Rnai constructs for inhibiting pnpla3 expression and methods of use thereof

Abstract

To provide RNAi constructs for reducing expression of the PNPLA3 gene.SOLUTION: An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 contiguous nucleotides differing by no more than 3 nucleotides from a specific antisense sequence, and wherein the RNAi construct inhibits the expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3).SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to compositions and methods for regulating hepatic expression of patatin-like phospholipase domain-containing 3 (PNPLA3). In particular, the present invention relates to nucleic acid-based therapeutics for reducing PNPLA3 expression by RNA interference and methods of using such nucleic acid-based therapeutics for treating or preventing liver disease, such as non-alcoholic fatty liver disease (NAFLD). [Background technology]

[0002] Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the world, including various liver pathologies, and its prevalence has doubled in the past 20 years, and it is currently estimated that about 20% of the world's population is affected (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). NAFLD begins with the accumulation of triglycerides in the liver and is defined by the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes in individuals who 1) have no history of significant alcohol consumption and 2) have excluded the diagnosis of other types of liver disease (Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7). In some individuals, the accumulation of ectopic fat in the liver, called steatosis, induces inflammation and hepatocellular injury, leading to a more advanced stage of the disease called non-alcoholic steatohepatitis (NASH) (Non-Patent Document 6). As of 2015, it is estimated that 75 to 100 million Americans have NAFLD; NASH accounts for approximately 10 to 30% of NAFLD diagnoses (6, 8).

[0003] Patatin-like phospholipase domain-containing 3 (PNPLA3), previously known as adiponutrin (ADPN) and calcium-independent phospholipase A2-epsilon (iPLA(2)ε), is a type II transmembrane protein (Non-Patent Document 9, Non-Patent Document 10). It was first identified in adipocytes as a membrane-bound, fat-rich protein induced during adipogenesis in mice, but has now been shown to be expressed in other tissues, including the liver (Non-Patent Document 9, Non-Patent Document 11, Non-Patent Document 12, Non-Patent Document 13, Non-Patent Document 14, Non-Patent Document 15). In cell-free biochemical systems, recombinant PNPLA3 protein can exhibit either triacylglycerol lipase or transacylation activity (Non-Patent Document 10, Non-Patent Document 16, Non-Patent Document 17). In hepatocytes, PNPLA3 is expressed on the endoplasmic reticulum and lipid membranes and mainly exhibits triacylglycerol hydrolase activity (17, 18, 19, 20). Although lacking a secretory signal, data indicate that PNPLA3 is secreted and can be found in human plasma as disulfide bond-dependent multimers (21). Thus, novel therapeutic agents targeting PNPLA3 function represent a novel approach to reduce PNPLA3 levels and treat hepatological diseases such as non-alcoholic fatty liver disease. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Sattar et al. (2014)BMJ 349:g459 [Non-Patent Document 2] Loomba and Sanyal(2013)Nature Reviews Gastroenterology & hepatology 10(11):686-690 [Non-Patent Document 3] Kim and Kim(2017)Clin Gastroenterol Hepatol 15(4):474-485

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Summary of the Invention

Means for Solving the Problems

[0005] The present invention is based in part on the design and generation of RNAi constructs that target the PNPLA3 gene and reduce PNPLA3 expression in liver cells. Sequence-specific inhibition of PNPLA3 expression is useful for treating or preventing conditions associated with PNPLA3 expression, such as liver-related diseases, such as simple fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis (irreversible advanced scarring of the liver), or PNPLA3-related obesity. Thus, in one embodiment, the present invention provides an RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence complementary to the mRNA sequence of PNPLA3. In certain embodiments, the antisense strand comprises a region having at least 15 consecutive nucleotides from the antisense sequence listed in Table 1 or Table 2. In some embodiments, the RNAi of the present invention selectively inhibits PNPLA3-rs738409, PNPLA3-rs738408, and / or PNPLA3-rs738409-rs738408 minor alleles over reference alleles that do not contain these alterations.

[0006] In some embodiments, the sense strand of the RNAi constructs described herein comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, each of the sense and antisense strands is about 15 to 30 nucleotides in length. In some embodiments, the RNAi construct comprises at least one blunt end. In other embodiments, the RNAi construct comprises at least one nucleotide overhang. Such a nucleotide overhang may comprise at least 1 to 6 unpaired nucleotides and may be located at the 3' end of the sense strand, the 3' end of the antisense strand, or the 3' ends of both the sense and antisense strands. In certain embodiments, the RNAi construct comprises an overhang of two unpaired nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct comprises an overhang of two unpaired nucleotides at the 3' end of the antisense strand and a blunt end at the 3' end of the sense strand / 5' end of the antisense strand.

[0007] The RNAi construct of the present invention may include one or more modified nucleotides, including nucleotides with modifications to the ribose ring, nucleobase, or phosphodiester backbone. In some embodiments, the RNAi construct includes one or more 2'-modified nucleotides. Such 2'-modified nucleotides may include 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), glycol nucleic acids (GNAs), inverted bases (e.g., inverted adenosines), or combinations thereof. In a particular embodiment, the RNAi construct includes one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof. In some embodiments, all nucleotides in the sense and antisense strands of the RNAi construct are modified nucleotides.

[0008] In some embodiments, the RNAi construct comprises at least one backbone modification, such as a modified internucleotide or internucleoside bond. In certain embodiments, the RNAi construct described herein comprises at least one phosphorothioate internucleotide bond. In certain embodiments, the phosphorothioate internucleotide bond can be located at the 3' or 5' end of the sense strand and / or the antisense strand.

[0009] In some embodiments, the antisense and / or sense strands of the RNAi constructs of the invention can comprise or consist of sequences from the antisense and sense sequences listed in Tables 1 or 2. In certain embodiments, the RNAi construct can be any one of the duplex compounds listed in any one of Tables 1-2. [Brief description of the drawings]

[0010] [Figure 1A-D] Screening of five siRNA molecules for both dose-dependent mRNA knockdown and functional durability in vivo is shown. [Figure 2A-G] The effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 3A-G] The effect of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 4A-D] The ability of PNPLA3 rs738409-rs738408-specific siRNA molecules to rescue disease-associated phenotypes by overexpression of PNPLA3 rs738409-rs738408, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 5A-L] 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 6a-6b] The complete sequence of the AAV plasmid containing the PNPLA3 minor allele target sequence is shown, with the mouse CMV promoter, firefly luciferase reporter, and the portion containing the target sequence underlined. [Figure 7a-7b] The full sequence of the AAV plasmid containing the PNPLA3 reference allele target sequence is shown, with the mouse CMV promoter, firefly luciferase reporter, and the portion containing the target sequence underlined. [Figure 8] Exemplary images are shown for mice injected with AAV expressing the human PNPLA3 reference allele target sequence (bottom row) versus mice injected with AVV expressing the human PNPLA3 minor allele target sequence (top row). After baseline images (first row) were acquired, both mice were injected with the same siRNA molecule (D-2878 at 3 mpk). Rows 2-5 are images captured at weeks 1, 2, 3, and 4, respectively. Images have been converted to grayscale using Living Image® software. Light regions are areas of low total flux [p / s], whereas dark regions are areas of high flux [p / s]. The relative knockdown percentages of siRNA-treated mice at each weekly time point, normalized to vehicle-treated mice, are shown in the inset (rows 2 and 4). [Figure 9A-F]Examples of siRNA molecule D-2419 demonstrating both dose-dependent and allele-selective mRNA knockdown in vivo as well as functional efficacy are shown. (A) siRNA molecule D-2419 was injected subcutaneously into the abdomen of mice at 3.0 and 10.0 milligrams per kilogram of body weight. (B) Data show the mean relative mRNA knockdown rate and standard error of PNPLA3WT versus human PNPLA3rs738409-rs738408 allele set relative to the vehicle-treated control group. (C) Livers from the 2-week treatment group were processed for triglyceride content to assess functional efficacy. (D) To control for efficient GalNAc-mediated delivery of siRNA molecule D-2787, siRNA cross-reactive against human and mouse HPRT and Hprt, respectively, was delivered at 10 milligrams per kilogram and livers were harvested after 2 weeks. Data show HPRT mRNA and Hprt mRNA copies in vehicle-treated (N=5) versus D-2787-treated (N=4) mice. (E) Data show the mean relative mRNA knockdown rate and standard error of human HPRT mRNA and mouse Hprt mRNA, respectively, set relative to the vehicle-treated control group. All normalized to human TBP. (F) To confirm expression of GalNAc receptors on stem cells of PXB mice®, mouse Asgr1 mRNA and human ASGR1 mRNA levels were assessed in the absence and presence of D-2419. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to compositions and methods for regulating the expression of the patatin-like phospholipase domain-containing 3 (PNPLA3) gene. In some embodiments, the gene can be in a subject, such as a cell or a mammal (e.g., human). In some embodiments, the composition of the present invention comprises an RNAi construct that targets the mRNA of PNPLA3 and reduces PNPLA3 expression in a cell or a mammal. Such RNAi constructs are useful for treating or preventing various forms of liver-related diseases, such as, for example, simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible advanced scarring of the liver), or PNPLA3-related obesity.

[0012] In 2008, a genome-wide association study (GWAS) searching for nonsynonymous sequence variations, or single nucleotide polymorphisms (SNPs), associated with NAFLD revealed that a variant in PNPLA3 (rs738409 [G] encoding I148M; which may be referred to as PNPLA3-rs738409, PNPLA3-ma or PNPLA3-minor allele) was significantly associated with hepatic fat content. Since this initial report, subsequent GWAS have confirmed that PNPLA3 rs738409 is a major genetic determinant of NAFLD that is significantly associated with 1) elevated levels of alanine transaminase (ALT), a serum biomarker of liver injury, 2) incidence, progression and severity of NAFLD, 3) both obese and lean individuals, and 4) all stages of NAFLD: steatosis, NASH, cirrhosis and hepatocellular carcinoma. The consensus of multiple GWAS shows that the association between PNPLA3 rs738409 and NAFLD is independent of age, sex, ethnicity, metabolic syndrome, body mass index, insulin resistance, and serum lipids. Furthermore, statistical analysis from multiple sources estimates that approximately 50% of NAFLD patients carry the PNPLA3 rs738409 mutation. Patients can be homozygous or heterozygous for the PNPLA3 rs738409 mutation. In addition, it has been found that patients with the PNPLA3 rs738409 mutation often also carry the rs738408 mutation, which is three base pairs away (Tian et al (2010) Nature Genetics 42:21-23). Thus, the patient may have a PNPLA3-rs738409 minor allele, a PNPLA3-rs738408 minor allele or a PNPLA3-rs738409-rs738408 double minor allele mutation (PNPLA3-dma).

[0013] The inventors developed a mouse model to explore PNPLA3 function in vivo. To date, no detectable metabolic phenotypes have been identified as a result of Pnpla3 deficiency or overexpression. In contrast, Pnpla3 in both transgenic and knock-in miceI148M Expression of Pnpla3 resulted in elevated hepatic triglyceride levels similar to those in NAFLD. Thus, taken together, the data from the in vivo mouse model support the conclusion that overexpression of the mutant Pnpla3, but not the wild-type protein, is the key to NAFLD-like hepatic triglyceride levels. I148M These findings, together with the high frequency of the minor allele in individuals with NAFLD and its strong association with disease, highlight PNPLA3 rs738409 as a key therapeutic target for NAFLD.

[0014] RNA interference (RNAi) is a process in which foreign RNA is introduced into cells to cause specific degradation of mRNAs encoding targeted proteins, resulting in reduced protein expression. Advances in both RNAi technology and delivery to the liver, as well as increasing success with other RNAi-based therapies, suggest RNAi as a compelling means to treat NAFLD by directly targeting PNPLA3I148M. Numerous GWAS have shown dose-dependent effects of PNPLA3 rs738409 on the incidence, progression and severity of NAFLD; odds ratios tend to be at least 2-fold higher for homozygous and heterozygous carriers, and at least 2-fold higher for heterozygous and wild-type individuals. Thus, silencing PNPLA3 using the discrimination specificity of the allele may be a potential means to reduce hepatic triglycerides in PNPLA3I148M carriers, while also presenting a scenario in which heterozygotes may benefit without silencing the wild-type allele. Along these lines, we have identified SNP-specific short interfering RNAs (siRNAs) for PNPLA3I148M and demonstrated in vitro proof of concept. Using both Hep3B (homozygous for reference allele PNPLA3I148I) and HEPG2 (homozygous for minor allele PNPLA3I148M) hepatocellular carcinoma cell lines, we have identified siRNA sequences that can specifically inhibit the expression of the PNPLA3I148M gene. The inhibitory effects of these sequences were confirmed by screening against Chinese Hamster Ovary (CHO) cells overexpressing either PNPLA3I148I or PNPLA3I148M. Next, using an adeno-associated virus (AAV) that overexpresses human PNPLA3I148M in vivo, we demonstrated that treatment with a minor allele-specific SNP not only specifically reduced expression of human PNPLA3I148M in mice, but also significantly reversed hepatic triglyceride accumulation induced by overexpression of human PNPLA3I148M.

[0015] As used herein, the term "RNAi construct" refers to an agent that comprises an RNA molecule that can downregulate the expression of a target gene (e.g., PNPLA3) through an RNA interference mechanism when introduced into a cell. RNA interference is a process in which a nucleic acid molecule induces the cleavage and degradation of a target RNA molecule (e.g., a messenger RNA or mRNA molecule) in a sequence-specific manner, for example, through the RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi construct comprises a double-stranded RNA molecule that comprises two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other and hybridize to form a double-stranded region. "Hybridize" or "hybridization" typically refers to the pairing of complementary polynucleotides through hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. The strand that comprises a region having a substantially complementary sequence to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand." "Sense strand" refers to the strand that contains a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand can contain a region that has substantially identical sequence to a target sequence.

[0016] In some embodiments, the present invention is an RNAi directed to PNPLA3. In some embodiments, the present invention is an RNAi that binds at the PNPLA3 rs738409 site. In some embodiments, the present invention is an RNAi that binds at the PNPLA3 rs738408 site. In some embodiments, the present invention is an RNAi that binds at both PNPLA3 rs738409 rs738408 sites. In some embodiments, the present invention is an RNAi that binds to PNPLA3 rs738409 preferentially over the native PNPLA3 sequence (PNPLA3-ref). In some embodiments, the present invention is an RNAi that binds to PNPLA3 rs738408 preferentially over the PNPLA3-ref sequence. In some embodiments, the present invention is an RNAi that binds to PNPLA3-dma preferentially over PNPLA3-ma. In some embodiments, the present invention is an RNAi molecule that contains any of the sequences found in Tables 1 or 2.

[0017] Double-stranded RNA molecules may contain chemical modifications to ribonucleotides, including modifications to the ribose sugar, the base, or the backbone components of the ribonucleotides, such as those described herein or known in the art. Any such modifications as used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.) are encompassed by the term "double-stranded RNA" for purposes of this disclosure.

[0018] As used herein, a first sequence is "complementary" to a second sequence if, under certain conditions, such as physiological conditions, the polynucleotide comprising the first sequence can hybridize to the polynucleotide comprising the second sequence to form a duplex region. Other such conditions can include moderate or stringent hybridization conditions known to those skilled in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if the polynucleotide comprising the first sequence base pairs with the polynucleotide comprising the second sequence over the entire length of one or both nucleotide sequences without mismatches. A sequence is "substantially complementary" to a target sequence if the sequence is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the target sequence. Percent complementarity can be calculated by dividing the number of bases in a first sequence that are complementary to the bases at corresponding positions in a second sequence or target sequence by the total length of the first sequence. A sequence can also be said to be substantially complementary to another sequence if there are no more than 5, 4, 3, 2 or 1 mismatches over a 30 base pair duplex region when the two sequences hybridize. In general, if any nucleotide overhangs as defined herein exist, the sequence of such overhangs is not taken into account in determining the degree of complementarity between two sequences. As an example, a 21 nucleotide long sense strand and a 21 nucleotide long antisense strand that hybridize to form a 19 base pair duplex region with a 2 nucleotide overhang at the 3' end of each strand would be considered to be fully complementary as this term is used herein.

[0019] In some embodiments, the region of the antisense strand comprises a sequence that is completely complementary to a region of the target RNA sequence (e.g., PNPLA3 mRNA). In such embodiments, the sense strand may comprise a sequence that is completely complementary to the sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to the sequence of the antisense strand, such as a sequence that has 1, 2, 3, 4 or 5 mismatches in the duplex region formed by the sense strand and the antisense strand. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g., within 6, 5, 4, 3, 2, or 1 nucleotide of the 5' and / or 3' ends of the strands). In one embodiment, any mismatches in the duplex region formed by the sense strand and the antisense strand occur within 6, 5, 4, 3, 2, or 1 nucleotide of the 5' end of the antisense strand.

[0020] In some embodiments, the sense strand and antisense strand of double-stranded RNA can be two separate molecules that hybridize to form a double-stranded region, but are not connected except for this region. Such double-stranded RNA molecules formed from two separate strands are called "small interfering RNA" or "short interfering RNA" (siRNA). Thus, in some embodiments, the RNAi construct of the present invention comprises siRNA.

[0021] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, the molecules do not have to be covalently linked, but can be. When the two strands are covalently linked by any means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of each other strand forming a duplex structure, the linking structure is called a "linker". The RNA strands may have the same or different number of nucleotides. The maximum number of base pairs in a duplex is the number of nucleotides of the shortest strand of dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi may include one or more nucleotide overhangs.

[0022] In other embodiments, the sense and antisense strands that hybridize to form a duplex region may be part of a single RNA molecule, i.e., the sense and antisense strands are part of a self-complementary region of a single RNA molecule. In such cases, the single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is joined to the 5' end of the antisense strand by a contiguous sequence of unpaired nucleotides that will form the loop region. The loop region is usually of sufficient length to allow the RNA molecule to fold back on itself so that the antisense strand can base pair with the sense strand to form the duplex or stem region. The loop region may comprise about 3 to about 25, about 5 to about 15, or about 8 to about 12 unpaired nucleotides. Such RNA molecules with at least a partially self-complementary region are referred to as "short hairpin RNAs" (shRNAs). In some embodiments, the loop region may comprise at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In some embodiments, the loop region may contain 10, 9, 8, 7, 6, 5, 4, 3, 2 or less unpaired nucleotides. In certain embodiments, the RNAi construct of the invention comprises an shRNA. The length of the single at least partially self-complementary RNA molecule may be about 35 nucleotides to about 100 nucleotides, about 45 nucleotides to about 85 nucleotides, or about 50 to about 60 nucleotides, and may include a duplex region and a loop region, each having a length as recited herein.

[0023] In some embodiments, the RNAi construct of the present invention comprises a sense strand and an antisense strand, and the antisense strand comprises a region having a sequence substantially or completely complementary to the messenger RNA (mRNA) sequence of PNPLA3. As used herein, "mRNA sequence of PNPLA3" refers to any messenger RNA sequence, including splice variants, that encodes the PNPLA3 protein, including variants or isoforms of the PNPLA3 protein from any species (e.g., mouse, rat, non-human primate, human). The PNPLA3 protein is also known as adiponutrin (ADPN) and calcium-independent phospholipase A2-epsilon (iPLA(2)ε).

[0024] The PNPLA3 mRNA sequence also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. The cDNA sequence refers to the sequence of the mRNA transcript expressed as DNA bases (e.g., guanine, adenine, thymine and cytosine) rather than RNA bases (e.g., guanine, adenine, uracil and cytosine). Thus, the antisense strand of the RNAi construct of the present invention can include a region having a substantially or completely complementary sequence to the target PNPLA3 mRNA sequence or PNPLA3 cDNA sequence. The PNPLA3 mRNA or cDNA sequence can include, but is not limited to, any PNPLA3 mRNA or cDNA sequence, such as may be derived from the NCBI reference sequence NM_025225.2.

[0025] The region of the antisense strand may be substantially or completely complementary to at least 15 contiguous nucleotides of the PNPLA3 mRNA sequence. In some embodiments, the target region of the PNPLA3 mRNA sequence to which the antisense strand comprises a region of complementarity may range from about 15 to about 30 contiguous nucleotides, about 16 to about 28 contiguous nucleotides, about 18 to about 26 contiguous nucleotides, about 17 to about 24 contiguous nucleotides, about 19 to about 25 contiguous nucleotides, about 19 to about 23 contiguous nucleotides, or about 19 to about 21 contiguous nucleotides. In certain embodiments, the region of the antisense strand that comprises a sequence substantially or completely complementary to the PNPLA3 mRNA sequence may, in some embodiments, comprise at least 15 contiguous nucleotides from the antisense sequences listed in Table 1 or Table 2. In other embodiments, the antisense sequence comprises at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In some embodiments, the sense and / or antisense sequence comprises at least 15 nucleotides from a sequence listed in Table 1 or Table 2 with no more than 1, 2, or 3 nucleotide mismatches.

[0026] The sense strand of an RNAi construct typically contains a sequence sufficiently complementary to that of the antisense strand such that the two strands hybridize under physiological conditions to form a duplex region. A "duplex region" refers to a region of two complementary or substantially complementary polynucleotides that base pair with each other by either Watson-Crick base pairing or other hydrogen bonding interactions to generate a duplex between the two polynucleotides. The duplex region of an RNAi construct should be of sufficient length to allow the RNAi construct to enter the RNA interference pathway, for example, by binding the Dicer enzyme and / or the RISC complex. For example, in some embodiments, the duplex region is about 15 to about 30 base pairs in length. Other lengths of the double-stranded region within this range are also suitable, such as about 15 to about 28 base pairs, about 15 to about 26 base pairs, about 15 to about 24 base pairs, about 15 to about 22 base pairs, about 17 to about 28 base pairs, about 17 to about 26 base pairs, about 17 to about 24 base pairs, about 17 to about 23 base pairs, about 17 to about 21 base pairs, about 19 to about 25 base pairs, about 19 to about 23 base pairs, or about 19 to about 21 base pairs. In one embodiment, the double-stranded region is about 17 to about 24 base pairs in length. In another embodiment, the double-stranded region is about 19 to about 21 base pairs in length.

[0027] In some embodiments, the RNAi agents of the invention contain a duplex region of about 24 to about 30 nucleotides that interacts with a target RNA sequence, e.g., a PNPLA3 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into a cell can be degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, 15 processes dsRNA into short interfering RNAs of 19 to 23 base pairs with characteristic 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to induce target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target 20 mRNA, one or more endonucleases in the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0028] For embodiments in which the sense and antisense strands are two separate molecules (e.g., the RNAi construct comprises an siRNA), the sense and antisense strands need not be the same length as the length of the duplex region. For example, one or both strands may be longer than the duplex region and may have one or more unpaired nucleotides or mismatches adjacent to the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, a "nucleotide overhang" refers to an unpaired nucleotide at the end of a strand or a nucleotide that extends beyond the duplex region. A nucleotide overhang is typically generated when the 3' end of one strand extends beyond the 5' end of the other strand or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of a nucleotide overhang is generally 1-6 nucleotides, 1-5 nucleotides, 1-4 nucleotides, 1-3 nucleotides, 2-6 nucleotides, 2-5 nucleotides, or 2-4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, the nucleotide overhang comprises 1-4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang can be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some embodiments, the overhang comprises a 5'-uridine uridine-3' (5'-UU-3') dinucleotide. In such embodiments, the UU dinucleotide can comprise a ribonucleotide or a modified nucleotide, such as a 2'-modified nucleotide. In other embodiments, the overhang comprises a 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3') dinucleotide.

[0029] The nucleotide overhang may be present at the 5'-end or 3'-end of one or both strands. For example, in one embodiment, the RNAi construct comprises a nucleotide overhang at the 5'-end and 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises a nucleotide overhang at the 5'-end and 3'-end of the sense strand. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 5'-end of the sense strand and the 5'-end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3'-end of the sense strand and the 3'-end of the antisense strand.

[0030] The RNAi construct may comprise a single nucleotide overhang at one end of the double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense and antisense strands are fully base-paired at the ends of the molecule, with no unpaired nucleotides extending beyond the duplex region. In some embodiments, the RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct comprises blunt ends at both ends of the double-stranded RNA molecule. In such embodiments, the sense and antisense strands have the same length, and the duplex region is the same length as the sense and antisense strands (i.e., the molecule is duplex throughout its entire length).

[0031] The sense strand and the antisense strand can each independently be about 15 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and the antisense strand are each about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and the antisense strand form a duplex region that has the same length but is shorter than the strands, such that the RNAi construct has a two nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each being 21 nucleotides long, (ii) a duplex region that is 19 base pairs long, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each being 23 nucleotides long, (ii) a duplex region that is 21 base pairs long, and (iii) a nucleotide overhang of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the sense strand and the antisense strand have the same length and form a duplex region throughout their length such that there are no nucleotide overhangs at either end of the duplex molecule. In such an embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand each being 21 nucleotides long, and (ii) a duplex region that is 21 base pairs long. In another such embodiment, the RNAi construct is blunt ended and comprises (i) sense and antisense strands, each of which is 23 nucleotides in length, and (ii) a duplex region that is 23 base pairs in length.

[0032] In other embodiments, the sense strand or antisense strand is longer than the other strand, and the two strands form a duplex region having a length equal to the length of the shorter strand, such that the RNAi construct comprises at least one nucleotide overhang.For example, in one embodiment, the RNAi construct comprises (i) a sense strand that is 19 nucleotides long, (ii) an antisense strand that is 21 nucleotides long, (iii) a duplex region that is 19 base pairs long, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand.In another embodiment, the RNAi construct comprises (i) a sense strand that is 21 nucleotides long, (ii) an antisense strand that is 23 nucleotides long, (iii) a duplex region that is 21 base pairs long, and (iv) a single nucleotide overhang of two unpaired nucleotides at the 3' end of the antisense strand.

[0033] The antisense strand of the RNAi construct of the present invention may comprise any one of the antisense sequences listed in Table 1 or Table 2, or the sequence of nucleotides 1-19 of any of these antisense sequences. Each of the antisense sequences listed in Tables 1 and 6 comprises a sequence of 19 contiguous nucleotides (the first 19 nucleotides counting from the 5' end) that is complementary to the PNPLA3 mRNA sequence, in addition to a 2-nucleotide overhang sequence. Thus, in some embodiments, the antisense strand comprises the sequence of nucleotides 1-19 of any one of SEQ ID NOs: 1-166 or 167-332.

[0034] Modified Nucleotides The RNAi construct of the present invention may include one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide that has one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotide does not include ribonucleotides that contain adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, and deoxyribonucleotides that contain deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, the RNAi construct may include a combination of modified nucleotides, ribonucleotides, and deoxyribonucleotides. The incorporation of modified nucleotides into one or both strands of a double-stranded RNA molecule can improve the in vivo stability of the RNA molecule, for example, by reducing the susceptibility of the molecule to nucleases and other degradative processes. The efficacy of the RNAi construct to reduce the expression of a target gene can also be enhanced by the incorporation of modified nucleotides.

[0035] In certain embodiments, modified nucleotides have modifications of the ribose sugar. These sugar modifications can include modifications at the 2' and / or 5' positions of the pentose ring as well as bicyclic sugar modifications. A 2'-modified nucleotide refers to a nucleotide having a pentose ring with a substituent at the 2' position other than H or OH. Such 2' modifications include, but are not limited to, 2'-O-alkyl (e.g., O-C1-C10 or O-C1-C10 substituted alkyl), 2'-O-allyl (O-CH2CH=CH2), 2'-C-allyl, 2'-fluoro, 2'-O-methyl (OCH3), 2'-O-methoxyethyl (O-(CH2)2OCH3), 2'-OCF3, 2'-O(CH2)2SCH3, 2'-O-aminoalkyl, 2'-amino (e.g., NH2), 2'-O-ethylamine, and 2'-azido. Modifications at the 5' position of the pentose ring include, but are not limited to, 5'-methyl (R or S), 5'-vinyl, and 5'-methoxy.

[0036] "Bicyclic sugar modification" refers to a modification of a pentose ring where a bridge connects two atoms of the ring to form a second ring resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides containing a sugar moiety having a bicyclic sugar modification are referred to herein as bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include α-L-methyleneoxy (4'-CH2-O-2') bicyclic nucleic acids (BNAs); β-D-methyleneoxy (4'-CH2-O-2') BNAs (also referred to as locked nucleic acids or LNAs); ethyleneoxy (4'-(CH2)2-O-2') BNAs; aminooxy (4'-CH2-ON(R)-2') BNAs; oxyamino (4'-CH2-N(R)-O-2') BNAs; methyl(methyleneoxy) (4'-CH These include, but are not limited to, 4'-CH-O-2') BNA (also referred to as constrained ethyl or cEt); methylene-thio (4'-CH-S-2') BNA; methylene-amino (4'-CH-N(R)-2') BNA; methyl carbocyclic (4'-CH-CH(CH)-2') BNA; propylene carbocyclic (4'-(CH)-2') BNA; and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also referred to as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the RNAi constructs of the invention are described in U.S. Pat. No. 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0037] In some embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In a particular embodiment, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0038] Both the sense strand and the antisense strand of the RNAi construct can contain one or more modified nucleotides. For example, in some embodiments, the sense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides can be 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or a combination thereof.

[0039] In some embodiments, all pyrimidine nucleotides preceding an adenosine nucleotide in the sense strand, antisense strand, or both strands are modified nucleotides. For example, when the sequence 5'-CA-3' or 5'-UA-3' occurs in either strand, the cytidine and uridine nucleotides are modified nucleotides, preferably 2'-O-methyl modified nucleotides. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides), and all 5' nucleotides of the sequence 5'-CA-3' or 5'-UA-3' occurring in the antisense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides). In other embodiments, all nucleotides in the duplex region are modified nucleotides. In such embodiments, the modified nucleotides are preferably 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or combinations thereof.

[0040] In embodiments where the RNAi construct comprises a nucleotide overhang, the nucleotide in the overhang can be a ribonucleotide, a deoxyribonucleotide, or a modified nucleotide. In one embodiment, the nucleotide in the overhang is a deoxyribonucleotide, such as deoxythymidine. In another embodiment, the nucleotide in the overhang is a modified nucleotide. For example, in some embodiments, the nucleotide in the overhang is a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-methoxyethyl modified nucleotide, or a combination thereof.

[0041] The RNAi construct of the present invention may also include one or more modified internucleotide bonds. As used herein, the term "modified internucleotide bond" refers to a bond between nucleotides other than the naturally occurring 3'-5' phosphodiester bond. In some embodiments, the modified internucleotide bond is a phosphorus-containing internucleotide bond, such as a phosphotriester, an aminoalkyl phosphotriester, an alkyl phosphonate (e.g., methyl phosphonate, 3'-alkylene phosphonate), a phosphinate, a phosphoramidate (e.g., 3'-amino phosphoramidate and aminoalkyl phosphoramidate), a phosphorothioate (P=S), a chiral phosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkyl phosphonate, a thionoalkyl phosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide bond is a 2'-5' phosphodiester bond. In other embodiments, the modified internucleotide linkage is a non-phosphorus-containing internucleotide linkage and may therefore also be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane linkages (-O-Si(H)2-O-); sulfide, sulfoxide and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (-CH2-N(CH3)-O-CH2-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S and CH2 component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to generate peptide nucleic acids or PNAs, such as those described in U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.Other suitable modified internucleotide and internucleoside linkages that can be used in the RNAi constructs of the present invention are described in U.S. Pat. Nos. 6,693,187, 9,181,551, U.S. Patent Application Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0042] In certain embodiments, the RNAi construct comprises one or more phosphorothioate internucleotide linkages. The phosphorothioate internucleotide linkages may be present in the sense strand, the antisense strand, or both strands of the RNAi construct. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages. In other embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages. In yet other embodiments, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide linkages. The RNAi construct may comprise one or more phosphorothioate internucleotide linkages at the 3' end, the 5' end, or both the 3' end and the 5' end of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 3'-end of the sense strand, the antisense strand, or both strands. In other embodiments, the RNAi construct comprises about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand, the antisense strand, or both strands. In one embodiment, the RNAi construct comprises a single phosphorothioate internucleotide linkage at the 3'-end of the sense strand and a single phosphorothioate internucleotide linkage at the 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3'-end of the antisense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at the 3'-end of the antisense strand). In another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3' and 5' ends of the antisense strand.In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3'-end and 5'-end of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand. In yet another embodiment, the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3'-end and 5'-end of the antisense strand and two consecutive phosphorothioate internucleotide linkages at both the 3'-end and 5'-end of the sense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5'-end and 3'-end of the antisense strand and phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5'-end and 3'-end of the sense strand). In any of the embodiments in which one or both strands comprise one or more phosphorothioate internucleotide linkages, the remaining internucleotide linkages in the strands may be native 3'-5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage in the sense and antisense strands is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0043] In embodiments where the RNAi construct comprises a nucleotide overhang, two or more of the unpaired nucleotides in the overhang can be linked by phosphorothioate internucleotide bonds. In certain embodiments, all of the unpaired nucleotides in the 3'-end nucleotide overhang of the antisense strand and / or the sense strand are linked by phosphorothioate internucleotide bonds. In other embodiments, all of the unpaired nucleotides in the 5'-end nucleotide overhang of the antisense strand and / or the sense strand are linked by phosphorothioate internucleotide bonds. In yet other embodiments, all of the unpaired nucleotides in any nucleotide overhang are linked by phosphorothioate internucleotide bonds.

[0044] In certain embodiments, modified nucleotides incorporated into one or both strands of an RNAi construct of the invention have a modification of the nucleobase (also referred to herein as "base"). A "modified nucleobase" or "modified base" refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases may be synthetic or naturally occurring modifications, and include the universal bases 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine (X), hypoxanthine (I), 2-aminoadenine, 6-methyladenine, 6-methylguanine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6 ... These may include, but are not limited to, azo-uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0045] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analogue that indiscriminately base pairs with all of the natural bases of RNA and DNA without changing the double helix structure of the resulting double-stranded region. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

[0046] Other suitable modified bases that can be incorporated into the RNAi constructs of the present invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol.10:297-310, 2000 and Peacock et al., J.Org.Chern., Vol.76:7295-7300, 2011, both of which are incorporated herein by reference in their entirety. Those skilled in the art will appreciate that guanine, cytosine, adenine, thymine, and uracil can be substituted by other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of polynucleotides that contain nucleotides with such substituted nucleobases.

[0047] In some embodiments of the RNAi construct of the present invention, the 5'-end of the sense strand, the antisense strand, or both the antisense strand and the sense strand comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to a terminal phosphate group, including unmodified phosphate (-OP=O)(OH)OH) and modified phosphate. Modified phosphate includes phosphates in which one or more of the O and OH groups are replaced with H, O, S, N(R) or alkyl, where R is H, an amino protecting group, or unsubstituted or substituted alkyl. Exemplary phosphate moieties include, but are not limited to, 5'-monophosphate; 5'-diphosphate; 5'-triphosphate; 5'-guanosine cap (7-methylated or unmethylated); 5'-adenosine cap or any other modified or unmodified nucleotide cap structure; 5'-monothiophosphate (phosphorothioate); 5'-monodithiophosphate (phosphorodithioate); 5'-α-thiotriphosphate; 5'-γ-thiotriphosphate; 5'-phosphoramidate; 5'-vinyl phosphate; 5'-alkyl phosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0048] Modified nucleotides that can be incorporated into the RNAi constructs of the present invention can have two or more chemical modifications as described herein. For example, modified nucleotides can have a modification to the ribose sugar and a modification to the nucleobase. By way of example, modified nucleotides can include a 2' sugar modification (e.g., 2'-fluoro or 2'-methyl) and can include a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, modified nucleotides can include a sugar modification combined with a modification to the 5' phosphate that will generate a modified internucleotide or internucleoside linkage when the modified nucleotide is incorporated into a polynucleotide. For example, in some embodiments, modified nucleotides can include sugar modifications such as a 2'-fluoro modification, a 2'-O-methyl modification, or a bicyclic sugar modification and a 5' phosphorothioate group. Thus, in some embodiments, one or both strands of the RNAi constructs of the present invention include a combination of 2' modified nucleotides or BNAs and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of the RNAi constructs of the invention comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs comprising modified nucleotides and internucleotide linkages are shown in Table 2.

[0049] Function of RNAi constructs Preferably, the RNAi construct of the present invention reduces or inhibits the expression of PNPLA3 in cells, particularly in liver cells.Thus, in one embodiment, the present invention provides a method for reducing PNPLA3 expression in cells by contacting cells with any RNAi construct described herein.The cells can be in vitro or in vivo.PNPLA3 expression can be assessed by measuring the amount or level of PNPLA3 mRNA, PNPLA3 protein, or another biomarker associated with PNPLA3 expression.The reduction of PNPLA3 expression in cells or animals treated with the RNAi construct of the present invention can be determined by comparing with PNPLA3 expression in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, the reduction of PNPLA3 expression is evaluated by (a) measuring the amount or level of PNPLA3 mRNA in liver cells treated with the RNAi construct of the present invention, (b) measuring the amount or level of PNPLA3 mRNA in liver cells treated with a control RNAi construct (e.g., an RNAi agent directed to an RNA molecule not expressed in liver cells or an RNAi construct with a nonsense or scrambled sequence) or without the construct, and (c) comparing the PNPLA3 mRNA level measured from the treated cells in (a) with the PNPLA3 mRNA level measured from the control cells in (b). Prior to comparison, the PNPLA3 mRNA levels in treated and control cells can be normalized to RNA levels for a control gene (e.g., 18S ribosomal RNA). PNPLA3 mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescent in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

[0050] In other embodiments, the reduction of PNPLA3 expression is evaluated by (a) measuring the amount or level of PNPLA3 protein in liver cells treated with the RNAi construct of the present invention, (b) measuring the amount or level of PNPLA3 protein in liver cells treated with a control RNAi construct (e.g., an RNAi agent that targets an RNA molecule that is not expressed in liver cells or an RNAi construct with a nonsense or scrambled sequence) or without the construct, and (c) comparing the protein level of PNPLA3 measured from the treated cells in (a) with the protein level of PNPLA3 measured from the control cells in (b). Methods for measuring the protein level of PNPLA3 are known to those skilled in the art and include Western blot, immunoassay (e.g., ELISA), and flow cytometry. An exemplary immunoassay-based method for evaluating protein expression of PNPLA3 is described in Example 2. Example 3 describes an exemplary method for measuring PNPLA3 mRNA using RNA FISH. Any method capable of measuring PNPLA3 mRNA or protein can be used to assess the effectiveness of the RNAi constructs of the invention.

[0051] In some embodiments, the method for evaluating the expression level of PNPLA3 is carried out in vitro in cells that naturally express PNPLA3 (e.g., liver cells) or cells engineered to express PNPLA3. In certain embodiments, these methods are carried out in vitro in liver cells. Suitable liver cells include, but are not limited to, primary liver cells (e.g., human, non-human primate, or rodent liver cells), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the liver cells are Hep3B cells. In another embodiment, the liver cells are HepG2 cells.

[0052] In other embodiments, the method for evaluating the expression level of PNPLA3 is carried out in vivo.RNAi construct and any control RNAi construct can be administered to animals (e.g., rodents or non-human primates), and PNPLA3 mRNA or protein level can be evaluated in liver tissue taken from treated animals.Alternatively or in addition, biomarkers or functional phenotypes associated with PNPLA3 expression can be evaluated in treated animals.

[0053] In certain embodiments, the expression of PNPLA3 is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% in liver cells by the RNAi construct of the present invention. In some embodiments, the expression of PNPLA3 is reduced by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% in liver cells by the RNAi construct of the present invention. In other embodiments, the expression of PNPLA3 is reduced by about 90% or more, for example 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more in liver cells by the RNAi construct of the present invention. The percent reduction of PNPLA3 expression can be measured by any of the methods described herein and other methods known in the art. For example, in certain embodiments, the RNAi construct of the present invention inhibits at least 45% of PNPLA3 expression at 5 nM in Hep3B cells in vitro (containing wild-type PNPLA3). In related embodiments, the RNAi construct of the present invention inhibits at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of PNPLA3 expression at 5 nM in Hep3B cells in vitro. In other embodiments, the RNAi construct of the present invention inhibits at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of PNPLA3 expression at 5 nM in Hep3B cells in vitro. In certain embodiments, the RNAi construct of the present invention inhibits at least 45% of PNPLA3 expression at 5 nM in HepG2 cells in vitro (containing PNPLA3-rs738409-rs738408 double minor allele). In related embodiments, an RNAi construct of the invention inhibits at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of PNPLA3 expression in HepG2 cells in vitro at 5 nM.In other embodiments, the RNAi constructs of the invention inhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of PNPLA3 expression at 5 nM in HepG2 cells in vitro. In certain embodiments, the RNAi constructs of the invention inhibit at least 45% of PNPLA3 expression at 5 nM in CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro. In related embodiments, the RNAi constructs of the invention inhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% of PNPLA3 expression at 5 nM in CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro. In other embodiments, the RNAi constructs of the invention inhibit at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% of PNPLA3 expression at 5 nM in CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro. Reduction of PNPLA3 can be measured using a variety of techniques, including RNA FISH or droplet digital PCR, as described in Examples 2 and 3.

[0054] In some embodiments, IC50 values ​​are calculated to evaluate the potency of the RNAi constructs of the present invention for inhibiting PNPLA3 expression in liver cells. "IC50 value" is the dose / concentration required to achieve 50% inhibition of biological or biochemical function. The IC50 value of any particular substance or antagonist can be determined by making a dose-response curve and testing the effect of different concentrations of the substance or antagonist on expression level or functional activity in any assay. IC50 values ​​can be calculated for a given antagonist or substance by determining the concentration required to inhibit half of the maximum biological response or native expression level. Thus, IC50 values ​​can be calculated for any RNAi construct by determining the concentration of RNAi construct required to inhibit half of the native expression level of PNPLA3 in liver cells (e.g., the expression level of PNPLA3 in control liver cells) in any assay, such as the immunoassay or RNA FISH assay or droplet digital PCR assay described in the examples. The RNAi construct of the present invention can inhibit PNPLA3 expression in liver cells (e.g., Hep3B cells) with an IC50 of less than about 20 nM. For example, the RNAi construct inhibits PNPLA3 expression in liver cells with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits PNPLA3 expression in liver cells (e.g., Hep3B cells) with an IC50 of about 1 nM to about 10 nM. The RNAi construct of the present invention can inhibit PNPLA3 expression in liver cells (e.g., HepG2 cells) with an IC50 of less than about 20 nM. For example, the RNAi construct inhibits PNPLA3 expression in liver cells with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM.In certain embodiments, the RNAi construct inhibits PNPLA3 expression in liver cells (e.g., HepG2 cells) with an IC50 of about 1 nM to about 10 nM. The RNAi construct of the present invention may inhibit PNPLA3 expression in liver cells (e.g., CHO transfected cells expressing human PNPLA3 I148I or I148M) with an IC50 of less than about 20 nM. For example, the RNAi construct inhibits PNPLA3 expression in liver cells with an IC50 of about 0.001 nM to about 20 nM, about 0.001 nM to about 10 nM, about 0.001 nM to about 5 nM, about 0.001 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 5 nM, or about 0.1 nM to about 1 nM. In certain embodiments, the RNAi construct inhibits PNPLA3 expression in liver cells (eg, CHO transfected cells expressing human PNPLA3 I148I or I148M) with an IC50 of about 1 nM to about 10 nM.

[0055] The RNAi construct of the present invention can be easily produced using techniques known in the art, for example, conventional solid-phase nucleic acid synthesis.The polynucleotide of the RNAi construct can be assembled on a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidites).Automated nucleic acid synthesizers are commercially available from several vendors, including the DNA / RNA synthesizer from Applied Biosystems (Foster City, CA), the MerMade synthesizer from BioAutomation (Irving, TX), and the OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, PA).

[0056] Oligonucleotides can be synthesized via phosphoramidite chemistry using 2' silyl protecting groups with acid-labile dimethoxytrityl (DMT) at the 5' position of the ribonucleoside. Final deprotection conditions are known not to significantly degrade the RNA product. All syntheses can be performed on any automated or manual synthesizer on a large, medium or small scale. Synthesis can be carried out in multiple well plates, columns or glass slides.

[0057] The 2'-O-silyl group can be removed through exposure to fluoride ions, which can include any source of fluoride ions, such as salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or salts containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluoride. Crown ether catalysts can be utilized in combination with inorganic fluorides in the deprotection reaction. The preferred fluoride ion source is tetrabutylammonium fluoride or amine hydrofluoride (e.g., triethylamine and aqueous HF combined in a dipolar aprotic solvent, such as dimethylformamide).

[0058] The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters towards fluoride: methyl protection of the phosphotriester or phosphite triester can stabilize the bond towards fluoride ion and improve process yields.

[0059] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in the RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, e.g., one that is stable to acid treatment. Silyl protecting groups meet this criteria and can be easily removed in a final fluoride deprotection step that may result in minimal RNA degradation.

[0060] Tetrazole catalysts can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, p-nitrophenyltetrazole.

[0061] As can be understood by those skilled in the art, further methods of synthesizing the RNAi constructs described herein will be apparent to those skilled in the art. In addition, various synthetic steps may be performed in an alternative order or sequence to obtain the desired compound. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present in bases) and protecting group techniques (protection and deprotection) useful in synthesizing the RNAi constructs described herein are known in the art, including, for example, those described in R.Larock, Comprehensive Organic Transformations, VCH Publishers (1989); TWGreene and PGMWuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L.Fieser and M.Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. Custom synthesis of RNAi agents is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, Colo.), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, Calif.).

[0062] The RNAi construct of the present invention may include a ligand. As used herein, "ligand" refers to any compound or molecule that can directly or indirectly interact with another compound or molecule. The interaction between another compound or molecule and a ligand may trigger a biological response (e.g., trigger a signal transduction cascade, induce receptor-mediated endocytosis), or may simply be a physical association. A ligand may modify one or more properties of the double-stranded RNA molecule that it binds to, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, intracellular uptake, charge and / or clearance properties of the RNA molecule.

[0063] The ligand may comprise a serum protein (e.g., human serum albumin, low density lipoprotein, globulin), a cholesterol moiety, a vitamin (biotin, vitamin E, vitamin B12), a folate moiety, a steroid, a bile acid (e.g., cholic acid), a fatty acid (e.g., palmitic acid, myristic acid), a carbohydrate (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), a glycoside, a phospholipid, or an antibody or binding fragment thereof (e.g., an antibody or binding fragment that targets the RNAi construct to a particular cell type such as the liver). Other examples of ligands are dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl groups, hexadecyl Examples of suitable glycerols include glycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenoic acid, dimethoxytrityl or phenoxazine, peptides (e.g., antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, polymers such as polyethylene glycols (PEG) (e.g., PEG-40K), polyamino acids and polyamines (e.g., spermine, spermidine).

[0064] In certain embodiments, the ligand has endosome destabilizing properties. The endosome destabilizing ligand promotes the lysis of endosomes and / or the transport of the RNAi construct of the present invention or its components from endosomes to the cytoplasm of cells. The endosome destabilizing ligand can be a polycationic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosome destabilizing ligand adopts its active conformation at endosomal pH. The "active" conformation is a conformation in which the endosome destabilizing ligand promotes the lysis of endosomes and / or the transport of the RNAi construct of the present invention or its components from endosomes to the cytoplasm of cells. Exemplary endosome destabilizing ligands include GALA peptides (Subbarao et al., Biochemistry, Vol. 26:2964-2972, 1987), EALA peptides (Vogel et al., J. Am. Chern. Soc., Vol. 118:1581-1586, 1996), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, Vol. 1559:56-68, 2002). In one embodiment, the endosome destabilizing component may contain chemical groups (e.g., amino acids) that undergo a change in charge or protonation in response to a change in pH. The endosome destabilizing component may be linear or branched.

[0065] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than oligonucleotides not conjugated with cholesterol (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12: 103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules are also described in U.S. Patent Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are incorporated herein by reference in their entirety. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated with a folate moiety can be taken up into cells via receptor-mediated endocytosis pathways. Such folate-polynucleotide conjugates are described in U.S. Patent No. 8,188,247, which is incorporated herein by reference in its entirety.

[0066] Given that PNPLA3 is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to specifically deliver the RNAi construct to those liver cells. In some embodiments, the RNAi construct can be specifically targeted to the liver by employing a ligand that binds to or interacts with a protein expressed on the surface of liver cells. For example, in certain embodiments, the ligand can include an antigen-binding protein (e.g., an antibody or a binding fragment thereof (e.g., Fab, scFv)) that specifically binds to a receptor expressed on hepatocytes.

[0067] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8 or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. In some embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from pentose, hexose, or heptose, as well as disaccharides and trisaccharides containing such monosaccharide units. In other embodiments, the carbohydrate incorporated into the ligand is an amino sugar, such as galactosamine, glucosamine, N-acetylgalactosamine, and N-acetylglucosamine.

[0068] In some embodiments, the ligand comprises a hexose or hexosamine. The hexose may be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine may be selected from fructosamine, galactosamine, glucosamine, or mannosamine. In certain embodiments, the ligand comprises glucose, galactose, galactosamine, or glucosamine. In one embodiment, the ligand comprises glucose, glucosamine, or N-acetylglucosamine. In another embodiment, the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine. In certain embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective in targeting compounds in liver cells. See, e.g., D'Souza and Devarajan, J. Control Release, Vol. 203:126-139, 2015. Examples of GalNAc or galactose-containing ligands that can be incorporated into the RNAi constructs of the invention are described in U.S. Pat. Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Publication No. 20030130186; and WO 2013166155, all of which are incorporated by reference in their entireties.

[0069] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety that comprises two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains composed of carbohydrates that can bind to two or more different molecules or to two or more different sites on the same molecule. The valency of a carbohydrate moiety refers to the number of individual binding domains within the carbohydrate moiety. For example, the terms "monovalent," "bivalent," "trivalent," and "tetravalent" with respect to a carbohydrate moiety refer to a carbohydrate moiety having one, two, three, and four binding domains, respectively. A multivalent carbohydrate moiety can comprise a multivalent lactose moiety, a multivalent galactose moiety, a multivalent glucose moiety, a multivalent N-acetyl-galactosamine moiety, a multivalent N-acetyl-glucosamine moiety, a multivalent mannose moiety, or a multivalent fucose moiety. In some embodiments, the ligand comprises a multivalent galactose moiety. In other embodiments, the ligand comprises a multivalent N-acetyl-galactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety is bivalent, trivalent, or tetravalent. In such embodiments, the polyvalent carbohydrate moiety can be biantennary or triantennary. In a particular embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another particular embodiment, the polyvalent galactose moiety is trivalent or tetravalent. Exemplary trivalent or tetravalent GalNAc-containing ligands for incorporation into the RNAi constructs of the invention are detailed below.

[0070] The ligand may be directly or indirectly bound or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is directly covalently bound to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently bound to the sense or antisense strand of the RNAi construct via a linker. The ligand may be bound to the nucleobase, sugar moiety, or internucleotide bond of the polynucleotide (e.g., sense or antisense strand) of the RNAi construct of the present invention. Conjugation or binding to the purine nucleobase or its derivative may occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is bound to the ligand. Conjugation or binding to the pyrimidine nucleobase or its derivative may also occur at any position. In some embodiments, the 2-, 5-, and 6-position of the pyrimidine nucleobase may be bound to the ligand. Conjugation or binding to the sugar moiety of the nucleotide may occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that may be attached to the ligand include the 2', 3', and 5' carbon atoms. The 1' position may also be attached to the ligand, such as at a basic residue. Internucleotide linkages may also facilitate attachment of the ligand. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand may be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the ligand may be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0071] In certain embodiments, the ligand may be attached to the 3' or 5' end of either the sense strand or the antisense strand. In certain embodiments, the ligand is covalently attached to the 5' end of the sense strand. In other embodiments, the ligand is covalently attached to the 3' end of the sense strand. For example, in some embodiments, the ligand is attached to the 3' terminal nucleotide of the sense strand. In certain such embodiments, the ligand is attached at the 3' position of the 3' terminal nucleotide of the sense strand. In alternative embodiments, the ligand is attached near the 3' end of the sense strand but before one or more terminal nucleotides (i.e., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2' position of the sugar of the 3' terminal nucleotide of the sense strand.

[0072] In certain embodiments, the ligand is attached to the sense or antisense strand via a linker. A "linker" is an atom or group of atoms that covalently attaches the ligand to the polynucleotide component of the RNAi construct. The linker can be about 1 to about 30 atoms long, about 2 to about 28 atoms long, about 3 to about 26 atoms long, about 4 to about 24 atoms long, about 6 to about 20 atoms long, about 7 to about 20 atoms long, about 8 to about 20 atoms long, about 8 to about 18 atoms long, about 10 to about 18 atoms long, and about 12 to about 18 atoms long. In some embodiments, the linker can include a bifunctional linking moiety, typically including an alkyl moiety having two functional groups. One of the functional groups is selected to attach to a compound of interest (e.g., the sense or antisense strand of the RNAi construct) and the other is selected to attach to essentially any selected group, such as a ligand, as described herein. In certain embodiments, the linker comprises a chain structure or an oligomer of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups commonly employed in bifunctional linking moieties include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, the bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), and the like.

[0073] Linkers that can be used to attach a ligand to the sense or antisense strand in an RNAi construct of the invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, or substituted or unsubstituted C2-C10 alkynyl. Preferred substituents for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0074] In certain embodiments, the linker is cleavable. A cleavable linker is one that is sufficiently stable outside a cell, but is cleaved after entry into a target cell to release the two moieties that the linker holds together. In some embodiments, the cleavable linker is cleaved at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 times faster or at least 100 times faster in a target cell or under a first reference condition (e.g., which can be selected to mimic or correspond to intracellular conditions) than in the subject's blood or under a second reference condition (e.g., which can be selected to mimic or correspond to conditions found in blood or serum).

[0075] Cleavable linkers are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are found to be more prevalent or at higher levels or activity inside cells than in serum or blood. Examples of such degradable agents include redox agents that are selected for specific substrates or have no substrate specificity, including oxidizing or reducing enzymes or reducing agents, such as mercaptans, that are present inside cells and can degrade redox cleavable linkers by reduction; esterases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid cleavable linkers by acting as general acids, peptidases (which may be substrate specific), and phosphatases.

[0076] The cleavable linker may contain a moiety that is sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have a cleavable group that is cleaved at a preferred pH, thereby releasing the RNA molecule from the ligand into the cell interior or desired compartment of the cell.

[0077] Linkers can include cleavable groups that can be cleaved by certain enzymes. The type of cleavable group incorporated into linkers can depend on the cells to be targeted. For example, liver targeting ligands can be linked to RNA molecules via linkers that contain ester groups. Liver cells are rich in esterases, and therefore linkers will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other types of cells that are rich in esterases include lung, renal cortex, and testicular cells. Linkers that contain peptide bonds can be used when targeting cells that are rich in peptidases, such as liver cells and synovial cells.

[0078] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degrading agent (or condition) to cleave the candidate linker. It is desirable to also test the candidate cleavable linker for its ability to resist cleavage when in blood or in contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between a first condition selected to be indicative of cleavage in target cells and a second condition selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. The evaluation can be performed in a cell-free system, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform an initial evaluation in a cell-free or cultured condition and confirm by further evaluation in a whole animal. In some embodiments, a useful candidate linker is cleaved at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 70-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0079] In other embodiments, redox cleavable linkers are utilized. Redox cleavable linkers are cleaved upon reduction or oxidation. One example of a reductively cleavable group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or suitable for use with, for example, a particular RNAi construct and a particular ligand, one or more methods described herein can be used. For example, a candidate linker can be evaluated by incubation with dithiothreitol (DTT) or other reducing agent known in the art that mimics the cleavage rate that would be observed in cells, for example, target cells. Candidate linkers can also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, a candidate linker is cleaved in blood at a maximum of 10%. In other embodiments, useful linker candidates are degraded at least 2, 4, 10, 20, 50, 70, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions).

[0080] In yet another embodiment, the phosphate-based cleavable linker is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that hydrolyzes the phosphate group in a cell is an enzyme such as a phosphatase in a cell. Examples of phosphate-based cleavable groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Particular embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, -OP(S)(H)-S-. Another particular embodiment is -OP(O)(OH)-O-. These linker candidates can be evaluated using methods similar to those described above.

[0081] In other embodiments, the linker may include an acid-cleavable group, which is a group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable group is cleaved in an acidic environment of about pH 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less) or by an agent such as an enzyme that can act as a general acid. Within a cell, certain low pH organelles such as endosomes and lysosomes may provide a cleavage environment for the acid-cleavable group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). A particular embodiment is where the carbon attached to the oxygen (alkoxy group) of the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethyl, pentyl, or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0082] In other embodiments, the linker may include an ester-based cleavable group that is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable groups have the general formula -C(O)O- or -OC(O)-. These potential linkers can be evaluated using methods similar to those described above.

[0083] In further embodiments, the linker may include a peptidic cleavable group that is cleaved by enzymes such as peptidases and proteases in cells. Peptidic cleavable groups are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptidic cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptidic cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptidic cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0084] Other types of linkers suitable for attaching a ligand to the sense or antisense strand in an RNAi construct of the invention are known in the art and can include those described in U.S. Pat. Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are incorporated herein by reference in their entireties.

[0085] In certain embodiments, the ligand covalently attached to the sense or antisense strand of an RNAi construct of the invention comprises a GalNAc moiety, e.g., a multivalent GalNAc moiety. In some embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 3'-end of the sense strand. In other embodiments, the multivalent GalNAc moiety is a trivalent GalNAc moiety and is attached to the 5'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3'-end of the sense strand. In yet other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5'-end of the sense strand.

[0086] In some embodiments, the RNAi construct of the present invention can be delivered to a cell or tissue of interest by administering a vector that encodes and controls the intracellular expression of the RNAi construct. A "vector" (also referred to herein as an "expression vector") is a composition of matter that can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. A vector can be replicated in a living cell or made synthetically.

[0087] Generally, a vector for expressing an RNAi construct of the present invention will include one or more promoters operably linked to a sequence encoding the RNAi construct. As used herein, the phrase "operably linked" or "under transcriptional control" means that the promoter is in the appropriate position and orientation relative to the polynucleotide sequence to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide sequence. "Promoter" refers to a sequence that is recognized by or introduced into the synthetic machinery of a cell and is required to initiate transcription of a particular gene sequence. Suitable promoters include, but are not limited to, RNA polI promoters, polII, H1 or U6 RNA polIII promoters, and viral promoters (e.g., human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, and Rous sarcoma virus long terminal repeat). In some embodiments, H1 or U6 RNA polIII promoters are preferred. The promoter may be a tissue-specific or inducible promoter. Of particular interest are liver-specific promoters, such as promoter sequences from the human α1-antitrypsin gene, albumin gene, hemopexin gene, and hepatic lipase gene. Inducible promoters include promoters regulated by ecdysone, estrogen, progesterone, tetracycline, and isopropyl-PD1-thiogalactopyranoside (IPTG).

[0088] In some embodiments where the RNAi construct comprises an siRNA, the two separate strands (sense and antisense strands) can be expressed from a single vector or two separate vectors. For example, in one embodiment, the sequence encoding the sense strand is operably linked to a promoter of a first vector, and the sequence encoding the antisense strand is operably linked to a promoter of a second vector. In such an embodiment, the first and second vectors are simultaneously introduced into a target cell, for example, by infection or transfection, such that the sense and antisense strands are transcribed and hybridize within the cell to form an siRNA molecule. In another embodiment, the sense and antisense strands are transcribed from two separate promoters located in a single vector. In some such embodiments, the sequence encoding the sense strand is operably linked to a first promoter, the sequence encoding the antisense strand is operably linked to a second promoter, and the first and second promoters are located in a single vector. In one embodiment, the vector comprises a first promoter operably linked to a sequence encoding an siRNA molecule and a second promoter operably linked to the same sequence in the reverse orientation, such that transcription of the sequence from the first promoter results in the synthesis of a sense strand of the siRNA molecule and transcription of the sequence from the second promoter results in the synthesis of an antisense strand of the siRNA molecule.

[0089] In other embodiments, where the RNAi construct comprises an shRNA, a sequence encoding a single at least partially self-complementary RNA molecule is operably linked to a promoter that generates a single transcript. In some embodiments, the sequence encoding the shRNA comprises an inverted repeat connected by a linker polynucleotide sequence, generating the stem and loop structure of the shRNA after transcription.

[0090] In some embodiments, the vector encoding the RNAi construct of the present invention is a viral vector.Various viral vector systems suitable for expressing the RNAi construct described herein include, but are not limited to, adenovirus vector, retrovirus vector (e.g., lentivirus vector, Moloney murine leukemia virus), adeno-associated virus vector; herpes simplex virus vector; SV40 vector; polyomavirus vector; papillomavirus vector; picornavirus vector; and poxvirus vector (e.g., vaccinia virus).In some embodiments, the viral vector is a retrovirus vector (e.g., lentivirus vector).

[0091] The various vectors suitable for use in the present invention, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into the vector, and methods for delivering the vector to cells of interest are within the skill of one in the art. See, e.g., Dornburg, Gene Therap., Vol. 2:301-310, 1995; Eglitis, Biotechniques, Vol. 6:608-614, 1988; Miller, HumGene Therap., Vol. 1:5-14, 1990; Anderson, Nature, Vol. 392:25-30, 1998; Rubinson DA et al., Nat. Genet., Vol. 33:401-406, 2003; Brummelkamp et al., Science, Vol. 296:550-553, 2002; Brummelkamp et al., Cancer Cell, Vol. 2:243-247, 2002; Lee et al., Nat. Biotechnol,Vol.20:500-505,2002;Miyagishi et al.,Nat Biotechnol,Vol.20:497-500,2002;Paddison et al.,GenesDev,Vol.16:948-958,2002;Paul et al.,Nat See Biotechnol, Vol. 20:505-508, 2002; Sui et al., Proc Natl Acad Sci USA, Vol. 99: 5515-5520, 2002; and Yu et al., Proc Natl Acad Sci USA, Vol. 99: 6047-6052, 2002.

[0092] The present invention also includes pharmaceutical compositions and formulations comprising the RNAi constructs described herein and pharma- ceutical acceptable carriers, excipients, or diluents.Such compositions and formulations are useful for reducing the expression of PNPLA3 in subjects in need.When considering clinical applications, pharmaceutical compositions and formulations will be made in a form suitable for intended applications.In general, this will entail making compositions that are essentially free of pyrogens and other impurities that may be harmful to humans or animals.

[0093] The phrases "pharmacologically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers, excipients or diluents" include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, and the like, that are acceptable for use in formulating a drug, such as a drug suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the RNAi construct of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition, provided that they do not inactivate the vector or RNAi construct of the composition.

[0094] The composition and method of formulation of the pharmaceutical composition depends on several conditions, including but not limited to the route of administration, the type and extent of the disease or disorder to be treated, or the dose to be administered. In some embodiments, the pharmaceutical composition is formulated based on the intended delivery route. For example, in certain embodiments, the pharmaceutical composition is formulated for parenteral delivery. Parenteral delivery forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (e.g., a GalNAc-containing ligand described herein).

[0095] In some embodiments, the pharmaceutical composition comprises an effective amount of the RNAi construct described herein. An "effective amount" is an amount sufficient to produce a beneficial or desired clinical outcome. In some embodiments, an effective amount is an amount sufficient to reduce PNPLA3 expression in liver cells of a subject. In some embodiments, an effective amount may be an amount sufficient to only partially reduce PNPLA3 expression, for example to a level equivalent to the expression of a wild-type PNPLA3 allele in a human heterozygote. It has been reported that heterozygous human carriers of a loss-of-function PNPLA3 variant allele have lower serum levels of non-HDL cholesterol and a lower risk of coronary artery disease and myocardial infarction compared to non-carriers (Nioi et al., New England Journal of Medicine, Vol. 374(22): 2131-2141, 2016). Thus, without being bound by theory, it is believed that a partial reduction in PNPLA3 expression may be sufficient to achieve beneficial reduction in serum non-HDL cholesterol and reduced risk of coronary artery disease and myocardial infarction.

[0096] An effective amount of the RNAi construct of the present invention may be about 0.01 mg / kg body weight to about 100 mg / kg body weight, about 0.05 mg / kg body weight to about 75 mg / kg body weight, about 0.1 mg / kg body weight to about 50 mg / kg body weight, about 1 mg / kg to about 30 mg / kg body weight, about 2.5 mg / kg body weight to about 20 mg / kg body weight, or about 5 mg / kg body weight to about 15 mg / kg body weight. In certain embodiments, an effective amount of the RNAi construct of the present invention per administration may be about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg. A pharmaceutical composition comprising an effective amount of the RNAi construct may be administered weekly, biweekly, monthly, quarterly, or semi-annually. The precise determination of what will be an effective dosage and frequency of administration can be based on several factors, including the size, age, and general condition of the patient, the type of disorder being treated (e.g., myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia), the particular RNAi construct employed, and the route of administration.Estimates of effective doses and in vivo half-lives for any particular RNAi construct of the invention can be confirmed using conventional methods and / or testing in appropriate animal models.

[0097] The administration of the pharmaceutical composition of the present invention can be via any common route, so long as the target tissue is available via that route. Such routes include, but are not limited to, parenteral (e.g., subcutaneous, intramuscular, intraperitoneal, or intravenous), oral, nasal, buccal, intradermal, transdermal, and sublingual routes, or direct injection into liver tissue or delivery via the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0098] Colloidal dispersion systems, such as oil-in-water emulsions, micelles, mixed micelles and macromolecular complexes, including liposomes, nanocapsules, microspheres, beads, and lipid-based systems, can be used as delivery vehicles for the RNAi constructs of the present invention or vectors encoding such constructs. Commercially available lipid emulsions suitable for delivering the nucleic acids of the present invention include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III, and other similar lipid emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the RNAi constructs of the present invention can be complexed with lipids, particularly cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC)), distearoylphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505; 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO 003 / 093449.

[0099] In some embodiments, the RNAi constructs of the present invention are fully encapsulated in lipid formulations to form, for example, SPLPs, pSPLPs, SNALPs, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle, including SPLPs. As used herein, the term "SPLP" refers to a nucleic acid-lipid particle that includes plasmid DNA encapsulated in lipid vesicles. SNALPs and SPLPs usually contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALPs and SPLPs are highly useful for systemic administration because they exhibit long circulation life after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the site of administration). SPLPs include "pSPLPs" that contain encapsulated condensing agent-nucleic acid complexes as described in WO 00 / 03683. The nucleic acid-lipid particles usually have an average diameter of about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm, and are substantially non-toxic. In addition, the nucleic acid present in the nucleic acid-lipid particles is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and WO 96 / 40964.

[0100] Pharmaceutical compositions suitable for injection use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that they are easily syringable. The preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. Prevention of microbial action can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents that delay absorption, for example, aluminum monostearate and gelatin.

[0101] Sterile solution for injection can be prepared by incorporating active compound in appropriate amount with any other desired components (for example, as listed above) in solvent, followed by sterilization by filtration.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle containing basic dispersion medium and other desired components, for example, as listed above.In the case of sterile powder for preparing sterile solution for injection, the preferred preparation method includes vacuum drying and freeze-drying techniques, which produce powder of active component and any additional desired components from its solution that has been previously sterilized and filtered.

[0102] The compositions of the present invention can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed by free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed by free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0103] For parenteral administration in aqueous solution, for example, the solution is usually suitably buffered and the liquid diluent is first rendered isotonic, for example, with sufficient saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, a sterile aqueous medium is employed, as known to those skilled in the art, particularly in light of the present disclosure. By way of illustration, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises or consists of sterile saline and the RNAi construct described herein. In other embodiments, the pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and sterile water (e.g., water for injection, WFI). In yet other embodiments, the pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and phosphate buffered saline (PBS).

[0104] In some embodiments, the pharmaceutical composition of the present invention is packaged or stored in a device for administration.Devices for injection formulation include, but are not limited to, injection ports, pre-filled syringes, automatic injection devices, injection pumps, wearable syringes, and injection pens.Devices for aerosolization or powder formulation include, but are not limited to, inhalers, inhalers, inhalers, etc.Therefore, the present invention includes an administration device that comprises the pharmaceutical composition of the present invention for treating or preventing one or more of the disorders described herein.

[0105] Methods for inhibiting PNPLA3 expression The present invention also provides a method for inhibiting the expression of PNPLA3 gene in cells. The method includes contacting the cell with an RNAi agent, for example, a double-stranded RNAi agent, in an amount effective to inhibit the expression of PNPLA3 in the cell, thereby inhibiting the expression of PNPLA3 in the cell. The contacting of the cell with the RNAi agent, for example, a double-stranded RNAi agent, can be performed in vitro or in vivo. Contacting the cell with the RNAi agent in vivo includes contacting a cell or a group of cells in a subject, for example, a human subject, with the RNAi agent. A combination of the in vitro and in vivo methods of contacting the cell is also possible.

[0106] The present invention provides methods of reducing or inhibiting the expression of PNPLA3 in a subject in need thereof, and methods of treating or preventing a condition, disease, or disorder associated with PNPLA3 expression or activity. "Condition, disease, or disorder associated with PNPLA3 expression" refers to a condition, disease, or disorder in which the expression level of PNPLA3 is altered or elevated expression levels of PNPLA3 are associated with an increased risk of developing the condition, disease, or disorder.

[0107] The contact of cells can be direct or indirect as described above.Furthermore, the contact of cells can be achieved through targeting ligand, including any ligand described herein or known in the art.In a preferred embodiment, the targeting ligand is a carbohydrate moiety, for example, GalNAc3 ligand, or any other ligand that guides RNAi agent to the site of interest.

[0108] In one embodiment, contacting a cell with an RNAi includes "introducing" or "delivering an RNAi to a cell" by facilitating or causing uptake or absorption into the cell. The uptake or uptake of the RNAi can occur by unassisted diffusive or active cellular processes, or by auxiliary agents or devices. The introduction of the RNAi into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the RNAi can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further techniques are described herein below and / or known in the art.

[0109] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0110] The phrase "inhibiting expression of PNPLA3" is intended to refer to the inhibition of expression of any PNPLA3 gene (such as, for example, a mouse PNPLA3 gene, a rat PNPLA3 gene, a monkey PNPLA3 gene, or a human PNPLA3 gene) and variants or mutants of the PNPLA3 gene. Thus, the PNPLA3 gene may be a wild-type PNPLA3 gene, a mutant PNPLA3 gene (such as a mutant PNPLA3 gene that causes amyloid deposition), or a transgenic PNPLA3 gene in the context of a genetically engineered cell, cell population, or organism.

[0111] "Inhibiting the expression of the PNPLA3 gene" includes any level of inhibition of the PNPLA3 gene, for example, suppressing at least a portion of the expression of the PNPLA3 gene. Expression of the PNPLA3 gene can be evaluated based on the level or change in the level of any variable associated with PNPLA3 gene expression, such as, for example, PNPLA3 mRNA level, PNPLA3 protein level, or the number or extent of amyloid deposits. The level can be evaluated in an individual cell or a group of cells, including, for example, a sample derived from a subject.

[0112] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with PNPLA3 expression compared to a control level, which can be any type of control level utilized in the art, such as, for example, a baseline level prior to administration, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive drug control). In some embodiments of the methods of the present invention, expression of the PNPLA3 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0113] Inhibition of expression of the PNPLA3 gene may be evidenced by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which the PNPLA3 gene is transcribed and which has been treated (e.g., by contacting the cell with an RNAi agent of the invention or by administering an RNAi agent of the invention to a subject in which the cell is or was present) such that expression of the PNPLA3 gene is inhibited, as compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been similarly treated. In a preferred embodiment, inhibition is achieved by a reduction in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which the PNPLA3 gene is transcribed and which has been treated (e.g., by contacting the cell with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cell is or was present) such that expression of the PNPLA3 gene is inhibited, as compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but has not been similarly treated.

number

[0114] Alternatively, inhibition of expression of the PNPLA3 gene may be assessed in terms of a reduction in a parameter functionally related to PNPLA3 gene expression, such as PNPLA3 protein expression or Hedgehog pathway protein activity. PNPLA3 gene silencing may be determined by any assay known in the art in any cell that constitutively or by genomic engineering expresses PNPLA3.

[0115] Inhibition of the expression of PNPLA3 protein can be demonstrated by a reduction in the level of PNPLA3 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived from a subject).As explained above, for the assessment of mRNA suppression, inhibition of protein expression levels in treated cells or cell groups can be similarly expressed as a percentage of the level of protein in control cells or cell groups.

[0116] The control cell or cell group that can be used to evaluate the inhibition of the expression of PNPLA3 gene includes the cell or cell group that has not yet been contacted with the RNAi agent of the present invention.For example, the control cell or cell group can be obtained from an individual subject (e.g., human or animal subject) before the subject is treated with the RNAi agent.

[0117] The level of PNPLA3 mRNA expressed by a cell or group of cells, or the level of circulating PNPLA3 mRNA, can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of PNPLA3 in a sample is determined by detecting a transcribed polynucleotide, such as, for example, the mRNA of the PNPLA3 gene, or a portion thereof. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kit (Qiagen) or PAXgene (PreAnalytix, Switzerland). Exemplary assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating PNPLA3 mRNA can be detected using the methods described in PCT / US2012 / 043584, the entire contents of which are incorporated herein by reference.

[0118] In one embodiment, the level of expression of PNPLA3 is determined using a nucleic acid probe. The term "probe" as used herein refers to any molecule that can selectively bind to a specific PNPLA3. Probes can be synthesized by those skilled in the art or derived from suitable biological preparations. Probes can also be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0119] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method of determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA of PNPLA3. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In an alternative embodiment, the probe is immobilized on a solid surface, for example in an Affymetrix gene chip array, and the mRNA is contacted with the probe. Those skilled in the art can easily adapt known mRNA detection methods for use in determining the level of PNPLA3 mRNA.

[0120] Alternative methods for determining the level of expression of PNPLA3 in a sample include, for example, RT-PCR (experimental embodiment described in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. (1989) Bio / Technology 6:1197), and the like. al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, for example, a step of nucleic acid amplification of mRNA in a sample and / or reverse transcriptase (to prepare cDNA), followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are useful for detecting nucleic acid molecules, especially when they are present in very low numbers. In a specific embodiment of the present invention, the level of expression of PNPLA3 is determined by quantitative fluorescent RT-PCR (i.e., TaqMan™ System). The expression level of PNPLA3 mRNA can be monitored using membrane blots (such as those used in hybridization analyses such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference. Determining the expression level of PNPLA3 can also include the use of a nucleic acid probe in solution.

[0121] In a preferred embodiment, the level of expression of mRNA is assessed using a branched DNA (bDNA) assay or real-time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.

[0122] The level of PNPLA3 protein expression can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), high diffusion chromatography, liquid or gel precipitin reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (one-way or two-way), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0123] In some embodiments, the effectiveness of the methods of the present invention can be monitored by detecting or monitoring a reduction in symptoms of PNPLA3 disease, such as reduced edematous swelling of the extremities, face, larynx, upper airway, abdomen, trunk, and genitals, prodrome, laryngeal edema, non-pruritic rash, nausea, vomiting, or abdominal pain. These symptoms can be assessed in vitro or in vivo using any method known in the art.

[0124] In some embodiments of the method of the present invention, the RNAi agent is administered to the subject so that the RNAi agent is delivered to a specific site in the subject. The inhibition of the expression of PNPLA3 can be evaluated using the measurement of the level or change in the level of PNPLA3 mRNA or PNPLA3 protein in a sample derived from a body fluid or tissue from a specific site in the subject. In a preferred embodiment, the site is selected from the group consisting of liver, choroid plexus, retina, and pancreas. The site can be a small unit or subgroup of cells from any one of the aforementioned sites. The site can also include cells that express a specific type of receptor.

[0125] Methods for treating or preventing PNPLA3-related diseases The present invention provides therapeutic and prophylactic methods, comprising administering a composition comprising an RNAi agent, or a pharmaceutical composition comprising an RNAi agent, or a vector comprising an RNAi agent of the present invention to a subject having a PNPLA3-related disease, disorder, and / or condition, or susceptible to developing a PNPLA3-related disease, disorder, and / or disorder. Non-limiting examples of PNPLA3-related diseases include, for example, fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, inflammation of the liver, hepatocyte necrosis, liver fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). In one embodiment, the PNPLA3-related disease is NAFLD. In another embodiment, the PNPLA3-related disease is NASH. In another embodiment, the PNPLA3-related disease is fatty liver (steatosis). In another embodiment, the PNPLA3-related disease is insulin resistance. In another embodiment, the PNPLA3-related disease is not insulin resistance.

[0126] In certain embodiments, the present invention provides a method for reducing the expression of PNPLA3 in a patient in need thereof, comprising administering any of the RNAi constructs described herein to the patient.As used herein, the term "patient" refers to a mammal, including human, and can be used interchangeably with the term "subject".Preferably, the expression level of PNPLA3 in the liver cells of the patient is reduced after administration of the RNAi construct, compared with the expression level of PNPLA3 of the patient who does not receive the RNAi construct.

[0127] The method of the present invention is useful for treating subjects with PNPLA3-related diseases, such as subjects who will benefit from reducing PNPLA3 gene expression and / or PNPLA3 protein production.In one aspect, the present invention provides a method for reducing the level of patatin-like phospholipase domain-containing 3 (PNPLA3) gene expression in subjects with non-alcoholic fatty liver disease (NAFLD).In another aspect, the present invention provides a method for reducing the level of PNPLA3 protein in subjects with NAFLD.The present invention also provides a method for reducing the level of Hedgehog pathway activity in subjects with NAFLD.

[0128] In another aspect, the present invention provides a method for treating a subject with NAFLD.In one aspect, the present invention provides a method for treating a subject with PNPLA3-related disease, such as fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), liver cirrhosis, fat accumulation in the liver, liver inflammation, hepatocyte necrosis, liver fibrosis, obesity, or nonalcoholic fatty liver disease (NAFLD).The treatment method (and use) of the present invention comprises administering to a subject, for example, a human, a therapeutically effective amount of the RNAi agent of the present invention that targets PNPLA3 gene or a pharmaceutical composition of the present invention that comprises the RNAi agent of the present invention that targets PNPLA3 gene or the vector of the present invention that comprises the RNAi agent of the present invention that targets PNPLA3 gene.

[0129] In one aspect, the present invention provides a method for preventing at least one symptom of the subject with NAFLD, such as the presence of increased Hedgehog signaling pathway, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider veins, yellowing of skin and eyes (jaundice), itching, water accumulation and swelling of legs (edema), abdominal distension (ascites) and mental confusion.This method includes administering a therapeutically effective amount of RNAi agent, such as the dsRNA of the present invention, pharmaceutical composition or vector to the subject, thereby preventing at least one symptom of the subject with disorder that will benefit from reducing PNPLA3 gene expression.

[0130] In another aspect, the present invention provides the use of a therapeutically effective amount of the RNAi agent of the present invention for treating a subject, for example, a subject that will benefit from reducing and / or inhibiting PNPLA3 gene expression.In a further aspect, the present invention provides the use of the RNAi agent of the present invention targeting the PNPLA3 gene, for example, a dsRNA, or a pharmaceutical composition comprising the RNAi agent targeting the PNPLA3 gene, in the manufacture of a medicament for treating a subject, such as a subject with a disorder that will benefit from reducing PNPLA3 gene expression, for example, a PNPLA3-related disease, for example, a subject that will benefit from reducing and / or inhibiting PNPLA3 gene expression and / or PNPLA3 protein production.

[0131] In another aspect, the present invention provides use of an RNAi, e.g., a dsRNA, of the present invention for preventing at least one symptom in a subject suffering from a disorder that would benefit from reducing and / or inhibiting PNPLA3 gene expression and / or PNPLA3 protein production.

[0132] In a further aspect, the present invention provides the use of an RNAi agent of the present invention in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reducing and / or inhibiting PNPLA3 gene expression and / or PNPLA3 protein production, such as a PNPLA3-related disease.

[0133] In one embodiment, the RNAi agent targeting PNPLA3 reduces or decreases expression of the PNPLA3 gene in cells, tissues, blood or other tissue or bodily fluid of the subject by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 108%, 109%, 109%, 109%, 104%, 105%, 106%, 107%, 108 The subject may be administered a PNPLA3-associated disease, such as non-alcoholic fatty liver disease (NAFLD), such that the PNPLA3-associated disease is reduced by 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0134] The methods and uses of the present invention include administering a composition as described herein such that expression of the target PNPLA3 gene is reduced for, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target PNPLA3 gene is reduced for an extended period of time, for example, at least about 2, 3, 4, 5, 6, 7 days or more, for example, about 1 week, 2 weeks, 3 weeks, or about 4 weeks or more.

[0135] The administration of dsRNA according to the method and use of the present invention can result in a reduction in the severity, signs, symptoms, and / or markers of PNPLA3-related diseases, such as non-alcoholic fatty liver disease (NAFLD), in patients with such diseases or disorders. "Reduction" in this context means a statistically significant reduction in such levels. Reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%. The effectiveness of disease treatment or prevention can be evaluated, for example, by measuring disease progression, disease remission, symptom severity, pain reduction, quality of life, the dose of medicine required to maintain the therapeutic effect, the level of disease markers, or any other measurable parameter level appropriate for the given disease being treated or targeted for prevention. It is well within the capabilities of a person skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one of these parameters or any combination of these parameters. For example, the effectiveness of treatment of NAFLD can be evaluated by, for example, regular monitoring of NAFLD symptoms, liver fat levels, or the expression of downstream genes. Comparison of the first reading with the subsequent reading provides the physician with an indication of whether the treatment is effective. It is well within the capabilities of a person skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one of these parameters or any combination of these parameters. In the context of administering an RNAi targeting PNPLA3 or a pharmaceutical composition thereof, "effective against" PNPLA3-related disease indicates that administration in a clinically relevant manner provides at least a statistically significant proportion of patients with a beneficial effect, such as symptom improvement, cure, disease reduction, life extension, improvement in quality of life, or other effects generally recognized as favorable by physicians familiar with the treatment of NAFLD and / or PNPLA3-related disease and related causes.

[0136] Treatment or prophylactic effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or by the absence of the aggravation or onset of symptoms that would otherwise be expected.As an example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more of the measurable parameters of the disease can indicate effective treatment.The efficacy of a given RNAi drug or a formulation of the drug can also be determined using an experimental animal model for a given disease known in the art.When using an experimental animal model, the efficacy of treatment is demonstrated when a statistically significant reduction in markers or symptoms is observed.

[0137] The subjects included dsRNA at approximately 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, 0.35 mg / kg, 0.4 mg / kg, 0.45 mg / kg, 0.5 mg / kg, 0.55 mg / kg, 0.6 mg / kg, 0.65 mg / kg, 0.7 mg / kg, 0.75 mg / kg, 0.8 mg / kg, 0.85 mg / kg, 0.9 mg / kg, 0.95 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg dsRNA, 2.6 mg / kg dsRNA, 2.7 mg / kg dsRNA, 2.8 mg / kg dsRNA, 2.9 mg / kg dsRNA, 3.0 mg / kg dsRNA, 3.1 mg / kg dsRNA, 3.2 mg / kg dsRNA, 3.3 mg / kg dsRNA, 3.4 mg / kg dsRNA, 3.5 mg / kg dsRNA, 3.6 mg / kg dsRNA, 3.7 mg / kg dsRNA, 3.8 mg / kg dsRNA, 3.9 mg / kg dsRNA, 4.0 mg / kg dsRNA, 4.1 mg / kg dsRNA, 4.2 mg / kg dsRNA, 4.3 mg / kg dsRNA, 4.4 mg / kg dsRNA, 4.5 mg / kg dsRNA, 4.6 mg / kg dsRNA, 4.7 mg / kg dsRNA, 4.8 mg / kg dsRNA, 4.9 mg / kg dsRNA, 5.0 mg / kg dsRNA, 5.1 mg / kg dsRNA, 5.2 mg / kg dsRNA, 5.3 mg / kg dsRNA, 5.4 mg / kg dsRNA, 5.5 mg / kg dsRNA, 5.6 mg / kg dsRNA, 5.7 mg / kg dsRNA, 5.8 mg / kg dsRNA, 5.9 mg / kg dsRNA, 6.0 mg / kg dsRNA, 6.1 mg / kg dsRNA, 6.2 mg / kg dsRNA, 6.3 mg / kg dsRNA, 6.4 mg / kg dsRNA, 6.5 mg / kg dsRNA, 6.6 mg / kg dsRNA, 6.7mg / kg dsRNA, 6.8mg / kg dsRNA, 6.9mg / kg dsRNA, 7.0mg / kg dsRNA, 7.1mg / kg dsRNA, 7.2mg / kg dsRNA, 7.3mg / kg dsRNA, 7.4mg / kg dsRNA, 7.5mg / kg dsRNA, 7.6mg / kg dsRNA, 7.7mg / kg dsRNA, 7.8mg / kg dsRNA, 7.9mg / kg dsRNA, 8.0mg / kg dsRNA, 8.1mg / kg dsRNA, 8.2mg / kg dsRNA, 8.3mg / kg dsRNA, 8.4mg / kg dsRNA, 8.5mg / kg dsRNA, 8.6mg / kg dsRNA, 8.7mg / kg dsRNA, 8.8mg / kg A therapeutic amount of RNAi may be administered, such as dsRNA, 8.9 mg / kg dsRNA, 9.0 mg / kg dsRNA, 9.1 mg / kg dsRNA, 9.2 mg / kg dsRNA, 9.3 mg / kg dsRNA, 9.4 mg / kg dsRNA, 9.5 mg / kg dsRNA, 9.6 mg / kg dsRNA, 9.7 mg / kg dsRNA, 9.8 mg / kg dsRNA, 9.9 mg / kg dsRNA, 9.0 mg / kg dsRNA, 10 mg / kg dsRNA, 15 mg / kg dsRNA, 20 mg / kg dsRNA, 25 mg / kg dsRNA, 30 mg / kg dsRNA, 35 mg / kg dsRNA, 40 mg / kg dsRNA, 45 mg / kg dsRNA, or about 50 mg / kg dsRNA. In one embodiment, a subject may be administered 0.5 mg / kg of dsRNA. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0138] Administration of RNAi, for example, may reduce the presence of PNPLA3 protein levels in cells, tissues, blood, urine, or other compartments of a patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 108%, 109%, 109%, 109%, 110%, 111%, %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0139] Prior to administration of the full dose of RNAi, the patient may be administered a smaller dose, such as a 5% infusion, and monitored for adverse effects, such as allergic reactions, hi another example, the patient may be monitored for unwanted immunostimulatory effects, such as elevated cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0140] Due to the inhibitory effect on PNPLA3 expression, the composition according to the present invention or a pharmaceutical composition prepared therefrom can improve the quality of life.

[0141] The RNAi of the present invention may be administered in a "naked" form, in which modified or unmodified RNAi agent is directly suspended in aqueous or suitable buffer solution as "free RNAi". Free RNAi is administered in the absence of pharmaceutical composition. Free RNAi may be in a suitable buffer solution. The buffer solution may include acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolality of the buffer solution containing RNAi may be adjusted to be suitable for administration to a subject.

[0142] Alternatively, the RNAi of the present invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

[0143] Subjects that would benefit from reducing and / or inhibiting PNPLA3 gene expression are those who have non-alcoholic fatty liver disease (NAFLD) and / or a PNPLA3-related disease or disorder as described herein.

[0144] Treatment of subjects that would benefit from reducing and / or inhibiting PNPLA3 gene expression includes therapeutic and prophylactic treatments.

[0145] The present invention further provides methods for treating subjects who would benefit from reducing and / or inhibiting PNPLA3 gene expression, e.g., subjects having a PNPLA3-related disease, and uses of RNAi agents or pharmaceutical compositions thereof, e.g., in combination with known pharmaceuticals and / or known therapies, e.g., those currently used to treat these disorders, along with other pharmaceuticals and / or therapies.

[0146] For example, in some embodiments, the RNAi that targets PNPLA3 gene is administered in combination with, for example, the agent useful for treating PNPLA3-related disease, as described elsewhere herein.For example, the additional therapeutic agent and treatment that is suitable for treating the subject that will benefit from the reduction of PNPLA3 expression, for example, the subject that has PNPLA3-related disease, include the RNAi agent that targets different parts of PNPLA3 gene, the therapeutic agent and / or treatment for treating PNPLA3-related disease, or any combination of the above.

[0147] In some embodiments, the first RNAi agent that targets PNPLA3 gene is administered in combination with the second RNAi agent that targets a different part of PNPLA3 gene.For example, the first RNAi agent comprises a first sense strand and a first antisense strand that form a double-stranded region, wherein substantially all of the nucleotides of the first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, and the first sense strand is conjugated to a ligand that is linked at its 3' end, and this ligand is one or more GalNAc derivatives that are linked via a bivalent or trivalent branched linker; and the second RNAi agent comprises a second sense strand and a second antisense strand that form a double-stranded region, wherein substantially all of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, and the second sense strand is conjugated to a ligand that is linked at its 3' end, and this ligand is one or more GalNAc derivatives that are linked via a bivalent or trivalent branched linker.

[0148] In one embodiment, all of the nucleotides of the first and second sense strands and / or all of the nucleotides of the first and second antisense strands comprise the modification.

[0149] In one embodiment, the at least one modified nucleotide is selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxy modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-O-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide comprising a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, and a nucleotide comprising a 5'-phosphate mimic.

[0150] In certain embodiments, the first RNAi agent that targets the PNPLA3 gene is administered in combination with the second RNAi agent that targets a gene different from the PNPLA3 gene.For example, the RNAi agent that targets the PNPLA3 gene can be administered in combination with the RNAi agent that targets the SCAP gene.The first RNAi agent that targets the PNPLA3 gene and the second RNAi agent that targets a gene different from the PNPLA3 gene, for example, the SCAP gene, can be administered as part of the same pharmaceutical composition.Alternatively, the first RNAi agent that targets the PNPLA3 gene and the second RNAi agent that targets a gene different from the PNPLA3 gene, for example, the SCAP gene, can be administered as part of different pharmaceutical compositions.

[0151] The RNAi agent and additional therapeutic agent and / or treatment may be administered simultaneously and / or in the same formulation, e.g., parenterally, or the additional therapeutic agent may be administered as part of a separate composition or separately, and / or by other methods known in the art or described herein.

[0152] The present invention also provides a method of using the RNAi agent of the present invention and / or a composition comprising the RNAi agent of the present invention to reduce and / or inhibit PNPLA3 expression in a cell. In another aspect, the present invention provides the RNAi of the present invention and / or a composition comprising the RNAi of the present invention for use in reducing and / or inhibiting PNPLA3 gene expression in a cell. In yet another aspect, the present invention provides the use of the RNAi of the present invention and / or a composition comprising the RNAi of the present invention for use in reducing and / or inhibiting PNPLA3 gene expression in a cell for the manufacture of a medicament for reducing and / or inhibiting PNPLA3 gene expression in a cell. In yet another aspect, the present invention provides the RNAi of the present invention and / or a composition comprising the RNAi of the present invention for use in reducing and / or inhibiting PNPLA3 protein production in a cell. In yet another aspect, the present invention provides the use of the RNAi of the present invention and / or a composition comprising the RNAi of the present invention for use in reducing and / or inhibiting PNPLA3 protein production in a cell for the manufacture of a medicament for reducing and / or inhibiting PNPLA3 protein production in a cell. The method and use include contacting a cell with the RNAi of the present invention, e.g., dsRNA, and maintaining the cell for a sufficient time to obtain degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting the expression of the PNPLA3 gene or inhibiting PNPLA3 protein production in the cell.

[0153] The reduction of gene expression can be evaluated by any method known in the art.For example, the reduction of the expression of PNPLA3 can be determined by using methods that are common to those skilled in the art, such as Northern blotting, qRT-PCR to determine the mRNA expression level of PNPLA3, by using methods that are common to those skilled in the art, such as Western blotting, immunological methods, flow cytometry, ELISA, etc., to determine the protein level of PNPLA3, and / or by determining the biological activity of PNPLA3.

[0154] In the methods and uses of the present invention, the cells may be contacted in vitro or in vivo, i.e. the cells may be within a subject.

[0155] The cell suitable for treatment using the method of the present invention can be any cell that expresses PNPLA3 gene, for example, a cell from a subject with NAFLD, or a cell that contains an expression vector that contains PNPLA3 gene or a part of PNPLA3 gene.The cell suitable for use in the method and use of the present invention can be a mammalian cell, for example, a primate cell (such as a human cell or a non-human primate cell, for example, a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster cell, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), an avian cell (such as a duck cell or a goose cell), or a whale cell.In one embodiment, the cell is a human cell.

[0156] PNPLA3 gene expression is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 108%, 109%, 109%, 109%, 109%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% inhibition.

[0157] PNPLA3 protein production is at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 109%, 108%. The expression level may be inhibited by 1%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

[0158] The in vivo method and use of the present invention may comprise administering to a subject a composition comprising an RNAi, the RNAi comprising a nucleotide sequence complementary to at least a portion of the RNA transcript of the PNPLA3 gene of the mammal to be treated.When the organism to be treated is a human, the composition can be administered by any means known in the art, including but not limited to subcutaneous, intravenous, oral, intraperitoneal, or intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intramuscular, transdermal, airway (spray), nasal, rectal, parenteral routes, and topical (including buccal and sublingual) administration.In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.In one embodiment, the composition is administered by subcutaneous injection.

[0159] In some embodiments, administration is via depot injection. Depot injection can release RNAi consistently over a long period of time. Thus, depot injection can reduce the frequency of administration required to obtain desired effect, for example, desired inhibition of PNPLA3, or therapeutic or prophylactic effect. Depot injection can also provide more consistent serum concentration. Depot injection can include subcutaneous injection or intramuscular injection. In a preferred embodiment, depot injection is subcutaneous injection.

[0160] In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers RNAi to a subject.

[0161] The mode of administration can be selected based on whether local or systemic treatment is desired, and based on the area to be treated. The route and site of administration can be selected to enhance targeting.

[0162] In one aspect, the present invention also provides a method for inhibiting the expression of PNPLA3 gene in mammals, such as humans.The present invention also provides a composition comprising RNAi, such as dsRNA that targets PNPLA3 gene in mammalian cells, for use in inhibiting the expression of PNPLA3 gene in mammals.In another aspect, the present invention provides the use of RNAi, such as dsRNA that targets PNPLA3 gene in mammalian cells, in the manufacture of the medicine that inhibits the expression of PNPLA3 gene in mammals.

[0163] The methods and uses include administering to a mammal, e.g., a human, a composition comprising RNAi, e.g., dsRNA targeting the PNPLA3 gene in a cell of the mammal, and maintaining the mammal for a period of time sufficient to obtain degradation of mRNA transcripts of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the mammal.

[0164] The reduction of gene expression can be evaluated in peripheral blood samples of RNAi-administered subjects by any method known in the art, for example, qRT-PCR as described herein.The reduction of protein production can be evaluated by any method and method known in the art, for example, ELISA or Western blotting as described herein.In one embodiment, tissue sample serves as tissue material for monitoring the reduction of PNPLA3 gene and / or protein expression.In another embodiment, blood sample serves as tissue material for monitoring the reduction of PNPLA3 gene and / or protein expression.

[0165] In one embodiment, validation of RISC-mediated cleavage of the target in vivo following administration of an RNAi agent is performed by performing 5'-RACE or modifications of protocols known in the art (Lasham A et al., (2010) Nucleic Acid Res., 38(3)p-el9) (Zimmermann et al. (2006) Nature 441:111-4).

[0166] It is understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by substituting uracil bases in the sequence with thymine bases.Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by substituting uracil bases in the sequence with thymine bases.Deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing mixtures of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein are included in the present invention.

[0167] In addition, any nucleic acid sequence disclosed herein may be modified by any combination of chemical modifications. Those skilled in the art will readily understand that in certain cases, such designations of "RNA" or "DNA" to describe modified polynucleotides are arbitrary. For example, a polynucleotide that includes a nucleotide with a 2'-OH substituent of the ribose sugar and a thymine base may be described as a DNA molecule with modified sugars (2'-OH for the natural 2'-H of DNA) or an RNA molecule with modified bases (thymine (methylated uracil) for the natural uracil of RNA).

[0168] Thus, the 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 with modified nucleic acid bases. As a further example, and without limitation, a polynucleotide having the sequence "ATCGATCG" encompasses any polynucleotide having such a sequence, whether modified or unmodified, including but not limited to such compounds including RNA bases, e.g., those with the sequence "AUCGAUCG", as well as those with some DNA and RNA bases such as "AUCGATCG", and polynucleotides with other modified bases such as "ATmeCGAUCG", in which meC denotes a cytosine base with a methyl group at the 5-position.

[0169] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

[0170] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. To the extent that definitions or terms provided in a reference incorporated by reference differ from the terms and discussion provided herein, the terms and definitions shall control.

[0171] Equivalent The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the present invention. The foregoing description and examples detail certain preferred embodiments of the present invention and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing is, it will be understood that the present invention can be practiced in many ways and that the present invention should be construed in accordance with the appended claims and their equivalents.

[0172] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0173] All animal studies described herein were approved by Amgen's Institutional Animal Care and Use Committee (IACUC) and maintained in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition (National Research Council (US). Committee for Update of the Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (US) and National Academies Press (US) (2011) Guide for the care and use of laboratory animals. 8th Ed., National Academies Press, Washington, DC). Mice were housed singly in an air-conditioned space at 22 ± 2 °C with a 12-h light; 12-h dark cycle (0600-1800 h). Animals were fed ad libitum with a regular chow diet (Envigo, 2920X, or diets specified otherwise) and water (reverse osmosis purified) by an automated watering system unless otherwise indicated. At termination, blood was collected by cardiac puncture under deep anesthesia and subsequently approved for use by the Association for Assessment and Accreditation of Laboratory Animals (AHA). Animals were euthanized by secondary physical methods in accordance with the American Academy of Clinical Nursing Care (AAALAC) guidelines. EXAMPLES

[0174] Example 1: Selection, design and synthesis of modified PNPLA3 siRNA molecules The identification and selection of optimal sequences for therapeutic siRNA molecules targeting patatin-like phospholipase domain containing 3 (PNPLA3) was confirmed using bioinformatics analysis of the human PNPLA3 transcript (NM_025225.2). Table 1 shows the sequences identified as having therapeutic properties. Across the various sequences, {INVAB} is inverted abasic, {INVDA} is inverted deoxythymidine, GNA is glycol nucleic acid, dT is deoxythymidine, and dC is deoxycytosine.

[0175] [Table 1]

[0176] [Table 2]

[0177] [Table 3]

[0178] [Table 4]

[0179] [Table 5]

[0180] [Table 6]

[0181] [Table 7]

[0182] [Table 8]

[0183]

Table 9

[0184]

Table 10

[0185]

Table 11

[0186]

Table 12

[0187]

Table 13

[0188]

Table 14

[0189]

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[0190]

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[0191]

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[0192]

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[0193]

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[0194]

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[0196]

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[0197]

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[0198]

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[0199]

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[0200]

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[0201]

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[0202]

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[0203]

Table 29

[0204]

Table 30

[0205]

Table 31

[0206]

Table 32

[0207] Chemical modifications were incorporated into PNPLA3 siRNA molecules to improve the potency and in vivo stability of PNPLA3 siRNA sequences. Specifically, 2'-O-methyl and 2'-fluoro modifications of ribose sugars were incorporated at specific positions within PNPLA3 siRNA. Phosphorothioate internucleotide linkages were also incorporated at the ends of antisense and / or sense sequences. Table 2 below shows the modifications in each of the sense and antisense sequences of modified PNPLA3 siRNAs. The nucleotide sequences in Table 2 and other parts of this application are listed according to the following notation: A, U, G, and C = corresponding ribonucleotides; dT = deoxythymidine; dA = deoxyadenosine; dC = deoxycytidine; dG = deoxyguanosine; invDT = inverted deoxythymidine; invDA = inverted deoxyadenosine; invDC = inverted deoxycytidine; invDG = inverted deoxyguanosine; a, u, g, and c = corresponding 2'-O-methyl ribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; Ab = abasic; MeO-I = 2' methoxyinosine; GNA = glycol nucleic acid; sGNA = glycol nucleic acid with 3' phosphorothioate; LNA = locked nucleic acid. The insertion of "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothiodiester group (e.g., a phosphorothioate internucleotide linkage). Unless otherwise indicated, all other nucleotides are linked by 3'-5' phosphodiester groups. Each of the siRNA compounds in Table 2 contains a 19 base pair duplex region with a 2 nucleotide overhang at the 3' end of both strands or a blunt end at one or both ends. GalNAc3K2AhxC6 is [ka] It is.

[0208] [Table 33]

[0209] [Table 34]

[0210]

Table 35

[0211]

Table 36

[0212]

Table 37

[0213]

Table 38

[0214]

Table 39

[0215]

Table 40

[0216]

Table 41

[0217]

Table 42

[0218]

Table 43

[0219]

Table 44

[0220]

Table 45

[0221]

Table 46

[0222]

Table 47

[0223]

Table 48

[0224]

Table 49

[0225]

Table 50

[0226]

Table 51

[0227]

Table 52

[0228]

Table 53

[0229]

Table 54

[0230] [Table 55]

[0231] [Table 56]

[0232] [Table 57]

[0233] Example 2: Efficacy of selected PNPLA3 siRNA molecules in an RNA FISH assay A series of fully chemically modified siRNAs, including siRNAs spanning the rs738409 and / or rs738408 SNPs in PNPLA3, were prepared and tested for efficacy and selectivity of mRNA knockdown in vitro. Each siRNA duplex consisted of two strands: a sense or "passenger" strand and an antisense or "guide" strand. The strands were 21 or 23 nucleotides long and had 19 complementary base pairs. In some cases, there was a 2-base pair 3' overhang. The siRNAs were prepared by replacing the natural 2'-OH in the ribose of each nucleotide with either a 2'-OMe or 2'-F group. Optionally, the phosphodiester internucleotide linkages in one or both strands were replaced with phosphorothioates to reduce exonuclease degradation.

[0234] The effectiveness of each of the siRNA molecules in reducing PNPLA3 expression was evaluated using a 384-well format in vitro siRNA transfection assay followed by a fluorescent in situ hybridization (RNA FISH) assay targeting ribonucleic acid molecules to determine IC50 and maximum activity values. This assay was performed on human hepatocellular carcinoma cell line Hep3B cells (ATCC HB-8064) and Chinese hamster ovary (CHO) cells expressing human PNPLA3 I148I. Human hepatocellular carcinoma HepB3 was maintained at 37°C and 5% CO2 in EMEM medium (ATCC 30-2003) supplemented with 10% fetal bovine serum and 1% antibiotic / antimycotic. CHO cells expressing human PNPLA3 I148I were maintained at 37°C and 5% CO2 in medium containing 50% CD-CHO (Life Technologies), 50% Ex-Cell CHO5 medium (Sigma), 8 mM L-glutamine, 1xHT, 1% antibiotic / antimycotic, and 10 μg / mL puromycin.

[0235] For Hep3B cell assays, transfection complexes of siRNA molecules and Lipofectamine RNAiMax transfection reagent (Life Technologies) in EMEM medium (ATCC 30-2003) were prepared at 10 μg per well in 384-well plates (PerkinElmer) according to the manufacturer's recommendations. For CHO human cell assays, transfection complexes of siRNA molecules and Lipofectamine RNAiMax transfection reagent in F12K medium (Mediatech) were prepared at 10 μg per well in 384-well plates according to the manufacturer's recommendations. Cells were diluted to 67,000 cells / ml in antibiotic / antimycotic-free medium and 30 μl was added to each well for a final density of 2000 cells / well in 40 μl medium. After 20 min of incubation at room temperature, plates were transferred to an incubator at 37° C. and 5% CO2. Hep3B cell transfection assays were incubated for 72 hours and CHO human PNPLA3 I148I transfection assays were incubated for 48 hours.

[0236] Upon harvesting, cells were fixed in 8% formaldehyde fixative (Thermo Scientific) for 15 minutes at room temperature. Plates were then dehydrated through successive 50%, 70%, and 100% ethanol washes. Plates were then sealed and stored at -20°C.

[0237] RNA FISH assays were performed using the Affymetrix QuantiGene® View RNA HC Screening Assay Kit (QVP0011), the Affymetrix View HC Signal Amplification Kit 3-plex (QVP0213), and the Affymetrix gene-specific probes: PNPLA3 Human 0.33 mL View RNA Type 6 (650 label) VA6-20279-01 and PPIB Human 0.44 mL View RNA Type 1 (488 label) VA1-10148-01.

[0238] First, the plates were rehydrated by successive 100%, 70%, and 50% ethanol washes. Next, the cells were washed with PBS, followed by permeabilization and protease digestion according to the kit's instructions. The Working Probe Set of interest was prepared according to the manufacturer's protocol, added to the wells, and incubated at 40°C for 3 hours. For sequential hybridization with the Working Probe Set, Working PreAmp, Working Amp, and Working LP, the manufacturer's protocol was followed. Finally, nuclear counterstain was applied (Hoechst 33342 and Cell Mask Blue; Molecular Probes). The plates were incubated at room temperature for 30 minutes, washed with PBS, and overlaid with 80 μl of PBS, and then the plates were sealed for imaging.

[0239] All plates were imaged on an Opera Phenix high content screening system (PerkinElmer) using the UV channel for Hoechst 33342 and Cell Mask Blue, the 488 channel for Type 1 probes, and the 647 channel for Type 6 probes.

[0240] RNA FISH data was analyzed using Columbus software, and images were generated using Genedata Screener. The results of the assay for PNPLA3 I148I transfected CHO are shown in Table 3. The results of the assay for PNPLA3 I148M transfected CHO are shown in Table 4. PNPLA3 knockdown provides the percentage of knockdown compared to control. Negative values ​​indicate a reduction in PNPLA3 levels.

[0241] [Table 58]

[0242]

Table 59

[0243]

Table 60

[0244] RNA FISH was also performed on a hepatic cell line containing the double mutant PNPLA3-rs738408-rs738409 and a control wild-type cell line, Hep3B. Hep3B and HepG2 cells (purchased from ATCC) were cultured in minimum essential medium (MEM from Corning for Hep3B and EMEM from ATCC for HepG2) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Corning). siRNA transfection was performed as follows: 1 μL of test siRNA and 4 μL of plain MEM or EMEM depending on the cell line were added to a PDL-coated CellCarrier-384 Ultra assay plate (PerkinElmer) by BioMek FX (Beckman Coulter). Next, 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) pre-diluted in plain MEM or EMEM (specifically, 0.035 μL of RNAiMAX in 5 μL of MEM for Hep3B and 0.06 μL of RNAiMAX in 5 μL of EMEM for HepG2) was dispensed into the assay plate by a Multidrop Combi reagent dispenser (Thermo Fisher Scientific). After 20 min incubation of the siRNA / RNAiMAX mixture at room temperature (RT), 30 μL of either Hep3B or HepG2 cells (2000 cells per well) in MEM or EMEM supplemented with 10% FBS and 1% PS were added to the transfection complex using the Multidrop Combi reagent dispenser. The assay plate was incubated for 20 min at RT before being placed in the incubator. The cells were then incubated at 37° C. and 5% CO2 for 72 h. The ViewRNA ISH cell assay was performed according to the manufacturer's protocol (Thermo Fisher Scientific) using an in-house automated FISH assay platform for liquid handling.Briefly, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 min at RT, permeabilized with detergent for 3 min at RT, followed by treatment with protease solution for 10 min at RT. Incubation of target-specific probe pairs (Thermo Fisher Scientific) was performed for 3 h, except for Preamplifier, Amplifier, and Label Probe (Thermo Fisher Scientific), which were each 1 h. All hybridization steps were performed at 40°C in a Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific). After the hybridization reaction, cells were stained with Hoechst and CellMask Blue (Thermo Fisher Scientific) for 30 min, and then imaged on an Opera Phenix (PerkinElmer). Images were analyzed using a Columbus image data archiving and analysis system (PerkinElmer) to obtain the average spot count per cell. Spot counts were normalized using high dose (containing phosphate buffered saline, Corning) and low dose (without the probe pair of interest) control wells. Normalized values ​​were plotted against total siRNA concentration, and data was fitted to a four-parameter sigmoidal model in Genedata Screener (Genedata) to obtain IC50 and maximum activity. The results of the assay for HepG2 cells are shown in Table 5, and the results of the assay for Hep3B cells are shown in Table 6. PNPLA3 knockdown provides the percentage of knockdown compared to the control. Negative values ​​indicate a reduction in PNPLA3 levels. In cases where duplexes were performed more than once, the average IC50 is shown with the standard deviation.

[0245] [Table 61]

[0246] [Table 62]

[0247]

Table 63

[0248]

Table 64

[0249]

Table 65

[0250]

Table 66

[0251]

Table 67

[0252]

Table 68

[0253]

Table 69

[0254]

Table 70

[0255]

Table 71

[0256]

Table 72

[0257] [Table 73]

[0258] [Table 74]

[0259] [Table 75]

[0260] [Table 76]

[0261] [Table 77]

[0262] [Table 78]

[0263] [Table 79]

[0264] Example 3: Droplet digital PCR assay of siRNA for PNPLA3-rs738409 and PNPLA3-rs738409-rs738408 Human primary hepatocytes (Xenotech / Sekisui donor lot #HC3-38) were thawed in OptiThaw medium (Xenotech cat#K8000) according to the manufacturer's protocol, cells were centrifuged and after medium aspirate, resuspended in OptiPlate hepatocyte medium (Xenotech cat#K8200) and plated in 96-well collagen-coated plates (Greiner cat#655950). After a 2-4 hour incubation period, the medium was removed and replaced with OptiCulture hepatocyte medium (Xenotech cat#K8300). 2-4 hours after the addition of OptiCulture medium, GalNAc-conjugated siRNA was delivered to the cells by free uptake (no transfection reagent). Cells were incubated at 37°C and 5% CO2 for 24-72 hours. Cells were subsequently lysed in Qiagen's RLT buffer (79216) supplemented with 1% 2-mercaptoethanol (Sigma, M-3148) and lysates were stored at -20°C. RNA was purified using a Qiagen QIACube HT instrument (9001793) and Qiagen's RNeasy 96 QIACube HT kit (74171) according to the manufacturer's instructions. Samples were analyzed using a QIAxpert system (9002340). cDNA was synthesized from RNA samples using Applied Biosystems' High Capacity cDNA Reverse Transcription Kit (4368813), reactions were assembled according to the manufacturer's instructions, and input RNA concentrations were varied depending on the sample. Reverse transcription was carried out on a BioRad quadruple thermal cycler (model no. PTC-0240G) under the following conditions: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes, followed by an optional hold at 4°C.

[0265] Droplet digital PCR (ddPCR) was performed using BioRad's QX200 AutoDG droplet digital PCR system according to the manufacturer's instructions, using BioRad's ddPCR Supermix for Probes (1863010) and fluorescently labeled qPCR assay for PNPLA3 (IDT Hs.PT.58.21464637, primer to probe ratio 3.6:1 and TBP (IDT Reactions were assembled in Eppendorf clear 96-well PCR plates (951020303) using Hs.PT.53a.20105486, primer to probe ratio 3.6:1, and RNase-free water (Ambion, AM9937). Final primer / probe concentrations were 900 nM / 250 nM, respectively, and input cDNA concentrations were varied between wells. Droplets were formed using a BioRad Auto DG droplet generator (1864101) set up with consumables recommended by the manufacturer (BioRad DG32 cartridge 1864108, BioRad tips 1864121, Eppendorf blue 96-well PCR plate 951020362, BioRad droplet generation oil for probes 1864110, and BioRad droplet plate assembly). Droplets were generated using a BioRad C1000 Amplification was performed on a touch thermal cycler (1851197) using the following conditions: enzyme activation 95°C for 10 min, denaturation 94°C for 30 s, followed by annealing / extension at 60°C for 1 min, 40 cycles with a ramp rate of 2°C / s, enzyme inactivation 98°C for 10 min, followed by (optional) a 4°C hold. Samples were then read on BioRad's QX200 droplet reader, which measures the FAM / HEX signal, which correlates to PNPLA3 or TBP concentration. Data were analyzed using BioRad's QuantaSoft software package. Samples were gated by channel (fluorescent label) to determine concentration per sample. Each sample is then expressed as a ratio of concentration of gene of interest (PNPLA3) / concentration of housekeeping gene (TBP) to control for differences in sample loading.The data was then imported into Genedata Screener, where each test siRNA was normalized to the median value of the neutral control wells (buffer only). IC50 values ​​are reported in Table 7.

[0266] [Table 80]

[0267] [Table 81]

[0268] [Table 82]

[0269] [Table 83]

[0270] [Table 84]

[0271] Example 4: Efficacy screening of selected PNPLA3 siRNA molecules in a humanized mouse model Adeno-associated virus (AAV; serotypes AAV8 or AAV7; endotoxin-free, produced in-house by Amgen) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) to 4e11 to 1e12 viral particles per animal was injected intravenously into the tail vein of C57BL / 6NCrl male mice (Charles River Laboratories Inc.) to induce expression of human PNPLA3 in the liver. WT (PNPLA3-WT), PNPLA3 rs738409 (PNPLA3-I148M), or PNPLA3 rs738409-rs738408Mice were generally 10-12 weeks old and contained n=4-6 animals per group. Every round of screening included at least two vehicle-treated control groups: vehicle-treated AAV-empty vector and AAV-PNPLA3 WT or PNPLA3 rs738409 and PNPLA3 rs738409-rs738408 All siRNAs contained AAV-PNPLA3 WT , PNPLA3 rs738409 , and / or PNPLA3 rs738409-rs738408 Two weeks after AAV injection, mice were treated with a single dose of siRNA D-2324 (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) via subcutaneous injection at 0.5, 1.0, 3.0 or 5.0 milligrams per kilogram of animal. Livers were collected from animals 8, 15, 22, 28 or 42 days after siRNA injection, flash frozen in liquid nitrogen and processed for purified RNA using a QIACube HT instrument (Qiagen, 9001793) and RNeasy 96 QIACube HT kit (Qiagen, 74171) according to the manufacturer's instructions. Samples were analyzed using a QIAxpert system (Qiagen, 9002340). RNA was treated with RQ1 RNase-free DNase (Promega, M6101) and purified using TaqMan™ RNA-to-C T The cells were prepared for real-time qPCR using the TaqMan™ 1-Step Kit (Applied Biosystems, 4392653). Real-time qPCR was performed on a QuantStudio real-time PCR instrument. Results are shown as the relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated control animals, based on gene expression of human PNPLA3 normalized to mouse Gapdh (TaqMan™ assay from Invitrogen, hs00228747_m1 and 4352932E, respectively). Endogenous mouse Pnpla3 expression was determined for comparison (Invitrogen, Mm00504420_m1).

[0272] For liver triglyceride content analysis, approximately 0.05-0.1 milligrams of frozen liver from animals were homogenized in 1 milliliter of isopropanol. After 1 hour of incubation on ice, samples were spun at 10,000 rpm in a microcentrifuge and the supernatant was transferred to a clear deep-well 96-well plate. Triglyceride content was determined using colorimetric Infinity Triglyceride Reagent (Thermo Fisher Scientific, TR22421) and triglyceride standards (Pointe Scientific, T7531-STD) according to the manufacturer's instructions. Results are presented as milligrams of triglyceride per milligram of tissue.

[0273] Figure 1A-D. An example of five siRNA molecules screened for both dose-dependent mRNA knockdown in vivo and functional durability. Human PNPLA3 rs738409-rs738408 Mice expressing human PNPLA3 were treated with siRNA 2 weeks after intravenous AAV injection. N=6 mice per group; data presented as mean and standard error of the mean. (A) siRNA was injected subcutaneously into the abdomen of mice at 0.5, 1.0, 3.0, or 5.0 milligrams per kilogram of body weight. Four weeks after siRNA treatment, mice were sacrificed and livers were harvested and processed for gene expression analysis. Data are for the expression of human PNPLA3 in each group set relative to the vehicle-treated control group. rs738409-rs738408 (B) Livers from the same 4-week treatment groups were also processed for triglyceride content to obtain functional efficacy. Data represent the average milligrams of triglyceride per gram of tissue processed. (C) siRNA was injected subcutaneously into the abdomen of a parallel cohort of animals at 1.0 and 3.0 milligrams per kilogram of body weight. Mice were harvested 6 weeks after siRNA treatment to compare the durability of siRNA molecules in vivo. Livers were harvested and processed for gene expression analysis. Data represent the average relative knockdown of human PNPLA3 in each group set relative to the vehicle-treated control group. rs738409-rs738408(D) Livers from the same 6-week treatment groups were also processed for triglyceride content to obtain functional efficacy over time. Data represent the average milligrams of triglyceride per gram of processed tissue.

[0274] Data on relative knockdown is shown in Tables 8-12, which show the relative knockdown at days 8, 15, 22, 28, and 42, respectively, and at various doses. PNPLA3 knockdown is a percentage with negative values ​​indicating reduced PNPLA3 levels.

[0275] [Table 85]

[0276] [Table 86]

[0277] [Table 87]

[0278] [Table 88]

[0279] [Table 89]

[0280] [Table 90]

[0281] [Table 91]

[0282] [Table 92]

[0283] [Table 93]

[0284] [Table 94]

[0285] [Table 95]

[0286] [Table 96]

[0287] Example 5: Humanized PNPLA3 rs738409-rs738408 Prevention and rescue of NAFLD by siRNA molecules in a mouse model The "American Lifestyle Induced Obesity," or ALIOS, mouse model for NAFLD / NASH is developed by feeding mice a diet high in trans fats (45% of total fat) and sugars (Tetri 2008). For these studies, 8-10 week old C57BL / 6NCrl male mice (Charles River Laboratories Inc.) were transfected with either AAV empty vector or AAV8-PNPLA3 as previously described. rs738409-rs738408 At the time of AAV injection, mice were either maintained on a regular chow diet until harvest or fed an ALIOS diet (Envigo, TD.06303) consisting of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively) complete with drinking water. rs738409-rs738408 We established that overexpression of both promotes and exacerbates the NAFLD phenotype in this context (data not shown).

[0288] Two weeks after AAV injection and initiation of diet, mice were treated with a single dose of siRNA D-2324 (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) at 5.0 milligrams per kilogram of animal or with solvent control via subcutaneous injection. Administration was repeated every two weeks until harvest. At harvest, body weights were collected, followed by serum collection via cardiac puncture under isoflurane anesthesia, followed by liver weight collection. The median lobe was fixed with 10% neutral buffered formalin, then paraffin processed and embedded. The remainder of the liver was snap frozen for content and gene expression analysis as previously described.

[0289] Snap-frozen liver tissue was processed for RNA and gene expression analysis as described above. Results are presented as both raw Ct values ​​and relative mRNA expression of the indicated genes normalized to mouse Gapdh. (TaqMan™ assays from Invitrogen: human PNPLA3, hs00228747_ml; mouse Pnpla3, Mm00504420_ml; mouse Gapdh, 4352932E).

[0290] Formalin-fixed tissues were processed for hemotoxylin and eosin staining (Dako, CS70030-2, CS70130-2, respectively) according to the manufacturer's instructions. Scoring for steatosis and inflammation was performed by a board-certified pathologist.

[0291] Serum analyses included TIMP1, a biomarker associated with NASH and NASH-associated fibrosis (Youssani 2011). TIMP1 ELISA (R&D Systems, MTM100) was performed according to the manufacturer's instructions.

[0292] Figure 2A-G. NAFLD and PNPLA3 rs738409-rs738408 PNPLA3 prevents the development of phenotypes associated with overexpression of rs738409-rs738408To evaluate the efficacy of the specific siRNA molecule D-2324, mice were transfected with AAV8 empty vector (EV) or AAV8-PNPLA3. rs738409-rs738408 Mice were given siRNA or vehicle and maintained on a normal chow diet or transitioned to the ALIOS diet. Two weeks after AAV injection, mice were treated with siRNA or vehicle every other week for 6 weeks (total of 3 injection rounds). Mice were harvested at the 8-week time point. Results are presented as group means and standard errors, N=8 per group. Asterisks indicate the mean and standard error of AAV8-PNPLA3, generated by one-way ANOVA with Dunnett's multiple comparison test. rs738409-rs738408 Statistical significance for cohorts is shown. (A) Ratio of liver weight (grams) to body weight (grams) at harvest. Adjusted P values: No AAV + vehicle, ****<0.0001; AAV-EV + vehicle, **=0.0018; AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001. (B) Confirmation of human PNPLA3 mRNA expression and silencing in liver by qPCR. (Left) Raw Ct values ​​and (Right) relative fold mRNA expression normalized to mouse Gapdh; ALIOS-administered PNPLA3 rs738409-rs738408 + Solvent and PNPLA3 rs738409-rs738408 + siRNA groups. (C) Analysis of mouse Pnpla3 mRNA expression in liver by qPCR shows that endogenous Pnpla3 is not significantly altered by AAV-mediated overexpression or siRNA silencing. (Left) Raw Ct values ​​and (right) relative fold mRNA expression normalized to mouse Gapdh; PNPLA3 in chow-fed AAV-free groups and ALIOS-fed groups. rs738409-rs738408 + Solvent and PNPLA3 rs738409-rs738408 (D) Liver triglyceride content, expressed as milligrams of triglyceride per gram of liver tissue. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, *=0.0393; AAV-PNPLA3 rs738409-rs738408+ siRNA, **=0.0063. (E) Serum TIMP1 shown as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, ****<0.0001; AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001. (F) Histological signs of steatosis based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, not significant; AAV-PNPLA3 rs738409-rs738408 + siRNA, **=0.0012. (G) Histological signs of inflammation based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, ****<0.0001; AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001.

[0293] Figure 3A-G. PNPLA3 after disease onset rs738409-rs738408 PNPLA3 prevents further progression of vector-borne disease rs738409-rs738408 To evaluate the potency of specific siRNA molecules, mice were transfected with AAV8 empty vector (EV) or AAV8-PNPLA3. rs738409-rs738408 Mice were fed either siRNA or vehicle and maintained on a normal chow diet or transitioned to the ALIOS diet. After 8 weeks of AAV injection and dietary change, mice were treated with siRNA or vehicle every other week for 8 weeks (4 injection rounds in total). Mice were harvested at the 16 week time point. No changes were observed in steatosis, but several other disease-related endpoints were significantly reduced when siRNA treatment was initiated after disease induction. Results are shown as mean and standard error, N=8 per group. Asterisks indicate the mean and standard error of AAV8-PNPLA3, generated by one-way ANOVA utilizing Dunnett's multiple comparison test. rs738409-rs738408Statistical significance for cohorts is shown. (A) Ratio of liver weight (grams) to body weight (grams) at harvest. Adjusted P values: No AAV + vehicle, ****<0.0001; AAV-EV + vehicle, not significant; AAV-PNPLA3 rs738409-rs738408 + siRNA, ***=0.0006. (B) Confirmation of human PNPLA3 mRNA expression and silencing in liver by qPCR. (Left) Raw Ct values ​​and (Right) relative fold mRNA expression normalized to mouse Gapdh; ALIOS-administered PNPLA3 rs738409-rs738408 + Solvent and PNPLA3 rs738409-rs738408 + siRNA groups. (C) Analysis of mouse Pnpla3 mRNA expression in liver by qPCR shows that endogenous Pnpla3 is not significantly altered by AAV-mediated overexpression or siRNA silencing. (Left) Raw Ct values ​​and (right) relative fold mRNA expression normalized to mouse Gapdh; PNPLA3 in chow-fed AAV-free groups and ALIOS-fed groups. rs738409-rs738408 + Solvent and PNPLA3 rs738409-rs738408 + siRNA groups. (D) Liver triglyceride content shown as milligrams of triglyceride per gram of liver tissue. Adjusted P values: AAV-EV + vehicle, not significant, AAV-PNPLA3 rs738409-rs738408 + siRNA, *=0.0403. (E) Serum TIMP1 shown as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, **=0.0027; AAV-PNPLA3 rs738409-rs738408 + siRNA, **=0.002. (F) Histological signs of steatosis based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, not significant; AAV-PNPLA3 rs738409-rs738408+ siRNA, not significant. (G) Histological signs of inflammation based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, not significant; AAV-PNPLA3 rs738409-rs738408 + siRNA, ** = 0.0068.

[0294] Figure 4A-D. PNPLA3 rs738409-rs738408 PNPLA3 rescues disease-associated phenotypes caused by overexpression of rs738409-rs738408 To evaluate the ability of specific siRNA molecules, ALIOS was administered 8 weeks after ingestion of AAV8-PNPLA3 rs738409-rs738408 Livers and serum from vehicle-treated mice were analyzed using ALIOS-infused vehicle- or siRNA-treated AAV8-PNPLA3 rs738409-rs738408 The liver and serum from 8 week AAV8-PNPLA3 mice were compared to control mice. No changes in steatosis were observed at this time point with siRNA treatment, but liver triglycerides, serum TIMP1, and inflammation were all statistically lower at week 16 compared to vehicle controls at week 8. Results are shown as mean and standard error, N=8 per group. Asterisks indicate mean values ​​generated by one-way ANOVA using Dunnett's multiple comparison test. rs738409-rs738408 Statistical significance versus vehicle-treated cohort is depicted. (A) Liver triglyceride content shown as milligrams of triglyceride per gram of liver tissue. Adjusted P-values: 16-week AAV-PNPLA3 rs738409-rs738408 + Vehicle, no significance; 16-week AAV-PNPLA3 rs738409-rs738408 + siRNA, ** = 0.0011. (B) Serum Timp1 shown as picograms per milliliter of serum. Adjusted P value: 16 week AAV-PNPLA3 rs738409-rs738408 + Vehicle, no significance; 16-week AAV-PNPLA3 rs738409-rs738408 + siRNA, *=0.0134. (C) Histological signs of steatosis based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: 16-week AAV-PNPLA3 rs738409-rs738408+ Vehicle, no significance; 16-week AAV-PNPLA3 rs738409-rs738408 + siRNA, not significant. (D) Histological signs of inflammation based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: 16-week AAV-PNPLA3 rs738409-rs738408 + Vehicle, no significance; 16-week AAV-PNPLA3 rs738409-rs738408 + siRNA, *=0.0112.

[0295] Example 6: Humanized PNPLA3 rs738409-rs738408 Prevention of liver fibrosis by siRNA molecules in a mouse model The "AMLN" diet, developed by Amylin Pharmaceuticals (Clapper 2013), is a modified version of the ALIOS diet. The diet contains a 10-fold increase in cholesterol (2%) and additional sucrose. Mice fed the "AMLN" diet develop mild to moderate fibrosis after 20-30 weeks (Clapper, Mells and Kristiansen papers). For this study, 8-10 week old C57BL / 6NCrl male mice (Charles River Laboratories Inc.) were transfected with either AAV empty vector or AAV-PNPLA3 vector as described above. rs738409-rs738408 At the time of AAV injection, mice were either continued on a normal chow diet until harvest or fed Envigo diet, TD.170748, complete with drinking water composed of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively).

[0296] Two weeks after AAV injection and initiation of diet, mice were treated with a single dose of siRNA D-2324 (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) at 5.0 milligrams per kilogram of animal or with solvent control via subcutaneous injection. Administration was repeated every two weeks until harvest. At harvest, body weights were collected, followed by serum collection via cardiac puncture under isoflurane anesthesia, followed by liver weight collection. The median lobe was fixed with 10% neutral buffered formalin, then paraffin processed and embedded. The remainder of the liver was snap frozen for gene expression analysis.

[0297] Snap-frozen liver tissue was processed for RNA and gene expression analysis as previously described. Results are presented as both raw Ct values ​​and relative mRNA expression of the indicated genes normalized to mouse Gapdh. (TaqMan™ assays from Invitrogen: human PNPLA3, hs00228747_m1; mouse Pnpla3, Mm00504420_m1; mouse Col1a1, Mm00801666_g1; mouse Col3a1, Mm01254471_g1; Col4a1, Mm01210125_m1; mouse Gapdh, 4352932E). Col1a1, Col3a1 and Col4a1 are extracellular matrix markers associated with hepatic stellate cell activation and liver fibrosis (Baiocchini 2016).

[0298] Formalin-fixed tissues were processed for hemotoxylin, eosin, and Masson's trichrome staining (Dako, CS70030-2, CS70130-2, AR17311-2, respectively) according to the manufacturer's instructions. Anti-smooth muscle actin staining was performed without antigen retrieval and using a DAKO automated stainer. Slides were processed using Peroxidazed 1 and Sniper (Biocare, PX968 and BS966, respectively) and stained with monoclonal anti-actin, α-smooth muscle antibody (Sigma, F3777), followed by rabbit anti-FITC (Invitrogen, 711900), Envision-rabbit HRP polymer (Dako, K4003), DAB+ (Dako, K3468), and hemotoxylin. Scoring for steatosis, inflammation, oval cell / bile duct hyperplasia, and amount of aSMA-positive cells was performed by a board-certified pathologist.

[0299] Serum was analyzed for mouse TIMP1 (R&D Systems, MTM100) and mouse cytokeratin 18-M30 (Cusabio, CSB-E14265m) according to the manufacturer's instructions. In addition to TIMP1, cytokine 18-M30 has been identified as a potential biomarker for NAFLD / NASH, including early fibrosis (Neuman 2014 and Yang 2015). Figure 5A-L. PNPLA3 prevents the development of early fibrosis. rs738409-rs738408 To evaluate the potency of specific siRNA molecules, mice were transfected with AAV8 empty vector (EV) or AAV8-PNPLA3. rs738409-rs738408 Mice were fed either siRNA D-2324 or vehicle and placed on a normal chow diet or transitioned to an AMLN diet. Two weeks after AAV injection, mice were treated with siRNA D-2324 or vehicle every other week for an additional 10 weeks (total of six injection rounds). Mice were harvested at the 10 week time point. Results are presented as mean and standard error, for chow-fed AAV no + vehicle and AMLN-fed AAV8-PNPLA3. rs738409-rs738408 + vehicle, N=8 per group; AMLN-infused AAV8-PNPLA3 rs738409-rs738408 + Solvent and AAV8-PNPLA3 rs738409-rs738408+ siRNA, N = 12 per group. Asterisks are generated by one-way ANOVA with Dunnett's multiple comparison test, AAV8-PNPLA3 rs738409-rs738408 Statistical significance relative to vehicle-treated cohort is shown. (A) Ratio of liver weight (grams) to body weight (grams) at harvest. Adjusted P values: No AAV + vehicle group, ****<0.0001, AAV-EV + vehicle, not significant, AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001. (B) Confirmation of human PNPLA3 mRNA expression and silencing in liver by qPCR. (Left) Raw Ct values ​​and (Right) relative fold mRNA expression normalized to mouse Gapdh;PNPLA3 rs738409-rs738408 + Solvent and PNPLA3 rs738409-rs738408 (C) Analysis of mouse Pnpla3 mRNA expression in liver by qPCR shows that endogenous Pnpla3 is not significantly altered by AAV-mediated overexpression or siRNA silencing. (Left) Raw Ct values ​​and (Right) relative fold mRNA expression normalized to mouse Gapdh; chow-fed AAV-free group and AMLN-fed PNPLA3 rs738409-rs738408 + Solvent and PNPLA3 rs738409-rs738408 (D) Serum Timp1 shown as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, ****<0.0001; AAV-PNPLA3 + siRNA group. rs738409-rs738408 + siRNA, ****<0.0001. (E) Serum CK18m30 shown as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, ****<0.0001; AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001. (F) Histological signs of inflammation based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, *=0.0108; AAV-PNPLA3 rs738409-rs738408+ siRNA, ****<0.0001. (G) Histological signs of oval cell / bile duct hyperplasia based on H&E staining, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, not significant; AAV-PNPLA3 rs738409-rs738408 + siRNA, **=0.0081. (H) Immunohistochemical staining for anti-smooth muscle actin, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, *=0.0101; AAV-PNPLA3 rs738409-rs738408 + siRNA, ***=0.0002. (I) Masson's trichrome staining for fibrosis, scored as within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, not significant; AAV-PNPLA3 rs738409-rs738408 + siRNA, not significant. (J) Mouse Col1a1 mRNA expression in liver by qPCR. Relative fold mRNA expression normalized to mouse Gapdh. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, ****<0.0001; AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001. (K) Mouse Col3a1 mRNA expression in liver by qPCR. Relative fold mRNA expression normalized to mouse Gapdh. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, ****<0.0001; AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001. (L) Mouse Col4a1 mRNA expression in liver by qPCR. Relative fold mRNA expression normalized to mouse Gapdh. Adjusted P values: No AAV + vehicle group, ****<0.0001; AAV-EV + vehicle, ***<0.0005; AAV-PNPLA3 rs738409-rs738408 + siRNA, **<0.0041.

[0300] Example 7: Screening of PNPLA3 siRNA molecules using bioluminescence imaging mouse model BALB / c male mice (Charles River Laboratories Inc.), typically 10-12 weeks old, were injected with adeno-associated virus (AAV; serotype AAVDJ8; endotoxin-free, produced in-house by Amgen) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). AAV was administered at 5e11 to a maximum of 7.5e11 viral particles per animal and was injected intravenously into the tail vein. AAV constructs were engineered with a mouse cytomegalovirus (CMV) promoter and a firefly luciferase reporter, and at their 3' ends were engineered the human PNPLA3 promoter and the human PNPLA3 promoter, as detailed in Figures 6 and 7. WT (reference allele) or human PNPLA3 rs738409-rs738408 (Minor allele) There is a string of nucleotides that contains the human PNPLA3 target sequence of interest spanning any stretch of the siRNA target sequence and other non-SNPs.

[0301] Two weeks after AAV injection, mice were injected with RediJect D-Luciferin (PerkinElmer, 770504) according to the manufacturer's instructions. The bioluminescence signals of mice were captured using an IVIS Spectrum In Vivo Imaging System (PerkinElmer) and analyzed using Living Image Software (PerkinElmer). Mice were then randomized into groups of n=5 based on the total luminous flux [photons / sec] signal in the liver region. After randomization into treatment groups, mice were given a single dose of siRNA (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). siRNA was administered by subcutaneous injection at the indicated milligrams per kilogram dose of animals. One vehicle-treated control group was included for each round of screening and for each AAV type.

[0302] Mice were imaged again at 1, 2, 3 and 4 weeks after siRNA injection, and total flux [p / s] measurements were collected using the same defined region of interest established for baseline reading. Relative knockdown percentages were determined for each animal by calculating the percent change in total flux at 1, 2, 3 or 4 weeks from the total flux at baseline for that animal, and normalizing to the average change in total flux from baseline for the vehicle control group at the same time point. For example, animals treated with siRNA have a relative knockdown calculated as follows: (total flux of animal at week 2) / (total flux of animal at baseline), normalized to the average (total flux of vehicle animals at week 2) / (total flux of vehicle animals at baseline). Figure 8 shows representative images of animals injected with either AAV expressing the human PNPLA3 minor allele target sequence or AAV expressing the reference allele target sequence, and the change in total flux over time both before and after siRNA treatment.

[0303] [Table 97]

[0304] [Table 98]

[0305] [Table 99]

[0306] [Table 100]

[0307] Example 8: PNPLA3 in a chimeric humanized liver mouse model rs738409-rs738408 Confirmation of efficacy of selected siRNA molecules To evaluate the efficacy of PNPLA3 minor allele-selective siRNA in human hepatocytes in vivo, chimeric humanized liver PXB-mice from Phoenix Bio Co., Ltd. (Japan) (Miyamoto et al. (2017) Xenobiotica 47(12):1052-1063; Tateno et al. (2015) PLoS One 10(11):e0142145.doi:10.1371 / journal.pone.0142145) were used. At the start of the study, male mice were approximately 4 months old and at least 3 months post-transplantation. The mice were determined by Phoenix Bio to have a human hepatocyte replacement index of 90-95% and, based on prior genotyping, were highly resistant to PNPLA3 rs738409The mice were heterozygous for siRNA (Lot BD195). Upon arrival, the mice were fed ProLab RMH 3000 chow as recommended by Phoenix Bio. After a one-week acclimation period, the diet was transitioned to a high-fat, fructose-rich NASH-inducing diet (Research Diets, D19021301). After one week on the NASH diet, the mice were randomized based on body weight measurements. Mice were treated with a single dose of siRNA (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) by subcutaneous injection at 3.0 or 10.0 milligrams per kilogram of animal, or received vehicle alone. Livers were collected from animals at 2 or 4 weeks after siRNA injection, flash frozen in liquid nitrogen, and processed for purified RNA using the QIAcube Automated DNA / RNA Isolation Purification System (Qiagen) and RNeasy Mini QIAcube Kit (Qiagen, 74116) according to the manufacturer's instructions. Samples were analyzed using a NanoDrop™ 8000 Spectrophotometer (Thermo Scientific, ND-8000-GL). RNA was treated with RQ1 RNase-free DNase (Promega, M6101) and prepared for digital droplet PCR (ddPCR) according to the manufacturer's instructions. AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Thermo Fisher, 200820) was used for the reverse transcription reaction, and ddPCR Supermix for Probes (BioRad, 1863010) was used to assemble the PCR reactions. ddPCR was performed using the AutoDG droplet digital PCR system (BioRad, QX200).The following TaqMan™ assays were purchased from Invitrogen: human PNPLA3 (Hs00228747_m1), human ASGR1 (Hs1005019_m1), mouse Asgr1 (Mm01245581_m1), and human PNPLA3 rs738409 minor / reference allele discrimination assay (C______7241_10). The following assays were purchased from Integrated DNA Technologies Inc.: human TBP (Hs.PT 53a.20105486; primer to probe ratio 3.6:1), human HPRT1 (Hs.PT.39a.22214821; primer to probe ratio 3.6:1), and mouse Hprt (Mm.PT.39a.22214828; primer to probe ratio 3.6:1). Results for human PNPLA3, HPRT, and ASGR1, and mouse Hprt and Asgr1 are shown as copies per 20 microliter reaction normalized to human TBP. Data for human PNPLA3, human HPRT, and mouse Hprt are also shown as relative knockdown of mRNA expression compared to vehicle-treated control animals.

[0308] To analyze hepatic triglyceride content, approximately 0.05-0.1 milligrams of frozen liver from mice were homogenized in 1 milliliter of isopropanol. After 1 hour of incubation on ice, samples were spun at 10,000 rpm in a microcentrifuge tube and the supernatant was transferred to a clear deep-well 96-well plate. Triglyceride content was measured using colorimetric Infinity Triglyceride Reagent (Thermo Fisher Scientific, TR22421) and Triglyceride Standard (Pointe Scientific, T7531-STD) and a SpectraMax Plus microplate reader with SoftMax Pro6 software (Molecular Devices) according to the manufacturer's instructions. Results are presented as milligrams of triglyceride per gram of liver tissue.

[0309] FIG. 9 shows an example of an siRNA molecule, D-2419, demonstrating both dose-dependent and allele-selective mRNA knockdown in vivo as well as functional efficacy. After one week on a NASH diet, human PNPLA3 rs738409-rs738408 Heterozygous mice were treated with siRNA or vehicle. (A) The siRNA molecule D-2419 was injected subcutaneously into the abdomen of the mice at 3.0 and 10.0 milligrams per kilogram of body weight. Mice were sacrificed 2 and 4 weeks after siRNA treatment and livers were harvested and processed for analysis. Using ddPCR and allele-selective TaqMan® 2 dye reagents to distinguish the reference allele from the minor allele of PNPLA3, the data showed that PNPLA3 rs738409-rs738408 Both dose-dependent and allele-selective knockdown of PNPLA3 was demonstrated. WT (B) Data are set relative to the vehicle-treated control group, showing no measurable changes in human PNPLA3. N=5 mice per group; data are presented as mean and standard error. 2-way ANOVA, **0.001, ***<0.001, ****<0.0001, NS=not significant. (C) Data are set relative to the vehicle-treated control group, showing no measurable changes in human PNPLA3. rs738409-rs738408 PNPLA3 for allele WT(C) Livers from the 2-week treatment groups were processed for triglyceride content to assess functional efficacy. Data represent milligrams of triglyceride per gram of liver. N=5 mice per group; data are presented as mean and standard error. One-way ANOVA, **0.01, NS=not significant. (D) To control for efficient GalNAc-mediated delivery of the siRNA molecule D-2787, siRNA cross-reactive against human and mouse HPRT and Hprt, respectively, was delivered at 10 milligrams per kilogram and livers were harvested 2 weeks later. Data show copies of HPRT mRNA and Hprt mRNA in vehicle-treated (N=5) versus D-2787-treated (N=4) mice. Data are presented as mean and standard error. One-way ANOVA, *0.01. (E) Data show the mean relative mRNA knockdown rate and standard error of human HPRT mRNA and mouse Hprt mRNA, respectively, set against the vehicle-treated control group. All normalized to human TBP. (F) To confirm the expression of GalNAc receptors on stem cells of PXB mice®, mouse Asgr1 mRNA and human ASGR1 mRNA levels were assessed in the absence and presence of D-2419 at both 2 and 4 weeks after siRNA injection. N=5 mice per group; data are presented as mean and standard error.

Claims

1. 1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from an antisense sequence comprising SEQ ID NO: 2618, 2978, 2986, 2988, 3002, 3034, 3118, 3124, 3128, 3130, or 3132, and wherein the RNAi construct inhibits expression of patatin-like phospholipase domain-containing 3 (PNPLA3).

2. The RNAi construct of claim 1 , wherein the antisense strand comprises a region that is complementary to a PNPLA3 mRNA sequence.

3. 2. The RNAi construct of claim 1, wherein the sense strand comprises a region having at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from an antisense sequence comprising SEQ ID NO: 2617, 2977, 2985, 2987, 3001, 3033, 3117, 3123, 3127, 3129, or 3131.

4. The RNAi construct of claim 1, wherein the construct preferentially inhibits the PNPLA3-rs738409 minor allele.

5. The RNAi construct of claim 4, wherein the construct has at least 10% greater inhibition of the PNPLA3-rs738409 minor allele compared to the major allele.

6. The RNAi construct of claim 1, wherein the construct preferentially inhibits the PNPLA3-rs738408 minor allele.

7. 7. The RNAi construct of claim 6, wherein the construct has at least 10% greater inhibition of the PNPLA3-rs738409-rs738408 double minor allele compared to the major allele.

8. The RNAi construct of any one of claims 1 to 7, wherein the sense strand comprises a sequence sufficiently complementary to a sequence of the antisense strand to form a duplex region of 15 to 30 base pairs in length.

9. The RNAi construct of claim 8, wherein the duplex region is 17 to 24 base pairs in length.

10. The RNAi construct of claim 8, wherein the duplex region is 19 to 21 base pairs in length.

11. The RNAi construct of claim 8, wherein the sense strand and the antisense strand are each 15 to 30 nucleotides in length.

12. The RNAi construct of claim 11, wherein the sense strand and the antisense strand are each 19 to 27 nucleotides in length.

13. The RNAi construct of claim 11, wherein the sense strand and the antisense strand are each 21 to 25 nucleotides in length.

14. The RNAi construct of claim 11, wherein the sense strand and the antisense strand are each 21 to 23 nucleotides in length.

15. The RNAi construct of claim 1 , comprising at least one blunt end.

16. The RNAi construct of claim 1, comprising at least one nucleotide overhang of 1 to 4 unpaired nucleotides.

17. The RNAi construct of claim 16, wherein the nucleotide overhang has two unpaired nucleotides.

18. The RNAi construct of claim 16, comprising a nucleotide overhang at the 3'-end of the sense strand, the 3'-end of the antisense strand, or the 3'-end of both the sense strand and the antisense strand.

19. The RNAi construct of claim 16, wherein the nucleotide overhang comprises a 5'-UU-3' dinucleotide or a 5'-dTdT-3' dinucleotide.

20. 20. The RNAi construct of any one of claims 1 to 7 or 9 to 19, comprising at least one modified nucleotide.

21. The RNAi construct of claim 20, wherein the modified nucleotide is a 2'-modified nucleotide.

22. 21. The RNAi construct of claim 20, wherein the modified nucleotide is a 2'-fluoro modified nucleotide, a 2'-O-methyl modified nucleotide, a 2'-O-methoxyethyl modified nucleotide, a 2'-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), a glycol nucleic acid (GNA), a 2'-methoxyinosine (MeO-1), a 2'-deoxy modified nucleotide, an abasic nucleotide, an inverted base, or a combination thereof.

23. 23. The RNAi construct of claim 22, wherein the modified nucleotides are 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

24. 21. The RNAi construct of claim 20, wherein all of the nucleotides in the sense strand and the antisense strand are modified nucleotides.

25. 25. The RNAi construct of claim 24, wherein the modified nucleotides are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, or a combination thereof.

26. The RNAi construct of claim 1 , comprising at least one phosphorothioate internucleotide linkage.

27. The RNAi construct of claim 26, comprising two consecutive phosphorothioate internucleotide linkages at the 3' end of the antisense strand.

28. The RNAi construct of claim 26, comprising two consecutive phosphorothioate internucleotide linkages at both the 3'-end and 5'-end of the antisense strand, and two consecutive phosphorothioate internucleotide linkages at the 5'-end of the sense strand.

29. a. SEQ ID NO:2618 and SEQ ID NO:2617 (duplex D-1698); b. SEQ ID NO: 2978 and SEQ ID NO: 2977 (duplex D-1878); c. SEQ ID NO: 2986 and SEQ ID NO: 2985 (duplex D-1882); d. SEQ ID NO: 2988 and SEQ ID NO: 2987 (duplex D-1883); e. SEQ ID NO: 2982 and SEQ ID NO: 2981 (duplex D-2698); f. SEQ ID NO: 3002 and SEQ ID NO: 3001 (duplex D-2708); g. SEQ ID NO: 3034 and SEQ ID NO: 3033 (duplex D-2724); h. SEQ ID NO: 3118 and SEQ ID NO: 3117 (duplex D-2766); i. SEQ ID NO:3124 and SEQ ID NO:3123 (duplex D-2769); j. SEQ ID NO: 3128 and SEQ ID NO: 3127 (duplex D-2771), or k. SEQ ID NO: 3132 and SEQ ID NO: 3131 (duplex D-2773) The RNAi construct of any one of claims 1 to 7, 9 to 22, or 24 to 28, comprising an antisense sequence and a sense sequence of each of the following:

30. The RNAi construct of any one of claims 1 to 29, which reduces the expression level of PNPLA3 in liver cells after incubation with the RNAi construct compared to the expression level of PNPLA3 in liver cells incubated with a control RNAi construct.

31. The RNAi construct of claim 30, wherein the liver cell is a Hep3B cell or a HepG2 cell.

32. The RNAi construct of claim 31, which inhibits at least 10% of PNPLA3 expression in Hep3B cells in vitro at 5 nM.

33. The RNAi construct of claim 31, which inhibits at least 10% of PNPLA3 expression in HepG2 cells in vitro at 5 nM.

34. The RNAi construct of claim 31, which inhibits PNPLA3 expression in Hep3B cells with an IC50 of less than 1 nM.

35. The RNAi construct of claim 31, which inhibits PNPLA3 expression in HepG2 cells with an IC50 of less than 1 nM.

36. A pharmaceutical composition comprising the RNAi construct of any one of claims 1 to 35 and a pharma- ceutically acceptable carrier, excipient or diluent.

37. A composition for use in reducing expression of PNPLA3 in a patient in need thereof, the composition comprising the RNAi construct of claim 1.

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