# Patent

## Title
Modulators of PNPLA3 expression

## Abstract

The present embodiments provide methods, compounds, and compositions useful for inhibiting PNPLA3 expression, which may be useful for treating, preventing, or ameliorating a disease associated with PNPLA3.

## Background
SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0317USLSEQ_ST25.txt created Sep. 13, 2018, which is 480 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD
The present embodiments provide methods, compounds, and compositions useful for inhibiting PNPLA3 (patatin like phospholipase domain containing 3; hypothetical protein dJ796I17.1; adiponutrin; DJ796I17.1) expression, and in certain instances, reducing the amount of PNPLA3 protein in a cell or animal, which can be useful for treating, preventing, or ameliorating a disease associated with PNPLA3.
BACKGROUND
Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of liver disease from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is defined as fat accumulation in the liver exceeding 5% by weight, in the absence of significant alcohol consumption, steatogenic medication, or hereditary disorders (Kotronen et al, Arterioscler Thromb. Vasc. Biol. 2008, 28: 27-38).
Non-alcoholic steatohepatitis (NASH) is NAFLD with signs of inflammation and hepatic injury. NASH is defined histologically by macrovesicular steatosis, hepatocellular ballooning, and lobular inflammatory infiltrates (Sanyal, Hepatol. Res. 2011. 41: 670-4). NASH is estimated to affect 2-3% of the general population. In the presence of other pathologies, such as obesity or diabetes, the estimated prevalence increases to 7% and 62% respectively (Hashimoto et al, J. Gastroenterol. 2011. 46(1): 63-69).
PNPLA3 is a 481 amino acid member of the patatin-like phospholipase domain-containing family that is expressed in the ER and on lipid droplets. In humans, PNPLA3 is highly expressed in the liver, whereas adipose tissue expression is five-fold less (Huang et al, Proc. Natl. Acad. Sci. USA 2010. 107: 7892-7).
SUMMARY
Certain embodiments provided herein are compounds and methods for reducing the amount or activity of PNPLA3 mRNA, and in certain embodiments, reducing the amount of PNPLA3 protein in a cell or animal. In certain embodiments, the animal has a liver disease. In certain embodiments, the disease is NASH. In certain embodiments, the disease is NAFLD. In certain embodiments, the disease is hepatic steatosis. In certain embodiments, the disease is liver cirrhosis. In certain embodiments, the disease is hepatocellular carcinoma. In certain embodiments, the disease is alcoholic liver disease. In certain embodiments, the disease is alcoholic steatohepatitis (ASH). In certain embodiments, the disease is HCV hepatitis. In certain embodiments, the disease is chronic hepatitis. In certain embodiments, the disease is hereditary hemochromatosis. In certain embodiments, the disease is primary sclerosing cholangitis. Certain compounds provided herein are directed to compounds and compositions that reduce liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an animal.
Certain embodiments provided herein are directed to potent and tolerable compounds and compositions useful for inhibiting PNPLA3 expression, which can be useful for treating, preventing, ameliorating, or slowing progression of liver diseases. Certain embodiments provided herein are directed to compounds and compositions that are more potent or have greater therapeutic value than compounds publicly disclosed.


DETAILED DESCRIPTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the embodiments, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and GenBank and NCBI reference sequence records are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Compounds described by ION number indicate 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 comprising 2′-H(H) furanosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).
“2′-O-methoxyethyl” (also 2′-MOE) refers to a 2′-O(CH2)2—OCH3) in the place of the 2′-OH group of a ribosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.
“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.
“2′-substituted nucleoside” or “2-modified nucleoside” means a nucleoside comprising a 2′-substituted or 2′-modified sugar moiety. As used herein, “2′-substituted” or “2-modified” in reference to a sugar moiety means a sugar moiety comprising at least one 2′-substituent group other than H or OH.
“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular compound.
“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular compound.
“5-methylcytosine” means a cytosine with a methyl group attached to the 5 position.
“About” means within +10% of a value. For example, if it is stated, “the compounds affected about 70% inhibition of PNPLA3”, it is implied that PNPLA3 levels are inhibited within a range of 60% and 80%.
“Administration” or “administering” refers to routes of introducing a compound or composition provided herein to an individual to perform its intended function. An example of a route of administration that can be used includes, but is not limited to parenteral administration, such as subcutaneous, intravenous, or intramuscular injection or infusion.
“Administered concomitantly” or “co-administration” means administration of two or more compounds in any manner in which the pharmacological effects of both are manifest in the patient. Concomitant administration does not require that both compounds be administered in a single pharmaceutical composition, in the same dosage form, by the same route of administration, or at the same time. The effects of both compounds need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive. Concomitant administration or co-administration encompasses administration in parallel or sequentially.
“Amelioration” refers to an improvement or lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression or severity of one or more indicators of a condition or disease. The progression or severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.
“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
“Antisense activity” means any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound to the target.
“Antisense compound” means a compound comprising an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, oligonucleotides, ribozymes, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
“Antisense oligonucleotide” means an oligonucleotide having a nucleobase sequence that is complementary to a target nucleic acid or region or segment thereof. In certain embodiments, an antisense oligonucleotide is specifically hybridizable to a target nucleic acid or region or segment thereof.
“Bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety. “Bicyclic sugar” or “bicyclic sugar moiety” means a modified sugar moiety comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming 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 comprise a furanosyl moiety.
“Branching group” means a group of atoms having at least 3 positions that are capable of forming covalent linkages to at least 3 groups. In certain embodiments, a branching group provides a plurality of reactive sites for connecting tethered ligands to an oligonucleotide via a conjugate linker and/or a cleavable moiety.
“Cell-targeting moiety” means a conjugate group or portion of a conjugate group that is capable of binding to a particular cell type or particular cell types.
“cEt” or “constrained ethyl” means a ribosyl bicyclic sugar moiety wherein the second ring of the bicyclic sugar is formed via a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH3)—O-2′, and wherein the methyl group of the bridge is in the S configuration.
“cEt nucleoside” means a nucleoside comprising a cEt modified sugar moiety.
“Chemical modification” in a compound describes the substitutions or changes through chemical reaction, of any of the units in the compound relative to the original state of such unit. “Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase. “Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
“Chemically distinct region” refers to a region of a compound that is in some way chemically different than another region of the same compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.
“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.
“Cleavable bond” means any chemical bond capable of being split. In certain embodiments, a cleavable bond is selected from among: an amide, a polyamide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, a di-sulfide, or a peptide.
“Cleavable moiety” means a bond or group of atoms that is cleaved under physiological conditions, for example, inside a cell, an animal, or a human.
“Complementary” in reference to an oligonucleotide means the nucleobase sequence of such oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof when the two nucleobase sequences are aligned in opposing directions. Nucleobase matches or complementary nucleobases, as 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-methyl cytosine (mC) and guanine (G) unless otherwise specified. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside and may include one or more nucleobase mismatches. By contrast, “fully complementary” or “100% complementary” in reference to oligonucleotides means that such oligonucleotides have nucleobase matches at each nucleoside without any nucleobase mismatches.
“Conjugate group” means a group of atoms that is attached to an oligonucleotide. Conjugate groups include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.
“Conjugate linker” means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.
“Conjugate moiety” means a group of atoms that is attached to an oligonucleotide via a conjugate linker.
“Contiguous” in the context of an oligonucleotide refers to nucleosides, nucleobases, sugar moieties, or internucleoside linkages that are immediately adjacent to each other. For example, “contiguous nucleobases” means nucleobases that are immediately adjacent to each other in a sequence.
“Designing” or “Designed to” refer to the process of designing a compound that specifically hybridizes with a selected nucleic acid molecule.
“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition can be a liquid, e.g. saline solution.
“Differently modified” means chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
“Dose” means a specified quantity of a compound or pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose may require a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In other embodiments, the compound or pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week or month.
“Dosing regimen” is a combination of doses designed to achieve one or more desired effects.
“Double-stranded antisense compound” means an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an oligonucleotide.
“Effective amount” means the amount of compound sufficient to effectuate a desired physiological outcome in an individual in need of the compound. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
“Efficacy” means the ability to produce a desired effect.
“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to, the products of transcription and translation.
“Gapmer” means an oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”
“Hybridization” means the annealing of oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an oligonucleotide and a nucleic acid target.
“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements of the same kind (e.g. no intervening nucleobases between the immediately adjacent nucleobases).
“Individual” means a human or non-human animal selected for treatment or therapy.
“Inhibiting the expression or activity” refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.
“Internucleoside linkage” means a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. “Modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring, phosphate internucleoside linkage. Non-phosphate linkages are referred to herein as modified internucleoside linkages.
“Lengthened oligonucleotides” are those that have one or more additional nucleosides relative to an oligonucleotide disclosed herein, e.g. a parent oligonucleotide.
“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.
“Linker-nucleoside” means a nucleoside that links an oligonucleotide to a conjugate moiety. Linker-nucleosides are located within the conjugate linker of a compound. Linker-nucleosides are not considered part of the oligonucleotide portion of a compound even if they are contiguous with the oligonucleotide.
“Mismatch” or “non-complementary” means a nucleobase of a first oligonucleotide that is not complementary to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned. For example, nucleobases including but not limited to a universal nucleobase, inosine, and hypoxanthine, are capable of hybridizing with at least one nucleobase but are still mismatched or non-complementary with respect to nucleobase to which it hybridized. As another example, a nucleobase of a first oligonucleotide that is not capable of hybridizing to the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligonucleotides are aligned is a mismatch or non-complementary nucleobase.
“Modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating PNPLA3 RNA can mean to increase or decrease the level of PNPLA3 RNA and/or PNPLA3 protein in a cell, tissue, organ or organism. A “modulator” effects the change in the cell, tissue, organ or organism. For example, a PNPLA3 compound can be a modulator that decreases the amount of PNPLA3 RNA and/or PNPLA3 protein in a cell, tissue, organ or organism.
“MOE” means methoxyethyl.
“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides.
“Motif” means the pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages, in an oligonucleotide.
“Natural” or “naturally occurring” means found in nature.
“Non-bicyclic modified sugar” or “non-bicyclic modified sugar moiety” means a modified sugar moiety that comprises a modification, such as a substituent, that does not form a bridge between two atoms of the sugar to form a second ring.
“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.
“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid. As used herein a “naturally occurring nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). A “modified nucleobase” is a naturally occurring nucleobase that is chemically modified. A “universal base” or “universal nucleobase” is a nucleobase other than a naturally occurring nucleobase and modified nucleobase, and is capable of pairing with any nucleobase.
“Nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage.
“Nucleoside” means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. “Modified nucleoside” means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase.
“Oligomeric compound” means a compound comprising a single oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group.
“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. Unless otherwise indicated, oligonucleotides consist of 8-80 linked nucleosides. “Modified oligonucleotide” means an oligonucleotide, wherein at least one sugar, nucleobase, or internucleoside linkage is modified. “Unmodified oligonucleotide” means an oligonucleotide that does not comprise any sugar, nucleobase, or internucleoside modification.
“Parent oligonucleotide” means an oligonucleotide whose sequence is used as the basis of design for more oligonucleotides of similar sequence but with different lengths, motifs, and/or chemistries. The newly designed oligonucleotides may have the same or overlapping sequence as the parent oligonucleotide.
“Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.
“Pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an individual. For example, a pharmaceutically acceptable carrier can be a sterile aqueous solution, such as PBS or water-for-injection.
“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds or oligonucleotides, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
“Pharmaceutical agent” means a compound that provides a therapeutic benefit when administered to an individual.
“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more compounds or salt thereof and a sterile aqueous solution.
“Phosphorothioate linkage” means a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. A phosphorothioate internucleoside linkage is a modified internucleoside linkage.
“Phosphorus moiety” means a group of atoms comprising a phosphorus atom. In certain embodiments, a phosphorus moiety comprises a mono-, di-, or tri-phosphate, or phosphorothioate.
“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an oligomeric compound.
“Prevent” refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely.
“Prodrug” means a compound in a form outside the body which, when administered to an individual, is metabolized to another form within the body or cells thereof. In certain embodiments, the metabolized form is the active, or more active, form of the compound (e.g., drug). Typically conversion of a prodrug within the body is facilitated by the action of an enzyme(s) (e.g., endogenous or viral enzyme) or chemical(s) present in cells or tissues, and/or by physiologic conditions.
“Reduce” means to bring down to a smaller extent, size, amount, or number.
“RefSeq No.” is a unique combination of letters and numbers assigned to a sequence to indicate the sequence is for a particular target transcript (e.g., target gene). Such sequence and information about the target gene (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 DNA Data Bank of Japan (the latter three forming the International Nucleotide Sequence Database Collaboration or INSDC).
“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
“RNAi compound” means an antisense compound that acts, at least in part, through RISC or Ago2, but not through RNase H, to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to double-stranded siRNA, single-stranded RNA (ssRNA), and microRNA, including microRNA mimics.
“Segments” are defined as smaller or sub-portions of regions within a nucleic acid.
“Side effects” means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.
“Single-stranded” in reference to a compound means the compound has only one oligonucleotide. “Self-complementary” means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligonucleotide, wherein the oligonucleotide of the compound 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 nucleobase positions within a target nucleic acid.
“Specifically hybridizable” refers to an oligonucleotide having a sufficient degree of complementarity between the oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids. In certain embodiments, specific hybridization occurs under physiological conditions.
“Specifically inhibit” with reference to a target nucleic acid means to reduce or block expression of the target nucleic acid while exhibiting fewer, minimal, or no effects on non-target nucleic acids. Reduction does not necessarily indicate a total elimination of the target nucleic acid's expression.
“Standard cell assay” means assay(s) described in the Examples and reasonable variations thereof.
“Standard in vivo experiment” means the procedure(s) described in the Example(s) and reasonable variations thereof.
“Stereorandom chiral center” in the context of a population of molecules of identical molecular formula means a chiral center having a random stereochemical configuration. For example, in a population of molecules comprising a stereorandom chiral center, the number of molecules having the (S) configuration of the stereorandom chiral center may be but is not necessarily the same as the number of molecules having the (R) configuration of the stereorandom chiral center. The stereochemical configuration of a chiral center is considered random when it is the result of a synthetic method that is not designed to control the stereochemical configuration. In certain embodiments, a stereorandom chiral center is a stereorandom phosphorothioate internucleoside linkage.
“Sugar moiety” means an unmodified sugar moiety or a modified sugar moiety. “Unmodified sugar moiety” or “unmodified sugar” means a 2′-OH(H) ribosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2′-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”). “Modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate. “Modified furanosyl sugar moiety” means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen or hydroxyl of an unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2′-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.
“Sugar surrogate” means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary compounds or nucleic acids.
“Synergy” or “synergize” refers to an effect of a combination that is greater than additive of the effects of each component alone at the same doses.
“PNPLA3” means any nucleic acid or protein of PNPLA3. “PNPLA3 nucleic acid” means any nucleic acid encoding PNPLA3. For example, in certain embodiments, a PNPLA3 nucleic acid includes a DNA sequence encoding PNPLA3, an RNA sequence transcribed from DNA encoding PNPLA3 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding PNPLA3. “PNPLA3 mRNA” means an mRNA encoding a PNPLA3 protein. The target may be referred to in either upper or lower case.
“PNPLA3 specific inhibitor” refers to any agent capable of specifically inhibiting PNPLA3 RNA and/or PNPLA3 protein expression or activity at the molecular level. For example, PNPLA3 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of PNPLA3 RNA and/or PNPLA3 protein.
“Target gene” refers to a gene encoding a target.
“Targeting” means the specific hybridization of a compound to a target nucleic acid in order to induce a desired effect.
“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by compounds described herein.
“Target region” means a portion of a target nucleic acid to which one or more compounds is targeted.
“Target segment” means the sequence of nucleotides of a target nucleic acid to which a compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.
“Terminal group” means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.
“Therapeutically effective amount” means an amount of a compound, pharmaceutical agent, or composition that provides a therapeutic benefit to an individual.
“Treat” refers to administering a compound or pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal.
Certain Embodiments
Certain embodiments provide methods, compounds and compositions for inhibiting PNPLA3 (PNPLA3) expression.
Certain embodiments provide compounds targeted to a PNPLA3 nucleic acid. In certain embodiments, the PNPLA3 nucleic acid has the sequence set forth in RefSeq or GENBANK Accession No. NM_025225.2 (incorporated by reference, disclosed herein as SEQ ID NO: 1); NC_000022.11 truncated from nucleotides 43921001 to U.S. Pat. No. 43,954,500 (incorporated by reference, disclosed herein as SEQ ID NO: 2); AK123806.1 (incorporated by reference, disclosed herein as SEQ ID NO: 3); BQ686328.1 (incorporated by reference, disclosed herein as SEQ ID NO: 4); BF762711.1 (incorporated by reference, disclosed herein as SEQ ID NO: 5); DA290491.1 (incorporated by reference, disclosed herein as SEQ ID NO: 6); and the sequences listed as SEQ ID Nos 7, 8, 9, and 10. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded.
In certain embodiments, the compound comprises a modified oligonucleotide 16 linked nucleosides in length. In certain embodiments, the compound is an antisense compound or oligomeric compound.
Certain embodiments provide a compound comprising a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.
Certain embodiments provide a compound comprising a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound is an antisense compound or oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded.
Certain embodiments provide a compound comprising a modified oligonucleotide 12 to 30 linked nucleosides in length and complementary within nucleobases 5567-5642, 5644-5731, 5567-5731, 5567-5620, 13697-13733, 20553-20676, 20664-20824, 20553-20824, and 25844-25912 of 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 oligomeric compound. In certain embodiments, the compound is single-stranded. In certain embodiments, the compound is double-stranded. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.
In certain embodiments, compounds target nucleotides 5567-5620 of a PNPLA3 nucleic acid. In certain embodiments, compounds target within nucleotides 5567-5642, 5644-5731, 5567-5731, 5567-5620 of a PNPLA3 nucleic acid having the nucleobase sequence of SEQ ID NO: 2. In certain embodiments, compounds have at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion within nucleotides 5567-5642, 5644-5731, 5567-5731, 5567-5620 of a PNPLA3 nucleic acid having the nucleobase sequence of SEQ ID NO: 2. In certain embodiments, these compounds are antisense compounds, oligomeric compounds, or oligonucleotides.
In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.
In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length.
In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899.
In certain embodiments, compounds targeted to PNPLA3 is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. Out of over 2,384 compounds that were screened as described in the Examples section below, ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, and 975612 emerged as the top lead compounds.
In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified internucleoside linkage, at least one modified sugar, and/or at least one modified nucleobase.
In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified sugar. In certain embodiments, at least one modified sugar comprises a 2′-O-methoxyethyl group. In certain embodiments, at least one modified sugar is a bicyclic sugar, such as a 4′-CH(CH3)-O-2′ group, a 4′-CH2-O-2′ group, or a 4′-(CH2)2-O-2′group.
In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.
In certain embodiments, any of the foregoing modified oligonucleotides comprises at least one modified nucleobase, such as 5-methylcytosine.
In certain embodiments, any of the foregoing modified oligonucleotides comprises:

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

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the modified oligonucleotide is 12 to 30 linked nucleosides in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the modified oligonucleotide is 16 to 30 linked nucleosides in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the modified oligonucleotide is 16 linked nucleosides in length having a nucleobase sequence consisting of the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899.

    
    


In certain embodiments, a compound comprises or consists of a modified oligonucleotide 12-30 linked nucleobases in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899, wherein the modified oligonucleotide comprises

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

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide consists of 16-30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides.

    
    


In certain embodiments, a compound comprises or consists of a modified oligonucleotide, wherein the modified oligonucleotide is 16 linked nucleosides in length and consists of the sequence of SEQ ID NO: 1089, wherein the modified oligonucleotide comprises:

    
    
        a gap segment consisting of ten linked deoxynucleosides;
        a 5′ wing segment consisting of three linked nucleosides; and
        a 3′ wing segment consisting of three linked nucleosides;
        wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.
    
    


In certain embodiments, a compound consists of a modified oligonucleotide and a conjugate group, wherein the modified oligonucleotide is 16 linked nucleosides in length and consists of the sequence of SEQ ID NO: 1089, wherein the modified oligonucleotide comprises:

    
    
        a gap segment consisting of ten linked deoxynucleosides;
        a 5′ wing segment consisting of three linked nucleosides; and
        a 3′ wing segment consisting of three linked nucleosides;
        wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; wherein each cytosine is a 5-methylcytosine; and wherein the conjugate group is positioned at the 5′end of the modified oligonucleotide and is
    
    






In certain embodiments, a compound comprises or consists of ION 916333 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):




In certain embodiments, a compound comprises or consists of ION 975616 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):




In certain embodiments, a compound comprises or consists of the sodium salt of 975616, having the following chemical structure (SEQ ID NO: 1089):




In certain embodiments, a compound comprises or consists of ION 975613 or salt thereof, having the following chemical structure (SEQ ID NO: 330):




In certain embodiments, a compound comprises or consists of the sodium salt of 975613, having the following chemical structure (SEQ ID NO: 330):




In certain embodiments, a compound comprises or consists of ION 975612 or salt thereof, having the following chemical structure (SEQ ID NO: 899):




In certain embodiments, a compound comprises or consists of the sodium salt of 975612, having the following chemical structure (SEQ ID NO: 899):




In certain embodiments, a compound comprises or consists of ION 916789 or salt thereof, having the following chemical structure (SEQ ID NO: 899):




In certain embodiments, a compound comprises or consists of the sodium salt of 916789, having the following chemical structure (SEQ ID NO: 330):




In certain embodiments, a compound comprises or consists of the sodium salt of 916789, having the following chemical structure (SEQ ID NO: 330):




In certain embodiments, a compound comprises or consists of the sodium salt of 916602, having the following chemical structure (SEQ ID NO: 330):




In any of the foregoing embodiments, the compound or oligonucleotide can be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 1000% complementary to a nucleic acid encoding PNPLA3.
In any of the foregoing embodiments, the compound can be single-stranded. In certain embodiments, the compound comprises deoxyribonucleotides. In certain embodiments, the compound is double-stranded. In certain embodiments, the compound is double-stranded and comprises ribonucleotides. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.
In any of the foregoing embodiments, the compound can be 8 to 80, 10 to 30, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked nucleosides in length. In certain embodiments, the compound comprises or consists of an oligonucleotide.
In certain embodiments, a compound comprises a modified oligonucleotide described herein and a conjugate group. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 5′ end of the modified oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide at the 3′ end of the modified oligonucleotide. In certain embodiments, the conjugate group comprises at least one N-Acetylgalactosamine (GalNAc), at least two N-Acetylgalactosamines (GalNAcs), or at least three N-Acetylgalactosamines (GalNAcs).
In certain embodiments, 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 as described herein are active by virtue of having at least one of an in vitro IC50 of 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, less than 0.1 μM, less than 0.05 μM, less than 0.04 μM, less than 0.03 μM, less than 0.02 μM, or less than 0.01 μM.
In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having at least one of an increase in alanine transaminase (ALT) or aspartate transaminase (AST) value of no more than 4 fold, 3 fold, or 2 fold over control animals, or an increase in liver, spleen, or kidney weight of no more than 30%, 20%, 15%, 12%, 10%, 5%, or 2% compared to control animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase of ALT or AST over control animals. In certain embodiments, the compounds or compositions as described herein are highly tolerable as demonstrated by having no increase in liver, spleen, or kidney weight over control animals.
Certain embodiments provide a composition comprising the compound of any of the aforementioned embodiments or any pharmaceutically acceptable salt thereof and at least one of a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition has a viscosity less than about 40 centipoise (cP), less than about 30 centipose (cP), less than about 20 centipose (cP), less than about 15 centipose (cP), or less than about 10 centipose (cP). In certain embodiments, the composition having any of the aforementioned viscosities comprises a compound provided herein at a concentration of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certain embodiments, the composition having any of the aforementioned viscosities and/or compound concentrations has a temperature of room temperature, or about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C.
Certain Indications
Certain embodiments provided herein relate to methods of inhibiting PNPLA3 expression, which can be useful for treating, preventing, or ameliorating a disease associated with PNPLA3 in an individual, by administration of a compound that targets PNPLA3. In certain embodiments, the compound can be a PNPLA3 specific inhibitor. In certain embodiments, the compound can be an antisense compound, an oligomeric compound, or an oligonucleotide targeted to PNPLA3.
Examples of diseases associated with PNPLA3 treatable, preventable, and/or ameliorable with the methods provided herein include liver disease, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. Certain compounds provided herein are directed to compounds and compositions that reduce liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an animal.
In certain embodiments, a method of treating, preventing, or ameliorating a disease associated with PNPLA3 in an individual comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby treating, preventing, or ameliorating the disease. In certain embodiments, the individual is identified as having, or at risk of having, a disease associated with PNPLA3. In certain embodiments, the disease is a liver disease. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound improves, preserves, or prevents liver damage, steatosis, liver fibrosis, cirrhosis, elevated transaminases, or hepatic fat accumulation in an animal.
In certain embodiments, a method of treating, preventing, or ameliorating liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an animal comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby treating, preventing, or ameliorating liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound improves, preserves, or prevents liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the individual is identified as having, or at risk of having, a disease associated with PNPLA3.
In certain embodiments, a method of inhibiting expression of PNPLA3 in an individual having, or at risk of having, a disease associated with PNPLA3 comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby inhibiting expression of PNPLA3 in the individual. In certain embodiments, administering the compound inhibits expression of PNPLA3 in the liver. In certain embodiments, the disease is a liver disease. In certain embodiments, the individual has, or is at risk of having, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the individual has, or is at risk of having, liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, administering the compound improves, preserves, or prevents liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation.
In certain embodiments, a method of inhibiting expression of PNPLA3 in a cell comprises contacting the cell with a compound comprising a PNPLA3 specific inhibitor, thereby inhibiting expression of PNPLA3 in the cell. In certain embodiments, the cell is a hepatocyte. In certain embodiments, the cell is in the liver. In certain embodiments, the cell is in the liver of an individual who has, or is at risk of having, liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.
In certain embodiments, a method of reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an individual having, or at risk of having, a disease associated with PNPLA3 comprises administering to the individual a compound comprising a PNPLA3 specific inhibitor, thereby reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in the individual. In certain embodiments, the individual has, or is at risk of having, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally. In certain embodiments, the individual is identified as having, or at risk of having, a disease associated with PNPLA3.
Certain embodiments are drawn to a compound comprising a PNPLA3 specific inhibitor for use in treating a disease associated with PNPLA3. In certain embodiments, the disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound. In certain embodiments, the compound is administered to the individual parenterally.
Certain embodiments are drawn to a compound comprising a PNPLA3 specific inhibitor for use in reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an individual having, or at risk of having, NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or oligomeric compound.
Certain embodiments are drawn to the use of a compound comprising a PNPLA3 specific inhibitor for the manufacture or preparation of a medicament for treating a disease associated with PNPLA3. Certain embodiments are drawn to the use of a compound comprising a PNPLA3 specific inhibitor for the preparation of a medicament for treating a disease associated with PNPLA3. In certain embodiments, the disease is a liver disease. In certain embodiments, the disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or an oligomeric compound.
Certain embodiments are drawn to the use of a compound comprising a PNPLA3 specific inhibitor for the manufacture or preparation of a medicament for reducing or inhibiting liver damage, steatosis, liver fibrosis, liver inflammation, liver scarring or cirrhosis, liver failure, liver enlargement, elevated transaminases, or hepatic fat accumulation in an individual having, or at risk of having, a liver disease associated with PNPLA3. In certain embodiments, the liver disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. Certain embodiments are drawn to use of a compound comprising a PNPLA3 specific inhibitor for the preparation of a medicament for treating a disease associated with PNPLA3. In certain embodiments, the disease is NAFLD, hepatic steatosis, non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma, alcoholic liver disease, alcoholic steatohepatitis (ASH), HCV hepatitis, chronic hepatitis, hereditary hemochromatosis, or primary sclerosing cholangitis. In certain embodiments, the compound comprises an antisense compound targeted to PNPLA3. In certain embodiments, the compound comprises an oligonucleotide targeted to PNPLA3. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 12 to 30 linked nucleosides in length and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, a compound comprises a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, a compound comprises a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899. In certain embodiments, the compound is ION 975616, 994284, 975605, 994282, 975613, 975617, 975735, 975736, or 975612. In any of the foregoing embodiments, the compound can be single-stranded or double-stranded. In any of the foregoing embodiments, the compound can be an antisense compound or an oligomeric compound.
In any of the foregoing methods or uses, the compound can be targeted to PNPLA3. In certain embodiments, the compound comprises or consists of a modified oligonucleotide, for example, a modified oligonucleotide 8 to 80 linked nucleosides in length, 10 to 30 linked nucleosides in length, 12 to 30 linked nucleosides in length, or 20 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to any of the nucleobase sequences recited in SEQ ID NOs: 1-10. In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar and/or at least one modified nucleobase. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage, the modified sugar is a bicyclic sugar or a 2′-O-methoxyethyl modified sugar, and the modified nucleobase is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; and a 3′ wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In any of the foregoing embodiments, the modified oligonucleotide is 12 to 30, 15 to 30, 15 to 25, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 19 to 24, 20 to 24, 19 to 22, 20 to 22, 16 to 20, or 16 or 20 linked nucleosides in length. In certain embodiments, the modified oligonucleotide is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to any of the nucleobase sequences recited in SEQ ID NOs: 1-10.
In any of the foregoing methods or uses, the compound comprises or consists of a modified oligonucleotide 16 to 30 linked nucleosides in length and having a nucleobase sequence comprising any one of SEQ ID NOs: 17-2169, wherein the modified oligonucleotide comprises:

    
    
        a gap segment consisting of linked 2′-deoxynucleosides;
        a 5′ wing segment consisting of linked nucleosides; and
        a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

    
    


In any of the foregoing methods or uses, the compound comprises or consists a modified oligonucleotide 16 linked nucleosides in length having a nucleobase sequence comprising the sequence recited in any one of SEQ ID NOs: 1089, 1757, 141, 1982, 330, 1665, 408, 830, and 899, wherein the modified oligonucleotide comprises

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

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage and wherein each cytosine is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide is 16-30 linked nucleosides in length.

    
    


In certain embodiments, a compound comprises or consists of ION 916333 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):




In certain embodiments, a compound comprises or consists of ION 975616 or salt thereof, having the following chemical structure (SEQ ID NO: 1089):




In certain embodiments, a compound comprises or consists of the sodium salt of 975616, having the following chemical structure (SEQ ID NO: 1089):




In certain embodiments, a compound comprises or consists of ION 975613 or salt thereof, having the following chemical structure (SEQ ID NO: 330):




In certain embodiments, a compound comprises or consists of the sodium salt of 975613, having the following chemical structure (SEQ ID NO: 330):




In certain embodiments, a compound comprises or consists of ION 975612 or salt thereof, having the following chemical structure (SEQ ID NO: 899):




In certain embodiments, a compound comprises or consists of the sodium salt of 975612, having the following chemical structure (SEQ ID NO: 899):




In certain embodiments, a compound comprises or consists of ION 916789 or salt thereof, having the following chemical structure (SEQ ID NO: 899):




In certain embodiments, a compound comprises or consists of the sodium salt of 916789, having the following chemical structure (SEQ ID NO: 899):




In certain embodiments, a compound comprises or consists of ION 916602 or salt thereof, having the following chemical structure (SEQ ID NO: 330):




In certain embodiments, a compound comprises or consists of the sodium salt of 916602, having the following chemical structure (SEQ ID NO: 330):




In any of the foregoing methods or uses, the compound can be administered parenterally. For example, in certain embodiments the compound can be administered through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.
Certain Compounds
In certain embodiments, compounds described herein can be antisense compounds. In certain embodiments, the antisense compound comprises or consists of an oligomeric compound. In certain embodiments, the oligomeric compound comprises a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to that of a target nucleic acid.
In certain embodiments, a compound described herein comprises or consists of a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to that of a target nucleic acid.
In certain embodiments, a compound or antisense compound is single-stranded. Such a single-stranded compound or antisense compound comprises or consists of an oligomeric compound. In certain embodiments, such an oligomeric compound comprises or consists of an oligonucleotide and optionally a conjugate group. In certain embodiments, the oligonucleotide is an antisense oligonucleotide. In certain embodiments, the oligonucleotide is modified. In certain embodiments, the oligonucleotide of a single-stranded antisense compound or oligomeric compound comprises a self-complementary nucleobase sequence.
In certain embodiments, compounds are double-stranded. Such double-stranded compounds comprise a first modified oligonucleotide having a region complementary to a 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 by a uracil nucleobase. In certain embodiments, compound comprises a conjugate group. In certain embodiments, one of the modified oligonucleotides is conjugated. In certain embodiments, both the modified oligonucleotides are conjugated. In certain embodiments, the first modified oligonucleotide is conjugated. In certain embodiments, the second modified oligonucleotide is conjugated. In certain embodiments, the first modified oligonucleotide is 16-30 linked nucleosides in length and the second modified oligonucleotide is 16-30 linked nucleosides in length. In certain embodiments, one of the modified oligonucleotides has a nucleobase sequence comprising at least 8 contiguous nucleobases of any of SEQ ID NOs: 17-2169.
In certain embodiments, antisense compounds are double-stranded. Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound. The first oligomeric compound of such double stranded antisense compounds typically comprises or consists of a modified oligonucleotide and optionally a conjugate group. The oligonucleotide of the second oligomeric compound of such a double-stranded antisense compound may be modified or unmodified. Either or both oligomeric compounds of a double-stranded antisense compound may comprise a conjugate group. The oligomeric compounds of double-stranded antisense compounds may include non-complementary overhanging nucleosides.
Examples of single-stranded and double-stranded compounds include, but are not limited to, oligonucleotides, siRNAs, microRNA targeting oligonucleotides, and single-stranded RNAi compounds, such as small hairpin RNAs (shRNAs), single-stranded siRNAs (ssRNAs), and microRNA mimics.
In certain embodiments, a compound described herein has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, a compound described herein comprises an oligonucleotide 12 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 12 to 22 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 14 to 30 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 14 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 15 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 15 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 16 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 16 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 18 to 30 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 18 to 20 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 20 to 30 linked subunits in length. In other words, such oligonucleotides are 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, or 20 to 30 subunits in length, respectively. In certain embodiments, a compound described herein comprises an oligonucleotide 14 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 16 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 17 linked subunits in length. In certain embodiments, compound described herein comprises an oligonucleotide 18 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 19 linked subunits in length. In certain embodiments, a compound described herein comprises an oligonucleotide 20 linked subunits in length. In other embodiments, a compound described herein comprises an oligonucleotide 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the compound described herein comprises an oligonucleotide 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 linked subunits in length, or a range defined by any two of the above values. In some embodiments the linked subunits are nucleotides, nucleosides, or nucleobases.
In certain embodiments, the compound may further comprise additional features 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 where a conjugate group comprises a nucleoside (i.e. a nucleoside that links the conjugate group to the oligonucleotide), the nucleoside of the conjugate group is not counted in the length of the oligonucleotide.
In certain embodiments, compounds may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated compound targeted to a PNPLA3 nucleic acid may have two subunits deleted from the 5′ end, or alternatively, may have two subunits deleted from the 3′ end of the compound. Alternatively, the deleted nucleosides may be dispersed throughout the compound.
When a single additional subunit is present in a lengthened compound, the additional subunit may be located at the 5′ or 3′ end of the compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in a compound having two subunits added to the 5′ end (5′ addition), or alternatively, to the 3′ end (3′ addition) of the compound. Alternatively, the added subunits may be dispersed throughout the compound.
It is possible to increase or decrease the length of a compound, such as an oligonucleotide, and/or introduce mismatch bases without eliminating activity (Woolf et al. Proc. Natl. Acad. Sci. USA 1992, 89:7305-7309; 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 oligonucleotide sequence, chemistry and motif can make large differences in one or more of the many properties required for clinical development (Seth et al. J Med. Chem. 2009, 52, 10; Egli et al. J Am. Chem. Soc. 2011, 133, 16642).
In certain embodiments, compounds described herein are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence-specific RNAi, for example, short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term “RNAi” is meant to be equivalent to other terms used to describe sequence-specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics.
In certain embodiments, a compound described herein can comprise any of the oligonucleotide sequences targeted to PNPLA3 described herein. In certain embodiments, the compound can be double-stranded. In certain embodiments, the compound comprises a first strand comprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion of any one of SEQ ID NOs: 17-2169 and a second strand. In certain embodiments, the compound comprises a first strand comprising the nucleobase sequence of any one of SEQ ID NOs: 17-2169 and a second strand. In certain embodiments, the compound comprises ribonucleotides in which the first strand has uracil (U) in place of thymine (T) in any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises (i) a first strand comprising a nucleobase sequence complementary to the site on PNPLA3 to which any of SEQ ID NOs: 17-2169 is targeted, and (ii) a second strand. In certain embodiments, the compound comprises one or more modified nucleotides in which the 2′ position of the sugar contains a halogen (such as fluorine group; 2′-F) or contains an alkoxy group (such as a methoxy group; 2′-OMe). In certain embodiments, the compound comprises at least one 2′-F sugar modification and at least one 2′-OMe sugar modification. In certain embodiments, the at least one 2′-F sugar modification and at least one 2′-OMe sugar modification are arranged in an alternating pattern 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 a strand of the dsRNA compound. In certain embodiments, the compound comprises one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The compounds may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the compound contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000.
In certain embodiments, the first strand of the compound is an siRNA guide strand and the second strand of the compound is an siRNA passenger strand. In certain embodiments, the second strand of the compound is complementary to the first strand. In certain embodiments, each strand of the compound is 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides in length. In certain embodiments, the first or second strand of the compound can comprise a conjugate group.
In certain embodiments, a compound described herein can comprise any of the oligonucleotide sequences targeted to PNPLA3 described herein. In certain embodiments, the compound is single stranded. In certain embodiments, such a compound is a single-stranded RNAi (ssRNAi) compound. In certain embodiments, the compound comprises at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises the nucleobase sequence of any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises ribonucleotides in which uracil (U) is in place of thymine (T) in any one of SEQ ID NOs: 17-2169. In certain embodiments, the compound comprises a nucleobase sequence complementary to the site on PNPLA3 to which any of SEQ ID NOs: 17-2169 is targeted. In certain embodiments, the compound comprises one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group; 2′-F) or contains an alkoxy group (such as a methoxy group; 2′-OMe). In certain embodiments, the compound comprises at least one 2′-F sugar modification and at least one 2′-OMe sugar modification. In certain embodiments, the at least one 2′-F sugar modification and at least one 2′-OMe sugar modification are arranged in an alternating pattern 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 a strand of the compound. In certain embodiments, the compound comprises one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The compounds may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the compound contains a capped strand, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000. In certain embodiments, the compound consists of 16, 17, 18, 19, 20, 21, 22, or 23 linked nucleosides. In certain embodiments, the compound can comprise a conjugate group.
Certain Mechanisms
In certain embodiments, compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, compounds described herein are antisense compounds. In certain embodiments, compounds comprise oligomeric compounds. In certain embodiments, compounds described herein are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, compounds described herein selectively affect one or more target nucleic acid. Such compounds comprise a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non-target nucleic acid in such a way that results in a significant undesired antisense activity.
In certain antisense activities, hybridization of a compound described herein to a target nucleic acid results in 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 the RNA strand of an RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not be unmodified DNA. In certain embodiments, compounds described herein are sufficiently “DNA-like” to elicit RNase H activity. Further, in certain embodiments, one or more non-DNA-like nucleoside in the gap of a gapmer is tolerated.
In certain antisense activities, compounds described herein or a portion of the compound is loaded into an RNA-induced silencing complex (RISC), ultimately resulting in cleavage of the target nucleic acid. For example, certain compounds described herein result in cleavage of the target nucleic acid by Argonaute. Compounds that are loaded into RISC are RNAi compounds. RNAi compounds may be double-stranded (siRNA) or single-stranded (ssRNA).
In certain embodiments, hybridization of compounds described herein to a target nucleic acid does not result in recruitment of a protein that cleaves that target nucleic acid. In certain such embodiments, hybridization of the compound to the target nucleic acid results in alteration of splicing of the target nucleic acid. In certain embodiments, hybridization of the compound to a target nucleic acid results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of the compound to a target nucleic acid results in alteration of translation of the target nucleic acid.
Antisense activities may be observed directly or indirectly. In certain embodiments, observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a 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, compounds described herein comprise or consist of an oligonucleotide comprising a region that is complementary to a 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 an mRNA and a pre-mRNA, including intronic, exonic 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 is entirely within an intron. In certain embodiments, the target region spans an intron/exon junction. In certain embodiments, the target region is at least 50% within an intron.
Nucleotide sequences that encode PNPLA3 include, without limitation, the following: RefSeq or GENBANK Accession Nos. NM_025225.2 (incorporated by reference, disclosed herein as SEQ ID NO: 1); GENBANK Accession No. NC_000022.11 truncated from nucleotides 43921001 to U.S. Pat. No. 43,954,500 (incorporated by reference, disclosed herein as SEQ ID NO: 2); AK123806.1 (incorporated by reference, disclosed herein as SEQ ID NO: 3); BQ686328.1 (incorporated by reference, disclosed herein as SEQ ID NO: 4); BF762711.1 (incorporated by reference, disclosed herein as SEQ ID NO: 5); DA290491.1 (incorporated by reference, disclosed herein as SEQ ID NO: 6); and the sequences listed as SEQ ID Nos. 7, 8, 9, and 10.
Hybridization
In some embodiments, hybridization occurs between a compound disclosed herein and a PNPLA3 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Hybridization conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the compounds provided herein are specifically hybridizable with a PNPLA3 nucleic acid.
Complementarity
An oligonucleotide is said to be complementary to another nucleic acid when the nucleobase sequence of such oligonucleotide or one or more regions thereof matches the nucleobase sequence of another oligonucleotide or nucleic acid or one or more regions thereof when the two nucleobase sequences are aligned in opposing directions. Nucleobase matches or complementary nucleobases, as 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-methyl cytosine (mC) and guanine (G), unless otherwise specified. Complementary oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside and may include one or more nucleobase mismatches. An oligonucleotide is fully complementary or 100% complementary when such oligonucleotides have nucleobase matches at each nucleoside without any nucleobase mismatches.
In certain embodiments, compounds described herein comprise or consist of modified oligonucleotides. In certain embodiments, compounds described herein are antisense compounds. In certain embodiments, compounds comprise oligomeric compounds. Non-complementary nucleobases between a compound and a PNPLA3 nucleic acid may be tolerated provided that the compound remains able to specifically hybridize to a target nucleic acid. Moreover, a compound may hybridize over one or more segments of a PNPLA3 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the compounds provided herein, or a specified portion thereof are at least, or are up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a PNPLA3 nucleic acid, a target region, target segment, or specified portion thereof. In certain embodiments, the compounds provided herein, or a specified portion thereof, are 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, 95% to 100%, or any number in between these ranges, complementary to a PNPLA3 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of a compound with a target nucleic acid can be determined using routine methods.
For example, a compound in which 18 of 20 nucleobases of the compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, a compound which is 18 nucleobases in length having four non-complementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid. Percent complementarity of a compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, compounds described herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, a compound may be fully complementary to a PNPLA3 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of a compound is complementary to the corresponding nucleobase of a target nucleic acid. For example, a 20 nucleobase compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the compound. “Fully complementary” can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase compound is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the 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 of the compound are also complementary to the target sequence.
In certain embodiments, compounds described herein comprise one or more mismatched nucleobases relative to the target nucleic acid. In certain such embodiments, antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount. Thus, in certain such embodiments, selectivity of the compound is improved. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide having a 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 wing region. In certain such embodiments, the mismatch is at position 4, 3, 2, or 1 from the 3′-end of the wing region. In certain embodiments, the mismatch is specifically positioned within an oligonucleotide not having a 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 a non-complementary nucleobase may be at the 5′ end or 3′ end of the compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer 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 comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PNPLA3 nucleic acid, or specified portion 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, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a PNPLA3 nucleic acid, or specified portion thereof.
In certain embodiments, compounds described herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of a compound. In certain embodiments, the compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 15 nucleobase portion of a target segment. In certain embodiments, the compounds are complementary to at least a 16 nucleobase portion of a target segment. Also contemplated are compounds that are complementary to at least a 9, 10, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
Identity
The compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific ION number, or portion thereof. In certain embodiments, compounds described herein are antisense compounds or oligomeric compounds. In certain embodiments, compounds described herein are modified oligonucleotides. As used herein, a compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the compounds described herein as well as compounds having non-identical bases relative to the compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the compound. Percent identity of an compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, compounds described herein, or portions thereof, are, or are at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the compounds or SEQ ID NOs, or a portion thereof, disclosed herein. In certain embodiments, compounds described herein are about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, or any percentage between such values, to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific ION number, or portion thereof, in which the compounds comprise an oligonucleotide having one or more mismatched nucleobases. 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, compounds described herein comprise or consist of antisense compounds. In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
In certain embodiments, compounds described herein comprise or consist of oligonucleotides. In certain embodiments, a portion of the oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
Certain Modified Compounds
In certain embodiments, compounds described herein comprise or consist of oligonucleotides consisting of linked nucleosides. Oligonucleotides may be unmodified oligonucleotides (RNA or DNA) or may be modified oligonucleotides. Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase) and/or at least one modified internucleoside linkage).
A. Modified Nucleosides
Modified nucleosides comprise a modified sugar moiety or a modified nucleobase or both a modified sugar moiety and a modified nucleobase.

    
    
        1. Modified Sugar Moieties
    
    


In certain embodiments, sugar moieties are non-bicyclic modified sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of other types of modified sugar moieties.
In certain embodiments, modified sugar moieties are non-bicyclic modified furanosyl sugar moieties comprising one or more acyclic substituent, including, but not limited, to substituents at the 2′,4′, and/or 5′ positions. In certain embodiments, the furanosyl sugar moiety is a ribosyl sugar moiety. In certain embodiments, one or more acyclic substituent of non-bicyclic modified sugar moieties is branched. Examples of 2′-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2′-F, 2′-OCH3 (“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3═(“MOE”). In certain embodiments, 2′-substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O—C1-C10 alkoxy, O—C1-C10 substituted alkoxy, O—C1-C10 alkyl, O—C1-C10 substituted alkyl, S-alkyl, N(Rm)-alkyl, O-alkenyl, S-alkenyl, N(Rm)-alkenyl, O-alkynyl, S-alkynyl, N(Rm)-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn) or OCH2C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat. No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al., U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groups suitable for linearly non-bicyclic modified sugar moieties include, but are not limited to, alkoxy (e.g., methoxy), alkyl, and those described in Manoharan et al., WO 2015/106128. Examples of 5′-substituent groups suitable for non-bicyclic modified sugar moieties include, but are not limited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certain embodiments, non-bicyclic modified sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties and the modified sugar moieties and modified nucleosides described in Migawa et al., WO 2008/101157 and Rajeev et al., US2013/0203836.
In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, NH2, N3, OCF3, OCH3, O(CH2)3NH2, CH2CH═CH2, OCH2CH═CH2, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2ON(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (OCH2C(═O)—N(Rm)(Rn)), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-C10 alkyl.
In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclic modified nucleoside comprises a sugar moiety comprising 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, a 2′-substituted nucleoside or 2′-non-bicyclic modified nucleoside comprises a sugar moiety comprising a linear 2′-substituent group selected from: F, OCH3, and OCH2CH2OCH3.
Nucleosides comprising modified sugar moieties, such as non-bicyclic modified sugar moieties, are referred to by the position(s) of the substitution(s) on the sugar moiety of the nucleoside. For example, nucleosides comprising 2′-substituted or 2′-modified sugar moieties are referred to as 2′-substituted nucleosides or 2′-modified nucleosides.
Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. In certain such embodiments, the furanose ring is a ribose ring. Examples of such 4′ to 2′ bridging sugar substituents include, but are not limited to: 4′-CH2-2′,4′-(CH2)2-2′,4′-(CH2)3-2′,4′-CH2—O-2′ (“LNA”), 4′-CH2—S-2′,4′-(CH2)2—O-2′ (“ENA”), 4′-CH(CH3)—O-2′ (referred to as “constrained ethyl” or “cEt” when in the S configuration), 4′-CH2—O—CH2-2′,4′-CH2—N(R)-2′,4′-CH(CH2OCH3)—O-2′ (“constrained MOE” or “cMOE”) and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhat et al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457, and Swayze et al., U.S. Pat. No. 8,022,193), 4′-C(CH3)(CH3)—O-2′ and analogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283), 4′-CH2—N(OCH3)-2′ and analogs thereof (see, e.g., Prakash et al., U.S. Pat. No. 8,278,425), 4′-CH2—O—N(CH3)-2′ (see, e.g., Allerson et al., U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745), 4′-CH2—C(H)(CH3)-2′ (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74, 118-134), 4′-CH2—C(═CH2)-2′ and analogs thereof (see e.g., Seth et al., U.S. Pat. No. 8,278,426), 4′-C(RaRb)—N(R)—O-2′,4′-C(RaRb)—O—N(R)-2′,4′-CH2—O—N(R)-2′, and 4′-CH2—N(R)—O-2′, wherein each R, Ra, and Rb is, independently, H, a protecting group, or C1-C12 alkyl (see, e.g. Imanishi et al., U.S. Pat. No. 7,427,672).
In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from: —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(Ra)═C(Rb), —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—;

    
    
        wherein:
        x is 0, 1, or 2;
        n is 1, 2, 3, or 4;
        each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting 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, 7731-7740, 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 Invens. 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. Pat. No. 7,053,207, Imanishi et al., U.S. Pat. No. 6,268,490, Imanishi et al. U.S. Pat. No. 6,770,748, Imanishi et al., U.S. RE44,779; Wengel et al., U.S. Pat. No. 6,794,499, Wengel et al., U.S. Pat. No. 6,670,461; Wengel et al., U.S. Pat. No. 7,034,133, Wengel et al., U.S. Pat. No. 8,080,644; Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No. 8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al., U.S. Pat. No. 6,525,191, Torsten et al., WO 2004/106356, Wengel et al., WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No. 7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat. No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S. Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al., U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa et al., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; Allerson et al., US2008/0039618; and Migawa et al., US2015/0191727.
In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, an LNA nucleoside (described herein) may be in the α-L configuration or in the β-D configuration.




α-L-methyleneoxy (4′-CH2—O—2′) or α-L-LNA bicyclic nucleosides have been incorporated into oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372). Herein, general descriptions of bicyclic nucleosides include both isomeric configurations. When the positions of specific bicyclic nucleosides (e.g., LNA or cEt) are identified in exemplified embodiments herein, they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars).
In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the sugar moiety is replaced, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moieties also comprise bridging and/or non-bridging substituents as described herein. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat et al., U.S. Pat. No. 7,939,677) and/or the 5′ position.
In certain embodiments, sugar surrogates comprise rings having other than 5 atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”) (see e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoro HNA:




(“F-HNA”, see e.g., Swayze et al., U.S. Pat. No. 8,088,904; Swayze et al., U.S. Pat. No. 8,440,803; and Swayze et al., U.S. Pat. No. 9,005,906) F-HNA can also be referred to as a F-THP or 3′-fluoro tetrahydropyran, and nucleosides comprising additional modified THP compounds having the formula:





wherein, independently, for each of said modified THP nucleoside:

    
    
        Bx is a nucleobase moiety;
        T3 and T4 are each, independently, an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide or one of T3 and T4 is an internucleoside linking group linking the modified THP nucleoside to the remainder of an oligonucleotide and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.
    
    


In certain embodiments, modified THP nucleosides are provided wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, 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, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligonucleotides have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton et al., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444; and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure:




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

In certain embodiments, sugar surrogates comprise acyclic moieties. Examples of nucleosides and oligonucleotides comprising such acyclic sugar surrogates include, but are not limited to: peptide nucleic acid (“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org. Biomol. Chem., 2013, 11, 5853-5865), and nucleosides and oligonucleotides described in Manoharan et al., US2013/130378.
Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used in modified nucleosides.

    
    
        2. Modified Nucleobases
    
    


Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds.
In certain embodiments, compounds described herein comprise modified oligonucleotides. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more nucleosides that does not comprise a nucleobase, referred to as an abasic nucleoside.
In certain embodiments, 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, modified nucleobases are selected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, 5-methylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (C═C—CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly, 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one, and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443.
Publications that teach the preparation of certain of the above noted 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. Pat. No. 4,845,205; Spielvogel et al., U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066; Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat. No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al., U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cook et al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No. 5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al., U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540; Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No. 5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S. Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook et al., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cook et al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903; Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No. 5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al., U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook et al., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No. 6,005,096.
In certain embodiments, compounds targeted to a PNPLA3 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
Modified Internucleoside Linkages
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. In certain embodiments, compounds described herein having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Representative internucleoside linkages having a chiral center include but are not limited to alkylphosphonates and phosphorothioates. Modified oligonucleotides comprising internucleoside linkages having a chiral center can be prepared as populations of modified oligonucleotides comprising stereorandom internucleoside linkages, or as populations of modified oligonucleotides comprising phosphorothioate linkages in particular stereochemical configurations. In certain embodiments, populations of modified oligonucleotides comprise phosphorothioate internucleoside linkages wherein all of the phosphorothioate internucleoside linkages are stereorandom. Such modified oligonucleotides can be generated using synthetic methods that result in random selection of the stereochemical configuration of each phosphorothioate linkage. Nonetheless, as is well understood by those of skill in the art, each individual phosphorothioate of each individual oligonucleotide molecule has a defined stereoconfiguration. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising one or more particular phosphorothioate internucleoside linkages in a particular, independently selected stereochemical configuration. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 65% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 70% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 80% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 90% of the molecules in the population. In certain embodiments, the particular configuration of the particular phosphorothioate linkage is present in at least 99% of the molecules in the population. Such chirally enriched populations of modified oligonucleotides can be generated using synthetic methods known in the art, e.g., methods described in Oka et al., JACS 125, 8307 (2003), Wan et al. Nuc. Acid. Res. 42, 13456 (2014), and WO 2017/015555. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one indicated phosphorothioate in the (Sp) configuration. In certain embodiments, a population of modified oligonucleotides is enriched for modified oligonucleotides having at least one phosphorothioate in the (Rp) configuration. In certain embodiments, modified oligonucleotides comprising (Rp) and/or (Sp) phosphorothioates comprise one or more of the following formulas, respectively, wherein “B” indicates a nucleobase:




Unless otherwise indicated, chiral internucleoside linkages of modified oligonucleotides described herein can be stereorandom or in a particular stereochemical configuration.

In certain embodiments, compounds targeted to a PNPLA3 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
In certain embodiments, compounds described herein comprise oligonucleotides. Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphates, which contain a phosphodiester bond (“P═O”) (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (“P═S”), and phosphorodithioates (“HS—P═S”). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH2—N(CH3)—O—CH2), thiodiester, thionocarbamate (—O—C(═O)(NH)—S—); siloxane (—O—SiH2—O—); and N,N′-dimethylhydrazine (—CH2—N(CH3)—N(CH3)—). Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
Neutral internucleoside linkages include, without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH2—N(CH3)—O—5′), amide-3 (3′-CH2—C(═O)—N(H)-5′), amide-4 (3′-CH2—N(H)—C(═O)-5′), formacetal (3′-O—CH2—O—5′), methoxypropyl, and thioformacetal (3′-S—CH2—O—5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See, for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments, the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The nucleoside motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped nucleoside motif and, if it does have a gapped nucleoside motif, the wing and gap lengths may or may not be the same.
In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.
In certain embodiments, oligonucleotides comprise one or more methylphosphonate linkages. In certain embodiments, oligonucleotides having a gapmer nucleoside motif comprise a linkage motif comprising all phosphorothioate linkages except for one or two methylphosphonate linkages. In certain embodiments, one methylphosphonate linkage is in the central gap of an oligonucleotide having a gapmer nucleoside motif.
In certain embodiments, it is desirable to arrange the number of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, it is desirable to arrange the number and position of phosphorothioate internucleoside linkages and the number and position of phosphodiester internucleoside linkages to maintain nuclease resistance. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased. In certain embodiments, the number of phosphorothioate internucleoside linkages may be decreased and the number of phosphodiester internucleoside linkages may be increased while still maintaining nuclease resistance. In certain embodiments, it is desirable to decrease the number of phosphorothioate internucleoside linkages while retaining nuclease resistance. In certain embodiments, it is desirable to increase the number of phosphodiester internucleoside linkages while retaining nuclease resistance.

    
    
        3. Certain Motifs
    
    


In certain embodiments, compounds described herein comprise oligonucleotides. Oligonucleotides can have a motif, e.g. a pattern of unmodified and/or modified sugar moieties, nucleobases, and/or internucleoside linkages. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified sugar. In certain embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In certain embodiments, modified oligonucleotides comprise one or more modified internucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or internucleoside linkages of a modified oligonucleotide define a pattern or motif. In certain embodiments, the patterns of sugar moieties, nucleobases, and internucleoside linkages are each independent of one another. Thus, a modified oligonucleotide may be described by its sugar motif, nucleobase motif and/or internucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the nucleobases independent of the sequence of nucleobases).

    
    
        a. Certain Sugar Motifs
    
    


In certain embodiments, compounds described herein comprise oligonucleotides. In certain embodiments, oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif. In certain instances, such sugar motifs include, but are not limited to, any of the sugar modifications discussed herein.
In certain embodiments, modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two external regions or “wings” and a central or internal region or “gap”. The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides, wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleosides having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5′-wing differs from the sugar motif of the 3′-wing (asymmetric gapmer).
In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides. In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides. In certain embodiments, the nucleosides of a gapmer are all modified nucleosides.
In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides. In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides. In certain embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiments, each nucleoside of the gap of a gapmer is an unmodified 2′-deoxy nucleoside.
In certain embodiments, the gapmer is a deoxy gapmer. In such embodiments, the nucleosides on the gap side of each wing/gap junction are unmodified 2′-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides. In certain such embodiments, each nucleoside of the gap is an unmodified 2′-deoxy nucleoside. In certain such embodiments, each nucleoside of each wing is a modified nucleoside.
In certain embodiments, a modified oligonucleotide has a fully modified sugar motif wherein each nucleoside of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif wherein each nucleoside of the region comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2′-modification.

    
    
        b. Certain Nucleobase Motifs
    
    


In certain embodiments, compounds described herein comprise oligonucleotides. In certain embodiments, oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases are 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 a modified oligonucleotide are 5-methylcytosines.
In certain embodiments, modified oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments, the block is within 3 nucleosides of 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 within 3 nucleosides of the 5′-end of the oligonucleotide.
In certain embodiments, oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase. In certain such embodiments, one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif. In certain such embodiments, the sugar moiety of said nucleoside is a 2′-deoxyribosyl moiety. In certain embodiments, the modified nucleobase is selected from: a 2-thiopyrimidine and a 5-propynepyrimidine.

    
    
        c. Certain Internucleoside Linkage Motifs
    
    


In certain embodiments, compounds described herein comprise oligonucleotides. In certain embodiments, oligonucleotides comprise modified and/or unmodified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or motif. In certain embodiments, essentially each internucleoside linking group is a phosphate internucleoside linkage (P═O). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is a phosphorothioate (P═S). In certain embodiments, each internucleoside linking group of a modified oligonucleotide is independently selected from a phosphorothioate and phosphate internucleoside linkage. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer and the internucleoside linkages within the gap are all modified. In certain such embodiments, some or all of the internucleoside linkages in the wings are unmodified phosphate linkages. In certain embodiments, the terminal internucleoside linkages are modified. In certain embodiments, the sugar motif of a modified oligonucleotide is a gapmer, and the internucleoside linkage motif comprises at least one phosphodiester internucleoside linkage in at least one wing, wherein the at least one phosphodiester linkage is not a terminal internucleoside linkage, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages. In certain such embodiments, all of the phosphorothioate linkages are stereorandom. In certain embodiments, all of the phosphorothioate linkages in the wings are (Sp) phosphorothioates, and the gap comprises at least one Sp, Sp, Rp motif. In certain embodiments, populations of modified oligonucleotides are enriched for modified oligonucleotides comprising such internucleoside linkage motifs.

    
    
        4. Certain Modified Oligonucleotides
    
    


In certain embodiments, compounds described herein comprise modified oligonucleotides. In certain embodiments, the above modifications (sugar, nucleobase, internucleoside linkage) are incorporated into a modified oligonucleotide. In certain embodiments, modified oligonucleotides are characterized by their modification, motifs, and overall lengths. In certain embodiments, such parameters are each independent of one another. Thus, unless otherwise indicated, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. For example, the internucleoside linkages within the wing regions of a sugar gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region of the sugar motif. Likewise, such gapmer oligonucleotides may comprise one or more modified nucleobases independent of the gapmer pattern of the sugar modifications. Furthermore, in certain instances, an oligonucleotide is described by an overall length or range and by lengths or length ranges of two or more regions (e.g., a regions of nucleosides having specified sugar modifications). In such circumstances, it may be possible to select numbers for each range that result in an oligonucleotide having an overall length falling outside the specified range. In such circumstances, both elements must be satisfied. For example, in certain embodiments, a modified oligonucleotide consists of 15-20 linked nucleosides and has a sugar motif consisting of three regions, A, B, and C, wherein region A consists of 2-6 linked nucleosides having a specified sugar motif, region B consists of 6-10 linked nucleosides having a specified sugar motif, and region C consists of 2-6 linked nucleosides having a specified sugar motif. Such embodiments do not include modified oligonucleotides where A and C each consist of 6 linked nucleosides and B consists of 10 linked nucleosides (even though those numbers of nucleosides are permitted within the requirements for A, B, and C) because the overall length of such oligonucleotide will be 22, which exceeds the upper limit of the overall length of the modified oligonucleotide (20). Herein, if a description of an oligonucleotide is silent with respect to one or more parameters, such parameter is not limited. Thus, a modified oligonucleotide described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase motif. Unless otherwise indicated, all modifications are independent of nucleobase sequence.
Certain Conjugated Compounds
In certain embodiments, the compounds described herein comprise or consist of an oligonucleotide (modified or unmodified) and, optionally, one or more conjugate groups and/or terminal groups. Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In certain embodiments, conjugate groups are attached to the 2′-position of a nucleoside of a modified oligonucleotide. In certain embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups. In certain such embodiments, conjugate groups or terminal groups are attached at the 3′ and/or 5′-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 3′-end of oligonucleotides. In certain embodiments, conjugate groups (or terminal groups) are attached at the 5′-end of oligonucleotides. In certain embodiments, conjugate groups are attached near the 5′-end of oligonucleotides.
In certain embodiments, the oligonucleotide is modified. In certain embodiments, the oligonucleotide of a compound has a nucleobase sequence that is complementary to a target nucleic acid. In certain embodiments, oligonucleotides are complementary to a messenger RNA (mRNA). In certain embodiments, oligonucleotides are complementary to a pre-mRNA. In certain embodiments, oligonucleotides are complementary to a sense transcript.
Examples of terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.
A. Certain Conjugate Groups
In certain embodiments, oligonucleotides are covalently attached to one or more conjugate groups. In certain embodiments, conjugate groups modify one or more properties of the attached oligonucleotide, including, but not limited to, pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance. In certain embodiments, conjugate groups impart a new property on the attached oligonucleotide, e.g., fluorophores or reporter groups that enable detection of the oligonucleotide.
Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic, a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, i, 923-937), a 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. Conjugate Moieties
    
    


Conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
In certain embodiments, a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial, or an antibiotic.

    
    
        2. Conjugate Linkers
    
    


Conjugate moieties are attached to oligonucleotides through conjugate linkers. In certain embodiments, a conjugate group is a single chemical bond (i.e. conjugate moiety is attached to an oligonucleotide via a conjugate linker through a single bond). In certain embodiments, the conjugate linker comprises a chain structure, such as a hydrocarbyl chain, or an oligomer of repeating units, such as ethylene glycol, nucleosides, or amino acid units.
In certain embodiments, a conjugate linker comprises one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino. In certain such embodiments, the conjugate linker comprises groups selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises groups selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus moiety. In certain embodiments, the conjugate linker comprises at least one phosphate group. In certain embodiments, the conjugate linker includes at least one neutral linking group.
In certain embodiments, conjugate linkers, including the conjugate linkers described above, are bifunctional linking moieties, e.g., those known in the art to be useful for attaching conjugate groups to parent compounds, such as the oligonucleotides provided herein. In general, a bifunctional linking moiety comprises at least two functional groups. One of the functional groups is selected to bind to a particular site on a compound and the other is selected to bind to a conjugate group. Examples of functional groups used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In certain embodiments, bifunctional linking moieties comprise one or more groups selected from amino, hydroxyl, carboxylic acid, thiol, alkyl, alkenyl, and alkynyl.
Examples of conjugate linkers include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, but are not limited to, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.
In certain embodiments, conjugate linkers comprise 1-10 linker-nucleosides. In certain embodiments, such linker-nucleosides are modified nucleosides. In certain embodiments, such linker-nucleosides comprise a modified sugar moiety. In certain embodiments, linker-nucleosides are unmodified. In certain embodiments, linker-nucleosides comprise an optionally protected heterocyclic base selected from a purine, substituted purine, pyrimidine or substituted pyrimidine. In certain embodiments, a cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine, 6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. It is typically desirable for linker-nucleosides to be cleaved from the compound after it reaches a target tissue. Accordingly, linker-nucleosides are typically linked to one another and to the remainder of the compound through cleavable bonds. In certain embodiments, such cleavable bonds are phosphodiester bonds.
Herein, linker-nucleosides are not considered to be part of the oligonucleotide. Accordingly, in embodiments in which a compound comprises an oligonucleotide consisting of a specified number or range of linked nucleosides and/or a specified percent complementarity to a reference nucleic acid and the compound also comprises a conjugate group comprising a conjugate linker comprising linker-nucleosides, those linker-nucleosides are not counted toward the length of the oligonucleotide and are not used in determining the percent complementarity of the oligonucleotide for the reference nucleic acid. For example, a compound may comprise (1) a modified oligonucleotide consisting of 8-30 nucleosides and (2) a conjugate group comprising 1-10 linker-nucleosides that are contiguous with the nucleosides of the modified oligonucleotide. The total number of contiguous linked nucleosides in such a compound is more than 30. Alternatively, a compound may comprise a modified oligonucleotide consisting of 8-30 nucleosides and no conjugate group. The total number of contiguous linked nucleosides in such a compound is no more than 30. Unless otherwise indicated, conjugate linkers comprise no more than 10 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 5 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 3 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 2 linker-nucleosides. In certain embodiments, conjugate linkers comprise no more than 1 linker-nucleoside.
In certain embodiments, it is desirable for a conjugate group to be cleaved from the oligonucleotide. For example, in certain circumstances, compounds comprising a particular conjugate moiety are better taken up by a particular cell type, but once the compound has been taken up, it is desirable that the conjugate group be cleaved to release the unconjugated or parent oligonucleotide. Thus, certain conjugates may comprise one or more cleavable moieties, typically within the conjugate linker. In certain embodiments, a cleavable moiety is a cleavable bond. In certain embodiments, a cleavable moiety is a group of atoms comprising at least one cleavable bond. In certain embodiments, a cleavable moiety comprises a group of atoms having one, two, three, four, or more than four cleavable bonds. In certain embodiments, a cleavable moiety is selectively cleaved inside a cell or subcellular compartment, such as a lysosome. In certain embodiments, a cleavable moiety is selectively cleaved by endogenous enzymes, such as nucleases.
In certain embodiments, a cleavable bond is selected from among: an amide, an ester, an ether, one or both esters of a phosphodiester, a phosphate ester, a carbamate, or a disulfide. In certain embodiments, a cleavable bond is one or both of the esters of a phosphodiester. In certain embodiments, a cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is a phosphate linkage between an oligonucleotide and a conjugate moiety or conjugate group.
In certain embodiments, a cleavable moiety comprises or consists of one or more linker-nucleosides. In certain such embodiments, one or more linker-nucleosides are linked to one another and/or to the remainder of the compound through cleavable bonds. In certain embodiments, such cleavable bonds are unmodified phosphodiester bonds. In certain embodiments, a cleavable moiety is 2′-deoxy nucleoside that is attached to either the 3′ or 5′-terminal nucleoside of an oligonucleotide by a phosphate internucleoside linkage and covalently attached to the remainder of the conjugate linker or conjugate moiety by a phosphate or phosphorothioate linkage. In certain such embodiments, the cleavable moiety is 2′-deoxyadenosine.

    
    
        3. Certain Cell-Targeting Conjugate Moieties
    
    


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




    
    
        wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2 or greater, 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, 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, conjugate groups comprise cell-targeting moieties that have at least one tethered ligand. In certain embodiments, cell-targeting moieties comprise two tethered ligands covalently attached to a branching group. In certain embodiments, cell-targeting moieties comprise three tethered ligands covalently attached to a branching group.
In certain embodiments, the cell-targeting moiety comprises a branching group comprising one or more groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain embodiments, the branching group comprises a branched aliphatic group comprising groups selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino, oxo, amide, and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl, amino, and ether groups. In certain such embodiments, the branched aliphatic group comprises groups selected from alkyl and ether groups. In certain embodiments, the branching group comprises a mono or polycyclic ring system.
In certain embodiments, each tether of a cell-targeting moiety comprises 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 tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, thioether, disulfide, amino, oxo, amide, and polyethylene glycol, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, phosphodiester, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, ether, amino, oxo, and amide, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl, amino, and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and oxo, in any combination. In certain embodiments, each tether is a linear aliphatic group comprising one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether comprises at least one phosphorus linking group or neutral linking group. In certain embodiments, each tether comprises a chain from about 6 to about 20 atoms in length. In certain embodiments, each tether comprises a chain from about 10 to about 18 atoms in length. In certain embodiments, each tether comprises about 10 atoms in chain length.
In certain embodiments, each ligand of a cell-targeting moiety has an affinity for at least one type of receptor on a 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 hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is, independently selected from galactose, N-acetyl galactoseamine (GalNAc), mannose, glucose, glucoseamine, and fucose. In certain embodiments, each ligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, the cell-targeting moiety comprises 3 GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises 2 GalNAc ligands. In certain embodiments, the cell-targeting moiety comprises 1 GalNAc ligand.
In certain embodiments, each ligand of a cell-targeting moiety is a carbohydrate, carbohydrate derivative, modified carbohydrate, polysaccharide, modified polysaccharide, or polysaccharide derivative. In certain such embodiments, the conjugate group comprises a carbohydrate cluster (see, e.g., Maier et al., “Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29, or Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004, 47, 5798-5808, 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, 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-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, and N-glycoloyl-α-neuraminic acid. For example, thio sugars may be selected from 5-Thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.
In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:




In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:




In certain embodiments, conjugate groups comprise a cell-targeting moiety having the formula:




In certain embodiments, compounds described herein comprise a conjugate group described herein as “LICA-1”. LICA-1 is shown below without the optional cleavable moiety at the end of the conjugate linker:




In certain embodiments, compounds described herein comprise LICA-1 and a cleavable moiety within the conjugate linker have the formula:




    
    
        wherein ‘oligo’ is an oligonucleotide.
    
    


Representative publications that teach the preparation of certain of the above noted conjugate groups and compounds comprising conjugate groups, tethers, conjugate linkers, branching groups, ligands, cleavable moieties as well as other modifications include, without limitation, U.S. Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177, 7,491,805, 8,106,022, 7,723,509, 9,127,276, US 2006/0148740, US 2011/0123520, WO 2013/033230 and WO 2012/037254, Biessen et al., J. Med. 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 by reference herein in its entirety.
In certain embodiments, compounds described herein comprise modified oligonucleotides comprising a gapmer or fully modified motif and a conjugate group comprising at least one, two, or three GalNAc ligands. In certain embodiments, compounds described herein comprise a conjugate group found in any of the following references: Lee, Carbohydr Res, 1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945; Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al., Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4, 317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessen et al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron, 1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490; Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol, 2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584; Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al., Glycoconj J, 2004, 21, 227-241; Lee et al., BioorgMed 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., BioorgMed 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., JAm Chem Soc, 2004, 126, 14013-14022; Lee et al., JOrg Chem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792; Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., Methods Enzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14, 18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan, Antisense Nucleic Acid Drug Dev, 2002, 12, 103-128; Merwin et al., Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013, 21, 5275-5281; International applications WO1998/013381; WO2011/038356; WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254; WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947; WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046; WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013; WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709; WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406; WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat. Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319; 8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812; 6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772; 8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182; 6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. Patent Application Publications US2011/0097264; US2011/0097265; US2013/0004427; US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730; US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814; US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393; US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075; US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938; US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968; US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132; each of which is incorporated by reference in its entirety.
Compositions and Methods for Formulating Pharmaceutical Compositions
Compounds described herein may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Certain embodiments provide pharmaceutical compositions comprising 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 a modified oligonucleotide. In certain such embodiments, the pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one compound and sterile water. In certain embodiments, the sterile water is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more compounds and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more compound and sterile PBS. In certain embodiments, the sterile PBS is pharmaceutical grade PBS. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
A compound described herein targeted to PNPLA3 nucleic acid can be utilized in pharmaceutical compositions by combining the compound with a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutically acceptable diluent is water, such as sterile water suitable for injection. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising a compound targeted to PNPLA3 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 provided herein.
Pharmaceutical compositions comprising compounds provided herein encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. 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 disclosure is also drawn to pharmaceutically acceptable salts of compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of a compound which are cleaved by endogenous nucleases within the body, to form the active compound.
In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.
Certain Selected Compounds
Approximately 2,384 newly designed compounds of various lengths, chemistries, and motifs were tested for their effect on human PNPLA3 mRNA in vitro in several cell types (Example 1). Of 2,384 compounds tested for potency at a single dose in vitro, over 400 selected compounds were tested for dose dependent inhibition in A431 cells (Example 2). Of the over 400 compounds tested by dose response assays, the compounds were further screened for high dose tolerability in a BALB/c mouse model and 87 oligonucleotides were selected for in vivo efficacy in a PNPLA3 transgenic mouse model.
Of the 87 oligonucleotides tested in the transgenic mouse model, 23 oligonucleotides were selected to be further tested for tolerability in preclinical rodel models. In the in vivo rodent tolerability models, body weights and organ weights, liver function markers (such as alanine transaminase, aspartate transaminase and bilirubin), and kidney function markers (such as BUN and creatinine) were measured. In the CD1 mouse model and in the Sprague-Dawley rat model, ION 975591, 975605, 975612, 975613, 975616, 975617, 975735, 975736, 994282, and 994284 were found tolerable (Examples 5 and 6).
These compounds were further tested for efficacy in multi-dose assays in PNPLA3 transgenic mice (Example 7).
IONs 994284, 97605, 975616, 994282, 975613, 975617, 975735, 975736, and 975612 were tested for tolerability in cynomolgus monkeys (Example 8). Treatment with the compounds was well tolerated in the monkeys.
Accordingly, provided herein are compounds with any one or more of the improved properties. In certain embodiments, the compounds as described herein are potent and tolerable.
EXAMPLES
The Examples below describe the screening process to identify lead compounds targeted to PNPLA3. ION 994284, 97605, 975616, 994282, 975613, 975617, 975735, 975736, and 975612 resulted in high potency and tolerability.
Non-Limiting Disclosure and Incorporation by Reference
Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).
Accordingly, nucleic acid sequences provided herein, including, but not limited to, those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to, such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and compounds having other modified nucleobases, such as “ATmCGAUCG,” wherein mC indicates a cytosine base comprising a methyl group at the 5-position.
Certain compounds described herein (e.g. modified oligonucleotides) have one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), as a or R, such as for sugar anomers, or as (D) or (L), such as for amino acids, etc. Compounds provided herein that are drawn or described as having certain stereoisomeric configurations include only the indicated compounds. Compounds provided herein that are drawn or described with undefined stereochemistry include all such possible isomers, including their stereorandom and optically pure forms. Likewise, all tautomeric forms of the compounds provided herein are included unless otherwise indicated. Unless otherwise indicated, oligomeric compounds and modified oligonucleotides described herein are intended to include corresponding salt forms.
Compounds described herein include variations in which one or more atoms are replaced with a non-radioactive isotope or radioactive isotope of the indicated element. For example, compounds herein that comprise hydrogen atoms encompass all possible deuterium substitutions for each of the 1H hydrogen atoms. Isotopic substitutions encompassed by the compounds herein include, but are not limited to: 2H or 3H in place of 1H, 13C or 14C in place of 12C, 15N in place of 14N, 17O or 18O in place of 16O, and 33S, 34S, 35S, or 36S in place of 32S.
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1: Antisense Inhibition of Human PNPLA3 in A431 Cells
Antisense oligonucleotides were designed targeting a PNPLA3 nucleic acid and were tested for their effects on PNPLA3 mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers. The gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human PNPLA3 mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_025225.2) or the human PNPLA3 genomic sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NC_000022.11 truncated from nucleotides 43921001 to 43954500). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.
Study 1
Cultured A431 cells at a density of 20,000 cells per well were transfected by free uptake with 4,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PNPLA3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS36070 (forward sequence CCTTGGTATGTTCCTGCTTCA, designated herein as SEQ ID NO: 11; reverse sequence GTTGTCACTCACTCCTCCATC, designated herein as SEQ ID NO: 12; probe sequence TGGCCTTATCCCTCCTTCCTTCAGA, designated herein as SEQ ID NO: 13) was used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.







TABLE 1







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















912709
27
42
2765
2780
GGCATTCCCAGCGCGA
0
17


 


912710
95
110
2833
2848
TCCTGATCCGCAGCAG
15
18


 


912711
103
118
2841
2856
GGCTCGGGTCCTGATC
0
19


 


912712
131
146
2869
2884
GTTAGGATCTGGGTCG
91
20


 


912713
164
179
2902
2917
GTACATGGCGGCGGCG
0
21


 


912714
183
198
2921
2936
TCCAGCCGCGCTCTGC
23
22


 


912715
196
211
2934
2949
GCGAAGGACAAGCTCC
60
23


 


912716
197
212
2935
2950
CGCGAAGGACAAGCTC
0
24


 


912717
272
287
3010
3025
GCGGAGGAGGTGCGGG
0
25


 


912718
273
288
3011
3026
CGCGGAGGAGGTGCGG
0
26


 


912719
274
289
3012
3027
TCGCGGAGGAGGTGCG
19
27


 


912720
290
305
3028
3043
GAACAACATGCGCGCG
0
28


 


912721
291
306
3029
3044
CGAACAACATGCGCGC
7
29


 


912722
292
307
3030
3045
CCGAACAACATGCGCG
21
30


 


912723
293
308
3031
3046
GCCGAACAACATGCGC
0
31


 


912724
294
309
3032
3047
CGCCGAACAACATGCG
0
32


 


912725
323
338
3061
3076
GCCGACGCAGTGCAAC
0
33


 


912726
324
339
3062
3077
CGCCGACGCAGTGCAA
0
34


 


912727
340
355
3078
3093
GGGATACCGGAGAGGA
43
35


 


912728
370
385
5944
5959
TCTGAGAGGACCTGCA
53
36


 


912729
375
390
5949
5964
CAAGATCTGAGAGGAC
64
37


 


912730
404
419
5978
5993
GCCAATGTTCCGACTC
71
38


 


912731
410
425
5984
5999
GAAGATGCCAATGTTC
51
39


 


912732
429
444
6003
6018
TTAAGTTGAAGGATGG
96
40


 


912733
432
447
6006
6021
TGCTTAAGTTGAAGGA
90
41


 


912734
478
493
6052
6067
TGGACATTGGCCGGGA
85
42


 


912735
479
494
6053
6068
GTGGACATTGGCCGGG
50
43


 


912736
484
499
6058
6073
AGCTGGTGGACATTGG
64
44


 


912737
528
543
6102
6117
CATCAGACACTCTGGT
5
45


 


912738
531
546
6105
6120
CCCCATCAGACACTCT
73
46


 


912739
552
567
6126
6141
AGTCAGACACCAGAAC
54
47


 


912755
693
708
11911
11926
TGGCATCAATGAAGGG
74
48


 


912756
698
713
11916
11931
TGTTTTGGCATCAATG
91
49


 


912757
746
761
11964
11979
TTTAGGGCAGATGTCG
89
50


 


912758
747
762
11965
11980
CTTTAGGGCAGATGTC
90
51


 


912759
795
810
12013
12028
GTAGACTGAGCTTGGT
98
52


 


912760
820
835
12038
12053
AGGTAGAGGTTCCCTG
0
53


 


912761
841
856
12059
12074
GGGACAAAAGCTCTCG
20
54


 


912762
873
888
13609
13624
GGCATATCTCTCCCAG
0
55


 


912763
874
889
13610
13625
AGGCATATCTCTCCCA
0
56


 


912764
886
901
13622
13637
AAATATCCTCGAAGGC
57
57


 


912765
888
903
13624
13639
CCAAATATCCTCGAAG
30
58


 


912766
889
904
13625
13640
TCCAAATATCCTCGAA
38
59


 


912767
894
909
13630
13645
ATGCATCCAAATATCC
58
60


 


912768
925
940
N/A
N/A
TTGCAGATGCCCTTCT
15
61


 


912769
968
983
16088
16103
ATCCATCCCTTCTGAG
34
62


 


912770
986
1001
16106
16121
GGGCATGGCGACCTCA
0
63


 


912771
1004
1019
16124
16139
ACTCATGTTTGCCCAG
67
64


 


912772
1068
1083
16188
16203
GGTCTAGCAGCTCATC
89
65


 


912773
1075
1090
16195
16210
CGCAGGTGGTCTAGCA
0
66


 


912774
1076
1091
16196
16211
ACGCAGGTGGTCTAGC
25
67


 


912775
1080
1095
16200
16215
TGAGACGCAGGTGGTC
50
68


 


912776
1086
1101
16206
16221
GGATGCTGAGACGCAG
67
69


 


912777
1172
1187
19012
19027
GTATCCACCTTTGTCT
78
70


 


912778
1178
1193
19018
19033
GCTCATGTATCCACCT
79
71


 


912779
1187
1202
19027
19042
GCAAATCTTGCTCATG
3
72


 


912780
1188
1203
19028
19043
TGCAAATCTTGCTCAT
13
73


 


912781
1189
1204
19029
19044
TTGCAAATCTTGCTCA
0
74


 


912782
1195
1210
19035
19050
AGCAAGTTGCAAATCT
77
75


 


912783
1199
1214
19039
19054
GGGTAGCAAGTTGCAA
74
76


 


912784
1205
1220
19045
19060
CCTAATGGGTAGCAAG
62
77


 


912785
1206
1221
19046
19061
TCCTAATGGGTAGCAA
79
78
















TABLE 2







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















912786
1207
1222
19047
19062
ATCCTAATGGGTAGCA
81
79


 


912787
1211
1226
19051
19066
CATTATCCTAATGGGT
46
80


 


912788
1212
1227
19052
19067
ACATTATCCTAATGGG
0
81


 


912789
1213
1228
19053
19068
GACATTATCCTAATGG
70
82


 


912790
1220
1235
19060
19075
TACATAAGACATTATC
34
83


 


912791
1224
1239
19064
19079
GCATTACATAAGACAT
86
84


 


912792
1245
1260
19085
19100
CCACAGGCAGGGTACA
76
85


 


912793
1246
1261
19086
19101
TCCACAGGCAGGGTAC
28
86


 


912794
1253
1268
19093
19108
GGCAGATTCCACAGGC
75
87


 


912795
1259
1274
19099
19114
CGCAATGGCAGATTCC
92
88


 


912796
1265
1280
19105
19120
GACAATCGCAATGGCA
64
89


 


912797
1266
1281
19106
19121
GGACAATCGCAATGGC
75
90


 


912798
1267
1282
19107
19122
TGGACAATCGCAATGG
73
91


 


912799
1285
1300
23690
23705
AGCCATGTCACCAGTC
67
92


 


912800
1289
1304
23694
23709
TGGAAGCCATGTCACC
24
93


 


912801
1290
1305
23695
23710
CTGGAAGCCATGTCAC
72
94


 


912802
1297
1312
23702
23717
GGCATATCTGGAAGCC
0
95


 


912803
1298
1313
23703
23718
GGGCATATCTGGAAGC
0
96


 


912804
1351
1366
23756
23771
AGCACTCGAGTGAACA
0
97


 


912805
1386
1401
N/A
N/A
GCATTTGGGACCTGGA
77
98


 


912806
1387
1402
N/A
N/A
GGCATTTGGGACCTGG
60
99


 


912807
1388
1403
25151
25166
TGGCATTTGGGACCTG
41
100


 


912808
1394
1409
25157
25172
GCTCACTGGCATTTGG
44
101


 


912809
1523
1538
25286
25301
GTTCAGGCTGGACCTG
49
102


 


912810
1547
1562
25310
25325
AGGTACTTTATTGCCC
11
103


 


912811
1550
1565
25313
25328
AGCAGGTACTTTATTG
64
104


 


912812
1653
1668
25416
25431
AACTTTAGCACCTCTG
91
105


 


912813
1655
1670
25418
25433
GAAACTTTAGCACCTC
88
106


 


912814
1656
1671
25419
25434
GGAAACTTTAGCACCT
53
107


 


912815
1669
1684
25432
25447
CTGCACAAAGATGGGA
80
108


 


912816
1671
1686
25434
25449
AGCTGCACAAAGATGG
45
109


 


912817
1685
1700
25448
25463
AGCAATGCGGAGGTAG
15
110


 


912818
1740
1755
25503
25518
ACCAACTCAGCTCAGA
85
111


 


912819
1741
1756
25504
25519
AACCAACTCAGCTCAG
79
112


 


912820
1757
1772
25520
25535
TCCTAGCTTTTCATAA
23
113


 


912821
1788
1803
25551
25566
TGCTGGACCGCTGCAC
0
114


 


912822
1796
1811
25559
25574
GAGTTAAGTGCTGGAC
93
115


 


912823
1802
1817
25565
25580
GTATTAGAGTTAAGTG
92
116


 


912824
1803
1818
25566
25581
TGTATTAGAGTTAAGT
79
117


 


912825
1806
1821
25569
25584
TGATGTATTAGAGTTA
92
118


 


912826
1808
1823
25571
25586
GCTGATGTATTAGAGT
80
119


 


912827
1821
1836
25584
25599
TGAATTAACGCATGCT
83
120


 


912828
1822
1837
25585
25600
CTGAATTAACGCATGC
78
121


 


912829
1870
1885
25633
25648
AGTAAGGGACCCTCTG
17
122


 


912830
1871
1886
25634
25649
CAGTAAGGGACCCTCT
28
123


 


912831
1872
1887
25635
25650
TCAGTAAGGGACCCTC
77
124


 


912832
1874
1889
25637
25652
AGTCAGTAAGGGACCC
51
125


 


912833
1893
1908
25656
25671
ATTAATAGGGCCACGA
80
126


 


912834
1895
1910
25658
25673
CCATTAATAGGGCCAC
90
127


 


912835
1896
1911
25659
25674
ACCATTAATAGGGCCA
81
128


 


912836
1906
1921
25669
25684
GAACAGTCTGACCATT
82
129


 


912837
1908
1923
25671
25686
TGGAACAGTCTGACCA
31
130


 


912838
1909
1924
25672
25687
CTGGAACAGTCTGACC
83
131


 


912839
1911
1926
25674
25689
TGCTGGAACAGTCTGA
72
132


 


912840
1916
1931
25679
25694
CCTCATGCTGGAACAG
83
133


 


912841
1928
1943
25691
25706
TCATTCTAAGAACCTC
96
134


 


912842
1945
1960
25708
25723
ACCCATCCAAACACCT
16
135


 


912843
1982
1997
25745
25760
ACACATGGGCCAGCCT
70
136


 


912844
1989
2004
25752
25767
CAAGATCACACATGGG
70
137


 


912845
2057
2072
25820
25835
GGGACGAACTGCACCC
0
138


 


912846
2098
2113
25861
25876
TATCATCTTTGCAGAC
81
139


 


912847
2116
2131
25879
25894
GTTTTTAGTAGTCAAG
91
140


 


912848
2117
2132
25880
25895
CGTTTTTAGTAGTCAA
91
141


 


912849
2145
2160
25908
25923
TATCATCTTGTTACCC
85
142


 


912850
2148
2163
25911
25926
GATTATCATCTTGTTA
70
143


 


912851
2150
2165
25913
25928
TAGATTATCATCTTGT
53
144


 


912852
2151
2166
25914
25929
GTAGATTATCATCTTG
80
145


 


912853
2152
2167
25915
25930
AGTAGATTATCATCTT
84
146


 


912854
2175
2190
25938
25953
GTGAAAAAGGTGTTCT
77
147


 


912855
2182
2197
25945
25960
TAGTTAGGTGAAAAAG
92
148


 


912856
2188
2203
25951
25966
TTATTTTAGTTAGGTG
88
149


 


912857
2190
2205
25953
25968
CATTATTTTAGTTAGG
86
150


 


912858
2273
2288
26036
26051
CTACTAACATCTCACT
55
151


 


912859
2274
2289
26037
26052
TCTACTAACATCTCAC
89
152


 


912860
2278
2293
26041
26056
TTATTCTACTAACATC
27
153


 


912861
2280
2295
26043
26058
GCTTATTCTACTAACA
79
154


 


912862
2281
2296
26044
26059
GGCTTATTCTACTAAC
81
155


 


912863
2632
2647
26395
26410
GGTGAATGCCCTGCAC
41
156
















TABLE 3







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















912864
2703
2718
26466
26481
TTCAAGTTGTGTGCTC
90
157


 


912865
2755
2770
26518
26533
GGGAGAAACTCACTGA
37
158


 


912866
N/A
N/A
4416
4431
TGCTACTTGCCCCAGC
2
159


 


912867
N/A
N/A
4421
4436
CACAATGCTACTTGCC
87
160


 


912868
N/A
N/A
4584
4599
CCCAATGGCAGGGCTT
58
161


 


912869
N/A
N/A
4592
4607
TGCTCCTACCCAATGG
46
162


 


912870
N/A
N/A
4766
4781
GACTTTTATTGTTGCT
95
163


 


912871
N/A
N/A
4883
4898
TTCTATACCAGAGTGA
89
164


 


912872
N/A
N/A
4884
4899
TTTCTATACCAGAGTG
89
165


 


912873
N/A
N/A
5405
5420
GTAGATGGCCTTAATG
83
166


 


912876
N/A
N/A
6155
6170
TACATCCACGACTTCG
94
167


 


912877
N/A
N/A
6156
6171
TTACATCCACGACTTC
76
168


 


912880
N/A
N/A
6606
6621
GGAACATTCAGGGTTT
13
169


 


912881
N/A
N/A
6834
6849
ATTACTTGGGTGCAGG
55
170


 


912884
N/A
N/A
6838
6853
GCAGATTACTTGGGTG
45
171


 


912885
N/A
N/A
6931
6946
TGCAGGACAGGTTCCT
30
172


 


912888
N/A
N/A
7549
7564
CACACTGGGTCACCAC
55
173


 


912889
N/A
N/A
7552
7567
AGTCACACTGGGTCAC
61
174


 


912928
N/A
N/A
12273
12288
GGTATATGTTCCCAGG
87
175


 


912929
N/A
N/A
12314
12329
TATAACCACAGCCTGG
29
176


 


912932
N/A
N/A
12321
12336
CTGACTATATAACCAC
81
177


 


912933
N/A
N/A
12666
12681
ATCTTAGTGGCTGGGT
91
178


 


912936
N/A
N/A
12767
12782
CTTACTATGGTAGAGT
88
179


 


912937
N/A
N/A
12768
12783
TCTTACTATGGTAGAG
74
180


 


912940
N/A
N/A
12835
12850
TGCATTGCATAGCCTT
97
181


 


912941
N/A
N/A
12836
12851
TTGCATTGCATAGCCT
96
182


 


912944
N/A
N/A
12907
12922
TGCTTATAAAGCACAC
61
183


 


912945
N/A
N/A
12988
13003
GGAATAAGCCTCCACC
14
184


 


912948
N/A
N/A
14055
14070
GAAATCTGATTGCTTC
59
185


 


912949
N/A
N/A
14393
14408
TACTTATCTGCTCACT
66
186


 


912952
N/A
N/A
14673
14688
TCTCTTAGTGTCCCCA
90
187





14707
14722





 


912953
N/A
N/A
14674
14689
ATCTCTTAGTGTCCCC
92
188





14708
14723





 


912956
N/A
N/A
15284
15299
TCACATTCATGCTTGC
82
189


 


912957
N/A
N/A
15291
15306
GATAACCTCACATTCA
0
190


 


912960
N/A
N/A
15712
15727
GAGCTAGGTGCTTCAC
6
191


 


912961
N/A
N/A
15753
15768
ATAACAACTGAACCAC
85
192


 


912964
N/A
N/A
15937
15952
GTTATTAGCCAAATGC
92
193


 


912965
N/A
N/A
16468
16483
GGAGACTTGGCAAGGT
87
194


 


912968
N/A
N/A
16960
16975
ATTCATGACAGCCCTT
46
195


 


912969
N/A
N/A
17128
17143
ATCGATTTTTCAGAGT
9
196


 


912972
N/A
N/A
17134
17149
ACAAACATCGATTTTT
52
197


 


912973
N/A
N/A
17769
17784
CTCTTTAATGACCTCG
90
198


 


912976
N/A
N/A
18865
18880
GTCAGAGGCACTCACA
25
199


 


912977
N/A
N/A
18959
18974
AGCTATTATCTCCCAC
0
200


 


912980
N/A
N/A
19315
19330
AGTTTCTGGGCTTGCA
90
201


 


912981
N/A
N/A
19382
19397
GGCAATCACAAGAGAC
73
202


 


912984
N/A
N/A
20286
20301
AGAGGAAGCCCAATCA
79
203





20316
20331





 


912985
N/A
N/A
20287
20302
CAGAGGAAGCCCAATC
93
204





20317
20332





 


912988
N/A
N/A
20658
20673
TAGAAATTGCAGTGCC
92
205


 


912989
N/A
N/A
20731
20746
TCCTATCCATATATTG
55
206


 


912992
N/A
N/A
21408
21423
GCAATTCTAGACATGG
88
207


 


912993
N/A
N/A
21558
21573
AGGACTTACACCAAGA
86
208


 


912996
N/A
N/A
21936
21951
TTCCTAATAAGAGCCC
24
209


 


912997
N/A
N/A
21946
21961
GTCAAACATCTTCCTA
66
210


 


913000
N/A
N/A
22077
22092
AAAACTGTAGGATAGG
47
211


 


913001
N/A
N/A
22162
22177
GTTACATCCATAAAAC
0
212


 


913004
N/A
N/A
22169
22184
AGAGAATGTTACATCC
62
213


 


913008
N/A
N/A
23083
23098
AAAGATTAATCAGGGC
61
214


 


913012
N/A
N/A
23788
23803
GTATTTACCTGGAGGC
0
215


 


913016
N/A
N/A
24426
24441
GGCCTATGATTTTCAG
0
216
















TABLE 4







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















912874
N/A
N/A
5869
5884
ATACTTTTGGCAAGGC
96
217


 


912875
N/A
N/A
5870
5885
AATACTTTTGGCAAGG
91
218


 


912878
N/A
N/A
6159
6174
TGCTTACATCCACGAC
12
219


 


912879
N/A
N/A
6296
6311
CATCATGTTGGTCTCG
54
220


 


912882
N/A
N/A
6835
6850
GATTACTTGGGTGCAG
39
221


 


912883
N/A
N/A
6837
6852
CAGATTACTTGGGTGC
69
222


 


912886
N/A
N/A
7083
7098
TTTAATGGTGTTTTGG
87
223


 


912887
N/A
N/A
7478
7493
TCAAATGCCGGTATTC
52
224


 


912890
N/A
N/A
7587
7602
GTGAACTTCAACTTCC
56
225


 


912930
N/A
N/A
12317
12332
CTATATAACCACAGCC
77
226


 


912931
N/A
N/A
12319
12334
GACTATATAACCACAG
92
227


 


912934
N/A
N/A
12670
12685
AATCATCTTAGTGGCT
91
228


 


912935
N/A
N/A
12765
12780
TACTATGGTAGAGTGG
80
229


 


912938
N/A
N/A
12786
12801
GTACATGGTCTGCAAA
84
230


 


912939
N/A
N/A
12787
12802
TGTACATGGTCTGCAA
57
231


 


912942
N/A
N/A
12843
12858
GCATGCATTGCATTGC
16
232


 


912943
N/A
N/A
12885
12900
ACCAATCCTGTTAGAC
93
233


 


912946
N/A
N/A
13557
13572
GGAGACACCAAGCACC
42
234


 


912947
N/A
N/A
13751
13766
GCACTAAGTGTTAGAA
79
235


 


912950
N/A
N/A
14396
14411
GCTTACTTATCTGCTC
0
236


 


912951
N/A
N/A
14501
14516
GGAGATCCATCCTGCA
0
237


 


912954
N/A
N/A
14675
14690
CATCTCTTAGTGTCCC
92
238





14709
14724





 


912955
N/A
N/A
15122
15137
TCCTAATGTCCTCAAC
9
239


 


912958
N/A
N/A
15293
15308
AAGATAACCTCACATT
33
240


 


912959
N/A
N/A
15294
15309
CAAGATAACCTCACAT
22
241


 


912962
N/A
N/A
15754
15769
TATAACAACTGAACCA
82
242


 


912963
N/A
N/A
15856
15871
GCTTTAAAGCAGGACA
8
243


 


912966
N/A
N/A
16774
16789
AAAATTGTGGGTTTAG
68
244


 


912967
N/A
N/A
16850
16865
ATCATTTGGACCATAG
81
245


 


912970
N/A
N/A
17130
17145
ACATCGATTTTTCAGA
83
246


 


912971
N/A
N/A
17133
17148
CAAACATCGATTTTTC
62
247


 


912974
N/A
N/A
17843
17858
GCTTTACAAGCTGGTC
0
248


 


912975
N/A
N/A
17879
17894
ATCTATGTTCTCCTAG
0
249


 


912978
N/A
N/A
19125
19140
ACCTAAAATGCTCACC
0
250


 


912979
N/A
N/A
19198
19213
CCAGACTACATGCCAC
79
251


 


912982
N/A
N/A
19446
19461
TCTACTAGGCATCTCT
63
252


 


912983
N/A
N/A
19447
19462
TTCTACTAGGCATCTC
42
253


 


912986
N/A
N/A
20288
20303
TCAGAGGAAGCCCAAT
92
254





20318
20333





 


912987
N/A
N/A
20656
20671
GAAATTGCAGTGCCCT
92
255


 


912990
N/A
N/A
21393
21408
GCCAACCTATCACTGA
60
256


 


912991
N/A
N/A
21400
21415
AGACATGGCCAACCTA
32
257


 


912994
N/A
N/A
21565
21580
TGAAATAAGGACTTAC
67
258


 


912995
N/A
N/A
21934
21949
CCTAATAAGAGCCCCA
31
259


 


912998
N/A
N/A
22041
22056
GAAATCTGTCAGAGCA
33
260


 


912999
N/A
N/A
22072
22087
TGTAGGATAGGACTAG
0
261


 


913002
N/A
N/A
22166
22181
GAATGTTACATCCATA
53
262


 


913003
N/A
N/A
22168
22183
GAGAATGTTACATCCA
80
263


 


913005
N/A
N/A
22605
22620
GTGATAAATCTGCAAG
70
264


 


913006
N/A
N/A
23081
23096
AGATTAATCAGGGCCA
8
265


 


913007
N/A
N/A
23082
23097
AAGATTAATCAGGGCC
30
266


 


913009
N/A
N/A
23325
23340
GGTCACATGTGAGCCC
0
267


 


913010
N/A
N/A
23496
23511
CACTTCTGGTTCAAGA
13
268


 


913011
N/A
N/A
23580
23595
CCAATCTGATGACTTC
80
269


 


913013
N/A
N/A
23790
23805
AAGTATTTACCTGGAG
0
270


 


913014
N/A
N/A
24028
24043
CACTCAAAGAGACTCA
65
271


 


913015
N/A
N/A
24425
24440
GCCTATGATTTTCAGG
0
272


 


913017
N/A
N/A
24633
24648
CACTACTGCCCTCTTC
50
273


 


913018
N/A
N/A
24983
24998
TGCTGGGCTGATGTCA
0
274


 


913019
N/A
N/A
25150
25165
GGCATTTGGGACCTGA
67
275
















TABLE 5







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















915343
1
16
2739
2754
GCCCCCCTCGGACCAT
0
276


 


915363
45
60
2783
2798
CCTCAGTGTCTCGGCC
0
277


 


915383
107
122
2845
2860
AATCGGCTCGGGTCCT
29
278


 


915403
190
205
2928
2943
GACAAGCTCCAGCCGC
64
279


 


915423
249
264
2987
3002
CGCTCAGGCAGCGGGT
0
280


 


915443
347
362
N/A
N/A
CTCCAGCGGGATACCG
6
281


 


915463
386
401
5960
5975
GGCCTTCCGCACAAGA
0
282


 


915483
416
431
5990
6005
TGGATGGAAGATGCCA
28
283


 


915503
452
467
6026
6041
GAGACCCTGTCGGAGG
45
284


 


915523
488
503
6062
6077
GATGAGCTGGTGGACA
70
285


 


915543
510
525
6084
6099
GAGAGATGCCTATTTT
92
286


 


915563
559
574
6133
6148
GACCGAAAGTCAGACA
7
287


 


915603
697
712
11915
11930
GTTTTGGCATCAATGA
94
288


 


915623
754
769
11972
11987
GACTTGACTTTAGGGC
98
289


 


915643
827
842
12045
12060
CGAGAGAAGGTAGAGG
97
290


 


915663
879
894
13615
13630
CTCGAAGGCATATCTC
66
291


 


915683
932
947
16052
16067
GGGCCTGTTGCAGATG
0
292


 


915703
985
1000
16105
16120
GGCATGGCGACCTCAG
6
293


 


915723
1037
1052
16157
16172
AGCCAAGGCAGCCGAC
0
294


 


915743
1132
1147
16252
16267
GCGAGCCTGGGCGAGA
0
295


 


915763
1177
1192
19017
19032
CTCATGTATCCACCTT
88
296


 


915783
1229
1244
19069
19084
GGGCAGCATTACATAA
73
297


 


915803
1286
1301
23691
23706
AAGCCATGTCACCAGT
34
298


 


915823
1348
1363
23753
23768
ACTCGAGTGAACACCT
12
299


 


915843
1405
1420
25168
25183
GCCTGTTGGCTGCTCA
1
300


 


915863
1473
1488
25236
25251
CTGCTGGACAGCCCTT
0
301


 


915883
1542
1557
25305
25320
CTTTATTGCCCAAGAA
72
302


 


915903
1601
1616
25364
25379
CAGACTCTTCTCTAGT
49
303


 


915923
1633
1648
25396
25411
AATCTGCTAGACTCGC
88
304


 


915943
1686
1701
25449
25464
CAGCAATGCGGAGGTA
80
305


 


915963
1768
1783
25531
25546
GAAAGGTTGCTTCCTA
84
306


 


915983
1789
1804
25552
25567
GTGCTGGACCGCTGCA
11
307


 


916003
1815
1830
25578
25593
AACGCATGCTGATGTA
69
308


 


916023
1848
1863
25611
25626
GCTTCCTGGTGTCATT
81
309


 


916043
1884
1899
25647
25662
GCCACGAAACAGTCAG
67
310


 


916063
1913
1928
25676
25691
CATGCTGGAACAGTCT
20
311


 


916083
1954
1969
25717
25732
AAGGCCCCCACCCATC
0
312


 


916103
1977
1992
25740
25755
TGGGCCAGCCTACCCC
0
313


 


916123
2026
2041
25789
25804
GGAAGTGGGATCATGC
55
314


 


916142
2100
2115
25863
25878
GTTATCATCTTTGCAG
57
315


 


916162
2139
2154
25902
25917
CTTGTTACCCCCGCCA
84
316


 


916182
2264
2279
26027
26042
TCTCACTGATTCACAT
83
317


 


916202
2624
2639
26387
26402
CCCTGCACACTAGATT
55
318


 


916222
2677
2692
26440
26455
GAGGCGGAAGCTCCTG
0
319


 


916242
2707
2722
26470
26485
CAGGTTCAAGTTGTGT
83
320


 


916282
N/A
N/A
4225
4240
AAATGTACGGAATCTC
79
321


 


916302
N/A
N/A
4822
4837
GTGTAAACATTTGTCC
74
322


 


916322
N/A
N/A
5414
5429
AGCTTTGGTGTAGATG
49
323


 


916342
N/A
N/A
5801
5816
TACTATGGGAGCCACA
42
324


 


916362
N/A
N/A
6866
6881
TGAAATTGTAACTGCC
70
325


 


916382
N/A
N/A
7492
7507
TAGATCGGTGCTGTTC
27
326


 


916402
N/A
N/A
7785
7800
GTTATAGGCGAGAGCA
0
327


 


916562
N/A
N/A
12316
12331
TATATAACCACAGCCT
58
328


 


916582
N/A
N/A
12932
12947
ATAAGAGCTGTCTCCT
94
329


 


916602
N/A
N/A
13703
13718
CTAGTAAATGCTTGTC
96
330


 


916622
N/A
N/A
14177
14192
CTAATATTTCTACAGC
0
331


 


916642
N/A
N/A
14672
14687
CTCTTAGTGTCCCCAT
95
332


 


916662
N/A
N/A
15542
15557
TTCCATCACAAGGCCT
50
333


 


916682
N/A
N/A
16317
16332
TCCATAATGCACAAGA
71
334


 


916702
N/A
N/A
17223
17238
TGTAGCTGGTTTGTGG
88
335


 


916722
N/A
N/A
18223
18238
AACAGCTACATCAGGC
44
336


 


916742
N/A
N/A
19249
19264
GGCATTGCACATAGAC
74
337


 


916761
N/A
N/A
20410
20425
GTAAGCAATGCAGCCA
88
338


 


916781
N/A
N/A
20659
20674
TTAGAAATTGCAGTGC
91
339


 


916801
N/A
N/A
20989
21004
AGGTATTAAACTGCCA
25
340


 


916821
N/A
N/A
21506
21521
GTCCTAAGAGCACTCA
57
341


 


916841
N/A
N/A
22603
22618
GATAAATCTGCAAGAG
49
342


 


916861
N/A
N/A
23472
23487
GGGACTTACACTGAAA
66
343


 


916881
N/A
N/A
24314
24329
GTCAACGCAGACTGCT
33
344
















TABLE 6







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















915344
2
17
2740
2755
CGCCCCCCTCGGACCA
0
345


 


915364
46
61
2784
2799
GCCTCAGTGTCTCGGC
0
346


 


915384
108
123
2846
2861
GAATCGGCTCGGGTCC
49
347


 


915404
191
206
2929
2944
GGACAAGCTCCAGCCG
9
348


 


915424
250
265
2988
3003
TCGCTCAGGCAGCGGG
0
349


 


915444
348
363
N/A
N/A
GCTCCAGCGGGATACC
0
350


 


915464
387
402
5961
5976
TGGCCTTCCGCACAAG
0
351


 


915484
428
443
6002
6017
TAAGTTGAAGGATGGA
96
352


 


915504
453
468
6027
6042
AGAGACCCTGTCGGAG
80
353


 


915524
489
504
6063
6078
AGATGAGCTGGTGGAC
81
354


 


915544
512
527
6086
6101
AAGAGAGATGCCTATT
77
355


 


915564
560
575
6134
6149
GGACCGAAAGTCAGAC
0
356


 


915604
700
715
11918
11933
GTTGTTTTGGCATCAA
91
357


 


915624
755
770
11973
11988
GGACTTGACTTTAGGG
81
358


 


915644
828
843
12046
12061
TCGAGAGAAGGTAGAG
24
359


 


915664
880
895
13616
13631
CCTCGAAGGCATATCT
41
360


 


915684
952
967
16072
16087
GATGACTTCAGGCCTG
0
361


 


915704
987
1002
16107
16122
TGGGCATGGCGACCTC
0
362


 


915724
1038
1053
16158
16173
CAGCCAAGGCAGCCGA
0
363


 


915744
1133
1148
16253
16268
AGCGAGCCTGGGCGAG
0
364


 


915764
1179
1194
19019
19034
TGCTCATGTATCCACC
56
365


 


915784
1230
1245
19070
19085
AGGGCAGCATTACATA
69
366


 


915804
1293
1308
23698
23713
TATCTGGAAGCCATGT
6
367


 


915824
1349
1364
23754
23769
CACTCGAGTGAACACC
0
368


 


915844
1406
1421
25169
25184
GGCCTGTTGGCTGCTC
0
369


 


915864
1477
1492
25240
25255
GTCTCTGCTGGACAGC
0
370


 


915884
1545
1560
25308
25323
GTACTTTATTGCCCAA
73
371


 


915904
1607
1622
25370
25385
GACTCACAGACTCTTC
92
372


 


915924
1634
1649
25397
25412
GAATCTGCTAGACTCG
65
373


 


915944
1687
1702
25450
25465
ACAGCAATGCGGAGGT
83
374


 


915964
1769
1784
25532
25547
CGAAAGGTTGCTTCCT
79
375


 


915984
1790
1805
25553
25568
AGTGCTGGACCGCTGC
38
376


 


916004
1816
1831
25579
25594
TAACGCATGCTGATGT
79
377


 


916024
1849
1864
25612
25627
GGCTTCCTGGTGTCAT
73
378


 


916044
1885
1900
25648
25663
GGCCACGAAACAGTCA
40
379


 


916064
1914
1929
25677
25692
TCATGCTGGAACAGTC
80
380


 


916084
1958
1973
25721
25736
TCACAAGGCCCCCACC
35
381


 


916104
1978
1993
25741
25756
ATGGGCCAGCCTACCC
0
382


 


916124
2053
2068
25816
25831
CGAACTGCACCCCTTC
38
383


 


916143
2101
2116
25864
25879
GGTTATCATCTTTGCA
81
384


 


916163
2140
2155
25903
25918
TCTTGTTACCCCCGCC
84
385


 


916183
2265
2280
26028
26043
ATCTCACTGATTCACA
86
386


 


916203
2625
2640
26388
26403
GCCCTGCACACTAGAT
65
387


 


916223
2678
2693
26441
26456
GGAGGCGGAAGCTCCT
0
388


 


916243
2709
2724
26472
26487
GCCAGGTTCAAGTTGT
62
389


 


916283
N/A
N/A
4226
4241
CAAATGTACGGAATCT
52
390


 


916303
N/A
N/A
4864
4879
TACTTTAGGCTCCTGG
90
391


 


916323
N/A
N/A
5422
5437
AGCATTAGAGCTTTGG
75
392


 


916343
N/A
N/A
5803
5818
TCTACTATGGGAGCCA
89
393


 


916363
N/A
N/A
6927
6942
GGACAGGTTCCTTGGA
0
394


 


916383
N/A
N/A
7493
7508
CTAGATCGGTGCTGTT
14
395


 


916403
N/A
N/A
7786
7801
AGTTATAGGCGAGAGC
0
396


 


916563
N/A
N/A
12318
12333
ACTATATAACCACAGC
90
397


 


916583
N/A
N/A
12936
12951
GACAATAAGAGCTGTC
0
398


 


916603
N/A
N/A
13704
13719
GCTAGTAAATGCTTGT
73
399


 


916623
N/A
N/A
14231
14246
CCAACTTTTAGTATTA
92
400


 


916643
N/A
N/A
14678
14693
AGCCATCTCTTAGTGT
50
401


 


916663
N/A
N/A
15566
15581
TCTGATGTCGAAGAGG
68
402


 


916683
N/A
N/A
16341
16356
TCCCATGTGGCAGTAC
0
403


 


916703
N/A
N/A
17239
17254
TCCAAATGCCCAACTC
37
404


 


916723
N/A
N/A
18241
18256
GCAAATAATGTGCACA
22
405


 


916743
N/A
N/A
19250
19265
GGGCATTGCACATAGA
59
406


 


916762
N/A
N/A
20413
20428
GTAGTAAGCAATGCAG
69
407


 


916782
N/A
N/A
20660
20675
CTTAGAAATTGCAGTG
91
408


 


916802
N/A
N/A
21002
21017
ATTTTAACAGCTCAGG
95
409


 


916822
N/A
N/A
21540
21555
TATGACATTTCAGAGT
88
410


 


916842
N/A
N/A
22629
22644
AGTACAAGCGCAGCCT
14
411


 


916862
N/A
N/A
23538
23553
ACAAGGACAAGCCCAC
37
412


 


916882
N/A
N/A
24339
24354
GAAGTAGCGGCATCCC
68
413
















TABLE 7







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















915345
3
18
2741
2756
CCGCCCCCCTCGGACC
0
414


 


915365
47
62
2785
2800
TGCCTCAGTGTCTCGG
0
415


 


915385
109
124
2847
2862
GGAATCGGCTCGGGTC
72
416


 


915405
193
208
2931
2946
AAGGACAAGCTCCAGC
41
417


 


915425
251
266
2989
3004
CTCGCTCAGGCAGCGG
0
418


 


915445
349
364
N/A
N/A
TGCTCCAGCGGGATAC
0
419


 


915465
388
403
5962
5977
CTGGCCTTCCGCACAA
16
420


 


915485
430
445
6004
6019
CTTAAGTTGAAGGATG
27
421


 


915505
454
469
6028
6043
CAGAGACCCTGTCGGA
72
422


 


915525
492
507
6066
6081
CGGAGATGAGCTGGTG
92
423


 


915545
513
528
6087
6102
TAAGAGAGATGCCTAT
57
424


 


915565
561
576
6135
6150
TGGACCGAAAGTCAGA
0
425


 


915605
701
716
11919
11934
GGTTGTTTTGGCATCA
97
426


 


915625
756
771
11974
11989
TGGACTTGACTTTAGG
93
427


 


915645
829
844
12047
12062
CTCGAGAGAAGGTAGA
0
428


 


915665
881
896
13617
13632
TCCTCGAAGGCATATC
0
429


 


915685
953
968
16073
16088
GGATGACTTCAGGCCT
0
430


 


915705
988
1003
16108
16123
CTGGGCATGGCGACCT
0
431


 


915725
1039
1054
16159
16174
ACAGCCAAGGCAGCCG
0
432


 


915745
1134
1149
16254
16269
TAGCGAGCCTGGGCGA
0
433


 


915765
1193
1208
19033
19048
CAAGTTGCAAATCTTG
0
434


 


915785
1231
1246
19071
19086
CAGGGCAGCATTACAT
74
435


 


915805
1300
1315
23705
23720
TCGGGCATATCTGGAA
21
436


 


915825
1350
1365
23755
23770
GCACTCGAGTGAACAC
0
437


 


915845
1407
1422
25170
25185
AGGCCTGTTGGCTGCT
0
438


 


915865
1480
1495
25243
25258
TTGGTCTCTGCTGGAC
21
439


 


915885
1546
1561
25309
25324
GGTACTTTATTGCCCA
62
440


 


915905
1609
1624
25372
25387
GTGACTCACAGACTCT
81
441


 


915925
1635
1650
25398
25413
AGAATCTGCTAGACTC
74
442


 


915945
1688
1703
25451
25466
CACAGCAATGCGGAGG
56
443


 


915965
1770
1785
25533
25548
GCGAAAGGTTGCTTCC
66
444


 


915985
1791
1806
25554
25569
AAGTGCTGGACCGCTG
71
445


 


916005
1817
1832
25580
25595
TTAACGCATGCTGATG
69
446


 


916025
1850
1865
25613
25628
GGGCTTCCTGGTGTCA
58
447


 


916045
1886
1901
25649
25664
GGGCCACGAAACAGTC
9
448


 


916065
1915
1930
25678
25693
CTCATGCTGGAACAGT
86
449


 


916085
1959
1974
25722
25737
ATCACAAGGCCCCCAC
82
450


 


916105
1979
1994
25742
25757
CATGGGCCAGCCTACC
0
451


 


916125
2054
2069
25817
25832
ACGAACTGCACCCCTT
84
452


 


916144
2102
2117
25865
25880
AGGTTATCATCTTTGC
90
453


 


916164
2141
2156
25904
25919
ATCTTGTTACCCCCGC
88
454


 


916184
2266
2281
26029
26044
CATCTCACTGATTCAC
91
455


 


916204
2626
2641
26389
26404
TGCCCTGCACACTAGA
47
456


 


916224
2680
2695
26443
26458
GAGGAGGCGGAAGCTC
0
457


 


916244
2710
2725
26473
26488
AGCCAGGTTCAAGTTG
71
458


 


916284
N/A
N/A
4227
4242
TCAAATGTACGGAATC
40
459


 


916304
N/A
N/A
4865
4880
GTACTTTAGGCTCCTG
89
460


 


916324
N/A
N/A
5429
5444
ACATATCAGCATTAGA
87
461


 


916344
N/A
N/A
5804
5819
GTCTACTATGGGAGCC
90
462


 


916364
N/A
N/A
6966
6981
GAAGATGCATAGAGGA
0
463


 


916384
N/A
N/A
7550
7565
TCACACTGGGTCACCA
43
464


 


916544
N/A
N/A
12135
12150
GGCAATCAGGGAGGCA
32
465


 


916564
N/A
N/A
12320
12335
TGACTATATAACCACA
92
466


 


916584
N/A
N/A
12951
12966
CCCAATTGCCACTAGG
83
467


 


916604
N/A
N/A
13718
13733
TCTTTACCAAGACCGC
92
468


 


916624
N/A
N/A
14245
14260
GACAAATTCATCAACC
87
469


 


916644
N/A
N/A
14778
14793
CTGTATCCAAAAGGCC
0
470


 


916664
N/A
N/A
15597
15612
ATACATAGCAGAGCCA
44
471


 


916684
N/A
N/A
16352
16367
CACCCTATCGCTCCCA
43
472


 


916704
N/A
N/A
17267
17282
AGTTATGTCTGACTCA
72
473


 


916724
N/A
N/A
18254
18269
AATATACCCCACAGCA
40
474


 


916744
N/A
N/A
19288
19303
GTGCATGTGTGGCTTG
82
475


 


916763
N/A
N/A
20414
20429
TGTAGTAAGCAATGCA
85
476


 


916783
N/A
N/A
20724
20739
CATATATTGCGGATGA
24
477


 


916803
N/A
N/A
21005
21020
GTTATTTTAACAGCTC
95
478


 


916823
N/A
N/A
21561
21576
ATAAGGACTTACACCA
83
479


 


916843
N/A
N/A
22679
22694
CAGCATGCAACCACCC
8
480


 


916863
N/A
N/A
23550
23565
TGGGATGCTAGGACAA
72
481


 


916883
N/A
N/A
24340
24355
GGAAGTAGCGGCATCC
0
482
















TABLE 8







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting


SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO

















915346
25
40
2763
2778
CATTCCCAGCGCGACG
0
483


 


915366
52
67
2790
2805
TACCCTGCCTCAGTGT
0
484


 


915386
112
127
2850
2865
TCGGGAATCGGCTCGG
26
485


 


915406
195
210
2933
2948
CGAAGGACAAGCTCCA
69
486


 


915426
252
267
2990
3005
GCTCGCTCAGGCAGCG
0
487


 


915446
350
365
N/A
N/A
CTGCTCCAGCGGGATA
0
488


 


915466
389
404
5963
5978
CCTGGCCTTCCGCACA
40
489


 


915486
431
446
6005
6020
GCTTAAGTTGAAGGAT
88
490


 


915506
455
470
6029
6044
GCAGAGACCCTGTCGG
32
491


 


915526
493
508
6067
6082
CCGGAGATGAGCTGGT
4
492


 


915546
514
529
6088
6103
GTAAGAGAGATGCCTA
94
493


 


915566
562
577
6136
6151
TTGGACCGAAAGTCAG
56
494


 


915606
702
717
11920
11935
TGGTTGTTTTGGCATC
99
495


 


915626
757
772
11975
11990
GTGGACTTGACTTTAG
89
496


 


915646
830
845
12048
12063
TCTCGAGAGAAGGTAG
0
497


 


915666
882
897
13618
13633
ATCCTCGAAGGCATAT
0
498


 


915686
956
971
16076
16091
TGAGGATGACTTCAGG
10
499


 


915706
989
1004
16109
16124
GCTGGGCATGGCGACC
10
500


 


915726
1064
1079
16184
16199
TAGCAGCTCATCTCCC
67
501


 


915746
1135
1150
16255
16270
GTAGCGAGCCTGGGCG
0
502


 


915766
1196
1211
19036
19051
TAGCAAGTTGCAAATC
78
503


 


915786
1232
1247
19072
19087
ACAGGGCAGCATTACA
87
504


 


915806
1302
1317
23707
23722
CGTCGGGCATATCTGG
53
505


 


915826
1352
1367
23757
23772
CAGCACTCGAGTGAAC
24
506


 


915846
1408
1423
25171
25186
GAGGCCTGTTGGCTGC
0
507


 


915866
1508
1523
25271
25286
GAGGATGGACCGCGGG
0
508


 


915886
1549
1564
25312
25327
GCAGGTACTTTATTGC
0
509


 


915906
1610
1625
25373
25388
AGTGACTCACAGACTC
35
510


 


915926
1636
1651
25399
25414
AAGAATCTGCTAGACT
69
511


 


915946
1689
1704
25452
25467
ACACAGCAATGCGGAG
69
512


 


915966
1771
1786
25534
25549
GGCGAAAGGTTGCTTC
58
513


 


915986
1792
1807
25555
25570
TAAGTGCTGGACCGCT
70
514


 


916006
1818
1833
25581
25596
ATTAACGCATGCTGAT
73
515


 


916026
1851
1866
25614
25629
TGGGCTTCCTGGTGTC
71
516


 


916046
1887
1902
25650
25665
AGGGCCACGAAACAGT
61
517


 


916066
1917
1932
25680
25695
ACCTCATGCTGGAACA
81
518


 


916086
1960
1975
25723
25738
CATCACAAGGCCCCCA
48
519


 


916106
1980
1995
25743
25758
ACATGGGCCAGCCTAC
54
520


 


916126
2055
2070
25818
25833
GACGAACTGCACCCCT
77
521


 


916145
2105
2120
25868
25883
TCAAGGTTATCATCTT
89
522


 


916165
2142
2157
25905
25920
CATCTTGTTACCCCCG
89
523


 


916185
2270
2285
26033
26048
CTAACATCTCACTGAT
66
524


 


916205
2627
2642
26390
26405
ATGCCCTGCACACTAG
62
525


 


916225
2681
2696
26444
26459
AGAGGAGGCGGAAGCT
25
526


 


916245
2711
2726
26474
26489
AAGCCAGGTTCAAGTT
83
527


 


916285
N/A
N/A
4240
4255
ATTAGGACAAGATTCA
75
528


 


916305
N/A
N/A
4866
4881
TGTACTTTAGGCTCCT
93
529


 


916325
N/A
N/A
5430
5445
AACATATCAGCATTAG
85
530


 


916345
N/A
N/A
5839
5854
CAAGGATGCCACCAAC
84
531


 


916365
N/A
N/A
6974
6989
TCATTATGGAAGATGC
0
532


 


916385
N/A
N/A
7602
7617
TTAACAACCCTGTCAG
1
533


 


916545
N/A
N/A
12151
12166
GTAACTGGTAGCTCCT
93
534


 


916565
N/A
N/A
12338
12353
ACCCATACTGCACCCC
79
535


 


916585
N/A
N/A
12957
12972
GCCTATCCCAATTGCC
70
536


 


916605
N/A
N/A
13719
13734
GTCTTTACCAAGACCG
23
537


 


916625
N/A
N/A
14248
14263
AACGACAAATTCATCA
84
538


 


916645
N/A
N/A
14788
14803
TGCAATCCCCCTGTAT
17
539


 


916665
N/A
N/A
15598
15613
AATACATAGCAGAGCC
68
540


 


916685
N/A
N/A
16366
16381
TGTCATGGTTGCCTCA
70
541


 


916705
N/A
N/A
17273
17288
ATAAGGAGTTATGTCT
80
542


 


916725
N/A
N/A
18255
18270
GAATATACCCCACAGC
58
543


 


916745
N/A
N/A
19295
19310
GTTACAGGTGCATGTG
75
544


 


916764
N/A
N/A
20435
20450
AGTCATCTGGAGTCAC
69
545


 


916784
N/A
N/A
20756
20771
TCAGACAACCCACTGA
24
546


 


916804
N/A
N/A
21046
21061
AGGAATCTGAATCCTA
0
547


 


916824
N/A
N/A
21640
21655
GATAATTTCCTAGAGC
29
548


 


916844
N/A
N/A
22699
22714
GAAATAAGTGCTCAGG
73
549


 


916864
N/A
N/A
23582
23597
CTCCAATCTGATGACT
53
550


 


916884
N/A
N/A
24347
24362
GAATTCAGGAAGTAGC
50
551
















TABLE 9







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915347
  26
  41
 2764
 2779
GCATTCCCAGCGCGAC
 0
552


 


915367
  58
  73
 2796
 2811
GCTCTCTACCCTGCCT
 9
553


 


915387
 113
 128
 2851
 2866
ATCGGGAATCGGCTCG
28
554


 


915407
 198
 213
 2936
 2951
CCGCGAAGGACAAGCT
 0
555


 


915427
 253
 268
 2991
 3006
TGCTCGCTCAGGCAGC
 0
556


 


915447
 351
 366
N/A
N/A
TCTGCTCCAGCGGGAT
 1
557


 


915467
 391
 406
 5965
 5980
CTCCTGGCCTTCCGCA
29
558


 


915487
 433
 448
 6007
 6022
TTGCTTAAGTTGAAGG
94
559


 


915507
 456
 471
 6030
 6045
TGCAGAGACCCTGTCG
31
560


 


915527
 494
 509
 6068
 6083
GCCGGAGATGAGCTGG
 0
561


 


915547
 515
 530
 6089
 6104
GGTAAGAGAGATGCCT
 0
562


 


915567
 563
 578
 6137
 6152
TTTGGACCGAAAGTCA
 0
563


 


915607
 703
 718
11921
11936
ATGGTTGTTTTGGCAT
35
564


 


915627
 758
 773
11976
11991
CGTGGACTTGACTTTA
85
565


 


915647
 831
 846
12049
12064
CTCTCGAGAGAAGGTA
 7
566


 


915667
 883
 898
13619
13634
TATCCTCGAAGGCATA
 0
567


 


915687
 959
 974
16079
16094
TTCTGAGGATGACTTC
39
568


 


915707
 996
1011
16116
16131
TTGCCCAGCTGGGCAT
 0
569


 


915727
1065
1080
16185
16200
CTAGCAGCTCATCTCC
58
570


 


915747
1136
1151
16256
16271
TGTAGCGAGCCTGGGC
16
571


 


915767
1197
1212
19037
19052
GTAGCAAGTTGCAAAT
80
572


 


915787
1233
1248
19073
19088
TACAGGGCAGCATTAC
71
573


 


915807
1316
1331
23721
23736
CAACCACAGGACATCG
 0
574


 


915827
1353
1368
23758
23773
TCAGCACTCGAGTGAA
 0
575


 


915847
1409
1424
25172
25187
GGAGGCCTGTTGGCTG
 0
576


 


915867
1509
1524
25272
25287
TGAGGATGGACCGCGG
14
577


 


915887
1553
1568
25316
25331
ACCAGCAGGTACTTTA
29
578


 


915907
1611
1626
25374
25389
AAGTGACTCACAGACT
29
579


 


915927
1637
1652
25400
25415
AAAGAATCTGCTAGAC
60
580


 


915947
1690
1705
25453
25468
TACACAGCAATGCGGA
69
581


 


915967
1772
1787
25535
25550
AGGCGAAAGGTTGCTT
 0
582


 


915987
1793
1808
25556
25571
TTAAGTGCTGGACCGC
82
583


 


916007
1819
1834
25582
25597
AATTAACGCATGCTGA
61
584


 


916027
1864
1879
25627
25642
GGACCCTCTGCACTGG
43
585


 


916047
1888
1903
25651
25666
TAGGGCCACGAAACAG
80
586


 


916067
1918
1933
25681
25696
AACCTCATGCTGGAAC
72
587


 


916087
1961
1976
25724
25739
CCATCACAAGGCCCCC
63
588


 


916107
1981
1996
25744
25759
CACATGGGCCAGCCTA
74
589


 


916127
2056
2071
25819
25834
GGACGAACTGCACCCC
 5
590


 


916146
2106
2121
25869
25884
GTCAAGGTTATCATCT
88
591


 


916166
2143
2158
25906
25921
TCATCTTGTTACCCCC
90
592


 


916186
2272
2287
26035
26050
TACTAACATCTCACTG
 1
593


 


916206
2628
2643
26391
26406
AATGCCCTGCACACTA
56
594


 


916226
2682
2697
26445
26460
GAGAGGAGGCGGAAGC
10
595


 


916246
2712
2727
26475
26490
TAAGCCAGGTTCAAGT
81
596


 


916286
N/A
N/A
 4244
 4259
TTTCATTAGGACAAGA
61
597


 


916306
N/A
N/A
 4867
 4882
GTGTACTTTAGGCTCC
97
598


 


916326
N/A
N/A
 5431
 5446
GAACATATCAGCATTA
52
599


 


916346
N/A
N/A
 5872
 5887
GTAATACTTTTGGCAA
75
600


 


916366
N/A
N/A
 7069
 7084
GGTATTACAAATTATC
10
601


 


916386
N/A
N/A
 7603
 7618
CTTAACAACCCTGTCA
 0
602


 


916546
N/A
N/A
12152
12167
AGTAACTGGTAGCTCC
88
603


 


916566
N/A
N/A
12343
12358
CTAATACCCATACTGC
84
604


 


916586
N/A
N/A
12966
12981
AACTTTGCAGCCTATC
85
605


 


916606
N/A
N/A
13739
13754
AGAACTAAGGCAAATC
85
606


 


916626
N/A
N/A
14257
14272
GTCTTGGCCAACGACA
 0
607


 


916646
N/A
N/A
14793
14808
CAGGATGCAATCCCCC
45
608


 


916666
N/A
N/A
15601
15616
GCCAATACATAGCAGA
75
609


 


916686
N/A
N/A
16630
16645
GTCCATGAAATCCAGG
 0
610


 


916706
N/A
N/A
17293
17308
TCTCTTAGGGCACCTC
87
611


 


916726
N/A
N/A
18256
18271
TGAATATACCCCACAG
24
612


 


916746
N/A
N/A
19337
19352
AGCTCTAGGAGTCCCC
63
613


 


916765
N/A
N/A
20513
20528
CCAGATTGAGTCTCCT
91
614


 


916785
N/A
N/A
20775
20790
AATCAAGTGCCCTCCA
73
615


 


916805
N/A
N/A
21211
21226
TGTAGCTGTGTGGTGG
85
616


 


916825
N/A
N/A
21760
21775
TACCATGATCAGGTCA
 0
617


 


916845
N/A
N/A
22713
22728
GTAAAGATGTGAGTGA
85
618


 


916865
N/A
N/A
23606
23621
GTTTACAAAAGCTGCC
17
619


 


916885
N/A
N/A
24375
24390
TGAACTCCGGCTCAGT
 0
620
















TABLE 10







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915348
  28
  43
 2766
 2781
GGGCATTCCCAGCGCG
 0
621


 


915368
  59
  74
 2797
 2812
CGCTCTCTACCCTGCC
 0
622


 


915388
 114
 129
 2852
 2867
GATCGGGAATCGGCTC
32
623


 


915408
 199
 214
 2937
 2952
CCCGCGAAGGACAAGC
 6
624


 


915428
 275
 290
 3013
 3028
GTCGCGGAGGAGGTGC
 0
625


 


915448
 352
 367
N/A
N/A
GTCTGCTCCAGCGGGA
 4
626


 


915468
 392
 407
 5966
 5981
ACTCCTGGCCTTCCGC
87
627


 


915488
 434
 449
 6008
 6023
CTTGCTTAAGTTGAAG
 0
628


 


915508
 457
 472
 6031
 6046
TTGCAGAGACCCTGTC
63
629


 


915528
 495
 510
 6069
 6084
TGCCGGAGATGAGCTG
 0
630


 


915548
 516
 531
 6090
 6105
TGGTAAGAGAGATGCC
18
631


 


915568
 564
 579
 6138
 6153
CTTTGGACCGAAAGTC
10
632


 


915608
 704
 719
11922
11937
GATGGTTGTTTTGGCA
98
633


 


915628
 772
 787
11990
12005
ACATGAAGAAAGTTCG
41
634


 


915648
 832
 847
12050
12065
GCTCTCGAGAGAAGGT
55
635


 


915668
 884
 899
13620
13635
ATATCCTCGAAGGCAT
33
636


 


915688
 962
 977
16082
16097
CCCTTCTGAGGATGAC
11
637


 


915708
 998
1013
16118
16133
GTTTGCCCAGCTGGGC
 0
638


 


915728
1067
1082
16187
16202
GTCTAGCAGCTCATCT
68
639


 


915748
1137
1152
16257
16272
CTGTAGCGAGCCTGGG
 0
640


 


915768
1198
1213
19038
19053
GGTAGCAAGTTGCAAA
90
641


 


915788
1234
1249
19074
19089
GTACAGGGCAGCATTA
69
642


 


915808
1317
1332
23722
23737
GCAACCACAGGACATC
51
643


 


915828
1354
1369
23759
23774
ATCAGCACTCGAGTGA
 0
644


 


915848
1410
1425
25173
25188
GGGAGGCCTGTTGGCT
17
645


 


915868
1510
1525
25273
25288
CTGAGGATGGACCGCG
53
646


 


915888
1554
1569
25317
25332
CACCAGCAGGTACTTT
 0
647


 


915908
1612
1627
25375
25390
CAAGTGACTCACAGAC
91
648


 


915928
1639
1654
25402
25417
TGAAAGAATCTGCTAG
59
649


 


915948
1691
1706
25454
25469
CTACACAGCAATGCGG
20
650


 


915968
1773
1788
25536
25551
CAGGCGAAAGGTTGCT
60
651


 


915988
1794
1809
25557
25572
GTTAAGTGCTGGACCG
86
652


 


916008
1820
1835
25583
25598
GAATTAACGCATGCTG
88
653


 


916028
1865
1880
25628
25643
GGGACCCTCTGCACTG
 0
654


 


916048
1889
1904
25652
25667
ATAGGGCCACGAAACA
75
655


 


916068
1919
1934
25682
25697
GAACCTCATGCTGGAA
72
656


 


916088
1962
1977
25725
25740
CCCATCACAAGGCCCC
37
657


 


916108
1984
1999
25747
25762
TCACACATGGGCCAGC
84
658


 


916128
2079
2094
25842
25857
CTGACAGGCAGTGTCG
10
659


 


916147
2107
2122
25870
25885
AGTCAAGGTTATCATC
81
660


 


916167
2144
2159
25907
25922
ATCATCTTGTTACCCC
88
661


 


916187
2276
2291
26039
26054
ATTCTACTAACATCTC
90
662


 


916207
2629
2644
26392
26407
GAATGCCCTGCACACT
72
663


 


916227
2691
2706
26454
26469
GCTCCAGTGGAGAGGA
14
664


 


916247
2713
2728
26476
26491
ATAAGCCAGGTTCAAG
88
665


 


916287
N/A
N/A
 4308
 4323
GTGAGAAACAAACCCT
92
666


 


916307
N/A
N/A
 4882
 4897
TCTATACCAGAGTGAG
84
667


 


916327
N/A
N/A
 5514
 5529
AGGAATGAGTCTCCCA
17
668


 


916347
N/A
N/A
 5873
 5888
GGTAATACTTTTGGCA
70
669


 


916367
N/A
N/A
 7106
 7121
CGCTTATGAAAGCATC
 0
670


 


916387
N/A
N/A
 7605
 7620
CCCTTAACAACCCTGT
28
671


 


916547
N/A
N/A
12167
12182
TTTGATTGTGCAGACA
98
672


 


916567
N/A
N/A
12345
12360
TCCTAATACCCATACT
74
673


 


916587
N/A
N/A
12969
12984
ACAAACTTTGCAGCCT
95
674


 


916607
N/A
N/A
13742
13757
GTTAGAACTAAGGCAA
94
675


 


916627
N/A
N/A
14301
14316
GAGCAGATAAATACAC
91
676


 


916647
N/A
N/A
14892
14907
TGGTATCTCGCTTCCT
 0
677


 


916667
N/A
N/A
15613
15628
TAAAGCCACGCAGCCA
46
678


 


916687
N/A
N/A
16656
16671
CCAGATGCAGGACCCC
 0
679


 


916707
N/A
N/A
17326
17341
AAACTAATGCACCTGG
43
680


 


916727
N/A
N/A
18257
18272
CTGAATATACCCCACA
75
681


 


916747
N/A
N/A
19360
19375
AGCTGCTATGTGAGGC
12
682


 


916766
N/A
N/A
20520
20535
TCAGTAACCAGATTGA
25
683


 


916786
N/A
N/A
20778
20793
TTTAATCAAGTGCCCT
81
684


 


916806
N/A
N/A
21216
21231
CAGGATGTAGCTGTGT
84
685


 


916826
N/A
N/A
21887
21902
TAAGATCCCATCTTAC
13
686


 


916846
N/A
N/A
22739
22754
AAAGTAAACACCCACC
42
687


 


916866
N/A
N/A
23625
23640
GCTTACAACACTACCC
57
688


 


916886
N/A
N/A
24393
24408
GTAATGGGAGCCAGGC
38
689
















TABLE 11







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915349
  29
  44
 2767
 2782
AGGGCATTCCCAGCGC
 0
690


 


915369
  60
  75
 2798
 2813
GCGCTCTCTACCCTGC
23
691


 


915389
 115
 130
 2853
 2868
GGATCGGGAATCGGCT
54
692


 


915409
 200
 215
 2938
 2953
GCCCGCGAAGGACAAG
32
693


 


915429
 276
 291
 3014
 3029
CGTCGCGGAGGAGGTG
 0
694


 


915449
 364
 379
 5938
 5953
AGGACCTGCAGAGTCT
21
695


 


915469
 394
 409
 5968
 5983
CGACTCCTGGCCTTCC
59
696


 


915489
 435
 450
 6009
 6024
ACTTGCTTAAGTTGAA
86
697


 


915509
 466
 481
 6040
 6055
GGGAGGCATTTGCAGA
57
698


 


915529
 496
 511
 6070
 6085
TTGCCGGAGATGAGCT
40
699


 


915549
 518
 533
 6092
 6107
TCTGGTAAGAGAGATG
61
700


 


915569
 565
 580
 6139
 6154
TCTTTGGACCGAAAGT
 9
701


 


915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
99
702


 


915629
 776
 791
11994
12009
GTCCACATGAAGAAAG
32
703


 


915649
 833
 848
12051
12066
AGCTCTCGAGAGAAGG
36
704


 


915669
 885
 900
13621
13636
AATATCCTCGAAGGCA
55
705


 


915689
 969
 984
16089
16104
GATCCATCCCTTCTGA
20
706


 


915709
 999
1014
16119
16134
TGTTTGCCCAGCTGGG
 5
707


 


915729
1077
1092
16197
16212
GACGCAGGTGGTCTAG
 0
708


 


915749
1138
1153
N/A
N/A
GCTGTAGCGAGCCTGG
71
709


 


915769
1200
1215
19040
19055
TGGGTAGCAAGTTGCA
81
710


 


915789
1235
1250
19075
19090
GGTACAGGGCAGCATT
88
711


 


915809
1318
1333
23723
23738
TGCAACCACAGGACAT
40
712


 


915829
1355
1370
23760
23775
CATCAGCACTCGAGTG
 0
713


 


915849
1424
1439
25187
25202
CTCAGGTGTGCATGGG
61
714


 


915869
1511
1526
25274
25289
CCTGAGGATGGACCGC
70
715


 


915889
1556
1571
25319
25334
AGCACCAGCAGGTACT
35
716


 


915909
1613
1628
25376
25391
TCAAGTGACTCACAGA
84
717


 


915929
1645
1660
25408
25423
CACCTCTGAAAGAATC
89
718


 


915949
1692
1707
25455
25470
ACTACACAGCAATGCG
33
719


 


915969
1774
1789
25537
25552
ACAGGCGAAAGGTTGC
88
720


 


915989
1795
1810
25558
25573
AGTTAAGTGCTGGACC
84
721


 


916009
1823
1838
25586
25601
GCTGAATTAACGCATG
67
722


 


916029
1866
1881
25629
25644
AGGGACCCTCTGCACT
15
723


 


916049
1890
1905
25653
25668
AATAGGGCCACGAAAC
52
724


 


916069
1920
1935
25683
25698
AGAACCTCATGCTGGA
85
725


 


916089
1963
1978
25726
25741
CCCCATCACAAGGCCC
20
726


 


916109
1985
2000
25748
25763
ATCACACATGGGCCAG
72
727


 


916129
2080
2095
25843
25858
CCTGACAGGCAGTGTC
15
728


 


916148
2108
2123
25871
25886
TAGTCAAGGTTATCAT
87
729


 


916168
2146
2161
25909
25924
TTATCATCTTGTTACC
82
730


 


916188
2279
2294
26042
26057
CTTATTCTACTAACAT
87
731


 


916208
2630
2645
26393
26408
TGAATGCCCTGCACAC
68
732


 


916228
2692
2707
26455
26470
TGCTCCAGTGGAGAGG
80
733


 


916248
2726
2741
26489
26504
GTCCCTGCAGAAAATA
 0
734


 


916288
N/A
N/A
 4337
 4352
AGCATACCACACCCCA
75
735


 


916308
N/A
N/A
 5086
 5101
GGACATGCTCAGCAGC
68
736


 


916328
N/A
N/A
 5533
 5548
TGCTGTAGGCCTCAGC
 0
737


 


916348
N/A
N/A
 5874
 5889
TGGTAATACTTTTGGC
86
738


 


916368
N/A
N/A
 7132
 7147
GTAAATGGAGTCCTTC
80
739


 


916388
N/A
N/A
 7612
 7627
CATAATCCCCTTAACA
32
740


 


916548
N/A
N/A
12195
12210
TTAACCATCAAGGACA
77
741


 


916568
N/A
N/A
12665
12680
TCTTAGTGGCTGGGTA
85
742


 


916588
N/A
N/A
12973
12988
CCTAACAAACTTTGCA
32
743


 


916608
N/A
N/A
13749
13764
ACTAAGTGTTAGAACT
76
744


 


916628
N/A
N/A
14338
14353
CTGCAGTATCCCTAGC
 0
745


 


916648
N/A
N/A
15012
15027
TCCCATCGGTCATTTC
45
746


 


916668
N/A
N/A
15682
15697
GAAACCACTATCATCA
62
747


 


916688
N/A
N/A
16671
16686
GTAATAGGCCAAGTCC
 0
748


 


916708
N/A
N/A
17327
17342
CAAACTAATGCACCTG
66
749


 


916728
N/A
N/A
18332
18347
CCAATATCATAGCTGA
85
750


 


916748
N/A
N/A
19376
19391
CACAAGAGACTGGACC
64
751


 


916767
N/A
N/A
20551
20566
TACTATGGGATGAGTA
 0
752


 


916787
N/A
N/A
20779
20794
TTTTAATCAAGTGCCC
38
753


 


916807
N/A
N/A
21218
21233
GGCAGGATGTAGCTGT
63
754


 


916827
N/A
N/A
21947
21962
AGTCAAACATCTTCCT
50
755


 


916847
N/A
N/A
22759
22774
CAGACTAACTTACTAA
77
756


 


916867
N/A
N/A
23626
23641
AGCTTACAACACTACC
13
757


 


916887
N/A
N/A
24505
24520
ATGCTACGGGCTCTCA
 0
758
















TABLE 12







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915350
  30
  45
 2768
 2783
CAGGGCATTCCCAGCG
 0
759


 


915370
  82
  97
 2820
 2835
CAGCTCCGCCCGGCGC
14
760


 


915390
 130
 145
 2868
 2883
TTAGGATCTGGGTCGG
88
761


 


915410
 201
 216
 2939
 2954
AGCCCGCGAAGGACAA
 0
762


 


915430
 295
 310
 3033
 3048
GCGCCGAACAACATGC
 0
763


 


915450
 366
 381
 5940
 5955
AGAGGACCTGCAGAGT
83
764


 


915470
 395
 410
 5969
 5984
CCGACTCCTGGCCTTC
68
765


 


915490
 436
 451
 6010
 6025
AACTTGCTTAAGTTGA
41
766


 


915510
 467
 482
 6041
 6056
CGGGAGGCATTTGCAG
44
767


 


915530
 497
 512
 6071
 6086
TTTGCCGGAGATGAGC
92
768


 


915550
 519
 534
 6093
 6108
CTCTGGTAAGAGAGAT
20
769


 


915570
 566
 581
 6140
 6155
GTCTTTGGACCGAAAG
19
770


 


915590
 627
 642
 7857
 7872
GAGGGATAAGGCCACT
83
771


 


915610
 706
 721
11924
11939
GTGATGGTTGTTTTGG
97
772


 


915630
 782
 797
12000
12015
GGTGATGTCCACATGA
87
773


 


915650
 834
 849
12052
12067
AAGCTCTCGAGAGAAG
44
774


 


915670
 887
 902
13623
13638
CAAATATCCTCGAAGG
 0
775


 


915690
 970
 985
16090
16105
GGATCCATCCCTTCTG
 0
776


 


915710
1003
1018
16123
16138
CTCATGTTTGCCCAGC
68
777


 


915730
1078
1093
16198
16213
AGACGCAGGTGGTCTA
 0
778


 


915750
1139
1154
N/A
N/A
TGCTGTAGCGAGCCTG
56
779


 


915770
1201
1216
19041
19056
ATGGGTAGCAAGTTGC
79
780


 


915790
1247
1262
19087
19102
TTCCACAGGCAGGGTA
48
781


 


915810
1320
1335
23725
23740
ACTGCAACCACAGGAC
22
782


 


915830
1356
1371
23761
23776
ACATCAGCACTCGAGT
 0
783


 


915850
1427
1442
25190
25205
CTGCTCAGGTGTGCAT
10
784


 


915870
1512
1527
25275
25290
ACCTGAGGATGGACCG
69
785


 


915890
1557
1572
25320
25335
CAGCACCAGCAGGTAC
62
786


 


915910
1617
1632
25380
25395
CTCCTCAAGTGACTCA
83
787


 


915930
1648
1663
25411
25426
TAGCACCTCTGAAAGA
55
788


 


915950
1693
1708
25456
25471
CACTACACAGCAATGC
74
789


 


915970
1775
1790
25538
25553
CACAGGCGAAAGGTTG
72
790


 


915990
1797
1812
25560
25575
AGAGTTAAGTGCTGGA
92
791


 


916010
1824
1839
25587
25602
AGCTGAATTAACGCAT
 0
792


 


916030
1867
1882
25630
25645
AAGGGACCCTCTGCAC
38
793


 


916050
1891
1906
25654
25669
TAATAGGGCCACGAAA
53
794


 


916070
1921
1936
25684
25699
AAGAACCTCATGCTGG
24
795


 


916090
1964
1979
25727
25742
CCCCCATCACAAGGCC
24
796


 


916110
1986
2001
25749
25764
GATCACACATGGGCCA
 0
797


 


916130
2081
2096
25844
25859
ACCTGACAGGCAGTGT
54
798


 


916149
2109
2124
25872
25887
GTAGTCAAGGTTATCA
87
799


 


916169
2154
2169
25917
25932
TAAGTAGATTATCATC
79
800


 


916189
2282
2297
26045
26060
AGGCTTATTCTACTAA
85
801


 


916209
2631
2646
26394
26409
GTGAATGCCCTGCACA
59
802


 


916229
2693
2708
26456
26471
GTGCTCCAGTGGAGAG
54
803


 


916249
2727
2742
26490
26505
GGTCCCTGCAGAAAAT
38
804


 


916289
N/A
N/A
 4338
 4353
AAGCATACCACACCCC
79
805


 


916309
N/A
N/A
 5278
 5293
AATCTTGGGATGCACA
95
806


 


916329
N/A
N/A
 5569
 5584
CATCATGGCTTCCAGT
79
807


 


916349
N/A
N/A
 5879
 5894
TGGGATGGTAATACTT
 0
808


 


916369
N/A
N/A
 7134
 7149
AAGTAAATGGAGTCCT
 5
809


 


916389
N/A
N/A
 7615
 7630
TTGCATAATCCCCTTA
33
810


 


916409
N/A
N/A
 8165
 8180
TTAACTAGATCACTGA
58
811


 


916429
N/A
N/A
 9109
 9124
TCCTAATGCGAGTCCC
86
812


 


916449
N/A
N/A
 9522
 9537
TGCTGCTGGGTGCACT
45
813


 


916469
N/A
N/A
10199
10214
GGTGATGACACAGCAT
94
814


 


916489
N/A
N/A
10382
10397
GCCATGTACAACTTTT
52
815


 


916509
N/A
N/A
11152
11167
TACAATTTGGACAGAG
71
816


 


916529
N/A
N/A
11546
11561
ACCTATAGGAGTGCCC
35
817


 


916549
N/A
N/A
12204
12219
TTATTTCCGTTAACCA
97
818


 


916569
N/A
N/A
12672
12687
AGAATCATCTTAGTGG
94
819


 


916589
N/A
N/A
12989
13004
CGGAATAAGCCTCCAC
 0
820


 


916609
N/A
N/A
13752
13767
GGCACTAAGTGTTAGA
57
821


 


916629
N/A
N/A
14375
14390
TCTCACAAGGCTGGCA
84
822


 


916649
N/A
N/A
15137
15152
GCCATACCGGCTCCCT
30
823


 


916669
N/A
N/A
15691
15706
GGCCTTACAGAAACCA
15
824


 


916689
N/A
N/A
16672
16687
AGTAATAGGCCAAGTC
16
825


 


916709
N/A
N/A
17328
17343
ACAAACTAATGCACCT
42
826


 


916729
N/A
N/A
18333
18348
TCCAATATCATAGCTG
32
827


 


916749
N/A
N/A
19445
19460
CTACTAGGCATCTCTA
32
828


 


916768
N/A
N/A
20553
20568
CTTACTATGGGATGAG
83
829


 


916788
N/A
N/A
20808
20823
TAATATTCAGACCAGG
94
830


 


916808
N/A
N/A
21252
21267
CCATGCATGGCACAGT
 4
831


 


916828
N/A
N/A
21968
21983
AGACAGGAATCCAACC
 0
832


 


916848
N/A
N/A
22767
22782
GGACATGACAGACTAA
96
833


 


916868
N/A
N/A
23637
23652
GCAGACACAACAGCTT
40
834


 


916888
N/A
N/A
24507
24522
CCATGCTACGGGCTCT
 0
835
















TABLE 13







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915351
  33
  48
 2771
 2786
GGCCAGGGCATTCCCA
 0
836


 


915371
  83
  98
 2821
 2836
GCAGCTCCGCCCGGCG
 2
837


 


915391
 132
 147
 2870
 2885
GGTTAGGATCTGGGTC
54
838


 


915411
 222
 237
 2960
 2975
GGTAGAAGCCCAGGAA
57
839


 


915431
 321
 336
 3059
 3074
CGACGCAGTGCAACGC
49
840


 


915451
 368
 383
 5942
 5957
TGAGAGGACCTGCAGA
 0
841


 


915471
 400
 415
 5974
 5989
ATGTTCCGACTCCTGG
82
842


 


915491
 437
 452
 6011
 6026
GAACTTGCTTAAGTTG
23
843


 


915511
 468
 483
 6042
 6057
CCGGGAGGCATTTGCA
 0
844


 


915531
 498
 513
 6072
 6087
TTTTGCCGGAGATGAG
84
845


 


915551
 520
 535
 6094
 6109
ACTCTGGTAAGAGAGA
 5
846


 


915571
 567
 582
 6141
 6156
CGTCTTTGGACCGAAA
64
847


 


915611
 708
 723
11926
11941
CGGTGATGGTTGTTTT
98
848


 


915631
 783
 798
12001
12016
TGGTGATGTCCACATG
 0
849


 


915651
 835
 850
12053
12068
AAAGCTCTCGAGAGAA
35
850


 


915671
 890
 905
13626
13641
ATCCAAATATCCTCGA
42
851


 


915691
 971
 986
16091
16106
AGGATCCATCCCTTCT
 0
852


 


915711
1005
1020
16125
16140
GACTCATGTTTGCCCA
73
853


 


915731
1079
1094
16199
16214
GAGACGCAGGTGGTCT
 0
854


 


915751
1140
1155
N/A
N/A
GTGCTGTAGCGAGCCT
 0
855


 


915771
1202
1217
19042
19057
AATGGGTAGCAAGTTG
80
856


 


915791
1248
1263
19088
19103
ATTCCACAGGCAGGGT
37
857


 


915811
1327
1342
23732
23747
GTCACCCACTGCAACC
52
858


 


915831
1357
1372
23762
23777
CACATCAGCACTCGAG
29
859


 


915851
1429
1444
25192
25207
TCCTGCTCAGGTGTGC
 2
860


 


915871
1513
1528
25276
25291
GACCTGAGGATGGACC
20
861


 


915891
1558
1573
25321
25336
TCAGCACCAGCAGGTA
68
862


 


915911
1620
1635
25383
25398
CGCCTCCTCAAGTGAC
48
863


 


915931
1652
1667
25415
25430
ACTTTAGCACCTCTGA
93
864


 


915951
1695
1710
25458
25473
GTCACTACACAGCAAT
84
865


 


915971
1776
1791
25539
25554
GCACAGGCGAAAGGTT
74
866


 


915991
1798
1813
25561
25576
TAGAGTTAAGTGCTGG
84
867


 


916011
1825
1840
25588
25603
CAGCTGAATTAACGCA
 0
868


 


916031
1868
1883
25631
25646
TAAGGGACCCTCTGCA
54
869


 


916051
1892
1907
25655
25670
TTAATAGGGCCACGAA
75
870


 


916071
1922
1937
25685
25700
TAAGAACCTCATGCTG
56
871


 


916091
1965
1980
25728
25743
CCCCCCATCACAAGGC
 9
872


 


916111
1987
2002
25750
25765
AGATCACACATGGGCC
26
873


 


916131
2084
2099
25847
25862
ACCACCTGACAGGCAG
80
874


 


916150
2110
2125
25873
25888
AGTAGTCAAGGTTATC
92
875


 


916170
2174
2189
25937
25952
TGAAAAAGGTGTTCTA
49
876


 


916190
2283
2298
26046
26061
AAGGCTTATTCTACTA
79
877


 


916210
2633
2648
26396
26411
AGGTGAATGCCCTGCA
71
878


 


916230
2694
2709
26457
26472
TGTGCTCCAGTGGAGA
75
879


 


916250
2728
2743
26491
26506
TGGTCCCTGCAGAAAA
79
880


 


916290
N/A
N/A
 4397
 4412
TGCCTACTGGCTCACA
14
881


 


916310
N/A
N/A
 5279
 5294
AAATCTTGGGATGCAC
94
882


 


916330
N/A
N/A
 5572
 5587
TGACATCATGGCTTCC
93
883


 


916350
N/A
N/A
 6158
 6173
GCTTACATCCACGACT
 0
884


 


916370
N/A
N/A
 7135
 7150
CAAGTAAATGGAGTCC
77
885


 


916390
N/A
N/A
 7620
 7635
ATCTATTGCATAATCC
86
886


 


916550
N/A
N/A
12205
12220
TTTATTTCCGTTAACC
96
887


 


916570
N/A
N/A
12694
12709
TTCTTGACCGTGTTTC
98
888


 


916590
N/A
N/A
12990
13005
CCGGAATAAGCCTCCA
47
889


 


916610
N/A
N/A
13822
13837
TGTACAATGGGACGGA
69
890


 


916630
N/A
N/A
14418
14433
ATCGACACAGCATCAC
92
891


 


916650
N/A
N/A
15138
15153
TGCCATACCGGCTCCC
 0
892


 


916670
N/A
N/A
15758
15773
GGTTTATAACAACTGA
89
893


 


916690
N/A
N/A
16722
16737
GCCTTGAGGTGGGTGG
 0
894


 


916710
N/A
N/A
17512
17527
AGTCATGGGATGTGCA
58
895


 


916730
N/A
N/A
18395
18410
ATGTTTGGAAGTCGCC
92
896


 


916750
N/A
N/A
19473
19488
AAGGATCCTGCTTCTA
 9
897


 


916769
N/A
N/A
20554
20569
GCTTACTATGGGATGA
62
898


 


916789
N/A
N/A
20809
20824
GTAATATTCAGACCAG
96
899


 


916809
N/A
N/A
21254
21269
ATCCATGCATGGCACA
72
900


 


916829
N/A
N/A
21979
21994
GTCAGACACGGAGACA
 0
901


 


916849
N/A
N/A
23110
23125
GGCTTTTGAAGGAGAG
84
902


 


916869
N/A
N/A
23787
23802
TATTTACCTGGAGGCG
 0
903


 


916889
N/A
N/A
24612
24627
CAAATCGGATCTTTGC
44
904
















TABLE 14







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915352
  34
  49
 2772
 2787
CGGCCAGGGCATTCCC
 0
905


 


915372
  86
 101
 2824
 2839
GCAGCAGCTCCGCCCG
48
906


 


915392
 135
 150
 2873
 2888
GCGGGTTAGGATCTGG
25
907


 


915412
 225
 240
 2963
 2978
CGTGGTAGAAGCCCAG
 2
908


 


915432
 322
 337
 3060
 3075
CCGACGCAGTGCAACG
31
909


 


915452
 371
 386
 5945
 5960
ATCTGAGAGGACCTGC
72
910


 


915472
 401
 416
 5975
 5990
AATGTTCCGACTCCTG
77
911


 


915492
 438
 453
 6012
 6027
GGAACTTGCTTAAGTT
55
912


 


915512
 469
 484
 6043
 6058
GCCGGGAGGCATTTGC
 3
913


 


915532
 499
 514
 6073
 6088
ATTTTGCCGGAGATGA
86
914


 


915552
 521
 536
 6095
 6110
CACTCTGGTAAGAGAG
 8
915


 


915612
 709
 724
11927
11942
ACGGTGATGGTTGTTT
87
916


 


915632
 787
 802
12005
12020
AGCTTGGTGATGTCCA
38
917


 


915652
 836
 851
12054
12069
AAAAGCTCTCGAGAGA
 0
918


 


915672
 891
 906
13627
13642
CATCCAAATATCCTCG
82
919


 


915692
 972
 987
16092
16107
CAGGATCCATCCCTTC
 0
920


 


915712
1006
1021
16126
16141
AGACTCATGTTTGCCC
77
921


 


915732
1081
1096
16201
16216
CTGAGACGCAGGTGGT
87
922


 


915752
1141
1156
N/A
N/A
AGTGCTGTAGCGAGCC
 1
923


 


915772
1203
1218
19043
19058
TAATGGGTAGCAAGTT
77
924


 


915792
1257
1272
19097
19112
CAATGGCAGATTCCAC
56
925


 


915812
1328
1343
23733
23748
GGTCACCCACTGCAAC
58
926


 


915832
1358
1373
23763
23778
ACACATCAGCACTCGA
66
927


 


915852
1430
1445
25193
25208
GTCCTGCTCAGGTGTG
52
928


 


915872
1518
1533
25281
25296
GGCTGGACCTGAGGAT
 0
929


 


915892
1570
1585
25333
25348
GTGGAGAGCCCCTCAG
47
930


 


915912
1621
1636
25384
25399
TCGCCTCCTCAAGTGA
 8
931


 


915932
1654
1669
25417
25432
AAACTTTAGCACCTCT
90
932


 


915952
1696
1711
25459
25474
GGTCACTACACAGCAA
82
933


 


915972
1777
1792
25540
25555
TGCACAGGCGAAAGGT
64
934


 


915992
1799
1814
25562
25577
TTAGAGTTAAGTGCTG
91
935


 


916012
1826
1841
25589
25604
CCAGCTGAATTAACGC
32
936


 


916032
1869
1884
25632
25647
GTAAGGGACCCTCTGC
73
937


 


916052
1894
1909
25657
25672
CATTAATAGGGCCACG
81
938


 


916072
1923
1938
25686
25701
CTAAGAACCTCATGCT
70
939


 


916092
1966
1981
25729
25744
ACCCCCCATCACAAGG
30
940


 


916112
1988
2003
25751
25766
AAGATCACACATGGGC
86
941


 


916132
2085
2100
25848
25863
GACCACCTGACAGGCA
61
942


 


916151
2111
2126
25874
25889
TAGTAGTCAAGGTTAT
88
943


 


916171
2176
2191
25939
25954
GGTGAAAAAGGTGTTC
84
944


 


916191
2284
2299
26047
26062
TAAGGCTTATTCTACT
76
945


 


916211
2634
2649
26397
26412
GAGGTGAATGCCCTGC
 0
946


 


916231
2695
2710
26458
26473
GTGTGCTCCAGTGGAG
87
947


 


916251
2729
2744
26492
26507
CTGGTCCCTGCAGAAA
67
948


 


916291
N/A
N/A
 4419
 4434
CAATGCTACTTGCCCC
68
949


 


916311
N/A
N/A
 5280
 5295
TAAATCTTGGGATGCA
94
950


 


916331
N/A
N/A
 5576
 5591
ACAATGACATCATGGC
97
951


 


916351
N/A
N/A
 6165
 6180
GCAAACTGCTTACATC
 0
952


 


916371
N/A
N/A
 7172
 7187
GTTAGACGCGCCAGGC
 7
953


 


916391
N/A
N/A
 7624
 7639
TCTCATCTATTGCATA
 0
954


 


916551
N/A
N/A
12206
12221
TTTTATTTCCGTTAAC
73
955


 


916571
N/A
N/A
12714
12729
TAAACTACCGAACGCA
96
956


 


916591
N/A
N/A
12991
13006
CCCGGAATAAGCCTCC
47
957


 


916611
N/A
N/A
13823
13838
CTGTACAATGGGACGG
23
958


 


916631
N/A
N/A
14422
14437
TCCCATCGACACAGCA
95
959


 


916651
N/A
N/A
15206
15221
GGAATATTGCCAGGTA
95
960


 


916671
N/A
N/A
15759
15774
TGGTTTATAACAACTG
29
961


 


916691
N/A
N/A
16746
16761
ATTAGGAGAGGTCTCA
55
962


 


916711
N/A
N/A
17602
17617
CTTGATAGTGAATGTG
90
963


 


916731
N/A
N/A
18859
18874
GGCACTCACAAAAGCG
10
964


 


916751
N/A
N/A
20182
20197
CCCTATGTTCTACTTT
54
965


 


916770
N/A
N/A
20572
20587
CAACATCTCTAGCTGG
82
966


 


916790
N/A
N/A
20810
20825
GGTAATATTCAGACCA
 0
967


 


916810
N/A
N/A
21265
21280
TGAAGCTACAGATCCA
74
968


 


916830
N/A
N/A
22042
22057
GGAAATCTGTCAGAGC
18
969


 


916850
N/A
N/A
23142
23157
GAATCTAGGAAGGCGA
77
970


 


916870
N/A
N/A
23789
23804
AGTATTTACCTGGAGG
 0
971


 


916890
N/A
N/A
24738
24753
AGCCTTAGGAAGCCTC
16
972
















TABLE 15







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915353
  35
  50
 2773
 2788
TCGGCCAGGGCATTCC
 0
 973


 


915373
  87
 102
 2825
 2840
CGCAGCAGCTCCGCCC
 0
 974


 


915393
 136
 151
 2874
 2889
CGCGGGTTAGGATCTG
 0
 975


 


915413
 239
 254
 2977
 2992
GCGGGTCGCCCCGACG
 0
 976


 


915433
 325
 340
 3063
 3078
ACGCCGACGCAGTGCA
 0
 977


 


915453
 372
 387
 5946
 5961
GATCTGAGAGGACCTG
24
 978


 


915473
 402
 417
 5976
 5991
CAATGTTCCGACTCCT
73
 979


 


915493
 441
 456
 6015
 6030
GGAGGAACTTGCTTAA
87
 980


 


915513
 470
 485
 6044
 6059
GGCCGGGAGGCATTTG
 0
 981


 


915533
 500
 515
 6074
 6089
TATTTTGCCGGAGATG
75
 982


 


915553
 522
 537
 6096
 6111
ACACTCTGGTAAGAGA
 0
 983


 


915613
 710
 725
11928
11943
CACGGTGATGGTTGTT
64
 984


 


915633
 788
 803
12006
12021
GAGCTTGGTGATGTCC
74
 985


 


915653
 837
 852
12055
12070
CAAAAGCTCTCGAGAG
 0
 986


 


915673
 892
 907
13628
13643
GCATCCAAATATCCTC
81
 987


 


915693
 973
 988
16093
16108
TCAGGATCCATCCCTT
10
 988


 


915713
1007
1022
16127
16142
CAGACTCATGTTTGCC
 0
 989


 


915733
1082
1097
16202
16217
GCTGAGACGCAGGTGG
64
 990


 


915753
1142
1157
N/A
N/A
CAGTGCTGTAGCGAGC
 0
 991


 


915773
1204
1219
19044
19059
CTAATGGGTAGCAAGT
72
 992


 


915793
1258
1273
19098
19113
GCAATGGCAGATTCCA
57
 993


 


915813
1329
1344
23734
23749
AGGTCACCCACTGCAA
56
 994


 


915833
1359
1374
23764
23779
GACACATCAGCACTCG
43
 995


 


915853
1431
1446
25194
25209
AGTCCTGCTCAGGTGT
66
 996


 


915873
1525
1540
25288
25303
AAGTTCAGGCTGGACC
54
 997


 


915893
1571
1586
25334
25349
GGTGGAGAGCCCCTCA
 0
 998


 


915913
1622
1637
25385
25400
CTCGCCTCCTCAAGTG
52
 999


 


915933
1660
1675
25423
25438
GATGGGAAACTTTAGC
85
1000


 


915953
1697
1712
25460
25475
GGGTCACTACACAGCA
78
1001


 


915973
1778
1793
25541
25556
CTGCACAGGCGAAAGG
35
1002


 


915993
1800
1815
25563
25578
ATTAGAGTTAAGTGCT
63
1003


 


916013
1827
1842
25590
25605
ACCAGCTGAATTAACG
66
1004


 


916033
1873
1888
25636
25651
GTCAGTAAGGGACCCT
52
1005


 


916053
1897
1912
25660
25675
GACCATTAATAGGGCC
51
1006


 


916073
1924
1939
25687
25702
TCTAAGAACCTCATGC
55
1007


 


916093
1967
1982
25730
25745
TACCCCCCATCACAAG
15
1008


 


916113
1990
2005
25753
25768
ACAAGATCACACATGG
72
1009


 


916133
2086
2101
25849
25864
AGACCACCTGACAGGC
79
1010


 


916152
2112
2127
25875
25890
TTAGTAGTCAAGGTTA
84
1011


 


916172
2177
2192
25940
25955
AGGTGAAAAAGGTGTT
88
1012


 


916192
2285
2300
26048
26063
TTAAGGCTTATTCTAC
82
1013


 


916212
2635
2650
26398
26413
TGAGGTGAATGCCCTG
58
1014


 


916232
2696
2711
26459
26474
TGTGTGCTCCAGTGGA
89
1015


 


916252
2730
2745
26493
26508
GCTGGTCCCTGCAGAA
44
1016


 


916272
N/A
N/A
 3328
 3343
GGGACGCACGAGAGTC
 0
1017


 


916292
N/A
N/A
 4432
 4447
GTCAATAGCTTCACAA
86
1018


 


916312
N/A
N/A
 5281
 5296
ATAAATCTTGGGATGC
92
1019


 


916332
N/A
N/A
 5577
 5592
CACAATGACATCATGG
95
1020


 


916352
N/A
N/A
 6170
 6185
GATAAGCAAACTGCTT
19
1021


 


916372
N/A
N/A
 7192
 7207
GAGGATGCAACTGGCT
84
1022


 


916392
N/A
N/A
 7644
 7659
TCGGACTTCAGGCCCA
 0
1023


 


916552
N/A
N/A
12208
12223
CCTTTTATTTCCGTTA
97
1024


 


916572
N/A
N/A
12745
12760
GCATACTAAAACCACC
85
1025


 


916592
N/A
N/A
13375
13390
GACTTTGCAGGCACCC
92
1026


 


916612
N/A
N/A
13909
13924
TGACATCCCAGTTCAA
30
1027


 


916632
N/A
N/A
14427
14442
TACTTTCCCATCGACA
81
1028


 


916652
N/A
N/A
15207
15222
AGGAATATTGCCAGGT
88
1029


 


916672
N/A
N/A
15768
15783
GGTTAGTGTTGGTTTA
92
1030


 


916692
N/A
N/A
16790
16805
CATTCGATGGAGGTTC
58
1031


 


916712
N/A
N/A
17629
17644
GGCGGATTTCCCCACT
11
1032


 


916732
N/A
N/A
18894
18909
TAAAATACGCCCGTCC
 7
1033


 


916752
N/A
N/A
20183
20198
TCCCTATGTTCTACTT
32
1034


 


916771
N/A
N/A
20574
20589
ATCAACATCTCTAGCT
46
1035


 


916791
N/A
N/A
20811
20826
GGGTAATATTCAGACC
43
1036


 


916811
N/A
N/A
21313
21328
TTTACTAGAGACTCTG
69
1037


 


916831
N/A
N/A
22071
22086
GTAGGATAGGACTAGA
45
1038


 


916851
N/A
N/A
23219
23234
ATAAATGCCTGACCAC
64
1039


 


916871
N/A
N/A
23861
23876
TGTTTCTAGAATGTCG
68
1040


 


916891
N/A
N/A
24873
24888
GCCTATCAGTTTCCCC
 0
1041
















TABLE 16







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915354
  36
  51
 2774
 2789
CTCGGCCAGGGCATTC
 0
1042


 


915374
  89
 104
 2827
 2842
TCCGCAGCAGCTCCGC
60
1043


 


915394
 137
 152
 2875
 2890
GCGCGGGTTAGGATCT
 0
1044


 


915414
 240
 255
 2978
 2993
AGCGGGTCGCCCCGAC
21
1045


 


915434
 337
 352
 3075
 3090
ATACCGGAGAGGACGC
85
1046


 


915454
 374
 389
 5948
 5963
AAGATCTGAGAGGACC
24
1047


 


915474
 403
 418
 5977
 5992
CCAATGTTCCGACTCC
95
1048


 


915494
 442
 457
 6016
 6031
CGGAGGAACTTGCTTA
93
1049


 


915514
 471
 486
 6045
 6060
TGGCCGGGAGGCATTT
 0
1050


 


915534
 501
 516
 6075
 6090
CTATTTTGCCGGAGAT
87
1051


 


915554
 523
 538
 6097
 6112
GACACTCTGGTAAGAG
26
1052


 


915614
 711
 726
11929
11944
ACACGGTGATGGTTGT
46
1053


 


915634
 791
 806
12009
12024
ACTGAGCTTGGTGATG
87
1054


 


915654
 838
 853
12056
12071
ACAAAAGCTCTCGAGA
 0
1055


 


915674
 900
 915
13636
13651
ACCTGAATGCATCCAA
93
1056


 


915694
 974
 989
16094
16109
CTCAGGATCCATCCCT
43
1057


 


915714
1008
1023
16128
16143
CCAGACTCATGTTTGC
 0
1058


 


915734
1083
1098
16203
16218
TGCTGAGACGCAGGTG
50
1059


 


915754
1143
1158
N/A
N/A
TCAGTGCTGTAGCGAG
42
1060


 


915774
1208
1223
19048
19063
TATCCTAATGGGTAGC
53
1061


 


915794
1260
1275
19100
19115
TCGCAATGGCAGATTC
67
1062


 


915814
1333
1348
23738
23753
TGTGAGGTCACCCACT
 0
1063


 


915834
1360
1375
23765
23780
AGACACATCAGCACTC
24
1064


 


915854
1432
1447
25195
25210
CAGTCCTGCTCAGGTG
54
1065


 


915874
1526
1541
25289
25304
GAAGTTCAGGCTGGAC
75
1066


 


915894
1572
1587
25335
25350
AGGTGGAGAGCCCCTC
 0
1067


 


915914
1623
1638
25386
25401
ACTCGCCTCCTCAAGT
 0
1068


 


915934
1661
1676
25424
25439
AGATGGGAAACTTTAG
84
1069


 


915954
1724
1739
25487
25502
GGCTGGGATCCTCCAC
24
1070


 


915974
1779
1794
25542
25557
GCTGCACAGGCGAAAG
56
1071


 


915994
1801
1816
25564
25579
TATTAGAGTTAAGTGC
75
1072


 


916014
1828
1843
25591
25606
AACCAGCTGAATTAAC
55
1073


 


916034
1875
1890
25638
25653
CAGTCAGTAAGGGACC
70
1074


 


916054
1898
1913
25661
25676
TGACCATTAATAGGGC
74
1075


 


916074
1925
1940
25688
25703
TTCTAAGAACCTCATG
22
1076


 


916094
1968
1983
25731
25746
CTACCCCCCATCACAA
 0
1077


 


916114
1992
2007
25755
25770
CCACAAGATCACACAT
 0
1078


 


916134
2087
2102
25850
25865
CAGACCACCTGACAGG
78
1079


 


916153
2113
2128
25876
25891
TTTAGTAGTCAAGGTT
93
1080


 


916173
2178
2193
25941
25956
TAGGTGAAAAAGGTGT
89
1081


 


916193
2306
2321
26069
26084
ACCCAACCGATTTTTT
61
1082


 


916213
2636
2651
26399
26414
CTGAGGTGAATGCCCT
73
1083


 


916233
2697
2712
26460
26475
TTGTGTGCTCCAGTGG
92
1084


 


916253
2746
2761
26509
26524
TCACTGACCATGTGGG
16
1085


 


916273
N/A
N/A
 3362
 3377
CTTCATGCACGGGCGC
37
1086


 


916293
N/A
N/A
 4462
 4477
GCATAATCTCCTGCCT
 0
1087


 


916313
N/A
N/A
 5284
 5299
GCCATAAATCTTGGGA
37
1088


 


916333
N/A
N/A
 5605
 5620
CTTTATTCAATGTGGC
97
1089


 


916353
N/A
N/A
 6529
 6544
TACAACTGCCTGTGTT
 0
1090


 


916373
N/A
N/A
 7218
 7233
AAAGCTTCCGCAAACA
51
1091


 


916393
N/A
N/A
 7657
 7672
CTAACATACACCCTCG
 0
1092


 


916553
N/A
N/A
12225
12240
AGCTTCTGGGACAAGC
10
1093


 


916573
N/A
N/A
12746
12761
GGCATACTAAAACCAC
55
1094


 


916593
N/A
N/A
13397
13412
TTGAATGTCACCCTTC
91
1095


 


916613
N/A
N/A
13914
13929
AGTCATGACATCCCAG
93
1096


 


916633
N/A
N/A
14442
14457
TCTCATTGGCACCTGT
86
1097


 


916653
N/A
N/A
15252
15267
CCCTATCAGATGCCCT
81
1098


 


916673
N/A
N/A
15799
15814
CATATCTGGTTTCATG
 0
1099


 


916693
N/A
N/A
16842
16857
GACCATAGCACTGTCT
 0
1100


 


916713
N/A
N/A
17737
17752
ATTAATCTGGTCATAT
 0
1101


 


916733
N/A
N/A
18898
18913
TCCATAAAATACGCCC
69
1102


 


916753
N/A
N/A
20195
20210
GAAAGATGGAATTCCC
86
1103


 


916772
N/A
N/A
20604
20619
TACGATCATCATTATT
91
1104


 


916792
N/A
N/A
20841
20856
GTATTAGCTCAATATT
 0
1105


 


916812
N/A
N/A
21314
21329
GTTTACTAGAGACTCT
64
1106


 


916832
N/A
N/A
22080
22095
GTAAAAACTGTAGGAT
 0
1107


 


916852
N/A
N/A
23220
23235
GATAAATGCCTGACCA
29
1108


 


916872
N/A
N/A
24011
24026
CCGACGGGAAGTCTTC
 0
1109


 


916892
N/A
N/A
24874
24889
GGCCTATCAGTTTCCC
 0
1110
















TABLE 17







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915355
  37
  52
 2775
 2790
TCTCGGCCAGGGCATT
 0
1111


 


915375
  90
 105
 2828
 2843
ATCCGCAGCAGCTCCG
52
1112


 


915395
 138
 153
 2876
 2891
GGCGCGGGTTAGGATC
 0
1113


 


915415
 241
 256
 2979
 2994
CAGCGGGTCGCCCCGA
 8
1114


 


915435
 338
 353
 3076
 3091
GATACCGGAGAGGACG
30
1115


 


915455
 378
 393
 5952
 5967
GCACAAGATCTGAGAG
72
1116


 


915475
 405
 420
 5979
 5994
TGCCAATGTTCCGACT
 0
1117


 


915495
 443
 458
 6017
 6032
TCGGAGGAACTTGCTT
69
1118


 


915515
 472
 487
 6046
 6061
TTGGCCGGGAGGCATT
 9
1119


 


915535
 502
 517
 6076
 6091
CCTATTTTGCCGGAGA
96
1120


 


915555
 524
 539
 6098
 6113
AGACACTCTGGTAAGA
 2
1121


 


915615
 712
 727
11930
11945
GACACGGTGATGGTTG
32
1122


 


915635
 792
 807
12010
12025
GACTGAGCTTGGTGAT
93
1123


 


915655
 839
 854
12057
12072
GACAAAAGCTCTCGAG
40
1124


 


915675
 901
 916
13637
13652
AACCTGAATGCATCCA
92
1125


 


915695
 975
 990
16095
16110
CCTCAGGATCCATCCC
 0
1126


 


915715
1011
1026
16131
16146
AATCCAGACTCATGTT
67
1127


 


915735
1088
1103
16208
16223
CAGGATGCTGAGACGC
86
1128


 


915755
1144
1159
N/A
N/A
CTCAGTGCTGTAGCGA
25
1129


 


915775
1209
1224
19049
19064
TTATCCTAATGGGTAG
23
1130


 


915795
1261
1276
19101
19116
ATCGCAATGGCAGATT
 0
1131


 


915815
1337
1352
23742
23757
CACCTGTGAGGTCACC
35
1132


 


915835
1361
1376
23766
23781
CAGACACATCAGCACT
54
1133


 


915855
1433
1448
25196
25211
CCAGTCCTGCTCAGGT
23
1134


 


915875
1530
1545
25293
25308
AGAAGAAGTTCAGGCT
81
1135


 


915895
1574
1589
25337
25352
AAAGGTGGAGAGCCCC
76
1136


 


915915
1624
1639
25387
25402
GACTCGCCTCCTCAAG
75
1137


 


915935
1674
1689
25437
25452
GGTAGCTGCACAAAGA
76
1138


 


915955
1726
1741
25489
25504
GAGGCTGGGATCCTCC
 0
1139


 


915975
1780
1795
25543
25558
CGCTGCACAGGCGAAA
 0
1140


 


915995
1805
1820
25568
25583
GATGTATTAGAGTTAA
82
1141


 


916015
1829
1844
25592
25607
CAACCAGCTGAATTAA
59
1142


 


916035
1876
1891
25639
25654
ACAGTCAGTAAGGGAC
81
1143


 


916055
1899
1914
25662
25677
CTGACCATTAATAGGG
49
1144


 


916075
1929
1944
25692
25707
GTCATTCTAAGAACCT
81
1145


 


916095
1969
1984
25732
25747
CCTACCCCCCATCACA
21
1146


 


916115
1995
2010
25758
25773
ACCCCACAAGATCACA
 0
1147


 


916135
2088
2103
25851
25866
GCAGACCACCTGACAG
44
1148


 


916154
2131
2146
25894
25909
CCCCGCCATGGAGACG
68
1149


 


916174
2180
2195
25943
25958
GTTAGGTGAAAAAGGT
90
1150


 


916194
2308
2323
26071
26086
GCACCCAACCGATTTT
83
1151


 


916214
2637
2652
26400
26415
GCTGAGGTGAATGCCC
52
1152


 


916234
2698
2713
26461
26476
GTTGTGTGCTCCAGTG
88
1153


 


916254
2747
2762
26510
26525
CTCACTGACCATGTGG
13
1154


 


916274
N/A
N/A
 3524
 3539
GCAAATCGGCCCCTCG
 3
1155


 


916294
N/A
N/A
 4463
 4478
GGCATAATCTCCTGCC
 0
1156


 


916314
N/A
N/A
 5324
 5339
TGGCATGCAAGACCAC
 0
1157


 


916334
N/A
N/A
 5606
 5621
ACTTTATTCAATGTGG
95
1158


 


916354
N/A
N/A
 6556
 6571
GTTTATGTCACTCTGG
68
1159


 


916374
N/A
N/A
 7245
 7260
GAACAGACAAGTGCTG
38
1160


 


916394
N/A
N/A
 7658
 7673
ACTAACATACACCCTC
31
1161


 


916554
N/A
N/A
12249
12264
ATAATCAGGGTGGTGC
 0
1162


 


916574
N/A
N/A
12747
12762
AGGCATACTAAAACCA
47
1163


 


916594
N/A
N/A
13500
13515
GAATCATGCAAGCTCT
50
1164


 


916614
N/A
N/A
13996
14011
TAAACTAAGGGTCACA
37
1165


 


916634
N/A
N/A
14497
14512
ATCCATCCTGCATGAG
76
1166


 


916654
N/A
N/A
15254
15269
GGCCCTATCAGATGCC
 0
1167


 


916674
N/A
N/A
15802
15817
CTACATATCTGGTTTC
 0
1168


 


916694
N/A
N/A
16844
16859
TGGACCATAGCACTGT
60
1169


 


916714
N/A
N/A
17738
17753
TATTAATCTGGTCATA
18
1170


 


916734
N/A
N/A
18926
18941
CCACTTTACTCTGTTG
64
1171


 


916754
N/A
N/A
20210
20225
AACTATGCCTAGAACG
43
1172


 


916773
N/A
N/A
20606
20621
TTTACGATCATCATTA
77
1173


 


916793
N/A
N/A
20842
20857
TGTATTAGCTCAATAT
 0
1174


 


916813
N/A
N/A
21319
21334
TGGGAGTTTACTAGAG
66
1175


 


916833
N/A
N/A
22118
22133
AGAGAGTACTCTTGGA
11
1176


 


916853
N/A
N/A
23222
23237
CTGATAAATGCCTGAC
78
1177


 


916873
N/A
N/A
24038
24053
ATCAATGCTGCACTCA
88
1178


 


916893
N/A
N/A
24889
24904
ACGAATCCCTGGAGGG
 0
1179
















TABLE 18







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915356
  38
  53
 2776
 2791
GTCTCGGCCAGGGCAT
 0
1180


 


915376
  93
 108
 2831
 2846
CTGATCCGCAGCAGCT
28
1181


 


915396
 165
 180
 2903
 2918
CGTACATGGCGGCGGC
 0
1182


 


915416
 242
 257
 2980
 2995
GCAGCGGGTCGCCCCG
 0
1183


 


915436
 339
 354
 3077
 3092
GGATACCGGAGAGGAC
64
1184


 


915456
 379
 394
 5953
 5968
CGCACAAGATCTGAGA
79
1185


 


915476
 406
 421
 5980
 5995
ATGCCAATGTTCCGAC
83
1186


 


915496
 444
 459
 6018
 6033
GTCGGAGGAACTTGCT
35
1187


 


915516
 473
 488
 6047
 6062
ATTGGCCGGGAGGCAT
 0
1188


 


915536
 503
 518
 6077
 6092
GCCTATTTTGCCGGAG
77
1189


 


915556
 525
 540
 6099
 6114
CAGACACTCTGGTAAG
62
1190


 


915616
 730
 745
11948
11963
TACTCCCCATAGAAGG
 8
1191


 


915636
 794
 809
12012
12027
TAGACTGAGCTTGGTG
90
1192


 


915656
 840
 855
12058
12073
GGACAAAAGCTCTCGA
60
1193


 


915676
 902
 917
13638
13653
GAACCTGAATGCATCC
72
1194


 


915696
 978
 993
16098
16113
CGACCTCAGGATCCAT
 0
1195


 


915716
1012
1027
16132
16147
GAATCCAGACTCATGT
 0
1196


 


915736
1089
1104
16209
16224
GCAGGATGCTGAGACG
55
1197


 


915756
1145
1160
N/A
N/A
ACTCAGTGCTGTAGCG
12
1198


 


915776
1210
1225
19050
19065
ATTATCCTAATGGGTA
28
1199


 


915796
1262
1277
19102
19117
AATCGCAATGGCAGAT
 0
1200


 


915816
1339
1354
23744
23759
AACACCTGTGAGGTCA
56
1201


 


915836
1365
1380
23770
23785
GGAGCAGACACATCAG
53
1202


 


915856
1434
1449
25197
25212
GCCAGTCCTGCTCAGG
21
1203


 


915876
1531
1546
25294
25309
AAGAAGAAGTTCAGGC
85
1204


 


915896
1575
1590
25338
25353
GAAAGGTGGAGAGCCC
78
1205


 


915916
1626
1641
25389
25404
TAGACTCGCCTCCTCA
32
1206


 


915936
1676
1691
25439
25454
GAGGTAGCTGCACAAA
91
1207


 


915956
1737
1752
25500
25515
AACTCAGCTCAGAGGC
46
1208


 


915976
1781
1796
25544
25559
CCGCTGCACAGGCGAA
 0
1209


 


915996
1807
1822
25570
25585
CTGATGTATTAGAGTT
93
1210


 


916016
1830
1845
25593
25608
CCAACCAGCTGAATTA
21
1211


 


916036
1877
1892
25640
25655
AACAGTCAGTAAGGGA
82
1212


 


916056
1900
1915
25663
25678
TCTGACCATTAATAGG
13
1213


 


916076
1930
1945
25693
25708
TGTCATTCTAAGAACC
40
1214


 


916096
1970
1985
25733
25748
GCCTACCCCCCATCAC
18
1215


 


916116
1996
2011
25759
25774
CACCCCACAAGATCAC
50
1216


 


916136
2089
2104
25852
25867
TGCAGACCACCTGACA
58
1217


 


916155
2132
2147
25895
25910
CCCCCGCCATGGAGAC
33
1218


 


916175
2224
2239
25987
26002
CGCTTCCTTACATTTT
89
1219


 


916195
2309
2324
26072
26087
TGCACCCAACCGATTT
64
1220


 


916215
2638
2653
26401
26416
GGCTGAGGTGAATGCC
 0
1221


 


916235
2699
2714
26462
26477
AGTTGTGTGCTCCAGT
85
1222


 


916255
2748
2763
26511
26526
ACTCACTGACCATGTG
 0
1223


 


916275
N/A
N/A
 3555
 3570
GGCCAAAGCCCCACTC
 0
1224


 


916295
N/A
N/A
 4464
 4479
GGGCATAATCTCCTGC
 0
1225


 


916315
N/A
N/A
 5342
 5357
GGCTGATCTGCACTCT
84
1226


 


916335
N/A
N/A
 5626
 5641
TAATTCTACCTGTGTC
92
1227


 


916355
N/A
N/A
 6557
 6572
AGTTTATGTCACTCTG
27
1228


 


916375
N/A
N/A
 7321
 7336
ACACTTTGCGAAGCAC
27
1229


 


916395
N/A
N/A
 7660
 7675
GAACTAACATACACCC
 1
1230


 


916555
N/A
N/A
12252
12267
CCCATAATCAGGGTGG
 0
1231


 


916575
N/A
N/A
12758
12773
GTAGAGTGGTAAGGCA
95
1232


 


916595
N/A
N/A
13502
13517
AAGAATCATGCAAGCT
34
1233


 


916615
N/A
N/A
13997
14012
TTAAACTAAGGGTCAC
65
1234


 


916635
N/A
N/A
14549
14564
TTAATGTGGATTCACG
76
1235


 


916655
N/A
N/A
15295
15310
CCAAGATAACCTCACA
64
1236


 


916675
N/A
N/A
15806
15821
CCATCTACATATCTGG
26
1237


 


916695
N/A
N/A
16854
16869
CACAATCATTTGGACC
72
1238


 


916715
N/A
N/A
17739
17754
GTATTAATCTGGTCAT
87
1239


 


916735
N/A
N/A
19113
19128
CACCTCTGGACAATCG
29
1240


 


916755
N/A
N/A
20212
20227
CAAACTATGCCTAGAA
70
1241


 


916774
N/A
N/A
20608
20623
ATTTTACGATCATCAT
91
1242


 


916794
N/A
N/A
20846
20861
TGCCTGTATTAGCTCA
90
1243


 


916814
N/A
N/A
21345
21360
CACATAAAGTCAAACG
87
1244


 


916834
N/A
N/A
22124
22139
AGAACAAGAGAGTACT
 3
1245


 


916854
N/A
N/A
23250
23265
CACATAAAGGACCCCC
54
1246


 


916874
N/A
N/A
24126
24141
CGCTATCTGACACTCC
87
1247


 


916894
N/A
N/A
24896
24911
TCCACCAACGAATCCC
50
1248
















TABLE 19







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915357
  39
  54
 2777
 2792
TGTCTCGGCCAGGGCA
 0
1249


 


915377
  94
 109
 2832
 2847
CCTGATCCGCAGCAGC
 0
1250


 


915397
 182
 197
 2920
 2935
CCAGCCGCGCTCTGCG
 0
1251


 


915417
 243
 258
 2981
 2996
GGCAGCGGGTCGCCCC
 0
1252


 


915437
 341
 356
 3079
 3094
CGGGATACCGGAGAGG
57
1253


 


915457
 380
 395
 5954
 5969
CCGCACAAGATCTGAG
71
1254


 


915477
 407
 422
 5981
 5996
GATGCCAATGTTCCGA
93
1255


 


915497
 445
 460
 6019
 6034
TGTCGGAGGAACTTGC
85
1256


 


915517
 474
 489
 6048
 6063
CATTGGCCGGGAGGCA
 0
1257


 


915537
 504
 519
 6078
 6093
TGCCTATTTTGCCGGA
44
1258


 


915557
 526
 541
 6100
 6115
TCAGACACTCTGGTAA
26
1259


 


915617
 732
 747
11950
11965
CGTACTCCCCATAGAA
66
1260


 


915637
 796
 811
12014
12029
CGTAGACTGAGCTTGG
98
1261


 


915657
 857
 872
12075
12090
CACCTTGAGATCCGGG
 0
1262


 


915677
 903
 918
13639
13654
AGAACCTGAATGCATC
78
1263


 


915697
 979
 994
16099
16114
GCGACCTCAGGATCCA
 1
1264


 


915717
1031
1046
16151
16166
GGCAGCCGACTCCGGG
19
1265


 


915737
1109
1124
16229
16244
CAGGATGCTCTCATCC
 0
1266


 


915757
1146
1161
N/A
N/A
CACTCAGTGCTGTAGC
33
1267


 


915777
1214
1229
19054
19069
AGACATTATCCTAATG
42
1268


 


915797
1263
1278
19103
19118
CAATCGCAATGGCAGA
49
1269


 


915817
1340
1355
23745
23760
GAACACCTGTGAGGTC
44
1270


 


915837
1398
1413
25161
25176
GGCTGCTCACTGGCAT
18
1271


 


915857
1435
1450
25198
25213
GGCCAGTCCTGCTCAG
 0
1272


 


915877
1534
1549
25297
25312
CCCAAGAAGAAGTTCA
76
1273


 


915897
1576
1591
25339
25354
GGAAAGGTGGAGAGCC
24
1274


 


915917
1627
1642
25390
25405
CTAGACTCGCCTCCTC
77
1275


 


915937
1679
1694
25442
25457
GCGGAGGTAGCTGCAC
16
1276


 


915957
1738
1753
25501
25516
CAACTCAGCTCAGAGG
61
1277


 


915977
1782
1797
25545
25560
ACCGCTGCACAGGCGA
34
1278


 


915997
1809
1824
25572
25587
TGCTGATGTATTAGAG
83
1279


 


916017
1831
1846
25594
25609
CCCAACCAGCTGAATT
42
1280


 


916037
1878
1893
25641
25656
AAACAGTCAGTAAGGG
92
1281


 


916057
1901
1916
25664
25679
GTCTGACCATTAATAG
64
1282


 


916077
1931
1946
25694
25709
CTGTCATTCTAAGAAC
41
1283


 


916097
1971
1986
25734
25749
AGCCTACCCCCCATCA
 0
1284


 


916117
1997
2012
25760
25775
CCACCCCACAAGATCA
 0
1285


 


916137
2090
2105
25853
25868
TTGCAGACCACCTGAC
65
1286


 


916156
2133
2148
25896
25911
ACCCCCGCCATGGAGA
54
1287


 


916176
2225
2240
25988
26003
ACGCTTCCTTACATTT
84
1288


 


916196
2310
2325
26073
26088
CTGCACCCAACCGATT
58
1289


 


916216
2639
2654
26402
26417
GGGCTGAGGTGAATGC
46
1290


 


916236
2700
2715
26463
26478
AAGTTGTGTGCTCCAG
86
1291


 


916256
2751
2766
26514
26529
GAAACTCACTGACCAT
41
1292


 


916276
N/A
N/A
 4068
 4083
GGAAACAACTTTCCTC
 0
1293


 


916296
N/A
N/A
 4730
 4745
GATCATGTGGCGGTCT
68
1294


 


916316
N/A
N/A
 5364
 5379
CACTTACTGGCCTGGC
30
1295


 


916336
N/A
N/A
 5645
 5660
ATATTGGGCTCAATGA
89
1296


 


916356
N/A
N/A
 6575
 6590
ATCACTGGAGGTGTAC
 0
1297


 


916376
N/A
N/A
 7328
 7343
CAGGATCACACTTTGC
17
1298


 


916396
N/A
N/A
 7661
 7676
GGAACTAACATACACC
 0
1299


 


916556
N/A
N/A
12272
12287
GTATATGTTCCCAGGT
81
1300


 


916576
N/A
N/A
12788
12803
GTGTACATGGTCTGCA
94
1301


 


916596
N/A
N/A
13529
13544
ATCATTGGAAGACCGC
89
1302


 


916616
N/A
N/A
13998
14013
GTTAAACTAAGGGTCA
85
1303


 


916636
N/A
N/A
14550
14565
CTTAATGTGGATTCAC
91
1304


 


916656
N/A
N/A
15351
15366
TCCAACTTCAGGCTGA
74
1305


 


916676
N/A
N/A
15819
15834
AGCTTTGTGGGCTCCA
69
1306


 


916696
N/A
N/A
16982
16997
GTTTAATAAGGGCACC
63
1307


 


916716
N/A
N/A
17740
17755
CGTATTAATCTGGTCA
93
1308


 


916736
N/A
N/A
19126
19141
CACCTAAAATGCTCAC
17
1309


 


916756
N/A
N/A
20213
20228
ACAAACTATGCCTAGA
58
1310


 


916775
N/A
N/A
20609
20624
AATTTTACGATCATCA
78
1311


 


916795
N/A
N/A
20927
20942
GACAGATCAGCACTCG
80
1312


 


916815
N/A
N/A
21407
21422
CAATTCTAGACATGGC
88
1313


 


916835
N/A
N/A
22338
22353
TGCACCTACCCTTTTC
39
1314


 


916855
N/A
N/A
23251
23266
ACACATAAAGGACCCC
48
1315


 


916875
N/A
N/A
24241
24256
GCATTACCAGGCACCT
61
1316


 


916895
N/A
N/A
24912
24927
GACATCACAGGTGTTG
 5
1317
















TABLE 20







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915358
  40
  55
 2778
 2793
GTGTCTCGGCCAGGGC
 0
1318


 


915378
  96
 111
 2834
 2849
GTCCTGATCCGCAGCA
 0
1319


 


915398
 184
 199
 2922
 2937
CTCCAGCCGCGCTCTG
14
1320


 


915418
 244
 259
 2982
 2997
AGGCAGCGGGTCGCCC
 0
1321


 


915438
 342
 357
 3080
 3095
GCGGGATACCGGAGAG
44
1322


 


915458
 381
 396
 5955
 5970
TCCGCACAAGATCTGA
41
1323


 


915478
 408
 423
 5982
 5997
AGATGCCAATGTTCCG
95
1324


 


915498
 446
 461
 6020
 6035
CTGTCGGAGGAACTTG
40
1325


 


915518
 475
 490
 6049
 6064
ACATTGGCCGGGAGGC
 5
1326


 


915538
 505
 520
 6079
 6094
ATGCCTATTTTGCCGG
61
1327


 


915558
 527
 542
 6101
 6116
ATCAGACACTCTGGTA
 0
1328


 


915618
 748
 763
11966
11981
ACTTTAGGGCAGATGT
87
1329


 


915638
 818
 833
12036
12051
GTAGAGGTTCCCTGTG
87
1330


 


915658
 859
 874
N/A
N/A
AGCACCTTGAGATCCG
 0
1331


 


915678
 926
 941
N/A
N/A
GTTGCAGATGCCCTTC
24
1332


 


915698
 980
 995
16100
16115
GGCGACCTCAGGATCC
 0
1333


 


915718
1032
1047
16152
16167
AGGCAGCCGACTCCGG
 8
1334


 


915738
1114
1129
16234
16249
GTGTCCAGGATGCTCT
41
1335


 


915758
1147
1162
N/A
N/A
TCACTCAGTGCTGTAG
59
1336


 


915778
1217
1232
19057
19072
ATAAGACATTATCCTA
85
1337


 


915798
1264
1279
19104
19119
ACAATCGCAATGGCAG
66
1338


 


915818
1342
1357
23747
23762
GTGAACACCTGTGAGG
58
1339


 


915838
1400
1415
25163
25178
TTGGCTGCTCACTGGC
79
1340


 


915858
1436
1451
25199
25214
GGGCCAGTCCTGCTCA
 0
1341


 


915878
1535
1550
25298
25313
GCCCAAGAAGAAGTTC
54
1342


 


915898
1590
1605
25353
25368
CTAGTGAAAAACTGGG
34
1343


 


915918
1628
1643
25391
25406
GCTAGACTCGCCTCCT
33
1344


 


915938
1680
1695
25443
25458
TGCGGAGGTAGCTGCA
 0
1345


 


915958
1756
1771
25519
25534
CCTAGCTTTTCATAAA
 0
1346


 


915978
1783
1798
25546
25561
GACCGCTGCACAGGCG
24
1347


 


915998
1810
1825
25573
25588
ATGCTGATGTATTAGA
86
1348


 


916018
1832
1847
25595
25610
TCCCAACCAGCTGAAT
 3
1349


 


916038
1879
1894
25642
25657
GAAACAGTCAGTAAGG
64
1350


 


916058
1902
1917
25665
25680
AGTCTGACCATTAATA
86
1351


 


916078
1933
1948
25696
25711
ACCTGTCATTCTAAGA
18
1352


 


916098
1972
1987
25735
25750
CAGCCTACCCCCCATC
41
1353


 


916118
1999
2014
25762
25777
CTCCACCCCACAAGAT
 0
1354


 


916138
2092
2107
25855
25870
CTTTGCAGACCACCTG
65
1355


 


916157
2134
2149
25897
25912
TACCCCCGCCATGGAG
57
1356


 


916177
2237
2252
26000
26015
CAACAGGTAACAACGC
88
1357


 


916197
2579
2594
26342
26357
GTCAGACTTTCACTCA
81
1358


 


916217
2659
2674
26422
26437
GTGCTTGGCTCCTGCC
43
1359


 


916237
2701
2716
26464
26479
CAAGTTGTGTGCTCCA
73
1360


 


916257
2769
2784
26532
26547
CATCGCCACACATGGG
61
1361


 


916277
N/A
N/A
 4105
 4120
AGGAAGGGTCCCAAAC
 0
1362


 


916297
N/A
N/A
 4731
 4746
TGATCATGTGGCGGTC
80
1363


 


916317
N/A
N/A
 5391
 5406
TGCTATCAGGTGCAGG
60
1364


 


916337
N/A
N/A
 5646
 5661
TATATTGGGCTCAATG
71
1365


 


916357
N/A
N/A
 6594
 6609
GTTTACAAACATGGAC
26
1366


 


916377
N/A
N/A
 7464
 7479
TCATTAGCATCACCGG
33
1367


 


916397
N/A
N/A
 7662
 7677
GGGAACTAACATACAC
 0
1368


 


916557
N/A
N/A
12274
12289
GGGTATATGTTCCCAG
 0
1369


 


916577
N/A
N/A
12830
12845
TGCATAGCCTTCTTTC
84
1370


 


916597
N/A
N/A
13530
13545
CATCATTGGAAGACCG
62
1371


 


916617
N/A
N/A
14016
14031
TCTTTAACTTCGGCCC
70
1372


 


916637
N/A
N/A
14551
14566
TCTTAATGTGGATTCA
88
1373


 


916657
N/A
N/A
15388
15403
TCAGACAACCACAGCT
66
1374


 


916677
N/A
N/A
15852
15867
TAAAGCAGGACACACG
74
1375


 


916697
N/A
N/A
17077
17092
AGACATGTTGGTGTCT
 0
1376


 


916717
N/A
N/A
17788
17803
CCCCAGTCTTTTATTC
 0
1377


 


916737
N/A
N/A
19140
19155
GGAAGACACGGAGCCA
20
1378


 


916757
N/A
N/A
20240
20255
CCTAACTGCTGGCTCT
85
1379


 


916776
N/A
N/A
20610
20625
TAATTTTACGATCATC
76
1380


 


916796
N/A
N/A
20939
20954
CTCTTTGTAGCAGACA
90
1381


 


916816
N/A
N/A
21439
21454
CAATATACTGAGAGGA
92
1382


 


916836
N/A
N/A
22392
22407
GTAGACATCCTTCCCG
65
1383


 


916856
N/A
N/A
23252
23267
GACACATAAAGGACCC
59
1384


 


916876
N/A
N/A
24242
24257
TGCATTACCAGGCACC
37
1385


 


916896
N/A
N/A
24913
24928
GGACATCACAGGTGTT
19
1386
















TABLE 21







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915359
  41
  56
 2779
 2794
AGTGTCTCGGCCAGGG
 0
1387


 


915379
  97
 112
 2835
 2850
GGTCCTGATCCGCAGC
 0
1388


 


915399
 185
 200
 2923
 2938
GCTCCAGCCGCGCTCT
26
1389


 


915419
 245
 260
 2983
 2998
CAGGCAGCGGGTCGCC
 0
1390


 


915439
 343
 358
 3081
 3096
AGCGGGATACCGGAGA
69
1391


 


915459
 382
 397
 5956
 5971
TTCCGCACAAGATCTG
71
1392


 


915479
 409
 424
 5983
 5998
AAGATGCCAATGTTCC
93
1393


 


915499
 448
 463
 6022
 6037
CCCTGTCGGAGGAACT
30
1394


 


915519
 477
 492
 6051
 6066
GGACATTGGCCGGGAG
88
1395


 


915539
 506
 521
 6080
 6095
GATGCCTATTTTGCCG
60
1396


 


915559
 529
 544
 6103
 6118
CCATCAGACACTCTGG
30
1397


 


915579
 595
 610
 7825
 7840
CAGGAACATACCAAGG
98
1398


 


915599
 674
 689
11892
11907
GTTGTCACTCACTCCT
98
1399


 


915619
 749
 764
11967
11982
GACTTTAGGGCAGATG
96
1400


 


915639
 819
 834
12037
12052
GGTAGAGGTTCCCTGT
92
1401


 


915659
 860
 875
N/A
N/A
CAGCACCTTGAGATCC
 0
1402


 


915679
 928
 943
N/A
N/A
CTGTTGCAGATGCCCT
58
1403


 


915699
 981
 996
16101
16116
TGGCGACCTCAGGATC
40
1404


 


915719
1033
1048
16153
16168
AAGGCAGCCGACTCCG
 0
1405


 


915739
1115
1130
16235
16250
GGTGTCCAGGATGCTC
40
1406


 


915759
1148
1163
N/A
N/A
TTCACTCAGTGCTGTA
29
1407


 


915779
1219
1234
19059
19074
ACATAAGACATTATCC
86
1408


 


915799
1268
1283
19108
19123
CTGGACAATCGCAATG
42
1409


 


915819
1344
1359
23749
23764
GAGTGAACACCTGTGA
81
1410


 


915839
1401
1416
25164
25179
GTTGGCTGCTCACTGG
85
1411


 


915859
1437
1452
25200
25215
AGGGCCAGTCCTGCTC
 0
1412


 


915879
1538
1553
25301
25316
ATTGCCCAAGAAGAAG
54
1413


 


915899
1591
1606
25354
25369
TCTAGTGAAAAACTGG
 0
1414


 


915919
1629
1644
25392
25407
TGCTAGACTCGCCTCC
72
1415


 


915939
1681
1696
25444
25459
ATGCGGAGGTAGCTGC
39
1416


 


915959
1764
1779
25527
25542
GGTTGCTTCCTAGCTT
87
1417


 


915979
1784
1799
25547
25562
GGACCGCTGCACAGGC
 0
1418


 


915999
1811
1826
25574
25589
CATGCTGATGTATTAG
35
1419


 


916019
1833
1848
25596
25611
TTCCCAACCAGCTGAA
 0
1420


 


916039
1880
1895
25643
25658
CGAAACAGTCAGTAAG
80
1421


 


916059
1905
1920
25668
25683
AACAGTCTGACCATTA
85
1422


 


916079
1934
1949
25697
25712
CACCTGTCATTCTAAG
45
1423


 


916099
1973
1988
25736
25751
CCAGCCTACCCCCCAT
53
1424


 


916119
2022
2037
25785
25800
GTGGGATCATGCTATT
76
1425


 


916139
2093
2108
25856
25871
TCTTTGCAGACCACCT
85
1426


 


916158
2135
2150
25898
25913
TTACCCCCGCCATGGA
 0
1427


 


916178
2238
2253
26001
26016
TCAACAGGTAACAACG
87
1428


 


916198
2620
2635
26383
26398
GCACACTAGATTATTT
66
1429


 


916218
2673
2688
26436
26451
CGGAAGCTCCTGCTGT
27
1430


 


916238
2702
2717
26465
26480
TCAAGTTGTGTGCTCC
91
1431


 


916258
2770
2785
26533
26548
TCATCGCCACACATGG
49
1432


 


916278
N/A
N/A
 4211
 4226
TCATTTCCAGGAGTAC
75
1433


 


916298
N/A
N/A
 4735
 4750
CAAATGATCATGTGGC
93
1434


 


916318
N/A
N/A
 5394
 5409
TAATGCTATCAGGTGC
95
1435


 


916338
N/A
N/A
 5648
 5663
GATATATTGGGCTCAA
97
1436


 


916358
N/A
N/A
 6596
 6611
GGGTTTACAAACATGG
75
1437


 


916378
N/A
N/A
 7465
 7480
TTCATTAGCATCACCG
78
1438


 


916398
N/A
N/A
 7686
 7701
GTTAATCCATGGGTCA
49
1439


 


916418
N/A
N/A
 8992
 9007
AGCCTAAACTTCCTCC
63
1440


 


916438
N/A
N/A
 9318
 9333
AGAAGAGCCGCCCTGC
77
1441


 


916458
N/A
N/A
 9795
 9810
GCAAGACTAGCAAGTG
85
1442


 


916478
N/A
N/A
10301
10316
AGCATGCGGTATGTAC
67
1443


 


916498
N/A
N/A
10849
10864
CACACAATTTCTAGGG
82
1444


 


916518
N/A
N/A
11346
11361
TTGACAATTAGAACCA
96
1445


 


916538
N/A
N/A
11711
11726
ACAAATCCTTACCGAG
54
1446


 


916558
N/A
N/A
12285
12300
GTTTTAGGTCTGGGTA
94
1447


 


916578
N/A
N/A
12831
12846
TTGCATAGCCTTCTTT
93
1448


 


916598
N/A
N/A
13660
13675
CATACATACCCTTCTC
 9
1449


 


916618
N/A
N/A
14025
14040
CGCAGAAACTCTTTAA
89
1450


 


916638
N/A
N/A
14552
14567
GTCTTAATGTGGATTC
93
1451


 


916658
N/A
N/A
15421
15436
AGCATTGGCACACTGG
70
1452


 


916678
N/A
N/A
15857
15872
GGCTTTAAAGCAGGAC
62
1453


 


916698
N/A
N/A
17079
17094
GCAGACATGTTGGTGT
 2
1454


 


916718
N/A
N/A
17839
17854
TACAAGCTGGTCCTTG
 0
1455


 


916738
N/A
N/A
19211
19226
GACAATCCAGGTCCCA
70
1456


 


916758
N/A
N/A
20285
20300
GAGGAAGCCCAATCAA
81
1457


 


916777
N/A
N/A
20611
20626
CTAATTTTACGATCAT
81
1458


 


916797
N/A
N/A
20984
20999
TTAAACTGCCAAGTCC
83
1459


 


916817
N/A
N/A
21440
21455
CCAATATACTGAGAGG
96
1460


 


916837
N/A
N/A
22406
22421
GGTAGCACCGCCAAGT
 0
1461


 


916857
N/A
N/A
23301
23316
CACCATGGAGAGGTCT
 0
1462


 


916877
N/A
N/A
24243
24258
TTGCATTACCAGGCAC
17
1463


 


916897
N/A
N/A
24934
24949
GCTACCTGGACACCTC
47
1464
















TABLE 22







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





841947
2094
2109
25857
25872
ATCTTTGCAGACCACC
89
1464


 


912986
N/A
N/A
20288
20303
TCAGAGGAAGCCCAAT
92
 254





20318
20333





 


915360
  42
  57
 2780
 2795
CAGTGTCTCGGCCAGG
 0
1466


 


915380
  98
 113
 2836
 2851
GGGTCCTGATCCGCAG
 0
1467


 


915400
 186
 201
 2924
 2939
AGCTCCAGCCGCGCTC
 0
1468


 


915420
 246
 261
 2984
 2999
TCAGGCAGCGGGTCGC
78
1469


 


915440
 344
 359
 3082
 3097
CAGCGGGATACCGGAG
72
1470


 


915460
 383
 398
 5957
 5972
CTTCCGCACAAGATCT
 0
1471


 


915480
 411
 426
 5985
 6000
GGAAGATGCCAATGTT
94
1472


 


915500
 449
 464
 6023
 6038
ACCCTGTCGGAGGAAC
40
1473


 


915520
 480
 495
 6054
 6069
GGTGGACATTGGCCGG
38
1474


 


915540
 507
 522
 6081
 6096
AGATGCCTATTTTGCC
76
1475


 


915560
 556
 571
 6130
 6145
CGAAAGTCAGACACCA
69
1476


 


915620
 750
 765
11968
11983
TGACTTTAGGGCAGAT
89
1477


 


915640
 821
 836
12039
12054
AAGGTAGAGGTTCCCT
10
1478


 


915660
 875
 890
13611
13626
AAGGCATATCTCTCCC
47
1479


 


915680
 929
 944
N/A
N/A
CCTGTTGCAGATGCCC
22
1480


 


915700
 982
 997
16102
16117
ATGGCGACCTCAGGAT
58
1481


 


915720
1034
1049
16154
16169
CAAGGCAGCCGACTCC
63
1482


 


915740
1121
1136
16241
16256
CGAGAGGGTGTCCAGG
 0
1483


 


915760
1149
1164
N/A
N/A
CTTCACTCAGTGCTGT
13
1484


 


915780
1226
1241
19066
19081
CAGCATTACATAAGAC
94
1485


 


915800
1270
1285
19110
19125
CTCTGGACAATCGCAA
55
1486


 


915820
1345
1360
23750
23765
CGAGTGAACACCTGTG
83
1487


 


915840
1402
1417
25165
25180
TGTTGGCTGCTCACTG
77
1488


 


915860
1470
1485
25233
25248
CTGGACAGCCCTTGGG
29
1489


 


915880
1539
1554
25302
25317
TATTGCCCAAGAAGAA
18
1490


 


915900
1598
1613
25361
25376
ACTCTTCTCTAGTGAA
67
1491


 


915920
1630
1645
25393
25408
CTGCTAGACTCGCCTC
88
1492


 


915940
1682
1697
25445
25460
AATGCGGAGGTAGCTG
 0
1493


 


915960
1765
1780
25528
25543
AGGTTGCTTCCTAGCT
55
1494


 


915980
1785
1800
25548
25563
TGGACCGCTGCACAGG
82
1495


 


916000
1812
1827
25575
25590
GCATGCTGATGTATTA
52
1496


 


916020
1837
1852
25600
25615
TCATTTCCCAACCAGC
94
1497


 


916040
1881
1896
25644
25659
ACGAAACAGTCAGTAA
79
1498


 


916060
1907
1922
25670
25685
GGAACAGTCTGACCAT
22
1499


 


916080
1936
1951
25699
25714
AACACCTGTCATTCTA
71
1500


 


916100
1974
1989
25737
25752
GCCAGCCTACCCCCCA
23
1501


 


916120
2023
2038
25786
25801
AGTGGGATCATGCTAT
 0
1502


 


916159
2136
2151
25899
25914
GTTACCCCCGCCATGG
47
1503


 


916179
2239
2254
26002
26017
TTCAACAGGTAACAAC
84
1504


 


916199
2621
2636
26384
26399
TGCACACTAGATTATT
 0
1505


 


916219
2674
2689
26437
26452
GCGGAAGCTCCTGCTG
 6
1506


 


916239
2704
2719
26467
26482
GTTCAAGTTGTGTGCT
85
1507


 


916259
2771
2786
26534
26549
CTCATCGCCACACATG
85
1508


 


916279
N/A
N/A
 4218
 4233
CGGAATCTCATTTCCA
 0
1509


 


916299
N/A
N/A
 4736
 4751
GCAAATGATCATGTGG
93
1510


 


916319
N/A
N/A
 5396
 5411
CTTAATGCTATCAGGT
83
1511


 


916339
N/A
N/A
 5649
 5664
GGATATATTGGGCTCA
96
1512


 


916359
N/A
N/A
 6597
 6612
AGGGTTTACAAACATG
32
1513


 


916379
N/A
N/A
 7466
 7481
ATTCATTAGCATCACC
52
1514


 


916399
N/A
N/A
 7687
 7702
GGTTAATCCATGGGTC
 0
1515


 


916559
N/A
N/A
12286
12301
AGTTTTAGGTCTGGGT
89
1516


 


916579
N/A
N/A
12833
12848
CATTGCATAGCCTTCT
96
1517


 


916599
N/A
N/A
13661
13676
CCATACATACCCTTCT
21
1518


 


916619
N/A
N/A
14077
14092
ACCCACACCTGACTGG
16
1519


 


916639
N/A
N/A
14572
14587
CGCTCCTACTTATCCC
96
1520


 


916659
N/A
N/A
15427
15442
TCTTACAGCATTGGCA
38
1521


 


916679
N/A
N/A
15973
15988
CATCTACCAAACTGCA
73
1522


 


916699
N/A
N/A
17135
17150
AACAAACATCGATTTT
47
1523


 


916719
N/A
N/A
17844
17859
AGCTTTACAAGCTGGT
 0
1524


 


916739
N/A
N/A
19213
19228
ACGACAATCCAGGTCC
14
1525


 


916778
N/A
N/A
20612
20627
TCTAATTTTACGATCA
92
1526


 


916798
N/A
N/A
20985
21000
ATTAAACTGCCAAGTC
72
1527


 


916818
N/A
N/A
21441
21456
ACCAATATACTGAGAG
92
1528


 


916838
N/A
N/A
22409
22424
AGCGGTAGCACCGCCA
 0
1529


 


916858
N/A
N/A
23323
23338
TCACATGTGAGCCCAG
46
1530


 


916878
N/A
N/A
24271
24286
GTACAACAGAGGGTGG
19
1531


 


916898
N/A
N/A
24978
24993
GGCTGATGTCACCACC
32
1532
















TABLE 23







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915361
  43
  58
 2781
 2796
TCAGTGTCTCGGCCAG
 0
1533


 


915381
 105
 120
 2843
 2858
TCGGCTCGGGTCCTGA
 0
1534


 


915401
 188
 203
 2926
 2941
CAAGCTCCAGCCGCGC
22
1535


 


915421
 247
 262
 2985
 3000
CTCAGGCAGCGGGTCG
48
1536


 


915441
 345
 360
 3083
 3098
CCAGCGGGATACCGGA
 0
1537


 


915461
 384
 399
 5958
 5973
CCTTCCGCACAAGATC
59
1538


 


915481
 414
 429
 5988
 6003
GATGGAAGATGCCAAT
83
1539


 


915501
 450
 465
 6024
 6039
GACCCTGTCGGAGGAA
26
1540


 


915521
 482
 497
 6056
 6071
CTGGTGGACATTGGCC
 0
1541


 


915541
 508
 523
 6082
 6097
GAGATGCCTATTTTGC
92
1542


 


915561
 557
 572
 6131
 6146
CCGAAAGTCAGACACC
 0
1543


 


915601
 691
 706
11909
11924
GCATCAATGAAGGGTA
91
1544


 


915621
 751
 766
11969
11984
TTGACTTTAGGGCAGA
85
1545


 


915641
 822
 837
12040
12055
GAAGGTAGAGGTTCCC
74
1546


 


915661
 876
 891
13612
13627
GAAGGCATATCTCTCC
32
1547


 


915681
 930
 945
16050
16065
GCCTGTTGCAGATGCC
 0
1548


 


915701
 983
 998
16103
16118
CATGGCGACCTCAGGA
 0
1549


 


915721
1035
1050
16155
16170
CCAAGGCAGCCGACTC
79
1550


 


915741
1122
1137
16242
16257
GCGAGAGGGTGTCCAG
 0
1551


 


915761
1150
1165
18990
19005
TCTTCACTCAGTGCTG
41
1552


 


915781
1227
1242
19067
19082
GCAGCATTACATAAGA
75
1553


 


915801
1273
1288
N/A
N/A
AGTCTCTGGACAATCG
 0
1554


 


915821
1346
1361
23751
23766
TCGAGTGAACACCTGT
52
1555


 


915841
1403
1418
25166
25181
CTGTTGGCTGCTCACT
80
1556


 


915861
1471
1486
25234
25249
GCTGGACAGCCCTTGG
 0
1557


 


915881
1540
1555
25303
25318
TTATTGCCCAAGAAGA
75
1558


 


915901
1599
1614
25362
25377
GACTCTTCTCTAGTGA
67
1559


 


915921
1631
1646
25394
25409
TCTGCTAGACTCGCCT
50
1560


 


915941
1683
1698
25446
25461
CAATGCGGAGGTAGCT
39
1561


 


915961
1766
1781
25529
25544
AAGGTTGCTTCCTAGC
71
1562


 


915981
1786
1801
25549
25564
CTGGACCGCTGCACAG
 0
1563


 


916001
1813
1828
25576
25591
CGCATGCTGATGTATT
73
1564


 


916021
1840
1855
25603
25618
GTGTCATTTCCCAACC
61
1565


 


916041
1882
1897
25645
25660
CACGAAACAGTCAGTA
34
1566


 


916061
1910
1925
25673
25688
GCTGGAACAGTCTGAC
82
1567


 


916081
1942
1957
25705
25720
CATCCAAACACCTGTC
69
1568


 


916101
1975
1990
25738
25753
GGCCAGCCTACCCCCC
 2
1569


 


916121
2024
2039
25787
25802
AAGTGGGATCATGCTA
26
1570


 


916140
2095
2110
25858
25873
CATCTTTGCAGACCAC
92
1571


 


916160
2137
2152
25900
25915
TGTTACCCCCGCCATG
51
1572


 


916180
2257
2272
26020
26035
GATTCACATAATACAA
87
1573


 


916200
2622
2637
26385
26400
CTGCACACTAGATTAT
 0
1574


 


916220
2675
2690
26438
26453
GGCGGAAGCTCCTGCT
 0
1575


 


916240
2705
2720
26468
26483
GGTTCAAGTTGTGTGC
69
1576


 


916260
2772
2787
26535
26550
TCTCATCGCCACACAT
72
1577


 


916280
N/A
N/A
 4220
 4235
TACGGAATCTCATTTC
80
1578


 


916300
N/A
N/A
 4791
 4806
GGCCACCTTGGGATAC
17
1579


 


916320
N/A
N/A
 5398
 5413
GCCTTAATGCTATCAG
67
1580


 


916340
N/A
N/A
 5650
 5665
TGGATATATTGGGCTC
97
1581


 


916360
N/A
N/A
 6603
 6618
ACATTCAGGGTTTACA
18
1582


 


916380
N/A
N/A
 7468
 7483
GTATTCATTAGCATCA
51
1583


 


916400
N/A
N/A
 7688
 7703
AGGTTAATCCATGGGT
29
1584


 


916560
N/A
N/A
12287
12302
GAGTTTTAGGTCTGGG
95
1585


 


916580
N/A
N/A
12905
12920
CTTATAAAGCACACGG
95
1586


 


916600
N/A
N/A
13683
13698
GGGCATGGCTGATCCT
 8
1587


 


916620
N/A
N/A
14099
14114
CAAACTTGTCTAGTGG
67
1588


 


916640
N/A
N/A
14600
14615
TCGCATCCATGGGTCC
83
1589


 


916660
N/A
N/A
15429
15444
GCTCTTACAGCATTGG
51
1590


 


916680
N/A
N/A
15974
15989
CCATCTACCAAACTGC
61
1591


 


916700
N/A
N/A
17195
17210
GACTTAGTCCGTGTTC
49
1592


 


916720
N/A
N/A
17883
17898
CAGCATCTATGTTCTC
67
1593


 


916740
N/A
N/A
19239
19254
ATAGACTGTGAGCTGT
82
1594


 


916759
N/A
N/A
20354
20369
GACCATTCTGCTCCCC
20
1595


 


916779
N/A
N/A
20632
20647
GCCCATACCTTTTATC
47
1596


 


916799
N/A
N/A
20987
21002
GTATTAAACTGCCAAG
81
1597


 


916819
N/A
N/A
21444
21459
CTAACCAATATACTGA
73
1598


 


916839
N/A
N/A
22506
22521
GGCTGGTGATGAAACA
 0
1599


 


916859
N/A
N/A
23345
23360
CCTCATGGTTTGCTGT
31
1600


 


916879
N/A
N/A
24273
24288
CAGTACAACAGAGGGT
81
1601


 


916899
N/A
N/A
25064
25079
CACATTGCCGGCCAGT
58
1602
















TABLE 24







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915362
  44
  59
 2782
 2797
CTCAGTGTCTCGGCCA
 0
1603


 


915382
 106
 121
 2844
 2859
ATCGGCTCGGGTCCTG
30
1604


 


915402
 189
 204
 2927
 2942
ACAAGCTCCAGCCGCG
24
1605


 


915422
 248
 263
 2986
 3001
GCTCAGGCAGCGGGTC
33
1606


 


915442
 346
 361
N/A
N/A
TCCAGCGGGATACCGG
 0
1607


 


915462
 385
 400
 5959
 5974
GCCTTCCGCACAAGAT
60
1608


 


915482
 415
 430
 5989
 6004
GGATGGAAGATGCCAA
60
1609


 


915502
 451
 466
 6025
 6040
AGACCCTGTCGGAGGA
75
1610


 


915522
 487
 502
 6061
 6076
ATGAGCTGGTGGACAT
74
1611


 


915542
 509
 524
 6083
 6098
AGAGATGCCTATTTTG
91
1612


 


915562
 558
 573
 6132
 6147
ACCGAAAGTCAGACAC
 0
1613


 


915602
 692
 707
11910
11925
GGCATCAATGAAGGGT
88
1614


 


915622
 752
 767
11970
11985
CTTGACTTTAGGGCAG
86
1615


 


915642
 824
 839
12042
12057
GAGAAGGTAGAGGTTC
81
1616


 


915662
 878
 893
13614
13629
TCGAAGGCATATCTCT
16
1617


 


915682
 931
 946
16051
16066
GGCCTGTTGCAGATGC
 0
1618


 


915702
 984
 999
16104
16119
GCATGGCGACCTCAGG
24
1619


 


915722
1036
1051
16156
16171
GCCAAGGCAGCCGACT
 0
1620


 


915742
1123
1138
16243
16258
GGCGAGAGGGTGTCCA
 0
1621


 


915762
1173
1188
19013
19028
TGTATCCACCTTTGTC
85
1622


 


915782
1228
1243
19068
19083
GGCAGCATTACATAAG
73
1623


 


915802
1283
1298
N/A
N/A
CCATGTCACCAGTCTC
59
1624


 


915822
1347
1362
23752
23767
CTCGAGTGAACACCTG
 0
1625


 


915842
1404
1419
25167
25182
CCTGTTGGCTGCTCAC
88
1626


 


915862
1472
1487
25235
25250
TGCTGGACAGCCCTTG
 0
1627


 


915882
1541
1556
25304
25319
TTTATTGCCCAAGAAG
38
1628


 


915902
1600
1615
25363
25378
AGACTCTTCTCTAGTG
60
1629


 


915922
1632
1647
25395
25410
ATCTGCTAGACTCGCC
86
1630


 


915942
1684
1699
25447
25462
GCAATGCGGAGGTAGC
38
1631


 


915962
1767
1782
25530
25545
AAAGGTTGCTTCCTAG
77
1632


 


915982
1787
1802
25550
25565
GCTGGACCGCTGCACA
79
1633


 


916002
1814
1829
25577
25592
ACGCATGCTGATGTAT
72
1634


 


916022
1841
1856
25604
25619
GGTGTCATTTCCCAAC
80
1635


 


916042
1883
1898
25646
25661
CCACGAAACAGTCAGT
87
1636


 


916062
1912
1927
25675
25690
ATGCTGGAACAGTCTG
78
1637


 


916082
1943
1958
25706
25721
CCATCCAAACACCTGT
64
1638


 


916102
1976
1991
25739
25754
GGGCCAGCCTACCCCC
 0
1639


 


916122
2025
2040
25788
25803
GAAGTGGGATCATGCT
71
1640


 


916141
2097
2112
25860
25875
ATCATCTTTGCAGACC
91
1641


 


916161
2138
2153
25901
25916
TTGTTACCCCCGCCAT
89
1642


 


916181
2259
2274
26022
26037
CTGATTCACATAATAC
91
1643


 


916201
2623
2638
26386
26401
CCTGCACACTAGATTA
59
1644


 


916221
2676
2691
26439
26454
AGGCGGAAGCTCCTGC
 0
1645


 


916241
2706
2721
26469
26484
AGGTTCAAGTTGTGTG
87
1646


 


916281
N/A
N/A
 4224
 4239
AATGTACGGAATCTCA
83
1647


 


916301
N/A
N/A
 4810
 4825
GTCCATGTGGGTGTCC
74
1648


 


916321
N/A
N/A
 5399
 5414
GGCCTTAATGCTATCA
13
1649


 


916341
N/A
N/A
 5711
 5726
TAGTATGAAATATCTC
96
1650


 


916361
N/A
N/A
 6862
 6877
ATTGTAACTGCCAGGC
 0
1651


 


916381
N/A
N/A
 7471
 7486
CCGGTATTCATTAGCA
 0
1652


 


916401
N/A
N/A
 7728
 7743
GAGCAGGGCAACAAAC
22
1653


 


916561
N/A
N/A
12315
12330
ATATAACCACAGCCTG
54
1654


 


916581
N/A
N/A
12906
12921
GCTTATAAAGCACACG
94
1655


 


916601
N/A
N/A
13702
13717
TAGTAAATGCTTGTCA
95
1656


 


916621
N/A
N/A
14123
14138
GGCAGAAATGTGCTCT
60
1657


 


916641
N/A
N/A
14632
14647
CTTCATGCCATCCTGT
83
1658


 


916661
N/A
N/A
15430
15445
TGCTCTTACAGCATTG
 0
1659


 


916681
N/A
N/A
16262
16277
GGTACCTGTAGCGAGC
 0
1660


 


916701
N/A
N/A
17197
17212
TTGACTTAGTCCGTGT
93
1661


 


916721
N/A
N/A
18220
18235
AGCTACATCAGGCTGG
 0
1662


 


916741
N/A
N/A
19244
19259
TGCACATAGACTGTGA
 0
1663


 


916760
N/A
N/A
20373
20388
GACTGCTGAGCCAAGC
61
1664


 


916780
N/A
N/A
20657
20672
AGAAATTGCAGTGCCC
92
1665


 


916800
N/A
N/A
20988
21003
GGTATTAAACTGCCAA
 0
1666


 


916820
N/A
N/A
21447
21462
TCACTAACCAATATAC
47
1667


 


916840
N/A
N/A
22602
22617
ATAAATCTGCAAGAGC
62
1668


 


916860
N/A
N/A
23369
23384
TCTCATGGTCAAGACC
52
1669


 


916880
N/A
N/A
24305
24320
GACTGCTAGGCTTCAC
54
1670


 


916900
N/A
N/A
25100
25115
CGCTGCTGCAGTGTGC
34
1671









Human primer probe set RTS36075 (forward sequence TGAGGCTGGAGGGAGATG, designated herein as SEQ ID NO: 14; reverse sequence GCTCATGTATCCACCTTTGTCT, designated herein as SEQ ID NO: 15; probe sequence CTAGACCACCTGCGTCTCAGCATC, designated herein as SEQ ID NO: 16) was also used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.







TABLE 25







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





898558
 581
 596
N/A
N/A
GGCATCCACGACTTCG
87
1672


 


912709
  27
  42
 2765
 2780
GGCATTCCCAGCGCGA
 0
  17


 


912710
  95
 110
 2833
 2848
TCCTGATCCGCAGCAG
 0
  18


 


912711
 103
 118
 2841
 2856
GGCTCGGGTCCTGATC
 0
  19


 


912712
 131
 146
 2869
 2884
GTTAGGATCTGGGTCG
76
  20


 


912713
 164
 179
 2902
 2917
GTACATGGCGGCGGCG
 0
  21


 


912714
 183
 198
 2921
 2936
TCCAGCCGCGCTCTGC
29
  22


 


912715
 196
 211
 2934
 2949
GCGAAGGACAAGCTCC
31
  23


 


912716
 197
 212
 2935
 2950
CGCGAAGGACAAGCTC
 0
  24


 


912717
 272
 287
 3010
 3025
GCGGAGGAGGTGCGGG
 0
  25


 


912718
 273
 288
 3011
 3026
CGCGGAGGAGGTGCGG
 0
  26


 


912719
 274
 289
 3012
 3027
TCGCGGAGGAGGTGCG
16
  27


 


912720
 290
 305
 3028
 3043
GAACAACATGCGCGCG
 0
  28


 


912721
 291
 306
 3029
 3044
CGAACAACATGCGCGC
 2
  29


 


912722
 292
 307
 3030
 3045
CCGAACAACATGCGCG
 0
  30


 


912723
 293
 308
 3031
 3046
GCCGAACAACATGCGC
 0
  31


 


912724
 294
 309
 3032
 3047
CGCCGAACAACATGCG
 0
  32


 


912725
 323
 338
 3061
 3076
GCCGACGCAGTGCAAC
 0
  33


 


912726
 324
 339
 3062
 3077
CGCCGACGCAGTGCAA
 0
  34


 


912727
 340
 355
 3078
 3093
GGGATACCGGAGAGGA
32
  35


 


912728
 370
 385
 5944
 5959
TCTGAGAGGACCTGCA
31
  36


 


912729
 375
 390
 5949
 5964
CAAGATCTGAGAGGAC
60
  37


 


912730
 404
 419
 5978
 5993
GCCAATGTTCCGACTC
52
  38


 


912731
 410
 425
 5984
 5999
GAAGATGCCAATGTTC
31
  39


 


912732
 429
 444
 6003
 6018
TTAAGTTGAAGGATGG
93
  40


 


912733
 432
 447
 6006
 6021
TGCTTAAGTTGAAGGA
82
  41


 


912734
 478
 493
 6052
 6067
TGGACATTGGCCGGGA
73
  42


 


912735
 479
 494
 6053
 6068
GTGGACATTGGCCGGG
44
  43


 


912736
 484
 499
 6058
 6073
AGCTGGTGGACATTGG
29
  44


 


912737
 528
 543
 6102
 6117
CATCAGACACTCTGGT
 0
  45


 


912738
 531
 546
 6105
 6120
CCCCATCAGACACTCT
55
  46


 


912739
 552
 567
 6126
 6141
AGTCAGACACCAGAAC
23
  47


 


912740
 582
 597
N/A
N/A
AGGCATCCACGACTTC
40
1673


 


912741
 584
 599
N/A
N/A
CAAGGCATCCACGACT
55
1674


 


912742
 591
 606
N/A
N/A
AACATACCAAGGCATC
59
1675


 


912743
 593
 608
N/A
N/A
GGAACATACCAAGGCA
69
1676


 


912744
 594
 609
 7824
 7839
AGGAACATACCAAGGC
85
1677


 


912745
 625
 640
 7855
 7870
GGGATAAGGCCACTGT
71
1678


 


912746
 626
 641
 7856
 7871
AGGGATAAGGCCACTG
12
1679


 


912747
 630
 645
 7860
 7875
AAGGAGGGATAAGGCC
 0
1680


 


912748
 652
 667
N/A
N/A
ACATATCGCACGCCTC
35
1681


 


912749
 653
 668
N/A
N/A
CACATATCGCACGCCT
 3
1682


 


912750
 654
 669
N/A
N/A
CCACATATCGCACGCC
27
1683


 


912751
 656
 671
N/A
N/A
ATCCACATATCGCACG
24
1684


 


912752
 660
 675
11878
11893
CTCCATCCACATATCG
87
1685


 


912753
 689
 704
11907
11922
ATCAATGAAGGGTACG
79
1686


 


912754
 690
 705
11908
11923
CATCAATGAAGGGTAC
63
1687


 


912755
 693
 708
11911
11926
TGGCATCAATGAAGGG
68
  48


 


912756
 698
 713
11916
11931
TGTTTTGGCATCAATG
88
  49


 


912757
 746
 761
11964
11979
TTTAGGGCAGATGTCG
75
  50


 


912758
 747
 762
11965
11980
CTTTAGGGCAGATGTC
82
  51


 


912759
 795
 810
12013
12028
GTAGACTGAGCTTGGT
96
  52


 


912760
 820
 835
12038
12053
AGGTAGAGGTTCCCTG
 0
  53


 


912761
 841
 856
12059
12074
GGGACAAAAGCTCTCG
 0
  54


 


912762
 873
 888
13609
13624
GGCATATCTCTCCCAG
 0
  55


 


912763
 874
 889
13610
13625
AGGCATATCTCTCCCA
 0
  56


 


912764
 886
 901
13622
13637
AAATATCCTCGAAGGC
71
  57


 


912765
 888
 903
13624
13639
CCAAATATCCTCGAAG
37
  58


 


912766
 889
 904
13625
13640
TCCAAATATCCTCGAA
 0
  59


 


912767
 894
 909
13630
13645
ATGCATCCAAATATCC
42
  60


 


912768
 925
 940
N/A
N/A
TTGCAGATGCCCTTCT
 5
  61


 


912769
 968
 983
16088
16103
ATCCATCCCTTCTGAG
 6
  62


 


912770
 986
1001
16106
16121
GGGCATGGCGACCTCA
 0
  63


 


912771
1004
1019
16124
16139
ACTCATGTTTGCCCAG
67
  64


 


912782
1195
1210
19035
19050
AGCAAGTTGCAAATCT
71
  75


 


912783
1199
1214
19039
19054
GGGTAGCAAGTTGCAA
37
  76


 


912784
1205
1220
19045
19060
CCTAATGGGTAGCAAG
25
  77


 


912785
1206
1221
19046
19061
TCCTAATGGGTAGCAA
64
  78
















TABLE 26







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





912786
1207
1222
19047
19062
ATCCTAATGGGTAGCA
65
 79


 


912787
1211
1226
19051
19066
CATTATCCTAATGGGT
43
 80


 


912788
1212
1227
19052
19067
ACATTATCCTAATGGG
 0
 81


 


912789
1213
1228
19053
19068
GACATTATCCTAATGG
59
 82


 


912790
1220
1235
19060
19075
TACATAAGACATTATC
 8
 83


 


912791
1224
1239
19064
19079
GCATTACATAAGACAT
86
 84


 


912792
1245
1260
19085
19100
CCACAGGCAGGGTACA
58
 85


 


912793
1246
1261
19086
19101
TCCACAGGCAGGGTAC
 5
 86


 


912794
1253
1268
19093
19108
GGCAGATTCCACAGGC
68
 87


 


912795
1259
1274
19099
19114
CGCAATGGCAGATTCC
84
 88


 


912796
1265
1280
19105
19120
GACAATCGCAATGGCA
63
 89


 


912797
1266
1281
19106
19121
GGACAATCGCAATGGC
54
 90


 


912798
1267
1282
19107
19122
TGGACAATCGCAATGG
59
 91


 


912799
1285
1300
23690
23705
AGCCATGTCACCAGTC
51
 92


 


912800
1289
1304
23694
23709
TGGAAGCCATGTCACC
32
 93


 


912801
1290
1305
23695
23710
CTGGAAGCCATGTCAC
44
 94


 


912802
1297
1312
23702
23717
GGCATATCTGGAAGCC
 0
 95


 


912803
1298
1313
23703
23718
GGGCATATCTGGAAGC
 0
 96


 


912804
1351
1366
23756
23771
AGCACTCGAGTGAACA
 6
 97


 


912805
1386
1401
N/A
N/A
GCATTTGGGACCTGGA
54
 98


 


912806
1387
1402
N/A
N/A
GGCATTTGGGACCTGG
33
 99


 


912807
1388
1403
25151
25166
TGGCATTTGGGACCTG
 0
100


 


912808
1394
1409
25157
25172
GCTCACTGGCATTTGG
 7
101


 


912809
1523
1538
25286
25301
GTTCAGGCTGGACCTG
17
102


 


912810
1547
1562
25310
25325
AGGTACTTTATTGCCC
30
103


 


912811
1550
1565
25313
25328
AGCAGGTACTTTATTG
55
104


 


912812
1653
1668
25416
25431
AACTTTAGCACCTCTG
87
105


 


912813
1655
1670
25418
25433
GAAACTTTAGCACCTC
85
106


 


912814
1656
1671
25419
25434
GGAAACTTTAGCACCT
26
107


 


912815
1669
1684
25432
25447
CTGCACAAAGATGGGA
66
108


 


912816
1671
1686
25434
25449
AGCTGCACAAAGATGG
41
109


 


912817
1685
1700
25448
25463
AGCAATGCGGAGGTAG
35
110


 


912818
1740
1755
25503
25518
ACCAACTCAGCTCAGA
76
111


 


912819
1741
1756
25504
25519
AACCAACTCAGCTCAG
77
112


 


912820
1757
1772
25520
25535
TCCTAGCTTTTCATAA
18
113


 


912821
1788
1803
25551
25566
TGCTGGACCGCTGCAC
 1
114


 


912822
1796
1811
25559
25574
GAGTTAAGTGCTGGAC
90
115


 


912823
1802
1817
25565
25580
GTATTAGAGTTAAGTG
86
116


 


912824
1803
1818
25566
25581
TGTATTAGAGTTAAGT
79
117


 


912825
1806
1821
25569
25584
TGATGTATTAGAGTTA
89
118


 


912826
1808
1823
25571
25586
GCTGATGTATTAGAGT
79
119


 


912827
1821
1836
25584
25599
TGAATTAACGCATGCT
73
120


 


912828
1822
1837
25585
25600
CTGAATTAACGCATGC
69
121


 


912829
1870
1885
25633
25648
AGTAAGGGACCCTCTG
 0
122


 


912830
1871
1886
25634
25649
CAGTAAGGGACCCTCT
44
123


 


912831
1872
1887
25635
25650
TCAGTAAGGGACCCTC
67
124


 


912832
1874
1889
25637
25652
AGTCAGTAAGGGACCC
50
125


 


912833
1893
1908
25656
25671
ATTAATAGGGCCACGA
78
126


 


912834
1895
1910
25658
25673
CCATTAATAGGGCCAC
72
127


 


912835
1896
1911
25659
25674
ACCATTAATAGGGCCA
65
128


 


912836
1906
1921
25669
25684
GAACAGTCTGACCATT
82
129


 


912837
1908
1923
25671
25686
TGGAACAGTCTGACCA
39
130


 


912838
1909
1924
25672
25687
CTGGAACAGTCTGACC
84
131


 


912839
1911
1926
25674
25689
TGCTGGAACAGTCTGA
72
132


 


912840
1916
1931
25679
25694
CCTCATGCTGGAACAG
84
133


 


912841
1928
1943
25691
25706
TCATTCTAAGAACCTC
87
134


 


912842
1945
1960
25708
25723
ACCCATCCAAACACCT
18
135


 


912843
1982
1997
25745
25760
ACACATGGGCCAGCCT
46
136


 


912844
1989
2004
25752
25767
CAAGATCACACATGGG
71
137


 


912845
2057
2072
25820
25835
GGGACGAACTGCACCC
 0
138


 


912846
2098
2113
25861
25876
TATCATCTTTGCAGAC
68
139


 


912847
2116
2131
25879
25894
GTTTTTAGTAGTCAAG
90
140


 


912848
2117
2132
25880
25895
CGTTTTTAGTAGTCAA
94
141


 


912849
2145
2160
25908
25923
TATCATCTTGTTACCC
87
142


 


912850
2148
2163
25911
25926
GATTATCATCTTGTTA
60
143


 


912851
2150
2165
25913
25928
TAGATTATCATCTTGT
50
144


 


912852
2151
2166
25914
25929
GTAGATTATCATCTTG
72
145


 


912853
2152
2167
25915
25930
AGTAGATTATCATCTT
79
146


 


912854
2175
2190
25938
25953
GTGAAAAAGGTGTTCT
64
147


 


912855
2182
2197
25945
25960
TAGTTAGGTGAAAAAG
77
148


 


912856
2188
2203
25951
25966
TTATTTTAGTTAGGTG
82
149


 


912857
2190
2205
25953
25968
CATTATTTTAGTTAGG
77
150


 


912858
2273
2288
26036
26051
CTACTAACATCTCACT
48
151


 


912859
2274
2289
26037
26052
TCTACTAACATCTCAC
91
152


 


912860
2278
2293
26041
26056
TTATTCTACTAACATC
37
153


 


912861
2280
2295
26043
26058
GCTTATTCTACTAACA
77
154


 


912862
2281
2296
26044
26059
GGCTTATTCTACTAAC
70
155


 


912863
2632
2647
26395
26410
GGTGAATGCCCTGCAC
42
156









Study 2

Cultured A431 cells at a density of 5,000 cells per well were transfected by free uptake with 1,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and PNPLA3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS36070 was used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREENR. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.







TABLE 27







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
97
 702


 


959270
 413
 428
 5987
 6002
ATGGAAGATGCCAATG
32
1688


 


959280
 491
 506
 6065
 6080
GGAGATGAGCTGGTGG
66
1689


 


959290
 793
 808
12011
12026
AGACTGAGCTTGGTGA
78
1690


 


959300
 899
 914
13635
13650
CCTGAATGCATCCAAA
69
1691


 


959310
1084
1099
16204
16219
ATGCTGAGACGCAGGT
 0
1692


 


959320
1256
1271
19096
19111
AATGGCAGATTCCACA
25
1693


 


959330
1642
1657
25405
25420
CTCTGAAAGAATCTGC
75
1694


 


959340
1659
1674
25422
25437
ATGGGAAACTTTAGCA
77
1695


 


959350
1839
1854
25602
25617
TGTCATTTCCCAACCA
79
1696


 


959360
2114
2129
25877
25892
TTTTAGTAGTCAAGGT
88
1697


 


959370
2223
2238
25986
26001
GCTTCCTTACATTTTT
85
1698


 


959380
2269
2284
26032
26047
TAACATCTCACTGATT
42
1699


 


959390
N/A
N/A
 4311
 4326
CTAGTGAGAAACAAAC
 0
1700


 


959400
N/A
N/A
 4761
 4776
TTATTGTTGCTAAACC
32
1701


 


959410
N/A
N/A
 4863
 4878
ACTTTAGGCTCCTGGG
60
1702


 


959420
N/A
N/A
 5285
 5300
AGCCATAAATCTTGGG
24
1703


 


959430
N/A
N/A
 5573
 5588
ATGACATCATGGCTTC
93
1704


 


959440
N/A
N/A
 5603
 5618
TTATTCAATGTGGCTT
95
1705


 


959450
N/A
N/A
 5640
 5655
GGGCTCAATGAAATTA
12
1706


 


959460
N/A
N/A
 5713
 5728
CTTAGTATGAAATATC
86
1707


 


959470
N/A
N/A
 5808
 5823
TACTGTCTACTATGGG
91
1708


 


959480
N/A
N/A
 6157
 6172
CTTACATCCACGACTT
35
1709


 


959660
N/A
N/A
12153
12168
CAGTAACTGGTAGCTC
74
1710


 


959670
N/A
N/A
12169
12184
TGTTTGATTGTGCAGA
95
1711


 


959680
N/A
N/A
12210
12225
CGCCTTTTATTTCCGT
92
1712


 


959690
N/A
N/A
12313
12328
ATAACCACAGCCTGGG
66
1713


 


959700
N/A
N/A
12675
12690
ATAAGAATCATCTTAG
 7
1714


 


959710
N/A
N/A
12711
12726
ACTACCGAACGCAGTT
41
1715


 


959720
N/A
N/A
12757
12772
TAGAGTGGTAAGGCAT
84
1716


 


959730
N/A
N/A
12793
12808
GGTTGGTGTACATGGT
96
1717


 


959740
N/A
N/A
12880
12895
TCCTGTTAGACAGCTT
93
1718


 


959750
N/A
N/A
12902
12917
ATAAAGCACACGGGAA
86
1719


 


959760
N/A
N/A
12931
12946
TAAGAGCTGTCTCCTC
85
1720


 


959770
N/A
N/A
12972
12987
CTAACAAACTTTGCAG
79
1721


 


959780
N/A
N/A
13392
13407
TGTCACCCTTCCACGG
15
1722


 


959790
N/A
N/A
13526
13541
ATTGGAAGACCGCAGA
43
1723


 


959800
N/A
N/A
13706
13721
CCGCTAGTAAATGCTT
44
1724


 


959810
N/A
N/A
13737
13752
AACTAAGGCAAATCTC
77
1725


 


959820
N/A
N/A
13915
13930
GAGTCATGACATCCCA
89
1726


 


959830
N/A
N/A
14299
14314
GCAGATAAATACACAT
93
1727


 


959840
N/A
N/A
14424
14439
TTTCCCATCGACACAG
78
1728


 


959850
N/A
N/A
14571
14586
GCTCCTACTTATCCCC
76
1729


 


959860
N/A
N/A
15202
15217
TATTGCCAGGTATCTG
64
1730


 


959870
N/A
N/A
15599
15614
CAATACATAGCAGAGC
23
1731


 


959880
N/A
N/A
17192
17207
TTAGTCCGTGTTCAGG
90
1732


 


959890
N/A
N/A
17222
17237
GTAGCTGGTTTGTGGG
20
1733


 


959900
N/A
N/A
17295
17310
CATCTCTTAGGGCACC
79
1734


 


959910
N/A
N/A
18393
18408
GTTTGGAAGTCGCCAT
77
1735


 


959920
N/A
N/A
20284
20299
AGGAAGCCCAATCAAG
85
1736


 


959930
N/A
N/A
20512
20527
CAGATTGAGTCTCCTG
10
1737


 


959940
N/A
N/A
20607
20622
TTTTACGATCATCATT
72
1738


 


959950
N/A
N/A
20661
20676
GCTTAGAAATTGCAGT
75
1739


 


959960
N/A
N/A
20812
20827
AGGGTAATATTCAGAC
86
1740


 


959970
N/A
N/A
20934
20949
TGTAGCAGACAGATCA
74
1741


 


959980
N/A
N/A
21000
21015
TTTAACAGCTCAGGTA
66
1742


 


959990
N/A
N/A
21405
21420
ATTCTAGACATGGCCA
51
1743


 


960000
N/A
N/A
21442
21457
AACCAATATACTGAGA
71
1744


 


960010
N/A
N/A
21545
21560
AGACATATGACATTTC
91
1745


 


960020
N/A
N/A
22765
22780
ACATGACAGACTAACT
55
1746


 


960030
N/A
N/A
24039
24054
CATCAATGCTGCACTC
13
1747
















TABLE 28







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
98
 702


 


959271
 425
 440
 5999
 6014
GTTGAAGGATGGATGG
90
1748


 


959281
 511
 526
 6085
 6100
AGAGAGATGCCTATTT
73
1749


 


959291
 813
 828
12031
12046
GGTTCCCTGTGCAGAG
79
1750


 


959301
 904
 919
13640
13655
AAGAACCTGAATGCAT
48
1751


 


959311
1085
1100
16205
16220
GATGCTGAGACGCAGG
 0
1752


 


959321
1602
1617
25365
25380
ACAGACTCTTCTCTAG
45
1753


 


959331
1643
1658
25406
25421
CCTCTGAAAGAATCTG
81
1754


 


959341
1673
1688
25436
25451
GTAGCTGCACAAAGAT
69
1755


 


959351
1842
1857
25605
25620
TGGTGTCATTTCCCAA
19
1756


 


959361
2115
2130
25878
25893
TTTTTAGTAGTCAAGG
91
1757


 


959371
2240
2255
26003
26018
ATTCAACAGGTAACAA
69
1758


 


959381
2271
2286
26034
26049
ACTAACATCTCACTGA
33
1759


 


959391
N/A
N/A
 4313
 4328
AGCTAGTGAGAAACAA
47
1760


 


959401
N/A
N/A
 4764
 4779
CTTTTATTGTTGCTAA
77
1761


 


959411
N/A
N/A
 4868
 4883
AGTGTACTTTAGGCTC
90
1762


 


959421
N/A
N/A
 5286
 5301
CAGCCATAAATCTTGG
 0
1763


 


959431
N/A
N/A
 5574
 5589
AATGACATCATGGCTT
74
1764


 


959441
N/A
N/A
 5604
 5619
TTTATTCAATGTGGCT
96
1765


 


959451
N/A
N/A
 5642
 5657
TTGGGCTCAATGAAAT
 0
1766


 


959461
N/A
N/A
 5714
 5729
GCTTAGTATGAAATAT
78
1767


 


959471
N/A
N/A
 5809
 5824
GTACTGTCTACTATGG
78
1768


 


959481
N/A
N/A
 6160
 6175
CTGCTTACATCCACGA
 4
1769


 


959661
N/A
N/A
12154
12169
ACAGTAACTGGTAGCT
72
1770


 


959671
N/A
N/A
12170
12185
CTGTTTGATTGTGCAG
50
1771


 


959681
N/A
N/A
12211
12226
GCGCCTTTTATTTCCG
14
1772


 


959691
N/A
N/A
12322
12337
CCTGACTATATAACCA
56
1773


 


959701
N/A
N/A
12689
12704
GACCGTGTTTCCAAAT
97
1774


 


959711
N/A
N/A
12712
12727
AACTACCGAACGCAGT
48
1775


 


959721
N/A
N/A
12759
12774
GGTAGAGTGGTAAGGC
95
1776


 


959731
N/A
N/A
12828
12843
CATAGCCTTCTTTCTT
93
1777


 


959741
N/A
N/A
12882
12897
AATCCTGTTAGACAGC
90
1778


 


959751
N/A
N/A
12903
12918
TATAAAGCACACGGGA
82
1779


 


959761
N/A
N/A
12933
12948
AATAAGAGCTGTCTCC
87
1780


 


959771
N/A
N/A
12974
12989
CCCTAACAAACTTTGC
62
1781


 


959781
N/A
N/A
13394
13409
AATGTCACCCTTCCAC
90
1782


 


959791
N/A
N/A
13527
13542
CATTGGAAGACCGCAG
76
1783


 


959801
N/A
N/A
13707
13722
ACCGCTAGTAAATGCT
35
1784


 


959811
N/A
N/A
13740
13755
TAGAACTAAGGCAAAT
65
1785


 


959821
N/A
N/A
13916
13931
GGAGTCATGACATCCC
42
1786


 


959831
N/A
N/A
14302
14317
TGAGCAGATAAATACA
75
1787


 


959841
N/A
N/A
14425
14440
CTTTCCCATCGACACA
79
1788


 


959851
N/A
N/A
14573
14588
GCGCTCCTACTTATCC
 0
1789


 


959861
N/A
N/A
15203
15218
ATATTGCCAGGTATCT
67
1790


 


959871
N/A
N/A
15763
15778
GTGTTGGTTTATAACA
13
1791


 


959881
N/A
N/A
17194
17209
ACTTAGTCCGTGTTCA
45
1792


 


959891
N/A
N/A
17224
17239
CTGTAGCTGGTTTGTG
42
1793


 


959901
N/A
N/A
17296
17311
CCATCTCTTAGGGCAC
53
1794


 


959911
N/A
N/A
18394
18409
TGTTTGGAAGTCGCCA
87
1795


 


959921
N/A
N/A
20289
20304
ATCAGAGGAAGCCCAA
84
1796


 


959931
N/A
N/A
20514
20529
ACCAGATTGAGTCTCC
91
1797


 


959941
N/A
N/A
20613
20628
CTCTAATTTTACGATC
70
1798


 


959951
N/A
N/A
20662
20677
AGCTTAGAAATTGCAG
 0
1799


 


959961
N/A
N/A
20813
20828
CAGGGTAATATTCAGA
87
1800


 


959971
N/A
N/A
20936
20951
TTTGTAGCAGACAGAT
18
1801


 


959981
N/A
N/A
21001
21016
TTTTAACAGCTCAGGT
71
1802


 


959991
N/A
N/A
21406
21421
AATTCTAGACATGGCC
25
1803


 


960001
N/A
N/A
21443
21458
TAACCAATATACTGAG
72
1804


 


960011
N/A
N/A
22023
22038
CGCAAAAAGACAACGA
16
1805


 


960021
N/A
N/A
22766
22781
GACATGACAGACTAAC
76
1806


 


960031
N/A
N/A
24040
24055
CCATCAATGCTGCACT
61
1807
















TABLE 29







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
97
 702


 


959272
 426
 441
 6000
 6015
AGTTGAAGGATGGATG
76
1808


 


959282
 517
 532
 6091
 6106
CTGGTAAGAGAGATGC
64
1809


 


959292
 815
 830
12033
12048
GAGGTTCCCTGTGCAG
81
1810


 


959302
 905
 920
13641
13656
CAAGAACCTGAATGCA
65
1811


 


959312
1087
1102
16207
16222
AGGATGCTGAGACGCA
66
1812


 


959322
1604
1619
25367
25382
TCACAGACTCTTCTCT
50
1813


 


959332
1644
1659
25407
25422
ACCTCTGAAAGAATCT
69
1814


 


959342
1675
1690
25438
25453
AGGTAGCTGCACAAAG
78
1815


 


959352
1903
1918
25666
25681
CAGTCTGACCATTAAT
77
1816


 


959362
2173
2188
25936
25951
GAAAAAGGTGTTCTAA
85
1817


 


959372
2242
2257
26005
26020
AAATTCAACAGGTAAC
62
1818


 


959382
2275
2290
26038
26053
TTCTACTAACATCTCA
80
1819


 


959392
N/A
N/A
 4732
 4747
ATGATCATGTGGCGGT
80
1820


 


959402
N/A
N/A
 4765
 4780
ACTTTTATTGTTGCTA
86
1821


 


959412
N/A
N/A
 4869
 4884
GAGTGTACTTTAGGCT
94
1822


 


959422
N/A
N/A
 5389
 5404
CTATCAGGTGCAGGAG
93
1823


 


959432
N/A
N/A
 5575
 5590
CAATGACATCATGGCT
90
1824


 


959442
N/A
N/A
 5607
 5622
TACTTTATTCAATGTG
 0
1825


 


959452
N/A
N/A
 5643
 5658
ATTGGGCTCAATGAAA
35
1826


 


959462
N/A
N/A
 5716
 5731
TGGCTTAGTATGAAAT
80
1827


 


959472
N/A
N/A
 5864
 5879
TTTGGCAAGGCCAGAA
 0
1828


 


959482
N/A
N/A
 6960
 6975
GCATAGAGGAAGCTCG
32
1829


 


959662
N/A
N/A
12155
12170
GACAGTAACTGGTAGC
92
1830


 


959672
N/A
N/A
12172
12187
TTCTGTTTGATTGTGC
97
1831


 


959682
N/A
N/A
12280
12295
AGGTCTGGGTATATGT
93
1832


 


959692
N/A
N/A
12323
12338
CCCTGACTATATAACC
32
1833


 


959702
N/A
N/A
12691
12706
TTGACCGTGTTTCCAA
94
1834


 


959712
N/A
N/A
12713
12728
AAACTACCGAACGCAG
92
1835


 


959722
N/A
N/A
12760
12775
TGGTAGAGTGGTAAGG
94
1836


 


959732
N/A
N/A
12829
12844
GCATAGCCTTCTTTCT
87
1837


 


959742
N/A
N/A
12883
12898
CAATCCTGTTAGACAG
13
1838


 


959752
N/A
N/A
12904
12919
TTATAAAGCACACGGG
87
1839


 


959762
N/A
N/A
12934
12949
CAATAAGAGCTGTCTC
81
1840


 


959772
N/A
N/A
13370
13385
TGCAGGCACCCCAGCA
 0
1841


 


959782
N/A
N/A
13395
13410
GAATGTCACCCTTCCA
90
1842


 


959792
N/A
N/A
13528
13543
TCATTGGAAGACCGCA
84
1843


 


959802
N/A
N/A
13708
13723
GACCGCTAGTAAATGC
57
1844


 


959812
N/A
N/A
13741
13756
TTAGAACTAAGGCAAA
78
1845


 


959822
N/A
N/A
13917
13932
TGGAGTCATGACATCC
 0
1846


 


959832
N/A
N/A
14303
14318
CTGAGCAGATAAATAC
74
1847


 


959842
N/A
N/A
14553
14568
AGTCTTAATGTGGATT
76
1848


 


959852
N/A
N/A
14667
14682
AGTGTCCCCATCCCCA
54
1849


 


959862
N/A
N/A
15204
15219
AATATTGCCAGGTATC
56
1850


 


959872
N/A
N/A
15765
15780
TAGTGTTGGTTTATAA
89
1851


 


959882
N/A
N/A
17196
17211
TGACTTAGTCCGTGTT
43
1852


 


959892
N/A
N/A
17225
17240
TCTGTAGCTGGTTTGT
61
1853


 


959902
N/A
N/A
17298
17313
TCCCATCTCTTAGGGC
24
1854


 


959912
N/A
N/A
18396
18411
TATGTTTGGAAGTCGC
91
1855


 


959922
N/A
N/A
20290
20305
AATCAGAGGAAGCCCA
40
1856


 


959932
N/A
N/A
20515
20530
AACCAGATTGAGTCTC
72
1857


 


959942
N/A
N/A
20614
20629
TCTCTAATTTTACGAT
23
1858


 


959952
N/A
N/A
20663
20678
CAGCTTAGAAATTGCA
24
1859


 


959962
N/A
N/A
20814
20829
CCAGGGTAATATTCAG
87
1860


 


959972
N/A
N/A
20937
20952
CTTTGTAGCAGACAGA
50
1861


 


959982
N/A
N/A
21003
21018
TATTTTAACAGCTCAG
94
1862


 


959992
N/A
N/A
21409
21424
TGCAATTCTAGACATG
12
1863


 


960002
N/A
N/A
21445
21460
ACTAACCAATATACTG
55
1864


 


960012
N/A
N/A
22541
22556
CAACAGATTACTGGAC
28
1865


 


960022
N/A
N/A
22768
22783
AGGACATGACAGACTA
66
1866


 


960032
N/A
N/A
24041
24056
ACCATCAATGCTGCAC
79
1867
















TABLE 30







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
98
 702


 


959273
 427
 442
 6001
 6016
AAGTTGAAGGATGGAT
86
1868


 


959283
 694
 709
11912
11927
TTGGCATCAATGAAGG
73
1869


 


959293
 816
 831
12034
12049
AGAGGTTCCCTGTGCA
80
1870


 


959303
 906
 921
13642
13657
CCAAGAACCTGAATGC
65
1871


 


959313
1090
1105
16210
16225
GGCAGGATGCTGAGAC
43
1872


 


959323
1605
1620
25368
25383
CTCACAGACTCTTCTC
81
1873


 


959333
1646
1661
25409
25424
GCACCTCTGAAAGAAT
51
1874


 


959343
1677
1692
25440
25455
GGAGGTAGCTGCACAA
73
1875


 


959353
1904
1919
25667
25682
ACAGTCTGACCATTAA
85
1876


 


959363
2179
2194
25942
25957
TTAGGTGAAAAAGGTG
91
1877


 


959373
2258
2273
26021
26036
TGATTCACATAATACA
71
1878


 


959383
2277
2292
26040
26055
TATTCTACTAACATCT
38
1879


 


959393
N/A
N/A
 4733
 4748
AATGATCATGTGGCGG
93
1880


 


959403
N/A
N/A
 4767
 4782
TGACTTTTATTGTTGC
87
1881


 


959413
N/A
N/A
 4870
 4885
TGAGTGTACTTTAGGC
94
1882


 


959423
N/A
N/A
 5392
 5407
ATGCTATCAGGTGCAG
 0
1883


 


959433
N/A
N/A
 5578
 5593
GCACAATGACATCATG
86
1884


 


959443
N/A
N/A
 5608
 5623
TTACTTTATTCAATGT
 9
1885


 


959453
N/A
N/A
 5644
 5659
TATTGGGCTCAATGAA
80
1886


 


959463
N/A
N/A
 5798
 5813
TATGGGAGCCACATGT
 4
1887


 


959473
N/A
N/A
 5866
 5881
CTTTTGGCAAGGCCAG
 0
1888


 


959483
N/A
N/A
 7199
 7214
TTAAACAGAGGATGCA
31
1889


 


959663
N/A
N/A
12156
12171
AGACAGTAACTGGTAG
75
1890


 


959673
N/A
N/A
12199
12214
TCCGTTAACCATCAAG
95
1891


 


959683
N/A
N/A
12282
12297
TTAGGTCTGGGTATAT
94
1892


 


959693
N/A
N/A
12324
12339
CCCCTGACTATATAAC
 0
1893


 


959703
N/A
N/A
12692
12707
CTTGACCGTGTTTCCA
97
1894


 


959713
N/A
N/A
12715
12730
TTAAACTACCGAACGC
95
1895


 


959723
N/A
N/A
12761
12776
ATGGTAGAGTGGTAAG
77
1896


 


959733
N/A
N/A
12832
12847
ATTGCATAGCCTTCTT
95
1897


 


959743
N/A
N/A
12884
12899
CCAATCCTGTTAGACA
83
1898


 


959753
N/A
N/A
12908
12923
CTGCTTATAAAGCACA
 2
1899


 


959763
N/A
N/A
12935
12950
ACAATAAGAGCTGTCT
85
1900


 


959773
N/A
N/A
13372
13387
TTTGCAGGCACCCCAG
63
1901


 


959783
N/A
N/A
13396
13411
TGAATGTCACCCTTCC
53
1902


 


959793
N/A
N/A
13531
13546
GCATCATTGGAAGACC
86
1903


 


959803
N/A
N/A
13713
13728
ACCAAGACCGCTAGTA
38
1904


 


959813
N/A
N/A
13743
13758
TGTTAGAACTAAGGCA
79
1905


 


959823
N/A
N/A
13919
13934
CCTGGAGTCATGACAT
 7
1906


 


959833
N/A
N/A
14304
14319
TCTGAGCAGATAAATA
39
1907


 


959843
N/A
N/A
14554
14569
AAGTCTTAATGTGGAT
84
1908


 


959853
N/A
N/A
14669
14684
TTAGTGTCCCCATCCC
78
1909


 


959863
N/A
N/A
15205
15220
GAATATTGCCAGGTAT
86
1910


 


959873
N/A
N/A
15766
15781
TTAGTGTTGGTTTATA
92
1911


 


959883
N/A
N/A
17198
17213
TTTGACTTAGTCCGTG
86
1912


 


959893
N/A
N/A
17226
17241
CTCTGTAGCTGGTTTG
82
1913


 


959903
N/A
N/A
17601
17616
TTGATAGTGAATGTGT
83
1914


 


959913
N/A
N/A
18397
18412
ATATGTTTGGAAGTCG
91
1915


 


959923
N/A
N/A
20291
20306
CAATCAGAGGAAGCCC
34
1916


 


959933
N/A
N/A
20516
20531
TAACCAGATTGAGTCT
66
1917


 


959943
N/A
N/A
20615
20630
GTCTCTAATTTTACGA
53
1918


 


959953
N/A
N/A
20664
20679
ACAGCTTAGAAATTGC
89
1919


 


959963
N/A
N/A
20843
20858
CTGTATTAGCTCAATA
67
1920


 


959973
N/A
N/A
20938
20953
TCTTTGTAGCAGACAG
84
1921


 


959983
N/A
N/A
21004
21019
TTATTTTAACAGCTCA
92
1922


 


959993
N/A
N/A
21410
21425
CTGCAATTCTAGACAT
29
1923


 


960003
N/A
N/A
21535
21550
CATTTCAGAGTATAAG
46
1924


 


960013
N/A
N/A
22708
22723
GATGTGAGTGAAATAA
69
1925


 


960023
N/A
N/A
22769
22784
AAGGACATGACAGACT
68
1926


 


960033
N/A
N/A
24043
24058
CCACCATCAATGCTGC
58
1927
















TABLE 31







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
 99
 702


 


959284
 695
 710
11913
11928
TTTGGCATCAATGAAG
 69
1928


 


959294
 817
 832
12035
12050
TAGAGGTTCCCTGTGC
 83
1929


 


959314
1091
1106
16211
16226
GGGCAGGATGCTGAGA
 22
1930


 


959324
1606
1621
25369
25384
ACTCACAGACTCTTCT
 68
1931


 


959334
1647
1662
25410
25425
AGCACCTCTGAAAGAA
 67
1932


 


959344
1678
1693
25441
25456
CGGAGGTAGCTGCACA
 85
1933


 


959354
1926
1941
25689
25704
ATTCTAAGAACCTCAT
 51
1934


 


959384
N/A
N/A
 4303
 4318
AAACAAACCCTCCGTC
 10
1935


 


959394
N/A
N/A
 4734
 4749
AAATGATCATGTGGCG
 94
1936


 


959404
N/A
N/A
 4768
 4783
CTGACTTTTATTGTTG
 74
1937


 


959414
N/A
N/A
 4871
 4886
GTGAGTGTACTTTAGG
 98
1938


 


959424
N/A
N/A
 5393
 5408
AATGCTATCAGGTGCA
 31
1939


 


959434
N/A
N/A
 5579
 5594
TGCACAATGACATCAT
 88
1940


 


959444
N/A
N/A
 5621
 5636
CTACCTGTGTCTTTTA
 90
1941


 


959454
N/A
N/A
 5647
 5662
ATATATTGGGCTCAAT
 81
1942


 


959474
N/A
N/A
 5867
 5882
ACTTTTGGCAAGGCCA
 17
1943


 


959484
N/A
N/A
 7211
 7226
CCGCAAACAAGGTTAA
 22
1944


 


959684
N/A
N/A
12283
12298
TTTAGGTCTGGGTATA
 93
1945


 


959694
N/A
N/A
12325
12340
CCCCCTGACTATATAA
 18
1946


 


959704
N/A
N/A
12693
12708
TCTTGACCGTGTTTCC
 97
1947


 


959734
N/A
N/A
12834
12849
GCATTGCATAGCCTTC
 96
1948


 


959744
N/A
N/A
12886
12901
AACCAATCCTGTTAGA
 59
1949


 


959754
N/A
N/A
12909
12924
TCTGCTTATAAAGCAC
100
1950


 


959774
N/A
N/A
13373
13388
CTTTGCAGGCACCCCA
 72
1951


 


959784
N/A
N/A
13398
13413
CTTGAATGTCACCCTT
 92
1952


 


959804
N/A
N/A
13715
13730
TTACCAAGACCGCTAG
 47
1953


 


959824
N/A
N/A
14228
14243
ACTTTTAGTATTAAAG
  0
1954


 


959844
N/A
N/A
14555
14570
AAAGTCTTAATGTGGA
 88
1955


 


959854
N/A
N/A
14670
14685
CTTAGTGTCCCCATCC
 76
1956


 


959874
N/A
N/A
15767
15782
GTTAGTGTTGGTTTAT
 95
1957


 


959884
N/A
N/A
17199
17214
GTTTGACTTAGTCCGT
 96
1958


 


959914
N/A
N/A
18398
18413
AATATGTTTGGAAGTC
 87
1959


 


959934
N/A
N/A
20518
20533
AGTAACCAGATTGAGT
 96
1960


 


959954
N/A
N/A
20665
20680
CACAGCTTAGAAATTG
 88
1961


 


959964
N/A
N/A
20844
20859
CCTGTATTAGCTCAAT
 92
1962


 


959974
N/A
N/A
20940
20955
CCTCTTTGTAGCAGAC
 84
1963


 


959984
N/A
N/A
21006
21021
GGTTATTTTAACAGCT
 85
1964


 


959994
N/A
N/A
21412
21427
ACCTGCAATTCTAGAC
 61
1965


 


960014
N/A
N/A
22710
22725
AAGATGTGAGTGAAAT
 70
1966


 


960024
N/A
N/A
22770
22785
AAAGGACATGACAGAC
 87
1967
















TABLE 32







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





959275
 440
 455
 6014
 6029
GAGGAACTTGCTTAAG
81
1968


 


959285
 696
 711
11914
11929
TTTTGGCATCAATGAA
62
1969


 


959305
1066
1081
16186
16201
TCTAGCAGCTCATCTC
64
1970


 


959335
1649
1664
25412
25427
TTAGCACCTCTGAAAG
72
1971


 


959345
1804
1819
25567
25582
ATGTATTAGAGTTAAG
77
1972


 


959355
1927
1942
25690
25705
CATTCTAAGAACCTCA
71
1973


 


959365
2183
2198
25946
25961
TTAGTTAGGTGAAAAA
81
1974


 


959375
2261
2276
26024
26039
CACTGATTCACATAAT
70
1975


 


959395
N/A
N/A
 4737
 4752
TGCAAATGATCATGTG
66
1976


 


959405
N/A
N/A
 4769
 4784
GCTGACTTTTATTGTT
84
1977


 


959415
N/A
N/A
 4872
 4887
AGTGAGTGTACTTTAG
94
1978


 


959425
N/A
N/A
 5395
 5410
TTAATGCTATCAGGTG
81
1979


 


959435
N/A
N/A
 5580
 5595
ATGCACAATGACATCA
86
1980


 


959445
N/A
N/A
 5624
 5639
ATTCTACCTGTGTCTT
97
1981


 


959455
N/A
N/A
 5651
 5666
TTGGATATATTGGGCT
97
1982


 


959475
N/A
N/A
 5868
 5883
TACTTTTGGCAAGGCC
70
1983


 


959485
N/A
N/A
 7697
 7712
GCACAGAGTAGGTTAA
72
1984


 


959655
N/A
N/A
12146
12161
TGGTAGCTCCTGGCAA
55
1985


 


959675
N/A
N/A
12201
12216
TTTCCGTTAACCATCA
94
1986


 


959695
N/A
N/A
12667
12682
CATCTTAGTGGCTGGG
93
1987


 


959705
N/A
N/A
12695
12710
GTTCTTGACCGTGTTT
97
1988


 


959715
N/A
N/A
12717
12732
GGTTAAACTACCGAAC
11
1989


 


959725
N/A
N/A
12783
12798
CATGGTCTGCAAATTT
89
1990


 


959745
N/A
N/A
12887
12902
AAACCAATCCTGTTAG
46
1991


 


959755
N/A
N/A
12910
12925
ATCTGCTTATAAAGCA
42
1992


 


959775
N/A
N/A
13374
13389
ACTTTGCAGGCACCCC
87
1993


 


959785
N/A
N/A
13399
13414
GCTTGAATGTCACCCT
94
1994


 


959805
N/A
N/A
13716
13731
TTTACCAAGACCGCTA
86
1995


 


959825
N/A
N/A
14230
14245
CAACTTTTAGTATTAA
 0
1996


 


959835
N/A
N/A
14417
14432
TCGACACAGCATCACC
62
1997


 


959855
N/A
N/A
14671
14686
TCTTAGTGTCCCCATC
78
1998


 


959865
N/A
N/A
15209
15224
TTAGGAATATTGCCAG
93
1999


 


959875
N/A
N/A
15769
15784
GGGTTAGTGTTGGTTT
94
2000


 


959895
N/A
N/A
17288
17303
TAGGGCACCTCAAGAA
 0
2001


 


959915
N/A
N/A
18400
18415
CAAATATGTTTGGAAG
50
2002


 


959925
N/A
N/A
20293
20308
CCCAATCAGAGGAAGC
41
2003


 


959935
N/A
N/A
20599
20614
TCATCATTATTACCTG
91
2004


 


959955
N/A
N/A
20803
20818
TTCAGACCAGGGTAAT
91
2005


 


959965
N/A
N/A
20845
20860
GCCTGTATTAGCTCAA
90
2006


 


959975
N/A
N/A
20941
20956
GCCTCTTTGTAGCAGA
 0
2007


 


959995
N/A
N/A
21434
21449
TACTGAGAGGAAATGA
64
2008


 


960015
N/A
N/A
22714
22729
TGTAAAGATGTGAGTG
78
2009


 


960025
N/A
N/A
22772
22787
TCAAAGGACATGACAG
80
2010


 


960037
N/A
N/A
22590
22605
GAGCAACGAGGAAGGA
68
2011
















TABLE 33







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
98
 702


 


959276
 447
 462
 6021
 6036
CCTGTCGGAGGAACTT
37
2012


 


959286
 699
 714
11917
11932
TTGTTTTGGCATCAAT
73
2013


 


959296
 895
 910
13631
13646
AATGCATCCAAATATC
 0
2014


 


959306
1069
1084
16189
16204
TGGTCTAGCAGCTCAT
25
2015


 


959316
1221
1236
19061
19076
TTACATAAGACATTAT
35
2016


 


959326
1614
1629
25377
25392
CTCAAGTGACTCACAG
85
2017


 


959336
1650
1665
25413
25428
TTTAGCACCTCTGAAA
24
2018


 


959346
1834
1849
25597
25612
TTTCCCAACCAGCTGA
60
2019


 


959356
2096
2111
25859
25874
TCATCTTTGCAGACCA
90
2020


 


959366
2184
2199
25947
25962
TTTAGTTAGGTGAAAA
57
2021


 


959376
2262
2277
26025
26040
TCACTGATTCACATAA
86
2022


 


959386
N/A
N/A
 4306
 4321
GAGAAACAAACCCTCC
 0
2023


 


959396
N/A
N/A
 4738
 4753
GTGCAAATGATCATGT
69
2024


 


959406
N/A
N/A
 4771
 4786
AAGCTGACTTTTATTG
57
2025


 


959416
N/A
N/A
 5276
 5291
TCTTGGGATGCACAGG
49
2026


 


959426
N/A
N/A
 5397
 5412
CCTTAATGCTATCAGG
 0
2027


 


959436
N/A
N/A
 5581
 5596
AATGCACAATGACATC
69
2028


 


959446
N/A
N/A
 5625
 5640
AATTCTACCTGTGTCT
82
2029


 


959456
N/A
N/A
 5652
 5667
TTTGGATATATTGGGC
95
2030


 


959466
N/A
N/A
 5802
 5817
CTACTATGGGAGCCAC
64
2031


 


959476
N/A
N/A
 5871
 5886
TAATACTTTTGGCAAG
29
2032


 


959486
N/A
N/A
 7784
 7799
TTATAGGCGAGAGCAC
 0
2033


 


959656
N/A
N/A
12147
12162
CTGGTAGCTCCTGGCA
44
2034


 


959666
N/A
N/A
12164
12179
GATTGTGCAGACAGTA
95
2035


 


959676
N/A
N/A
12202
12217
ATTTCCGTTAACCATC
93
2036


 


959686
N/A
N/A
12288
12303
TGAGTTTTAGGTCTGG
96
2037


 


959696
N/A
N/A
12669
12684
ATCATCTTAGTGGCTG
91
2038


 


959706
N/A
N/A
12696
12711
TGTTCTTGACCGTGTT
98
2039


 


959716
N/A
N/A
12719
12734
AAGGTTAAACTACCGA
 6
2040


 


959726
N/A
N/A
12785
12800
TACATGGTCTGCAAAT
90
2041


 


959736
N/A
N/A
12838
12853
CATTGCATTGCATAGC
96
2042


 


959746
N/A
N/A
12888
12903
AAAACCAATCCTGTTA
47
2043


 


959756
N/A
N/A
12911
12926
CATCTGCTTATAAAGC
81
2044


 


959766
N/A
N/A
12967
12982
AAACTTTGCAGCCTAT
93
2045


 


959776
N/A
N/A
13376
13391
AGACTTTGCAGGCACC
90
2046


 


959786
N/A
N/A
13400
13415
GGCTTGAATGTCACCC
69
2047


 


959796
N/A
N/A
13697
13712
AATGCTTGTCAAAAGG
70
2048


 


959806
N/A
N/A
13717
13732
CTTTACCAAGACCGCT
82
2049


 


959816
N/A
N/A
13747
13762
TAAGTGTTAGAACTAA
30
2050


 


959826
N/A
N/A
14232
14247
ACCAACTTTTAGTATT
79
2051


 


959836
N/A
N/A
14419
14434
CATCGACACAGCATCA
59
2052


 


959846
N/A
N/A
14557
14572
CCAAAGTCTTAATGTG
53
2053


 


959856
N/A
N/A
14676
14691
CCATCTCTTAGTGTCC
88
2054


 


959866
N/A
N/A
15210
15225
CTTAGGAATATTGCCA
87
2055


 


959876
N/A
N/A
15770
15785
AGGGTTAGTGTTGGTT
89
2056


 


959886
N/A
N/A
17202
17217
TCTGTTTGACTTAGTC
70
2057


 


959896
N/A
N/A
17290
17305
CTTAGGGCACCTCAAG
30
2058


 


959906
N/A
N/A
17735
17750
TAATCTGGTCATATGG
43
2059


 


959916
N/A
N/A
18445
18460
TGCTTACGGAGCATAG
 0
2060


 


959926
N/A
N/A
20472
20487
CTCTAGACGGGAAGCT
31
2061


 


959936
N/A
N/A
20601
20616
GATCATCATTATTACC
71
2062


 


959946
N/A
N/A
20652
20667
TTGCAGTGCCCTGGCC
31
2063


 


959956
N/A
N/A
20804
20819
ATTCAGACCAGGGTAA
87
2064


 


959966
N/A
N/A
20847
20862
ATGCCTGTATTAGCTC
65
2065


 


959976
N/A
N/A
20942
20957
AGCCTCTTTGTAGCAG
 5
2066


 


959986
N/A
N/A
21008
21023
GAGGTTATTTTAACAG
69
2067


 


959996
N/A
N/A
21435
21450
ATACTGAGAGGAAATG
68
2068


 


960006
N/A
N/A
21539
21554
ATGACATTTCAGAGTA
88
2069


 


960016
N/A
N/A
22715
22730
GTGTAAAGATGTGAGT
84
2070


 


960026
N/A
N/A
24033
24048
TGCTGCACTCAAAGAG
 0
2071


 


960038
N/A
N/A
19377
19392
TCACAAGAGACTGGAC
31
2072
















TABLE 34







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
98
 702


 


959277
 481
 496
 6055
 6070
TGGTGGACATTGGCCG
 5
2073


 


959287
 707
 722
11925
11940
GGTGATGGTTGTTTTG
85
2074


 


959297
 896
 911
13632
13647
GAATGCATCCAAATAT
 0
2075


 


959307
1070
1085
16190
16205
GTGGTCTAGCAGCTCA
66
2076


 


959317
1223
1238
19063
19078
CATTACATAAGACATT
46
2077


 


959327
1615
1630
25378
25393
CCTCAAGTGACTCACA
83
2078


 


959337
1651
1666
25414
25429
CTTTAGCACCTCTGAA
71
2079


 


959347
1835
1850
25598
25613
ATTTCCCAACCAGCTG
49
2080


 


959357
2099
2114
25862
25877
TTATCATCTTTGCAGA
48
2081


 


959367
2185
2200
25948
25963
TTTTAGTTAGGTGAAA
43
2082


 


959377
2263
2278
26026
26041
CTCACTGATTCACATA
79
2083


 


959387
N/A
N/A
 4307
 4322
TGAGAAACAAACCCTC
 0
2084


 


959397
N/A
N/A
 4739
 4754
TGTGCAAATGATCATG
82
2085


 


959407
N/A
N/A
 4859
 4874
TAGGCTCCTGGGACCT
 0
2086


 


959417
N/A
N/A
 5277
 5292
ATCTTGGGATGCACAG
89
2087


 


959427
N/A
N/A
 5567
 5582
TCATGGCTTCCAGTGT
78
2088


 


959437
N/A
N/A
 5600
 5615
TTCAATGTGGCTTCTA
96
2089


 


959447
N/A
N/A
 5627
 5642
TTAATTCTACCTGTGT
72
2090


 


959457
N/A
N/A
 5706
 5721
TGAAATATCTCATTAG
77
2091


 


959467
N/A
N/A
 5805
 5820
TGTCTACTATGGGAGC
83
2092


 


959477
N/A
N/A
 5875
 5890
ATGGTAATACTTTTGG
75
2093


 


959657
N/A
N/A
12148
12163
ACTGGTAGCTCCTGGC
78
2094


 


959667
N/A
N/A
12165
12180
TGATTGTGCAGACAGT
97
2095


 


959677
N/A
N/A
12203
12218
TATTTCCGTTAACCAT
91
2096


 


959687
N/A
N/A
12289
12304
CTGAGTTTTAGGTCTG
96
2097


 


959697
N/A
N/A
12671
12686
GAATCATCTTAGTGGC
94
2098


 


959707
N/A
N/A
12697
12712
TTGTTCTTGACCGTGT
98
2099


 


959717
N/A
N/A
12753
12768
GTGGTAAGGCATACTA
35
2100


 


959727
N/A
N/A
12789
12804
GGTGTACATGGTCTGC
97
2101


 


959737
N/A
N/A
12839
12854
GCATTGCATTGCATAG
92
2102


 


959747
N/A
N/A
12890
12905
GGAAAACCAATCCTGT
69
2103


 


959757
N/A
N/A
12927
12942
AGCTGTCTCCTCTACT
70
2104


 


959767
N/A
N/A
12968
12983
CAAACTTTGCAGCCTA
95
2105


 


959777
N/A
N/A
13377
13392
GAGACTTTGCAGGCAC
88
2106


 


959787
N/A
N/A
13402
13417
TCGGCTTGAATGTCAC
67
2107


 


959797
N/A
N/A
13700
13715
GTAAATGCTTGTCAAA
91
2108


 


959807
N/A
N/A
13720
13735
AGTCTTTACCAAGACC
 0
2109


 


959817
N/A
N/A
13911
13926
CATGACATCCCAGTTC
29
2110


 


959827
N/A
N/A
14233
14248
AACCAACTTTTAGTAT
27
2111


 


959837
N/A
N/A
14420
14435
CCATCGACACAGCATC
89
2112


 


959847
N/A
N/A
14567
14582
CTACTTATCCCCAAAG
16
2113


 


959857
N/A
N/A
14677
14692
GCCATCTCTTAGTGTC
36
2114


 


959867
N/A
N/A
15211
15226
CCTTAGGAATATTGCC
75
2115


 


959877
N/A
N/A
15771
15786
GAGGGTTAGTGTTGGT
93
2116


 


959887
N/A
N/A
17218
17233
CTGGTTTGTGGGTTCT
75
2117


 


959897
N/A
N/A
17291
17306
TCTTAGGGCACCTCAA
53
2118


 


959907
N/A
N/A
17736
17751
TTAATCTGGTCATATG
 0
2119


 


959917
N/A
N/A
18852
18867
ACAAAAGCGACAAGGT
33
2120


 


959927
N/A
N/A
20508
20523
TTGAGTCTCCTGACCA
65
2121


 


959937
N/A
N/A
20602
20617
CGATCATCATTATTAC
87
2122


 


959947
N/A
N/A
20653
20668
ATTGCAGTGCCCTGGC
69
2123


 


959957
N/A
N/A
20805
20820
TATTCAGACCAGGGTA
89
2124


 


959967
N/A
N/A
20848
20863
GATGCCTGTATTAGCT
72
2125


 


959977
N/A
N/A
20944
20959
GCAGCCTCTTTGTAGC
 0
2126


 


959987
N/A
N/A
21010
21025
CTGAGGTTATTTTAAC
40
2127


 


959997
N/A
N/A
21436
21451
TATACTGAGAGGAAAT
47
2128


 


960007
N/A
N/A
21541
21556
ATATGACATTTCAGAG
91
2129


 


960017
N/A
N/A
22716
22731
CGTGTAAAGATGTGAG
87
2130


 


960027
N/A
N/A
24035
24050
AATGCTGCACTCAAAG
31
2131


 


960039
N/A
N/A
20215
20230
TAACAAACTATGCCTA
44
2132
















TABLE 35







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
98
 702


 


959274
 439
 454
 6013
 6028
AGGAACTTGCTTAAGT
73
2133


 


959284
 695
 710
11913
11928
TTTGGCATCAATGAAG
60
1928


 


959294
 817
 832
12035
12050
TAGAGGTTCCCTGTGC
77
1929


 


959304
1063
1078
16183
16198
AGCAGCTCATCTCCCT
62
2134


 


959314
1091
1106
16211
16226
GGGCAGGATGCTGAGA
13
1930


 


959324
1606
1621
25369
25384
ACTCACAGACTCTTCT
72
1931


 


959334
1647
1662
25410
25425
AGCACCTCTGAAAGAA
65
1932


 


959344
1678
1693
25441
25456
CGGAGGTAGCTGCACA
86
1933


 


959354
1926
1941
25689
25704
ATTCTAAGAACCTCAT
54
1934


 


959364
2181
2196
25944
25959
AGTTAGGTGAAAAAGG
92
2135


 


959374
2260
2275
26023
26038
ACTGATTCACATAATA
78
2136


 


959384
N/A
N/A
 4303
 4318
AAACAAACCCTCCGTC
 2
1935


 


959394
N/A
N/A
 4734
 4749
AAATGATCATGTGGCG
94
1936


 


959404
N/A
N/A
 4768
 4783
CTGACTTTTATTGTTG
72
1937


 


959414
N/A
N/A
 4871
 4886
GTGAGTGTACTTTAGG
97
1938


 


959424
N/A
N/A
 5393
 5408
AATGCTATCAGGTGCA
31
1939


 


959434
N/A
N/A
 5579
 5594
TGCACAATGACATCAT
87
1940


 


959444
N/A
N/A
 5621
 5636
CTACCTGTGTCTTTTA
90
1941


 


959454
N/A
N/A
 5647
 5662
ATATATTGGGCTCAAT
80
1942


 


959464
N/A
N/A
 5799
 5814
CTATGGGAGCCACATG
24
2137


 


959474
N/A
N/A
 5867
 5882
ACTTTTGGCAAGGCCA
23
1943


 


959484
N/A
N/A
 7211
 7226
CCGCAAACAAGGTTAA
 0
1944


 


959664
N/A
N/A
12157
12172
CAGACAGTAACTGGTA
96
2138


 


959674
N/A
N/A
12200
12215
TTCCGTTAACCATCAA
97
2139


 


959684
N/A
N/A
12283
12298
TTTAGGTCTGGGTATA
93
1945


 


959694
N/A
N/A
12325
12340
CCCCCTGACTATATAA
26
1946


 


959704
N/A
N/A
12693
12708
TCTTGACCGTGTTTCC
98
1947


 


959714
N/A
N/A
12716
12731
GTTAAACTACCGAACG
35
2140


 


959724
N/A
N/A
12763
12778
CTATGGTAGAGTGGTA
93
2141


 


959734
N/A
N/A
12834
12849
GCATTGCATAGCCTTC
97
1948


 


959744
N/A
N/A
12886
12901
AACCAATCCTGTTAGA
55
1949


 


959754
N/A
N/A
12909
12924
TCTGCTTATAAAGCAC
 0
1950


 


959764
N/A
N/A
12937
12952
GGACAATAAGAGCTGT
91
2142


 


959774
N/A
N/A
13373
13388
CTTTGCAGGCACCCCA
72
1951


 


959784
N/A
N/A
13398
13413
CTTGAATGTCACCCTT
92
1952


 


959794
N/A
N/A
13532
13547
AGCATCATTGGAAGAC
92
2143


 


959804
N/A
N/A
13715
13730
TTACCAAGACCGCTAG
57
1953


 


959814
N/A
N/A
13744
13759
GTGTTAGAACTAAGGC
94
2144


 


959824
N/A
N/A
14228
14243
ACTTTTAGTATTAAAG
 0
1954


 


959834
N/A
N/A
14306
14321
TTTCTGAGCAGATAAA
66
2145


 


959844
N/A
N/A
14555
14570
AAAGTCTTAATGTGGA
87
1955


 


959854
N/A
N/A
14670
14685
CTTAGTGTCCCCATCC
77
1956


 


959864
N/A
N/A
15208
15223
TAGGAATATTGCCAGG
89
2146


 


959874
N/A
N/A
15767
15782
GTTAGTGTTGGTTTAT
94
1957


 


959884
N/A
N/A
17199
17214
GTTTGACTTAGTCCGT
95
1958


 


959894
N/A
N/A
17228
17243
AACTCTGTAGCTGGTT
41
2147


 


959904
N/A
N/A
17603
17618
TCTTGATAGTGAATGT
73
2148


 


959914
N/A
N/A
18398
18413
AATATGTTTGGAAGTC
89
1959


 


959924
N/A
N/A
20292
20307
CCAATCAGAGGAAGCC
58
2149


 


959934
N/A
N/A
20518
20533
AGTAACCAGATTGAGT
81
1960


 


959944
N/A
N/A
20617
20632
CTGTCTCTAATTTTAC
75
2150


 


959954
N/A
N/A
20665
20680
CACAGCTTAGAAATTG
87
1961


 


959964
N/A
N/A
20844
20859
CCTGTATTAGCTCAAT
92
1962


 


959974
N/A
N/A
20940
20955
CCTCTTTGTAGCAGAC
83
1963


 


959984
N/A
N/A
21006
21021
GGTTATTTTAACAGCT
85
1964


 


959994
N/A
N/A
21412
21427
ACCTGCAATTCTAGAC
54
1965


 


960004
N/A
N/A
21537
21552
GACATTTCAGAGTATA
93
2151


 


960014
N/A
N/A
22710
22725
AAGATGTGAGTGAAAT
69
1966


 


960024
N/A
N/A
22770
22785
AAAGGACATGACAGAC
75
1967


 


960034
N/A
N/A
24613
24628
GCAAATCGGATCTTTG
32
2152
















TABLE 36







Inhibition of PNPLA3 mRNA by 3-10-3 cEt gapmers targeting SEQ ID NO: 1 and 2















SEQ
SEQ
SEQ
SEQ






ID
ID
ID
ID






NO: 1
NO: 1
NO: 2
NO: 2

PNPLA3
SEQ


Compound
Start
Stop
Start
Stop

%
ID


Number
Site
Site
Site
Site
Sequence (5′ to 3′)
Inhibition
NO





915609
 705
 720
11923
11938
TGATGGTTGTTTTGGC
98
 702


 


959275
 440
 455
 6014
 6029
GAGGAACTTGCTTAAG
80
1968


 


959285
 696
 711
11914
11929
TTTTGGCATCAATGAA
63
1969


 


959295
 823
 838
12041
12056
AGAAGGTAGAGGTTCC
48
2153


 


959305
1066
1081
16186
16201
TCTAGCAGCTCATCTC
66
1970


 


959315
1093
1108
16213
16228
CAGGGCAGGATGCTGA
 2
2154


 


959325
1608
1623
25371
25386
TGACTCACAGACTCTT
60
2155


 


959335
1649
1664
25412
25427
TTAGCACCTCTGAAAG
54
1971


 


959345
1804
1819
25567
25582
ATGTATTAGAGTTAAG
79
1972


 


959355
1927
1942
25690
25705
CATTCTAAGAACCTCA
68
1973


 


959365
2183
2198
25946
25961
TTAGTTAGGTGAAAAA
69
1974


 


959375
2261
2276
26024
26039
CACTGATTCACATAAT
73
1975


 


959385
N/A
N/A
 4305
 4320
AGAAACAAACCCTCCG
70
2156


 


959395
N/A
N/A
 4737
 4752
TGCAAATGATCATGTG
69
1976


 


959405
N/A
N/A
 4769
 4784
GCTGACTTTTATTGTT
83
1977


 


959415
N/A
N/A
 4872
 4887
AGTGAGTGTACTTTAG
94
1978


 


959425
N/A
N/A
 5395
 5410
TTAATGCTATCAGGTG
82
1979


 


959435
N/A
N/A
 5580
 5595
ATGCACAATGACATCA
84
1980


 


959445
N/A
N/A
 5624
 5639
ATTCTACCTGTGTCTT
95
1981


 


959455
N/A
N/A
 5651
 5666
TTGGATATATTGGGCT
97
1982


 


959465
N/A
N/A
 5800
 5815
ACTATGGGAGCCACAT
26
2157


 


959475
N/A
N/A
 5868
 5883
TACTTTTGGCAAGGCC
69
1983


 


959485
N/A
N/A
 7697
 7712
GCACAGAGTAGGTTAA
70
1984


 


959655
N/A
N/A
12146
12161
TGGTAGCTCCTGGCAA
50
1985


 


959665
N/A
N/A
12162
12177
TTGTGCAGACAGTAAC
87
2158


 


959675
N/A
N/A
12201
12216
TTTCCGTTAACCATCA
95
1986


 


959685
N/A
N/A
12284
12299
TTTTAGGTCTGGGTAT
81
2159


 


959695
N/A
N/A
12667
12682
CATCTTAGTGGCTGGG
91
1987


 


959705
N/A
N/A
12695
12710
GTTCTTGACCGTGTTT
97
1988


 


959715
N/A
N/A
12717
12732
GGTTAAACTACCGAAC
26
1989


 


959725
N/A
N/A
12783
12798
CATGGTCTGCAAATTT
89
1990


 


959735
N/A
N/A
12837
12852
ATTGCATTGCATAGCC
95
2160


 


959745
N/A
N/A
12887
12902
AAACCAATCCTGTTAG
54
1991


 


959755
N/A
N/A
12910
12925
ATCTGCTTATAAAGCA
43
1992


 


959765
N/A
N/A
12964
12979
CTTTGCAGCCTATCCC
95
2161


 


959775
N/A
N/A
13374
13389
ACTTTGCAGGCACCCC
86
1993


 


959785
N/A
N/A
13399
13414
GCTTGAATGTCACCCT
95
1994


 


959795
N/A
N/A
13534
13549
TCAGCATCATTGGAAG
60
2162


 


959805
N/A
N/A
13716
13731
TTTACCAAGACCGCTA
82
1995


 


959815
N/A
N/A
13745
13760
AGTGTTAGAACTAAGG
93
2163


 


959825
N/A
N/A
14230
14245
CAACTTTTAGTATTAA
 9
1996


 


959835
N/A
N/A
14417
14432
TCGACACAGCATCACC
59
1997


 


959845
N/A
N/A
14556
14571
CAAAGTCTTAATGTGG
84
2164


 


959855
N/A
N/A
14671
14686
TCTTAGTGTCCCCATC
78
1998


 


959865
N/A
N/A
15209
15224
TTAGGAATATTGCCAG
91
1999


 


959875
N/A
N/A
15769
15784
GGGTTAGTGTTGGTTT
93
2000


 


959885
N/A
N/A
17200
17215
TGTTTGACTTAGTCCG
96
2165


 


959895
N/A
N/A
17288
17303
TAGGGCACCTCAAGAA
 0
2001


 


959905
N/A
N/A
17734
17749
AATCTGGTCATATGGT
42
2166


 


959915
N/A
N/A
18400
18415
CAAATATGTTTGGAAG
55
2002


 


959925
N/A
N/A
20293
20308
CCCAATCAGAGGAAGC
57
2003


 


959935
N/A
N/A
20599
20614
TCATCATTATTACCTG
93
2004


 


959945
N/A
N/A
20651
20666
TGCAGTGCCCTGGCCT
40
2167


 


959955
N/A
N/A
20803
20818
TTCAGACCAGGGTAAT
93
2005


 


959965
N/A
N/A
20845
20860
GCCTGTATTAGCTCAA
88
2006


 


959975
N/A
N/A
20941
20956
GCCTCTTTGTAGCAGA
 0
2007


 


959985
N/A
N/A
21007
21022
AGGTTATTTTAACAGC
94
2168


 


959995
N/A
N/A
21434
21449
TACTGAGAGGAAATGA
65
2008


 


960005
N/A
N/A
21538
21553
TGACATTTCAGAGTAT
87
2169


 


960015
N/A
N/A
22714
22729
TGTAAAGATGTGAGTG
75
2009


 


960025
N/A
N/A
22772
22787
TCAAAGGACATGACAG
78
2010


 


960037
N/A
N/A
22590
22605
GAGCAACGAGGAAGGA
64
2011









Example 2: Dose-Dependent Antisense Inhibition of Human PNPLA3 in A431 Cells
Gapmers from Example 1 exhibiting significant in vitro inhibition of PNPLA3 mRNA were selected and tested at various doses in A431 cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 10,000 cells per well and transfected free uptake with different concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and PNPLA3 mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS36070 was used to measure mRNA levels. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of PNPLA3, relative to untreated control cells.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented. PNPLA3 mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.







TABLE 37







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912712
27
67
76
74
0.2



912732
54
78
88
87
<0.1



912733
45
74
85
88
<0.1



912734
33
64
80
83
0.1



912756
46
72
89
92
<0.1



912757
31
62
78
86
0.2



912758
38
70
85
90
0.1



912759
66
92
97
98
<0.1



912772
46
63
79
88
0.1



912795
40
64
83
84
0.1



912812
43
81
88
88
<0.1



912822
81
83
92
86
<0.1



912823
67
80
91
86
<0.1



912825
58
80
86
88
<0.1



912834
37
75
81
84
0.1



912841
17
62
79
69
0.3



912847
70
83
90
91
<0.1



912848
80
89
90
90
<0.1



912855
48
62
77
80
0.1

















TABLE 38







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
68
94
94
98
<0.1



912813
57
84
90
87
<0.1



912856
60
81
91
88
<0.1



912859
48
79
81
72
<0.1



912864
60
88
90
90
<0.1



912870
67
81
91
94
<0.1



912871
21
67
84
89
0.2



912872
18
73
90
92
0.2



912876
43
70
87
92
0.1



912933
68
89
90
90
<0.1



912940
86
91
95
96
<0.1



912941
87
94
96
96
<0.1



912952
68
85
90
91
<0.1



912953
80
90
95
93
<0.1



912964
59
78
88
91
<0.1



912973
53
70
87
91
<0.1



912980
54
77
84
88
<0.1



912985
23
61
81
87
0.2



912988
65
83
86
89
<0.1

















TABLE 39







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
72
95
97
99
<0.1



912874
78
90
96
97
<0.1



912875
64
83
92
94
<0.1



912886
49
78
85
92
<0.1



912931
68
88
94
95
<0.1



912934
57
83
90
92
<0.1



912936
50
78
89
89
<0.1



912938
57
73
85
87
<0.1



912943
64
84
90
93
<0.1



912954
80
92
93
94
<0.1



912970
44
73
86
90
<0.1



912986
56
78
91
92
<0.1



912987
79
90
92
88
<0.1



912992
21
59
74
81
0.3



915603
50
88
96
98
<0.1



915623
81
96
98
98
<0.1



915643
67
89
94
96
<0.1



916602
79
92
95
96
<0.1



916642
44
83
91
93
<0.1

















TABLE 40







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
73
94
98
99
<0.1



915484
67
87
93
95
<0.1



915543
34
69
87
90
0.1



915604
54
78
91
95
<0.1



915763
63
80
87
87
<0.1



915904
50
83
92
94
<0.1



915923
63
74
82
87
<0.1



916183
33
78
89
91
0.1



916303
58
73
84
91
<0.1



916343
15
72
76
87
0.2



916563
46
74
90
95
<0.1



916582
48
74
89
91
<0.1



916623
64
81
91
94
<0.1



916702
45
70
78
79
<0.1



916761
46
75
85
88
<0.1



916781
55
79
86
87
<0.1



916782
62
87
91
93
<0.1



916802
66
88
94
91
<0.1



916822
29
72
83
87
0.1

















TABLE 41







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
72
95
98
99
<0.1



915525
51
76
88
84
<0.1



915546
39
79
90
94
0.1



915605
59
84
96
96
<0.1



915606
74
94
99
98
<0.1



915625
72
82
91
95
<0.1



915944
36
71
75
83
0.1



916065
36
62
78
79
0.1



916144
71
86
90
92
<0.1



916163
36
67
81
74
0.1



916164
82
88
89
92
<0.1



916184
60
79
87
89
<0.1



916304
46
65
80
84
0.1



916324
57
77
87
92
<0.1



916344
41
70
83
88
0.1



916564
38
66
88
94
0.1



916604
67
87
95
96
<0.1



916624
43
59
79
87
0.1



916803
67
84
93
92
<0.1

















TABLE 42







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
70
94
98
99
<0.1



915486
35
64
82
90
0.1



915487
62
89
94
95
<0.1



915626
67
83
92
94
<0.1



915786
65
84
88
88
<0.1



916145
53
66
85
87
<0.1



916146
62
77
86
86
<0.1



916165
71
86
89
88
<0.1



916166
71
83
87
88
<0.1



916305
57
86
90
92
<0.1



916306
86
96
98
98
<0.1



916325
59
78
83
86
<0.1



916345
21
47
67
73
0.4



916545
63
88
95
94
<0.1



916546
66
85
92
95
<0.1



916625
47
71
84
92
<0.1



916706
22
65
80
85
0.2



916765
67
85
92
93
<0.1



916845
38
71
80
87
0.1

















TABLE 43







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
97
99
100
100
<0.1



915608
66
91
97
97
<0.1



915609
71
97
99
99
<0.1



915627
0
26
53
62
1.3



915768
39
69
86
91
0.1



915908
49
70
80
85
<0.1



915987
47
60
75
78
0.1



916008
45
69
84
83
<0.1



916187
71
82
88
92
<0.1



916247
34
72
83
84
0.1



916287
31
70
90
91
0.1



916547
79
93
97
97
<0.1



916566
8
45
73
81
0.5



916586
47
67
89
91
<0.1



916587
48
81
90
94
<0.1



916606
18
64
87
90
0.2



916607
72
94
94
95
<0.1



916627
18
51
82
79
0.3



916805
18
65
78
81
0.2

















TABLE 44







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells










PNPLA3 % Inhibition













Compound
62.5
250
1,000
4,000
IC50


Number
nM
nM
nM
nM
(μM)















912759
64
92
97
99
<0.1


915610
74
94
98
99
<0.1


915789
35
72
82
85
0.1


915909
52
69
82
86
<0.1


915929
13
32
60
59
1.0


915969
39
54
74
74
0.2


915989
46
67
81
86
0.1


916069
24
59
75
56
0.3


916148
42
71
85
80
<0.1


916168
28
54
74
68
0.3


916188
22
42
72
70
0.4


916309
30
77
91
96
0.1


916348
41
57
65
73
0.1


916549
64
85
94
96
<0.1


916568
54
66
81
87
<0.1


916569
60
86
92
95
<0.1


916728
22
50
68
73
0.4


916788
60
89
94
96
<0.1


916848
49
75
89
95
<0.1
















TABLE 45







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
81
93
98
100
<0.1



915390
9
40
67
77
0.6



915611
46
80
93
88
<0.1



915630
52
69
81
82
<0.1



915910
44
64
79
80
0.1



915931
83
88
89
86
<0.1



916149
73
87
89
83
<0.1



916150
51
68
77
84
<0.1



916189
60
73
77
79
<0.1



916310
45
77
88
95
<0.1



916330
48
67
84
86
<0.1



916550
62
85
94
97
<0.1



916570
89
96
98
98
<0.1



916629
26
53
73
86
0.3



916630
52
68
87
91
<0.1



916670
43
77
78
85
<0.1



916730
61
74
82
86
<0.1



916768
35
57
67
72
0.2



916789
79
92
96
96
<0.1

















TABLE 46







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
76
94
96
99
<0.1



915532
31
66
82
92
0.1



915612
54
77
86
90
<0.1



915732
42
63
80
84
0.1



915932
45
71
88
89
<0.1



915951
26
58
71
74
0.3



915991
67
84
85
85
<0.1



915992
54
78
86
87
<0.1



916112
35
67
76
78
0.1



916151
51
79
87
90
<0.1



916311
36
70
81
87
0.1



916331
56
85
93
95
<0.1



916332
82
91
94
96
<0.1



916390
30
41
68
64
0.5



916552
79
93
96
97
<0.1



916571
53
78
90
94
<0.1



916631
48
77
86
90
<0.1



916651
81
89
94
95
<0.1



916711
37
66
85
91
0.1

















TABLE 47







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
58
90
98
99
<0.1



915474
51
79
90
93
<0.1



915493
48
58
83
80
0.1



915494
46
73
86
90
<0.1



915674
49
72
89
93
<0.1



915933
40
63
75
79
0.1



916153
68
86
89
91
<0.1



916172
85
89
87
87
<0.1



916173
81
90
91
88
<0.1



916292
64
83
92
92
<0.1



916312
60
84
91
92
<0.1



916333
75
92
96
96
<0.1



916572
29
62
79
88
0.2



916592
52
74
89
90
<0.1



916593
25
67
83
93
0.2



916613
46
75
89
92
<0.1



916652
65
83
91
88
<0.1



916672
73
89
93
90
<0.1



916772
50
61
83
89
<0.1

















TABLE 48







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
50
89
96
99
<0.1



915534
0
33
66
57
1.1



915535
51
81
92
96
<0.1



915634
18
67
79
84
0.2



915635
44
72
86
91
0.1



915675
36
68
82
90
0.1



915735
45
68
73
84
0.1



915936
36
67
78
83
0.1



915995
78
87
90
89
<0.1



915996
83
91
93
92
<0.1



916174
80
84
86
81
<0.1



916175
55
82
86
89
<0.1



916334
50
82
92
94
<0.1



916335
52
76
89
93
<0.1



916575
62
88
93
93
<0.1



916753
49
69
76
74
<0.1



916774
49
72
86
91
<0.1



916794
26
64
85
85
0.2



916873
16
49
72
82
0.4

















TABLE 49







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
57
90
97
99
<0.1



915477
47
65
88
93
0.1



915478
47
78
90
95
<0.1



915497
63
68
79
86
<0.1



915637
67
91
97
98
<0.1



916037
15
47
70
61
0.6



916236
80
87
90
88
<0.1



916336
52
67
81
87
<0.1



916576
50
76
89
93
<0.1



916596
55
82
93
94
<0.1



916636
42
71
87
90
0.1



916637
56
85
90
93
<0.1



916715
27
38
68
68
0.5



916716
35
77
89
93
0.1



916796
14
62
84
89
0.3



916814
22
44
70
79
0.4



916815
56
79
87
89
<0.1



916816
33
72
83
93
0.1



916874
5
34
61
70
0.8

















TABLE 50







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
56
91
97
100
<0.1



915479
38
70
89
94
0.1



915618
42
63
75
85
0.1



915619
65
87
96
97
<0.1



915638
31
64
80
82
0.2



915639
33
78
88
93
0.1



915778
41
50
78
87
0.2



916058
26
34
73
81
0.4



916177
38
55
83
82
0.1



916238
84
91
93
93
<0.1



916298
79
87
92
94
<0.1



916318
59
71
91
94
<0.1



916338
71
91
94
92
<0.1



916558
73
89
94
94
<0.1



916577
41
66
78
82
0.1



916578
69
85
91
93
<0.1



916638
46
84
90
92
<0.1



916757
33
60
82
88
0.2



916817
31
67
82
87
0.1

















TABLE 51







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells










PNPLA3 % Inhibition













Compound
62.5
250
1,000
4,000
IC50


Number
nM
nM
nM
nM
(μM)















841947
50
78
89
92
<0.1


912759
75
50
85
99
<0.1


912986
54
78
90
95
<0.1


915480
61
87
94
97
<0.1


915519
47
77
86
85
<0.1


915620
46
75
88
91
<0.1


915780
24
76
92
94
0.1


915920
23
65
79
82
0.2


916020
45
80
85
81
<0.1


916299
59
87
92
93
<0.1


916339
88
95
97
97
<0.1


916340
83
96
97
98
<0.1


916559
41
68
83
89
0.1


916579
71
86
96
96
<0.1


916580
59
90
95
96
<0.1


916618
10
47
70
78
0.5


916639
38
58
80
86
0.1


916778
68
87
92
94
<0.1


916818
60
77
90
91
<0.1
















TABLE 52







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
62.5
250
1,000
4,000
IC50



Number
nM
nM
nM
nM
(μM)


















912759
60
0
85
99
0.3



915541
48
71
89
91
<0.1



915542
50
72
86
93
<0.1



915601
8
53
84
84
0.3



915602
1
56
77
91
0.4



915621
21
54
75
80
0.3



915622
0
44
73
84
0.5



915922
27
64
79
85
0.2



916042
6
57
89
88
0.3



916140
43
82
90
89
<0.1



916141
72
88
93
91
<0.1



916180
33
62
69
83
0.2



916181
53
80
89
92
<0.1



916341
0
78
94
94
0.3



916560
72
91
95
94
<0.1



916581
38
76
91
91
0.1



916601
44
80
88
90
<0.1



916701
61
83
91
93
<0.1



916780
75
91
93
94
<0.1

















TABLE 53







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
15.6
62.5
250
1,000
IC50



Number
nM
nM
nM
nM
(μM)


















915609
52
86
96
99
<0.01



959430
42
76
90
95
<0.01



959440
39
73
92
98
0.02



959470
46
73
89
94
<0.01



959670
52
90
96
98
<0.01



959680
50
75
91
96
<0.01



959730
83
96
98
98
<0.01



959740
50
70
90
96
<0.01



959820
40
69
85
92
0.02



959830
46
69
93
97
0.02



959880
34
62
85
93
0.03



960010
48
78
92
95
<0.01

















TABLE 54







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells










PNPLA3 % Inhibition













Compound
15.6
62.5
250
1,000
IC50


Number
nM
nM
nM
nM
(μM)















915609
53
87
98
99
<0.01


959271
55
74
91
91
<0.01


959360
7
43
79
85
0.1


959361
60
87
93
94
<0.01


959411
56
76
91
94
<0.01


959441
50
81
93
97
<0.01


959460
0
29
75
90
0.2


959701
62
91
97
98
<0.01


959721
80
94
97
97
<0.01


959731
25
64
82
91
0.05


959741
41
65
83
91
0.02


959750
0
26
65
87
0.2


959761
28
60
84
91
0.05


959781
39
58
75
87
0.04


959911
20
54
78
90
0.1


959921
37
61
83
91
0.03


959931
48
72
89
92
<0.01


959960
11
51
79
90
0.1


959961
38
64
85
92
0.03
















TABLE 55







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
15.6
62.5
250
1,000
IC50



Number
nM
nM
nM
nM
(μM)


















915609
11
71
93
98
0.1



959412
52
77
90
94
<0.01



959413
34
82
95
97
0.02



959422
15
50
80
87
0.1



959432
33
60
86
95
0.04



959662
0
53
84
92
0.1



959672
54
85
95
97
<0.01



959673
18
62
88
95
0.1



959682
46
77
90
91
<0.01



959702
39
71
91
96
0.02



959703
81
96
99
99
<0.01



959712
4
30
75
92
0.1



959713
0
53
86
96
0.1



959722
33
80
90
94
0.02



959733
31
68
92
96
0.03



959782
35
63
86
94
0.03



959872
29
64
77
89
0.04



959912
25
69
89
92
0.04



959982
21
61
83
91
0.1

















TABLE 56







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
15.6
62.5
250
1,000
IC50



Number
nM
nM
nM
nM
(μM)


















915609
2
73
93
98
0.1



959363
49
82
91
91
<0.01



959393
38
71
87
95
0.02



959394
27
73
91
97
0.03



959414
69
94
98
99
<0.01



959664
51
77
95
98
<0.01



959674
43
74
95
98
0.02



959683
14
71
90
96
0.05



959704
57
92
98
99
<0.01



959724
0
68
90
95
0.1



959734
71
93
98
98
<0.01



959814
24
76
90
95
0.03



959873
38
53
83
90
0.04



959874
51
82
95
97
<0.01



959884
44
77
94
97
<0.01



959913
18
50
85
92
0.1



959953
6
51
85
92
0.1



959983
22
54
81
92
0.1



960004
10
71
92
96
0.1

















TABLE 57







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
15.6
62.5
250
1,000
IC50



Number
nM
nM
nM
nM
(μM)


















915609
12
69
93
98
0.1



959364
32
72
89
91
0.03



959415
21
70
91
96
0.04



959444
6
47
82
91
0.1



959445
32
70
92
97
0.03



959455
61
87
95
97
<0.01



959675
20
56
80
94
0.1



959684
8
47
83
86
0.1



959705
77
95
98
99
<0.01



959735
12
67
90
95
0.1



959764
4
32
80
92
0.1



959765
1
59
88
93
0.1



959784
3
35
75
90
0.1



959785
27
72
92
96
0.03



959794
0
0
53
83
0.3



959864
26
61
84
91
0.05



959885
49
81
95
96
<0.01



959914
7
43
76
89
0.1



959964
17
55
83
91
0.1

















TABLE 58







Multi-dose assay of 3-10-3 cEt gapmers in A431 cells












PNPLA3 % Inhibition















Compound
15.6
62.5
250
1,000
IC50



Number
nM
nM
nM
nM
(μM)


















915609
0
73
95
97
0.1



959456
66
90
97
98
<0.01



959666
29
60
89
97
0.04



959676
15
44
81
93
0.1



959686
71
92
97
97
<0.01



959695
40
75
91
93
0.02



959696
21
81
90
92
0.03



959706
81
95
98
98
<0.01



959725
8
55
76
84
0.1



959726
0
59
88
91
0.1



959736
46
84
94
98
<0.01



959766
22
57
83
94
0.1



959776
1
53
87
93
0.1



959815
31
67
89
91
0.03



959865
6
49
84
91
0.1



959875
34
74
91
92
0.02



959935
22
55
84
94
0.1



959955
0
55
83
89
0.1



959985
29
71
88
93
0.03

















TABLE 59







Multi-dose assay of 3-10-3 cEt gapmers


in A431 cells










PNPLA3 % Inhibition













Compound
15.6
62.5
250
1,000
IC50


Number
nM
nM
nM
nM
(μm)















915609
37
80
96
99
0.02


959356
40
71
87
88
0.02


959417
25
58
83
92
0.1


959437
65
88
94
95
<0.01


959667
37
69
90
95
0.02


959677
29
56
82
92
0.05


959687
51
79
93
97
<0.01


959697
51
75
93
95
<0.01


959707
81
94
98
98
<0.01


959727
71
92
96
96
<0.01


959737
45
75
89
94
<0.01


959767
47
76
93
96
<0.01


959797
32
59
87
94
0.04


959856
13
35
67
80
0.1


959876
38
75
89
90
0.02


959877
40
81
89
94
<0.01


959956
25
25
66
85
0.1


960006
13
40
68
83
0.1


960007
24
59
88
91
0.05









Example 3: Tolerability of Modified Oligonucleotides Targeting Human PNPLA3 in BALB/c Mice
BALB/c mice are a multipurpose mouse model frequently utilized for safety and efficacy testing. The mice were treated with antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Ionis oligonucleotides selected from the studies above were conjugated with 3′-THA-C6-GalNAC3-(3R,5S)-5-(hydroxymethyl) pyrrolidin-3-ol phosphate endcap (henceforth referred to as 3′-THA).
Treatment
Groups of 6- to 7-week-old male mice were injected subcutaneously once with 200 mg/kg of modified oligonucleotides. One group of male BALB/c mice was injected with PBS. Mice were euthanized 72-96 hours after the single dose and plasma was harvested for further analysis.
To evaluate the effect of modified oligonucleotides on liver function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). Modified oligonucleotides that caused changes in the levels of transaminases outside the expected range for antisense oligonucleotides were excluded in further studies. The oligonucleotides which were considered tolerable in this study and were selected for further evaluation are presented in the Table below. ‘Parent Oligo’ indicates the Ionis oligonucleotide that has been described in the studies above and that was conjugated with 3′-THA and tested in this study.







TABLE 60







Antisense oligonucleotides


in BALB/c mouse study










Compound
Parent oligo



ID
ID







975746
916339



975747
912941



975748
916306



975755
916332



975760
912848



975764
916298



975766
916552



975767
916789



975768
916602



975770
912874



975771
916333



975772
916780



975775
916672



975777
916558



975780
916607



975783
916338



975788
912847



975790
916778



975792
912870



975794
916802



975797
916637



975799
912732



975800
912733



975803
912813



975804
912823



975805
912834



975806
912855



975807
912856



975808
912864



975809
912871



975810
912872



975811
912875



975813
912931



975814
912934



975815
912936



975816
912938



975817
912943



975820
912988



975822
915486



975829
915619



975836
915780



975840
915989



975844
916151



975849
916292



975850
916299



975851
916303



975852
916309



975853
916310



975854
916312



975855
916318



975856
916324



975857
916331



975858
916334



975859
916335



975860
916336



975861
916549



975862
916550



975864
916563



975865
916564



975866
916568



975868
916571



975869
916575



975870
916580



975871
916581



975873
916586



975875
916601



975878
916624



975879
916625



975880
916636



975881
916638



975883
916670



975886
916711



975887
916716



975888
916774



975889
916781



975890
916782



975891
916788



975893
916815



975894
916816



975895
916817



975896
916818



975897
916822



975898
916845



994288
959455



994289
960010



994290
959361



994291
959271










Example 4: Effect of Antisense Inhibition of PNPLA3 in Transgenic Mouse Model
A PNPLA3 transgenic mouse model from wild-type C57BL/6 generated by the University of California, Irvine was used. The mouse model comprises a genomic construct containing the entire PNPLA3 gene fosmid, generously provided by the University of Washington. The efficacy of Ionis oligonucleotides was evaluated in this model.
Treatment
Transgenic mice were maintained on a 12-hour light/dark cycle and were fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
The hPNPLA3 Tg mice were divided into groups of 2 mice each. Groups received subcutaneous injections of Ionis oligonucleotide at a dose of 2.5 mg/kg on days 1 and 8. One group of 4 mice received subcutaneous injections of PBS on days 1 and 8. The saline-injected group served as the control group to which oligonucleotide-treated groups were compared.
RNA Analysis
On day 10, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 and RTS36075 were both used to measure PNPLA3 mRNA levels. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As presented in the Table below, treatment with Ionis antisense oligonucleotides resulted in significant reduction of PNPLA3 mRNA in comparison to the PBS control. ‘0’ indicates that the oligonucleotides did not inhibit mRNA expression.







TABLE 61







Percent inhibition of PNPLA3 mRNA in the 


transgenic mice liver relative to the PBS control










Inhibition
Inhibition



(%)
(%)



measured
measured


Compound
with
with


ID
RTS36070
RTS36075












975746
99
99


975747
99
99


975748
98
98


975755
99
99


975760
96
97


975764
75
83


975766
99
99


975767
98
98


975768
98
98


975770
97
97


975771
98
99


975772
96
96


975775
90
91


975777
85
89


975780
44
63


975783
87
90


975788
0
26


975790
0
0


975792
9
34


975794
44
50


975797
0
0


975799
0
0


975800
0
5


975803
68
68


975804
11
38


975805
0
0


975806
0
0


975807
0
0


975808
47
58


975809
0
19


975810
12
22


975811
19
32


975813
36
39


975814
48
54


975815
78
77


975816
56
56


975817
84
86


975820
35
45


975822
0
0


975829
98
98


975836
85
91


975840
19
44


975844
21
31


975849
88
89


975850
41
48


975851
5
18


975852
24
41


975853
0
0


975854
0
0


975855
0
0


975856
45
31


975857
73
67


975858
58
40


975860
92
92


975861
66
49


975862
46
36


975864
16
21


975865
0
0


975866
40
41


975868
56
48


975869
30
19


975870
0
14


975871
0
0


975875
75
73


975878
18
12


975879
7
0


975880
0
0


975881
54
54


975883
77
80


975886
18
28


975887
49
57


975888
10
9


975889
90
91


975890
96
98


975891
97
98


975893
95
95


975894
85
87


975895
89
89


975896
91
89


975898
94
95


975897
96
97


975873
99
99


994288
99
99


994289
98
99


994290
98
99


994291
95
95


975859
95
96









Example 5: Tolerability of Modified Oligonucleotides Targeting Human PNPLA3 in CD1 Mice
CD1 ® mice (Charles River, MA) are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with Ionis antisense oligonucleotides selected from studies described above and evaluated for changes in the levels of various plasma chemistry markers.
Ionis oligonucleotides selected from the studies above were conjugated with 5′-Trishexylamino-(THA)-C6GalNAC3 endcap (henceforth referred to as 5′-THA). The Ionis oligonucleotides tested are presented in the Table below. ‘Unconjugated parent ION No.’ refers to the Ionis oligonucleotide described in the in vitro studies above with the same sequence. ‘3′-THA counterpart ION No.’ refers to the 3′-THA conjugated oligonucleotide with the same sequence and evaluated in the mice studies above.







TABLE 62







5′-THA oligonucleotides tested in CD1 mice tolerability study












Unconjugated
3′-THA

SEQ


Compound
parent ION
counterpart

ID


ID
No.
ION No
Sequence
NO





975591
916339
975746
GGATATATTGGGCTCA
1512


 


975592
912941
975747
TTGCATTGCATAGCCT
 182


 


975593
916306
975748
GTGTACTTTAGGCTCC
 598


 


975600
916332
975755
CACAATGACATCATGG
1020


 


975605
912848
975760
CGTTTTTAGTAGTCAA
 141


 


975611
916552
975766
CCTTTTATTTCCGTTA
1024


 


975612
916789
975767
GTAATATTCAGACCAG
 899


 


975613
916602
975768
CTAGTAAATGCTTGTC
 330


 


975615
912874
975770
ATACTTTTGGCAAGGC
 217


 


975616
916333
975771
CTTTATTCAATGTGGC
1089


 


975617
916780
975772
AGAAATTGCAGTGCCC
1665


 


975674
915619
975829
GACTTTAGGGCAGATG
1400


 


975704
916335
975859
TAATTCTACCTGTGTC
1227


 


975718
916586
975873
AACTTTGCAGCCTATC
 605


 


975735
916782
975890
CTTAGAAATTGCAGTG
 408


 


975736
916788
975891
TAATATTCAGACCAGG
 830


 


975738
916815
975893
CAATTCTAGACATGGC
1313


 


975742
916822
975897
TATGACATTTCAGAGT
 410


 


975743
916845
975898
GTAAAGATGTGAGTGA
 618


 


994282
959455
994288
TTGGATATATTGGGCT
1982


 


994283
960010
994289
AGACATATGACATTTC
1745


 


994284
959361
994290
TTTTTAGTAGTCAAGG
1757


 


994285
959271
994291
GTTGAAGGATGGATGG
1748









Treatment

Groups of four CD1 mice each were weekly injected subcutaneously with 15 mg/kg of Ionis oligonucleotides for 6 weeks, with one loading dose at day 4 (total 8 doses). One group of male CD1 mice was injected subcutaneously for 6 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
To evaluate the effect of Ionis oligonucleotides on liver and kidney function, plasma levels of transaminases (ALT and AST), albumin, total bilirubin, and creatinine were measured at week 3 using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). The results are presented in the Table below. Ionis oligonucleotides that caused changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides were excluded in further studies.







TABLE 63







Plasma chemistry marker levels in CD1 mice at week 3
















Total




Albumin
ALT
AST
bilirubin
Creatinine



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















PBS
2.9
31
64
0.4
0.1


975611
2.7
640
385
0.3
0.1


994282
2.4
76
83
0.3
0.1


975592
3.0
786
942
0.5
0.1


975600
2.7
334
431
0.3
0.1


975591
2.6
62
115
0.4
0.1


975718
2.4
1717
2183
1.2
0.1


994284
2.7
41
97
0.3
0.1


994283
2.8
216
154
0.3
0.1


975616
3.0
69
137
0.3
0.1


975612
2.7
47
218
0.4
0.1


975674
2.9
134
114
0.4
0.1


975613
2.8
60
277
0.3
0.1


975593
2.7
429
405
0.4
0.1


975736
2.9
46
63
0.2
0.2


975735
2.5
46
79
0.2
0.1


975742
2.6
152
96
0.2
0.1


975615
2.9
207
189
0.4
0.1


975617
2.9
65
70
0.3
0.1


975605
2.9
67
92
0.3
0.1


975704
2.4
33
61
0.2
0.1


975738
2.6
43
67
0.2
0.1


975743
2.9
119
126
0.4
0.1


994285
2.8
400
353
0.2
0.1









Hematology Assays

Blood obtained from selected mouse groups at week 6 were sent to IDEXX BioResearch for measurement of platelet count. The results are presented in the tables below. Ionis oligonucleotides that caused changes in the platelet count outside the expected range for antisense oligonucleotides were excluded in further studies.







TABLE 64







Platelet count in


CD1 mice











Platelet




(×103/μL)














PBS
1067



975605
1202



975612
1200



975613
1417



975616
1178



975617
922



975674
618



975591
941



975743
1127



994282
1384



994284
1255



975704
939



975735
1039



975736
1116



975738
1126



975742
808










Example 6: Tolerability of Modified Oligonucleotides Targeting Human PNPLA3 in Sprague-Dawley Rats
Sprague-Dawley rats are a multipurpose model used for safety and efficacy evaluations. The rats were treated with Ionis antisense oligonucleotides from the studies described in the Examples above and evaluated for changes in the levels of various plasma chemistry markers.
Treatment
Male Sprague-Dawley rats were maintained on a 12-hour light/dark cycle and fed ad libitum with Purina normal rat chow, diet 5001. Groups of 4 Sprague-Dawley rats each were weekly injected subcutaneously with 15 mg/kg of Ionis oligonucleotide for 6 weeks, with one loading dose on day 4 (total 8 doses). Forty eight hours after the last dose, rats were euthanized and organs and plasma were harvested for further analysis.
Plasma Chemistry Markers
To evaluate the effect of Ionis oligonucleotides on hepatic function, plasma levels of transaminases were measured using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). Plasma levels of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in the Table below expressed in IU/L. Plasma levels of bilirubin, creatinine, albumin, and BUN were also measured using the same clinical chemistry analyzer and the results are also presented in the Table below expressed in mg/dL. Ionis oligonucleotides that caused changes in the levels of any markers of liver function outside the expected range for antisense oligonucleotides were excluded in further studies.







TABLE 65







Plasma chemistry markers in Sprague-Dawley rats

















Total





Albumin
ALT
AST
bilirubin
Creatinine
BUN



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
















PBS
3
35
81
0.2
0.2
12


975591
3
57
161
0.2
0.3
14


975605
4
62
176
0.3
0.2
14


975612
3
106
153
0.2
0.3
13


975613
3
32
94
0.2
0.2
12


975616
4
31
106
0.2
0.3
13


975617
3
49
263
0.2
0.2
12


975735
3
44
128
0.2
0.2
14


975736
3
73
293
0.3
0.3
14


994282
3
41
135
0.1
0.3
12


994284
3
32
95
0.1
0.2
13









Kidney Function

To evaluate the effect of Ionis oligonucleotides on kidney function, urinary levels of protein and creatinine were measured using an automated clinical chemistry analyzer (Beckman Coulter AU480, Brea, CA). The ratios of total protein to creatinine are presented in the Table below. Ionis oligonucleotides that caused changes in the levels of the ratio outside the expected range for antisense oligonucleotides were excluded in further studies.







TABLE 66





Total protein to creatinine 


ratio in Sprague-Dawley rats


















PBS
1.5



975591
2.0



975605
1.6



975612
1.9



975613
2.3



975616
2.0



975617
1.4



975735
2.2



975736
1.1



994282
2.1



994284
2.1










Organ Weights

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







TABLE 67







Organ weights (g)













Liver
Kidney
Spleen
















Saline
16
3
1



975591
16
4
1



975605
21
3
1



975612
12
3
1



975613
16
3
1



975616
15
3
1



975617
19
4
2



975735
14
4
1



975736
15
3
1



994282
14
3
1



994284
15
3
1










Example 7: Effect of Antisense Inhibition of PNPLA3 in Transgenic Mouse Model
Ionis oligonucleotides were tested in a multi-dose assay in the hPNPLA3 Tg model.
Treatment
Transgenic mice were maintained on a 12-hour light/dark cycle and were fed ad libitum normal Purina mouse chow. Animals were acclimated for at least 7 days in the research facility before initiation of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.
Study 1
The hPNPLA3 Tg mice were divided into groups of 4 mice each. Groups received subcutaneous injections of Ionis oligonucleotide at a weekly dose of 5 mg/kg, 1 mg/kg, or 0.25 mg/kg administered on days 1, 5, 8, 15, and 23. One group of 4 mice received subcutaneous injections of PBS on days 1, 5, 8, 15, and 23. The saline-injected group served as the control group to which oligonucleotide-treated groups were compared.
RNA Analysis
On day 26, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 and RTS36075 were both used to measure PNPLA3 mRNA levels. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN©. As presented in the Table below, treatment with Ionis antisense oligonucleotides resulted in significant dose-dependent reduction of PNPLA3 mRNA in comparison to the PBS control.







TABLE 68







Percent inhibition of PNPLA3 mRNA in the transgenic


mice liver relative to the PBS control















Inhibition
Inhibition






measured
measured






by
by
EC50 




mg/kg/wk
RTS36070
RTS36075
(μg/g)

















975605
5
93
90
2.2




1
66
57





0.25
45
46




975612
5
98
99
3.1




1
89
88





0.25
34
44




975613
5
98
97
1.0




1
87
85





0.25
58
56




975616
5
93
93
0.5




1
85
87





0.25
60
63




975617
5
97
97
0.3




1
76
78





0.25
55
53




975735
5
97
98
1.5




1
74
75





0.25
29
33




975736
5
98
98
0.9




1
73
71





0.25
44
45




994282
5
98
98
0.2




1
91
80





0.25
62
58




994284
5
99
100
0.3




1
89
88





0.25
53
47










Study 2

The hPNPLA3 Tg mice were divided into groups of 4 mice each. Groups received subcutaneous injections of Ionis oligonucleotide at a weekly dose of 5 mg/kg, 2.5 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.25 mg/kg administered on days 1, 5, 8, 15, and 23. One group of 4 mice received subcutaneous injections of PBS on days 1, 5, 8, 15, and 23. The saline-injected group served as the control group to which oligonucleotide-treated groups were compared.
RNA Analysis
On day 26, RNA was extracted from liver for real-time PCR analysis of measurement of mRNA expression of PNPLA3. Primer probe sets RTS36070 and RTS36075 were both used to measure PNPLA3 mRNA levels. Results are presented as percent change of mRNA, relative to PBS control, normalized with RIBOGREEN®. As presented in the Table below, treatment with Ionis antisense oligonucleotides resulted in significant dose-dependent reduction of PNPLA3 mRNA in comparison to the PBS control.







TABLE 69







Percent inhibition of PNPLA3 mRNA in the transgenic


mice liver relative to the PBS control














Inhibition
Inhibition






measured
measured






by
by
EC50 
EC90 



mg/kg/wk
RTS36070
RTS36075
(μg/g)
(μg/g)















975612
5
96
97
1.0
8.6



2.5
98
98





1
95
96





0.5
82
83





0.25
43
44




975613
5
99
99
0.9
7.7



2.5
99
99





1
91
91





0.5
82
83





0.25
69
74




975616
5
96
96
1.0
9.4



2.5
94
93





1
89
89





0.5
81
81





0.25
73
60









Example 8: Effect of Modified Oligonucleotides Targeting Human PNPLA3 in Cynomolgus Monkeys
Cynomolgus monkeys were treated with Ionis antisense oligonucleotides selected from studies described in the Examples above. Antisense oligonucleotide tolerability was evaluated.
Treatment
Prior to the study, the monkeys were kept in quarantine during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed 2-4 kg. Nine groups of 5 randomly assigned male cynomolgus monkeys each were injected subcutaneously with Ionis oligonucleotide or PBS in a clock-wise rotation between four different sites on the back. The monkeys were dosed twice per week (days 1, 5, 9, and 14) for the first 2 weeks, and then subsequently once a week for 10 weeks with 10 mg/kg of Ionis oligonucleotide on days 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84. A control group of 5 cynomolgus monkeys was injected with PBS in a similar manner and served as the control group.
During the study period, the monkeys were observed twice daily for signs of illness or distress. Any animal experiencing more than momentary or slight pain or distress due to the treatment, injury or illness was treated by the veterinary staff with approved analgesics or agents to relieve the pain after consultation with the Study Director. Any animal in poor health or in a possible moribund condition was identified for further monitoring and possible euthanasia. Scheduled euthanasia of the animals was conducted on day 86 approximately 48 hours after the last dose by exsanguination while under deep anesthesia. The protocols described in the Example were approved by the Institutional Animal Care and Use Committee (IACUC).
Body and Organ Weight Measurements
To evaluate the effect of Ionis oligonucleotides on the overall health of the animals, body and organ weights were measured. Body weights and organ weights were measured on day 86 and the data is presented in the Table below. The results indicate that effect of treatment with antisense oligonucleotides on body and organ weights was within the expected range for antisense oligonucleotides. Specifically, treatment with ION 945616 was well tolerated in terms of the body and organ weights of the monkeys.







TABLE 70







Final body and organ weights in cynomolgus monkey














Body 


Liver with




Wt
Spleen
Kidney
gallbladder




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

















PBS Control
2797
2.6
13.1
53



994284
2789
3.3
14.7
69



975605
2685
4.1
12.2
58



975616
2868
3.1
12.9
63



994282
2782
4.4
12.1
62



975613
2704
3.0
13.5
60



975617
2761
3.8
14.1
61



975735
2765
4.1
15.5
67



975736
2844
3.0
14.1
66



975612
2711
2.8
13.2
60










Liver Function

To evaluate the effect of Ionis oligonucleotides on hepatic function, blood samples were collected from all the study groups on day 86. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged at 3000 rpm for 10 minutes to obtain serum. Levels of various liver function markers were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Plasma levels of ALT and AST were measured and the results are presented in the Table below, expressed in IU/L. Bilirubin, a liver function marker, was similarly measured and is presented in the Table below, expressed in mg/dL. The results indicate that antisense oligonucleotides had no effect on liver function outside the expected range for antisense oligonucleotides.







TABLE 71







Liver function markers in cynomolgus monkey plasma














ALT
AST
Bilirubin
Albumin




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

















PBS Control
38
55
0.2
4.3



994284
64
48
0.2
3.7



975605
48
54
0.3
4.0



975616
54
57
0.3
3.9



994282
89
53
0.3
4.0



975613
60
71
0.4
4.0



975617
65
61
0.3
4.0



975735
59
79
0.3
4.1



975736
70
56
0.3
3.9



975612
61
66
0.3
3.9










Kidney Function

To evaluate the effect of Ionis oligonucleotides on kidney function, blood samples were collected from all the study groups on day 86. The monkeys were fasted overnight prior to blood collection. Blood was collected in tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 minutes and then centrifuged at 3000 rpm for 10 minutes to obtain serum. Levels of BUN and creatinine were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan). Results are presented in the Table below, expressed in mg/dL.
The plasma chemistry data indicate that most of the Ionis oligonucleotides did not have any effect on the kidney function outside the expected range for antisense oligonucleotides.







TABLE 72







Plasma BUN and creatinine levels


(mg/dL) in cynomolgus monkeys












BUN
Creatinine







PBS Control
23
0.8



994284
24
0.8



975605
27
0.7



975616
21
0.8



994282
24
0.8



975613
23
0.9



975617
21
0.7



975735
20
0.8



975736
23
0.8



975612
20
0.8










Hematology

To evaluate any effect of Ionis oligonucleotides in cynomolgus monkeys on hematologic parameters, blood samples of approximately 0.5 mL of blood was collected from each of the available study animals on day 86. The samples were collected in tubes containing K2-EDTA. Samples were analyzed for red blood cell (RBC) count, white blood cells (WBC) count, individual white blood cell counts, such as that of monocytes, neutrophils, lymphocytes, as well as for platelet count, hemoglobin content and hematocrit, using an ADVIA2120i hematology analyzer (Siemens, USA).
The data indicate the oligonucleotides did not cause any changes in hematologic parameters outside the expected range for antisense oligonucleotides at this dose.







TABLE 73







Blood cell counts in cynomolgus monkeys














RBC
Platelets
WBC
Neutrophils
Lymphocytes
Monocytes



(×106/μL)
(×103/μL)
(×103/μL)
(×103/μL)
(×103/μL)
(×103/μL)
















PBS Control
6.0
342
12
3.2
7.8
0.3


994284
6.0
410
10
2.7
6.7
0.3


975605
5.8
326
10
4.8
4.5
0.4


975616
6.0
362
10
3.4
5.8
0.3


994282
5.8
359
10
3.9
5.5
0.3


975613
5.5
327
8
2.6
5.5
0.2


975617
6.1
358
10
3.1
6.4
0.3


975735
5.9
241
13
5.4
6.6
0.4


975736
5.8
360
10
3.5
6.4
0.2


975612
6.2
421
11
5.1
5.7
0.2
















TABLE 74







Hematologic parameters in


cynomolgus monkeys












Hemoglobin
HCT




(g/dL)
(%)







PBS Control
14
49



994284
14
48



975605
14
46



975616
14
49



994282
14
47



975613
13
46



975617
14
49



975735
14
48



975736
14
48



975612
14
49










Pro-Inflammatory Proteins Analysis

To evaluate any inflammatory effect of Ionis oligonucleotides in cynomolgus monkeys, blood samples were taken for analysis. The monkeys were fasted overnight prior to blood collection. Approximately 1.5 mL of blood was collected from each animal and put into tubes without anticoagulant for serum separation. The tubes were kept at room temperature for a minimum of 90 min and then centrifuged at 3,000 rpm for 10 min at room temperature to obtain serum. C-reactive protein (CRP), which is synthesized in the liver and which serves as a marker of inflammation, and complement C3 were measured using a Toshiba 200FR NEO chemistry analyzer (Toshiba Co., Japan).
Example 9: Measurement of Viscosity of Antisense Oligonucleotides Targeting Human PNPLA3
The viscosity of select antisense oligonucleotides from the studies described above was measured with the aim of screening out antisense oligonucleotides which have a viscosity of more than 40 centipoise (cP). Oligonucleotides having a viscosity greater than 40 cP would 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 into solution by heating the vial at 50° C. Part (75 μL) of the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometer was set to 25° C. and the viscosity of the sample was measured. Another part (20 μL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UV instrument). The results are presented in the Table below, where the concentration of each antisense oligonucleotide was 200 mg/ml, and indicate that most of the antisense oligonucleotides solutions are optimal in their viscosity under the criterion stated above.







TABLE 75







Viscosity of antisense 


oligonucleotides at 200 mg/mL











Viscosity 




(cP)







994284
21



975605
19



975616
20



994282
30



975613
24



975617
22



975735
15



975736
49



975612
25










Example 10: Design of Oligonucleotides at the Site of ION 975616
Additional antisense oligonucleotides were designed targeting a PNPLA3 nucleic acid that overlap the target site of ION 916333, which is the unconjugated version of ION 975616, and with different chemical modifications and motifs.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers or deoxy, MOE, and cEt oligonucleotides. The 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt sugar modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. The deoxy, MOE and (S)-cEt oligonucleotides are 16 nucleosides in length wherein the nucleoside have either a MOE sugar modification, an (S)-cEt sugar modification, or a deoxy modification. The ‘Chemistry’ column describes the sugar modifications of each oligonucleotide. ‘k’ indicates an (S)-cEt sugar modification; ‘d’ indicates deoxyribose; the number after the ‘d’ indicates the number of deoxyribose; and ‘e’ indicates a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence (SEQ ID NO: 2).







TABLE 76







Modified oligonucleotides targeting human PNPLA3












Start
Stop

Compound

SEQ


Site
Site
Sequence
Number
Chemistry
ID NO





5599
5614
TCAATGTGGCTTCTAG
995553
kkk-d10-kkk
2170


 


5600
5615
TTCAATGTGGCTTCTA
959437
kkk-d10-kkk
2089


 


5601
5616
ATTCAATGTGGCTTCT
959438
kkk-d10-kkk
2171


 


5602
5617
TATTCAATGTGGCTTC
959439
kkk-d10-kkk
2172


 


5603
5618
TTATTCAATGTGGCTT
959440
kkk-d10-kkk
1705


 


5603
5618
TTATTCAATGTGGCTT
995696
k-d10-kekek
1705


 


5603
5618
TTATTCAATGTGGCTT
995906
kk-d9-eeekk
1705


 


5603
5618
TTATTCAATGTGGCTT
996116
kk-d9-ekeke
1705


 


5604
5619
TTTATTCAATGTGGCT
959441
kkk-d10-kkk
1765


 


5604
5619
TTTATTCAATGTGGCT
995697
k-d10-kekek
1765


 


5604
5619
TTTATTCAATGTGGCT
995907
kk-d9-eeekk
1765


 


5604
5619
TTTATTCAATGTGGCT
996117
kk-d9-ekeke
1765


 


5605
5620
CTTTATTCAATGTGGC
916333
kkk-d10-kkk
1089


 


5605
5620
CTTTATTCAATGTGGC
995698
k-d10-kekek
1089


 


5605
5620
CTTTATTCAATGTGGC
995908
kk-d9-eeekk
1089


 


5605
5620
CTTTATTCAATGTGGC
996118
kk-d9-ekeke
1089


 


5605
5620
CTTTATTCAATGTGGC
996277
kek-d9-eekk
1089


 


5606
5621
ACTTTATTCAATGTGG
916334
kkk-d10-kkk
1158


 


5606
5621
ACTTTATTCAATGTGG
995699
k-d10-kekek
1158


 


5606
5621
ACTTTATTCAATGTGG
995909
kk-d9-eeekk
1158


 


5606
5621
ACTTTATTCAATGTGG
996119
kk-d9-ekeke
1158


 


5607
5622
TACTTTATTCAATGTG
959442
kkk-d10-kkk
1825


 


5608
5623
TTACTTTATTCAATGT
959443
kkk-d10-kkk
1885









The oligonucleotides were tested in a series of experiments. Cultured A-431 cells at a density of 10,000 cells per well were treated using free uptake with modified oligonucleotides diluted to different concentrations. After a treatment period of approximately 48 hours, PNPLA3 mRNA levels were measured as previously described using the Human PNPLA3 primer-probe set RTS36070. PNPLA3 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The IC50 ratios of the assays are presented in the tables below, which is the ratio of the IC50 of a benchmark oligonucleotide to the IC50 of the oligonucleotide. Hence, a bigger value of the ratio indicates that the oligonucleotide is more active than the benchmark.







TABLE XX







Efficacy of modified oligonucleotides targeting


human PNPLA3











Start
Stop
Compound

IC50


Site
Site
Number
Chemistry
ratio





5600
5615
959437
kkk-d10-kkk
1.42


5601
5616
959438
kkk-d10-kkk
0.49


5602
5617
959439
kkk-d10-kkk
0.36


5603
5618
959440
kkk-d10-kkk
0.55


5603
5618
995906
kk-d9-eeekk
1.42


5604
5619
959441
kkk-d10-kkk
1.66


5605
5620
916333
kkk-d10-kkk
1.96


5605
5620
995908
kk-d9-eeekk
0.70


5606
5621
916334
kkk-d10-kkk
0.95


5606
5621
995909
kk-d9-eeekk
1.47

## Claims

1. A compound comprising a modified oligonucleotide 16 to 30 linked nucleosides in length having a nucleobase sequence comprising the nucleobase sequence SEQ ID NO: 1089, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the modified oligonucleotide is 16 linked nucleosides having a nucleobase sequence comprising the nucleobase sequence SEQ ID NO: 1089, or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, and/or at least one modified sugar, and/or at least one modified nucleobase.

4. The compound of claim 3, wherein the at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.

5. The compound of claim 3, wherein the at least one modified sugar is a bicyclic sugar.

6. The compound of claim 5, wherein the bicyclic sugar comprises a
a 4′-CH2—O—2′ group or a 4′-CH(CH3)—O—2′ group or a 4′-(CH2)2—O-2′ group.

7. The compound of claim 3, wherein the at least one modified sugar comprises a 2′-O(CH)2—OCH3 group or a 2′-O—CH3 group.

8. The compound of claim 3, wherein the at least one modified nucleobase is a 5-methylcytosine.

9. The compound of claim 1, wherein the modified oligonucleotide comprises:
a gap segment consisting of ten linked deoxynucleosides;
a 5′ wing segment consisting of three linked nucleosides; and
a 3′ wing segment consisting of three linked nucleosides; and
wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; wherein each cytosine is a 5-methylcytosine.

10. The compound of claim 1, wherein the compound further comprises a conjugate group.

11. The compound of claim 10, wherein the conjugate group is positioned at the 5′end of the modified oligonucleotide and is

12. The compound of claim 1, wherein the pharmaceutically acceptable salt is a sodium salt.

13. The compound of claim 1, wherein the pharmaceutically acceptable salt is a potassium salt.

14. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.