCAG CTCT 34 18645 1866 0 1045 849217 N/A N/A AGGCACCAAGAG GATG 20 19370 1938 5 1046 849233 N/A N/A GCGGATTCAGA CTTG 80 19613 1962 8 1047 848408 1304 1319 CCGGAAGTTGCC GGCA 3 20135 2015 0 1048 849249 N/A N/A CGTGCCACAAGA AGCC 21 20269 2028 4 1049 20287 2030 2 849265 N/A N/A GCCTTTAAATTCG GCC 0 20963 2097 8 1050 848424 1435 1450 GCAAAGAGGTCC ACAC 0 21407 2142 2 1051

- 116 042765

849281 N/A N/A GGCACTGGAGGT CCCG 31 21583 2159 8 1052 848440 1582 1597 CTCAACTCGGCC AGGG 43 22075 2209 0 1053 849297 N/A N/A TAGGAGAGAGGA GGGC 21 22410 2242 5 1054 848456 1718 1733 GCAAAACAGCTG CCAA 24 22500 2251 5 1055 848472 1778 1793 GCAGCGGGCGAC GGCT 0 22560 2257 5 1056 849313 N/A N/A CTCTCACACGAG CATT eleven 23177 2319 2 1057 848486 1933 1948 AGCAGGCAGCAC CTGG 35 23553 2356 8 1058 848502 2003 2018 GCAGTGGACACG GGTC 43 23623 2363 8 1059 849329 N/A N/A GTATGGAACTGC AAGT 0 23772 2378 7 1060 849345 N/A N/A ATTTTAAAGCAA CGGG 75 24175 2419 0 1061 849361 N/A N/A AAACCTAAAATA GTGG 4 24928 2494 3 1062 849377 N/A N/A GGGCAGAGGAAA GCCA 12 25356 2537 1 1063 848518 2190 2205 TTCCATGCTCCTT GAC eleven 25521 2553 6 1064 849393 N/A N/A ATACACTCAGGTT TCT 45 26199 2621 4 1065 849409 N/A N/A ACCCAGGCGGTT CTGC 10 27185 2720 0 1066

- 117 042765

848534 2427 2442 GTCACTGGAGCT CCTG 59 27570 2758 5 1067 848550 2554 2569 AAAGGCGGAAGC ATCC 23 27697 2771 2 1068 848566 2677 2692 GTGAAATGCCCC ACAG 34 27820 2783 5 1069 848581 2729 2744 GCACATTGGGAG CAGC 58 27872 2788 7 1070 848597 2773 2788 ACGGAACAGAG CTCA 77 27916 2793 1 1071 848613 2869 2884 CAACGATGTTTGT CCC 67 28012 2802 7 1072 848629 2962 2977 GAAAGCTAAGCC TCCA 69 28105 2812 0 1073 848644 3059 3074 TGGCATAGAGCA GAGT 49 28202 2821 7 1074 848659 3129 3144 CTGCACACTGCC GAGT 48 28272 2828 7 1075 848675 3216 3231 TCCAGGTTTCTC CTC 63 28359 2837 4 1076 848690 3247 3262 GTGTGAGCTTGG CAGG 78 28390 2840 5 1077 848706 3269 3284 CGTTTCTGGCTCA GTT 55 28412 2842 7 1078 848722 3342 3357 TGTTACCCGTAAA AAT 0 28485 2850 0 1079 848738 3427 3442 TAACGGAAAAAAG TTCC 64 28570 2858 5 1080 848753 3513 3528 TGCTCAAGGAGG GACA 27 28656 2867 1 1081 848769 3601 3616 CAAAACAGGTCT AGAA 43 28744 2875 9 1082

- 118 042765

Table 15

Suppression of PCSK9 mRNA. with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, old site SEQ ID2: stopsite SEQ ID NO 84830 2 755 770 CTTCAAGGCCA GCTCC 0 N/A N/A 1083 84834 7 1016 1031 ACACTTGCTGG CCTGT 0 N/A N/A 1084 84821 3 85 100 CTGCAACCATG AGCGC 4 3560 3575 1085 84822 9 309 324 CTAGAGGCCGT GCGCG 18 3784 3799 1086 84824 5 483 498 GCAAGGCTAGC ACCAG 45 3958 3973 1087 84878 6 N/A N/A GAAACTGGGAA ATCTG 0 4370 4385 1088 84880 2 N/A N/A GTCAAGCACCA CAGCT 54 5009 5024 1089 84881 8 N/A N/A CCTCAAGGGCT TGGTT 0 5509 5524 1090

- 119 042765

84883 4 N/A N/A AATCTATGCAG CAAAA 0 6407 6422 1091 84885 0 N/A N/A AACAAATTATA GCAGC 75 6766 6781 1092 84888 2 N/A N/A GACCACAGTAC CTATG thirty 7332 7347 1093 84889 8 N/A N/A ACCAAATGCGG ACCAA 66 7696 7711 1094 84826 0 571 586 GGCAACCTCCA CGGAT 53 7844 7859 1095 46684 7 671 686 GAGGTATCCCC GGCGG 73 7944 7959 1096 84828 7 687 702 CATGCAGGATC TTGGT 5 7960 7975 1097 84891 4 N/A N/A TGCTTGGTACCCC GATA 39 8161 8176 1098 84893 0 N/A N/A CCTAAAGAAGG GACTT 18 8479 8494 1099 84894 6 N/A N/A AGGCATTGACT TGTCA 46 8956 8971 1100 84896 2 N/A N/A TACCTAGCATCT GCTG 0 9437 9452 1101 84897 8 N/A N/A ACAGACTAGGA GCCTG 0 1036 1 10376 1102 84831 7 852 867 CCGCCCGGTAC CGTGG 0 1061 3 10628 1103 84899 4 N/A N/A AGGCATCCCAG ACAGG 52 1104 2 11057 1104 84901 0 N/A N/A GCACACATGAC CGACC 0 1177 5 11790 1105

- 120 042765

84902 6 N/A N/A CCCCATGCCAG CCCAA 33 1260 4 12619 1106 84904 2 N/A N/A ATTAATCTTCTG GATA 13 1301 6 13031 1107 84904 4 N/A N/A GGATACACAGG CTCGC 82 1310 8 13123 337 84905 8 N/A N/A GGACAGGGTCA GGAGG 22 1372 6 13741 1108 84907 4 N/A N/A GGATACAGGTG CTCAG 55 1418 8 14203 1109 84909 0 N/A N/A ACACACACTGT CTACC 38 1462 9 14644 1110 84910 6 N/A N/A CCTCAGGTGGA ATCAG 56 1530 6 15321 1111 84912 2 N/A N/A GAATAACAGTG ATGTC 17 1585 2 15867 1112 84913 8 N/A N/A ACCTTATAGTTA ACAC 4 1621 4 16229 1113 84833 2 982 997 TCCTCCTCGGGC ACAT 21 1637 4 16389 1114 84836 2 1059 1074 CCCGGCCCGCTG ACCAC 0 1668 9 16704 1115 46846 0 1075 1090 TTGGCCACGCC GGCAT 0 1670 5 16720 48 84837 7 1083 1098 TGGCACCCTTG GCCAC 0 1671 3 16728 1116 84915 4 N/A N/A GGTCAAGGCTG AACTC 27 1701 4 17029 1117 84917 0 N/A N/A TAGGAAAGTCT CAGGG 36 1768 2 17697 1118

- 121 042765

84918 6 N/A N/A AGGCAATAGTG ACTGT 52 1798 5 18000 1119 84920 2 N/A N/A CTGGAGACTGG AGGCC 0 1865 7 18672 1120 84921 8 N/A N/A GAAAGAGAGGC ACCAA 0 1937 7 19392 1121 84923 4 N/A N/A TGCGGATTCA GACTT 24 1961 4 19629 1122 84839 3 1161 1176 GAATAAACTCC AGGCC 0 1999 2 20007 1123 84840 9 1322 1337 GTAGAGGCAGG CATCG 42 2015 3 20168 1124 84925 0 N/A N/A CAGAAGGGTTC GGCCT 32 2031 0 20325 1125 84926 6 N/A N/A GGCTTTGTTTGGC TTGA 68 2098 0 20995 1126 84842 5 1437 1452 GGGCAAAGAGG TCCAC 0 2140 9 21424 1127 84928 2 N/A N/A CATCAGGCCTA CTTCA 0 2162 4 21639 1128 84844 1 1603 1618 GAGAAGTGGAT CAGTC 0 2209 6 22111 1129 84929 8 N/A N/A TGCAAAAAGGG CCTGG 0 2248 2 22497 ISO 84845 7 1720 1735 CTGCAAAACAG CTGCC 13 2250 2 22517 1131 84847 3 1780 1795 GCGCAGCGGGC GACGG 0 2256 2 22577 1132 84931 4 N/A N/A ATCCAGCCAGC TCCAC 7 2320 7 23222 1133

- 122 042765

84848 7 1934 1949 TAGCAGGCAGC ACCTG 0 2355 4 23569 1134 84850 3 2004 2019 GGCAGTGGACA CGGGT 26 2362 4 23639 1135 84933 0 N/A N/A GGCAGAACCAG AGTAT 0 2378 4 23799 1136 84934 6 N/A N/A CAATAGTTGCCT ATAC 0 2420 1 24216 1137 84936 2 N/A N/A AGACACACCCA TTGGC 8 2494 8 24963 1138 84851 9 2198 2213 GGCCGGGATTC CATGC 0 2552 9 25544 1139 84937 8 N/A N/A ACGCAGCACCC CACCC 0 2558 1 25596 1140 84939 4 N/A N/A AAGGAGAGTTA TACAC 53 2620 9 26224 1141 84941 0 N/A N/A GGACACAACCG TGTAT 0 2722 0 27235 1142 84853 5 2447 2462 ACCCATCCTGG GATGG 0 2759 0 27605 1143 84855 1 2556 2571 GGAAAAGGCGGA AGCAT 38 2769 9 27714 1144 84856 7 2684 2699 TTGAATGGTGA AATGC 65 2782 7 27842 1145 84858 2 2731 2746 CGGCACATTGG GAGCA 33 2787 4 27889 1146 84859 8 2774 2789 CACGGAACAAG AGCTC 68 2791 7 27932 1147 84861 4 2872 2887 CCCCAACGATG TTTGT 32 2801 5 28030 1148

- 123 042765

84863 0 2964 2979 CAGAAAGCTAA GCCTC 72 2810 7 28122 1149 46685 3 3067 3082 GCACAGCCTGG CATAG 51 2821 0 28225 1150 84866 0 3130 3145 ACTGCACACTG CCGAG 1 2827 3 28288 1151 84867 6 3218 3233 GTTCAGGTTTC TTSS 43 2836 1 28376 1152 84869 1 3248 3263 TGTGTGAGCTTG GCAG 22 2839 1 28406 1153 84870 7 3284 3299 AGCCAGCCCAA TCTGC 0 2842 7 28442 1154 84872 3 3348 3363 CCTCACTGTTAC CCGT 36 2849 1 28506 1155 84873 9 3429 3444 GATAACGGAAA AAGTT 0 2857 2 28587 1156 84875 4 3543 3558 ATAAATGTCTG CTTGC 52 2868 6 28701 1157 84877 0 3603 3618 AGCAAAACAGG TCTAG 60 2874 6 28761 1158

Table 16

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Sequential th % depressed SEQ ID: 2, start site SEQ ID2: stopsite SEQ ID NO 8483 03 756 771 ACTTCAAGGCCA GCTC 43 N/A N/A 1159 8482 14 105 120 AACTGAACGGCG GCGC 0 3580 3595 1160 8482 thirty 311 326 ACCTAGAGGCCG TGCG thirty 3786 3801 1161 8482 46 484 499 CGCAAGGCTAGC ACCA 45 3959 3974 1162 8487 87 N/A N/A GGCGAGGCAGA AACTG 29 4379 4394 1163 8488 03 N/A N/A TCAAGGATAAGT GACT 42 5026 5041 1164 8488 19 N/A N/A AGAAACATCCTC AAGG 38 5517 5532 1165 8488 35 N/A N/A TAATCTATGCAG SAAA 0 6408 6423 1166 8488 51 N/A N/A SSAAATTTSSAS ATGA 41 6797 6812 1167

- 124 042765

8488 67 N/A N/A AAGGAATACCTG AAGG 22 7153 7168 1168 8488 83 N/A N/A CCTCAGATACCCT CTGC 17 7359 7374 1169 8488 99 N/A N/A GTATAGTAGATG ATAA 0 7719 7734 1170 8482 61 576 591 TGCCAGGCAACC TCCA 14 7849 7864 1171 8482 74 672 687 TGAGGTATCCCCC ggcg 14 7945 7960 1172 8482 88 688 703 ACATGCAGGATC TTGG 16 7961 7976 1173 8489 15 N/A N/A AGCCAGTAGTTA CTGT 9 8176 8191 1174 8489 31 N/A N/A GAAGATGTGACA TCCA 28 8517 8532 1175 8489 47 N/A N/A GAGCAGACTGAT GGAA 4 8971 8986 1176 8489 63 N/A N/A GTGGTAACAGCC TCCT 26 9526 9541 1177 8489 79 N/A N/A GGACAGACTAG GAGCC 4 10363 1037 8 1178 8483 18 858 873 ATTCATCCGCCC GGTA 0 10619 1063 4 1179 8489 95 N/A N/A AGGTAGGCAGA GGCAT 20 11052 1106 7 1180 8490 AND N/A N/A GGCCGGGTCAGC ACAC 0 11785 1180 0 1181 8490 27 N/A N/A GGCCCATGCTTG TGGC 0 12663 1267 8 1182 8490 43 N/A N/A AGGAATGGATTA ATCT 48 13024 1303 9 1183 8490 44 N/A N/A GGATACACAGGC TCGC 81 13108 1312 3 337 8490 59 N/A N/A TCTAAGTGCAGG CGGT 23 13776 1379 1 1184 8490 75 N/A N/A CAGCAAGGTGG GCAGA 10 14274 1428 9 1185 8490 91 N/A N/A GCACAGTCCGAA CTGT 0 14660 1467 5 1186 8491 07 N/A N/A GCGGAAATGGCC TGGC 0 15353 1536 8 1187 8491 23 N/A N/A GGAGAATAACA GTGAT 62 15855 1587 0 1188

- 125 042765

8491 39 N/A N/A AACCTTATAGTT AACA 0 16215 1623 0 1189 8483 33 983 998 GTCCTCCTCGGG CACA 18 16375 1639 0 1190 4684 57 1017 1032 CACACTTGCTGG CCTG 0 16647 1666 2 1191 8483 63 1060 1075 TCCCGGCCCGCTG ACCA thirty 16690 1670 5 1192 4684 60 1075 1090 TTGGCCCACGCCG GCAT 0 16705 1672 0 48 8483 78 1084 1099 CTGGCACCCTTG GCCA 0 16714 1672 9 1193 8491 55 N/A N/A AGACATCACCGG ATTT 42 17074 1708 9 1194 8491 71 N/A N/A CTGTAGGAAAGT CTCA 49 17685 1770 0 1195 8491 87 N/A N/A GGACACACAATC ACCT 14 18033 1804 8 1196 8492 03 N/A N/A CAGTAACAGTTC TAAC 0 18706 1872 1 1197 8492 19 N/A N/A CTTATTATCCCTT TCC 29 19395 1941 0 1198 8492 35 N/A N/A TCAAATTCTGCA GGAA 0 19653 1966 8 1199 8483 94 1163 1178 CCGAATAAACTC CAGG 42 19994 2000 9 1200 8484 10 1324 1339 GAGTAGAGGCA GGCAT 0 20155 2017 0 1201 8492 51 N/A N/A CCAAAGCCAGA AGGGT 32 20317 2033 2 1202

- 126 042765

8492 67 N/A N/A TTTCTAAGTGCCA CGGG 66 21029 2104 4 1203 8484 26 1455 1470 CAATGATGTCCT CCCC 0 21427 2144 2 1204 8492 83 N/A N/A TCCCGATCAAAT GTCC 0 21772 2178 7 1205 8484 42 1606 1621 GCAGAGAAGTG GATCA 0 22099 2211 4 1206 8492 99 N/A N/A TGCCAACCTGCA AAAA 0 22490 2250 5 1207 8484 58 1728 1743 ATACAGTCCTGC AAAA 0 22510 2252 5 1208 8484 74 1803 1818 AGCTCAGCAGCT CCTC eleven 22585 2260 0 1209 8493 15 N/A N/A ATCCAGGAGGCC AAAG 0 23228 2324 3 1210 8484 88 1937 1952 GGGTAGCAGGC AGCAC 0 23557 2357 2 1211 8485 04 2005 2020 TGGCAGTGGACA CGGG 17 23625 2364 0 1212 8493 31 N/A N/A GGAGAAGTAAG GTCAC 52 23827 2384 2 1213 8493 47 N/A N/A GTCAATAGTTGC STAT 33 24203 2421 8 1214 8493 63 N/A N/A GTTCAGGGTAA GCCG 25 25011 2502 6 1215 8493 79 N/A N/A TGGCACAAACTG ACAC 0 25619 2563 4 1216 8493 95 N/A N/A TTAAGAGTGGAC TCCT 40 26260 2627 5 1217

- 127 042765

8494 AND N/A N/A CCAAAGTGCAGA CGGC 0 27260 2727 5 1218 8485 20 2222 2237 GGCCACGGTCAC CTGC 0 27365 2738 0 1219 8485 36 2455 2470 CCCCAGACACCC ATCC 26 27598 2761 3 1220 8485 52 2558 2573 CCGGAAAAGGCG GAAGC 0 27701 2771 6 1221 8485 68 2700 2715 GCACAGCTCGAC CTGT 3 27843 2785 8 1222 8485 83 2737 2752 GGACATCGGCAC ATTG 68 27880 2789 5 1223 8485 99 2775 2790 GCACGGAACAA GAGCT 19 27918 2793 3 1224 8486 15 2885 2900 TTTCACACTCAC CCCC 7 28028 2804 3 1225 8486 31 2967 2982 ATCCAGAAAGCT AAGC 1 28110 2812 5 1226 8486 45 3072 3087 TGCTAGCACAGC CTGG 19 28215 2823 0 1227 8486 61 3131 3146 CACTGCACACTG CCGA eleven 28274 2828 9 1228 8486 77 3219 3234 GGTTCCAGGTTTT CTTC 61 28362 2837 7 1229 8486 92 3249 3264 CTGTGTGGCTT GGCA 69 28392 2840 7 1230 8487 08 3288 3303 TCAGAGCCAGCC CAAT 8 28431 2844 6 1231 8487 24 3378 3393 GAGCTTCCTGGT CTGT 47 28521 2853 6 1232 8487 40 3431 3446 GTGATAACGGAA AAAG 65 28574 2858 9 1233 8487 55 3545 3560 AGATAAATGTCT GCTT 14 28688 2870 3 1234 8487 71 3616 3631 TTCAAGTTACAA AAGC 57 28759 2877 4 1235

- 128 042765

Table 17

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Sequential th % suppression SEQ ID: 2, start site SEQ ID2: stopsite SEQ ID NO 84830 6 759 774 GCAACTTCAAGG CCAG 84 N/A N/A 1236 84821 7 149 164 AGCCAGTCTCAC TGCC thirty 3624 3639 1237 84823 3 362 377 GCTGACGGTGCC CATG 51 3837 3852 1238 84824 9 487 502 GAACGCAAGGCT AGCA 5 3962 3977 1239 84879 0 N/A N/A CCAAATCGGAAC CCAC 88 4478 4493 1240 84880 6 N/A N/A ACAGAACTTTCC CTTC 57 5114 5129 1241 84882 2 N/A N/A TAAATTCGATTC CCAC 32 5600 5615 1242

- 129 042765

84883 8 N/A N/A AGGCAGTAATGG GCAA 92 6461 6476 1243 84885 4 N/A N/A TCCAACACTGAG GACC 93 6811 6826 1244 84887 0 N/A N/A AAAGGCAACACT TCTT 61 7222 7237 1245 84888 6 N/A N/A CAATATTTACTG GTTG 57 7409 7424 1246 84890 2 N/A N/A CTTACAAATTAC AACA 59 7751 7766 1247 84826 4 622 637 TCTGACTGCGAG AGGT 71 7895 7910 1248 46684 9 675 690 TGGTGAGGTATC CCCG 48 7948 7963 1249 46844 4 691 706 AAGACATGCAGG ATCT thirty 7964 7979 1250 84891 8 N/A N/A GACCAATGGGTT TGAT 13 8311 8326 1251 84893 4 N/A N/A GAGAATACCCAG TCCC 50 8550 8565 1252 84895 0 N/A N/A GCTCAAGGGAAA GGCC 3 9044 9059 1253 84896 6 N/A N/A GGACAAGAGTGC ATCA 6 10011 1002 6 1254 84898 2 N/A N/A GGCCTTACCTGA TCAA 12 10437 1045 2 1255 84899 8 N/A N/A CTAGAACCCTTC ATTC 0 11304 1131 9 1256 84901 4 N/A N/A TCTCACCTGGTT GGAA 31 11856 1187 1 1257

- 130 042765

84903 0 N/A N/A GTTAAGAGTGCA GGGT 90 12708 1272 3 1258 84904 4 N/A N/A GGATACACAGGC TCGC 92 13108 1312 3 337 84904 6 N/A N/A CCGTAGGTCTTG GCTA 65 13166 1318 1 1259 84906 2 N/A N/A AGCTGTACCTGG GTTC 79 13888 1390 3 1260 84907 8 N/A N/A CCTAGAGGAACC ACTA 51 14340 1435 5 1261 84909 4 N/A N/A ACATGAATTTCA GGCA 73 14731 1474 6 1262 84911 0 N/A N/A TCTCAGCCAGGC CAAA 0 15411 1542 6 1263 84912 6 N/A N/A CCCTATTATAGC CTTT 67 15920 1593 5 1264 84832 1 890 905 CTCCACCAGGCT GCCT 0 16282 1629 7 1265 84833 6 986 1001 CCCGTCCTCCTC GGGC 0 16378 1639 3 1266 84914 2 N/A N/A CCATCAGACGGC CGTG 48 16417 1643 2 1267 84835 0 1020 1035 TGTCACACTTGC TGGC 45 16650 1666 5 1268 84836 5 1064 1079 GGCATCCCGGCC GCTG 62 16694 1670 9 1269 46846 0 1075 1090 TTGGCCCACGCCG GCAT 0 16705 1672 0 48 84838 1 1098 1113 GCAGGCTGCGCA TGCT 42 16728 1674 3 1270

- 131 042765

84915 8 N/A N/A TGCAAGCAGAAG ATAG 39 17153 1716 8 1271 84917 4 N/A N/A CAGGAACTGACC TGAC 21 17808 1782 3 1272 84919 0 N/A N/A TACAGCTGCTAG TTAT 59 18086 1810 1 1273 18399 1841 4 84920 6 N/A N/A GTGCAGGCCATG GTCT 68 19054 1906 9 1274 84922 2 N/A N/A ATACGAACTC AGGG 0 19426 1944 1 1275 84923 8 N/A N/A GGCCTTAAGAGA ACAG 46 19734 1974 9 1276 84839 7 1178 1193 GACCAGCTGGCT TTTC 2 20009 2002 4 1277 84925 4 N/A N/A CCCAAAGAGTAT TGGG 0 20552 2056 7 1278 84927 0 N/A N/A AACTATTCGGTG TATC 67 21051 2106 6 1279 84841 3 1358 1373 CCCAACTGTGAT GACC 0 21330 2134 5 1280 84842 9 1485 1500 CAAAGCAGGTGC TGCA 36 21457 2147 2 1281 84928 6 N/A N/A AGGTAAGACAGC CTCC 53 21856 2187 1 1282 84844 5 1619 1634 GATGACATCTTT GGCA 59 22112 2212 7 1283 84846 1 1732 1747 GACCATACAGTC CTGC 17 22514 2252 9 1284

- 132 042765

84847 7 1816 1831 GAGAAACTGGA GCAGC 0 22598 2261 3 1285 84930 2 N/A N/A TAGCAGATCGCT GACC 67 22948 2296 3 1286 84931 8 N/A N/A ACTCAAGCACCC TCAT 0 23394 2340 9 1287 84849 1 1985 2000 CATGCTGGCCTC AGCT eleven 23605 2362 0 1288 84850 7 2008 2023 TGGTGGCAGTGG ACAC 71 23628 2364 3 1289 84933 4 N/A N/A AATGAGGCAGGT AATA 19 23879 2389 4 1290 84935 0 N/A N/A CCTCAGGGCACC ATCC 72 24293 2430 8 1291 84936 6 N/A N/A GGGAAAAACCC ATCTT 31 25120 2513 5 1292 84938 2 N/A N/A AGCATCTACCTG GCAA 39 25666 2568 1 1293 84939 8 N/A N/A GTGTACCCAGGG CAAG 59 26373 2638 8 1294 84941 4 N/A N/A AAGCACATGTCT AGCG 8 27331 2734 6 1295 84852 3 2246 2261 AGTCAGGGTCCA GCCC 63 27389 2740 4 1296 84853 9 2487 2502 CATTTTAAAGCT CAGC 87 27630 2764 5 1297 84855 5 2580 2595 CTCAAGGGCCAG GCCA 70 27723 2773 8 1298 84857 1 2708 2723 GCACCCGAGCAC AGCT 75 27851 2786 6 1299

- 133 042765

84858 6 2743 2758 GCCCACGGACAT CGGC 58 27886 2790 1 1300 84860 2 2778 2793 CTGGCACGGAAC AAGA 8 27921 2793 6 1301 84861 8 2904 2919 GATGAGGGCCAT CAGC 87 28047 2806 2 1302 84863 3 2978 2993 GGCTAGATGCCA TCCA 64 28121 2813 6 1303 84864 8 3093 3108 CCGCAGGCCACC TTTG 12 28236 2825 1 1304 84866 4 3146 3161 AGACAGTGCATG CACC 10 28289 2830 4 1305 84868 0 3222 3237 TCTGGTTCCAGG TTTC 89 28365 2838 0 1306 84869 5 3255 3270 TTCCTGCTGTGT GAGC 63 28398 2841 3 1307 84871 1 3304 3319 AAGAAGAGGCTT GGCT 38 28447 2846 2 1308 84872 7 3382 3397 CACCGAGCTTCC TGGT 66 28525 2854 0 1309 84874 3 3446 3461 GTGAATCAGGCC TGGG 31 28589 2860 4 1310 84875 8 3558 3573 GGACAGACCCAA AAGA 71 28701 2871 6 1311 84877 4 3641 3656 TGCTACAAAACC CAGA 95 28784 2879 9 1312

- 134 042765

Table 18

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, start site SEQ ID2: stopsite SEQ ID NO 8482 18 153 168 CCCGAGCCAG TCTCAC 1 3628 3643 13 13 8482 34 368 383 CCTGGAGCTG ACGGTG 18 3843 3858 13 14 8482 50 488 503 GGAACGCAAG GCTAGC 77 3963 3978 13 15 8487 91 N/A N/A TTCCAAACCA AATCGG 46 4485 4500 13 16 8488 07 N/A N/A GGACAAAACT GCAAGT 42 5170 5185 13 17 8488 23 N/A N/A TTCTAAATTCG ATTCC 61 5603 5618 13 18 8488 39 N/A N/A TTAGAAAGGC AGTAAT thirty 6467 6482 13 19 8488 55 N/A N/A CAAGGACGGC ACCAAG 37 6881 6896 13 20 8488 71 N/A N/A GAAGGGATGA CTAAGT 0 7242 7257 13 21 8488 87 N/A N/A AGGAAACCGT GGACCT 60 7437 7452 13 22 8489 03 N/A N/A CCTACTTACA AATTAC 4 7755 7770 13 23 8482 65 633 648 GGGCAGTGCG CTCTGA 78 7906 7921 13 24 8482 76 676 691 TTGGTGAGGT ATCCCC 66 7949 7964 13 25

- 135 042765

8482 91 692 707 GAAGACATGC AGGATC 61 7965 7980 13 26 8489 19 N/A N/A GGCAGACCAA TGGGTT 10 8315 8330 13 27 8489 35 N/A N/A TAAGATTTGA AGCACT 60 8580 8595 13 28 8489 51 N/A N/A CTGAACTGTA AGCTCA 46 9055 9070 13 29 8489 67 N/A N/A GGCACTAGCA GGAGTT 62 10031 10046 13 thirty 8489 83 N/A N/A ATGGATTCAG CTCAGA 66 10680 10695 13 31 8489 99 N/A N/A TCCAACTAGA ACCCTT 87 11309 11324 13 32 8490 15 N/A N/A CACTGAAGAG GTCTCA 17 11867 11882 13 33 8490 31 N/A N/A AGGAACCTGA AGATCA 72 12756 12771 13 34 8490 44 N/A N/A GGATACACAG GCTCGC 85 13108 13123 33 7 8490 47 N/A N/A TAATAATGCC CCCGTA 49 13177 13192 13 35 8490 63 N/A N/A AGTTAGCCCA GGTGAG 6 13951 13966 13 36 8490 79 N/A N/A CGCCACCCTA GAGGAA 0 14346 14361 13 37 8490 95 N/A N/A AGCAATTCAG TGGCTG 49 14759 14774 13 38 8491 AND N/A N/A CTCCAGCGCA GGTCTC 35 15433 15448 13 39

- 136 042765

8491 27 N/A N/A GTGTAAAATA AAGCCC 77 15934 15949 13 40 8483 22 900 915 GGAGATACAC CTCCAC 15 16292 16307 13 41 8483 37 987 1002 TCCCGTCCTCC TCGGG 8 16379 16394 13 42 8491 43 N/A N/A GGACAGTGAC AGCTGG 39 16524 16539 13 43 8483 51 1021 1036 CTGTCACACTT GCTGG 8 16651 16666 13 44 8483 66 1070 1085 CACGCCGGCA TCCCGG 3 16700 16715 13 45 4684 60 1075 1090 TTGGCCACGC CGGCAT 0 16705 16720 48 8483 82 1100 1115 GCGCAGGCTG CGCATG 0 16730 16745 13 46 8491 59 N/A N/A ATGCAAGTGA GTGAGT 74 17187 17202 13 47 8491 75 N/A N/A GGAAAGAACA GCCTCC 44 17842 17857 13 48 8491 91 N/A N/A GATACAGACA CCCACC 37 18250 18265 13 49 8492 07 N/A N/A TACCATAACT CCCCAC 37 19123 19138 13 50 8492 23 N/A N/A ATCAAACTAG CCAACC 0 19482 19497 13 51 8492 39 N/A N/A GAAGACGGAG TAAGGC 81 19795 19810 13 52 8483 98 1183 1198 GGCTGGACCA GCTGGC 0 20014 20029 13 53

- 137 042765

8492 55 N/A N/A GGCCCAAAGA GTATTG 0 20554 20569 13 54 8492 71 N/A N/A GAAACTATTC GGTGTA 38 21053 21068 13 55 8484 14 1370 1385 GGCATTGGTG GCCCCA 19 21342 21357 13 56 8484 thirty 1492 1507 TGTGACACAA AGCAGG 84 21464 21479 13 57 8492 87 N/A N/A CGAAGGTAAG CCGCCT 56 21889 21904 13 58 8484 46 1626 1641 CCTCATTGATG ACATC 0 22119 22134 13 59 8484 62 1733 1748 TGACCATACA GTCCTG 19 22515 22530 13 60 8484 78 1842 1857 GCTCGCCCCG CCGCTT 35 22624 22639 13 61 8493 03 N/A N/A GGCTGAGTCC AGAGTA 89 22962 22977 13 62 8493 19 N/A N/A GAAATGGGAA TCTGCT 0 23454 23469 13 63 8484 92 1986 2001 CCATGCTGGC CTCAGC 32 23606 23621 13 64 8493 35 N/A N/A CAGCTATAGA GCGGCA 0 24045 24060 13 65 8493 51 N/A N/A GAACAGCTCA GCCTCA 0 24304 24319 13 66 8493 67 N/A N/A AGCCTTAGTTTT CTCAG 25 25135 25150 13 67 8485 08 2047 2062 TCCCAGTGGG AGCTGC 0 25378 25393 13 68

- 138 042765

8493 83 N/A N/A CCACAGCATC TACCTG 80 25670 25685 13 69 8493 99 N/A N/A CCAGGAAGAG CACCTG 17 26406 26421 13 70 8494 15 N/A N/A AGAAAGCACA TGTCTA 0 27334 27349 13 71 8485 24 2250 2265 AGCCAGTCAG GGTCCA 55 27393 27408 13 72 8485 40 2490 2505 AACCATTTTA AAGCTC 45 27633 27648 13 73 8485 56 2582 2597 CACTCAAGGG CCAGGC 37 27725 27740 13 74 8485 72 2709 2724 AGCACCCGAG CACAGC 89 27852 27867 13 75 8485 87 2752 2767 AGTCATTCTGC CCACG 78 27895 27910 13 76 8486 03 2779 2794 CCTGGCACGG AACAAG 51 27922 27937 13 77 8486 19 2906 2921 GAGATGAGGG CCATCA 80 28049 28064 13 78 8486 34 2999 3014 GCGCACCTGT CTCCAG 0 28142 28157 13 79 8486 49 3110 3125 TCCTAGGTGA TGGCTC 80 28253 28268 13 80 8486 65 3148 3163 TGAGACAGTG CATGCA 37 28291 28306 13 81 8486 81 3223 3238 CTCTGGTTCCA GGTTT 43 28366 28381 13 82 8486 96 3256 3271 GTTCCTGCTGT GTGAG 78 28399 28414 13 83 8487 12 3306 3321 GTAAGAGAG GCTTGG 75 28449 28464 13 84 8487 28 3383 3398 TCACCGAGCT TCCTGG 50 28526 28541 13 85 8487 44 3451 3466 GGCCAGTGAA TCAGGC 17 28594 28609 13 86 8487 59 3563 3578 AGAGAGGACA GACCCA 83 28706 28721 13 87 8487 75 3644 3659 AAATGCTACA AAACCC 67 28787 28802 13 88 8483 07 760 775 GGCAACTTCA AGGCCA 0 N/A N/A 13 89

- 139 042765

Table 19

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, starting site SEQ ID: 1, stop site Subsequence % suppression SEQ ID: 2, starting site SEQ ID2: stopsite SEQ ID NO 8482 19 181 196 CGCTGCTGCAA CGACG 66 3656 3671 1390 8482 35 378 393 ACCAGGACCGC CTGGA 0 3853 3868 1391 8482 51 489 504 CGGAACGCAAG GCTAG 62 3964 3979 1392 8487 92 N/A N/A GTCATAAAGAA ATTGC 47 4593 4608 1393 8488 08 N/A N/A TGGCAGAATTTT CCCC 17 5249 5264 1394 8488 24 N/A N/A TTTCATTCTAAA TTCG 58 5608 5623 1395 8488 40 N/A N/A CTTTACCCAAA GCCTT 0 6523 6538 1396 8488 56 N/A N/A CATAGCGGGAG AACTT 51 6908 6923 1397 8488 72 N/A N/A GCAGAAGGGAT GACTA 53 7245 7260 1398 8488 88 N/A N/A AGCCAGGAAAC CGTGG 63 7441 7456 1399 8489 04 N/A N/A CCCTACTTACAAA ATTA 40 7756 7771 1400 8482 66 640 655 AGGCGGCGGGC AGTGC 31 7913 7928 1401 8482 77 677 692 CTTGGTGAGGT ATCCC 65 7950 7965 1402 8482 92 693 708 GGAAGACATGC AGGAT 64 7966 7981 1403 8489 20 N/A N/A AGGCAGACAA TGGGT 63 8316 8331 1404 8489 36 N/A N/A CTAGAAGGTGG TGCAG 0 8619 8634 1405 8489 52 N/A N/A CCATAGTCAAC TGTAC 7 9097 9112 1406 8489 68 N/A N/A AGCCAATGGGA GGCAC 15 10042 1005 7 1407 8483 08 761 776 GGGCAACTTCA AGGCC 7 10522 1053 7 1408

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8489 84 N/A N/A GGGCAGAGCAA ATGGA 0 10691 1070 6 1409 8490 00 N/A N/A GTTCACCCCAAA GCTCT 29 11420 1143 5 1410 8490 16 N/A N/A CTCCACTGAAG AGGTC 66 11870 1188 5 1411 8490 32 N/A N/A GATTTAACCCTC CAAA 0 12814 1282 9 1412 8490 44 N/A N/A GGATACACAGG CTCGC 86 13108 1312 3 337 8490 48 N/A N/A GTTAATAATGC CCCCG 80 13179 1319 4 1413 8490 64 N/A N/A GCTGAGTTAGC CCAGG 51 13955 1397 0 1414 8490 80 N/A N/A AAACAGTGCTC GCCAC 29 14356 1437 1 1415 8490 96 N/A N/A TACAGAGCAAT TCAGT 33 14764 1477 9 1416 8491 12 N/A N/A TGCCAGGCAGG TCCAG 0 15506 1552 1 1417 8491 28 N/A N/A CAATATCTAAC AATAA 0 15977 1599 2 1418 8483 23 905 920 GTCTAGGAGAT ACACC 72 16297 1631 2 1419 8483 38 988 1003 GTCCCGTCCTCC TCGG 33 16380 1639 5 1420 8491 44 N/A N/A AATCAGCAGGT GGCTG 0 16579 1659 4 1421 8483 52 1022 1037 ACTGTCACACTT GCTG 45 16652 1666 7 1422

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8483 67 1072 1087 GCCACGCCGGC ATCCC 27 16702 1671 7 1423 4684 60 1075 1090 TTGGCCACGCC GGCAT 2 16705 1672 0 48 8483 83 1105 1120 AGCACGCGCAG GCTGC 0 16735 1675 0 1424 8491 60 N/A N/A CCACATCTGCCT GGCC 0 17226 1724 1 1425 8491 76 N/A N/A TGCCAGGTCAT GCAAT 0 17954 1796 9 1426 8491 92 N/A N/A GGGATACAGAC ACCCA 48 18252 1826 7 1427 8492 08 N/A N/A CCATACCATAA CTCCC 36 19126 1914 1 1428 8492 24 N/A N/A CAATTACACCA GCAAT 64 19496 1951 1 1429 8492 40 N/A N/A CTGCGAAAATA 32 19835 1985 0 1430 8483 99 1188 1203 CCACAGGCTGG ACCAG 8 20019 2003 4 1431 8492 56 N/A N/A CCTCAACCGCTC CCAT 15 20631 2064 6 1432 8492 72 N/A N/A GAGATAGGAAA CTATT 19 21060 2107 5 1433 8484 15 1388 1403 GGTCACCGGCT GGTCT 0 21360 2137 5 1434 8484 31 1500 1515 TCCCACTCTGTG ACAC 93 21472 2148 7 1435 8492 88 N/A N/A ACAGACACTAA GCTCT 76 21910 2192 5 1436

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8484 47 1628 1643 GGCCTCATTGAT GACA 0 22121 2213 6 1437 8484 63 1734 1749 CTGACCATACA GTCCT 15 22516 2253 1 1438 8484 79 1850 1865 CTCCATGCGCTC GCCC 39 22632 2264 7 1439 8493 04 N/A N/A CTAGAGATGGC TGAGT 56 22970 2298 5 1440 8493 20 N/A N/A ACGGAAATGGG AATCT 0 23457 2347 2 1441 8484 93 1990 2005 GTCCCCATGCTG GCCT 33 23610 2362 5 1442 8493 36 N/A N/A ATACAGAGATG TTAAG thirty 24079 2409 4 1443 8493 52 N/A N/A TCTTATTCCAGG CTGG 23 24362 2437 7 1444 8493 68 N/A N/A AGCTGACCAGC TGTGT 0 25180 2519 5 1445 8485 09 2064 2079 TGCCAAGGTCC TCCAC 18 25395 2541 0 1446 8493 84 N/A N/A CAAAAACAGAC CCAGC eleven 25733 2574 8 1447 8494 00 N/A N/A AAATAGATGCT CCAGG 35 26465 2648 0 1448 8494 16 N/A N/A CGAGGAAAAAGA AAGCA 20 27342 2735 7 1449 8485 25 2254 2269 CTGCAGCCAGT CAGGG 1 27397 2741 2 1450 8485 41 2494 2509 TCGGAACCATTT TAAA 0 27637 2765 2 1451

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8485 57 2585 2600 CCCCACTCAAG GGCCA 0 27728 2774 3 1452 8485 73 2710 2725 CAGCACCCGAG CACAG 23 27853 2786 8 1453 8485 88 2755 2770 AAAAGTCATTC TGCCC 62 27898 2791 3 1454 8486 04 2780 2795 GCCTGGCACGG AACAA 55 27923 2793 8 1455 8486 20 2909 2924 CTGGAGATGAG GGCCA 24 28052 2806 7 1456 8486 35 3021 3036 GCACAGCCTGT GACCA 71 28164 2817 9 1457 8486 50 3120 3135 GCCGAGTCAGT CCTAG 23 28263 2827 8 1458 8486 66 3150 3165 GCTGAGACAGT GCATG 47 28293 2830 8 1459 4684 97 3224 3239 CCTCTGGTTCCA GGTT 77 28367 2838 2 1460 8486 97 3257 3272 AGTTCCTGCTGT GTGA 44 28400 2841 5 1461 8487 13 3308 3323 AAGTAAGAAGA GGCTT 0 28451 2846 6 1462 8487 29 3384 3399 CTCACCGAGCTT CCTG 76 28527 2854 2 1463 8487 45 3456 3471 CGCCAGGCCAG TGAAT 44 28599 2861 4 1464 8487 60 3565 3580 ACAGAGAGGAC AGACC 45 28708 2872 3 1465 8487 76 3661 3676 AGTCACCATATTT AATA 63 28804 2881 9 1466

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Table 20

Suppression of PCSK9 mRNA with cEt gapmers 3-10-3 targeting SEQ ID NOs: 1 and 2

No. ISIS SEQ ID: 1, start site SE Q ID: 1 CTO II- sai T Subsequence % depressed SEQ ID: 2, start site SEQ ID 2: stop website SE Q ID NO 84843 2 1535 155 0 TGCAATGCCAGCC ACG 0 N/A N/A 146 7 84822 0 184 199 AGCCGCTGCTGCA ACG 57 3659 3674 146 8 84823 6 381 396 GCCACCAGGACCG CCT 28 3856 3871 146 9 84825 2 492 507 CCTCGGAACGCAA GGC 38 3967 3982 147 0 84879 3 N/A N/A AGCATGAGTTCTGT GT 43 4607 4622 147 1 84880 9 N/A N/A CAGCACACTCAGA CAG 47 5288 5303 147 2 84882 5 N/A N/A AAAAGGATTGGTC TAA 0 5652 5667 147 3 84884 1 N/A N/A CCCTTTACCCAAAG CC 71 6525 6540 147 4 84885 7 N/A N/A AATCAGCCTTCAAG GG 39 6928 6943 147 5

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84887 3 N/A N/A GGCAGAAGGGATG ACT 35 7246 7261 147 6 84888 9 N/A N/A TCGACAACAGGTTT TC 58 7576 7591 147 7 84890 5 N/A N/A TGACATGGAAGAA ACC 34 7801 7816 147 8 84826 7 642 657 GCAGGCGGCGGGC AGT 0 7915 7930 147 9 84827 8 678 693 TCTTGGTGAGGTAT CC 52 7951 7966 148 0 84829 3 694 709 TGGAAGACATGCA GGA 26 7967 7982 148 1 84892 1 N/A N/A TGCATTGGCACAA GAA 44 8340 8355 148 2 84893 7 N/A N/A AGTTAGAGGCCAG GAA 25 8645 8660 148 3 84895 3 N/A N/A TCCCATAGTCAACT GT eleven 9099 9114 148 4 84896 9 N/A N/A TTGCAAAGCTTCCA GT 7 10065 1008 0 148 5 46844 6 775 790 ATGTAGTCGACATG GG 38 10536 1055 1 148 6 84898 5 N/A N/A CGAGAAGTGGAAA CCA 0 10727 1074 2 148 7 84900 1 N/A N/A GTGAAAGCTGAGT TCA 6 11431 1144 6 148 8 84901 7 N/A N/A GGGTGGTAATTTGT CA 15 12045 1206 0 148 9 84903 3 N/A N/A AATAACTGATTTAA CC 12 12821 1283 6 149 0

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84904 4 N/A N/A GGATACACAGGCT CGC 82 13108 1312 3 337 84904 9 N/A N/A GTCATTCCACTGC TT thirty 13216 1323 1 149 1 84906 5 N/A N/A CAACGCACATCGA GCA 39 14038 1405 3 149 2 84908 1 N/A N/A ACCAAAACAGTGCT CGC 48 14359 1437 4 149 3 84909 7 N/A N/A AATAAGGTCTGGCT CA 43 14829 1484 4 149 4 84911 3 N/A N/A ACAGATGCCAGGC AGG 10 15511 1552 6 149 5 84912 9 N/A N/A TCAATATCTAACAA TA 0 15978 1599 3 149 6 84832 4 917 932 CTGTATGCTGGTGT CT 0 16309 1632 4 149 7 84833 9 989 100 4 GGTCCCGTCCCTCCT CG 0 16381 1639 6 149 8 84914 5 N/A N/A CATGAGAAAGACC CCC 0 16611 1662 6 149 9 84835 3 1024 103 9 TGACTGTCACACTT GC 56 16654 1666 9 150 0 84836 8 1073 108 8 GGCCACGCCGGCA TCC 0 16703 1671 8 150 1 46846 0 1075 109 0 TTGGCCCACGCCGGC AT 0 16705 1672 0 48 84838 4 1109 112 4 GTTGAGCACGCGC AGG 58 16739 1675 4 150 2 84916 1 N/A N/A CTCCACCACATCTG CC thirty 17231 1724 6 150 3

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84917 7 N/A N/A CGTGCCAGGTCATG CA 74 17956 1797 1 150 4 84919 3 N/A N/A TGGGATACAGACA CCC 5 18253 1826 8 150 5 84920 9 N/A N/A TAAAAGACTCCAT GCC 0 19151 1916 6 150 6 84922 5 N/A N/A GCAATTACACCAG CAA 84 19497 1951 2 150 7 84924 1 N/A N/A GCACAGAGTGATG GTT 66 19908 1992 3 150 8 84840 0 1190 120 5 CCCCACAGGCTGG ACC 34 20021 2003 6 150 9 84925 7 N/A N/A CTGCACCGGGCAT GCG 0 20686 2070 1 151 0 84927 3 N/A N/A CCCTACCATAGCCA GG 36 21118 2113 3 151 1 84841 6 1394 140 9 CCCCAGGGTCACC GGC 38 21366 2138 1 151 2 84928 9 N/A N/A GCACACAGACACT AAG 0 21914 2192 9 151 3 84844 8 1633 164 8 AACCAGGCCTCATT GA 0 22126 2214 1 151 4 84846 4 1735 175 0 GCTGACCATACAGT CC 9 22517 2253 2 151 5 84930 5 N/A N/A CTATCCTGTAGCAT CA 73 23060 2307 5 151 6 84932 1 N/A N/A GACGGAAATGGGA ATS 0 23458 2347 3 151 7 84848 0 1875 189 0 GGCAGACCAGCTT GCC 0 23495 2351 0 151 8

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84849 4 1991 200 6 GGTCCCCATGCTGG CC 0 23611 2362 6 151 9 84933 7 N/A N/A TAGCACTCATCATT TC 33 24098 2411 3 152 0 84935 3 N/A N/A GACGAGAATCAAC TCT 6 24383 2439 8 152 1 84936 9 N/A N/A CACCAGGACTCCTG TG 4 25209 2522 4 152 2 84851 0 2074 208 9 GGCTTGTGGGTGCC AA 15 25405 2542 0 152 3 84938 5 N/A N/A CTCCAAGTGGAGT GGG 0 25786 2580 1 152 4 84940 1 N/A N/A ACTAAAATAGATG CTC 34 26469 2648 4 152 5 84941 7 N/A N/A TGCCAGAGCCCGA GGA 0 27352 2736 7 152 6 84852 6 2259 227 4 GGGCACTGCAGCC AGT 0 27402 2741 7 152 7 84854 2 2500 251 5 GACAAGTCGGAAC CAT 77 27643 2765 8 152 8 84855 8 2598 261 3 GGCAAGGAGGCTG CCC 0 27741 2775 6 152 9 84857 4 2711 272 6 GCAGCACCCGAGC ASA 70 27854 2786 9 153 0 84858 9 2756 277 1 TAAAAGTCATTCTG CC 56 27899 2791 4 153 1 84860 5 2781 279 6 TGCCTGGCACGGA ASA 44 27924 2793 9 153 2 84862 1 2915 293 0 AGTTAGCTGGAGA TGA 0 28058 2807 3 153 3

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84863 6 3027 304 2 ACCAAGGCACAGC CTG 70 28170 2818 5 153 4 84865 1 3121 313 6 TGCCGAGTCAGTCC TA 69 28264 2827 9 153 5 84866 7 3163 317 8 GTGGAGCGGGTTG GCT 26 28306 2832 1 153 6 84868 2 3225 324 0 CCCTCTGGTTCCAG GT 23 28368 2838 3 153 7 84869 8 3259 327 4 TCAGTTCCTGCTGT GT thirty 28402 2841 7 153 8 84871 4 3311 332 6 GTGAAGTAAGAAG AGG 47 28454 2846 9 153 9 84873 0 3385 340 0 ACTCACCGAGCTTC CT 63 28528 2854 3 154 0 84874 6 3469 348 4 TTAGAAGCATCTCC GC 44 28612 2862 7 154 1 84876 1 3569 358 4 GGCAACAGAGAGG ASA 79 28712 2872 7 154 2 84877 7 3666 368 1 TAAAAAGTCACCA TAT 9 28809 2882 4 154 3

Example 2 Dose-dependent antisense inhibition of human PCSK9 in HepG2 cells by cEt gapmers 3-10-3.

Selected gapmers from Example 1 showing downregulation of PCSK9 mRNA in vitro were tested at various doses in HepG2 cells. Antisense oligonucleotides were tested in a series of experiments in which culture conditions were similar. The results of each experiment are presented in the individual tables shown below.

Research 1.

Cells were seeded at a density of 20,000 cells per well and transfected by electroporation with antisense oligonucleotide at concentrations of 46.88 nM, 187.5 nM, 750 nM, and 3000 nM, as indicated in the tables below. ISIS 405879 previously disclosed in WO2008066776 was also included in the study as a comparison oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer and probe kit from ABI (ID no. Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels were corrected according to total RNA measured with RIBOGREEN®. Results are presented as percent suppression of PCSK9 compared to untreated control cells.

Also shown is the half-maximal inhibitory concentration (IC ₅₀ ) for each oligonucleotide. PCSK9 mRNA levels decreased in a dose dependent manner in cells treated with the antisense oligonucleotide. Some of the newly designed antisense oligonucleotides showed better performance compared to the previously disclosed ISIS 405879 oligonucleotide. compared to the previously disclosed oligonucleotide ISIS 405879.

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Table 21

No. ISIS 46.875 nM 187.5 nM 750 nM 3000 nM 1C ₅₀ (µM) 405879 13 6 3 29 >3.00 466847 1 19 65 94 0.47 468497 12 27 60 83 0.48 848219 0 0 54 88 0.91 848241 0 5 31 63 1.78 848283 18 27 54 85 0.48 848306 0 19 53 91 0.62 848323 0 0 0 50 >3.00 848376 0 3 12 43 >3.00 848430 0 thirty 52 81 0.62 848431 67 79 92 96 <0.05 848500 6 thirty 55 82 0.54 848539 0 0 0 55 >3.00 848542 10 thirty 66 86 0.41 848560 12 32 76 91 0.33 848567 0 1 42 89 0.85 848570 23 27 67 87 0.35 848572 18 44 71 81 0.30 848574 0 5 68 95 0.55 848575 0 17 51 80 0.75 848576 AND 18 45 74 0.86 848583 3 18 62 88 0.52 848584 0 22 54 89 0.57 848585 1 22 67 92 0.45 848593 0 25 53 83 0.63 848596 18 40 76 93 0.26 848597 22 42 78 87 0.24 848598 0 18 62 95 0.53 848616 0 14 55 88 0.66 848618 0 0 56 77 0.96 848619 3 7 41 74 1.07 848629 21 34 78 78 0.31 848630 7 22 65 83 0.50 848632 25 34 68 89 0.29 848635 0 0 49 83 0.92 848636 5 34 77 76 0.42 848649 5 10 43 78 0.89

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848651 4 0 48 88 0.88 848652 0 6 28 80 1.16 848656 1 13 52 80 0.73 848670 0 38 84 85 0.39 848671 26 28 70 81 0.34 848672 0 AND 27 73 1.37 848674 36 31 69 89 0.24 848677 2 32 69 90 0.41 848679 0 AND 55 81 0.73 848680 AND 20 64 81 0.52 848685 0 14 33 62 1.63 848687 0 25 63 90 0.51 848688 0 23 61 89 0.55 848690 15 21 78 84 0.39 848692 31 45 80 97 0.17 848693 8 23 69 88 0.44 848694 27 43 75 93 0.21 848701 0 10 39 77 1.02 848704 AND 5 43 78 0.94 848725 0 9 51 81 0.79 848729 26 33 63 92 0.30 848731 0 15 60 89 0.55 848732 12 71 67 90 0.21 848736 6 18 40 73 1.00 848740 2 18 59 86 0.57 848748 9 42 60 85 0.39 848756 3 1 55 85 0.72 848759 18 37 75 94 0.28 848761 20 35 68 81 0.35

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848764 0 8 37 64 1.52 848770 0 4 63 90 0.63 848771 3 23 57 93 0.50 848772 15 32 66 82 0.40 848774 0 8 64 90 0.61 848790 0 0 33 73 1.49 848795 9 42 62 91 0.35 848811 7 5 46 86 0.75 848814 17 20 64 89 0.43 848826 7 57 77 93 0.24 848830 0 35 45 86 0.62 848832 0 40 71 78 0.52 848833 19 43 68 81 0.32 848838 0 36 72 89 0.39 848841 5 24 62 86 0.50 848844 0 22 61 89 0.53 848849 9 48 79 92 0.26 848850 0 38 68 97 0.43 848854 2 7 58 84 0.66 848875 3 17 58 79 0.65 848890 0 6 27 65 1.74 848898 7 14 79 87 0.43 848927 12 25 75 95 0.35 848928 0 0 45 78 ι, οι 848971 0 21 54 82 0.65 848999 6 29 34 64 1.38 849007 0 4 41 78 1.02 849008 0 39 74 90 0.42 849013 4 17 47 86 0.67

- 153 042765

849016 0 23 56 79 0.65 849020 0 13 40 67 1.28 849021 10 34 65 80 0.44 849030 2 28 58 86 0.52 849036 2 1 37 76 1.16 849040 28 55 79 96 0.15 849044 26 39 72 91 0.28 849048 0 thirty 60 79 0.61 849069 12 12 40 74 1.02 849071 2 31 65 92 0.42 849084 0 0 0 43 >3.00 849085 20 39 64 89 0.31 849086 13 40 69 81 0.36 849106 20 24 55 80 0.53 849120 0 15 61 80 0.64 849123 9 48 82 84 0.27 849133 0 0 29 70 1.62 849135 25 54 79 95 0.17 849162 0 4 35 75 1.19 849169 34 63 72 83 0.12 849171 9 60 63 83 0.30 849177 0 16 69 75 0.68 849178 0 22 53 83 0.65 849181 6 8 39 84 0.86 849189 24 44 81 90 0.21 849200 0 0 34 70 1.48 849205 7 29 51 88 0.52 849216 0 15 28 66 1.66 849225 23 47 78 94 0.21

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849227 7 35 74 94 0.34 849233 9 42 68 93 0.32 849236 0 1 57 88 0.73 849239 18 38 74 86 0.30 849266 0 22 66 86 0.51 849267 18 69 72 94 0.17 849285 3 18 45 84 0.71 849288 0 0 0 52 >3.00 849303 18 33 57 72 0.54 849305 25 28 62 82 0.40 849309 10 18 59 82 0.57 849331 0 10 65 93 0.57 849332 0 0 48 76 1.09 849345 0 0 62 80 0.78 849354 0 0 43 64 1.49 849357 5 21 45 75 0.84 849358 0 10 45 79 0.88 849359 3 22 46 88 0.62 849372 0 15 29 82 1.04 849383 2 0 33 71 1.43 849386 AND 18 51 84 0.60

Research 2.

Cells were seeded at a density of 20,000 cells per well and transfected by electroporation with antisense oligonucleotide at concentrations of 78.13, 312.5, 1250 and 5000 nM, as indicated in the tables below. ISIS 431131 previously disclosed in WO2014179620 was also included in the study as a comparison oligonucleotide. After a treatment period of approximately 16 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer and probe kit from ABI (ID no. Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels were adjusted according to total RNA measured with RIBOGREEN®. Results are presented as percent suppression of PCSK9 compared to untreated control cells.

Also shown is the half-maximal inhibitory concentration (IC ₅₀ ) for each oligonucleotide. PCSK9 mRNA levels decreased in a dose dependent manner in cells treated with the antisense oligonucleotide. Some of the newly designed antisense oligonucleotides showed better performance than the previously disclosed ISIS 431131 oligonucleotide.

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Table 22

No. ISIS 78.125 nM 312.5 nM 1250 nM 5000 nM 1C ₅₀ (µM) 431131 18 16 thirty 55 >5 849169 23 57 86 92 0.28 859341 24 48 69 85 1.54 859346 27 54 80 94 0.27 859358 0 thirty 69 89 0.76 859361 28 53 73 94 0.30 859363 38 50 79 82 0.20 859384 19 59 76 94 0.30 859387 36 66 79 96 0.16 859392 14 33 63 81 0.96 859393 20 59 90 96 0.27 859394 43 65 80 96 0.12 859395 37 51 82 91 0.21 859401 27 64 88 94 0.20 859408 0 59 61 92 0.55 859419 22 58 77 84 0.31 859459 33 55 91 93 0.19 859462 31 56 78 96 0.24 859469 26 48 76 83 0.36 859472 19 35 67 87 0.56 859504 19 47 59 80 0.57 859529 19 50 88 94 0.32 859531 33 52 76 69 0.29 859532 24 55 79 90 0.30 859533 9 40 77 91 0.51 859534 25 49 85 85 0.70 859551 25 41 51 78 0.69 859552 0 17 53 78 1.36 859553 24 45 79 94 0.35 859561 28 54 71 92 0.30 859571 35 34 76 73 0.43 859584 19 51 85 95 0.32 859598 33 55 73 83 0.25 859601 23 39 63 90 0.50 859603 0 6 70 71 1.25 859605 9 42 67 83 0.62 859611 21 22 47 76 1.16

Example 3 Tolerance of 3'-terminally conjugated cEt gapmers 3-10-3 targeting human PCSK9 in BALB/c mice.

The ISIS oligonucleotides selected after the above studies were conjugated to the terminal cap structure 3'-THA-C6-GalNAc3-(3R,5S)-5-(hydroxymethyl)pyrrolidin-3-olphosphate (hereinafter referred to as 3'-THA). One hundred and sixty-two 3'-THA-conjugated ISIS antisense oligonucleotides that were evaluated for changes in plasma biochemical marker levels are shown in the table below. The parent oligonucleotide points to the ISIS oligonucleotide which was described in the above studies and which was conjugated to 3'THA and tested in this study.

Table 23

3'-conjugated cEt gapmers 3-10-3 selected for tolerability assessment in BALB/c mice

No. ISIS Source oligonucleotide SEQ ID NO 863413 466847 1096 863415 848219 1390 863416 848241 777 863418 848306 1236 863419 848323 1419 863421 848430 1357 863422 848431 1435 863423 848500 905 863424 848539 1297 863425 848542 1528 863426 848560 607 863427 848567 1145 863428 848570 451 863429 848572 1375 863433 848583 1223 863434 848584 377 863435 848585 452 863436 848593 763 863437 848596 994 863438 848597 1071 863439 848598 1147 863440 848616 379 863441 848618 1302 863442 848619 1378

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863443 848629 1073 863444 848630 1149 863445 848632 455 863446 848635 1457 863447 848636 1534 863448 848649 1380 863449 848651 1535 863451 848656 844 863452 848670 691 863453 848671 768 863457 848679 458 863458 848680 1306 863464 848693 384 863465 848694 459 863468 848725 386 863472 848736 926 863473 848740 1233 863474 848748 619 863475 848756 388 863476 848759 1387 863477 848761 1542 863478 848764 697 863479 848770 1158 863480 848771 1235 863481 848772 389 863482 848774 1312 863483 848790 1240 863484 848795 549 863485 848811 550

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863486 848814 781 863487 848826 474 863488 848830 782 863489 848832 939 863490 848833 1016 863491 848838 1243 863492 848841 1474 863493 848844 629 863494 848849 1017 863495 848850 1092 863496 848854 1244 863497 848875 554 863498 848890 478 863499 848898 1094 863500 848927 871 863501 848928 947 863502 848971 563 863503 848999 1332 863504 849007 877 863505 849008 953 863506 849013 410 863507 849016 1411 863508 849020 644 863509 849021 721 863510 849030 1258 863511 849036 645 863512 849040 955 863513 849044 337 863514 849048 1413

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863515 849069 724 863516 849071 881 863517 849084 648 863518 849085 725 863519 849086 802 863520 849106 1111 863521 849120 960 863522 849123 1188 863523 849133 728 863524 849135 885 863525 849162 504 863526 849169 1043 863527 849171 1195 863531 849189 426 863532 849200 968 863533 849205 427 863535 849225 1507 863536 849227 585 863537 849233 1047 863538 849236 353 863539 849239 1352 863540 849266 1126 863541 849267 1203 863542 849285 434 863543 849288 1436 863544 849303 1362 863545 849305 1516 863546 849309 749 863547 849331 1213

- 160 042765

863548 849332 367 863549 849345 1061 863550 849354 523 863551 849357 754 863552 849358 831 863553 849359 908 863554 849372 678 863555 849383 1369 863556 849386 526 884269 859561 55 884270 859459 180 884271 859395 164 884272 859603 218 884273 859504 284 884274 859394 87 884275 859598 141 884276 859531 198 884277 859469 28 884278 859346 152 884279 859361 79 884280 859472 276

BALB/c mice are a multipurpose mouse model often used for safety and efficacy testing. Mice were injected with a single dose of the oligonucleotide. Plasma transaminase levels were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). ISIS oligonucleotides that caused changes in ALT and/or AST levels outside the expected range for antisense oligonucleotides were excluded from further studies. ISIS 863413, ISIS 863419, ISIS 863424, ISIS 863425, ISIS 863427, ISIS 863433, ISIS 863434, ISIS 863436, ISIS 863437, ISIS 863438, ISIS 863439, ISIS 863438

ISIS 863444, ISIS 863445, ISIS 863448, ISIS 863452, ISIS 863472, ISIS 863473, ISIS 863474, ISIS 863475,

ISIS 863477, ISIS 863479, ISIS 863480, ISIS 863481, ISIS 863482, ISIS 863483, ISIS 863484, ISIS 863485,

ISIS 863486, ISIS 863489, ISIS 863490, ISIS 863491, ISIS 863493, ISIS 863494, ISIS 863495, ISIS 863496,

ISIS 863497, ISIS 863498, ISIS 863499, ISIS 863502, ISIS 863506, ISIS 863507, ISIS 863509, ISIS 863510,

ISIS 863511, ISIS 863512, ISIS 863514, ISIS 863516, ISIS 863517, ISIS 863518, ISIS 863520, ISIS 863522,

ISIS 863524, ISIS 863525, ISIS 863526, ISIS 863527, ISIS 863531, ISIS 863533, ISIS 863536, ISIS 863537,

ISIS 863538, ISIS 863539, ISIS 863541, ISIS 863545, ISIS 863547, ISIS 863548, ISIS 863549, ISIS 863550,

ISIS 863552 and ISIS 863553 were considered portable in this study and were selected for further evaluation.

Example 4 Effect of antisense suppression of PCSK9 in a transgenic mouse model.

A transgenic mouse model was developed at UCI using the human PCSK9 genomic construct contained in ABC7-611722G24 fosmid and digested with Nhe1 to obtain a DNA fragment containing the entire genomic sequence plus 8 kb. 5' non-coding and 0.4 t. 3' non-coding sequence. Mice transgenic for PCSK9 were obtained by random insertion via nuclear microinjection. The offspring expressed human PCSK9 mRNA in the liver and secreted human PCSK9 plasma protein.

This model (herein referred to as Tg mice) evaluated the efficacy of ISIS oligonucleotides conjugated to 3'-THA. The ISIS oligonucleotides tested are shown in the table below. The ISIS No. of the parent compound indicates the ISIS oligonucleotide that was described in the above studies and which was conjugated to 3'-THA and tested in this study.

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Table 24

ISIS oligonucleotides tested in Tg mice

No. ISIS Subsequence No. ISIS original connection SEQ ID NO 863413 GAGGTATCCCCGGCGG 466847 1096 863419 GTCTAGGAGATACACC 848323 1419 863424 CATTTTAAAAGCTCAGC 848539 1297 863425 GACAAGTCGGAACCAT 848542 1528 863427 TTGAATGGTGAAATGC 848567 1145 863433 GGACATCGGCACATTG 848583 1223 863434 ACGGACATCGGCACAT 848584 377 863436 AGCTCAATAAAAGTCA 848593 763 863437 CGGAACAAGAGCTCAA 848596 994 863438 ACGGAACAAGAGCTCA 848597 1071 863439 CACGGAACAAGAGCTC 848598 1147 863441 GATGAGGGCCATCAGC 848618 1302 863444 CAGAAAGCTAAGCCTC 848630 1149 863445 CTAGATGCCATCCAGA 848632 455 863448 TCCTAGGTGATGGCTC 848649 1380 863452 GCGAATGTGTACCCTG 848670 691 863472 GGAAAAAAGTTCCATGC 848736 926 863473 GTGATAACGGAAAAAAG 848740 1233 863474 TGCCTTAGAAGCATCT 848748 619 863475 AAAGATAAATGTCTGC 848756 388 863477 GGCAACAGAGAGGACA 848761 1542 863479 AGCAAAACAGGTCTAG 848770 1158 863480 TTCAAGTTACAAAAGC 848771 1235 863481 CCCAGAATAAATATCT 848772 389 863482 TGCTACAAAACCCAGA 848774 1312 863483 CCAAATCGGAACCCAC 848790 1240

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863484 CGAGAATACCTCCGCC 848795 549 863485 GCTGAGTAAGGACTTG 848811 550 863486 AGAAAGTCAAAGGCTC 848814 781 863489 TACATTTCAGACGGTG 848832 939 863490 AATAATCTCATGTCAG 848833 1016 863491 AGGCAGTAATGGGCAA 848838 1243 863493 TCATGAATCAAGTCCA 848844 629 863494 CAAATTATAGCAGCCA 848849 1017 863495 AACAAATTATAGCAGC 848850 1092 863496 TCCAACACTGAGGACC 848854 1244 863497 CTACAAATGCAGGCAG 848875 554 863498 CTCGACAACAGGTTTT 848890 478 863499 ACCAAATGCGGACCAA 848898 1094 863502 GCAATTCGGTTTGTCC 848971 563 863506 GGAAAGGAACAGGCTC 849013 410 863507 CTCCACTGAAGAGGTC 849016 1411 863509 GACAATGAAGAGGAGA 849021 721 863510 GTTAAGAGTGCAGGGT 849030 1258 863511 ACAGAGAAAATGCATGC 849036 645 863512 GTTATTATTGAATGGT 849040 955 863514 GTTAATAATGCCCCCG 849048 1413 863516 ACAACTGGATACATTG 849071 881 863517 CAATAGGCATCTACCA 849084 648 863518 ACTCATCAATAGGCAT 849085 725 863520 CCTCAGGTGGAATCAG 849106 1111 863522 GGAGAATAACAGTGAT 849123 1188 863524 ATAGACAAGGAAAGGG 849135 885 863525 GTCTAGAAAAAGTCCT 849162 504 863526 AGGAAAGTCTCAGGGC 849169 1043 863527 CTGTAGGAAAGTCTCA 849171 1195 863531 ACAGCTGCTAGTTTATT 849189 426 863533 TGCTACTGTCAACAGT 849205 427 863536 GGAAGATATTAGCAAT 849227 585 863537 GCGGATTTCAGACTTG 849233 1047 863538 CAACATCAAATTCTGC 849236 353 863539 GAAGACGGAGTAAGGC 849239 1352 863541 TTCTAAGTGCCACGGG 849267 1203 863545 CTATCCTGTAGCATCA 849305 1516 863547 GGAGAAGTAAGGTCAC 849331 1213 863548 AGGAGAAGTAAGGTCA 849332 367 863549 ATTTTAAAGCAACGGG 849345 1061 863550 CCTACATGCCAAGCCTG 849354 523 863552 TCTGAACATGGTAGGG 849358 831 863553 GTAAGATGGAAAGAGA 849359 908

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Treatment.

PCSK9 transgenic mice were maintained under a 12 hour light/dark cycle and fed standard Purina mouse chow ad libitum. Animals were acclimatized for at least 7 days in the research laboratory before the start of the experiment. Antisense oligonucleotides (ASO) were prepared in buffered saline (PBS) and sterilized by filtration through a 0.2 micron filter. The oligonucleotides were dissolved in 0.9% PBS for injection.

Mice transgenic for PCSK9 received subcutaneous injections of ISIS oligonucleotide at a dose of 2.5 mg/kg twice a week for 2 weeks. Two groups of mice received subcutaneous injections of PBS for 2 weeks. The saline injection groups served as controls against which the oligonucleotide-treated groups were compared.

Biochemical markers of blood plasma.

To assess the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases (ALT and AST), cholesterol (CHOL), HDL cholesterol (HDL), LDL cholesterol (LDL) and triglycerides (TRIG) were measured in these mice on day 12 using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in the table below. ISIS oligonucleotides that caused changes in the levels of any markers of liver or kidney function outside the expected range for antisense oligonucleotides were excluded from further studies.

Table 25

Biochemical markers of blood plasma in the blood plasma of Tg mice

Treatment ALT (Chalk) AST (IU/l) CHOL (mg/dl) HDL (mg/dl) LDL (mg/dl) TRIG (mg/dl) PBS 17 37 114 73 35 65 PBS 23 102 105 68 32 76 863413 19 72 122 88 24 89 863419 15 44 115 73 31 104 863424 18 34 197 112 79 105 863425 25 53 143 101 36 104 863427 22 96 FROM 75 28 127 863433 17 54 92 67 19 61 863434 24 36 132 98 35 102 863436 17 50 124 81 31 96 863437 21 69 126 91 thirty 81 863438 18 66 57 39 15 54 863439 42 173 111 78 22 107 863441 20 57 84 58 20 56 863444 19 41 149 101 42 116 863445 20 36 169 111 48 111 863448 24 44 107 69 29 79

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863452 20 67 114 78 23 100 863472 35 55 105 71 26 89 863473 23 67 152 105 38 130 863474 21 115 118 78 32 45 863475 23 87 179 112 62 92 863477 67 142 222 137 73 69 863479 29 60 142 96 32 90 863480 19 52 122 88 25 53 863481 28 50 223 132 70 115 863482 20 43 168 105 46 129 863483 19 34 174 110 47 139 863484 25 71 64 38 21 66 863485 19 71 170 FROM 42 110 863486 18 40 117 78 28 121 863489 38 68 90 66 18 89 863490 19 41 99 71 19 FROM 863491 20 40 148 99 35 146 863493 18 74 107 73 26 59 863494 19 41 149 106 32 104 863495 17 34 136 95 32 116 863496 545 275 136 97 31 29 863497 36 49 121 84 28 53 863498 23 44 181 122 42 98 863499 21 51 128 102 18 58 863502 34 144 116 84 22 86 863506 thirty 44 188 119 51 108 863507 19 46 108 78 18 100 863509 23 63 121 80 28 115 863510 40 71 149 111 28 51

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863511 31 61 101 70 23 83 863512 21 52 83 59 14 103 863514 22 65 99 69 23 64 863516 18 52 117 73 33 122 863517 25 98 FROM 74 27 125 863518 35 116 96 65 23 70 863520 21 102 107 68 31 76 863522 31 55 89 56 24 77 863524 49 61 154 104 43 94 863525 20 58 208 116 66 155 863526 32 82 159 114 37 59 863527 25 112 98 66 23 101 863531 835 454 268 149 67 84 863533 0 109 276 152 80 98 863536 22 68 120 75 28 181 863537 29 102 134 97 27 52 863538 16 42 126 76 39 89 863539 22 53 100 65 25 64 863541 19 41 98 66 23 115 863545 28 53 109 73 25 114 863547 22 41 119 80 thirty 71 863548 550 433 188 125 33 60 863549 17 36 109 70 33 72 863550 32 62 158 98 43 77 863552 18 55 FROM 71 33 79 863553 18 43 103 62 thirty 86

Example 5 Tolerability of ISIS Oligonucleotides Targeting Human PCSK9 in CD1 Mice.

The ISIS oligonucleotides selected after the above studies were conjugated to the terminal cap structure 5'-trishexulamino-(THA)-C6GalNAC3 (hereinafter referred to as 5'-THA). CD1® mice (Charles River, Massachusetts) are a multipurpose mouse model often used for safety and efficacy testing. Mice were treated with 5'THA-conjugated ISIS antisense oligonucleotides, selected after the studies described above, and assessed for changes in the levels of various plasma biochemical markers.

Research 1.

Treatment.

Groups of male CD1 mice received a subcutaneous injection of 15 mg/kg ISIS oligonucleotides once a week for 6 weeks. One group of male CD1 mice received a subcutaneous injection of PBS once a week for 6 weeks. Mice were euthanized 48 hours after the last dose, and organs and blood plasma were collected for further analysis.

The ISIS oligonucleotides tested are shown in the table below. The ISIS No. of the unconjugated parent compound refers to the ISIS oligonucleotide described in the above in vitro studies that has the same sequence. ISIS No. 3'-THA conjugated analog refers to a 3'-THA conjugated oligonucleotide that has the same sequence and was evaluated higher in a transgenic mouse study.

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Table 26

ISIS Oligonucleotides Tested in the Tolerance Study in CD1 Mice

No. ISIS No. ISIS unconjugated parent compound No. ISIS analogue conjugated with 3'-THA Subsequence SEQ ID NO 863576 848583 863433 GGACATCGGCACATT G 122 3 863633 848833 863490 AATAATCTCATGTCA G 101 6 863655 849040 863512 GTTATTATTGAATGGT 955 863670 849171 863527 CTGTAGGAAAGTCTC A 119 5 863681 849236 863538 CAACATCAAAATTCTG WITH 353

Biochemical markers of blood plasma.

To assess the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results are presented in the table below. Treatment with ISIS oligonucleotides did not cause any changes in the levels of any markers of liver or kidney function outside the expected range for antisense oligonucleotides.

Table 27

Biochemical markers of blood plasma in the blood plasma of CD1 mice at day 45

cytes, neutrophils, lymphocytes and monocytes, as well as hemoglobin, HCT and MCV levels. The results are presented in the table below. Treatment with ISIS oligonucleotides did not cause any changes in the levels of any hematological markers outside the expected range for antisense oligonucleotides.

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Table 28

Number of blood cells in CD1 mice per day 45

RBC platelets WBC Neutrophils (x 10 ³ / μl) Lymphocytes (x 10 ³ / μl) Monocytes (x 10 ³ / µl) (x 10 ⁶ / µl) (x 10 ³ / µl) (X 10 ³ /µl) PBS 9.0 1288 from 12.6 9128 515 863576 8.3 1251 9.4 18.8 6643 316 863633 9.4 1338 Yu,1 13.5 8009 698 863655 8.9 1320 8.0 10.0 5919 473 863670 9.7 1335 8.3 21.3 6514 402 863681 9.5 1329 7.6 But 6376 310

Table 29

Hematologic markers in CD1 mice at day 45

Hemoglobin (g/dl) PBS 14.0 863576 12.9 863633 14.6 863655 14.1 863670 14.7 863681 14.6

No (%) MCV (fl) 43 48 40 48 45 49 44 50 44 46 45 47

Measurements of body weight and organs.

To evaluate the effect of ISIS oligonucleotides on the general health of animals, body and organ weights were measured. Body weights were measured every week and are shown in the table below. Organ weights were measured and the data are also presented in the table below. The results indicate that the effect of antisense oligonucleotide treatment on body and organ weight scores was within the expected range for antisense oligonucleotides.

Table 30

Body weight (g) in CD1 mice

Baseline (Day 1) Week 1(Day 8) Week 2 (Day 15) Week 3 (day 22) Week 4 (Day 29) Week 5 (Day 36) Week 6 (Day 43) PBS 46 48 49 48 50 49 49 863576 29 33 35 36 38 38 39 863633 27 32 33 34 36 37 39 863655 28 33 33 35 37 38 39 863670 27 31 32 33 36 36 38 863681 28 33 33 35 37 38 40

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Table 31

Organ weights (g) in CD 1 mice

Liver Spleen Bud PBS 2.25 0.16 0.63 863576 2.32 0.19 0.59 863633 2.25 oh p 0.50 863655 2.55 0.15 0.61 863670 1.89 oh p 0.51 863681 2.25 0.10 0.47

Research 2.

Treatment.

Groups of male CD1 mice received a subcutaneous injection of 15 mg/kg ISIS oligonucleotides once a week for 6 weeks. One group of male CD1 mice received a subcutaneous injection of PBS once a week for 6 weeks. Mice were euthanized 48 hours after the last dose, and organs and blood plasma were collected for further analysis.

The ISIS oligonucleotides tested are shown in the table below. The ISIS No. of the unconjugated parent compound refers to the ISIS oligonucleotide described in the above in vitro studies that has the same sequence. ISIS No. 3'-THA conjugated analog refers to a 3'-THA conjugated oligonucleotide that has the same sequence and is described above in the transgenic mouse study. Table 32

ISIS Oligonucleotides Tested in the Tolerance Study in CD1 Mice

No. ISIS ISIS No. of unconjugated parent compound No. ISIS analogue conjugated with 3'-THA Subsequence SEQ ID NO 863568 848542 863425 GACAAGTCGGAACCA T 152 8 863581 848597 863438 ACGGAACAAGAGCTC A 107 1 863582 848598 863439 CACGGAACAAGAGCT WITH 114 7 863587 848630 863444 CAGAAAGCTAAGCCT WITH 114 9

Biochemical markers of blood plasma.

To assess the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY).

The results are presented in the table below. Treatment with ISIS oligonucleotides did not cause any changes in the levels of any markers of liver or kidney function outside the expected range for antisense oligonucleotides.

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Table 33

Biochemical markers of blood plasma in the blood plasma of CD1 mice at day 45

cytes, neutrophils, lymphocytes and monocytes, as well as hemoglobin, HCT and MCV levels. The results are presented in the table below. Treatment with ISIS oligonucleotides did not cause any changes in the levels of any hematological markers outside the expected range for antisense oligonucleotides.

Table 34

Number of blood cells in CD1 mice per day 45

RBC (x 10 ⁶ /µl) Platelets (x 10 ³ / μl) WBC (X 10 ³ /µl) Neutrophils (x 10 ³ / μl) Lymphocytes (x 10 ³ / μl) Monocytes (x 10 ³ / µl) PBS 9.6 853 3.9 12 3174 129 86356 8 10.3 859 7.8 15 6060 347 86358 1 9.4 812 8.0 17 6162 359 86358 2 9.1 868 10.5 21 6671 713 86358 7 9.6 903 9.8 10 8233 344

Table 35

Hematologic markers in CD1 mice at day 45

Hemoglobin (g/dl) No (%) MCV (fl) PBS 14.5 44 46 863568 16.2 47 46 863581 14.5 43 46 863582 13.5 41 45 863587 15.0 45 47

Measurements of body weight and organs.

To evaluate the effect of ISIS oligonucleotides on the general health of animals, body and organ weights were measured. Body weights were measured every week and are shown in the table below. Organ weights were measured and the data are also presented in the table below. The results indicate that the effect of treatment with antisense oligonucleotides on body and organ weights was in the range

- 170 042765 expected range for antisense oligonucleotides.

Table 36

Body weight (g) in mice CD 1

Baseline (Day 1) Week 1 (day 9) Week 2 (Day 16) Week 3 (Day 23) Week 4 (day 30) Week 5 (Day 37) Week 6 (Day 43) PBS thirty 33 36 36 37 39 39 863568 29 32 34 34 36 39 38 863581 29 33 35 36 36 39 39 863582 31 34 36 36 37 40 41 863587 thirty 35 37 37 39 41 43

Table 37

Organ weights (g) in CD 1 mice

Liver Spleen Bud PBS 2.06 oh and 0.59 863568 2.46 0.10 0.54 863581 2.23 0.13 0.56 863582 2.52 0.16 0.63 863587 2.94 0.16 0.64

Research 3.

Treatment.

Groups of male CD1 mice received a subcutaneous injection of 5 mg/kg or 15 mg/kg of oligonucleotides

ISIS once a week for 6 weeks. One group of male CD1 mice received a subcutaneous injection of PBS once a week for 6 weeks. Mice were euthanized 48 hours after the last dose, and organs and blood plasma were collected for further analysis.

The ISIS oligonucleotides tested are shown in the table below. The ISIS No. of the unconjugated parent compound refers to the ISIS oligonucleotide described in the above in vitro studies that has the same sequence. ISIS No. 3'-THA conjugated analog refers to a 3'-THA conjugated oligonucleotide that has the same sequence and is described above in the transgenic mouse study.

Table 38

ISIS Oligonucleotides Tested in the Tolerance Study in CD1 Mice

No. ISIS ISIS No. of unconjugated parent compound No. ISIS analogue conjugated with 3'-THA Subsequence SEQ ID NO 845219 466847 863413 GAGGTATCCCCGGCGG 109 6 863577 848584 863434 ACGGACATCGGCACAT 377 863579 848593 863436 AGCTCAATAAAAGTCA 763 863637 848849 863494 CAAATTATAGCAGCCA 101 7 863682 849239 863539 GAAGACGGAGTAAGG WITH 135 2

Biochemical markers of blood plasma.

To assess the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, creatinine, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY).

The results are presented in the table below. ISIS oligonucleotides that caused changes in the levels of any markers of liver function or

- 171 042765 kidneys outside the expected range for antisense oligonucleotides. Table 39

Biohit mic mg PBS 845219 863577 archers Dose (mg/kg) 5 15 5 15 plasma ALT (Chalk) 26 96 187 176 621 blood in AST (Chalk) 62 64 115 160 492 > plasma ALB (g/dl) 2.5 2.4 2.3 2.5 2.5 e blood bun (mg/dl) 19.5 19.3 22.0 17.2 15.6 CRE mice (mg/dl) 0.08 0.08 0.07 0.09 0.10 CD1 i d TBIL (mg/dL) 0.18 0.15 0.17 0.13 0.20 [Day 45 863579 5 50 50 2.5 19.9 0.10 0.13 15 87 70 2.4 20.3 oh and 0.13 863637 5 321 301 1.7 19.3 0.05 0.19 15 508 472 2.4 20.5 0.09 0.22 863682 5 306 156 2.3 20.0 0.06 0.13 15 373 212 2.4 21.0 0.06 0.14

hematological analyses.

Blood obtained from all groups of mice was analyzed for the number of RBC, WBC, platelets, neutrophils, lymphocytes and monocytes, as well as the levels of hemoglobin, HCT and MCV. The results are presented in the table below. ISIS oligonucleotides that caused changes in the levels of any hematological markers outside the expected range for antisense oligonucleotides were excluded from further studies. Table 40

Number of blood cells in CD1 mice per day 45

Dose (mg/kg) RBC (x 10 ⁶ /µl) Platelets (x 10 ³ / µl) WBC (x 10 ³ /µl) Neutrophils (x 10 ³ / µl) Lymphocytes (x 10 ³ / μl) Monocytes (x 10 ³ /µl) PBS - 9.4 1218 8.8 13 7078 367 845219 5 9.4 1349 7.2 13 5749 444 15 9.3 1263 7.2 13 5664 511 863577 5 9.2 1401 8.0 17 5851 650 15 8.2 1299 10.0 10 7718 1034 863579 5 10.0 1252 9.0 16 6876 587 15 9.6 1256 8.2 AND 6645 415 863637 5 9.0 956 5.7 14 4266 448 15 9.3 843 6.8 13 5044 696 863682 5 9.4 1040 5.9 13 4581 447 15 9.0 800 8.3 8 6757 694

Table 41

Hematologic markers in mice CD 1 at day 45

Dose (mg/kg) Hemoglobin (g/dl) No (%) MCV (fl) PBS - 14D 45 48 845219 5 14.2 45 49 15 13.8 43 46 863577 5 13.9 45 49

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Measurements of body weight and organs.

To evaluate the effect of ISIS oligonucleotides on the general health of animals,

15 12.2 39 48 863579 5 14.9 47 48 15 15.3 48 50 863637 5 13.5 42 47 15 13.8 43 47 863682 5 14.5 45 48 15 13.6 43 48

body and organ weights were measured. Body weights were measured every week and are shown in the table below. Organ weights were measured and the data are also presented in the table below. The results indicate that the effect of antisense oligonucleotide treatment on body and organ weight scores was within the expected range for antisense oligonucleotides. Table 42

Body weight (g) in mice CD 1

Dose (mg/kg) original A week A week A week A week A week A week level 1 day 2(day 3(day 4(day 5(day 6 (day (day 1) 8) 15) 22) 29) 36) 42) PBS - 27 thirty 32 32 33 35 35 5 28 33 34 35 37 40 39 845219 15 29 33 34 35 36 38 38 5 26 31 32 33 34 37 35 863577 15 27 31 31 32 34 36 35 5 28 32 33 32 36 38 36 863579 15 27 32 33 32 36 38 37 863637 5 28 33 34 35 36 37 38 15 27 32 34 34 36 38 38 863682 5 26 32 33 32 35 37 36 15 27 31 32 33 34 36 36

Table 43

Organ weights (g) in CD1 mice

Dose (mg/kg) Liver Spleen Bud PBS - 1.84 0.08 0.50 845219 5 2.42 0.13 0.54 15 2.41 0.15 0.54 863577 5 2.47 0.11 0.58 15 2.62 0.15 0.59 863579 5 2.53 0.13 0.57 15 2.82 0.12 0.54 863637 5 2.38 0.17 0.52 15 2.67 0.22 0.58 863682 5 1.79 0.11 0.54 15 2.13 0.21 0.53

Example 6 Tolerance of ISIS oligonucleotides targeting human PCSK9 in Sprague-Dawley rats.

The Sprague-Dawley rat is a multipurpose model used to assess safety and efficacy. Rats were treated with ISIS antisense oligonucleotides from the studies described in the examples above and assessed for changes in the levels of various plasma biochemical markers.

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Treatment.

Sprague-Dawley rats were maintained on a 12 hour light/dark cycle and fed standard Purina rat chow, Diet 5001, ad libitum. Groups of 4 Sprague-Dawley rats each received a subcutaneous injection of 15 mg/kg ISIS oligonucleotide once a week for 6 weeks. Rats were euthanized 48 hours after the last dose, and organs and blood plasma were collected for further analysis.

Liver function.

To evaluate the effect of ISIS oligonucleotides on liver function, plasma transaminase levels were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results, expressed in IU/l, are shown in the table below. Plasma bilirubin (TBIL), BUN, albumin (ALB) and creatinine (CRE) levels were also measured using the same clinical chemistry analyzer and the results expressed in mg/dl are also presented in the table below. Plasma albumin levels were also measured using the same clinical chemistry analyzer and the results, expressed in g/dl, are also presented in the table below.

Table 44

Liver function markers in Sprague-Dawley rats

ALT (IU/l) AST (IU/l) ALB (g/dl) BUN (mg/dl) CRE (mg/dl) TBIL (mg/dl) PBS 20 64 3.1 13 0.2 0.1 863568 40 70 3.5 14 0.2 0.2 863576 39 94 3.7 16 oh, s 0.1 863579 33 91 h, h 14 0.2 0.1 863581 36 154 2.8 18 oh, s 0.2 863582 38 122 2.9 20 0.3 0.1 863587 38 87 h, o 12 0.2 0.1 863633 26 78 h, o 16 oh, s 0.1 863655 27 84 3.1 15 0.2 0.1 863670 thirty 91 3.2 15 0.2 0.1 863681 29 81 3.2 14 0.2 0.1

Kidney function.

To assess the effect of ISIS oligonucleotides on kidney function, blood creatinine and urinary total protein levels were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results, expressed in mg/dL, are shown in the table below. ISIS oligonucleotides that caused changes in the levels of any markers of kidney function outside the expected range for antisense oligonucleotides were excluded from further studies. N/A indicates that there are no data available for this group.

Table 45

Renal function markers (mg/dl) in Sprague-Dawley rats

CRE (mg/dl) FR (mg/dl) PBS 79 59 863568 111 137 863576 N/A N/A 863579 93 117 863581 54 75 863582 71 91 863587 64 86 863633 100 85 863655 85 117 863670 89 104 863681 144 142

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Indicators of body weight and organs.

To evaluate the effect of ISIS oligonucleotides on the general health of animals, body and organ weights were measured. Body weights were measured every week and are shown in the table below. Organ weights were measured and the data are also presented in the table below. The results indicate that the effect of antisense oligonucleotide treatment on body and organ weight was within the expected range for antisense oligonucleotides.

Table 46 Body weight indicators (g)

Baseline (Day 1) Week 1(Day 8) Week 2 (Day 15) Week 3 (Day 22) Week 4 (Day 29) Week 5 (Day 36) Week 6 (Day 43) PBS 201 267 344 389 433 451 470 863568 200 265 334 371 404 419 435 863576 272 327 361 389 417 424 435 863579 264 313 357 379 407 420 430 863581 195 258 329 361 397 411 430 863582 204 273 350 387 423 440 458 863587 205 273 353 398 439 459 474 863633 271 319 361 377 401 406 417 863655 195 256 330 366 406 417 435 863670 189 249 319 351 384 394 406 863681 275 312 364 386 415 426 435

Table 47 Indicators of mass of organs (g)

Liver Spleen Bud PBS 12 0.8 3.4 863568 16 1.2 3.3 863576 12 1.3 3.0 863579 AND 1.0 3.0 863581 13 2.0 3.1 863582 14 2.1 3.5 863587 14 1.1 3.4 863633 12 1.6 3.2 863655 AND 1.7 3.4 863670 12 1.0 2.9 863681 12 1.1 3.3

Example 7 Effect of antisense suppression of PCSK9 in a transgenic mouse model.

The transgenic mouse model was developed at the UCI and has never been described in the literature. The human PCSK9 genomic construct contained in ABC7-611722G24 fosmid was subjected to restriction under the action of Nhel to obtain a DNA fragment containing the entire genomic sequence, as well as 8 kb. 5'-noncoding and 0.4 t. 3' non-coding sequence. Mice transgenic for PCSK9 were obtained by random insertion via nuclear microinjection. The offspring expressed human PCSK9 mRNA in the liver and secreted human PCSK9 plasma protein.

Treatment.

The transgenic mice were maintained under a 12 hour light/dark cycle and fed standard Purina mouse chow ad libitum. Animals were acclimatized for at least 7 days in the research laboratory before the start of the experiment. Antisense oligonucleotides (ASO) were prepared in buffered saline (PBS) and sterilized by filtration through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

- 175042765 tsy.

Tg mice were divided into 27 groups of 3 mice each. The groups received subcutaneous injections of ISIS oligonucleotide at 0.25 mg/kg, 1.00 mg/kg, or 5.00 mg/kg once a week for 4 weeks. One group of 4 mice received subcutaneous injections of PBS once a week for 4 weeks. The saline injection group served as a control group against which the oligonucleotide treated groups were compared.

RNA analysis.

On day 26, RNA was extracted from the liver for real-time PCR analysis to measure PCSK9 mRNA expression. Results are presented as percentage change in mRNA level compared to control PBS, normalized with RIBOGREEN®. As shown in the table below, treatment with ISIS antisense oligonucleotides resulted in a significant decrease in PCSK9 mRNA levels compared to control PBS.

Table 48

Percent downregulation of PCSK9 mRNA in the liver of transgenic mice compared to control PBS

Weekly dose (mg/kg) % suppression EDso 863568 0.25 26 0.5 1 76 5 95 863579 0.25 9 1.4 1 41 5 84 863581 0.25 12 0.6 1 73 5 80 863582 0.25 32 0.5 1 71 5 91 863587 0.25 27 0.5 1 71 5 87 863633 0.25 60 0.2 1 96 5 99 863655 0.25 37 oh, s 1 94 5 99 863670 0.25 0 1.0 1 62 5 87 863681 0.25 25 0.9 1 45 5 94

Protein analysis.

Plasma protein PCSK9 was measured using a human specific ELISA kit (R&D Systems). Results are presented as percentage change in protein levels compared to control PBS. As shown in the table below, treatment with ISIS antisense oligonucleotides resulted in a significant decrease in PCSK9 plasma protein levels compared to control PBS.

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Table 49

Percent downregulation of plasma protein PCSK9 in transgenic mice compared to control PBS

Weekly dose (mg/kg) % suppression EDso 863568 0.25 37 0.4 1 74 5 97 863579 0.25 5 1.5 1 41 5 81 863581 0.25 31 0.6 1 59 5 91 863582 0.25 25 0.5 1 73 5 95 863587 0.25 33 0.4 1 77 5 93 863633 0.25 50 oh, s 1 97 5 99 863655 0.25 44 oh, s 1 93 5 99 863670 0.25 23 1.6 1 38 5 72 863681 0.25 28 0.8 1 54 5 89

LDL cholesterol levels.

Plasma LDL cholesterol levels were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The levels of LDL receptor protein (LDLr) in the liver were also measured. Results are presented as percentage change in levels compared to control PBS. As shown in the table below, treatment with several ISIS antisense oligonucleotides resulted in a significant reduction in plasma LDL cholesterol levels compared to control PBS. Accordingly, it was observed that treatment with ISIS antisense oligonucleotides resulted in a dose-dependent increase in liver LDLr protein levels in mice.

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Table 50

Suppression of plasma levels of LDL cholesterol compared to control PBS

Weekly dose (mg/kg) % suppression 863568 0.25 7 1 41 5 59 863579 0.25 0 1 15 5 33 863581 0.25 thirty 1 17 5 41 863582 0.25 23 1 28 5 50 863587 0.25 20 1 38 5 38 863633 0.25 27 1 66 5 71 863655 0.25 29 1 58 5 74 863670 0.25 19 1 0 5 39 863681 0.25 21 1 23 5 35

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Table 51

Percentage increase in liver LDLr levels in transgenic mice by . compared to control PBS

Weekly dose (mg/kg) % increase 863568 0.25 3 1 61 5 245 863579 0.25 -46 1 -5 5 132 863581 0.25 -44 1 6 5 73 863582 0.25 -19 1 93 5 191 863587 0.25 -6 1 -10 5 95 863633 0.25 -22 1 131 5 269 863655 0.25 0 1 129 5 207 863670 0.25 9 1 81 5 78 863681 0.25 123 1 360 5 222

Example 8 Viscosity measurement of antisense oligonucleotides targeting human PCSK9.

The viscosity of the antisense oligonucleotides selected after the studies described above was measured to screen out antisense oligonucleotides that have a viscosity greater than 40 centipoise (cP). Oligonucleotides having a viscosity greater than 40 cps will have less than optimal viscosity.

Oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μl of water was added, and the antisense oligonucleotide was dissolved in solution by heating the vial at 50°C. A portion (75 μl) of the preheated sample was pipetted into a microviscometer (Cambridge). The temperature in the microviscometer was set to 25°C and the viscosity of the sample was measured. Another portion (20 μl) of the pre-warmed sample was pipetted into 10 ml water for UV reading at 260 nm and 85° C. (Cary UV instrument). The results are presented in the table below, where the concentration of each antisense oligonucleotide was 350 mg/ml, and indicate that all solutions of antisense oligonucleotides had optimal viscosity in accordance with the criterion above.

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Table 52

Viscosity of antisense oligonucleotides targeting human PCSK9

Example 9 Effect of ISIS antisense oligonucleotides targeting human PCSK9 in cynomolgus monkeys.

Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected after the studies described in the examples above. The efficacy and tolerability of antisense oligonucleotides, as well as their pharmacokinetic profile in the liver and kidneys, were evaluated.

At the time of this study, the cynomolgus monkey genomic sequence was not available from the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the ISIS antisense oligonucleotide sequences that were used in cynomolgus monkeys were compared with the rhesus monkey sequence for homology. The ISIS oligonucleotides, homologous to the rhesus monkey sequence, are also expected to fully cross-react with the cynomolgus monkey sequence. The human antisense oligonucleotides tested cross-react with the genomic sequence of the rhesus monkey (sequence complementary to GENBANK accession no. NW_005092960.1 truncated nucleotides 83474000-83501000, referred to herein as SEQ ID NO: 1544). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the greater the likelihood that the human oligonucleotide can cross-react with the rhesus monkey sequence. Start sites and stop sites for each oligonucleotide targeting SEQ ID NO: 1544 are shown in the table below. The start site indicates the most 5' end nucleotide targeted by the gapmer in the rhesus monkey gene sequence.

Table 53

Antisense oligonucleotides complementary to the rhesus monkey PCSK9 genomic sequence (SEQ ID NO: 1544)

No. ISIS Start sitetarget Mismatches Stopsite- target SEQ ID NO 863568 25252 1 25267 1528 863579 25510 2 25525 763 863581 25520 1 25535 1071 863582 25521 1 25536 1147 863587 25712 2 25727 1149 863633 3911 0 3926 353 863655 10464 1 10479 1195 863670 15306 0 15321 1223 863681 17270 0 17285 955

Treatment.

Prior to the start of the study, monkeys were kept in quarantine, during which daily

- 180 042765 Animal health survey. The monkeys were 2-4 years old and weighed 2-4 kg. Nine groups of 5 randomly assigned male cynomolgus monkeys each received subcutaneous injections of ISIS or PBS oligonucleotide, moving clockwise between four different sites on the backs of the monkeys (i.e., left side, right side, upper side, and lower side) in every day of dosing; one site per dose. Monkeys were dosed with 10 mg/kg of ISIS oligonucleotide twice a week (days 1, 5, 9 and 14) for the first two weeks, and thereafter once a week for 10 weeks (on days 21, 28, 35, 42 , 49, 56, 63, 70, 77 and 84). A control group of 5 cynomolgus monkeys received PBS injections in a similar manner and served as a control group.

During the study period, the monkeys were examined twice daily for signs of illness or distress. Any animal exhibiting more than brief or mild pain or distress due to handling, injury, or illness was treated by veterinary staff with approved analgesics or painkillers in consultation with the Study Director. Any animal in unsatisfactory health or in a possible agonal state was identified for additional control and possible euthanasia. Scheduled euthanasia of the animals was performed on day 86 approximately 48 hours post-dose by bloodletting under deep anesthesia. The protocols described in this example have been approved by the Institutional Animal Care and Use Committee (IACUC).

Lowering the target level.

RNA analysis.

On day 86, RNA was extracted from the liver for real-time PCR analysis to measure PCSK9 mRNA expression. Results are presented as percentage change in mRNA level compared to control PBS, normalized with RIBOGREEN®. As shown in the table below, treatment with antisense oligonucleotides ISIS 863633, ISIS 863670 and ISIS 863681, which did not mismatch the rhesus monkey gene, resulted in a significant decrease in PCSK9 mRNA levels compared to control PBS.

Table 54

Percent downregulation of PCSK9 mRNA in cynomolgus monkey liver compared to control PBS

% suppression 863655 46 863633 88 863568 74 863579 35 863582 55 863581 53 863587 36 863670 84 863681 87

Protein analysis.

Approximately 1 ml of blood was collected from all available animals on day -7 (pre-treatment), days 16, 30, 58, and 86 (approximately 48 hours post-dose on days 14, 28, 56, and 84, respectively) and placed in tubes containing the potassium salt of EDTA. The tubes were centrifuged (3000 rpm for 10 min at 4°C) to obtain blood plasma. The results are presented in the table below as a percentage increase or decrease relative to levels on day -7 (pre-treatment levels). As shown in the table below, treatment with antisense oligonucleotides ISIS 863633, ISIS 863670 and ISIS 863681, which did not mismatch the rhesus monkey gene, resulted in a significant decrease in PCSK9 protein levels.

Liver tissue was also analyzed for inducing levels of LDL protein receptors (LDLr). The results are presented in the table below. The data demonstrate that treatment with three antisense oligonucleotides homologous to the genomic sequence of the rhesus monkey resulted in the induction of liver LDLr protein after 12 weeks of treatment.

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Table 55

Plasma protein levels (% of levels before treatment) in cynomolgus monkeys

Day 16 Day 30 Day 86 PBS 34 36 18 863655 -13 -2 -36 863633 -90 -91 -91 863568 -19 -14 -22 863579 34 36 18 863582 8 2 35 863581 16 8 AND 863587 -3 1 -12 863670 -79 -83 -86 863681 -80 -82 -86

Table 56

Liver LDLr protein levels (% of control PBS) in cynomolgus monkeys

% increase 863655 98 863633 399 863568 243 863579 163 863582 115 863581 156 863587 158 863670 521 863681 417

Plasma lipid levels.

To evaluate the effect of ISIS oligonucleotides on total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides, monkeys were fasted overnight prior to blood sampling. Approximately 1.7 ml of blood samples were taken from all study groups. Blood was collected in test tubes without anticoagulant to separate blood serum. The tubes were kept at room temperature for at least 90 minutes and then centrifuged at 3000 rpm for 10 minutes at room temperature to obtain blood serum. Levels were measured using a Toshiba 200FR NEO biochemical analyzer (Toshiba Co., Japan). The data demonstrate that treatment with three antisense oligonucleotides homologous to the genomic sequence of the rhesus monkey resulted in a significant reduction in plasma levels of total cholesterol and LDL cholesterol. As expected, lowering human PCSK9 levels did not affect plasma HDL cholesterol or triglyceride levels.

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Table 57

Plasma lipid levels (mg/dl) in cynomolgus monkeys per day 86

total cholesterol Cholesterol LDL Cholesterol HDL Triglycerides PBS 146 37 110 71 863655 152 73 71 46 863633 115 17 99 57 863568 128 32 99 51 863579 109 44 71 45 863582 137 38 103 37 863581 174 51 122 34 863587 140 45 102 35 863670 95 17 83 45 863681 109 19 99 29

Tolerance studies.

Clinical and biochemical parameters.

To evaluate the effect of ISIS oligonucleotides on liver function, monkeys were fasted overnight prior to blood sampling. Approximately 1.7 ml of blood samples were taken from all study groups. Blood was collected in test tubes without anticoagulant to separate blood serum. The tubes were kept at room temperature for at least 90 minutes and then centrifuged at 3000 rpm for 10 minutes at room temperature to obtain blood serum. Levels of total bilirubin (TBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and albumin (ALB) were measured using a Toshiba 200FR NEO biochemical analyzer (Toshiba Co., Japan). The results indicate that the antisense oligonucleotides have no effect on liver function outside the expected range for antisense oligonucleotides. In particular, ISIS 863633 was observed to be well tolerated.

Table 58

Biochemical markers of blood plasma in cynomolgus monkeys on day 86

ALT (IU/l) AST (IU/L) Bilirubin (mg/dl) ALB (g/dl) PBS 65 64 0.2 4.1 863655 63 59 oh, s 3.5 863633 47 70 oh, s 4.0 863568 44 64 oh, s 4.1 863579 50 52 0.2 4.1 863582 42 57 0.2 4.2 863581 55 61 oh, s 4.2 863587 110 76 oh, s 3.9 863670 42 64 oh, s 4.1 863681 62 60 oh, s 4.1

Kidney function.

To evaluate the effect of ISIS oligonucleotides on kidney function, monkeys were fasted overnight prior to blood sampling. Approximately 1.7 ml of blood samples were taken from all study groups. Blood was collected in test tubes without anticoagulant to separate blood serum. The tubes were kept at room temperature for at least 90 minutes and then centrifuged at 3000 rpm for 10 minutes at room temperature to obtain blood serum. BUN and creatinine levels were measured using a Toshiba 200FR NEO biochemical analyzer (Toshiba Co., Japan). The results, expressed in mg/dL, are shown in the table below.

Plasma chemistry data indicate that most of the ISIS oligonucleotides did not have any effect on kidney function beyond the expected range for antisense oligonucleotides. In particular, ISIS 863633 was observed to be well tolerated.

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Table 59

Plasma BUN and creatinine levels (mg/dl) in cynomolgus monkeys per day 86

BUN Creatinine PBS 31 0.8 863655 26 0.8 863633 27 0.8 863568 27 0.9 863579 24 0.8 863582 24 0.8 863581 27 0.8 863587 24 0.8 863670 26 1.0 863681 23 0.8

Hematology.

To evaluate any effects of ISIS oligonucleotides in cynomolgus monkeys on hematological parameters, approximately 0.5 ml of blood was collected from each of the available test animals in tubes containing K2-EDTA. Samples were analyzed for red blood cell count (RBC), white blood cell count (WBC), number of individual types of white blood cells such as monocytes, neutrophils, lymphocytes, as well as platelet count, hemoglobin content and hematocrit using the ADVIA120 hematology analyzer (Bayer, USA). The data are presented in the tables below.

The data indicate that the oligonucleotides did not cause any changes in hematological parameters beyond the expected range for antisense oligonucleotides at this dose. In particular, ISIS 863633 was observed to be well tolerated.

Table 60

The number of blood cells in cynomolgus monkeys per day is 86

RBC (x 10 ⁶ /µl) Platelets (x 10 ³ / μl) WBC (χ 10 ³ /µl) Neutrophils (x 10 ³ / μl) Lymphocytes (x 10 ³ / μl) Monocytes (x 10 ³ / µl) PBS 5.5 340 9.0 3.1 5.4 oh, s 863655 5.6 361 9.7 3.7 5.4 oh, s 863633 5.6 395 I,4 4.5 6.5 oh, s 863568 5.8 409 12.1 602 5.4 oh, s 863579 5.9 390 8.0 2.2 5.3 oh, s 863582 5.6 333 8.9 h, o 5.5 oh, s 863581 5.8 382 8.2 2.4 5.3 oh, s 863587 6.0 348 8.8 2.3 6.0 0.2 863670 5.4 417 10.7 4.3 5.9 oh, s 863681 5.6 408 9.0 3.1 5.5 0.2

Table 61

Hematological per day 86 macaques-

Hemoglobin No (g/dl) (%) PBS 12.8 45 863655 13.0 43 863633 12.9 43 863568 13.0 44 863579 13.5 46 863582 12.4 43

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863581 13.0 44 863587 13.5 46 863670 12.7 43 863681 12.7 43

Overall, the results of the study indicate that ISIS 863633 is the most effective and well-tolerated compound tested for PCSK9 suppression and is an important candidate for the treatment of cardiovascular disease, in particular for lowering LDL cholesterol levels.

Example 10 Comparative Inhibition by Antisense Oligonucleotides Targeting Human PCSK9 in a Dose Response Assay.

The unconjugated parent oligonucleotides from the antisense oligonucleotides tested in the monkey study were compared to previously engineered compounds. ISIS 848542, ISIS 848593, ISIS 848597, ISIS 848598, ISIS 848630, ISIS 848833, ISIS 849040, ISIS 849171 and ISIS 849236 were tested for efficacy along with ISIS 405879 and ISIS 405995, which were previously identified as one of the most effective antisense compounds in vitro (see, for example, US Pat. No. 8,084,437) as well as ISIS 431,131 and ISIS 480,604 which were previously described in US Pat. No. 9,127,276.

The cells were seeded at a density of 10,000 cells per well, and the antisense oligonucleotide was added to the medium at concentrations of 39.1 nM, 156.25 nM, 625 nM, 2500 nM, and 10,000 nM for free uptake of the oligonucleotide by the cells. After a treatment period of approximately 48 hours, RNA was isolated from the cells and PCSK9 mRNA levels were measured by quantitative real-time PCR. Human PCSK9 primer and probe kit from ABI (ID no. Hs03037355_m1) was used to measure mRNA levels. PCSK9 mRNA levels were corrected according to total RNA measured with RIBOGREEN®. Results are presented as percent suppression of PCSK9 compared to untreated control cells.

Also shown is the half-maximal inhibitory concentration (IC ₅₀ ) for each oligonucleotide. PCSK9 mRNA levels decreased in a dose dependent manner in cells treated with the antisense oligonucleotide. ISIS 848542, ISIS 848593, ISIS 848597, ISIS 848598, ISIS 848630, ISIS 848833, ISIS 849040, ISIS 849171 and ISIS 849236 showed better performance than all previously disclosed oligonucleotides.

Table 62

No. ISIS 39.1 nM 156.25 nM 625.0 nM 2500.0 nM 10000.0 nM IC50 (µM) 405879 0 0 0 0 0 >10 405995 0 0 0 0 0 >10 431131 0 3 0 0 5 >10 480604 0 1 19 31 36 >10 848542 9 37 70 83 88 0.4 848593 19 36 51 53 69 1.1 848597 41 67 78 86 91 0.04 848598 41 64 76 85 90 0.1 848630 0 17 59 79 85 0.9 848833 4 66 88 93 94 0.2 849040 41 93 98 99 98 <0.03 849171 0 52 86 94 96 oh, s 849236 0 26 71 89 95 0.6

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Claims (8)

CLAIM or a pharmaceutically acceptable salt thereof. 2. The compound of claim 1 wherein the pharmaceutically acceptable salt is the sodium salt. 3. The compound of claim 1 wherein the pharmaceutically acceptable salt is the potassium salt. 4. A compound or pharmaceutically acceptable salt thereof, consisting of a modified oligonucleotide of length 16 linked nucleosides having the nucleotide sequence shown in SEQ ID NO: 1016, where the modified oligonucleotide contains: a gap segment consisting of ten linked deoxynucleosides; 5' end flank segment consisting of three linked nucleosides; And 3'-terminal flank segment, consisting of three linked nucleosides; where the gap segment is located between the 5'-terminal flank segment and the 3'-terminal flank segment, where each nucleoside of each flank segment contains cEt-caxap; where each internucleoside bond is a phosphothioate bond; and where each cytosine is 5 methyl cytosine. 5. A compound or a pharmaceutically acceptable salt thereof, comprising a modified oligonucleotide having a length of 16 linked nucleosides, which has a nucleobase sequence consisting of SEQ ID NO: 1016, wherein the modified oligonucleotide contains: a gap segment consisting of linked deoxynucleosides; 5'-terminal flank segment, consisting of linked nucleosides; And 3'-terminal flank segment consisting of linked nucleosides; where the gap segment is located between the 5'-end flank segment and the 3'-end flank segment, where the 5'-end flank segment and the 3'-end flank segment contain cEt-caxapa; where each internucleoside bond is a phosphothioate bond; and where each cytosine is 5-methylcytosine. 6. The use of a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5 in therapy. 7. Use according to claim 6 in the treatment, prevention, or amelioration of a disease associated with PCSK9. 8. Use according to claim 7, wherein the disease is hypercholesterolemia, dyslipidemia, or mixed dyslipidemia.