Rnai constructs for inhibiting pnpla3 expression

Abstract

To provide a novel approach to reducing PNPLA3 levels and treating hepatologic diseases, such as nonalcoholic fatty liver disease.SOLUTION: Provided are An RNAi construct comprising a sense strand and an antisense strand, the antisense strand comprising a region having at least 15 contiguous nucleotides differing by no more than 3 nucleotides from a specific antisense sequence, and the RNAi construct inhibiting the expression of Patatin-Like Phospholipase Domain Containing 3 (PNPLA3); a pharmaceutical composition comprising the RNAi construct; and a method for reducing the expression of PNPLA3 in a patient in need thereof, comprising administering the RNAi construct to the patient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 597,841, filed December 12, 2017, the contents of which are incorporated herein by reference.

[0002] Sequence Listing This application includes a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on December 12, 2018, is named A-2219-WO-PCT_SL.txt and is 708,961 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present invention relates to compositions and methods for modulating 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 diseases, such as non-alcoholic fatty liver disease (NAFLD).

[0004] This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy created on December 12, 2018, is named A-2219-WO-PCT_SL.txt and is 708,961 bytes in size. [Background technology]

[0005] Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, encompassing a variety of liver pathologies. Its prevalence has doubled over the past 20 years, and it is now estimated to affect approximately 20% of the global population (Sattar et al. (2014) BMJ 349:g4596; Loomba and Sanyal (2013) Nature Reviews Gastroenterology & Hepatology 10(11):686-690; Kim and Kim (2017) Clin Gastroenterol Hepatol 15(4):474-485; Petta et al. (2016) Dig Liver Dis 48(3):333-342). 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 other types of liver disease (Zhu et al. (2016) World J Gastroenterol 22(36):8226-33; Rinella (2015) JAMA 313(22):2263-73; Yki-Jarvinen (2016) Diabetologia 59(6):1104-11). 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 nonalcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, it is estimated that 75 to 100 million Americans have NAFLD; NASH accounts for approximately 10 to 30% of NAFLD diagnoses (Rinella, supra; Younossi et al (2016) Hepatology 64(5):1577-1586).

[0006] Patatin-like phospholipase domain-containing 3 (PNPLA3), formerly known as adiponutrin (ADPN) and calcium-independent phospholipase A2-epsilon (iPLA(2)ε), is a type II transmembrane protein (Wilson et al (2006) J Lipid Res 47(9):1940-9; Jenkins et al (2004) J Biol Chem 279(47):48968-75). It was first identified in adipocytes as a membrane-bound, lipid-enriched protein induced during adipogenesis in mice, but is now known to be expressed in other tissues, including the liver (Wilson et al., supra; Baulande et al. (2001) J Biol Chem 276(36):3336-44; Moldes et al. (2006) Eur J Endocrinol 155(3):461-8; Faraj et al. (2006) J Endocrinol 191(2):427-35; Liu et al. (2004) J Clin Endocrinol Metab 89(6):2684-9; Lake et al. (2005) J Lipid Res 46(11):2477-87). In cell-free biochemical systems, recombinant PNPLA3 protein can exhibit either triacylglycerol lipase or transacylation activity (Jenkins et al., supra; Kumari et al (2012) Cell Metab 15(5):691-702; He et al (2010) J Biol Chem 285(9):6706-15). In hepatocytes, PNPLA3 is expressed in the endoplasmic reticulum and on lipid membranes and primarily exhibits triacylglycerol hydrolase activity (He et al., supra; Huang et al. (2010) Proc Natl Acad Sci USA 107(17):7892-7; Ruhanen et al. (2014) J Lipid Res 55(4):739-46; Pingitore et al. (2014) Biochim Biophys Acta 1841(4):574-80).Although it lacks a secretory signal, data indicate that PNPLA3 is secreted and can be found in human plasma as a disulfide bond-dependent multimer (Winberg et al. (2014) Biochem Biophys Res Commun 446(4):1114-9). Therefore, novel therapeutic agents targeting PNPLA3 function provide a novel approach to reduce PNPLA3 levels and treat hepatological diseases such as nonalcoholic fatty liver disease. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Sattar et al. (2014)BMJ 349:g4596 [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 [Non-patent document 4] Petta et al. (2016) Dig Liver Dis 48(3):333-342 [Non-patent document 5] Zhu et al (2016) World J Gastroenterol 22(36):8226-33 [Non-patent document 6] Rinella (2015) JAMA 313(22):2263-73 [Non-Patent Document 7] Yki-Jarvinen (2016) Diabetologia 59(6):1104-11 [Non-patent document 8] Younossi et al (2016) Hepatology 64(5):1577-1586

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[0008] 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 PNPLA3 mRNA sequence. In certain embodiments, the antisense strand comprises a region having at least 15 consecutive nucleotides from an antisense sequence listed in Table 1 or Table 2. In some embodiments, the RNAi of the present invention selectively inhibits the PNPLA3-rs738409, PNPLA3-rs738408, and / or PNPLA3-rs738409-rs738408 minor alleles over reference alleles that do not contain these alterations.

[0009] In some embodiments, the sense strand of an RNAi construct described herein comprises a sequence sufficiently complementary to the sequence of the antisense strand to form a duplexed region of about 15 to about 30 base pairs in length. In these and other embodiments, the sense and antisense strands are each 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 two unpaired nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand. In other embodiments, the RNAi construct comprises two unpaired nucleotide overhangs at the 3'-end of the antisense strand and blunt ends at the 3'-end of the sense strand / 5'-end of the antisense strand.

[0010] The RNAi constructs of the present invention may contain one or more modified nucleotides, including nucleotides with modifications to the ribose ring, nucleobase, or phosphodiester backbone. In some embodiments, the RNAi constructs contain 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 specific embodiment, the RNAi constructs contain 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.

[0011] 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.

[0012] In some embodiments, the antisense and / or sense strands of an RNAi construct 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 explanation of the drawings]

[0013] [Figure 1A] Screening of five siRNA molecules for both dose-dependent mRNA knockdown and functional durability in vivo is shown. [Figure 1B] Screening of five siRNA molecules for both dose-dependent mRNA knockdown and functional durability in vivo is shown. [Figure 1C] Screening of five siRNA molecules for both dose-dependent mRNA knockdown and functional durability in vivo is shown. [Figure 1D] Screening of five siRNA molecules for both dose-dependent mRNA knockdown and functional durability in vivo is shown. [Figure 2A] 1 shows the effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation. [Figure 2B]1 shows the effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation. [Figure 2C] 1 shows the effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation. [Figure 2D] 1 shows the effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation. [Figure 2E] 1 shows the effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation. [Figure 2F] 1 shows the effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation. [Figure 2G] 1 shows the effect of PNPLA3 siRNA molecules in vivo in mice, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation. [Figure 3A] The effects of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 3B] The effects of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 3C]The effects of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 3D] The effects of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 3E] The effects of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 3F] The effects of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 3G] The effects of PNPLA3 siRNA molecules in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 4A] The ability of PNPLA3rs738409-rs738408-specific siRNA molecules to rescue disease-associated phenotypes due to overexpression of PNPLA3rs738409-rs738408, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 4B] The ability of PNPLA3rs738409-rs738408-specific siRNA molecules to rescue disease-associated phenotypes due to overexpression of PNPLA3rs738409-rs738408, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 4C] The ability of PNPLA3rs738409-rs738408-specific siRNA molecules to rescue disease-associated phenotypes due to overexpression of PNPLA3rs738409-rs738408, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 4D] The ability of PNPLA3rs738409-rs738408-specific siRNA molecules to rescue disease-associated phenotypes due to overexpression of PNPLA3rs738409-rs738408, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation are shown. [Figure 5A] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5B] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5C] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5D] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5E] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5F] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5G] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5H] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5I] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5J] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5K]Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. [Figure 5L] Figure 1 shows the ability of PNPLA3 rs738409-rs738408 specific siRNA molecules to prevent the development of early fibrosis. DETAILED DESCRIPTION OF THE INVENTION

[0014] 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., a human). In some embodiments, the compositions of the present invention include an RNAi construct that targets PNPLA3 mRNA and reduces PNPLA3 expression in a cell or mammal. Such RNAi constructs are useful for treating or preventing various forms of liver-related diseases, such as simple fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible, advanced scarring of the liver), or PNPLA3-associated obesity.

[0015] 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; also referred to as PNPLA3-rs738409, PNPLA3-ma, or PNPLA3-minor allele) was significantly associated with liver fat content. Since this initial report, subsequent GWAS studies have confirmed that PNPLA3 rs738409 is a major genetic determinant of NAFLD, significantly associated with 1) elevated levels of alanine transaminase (ALT), a serum biomarker of liver injury; 2) the incidence, progression, and severity of NAFLD; 3) both obese and lean individuals; and 4) all stages of NAFLD: steatosis, NASH, cirrhosis, and hepatocellular carcinoma. Consensus from multiple GWAS studies indicates 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 may be homozygous or heterozygous for the PNPLA3 rs738409 mutation. Furthermore, it has been found that patients with the PNPLA3 rs738409 mutation often also carry the rs738408 mutation, located three base pairs away (Tian et al. (2010) Nature Genetics 42:21-23). Thus, patients may have a PNPLA3-rs738409 minor allele, a PNPLA3-rs738408 minor allele, or a PNPLA3-rs738409-rs738408 double minor allele mutation (PNPLA3-dma).

[0016] We 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 Pnpla3 overexpression. In contrast, Pnpla3 expression in both transgenic and knock-in mice was significantly reduced.I148M 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 responsible for the elevated hepatic triglyceride levels. I148M These findings, along with the high frequency of the minor allele in individuals with NAFLD and its strong association with the disease, highlight PNPLA3 rs738409 as a key therapeutic target for NAFLD.

[0017] RNA interference (RNAi) is a process in which foreign RNA is introduced into cells to cause the specific degradation of mRNA encoding a targeted protein, resulting in reduced protein expression. Advances in both RNAi technology and liver delivery, as well as increasing success with other RNAi-based therapies, suggest RNAi as a compelling approach to treating NAFLD by directly targeting PNPLA3I148M. Numerous GWAS studies have demonstrated a dose-dependent effect of PNPLA3 rs738409 on the incidence, progression, and severity of NAFLD; odds ratios tend to be at least 2-fold higher for homozygous versus heterozygous carriers, and at least 2-fold higher for heterozygous versus wild-type individuals. Therefore, silencing PNPLA3 using allele specificity 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 the reference allele PNPLA3I148I) and HEPG2 (homozygous for the minor allele PNPLA3I148M) hepatocellular carcinoma cell lines, we identified siRNA sequences that can specifically inhibit PNPLA3I148M gene expression. The inhibitory effects of these sequences were confirmed by screening Chinese hamster ovary (CHO) cells overexpressing either PNPLA3I148I or PNPLA3I148M. Next, using adeno-associated virus (AAV) that overexpresses human PNPLA3I148M in vivo, we demonstrated that treatment with a minor allele-specific SNP not only specifically reduced the expression of human PNPLA3I148M in mice, but also significantly reversed hepatic triglyceride accumulation induced by overexpression of human PNPLA3I148M.

[0018] As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, can downregulate the expression of a target gene (e.g., PNPLA3) via the RNA interference mechanism. 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, via the RNA-induced silencing complex (RISC) pathway. In some embodiments, an RNAi construct comprises a double-stranded RNA molecule comprising two antiparallel strands of consecutive nucleotides that are sufficiently complementary to each other and hybridize to form a duplex region. "Hybridizing" or "hybridization" typically refers to the pairing of complementary polynucleotides via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonds) between complementary bases in two polynucleotides. The strand containing a region having a sequence substantially complementary to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand." "Sense strand" refers to the strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand can include a region that has substantial sequence identity to a target sequence.

[0019] 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 the PNPLA3 rs738409 and rs738408 sites. In some embodiments, the present invention is an RNAi that binds preferentially to PNPLA3 rs738409 over the native PNPLA3 sequence (PNPLA3-ref). In some embodiments, the present invention is an RNAi that binds preferentially to PNPLA3 rs738408 over the PNPLA3-ref sequence. In some embodiments, the present invention is an RNAi that binds preferentially to PNPLA3-dma over PNPLA3-ma. In some embodiments, the present invention is an RNAi molecule containing any of the sequences found in Table 1 or 2.

[0020] Double-stranded RNA molecules can contain chemical modifications to ribonucleotides, including modifications to the ribose sugar, the base, or the backbone components of 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.

[0021] As used herein, a first sequence is "complementary" to a second sequence if, under certain conditions, such as physiological conditions, a polynucleotide comprising the first sequence can hybridize to a 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 without mismatches over the entire length of one or both nucleotide sequences. 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. The 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.When two sequences hybridize, if there are 5, 4, 3, 2, or 1 or fewer mismatches across a 30-base pair duplex region, the sequence can be said to be substantially complementary to another sequence.Generally, if any nucleotide overhangs, as defined herein, exist, the sequence of such overhangs is not taken into account when determining the degree of complementarity between two sequences.For example, a 21-nucleotide sense strand and a 21-nucleotide 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 fully complementary as this term is used herein.

[0022] 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, for example, 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' end of the strand). 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.

[0023] In certain embodiments, the sense strand and antisense strand of the double-stranded RNA hybridize to form a double-stranded region, but can be two separate molecules that 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.

[0024] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules do not need 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 that forms a duplex structure, this bond 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 also include one or more nucleotide overhangs.

[0025] In other embodiments, the sense and antisense strands that hybridize to form the 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 the 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 connected to the 5' end of the antisense strand by a contiguous sequence of unpaired nucleotides, which form the loop region. The loop region is typically 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 can contain 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 can contain at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In some embodiments, the loop region can contain 10, 9, 8, 7, 6, 5, 4, 3, 2 or fewer unpaired nucleotides. In certain embodiments, the RNAi constructs of the invention comprise shRNAs. The length of the single, at least partially self-complementary RNA molecule can be about 35 to about 100 nucleotides, about 45 to about 85 nucleotides, or about 50 to about 60 nucleotides, and can include a duplex region and a loop region, each having a length as recited herein.

[0026] In some embodiments, the RNAi constructs of the present invention comprise 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, "PNPLA3 mRNA sequence" refers to any messenger RNA sequence, including splice variants, encoding 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).

[0027] The PNPLA3 mRNA sequence also includes the transcript sequence expressed as its complementary DNA (cDNA) sequence. A cDNA sequence refers to the sequence of an 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 an RNAi construct of the present invention can include a region having a sequence substantially or completely complementary 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 those derived from the NCBI reference sequence NM_025225.2.

[0028] The region of the antisense strand can be substantially complementary or fully 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 can 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 comprising a sequence substantially or fully complementary to the PNPLA3 mRNA sequence can, in some embodiments, comprise at least 15 contiguous nucleotides from an antisense sequence 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.

[0029] The sense strand of an RNAi construct typically contains a sequence sufficiently complementary to that of the antisense strand so 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 through 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, for example, binding of the Dicer enzyme and / or the RISC complex, thereby allowing the RNAi construct to enter the RNA interference pathway. 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.

[0030] In some embodiments, the RNAi agents of the present 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 being bound by theory, long double-stranded RNA introduced into cells can be degraded into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a RNase III-like enzyme, processes dsRNA into short interfering RNAs of 19 to 23 base pairs with characteristic two-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 mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0031] 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 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, "nucleotide overhang" refers to an unpaired nucleotide at the end of a strand or a nucleotide that extends beyond the duplex region. Nucleotide overhangs are 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 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, or 2 to 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In a particular embodiment, the nucleotide overhang comprises 1 to 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.

[0032] The nucleotide overhangs may be present at the 5'-end or 3'-end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5'-end and 3'-end of the sense strand. In some embodiments, the RNAi construct comprises nucleotide overhangs at the 5'-end of the sense strand and the 5'-end of the antisense strand. In other embodiments, the RNAi construct comprises nucleotide overhangs at the 3'-end of the sense strand and the 3'-end of the antisense strand.

[0033] An 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 perfectly base-paired at the ends of the molecule, with no unpaired nucleotides extending beyond the duplex region. In some embodiments, an 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, an 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, an 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).

[0034] The sense strand and 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 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 antisense strand form a duplexed region of the same length but shorter than the other 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 21 nucleotides in length, (ii) a duplex region that is 19 base pairs in length, 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 23 nucleotides in length, (ii) a duplex region that is 21 base pairs in length, 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 entire length, such that there are no nucleotide overhangs at either end of the duplex molecule. In one such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand, each 21 nucleotides in length, and (ii) a duplex region that is 21 base pairs in length. In another such embodiment, the RNAi construct is blunt-ended and comprises (i) sense and antisense strands that are each 23 nucleotides in length, and (ii) a duplex region that is 23 base pairs in length.

[0035] 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.

[0036] The antisense strand of an RNAi construct of the invention can 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 two-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.

[0037] Modified Nucleotides The RNAi construct of the present invention may contain one or more modified nucleotides. "Modified nucleotide" refers to a nucleotide having one or more chemical modifications to the nucleoside, nucleobase, pentose ring, or phosphate group. As used herein, modified nucleotides do not include ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, as well as deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. However, an RNAi construct may contain a combination of modified nucleotides, ribonucleotides, and deoxyribonucleotides. 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 molecule's susceptibility to nucleases and other degradation processes. Incorporation of modified nucleotides can also enhance the efficacy of an RNAi construct for reducing target gene expression.

[0038] 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. 2'-modified nucleotides refer to nucleotides 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.

[0039] "Bicyclic sugar modification" refers to a modification of a pentose ring in which a bridge connects two atoms of the ring to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, a bicyclic sugar modification comprises a bridge between the 4' and 2' carbons of the pentose ring. Nucleotides comprising 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(CH3)-O-2′) BNAs (constrained ethyl methylene-thio (4'-CH2-S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA; propylene carbocyclic (4'-(CH2)3-2') 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 Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated by reference in their entireties.

[0040] In some embodiments, an 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, an 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, an RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0041] Both the sense strand and the antisense strand of an 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 of the 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 of the nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all of the nucleotides in the sense strand and all of the 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.

[0042] In some embodiments, all pyrimidine nucleotides preceding an adenosine nucleotide in the sense strand, the antisense strand, or both strands are modified nucleotides. For example, when the sequence 5'-CA-3' or 5'-UA-3' appears 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 the 5' nucleotide of all sequences 5'-CA-3' or 5'-UA-3' present in the antisense strand is 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 a combination thereof.

[0043] In embodiments in which 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.

[0044] The RNAi constructs of the present invention may also contain one or more modified internucleotide linkages. As used herein, the term "modified internucleotide linkage" refers to a linkage between nucleotides other than the naturally occurring 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage, such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiral phosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. 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)-O-); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimino (-CH-N(CH)-O-CH-) and methylenehydrazino linkages; sulfonate and sulfonamide linkages; amide linkages; and others having mixed N, O, S, and CH component moieties. In one embodiment, the modified internucleoside linkage is a peptide-based linkage (e.g., aminoethylglycine) to produce 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 Deleaviey and Damha, Chemistry and Biology, Vol. 19:937-954, 2012, all of which are incorporated herein by reference in their entireties.

[0045] In certain embodiments, an 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 still other embodiments, both strands comprise 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages. An RNAi construct may comprise one or more phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or both the 3'-end and 5'-end of the sense strand, the antisense strand, or both strands. For example, in certain embodiments, an 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, an 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, an 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, an 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'- and 5'-ends 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'- and 5'-ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at both the 3'- and 5'-ends of the sense strand (i.e., phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5'- and 3'-ends of the antisense strand, and phosphorothioate internucleotide linkages at the first and second internucleotide linkages at both the 5'- and 3'-ends 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 within the strand may be native 3'-5' phosphodiester linkages. For example, in some embodiments, each internucleotide linkage in the sense strand and the antisense strand is selected from phosphodiester and phosphorothioate, and at least one internucleotide linkage is phosphorothioate.

[0046] In embodiments in which the RNAi construct comprises a nucleotide overhang, two or more of the unpaired nucleotides in the overhang may be linked by phosphorothioate internucleotide bonds. In certain embodiments, all unpaired nucleotides in the 3'-end nucleotide overhang of the antisense strand and / or sense strand are linked by phosphorothioate internucleotide bonds. In other embodiments, all unpaired nucleotides in the 5'-end nucleotide overhang of the antisense strand and / or sense strand are linked by phosphorothioate internucleotide bonds. In still other embodiments, all unpaired nucleotides in any nucleotide overhang are linked by phosphorothioate internucleotide bonds.

[0047] In certain embodiments, modified nucleotides incorporated into one or both strands of an RNAi construct of the invention have a nucleobase (also referred to herein as a "base") modification. 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 synthetically or naturally occurring modifications and include 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-azouracil, These may include, but are not limited to, 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.

[0048] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that indiscriminately base pairs with all natural bases in RNA and DNA without altering the resulting double-stranded region's double helix structure. 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.

[0049] 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 with other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of polynucleotides containing nucleotides having such substituted nucleobases.

[0050] In some embodiments of the RNAi constructs 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 phosphates include 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'-alkylphosphonates (e.g., alkyl = methyl, ethyl, isopropyl, propyl, etc.); and 5'-alkyl ether phosphonates (e.g., alkyl ether = methoxymethyl, ethoxymethyl, etc.).

[0051] Modified nucleotides that can be incorporated into RNAi constructs of the present invention can have two or more chemical modifications 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 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, which 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 an RNAi construct 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.

[0052] Function of RNAi constructs Preferably, the RNAi construct of the present invention reduces or inhibits the expression of PNPLA3 in cells, particularly liver cells. Thus, in one embodiment, the present invention provides a method for reducing PNPLA3 expression in cells by contacting cells with any of the RNAi constructs 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 compared to 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 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 PNPLA3 mRNA level measured from the treated cells in (a) with the PNPLA3 mRNA level measured from the control cells in (b).Before comparison, the PNPLA3 mRNA levels in treated cells and control cells can be normalized to the RNA level 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 assay, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

[0053] In another embodiment, the reduction of PNPLA3 expression is assessed by (a) measuring the amount or level of PNPLA3 protein in liver cells treated with an 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 targeting an RNA molecule not expressed in liver cells or an RNAi construct having a nonsense or scrambled sequence) or without the construct, and (c) comparing the PNPLA3 protein level measured from the treated cells in (a) with the PNPLA3 protein level measured from the control cells in (b). Methods for measuring PNPLA3 protein levels 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 assessing PNPLA3 protein expression 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 present invention.

[0054] In some embodiments, the methods for assessing the expression level of PNPLA3 are performed in vitro in cells that naturally express PNPLA3 (e.g., liver cells) or cells engineered to express PNPLA3. In certain embodiments, these methods are performed 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.

[0055] 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 the liver tissue collected from the treated animals.Alternatively or additionally, biomarkers or functional phenotypes associated with PNPLA3 expression can be evaluated in the treated animals.

[0056] In certain embodiments, the expression of PNPLA3 is reduced in liver cells 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% by the RNAi constructs of the present invention. In some embodiments, the expression of PNPLA3 is reduced in liver cells by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% by the RNAi constructs of the present invention. In other embodiments, the expression of PNPLA3 is reduced in liver cells by about 90% or more, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more by the RNAi constructs of the present invention. The percentage reduction in PNPLA3 expression can be measured by any of the methods described herein and other methods known in the art. For example, in certain embodiments, an RNAi construct of the present invention inhibits at least 45% of PNPLA3 expression in Hep3B cells (containing wild-type PNPLA3) in vitro at 5 nM. In related embodiments, an 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 in Hep3B cells in vitro at 5 nM. In other embodiments, an 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 in Hep3B cells in vitro at 5 nM. In certain embodiments, an RNAi construct of the present invention inhibits at least 45% of PNPLA3 expression in HepG2 cells (containing the PNPLA3-rs738409-rs738408 double minor allele) in vitro at 5 nM. In related embodiments, an RNAi construct of the invention inhibits PNPLA3 expression by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% at 5 nM in HepG2 cells in vitro.In other embodiments, an RNAi construct of the invention inhibits PNPLA3 expression by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% in HepG2 cells in vitro at 5 nM. In certain embodiments, an RNAi construct of the invention inhibits PNPLA3 expression by at least 45% in CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro at 5 nM. In related embodiments, an RNAi construct of the invention inhibits PNPLA3 expression by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro at 5 nM. In other embodiments, the RNAi constructs of the invention inhibit PNPLA3 expression by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% at 5 nM in CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro. Reduction of PNPLA3 can be measured using various techniques, including RNA FISH or droplet digital PCR, as described in Examples 2 and 3.

[0057] In some embodiments, the efficacy of the RNAi constructs of the present invention in inhibiting PNPLA3 expression in liver cells is evaluated by calculating an IC50 value. The "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 constructing a dose-response curve and testing the effect of different concentrations of the substance or antagonist on expression levels or functional activity in any assay. The IC50 value 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. Therefore, the IC50 value for any RNAi construct can be calculated by determining the concentration of the RNAi construct required to inhibit half of the native PNPLA3 expression level in liver cells (e.g., the PNPLA3 expression level in control liver cells) in any assay, such as the immunoassay, RNA FISH assay, or droplet digital PCR assay described in the Examples. The RNAi constructs 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 constructs inhibit 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 constructs inhibit PNPLA3 expression in liver cells (e.g., Hep3B cells) with an IC50 of about 1 nM to about 10 nM. The RNAi constructs 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 constructs inhibit PNPLA3 expression in liver cells (e.g., HepG2 cells) with an IC50 of about 1 nM to about 10 nM. The RNAi constructs of the present invention can 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 constructs inhibit 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.

[0058] The RNAi construct of the present invention can be easily produced using techniques known in the art, such as 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).

[0059] Oligonucleotides can be synthesized via phosphoramidite chemistry using a 2' silyl protecting group with an 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, at large, medium, or small scales. Synthesis can be performed in multiple well plates, columns, or glass slides.

[0060] The 2'-O-silyl group can be removed by 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 used in combination with inorganic fluorides in the deprotection reaction. Preferred fluoride ion sources are tetrabutylammonium fluoride or amine hydrofluorides (e.g., triethylamine and aqueous HF combined in a dipolar aprotic solvent, such as dimethylformamide).

[0061] The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters to fluoride. Methyl protection of the phosphotriester or phosphite triester can stabilize the bond to fluoride ions and improve process yields.

[0062] Because ribonucleosides have a reactive 2' hydroxyl substituent, it may be desirable to protect the reactive 2' position in 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 requirement and can be easily removed in a final fluoride deprotection step, which may result in minimal RNA degradation.

[0063] Tetrazole catalysts can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyl-tetrazole, benzylthiotetrazole, p-nitrophenyltetrazole.

[0064] As can be appreciated by those skilled in the art, additional methods for 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 alternate 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); T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley & Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley & Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons (1995), and subsequent editions thereof. Custom synthesis of RNAi agents is also available from several commercial vendors, including Dharmacon, Inc. (Lafayette, CO), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, CA).

[0065] The RNAi construct of the present invention may contain 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 can induce a biological response (e.g., trigger a signal transduction cascade, induce receptor-mediated endocytosis), or it can simply be a physical association. A ligand can modify one or more properties of the double-stranded RNA molecule to which it is bound, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties of the RNA molecule.

[0066] 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 specific cell type, such as the liver). Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecyl These 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 glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (e.g., spermine, spermidine).

[0067] In certain embodiments, the ligand has endosome-destabilizing properties. The endosome-destabilizing ligand promotes endosomal lysis and / or transport of the RNAi construct of the present invention or its components from the endosome to the cytoplasm of the cell. 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 one in which the endosome-destabilizing ligand promotes endosomal lysis and / or transport of the RNAi construct of the present invention or its components from the endosome to the cytoplasm of the cell. Exemplary endosome-destabilizing ligands include GALA peptide (Subbarao et al., Biochemistry, Vol. 26:2964-2972, 1987), EALA peptide (Vogel et al., J. Am. Chern. Soc., Vol. 118:1581-1586, 1996), and their derivatives (Turk et al., Biochem. Biophys. Acta, Vol. 1559:56-68, 2002). In one embodiment, the endosome-destabilizing component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosome-destabilizing component may be linear or branched.

[0068] 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 their unconjugated counterparts (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. Pat. Nos. 7,851,615; 7,745,608; and 7,833,992, all of which are incorporated herein by reference in their entireties. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to a folate moiety can be taken up into cells via receptor-mediated endocytosis. Such folate-polynucleotide conjugates are described in U.S. Pat. No. 8,188,247, which is incorporated herein by reference in its entirety.

[0069] Given that PNPLA3 is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to specifically deliver an 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.

[0070] 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), as well as 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.

[0071] 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 particular embodiments, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose, and N-acetyl-galactosamine (GalNAc) are particularly effective for targeting compounds to 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 Application Publication No. 20030130186; and WO 2013166155, all of which are incorporated by reference in their entireties.

[0072] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety containing two or more carbohydrate units that can independently bind or interact with other molecules. For example, a multivalent carbohydrate moiety contains 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 carbohydrate moieties 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 specific embodiment, the polyvalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another specific 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 described in detail below.

[0073] The ligand can be directly or indirectly bound or conjugated to the RNA molecule of the RNAi construct. For example, in some embodiments, the ligand is covalently bound directly 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 can be bound to the nucleobase, sugar moiety, or internucleotide linkage of the polynucleotide (e.g., the sense or antisense strand) of the RNAi construct of the present invention. Conjugation or binding to a purine nucleobase or a derivative thereof can 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 a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of the pyrimidine nucleobase can be bound to the ligand. Conjugation or binding to the sugar moiety of the nucleotide can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be attached to a ligand include the 2', 3', and 5' carbon atoms. The 1' position can also be attached to a ligand, such as at a basic residue. Internucleotide linkages can also facilitate ligand attachment. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand can 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 can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0074] In certain embodiments, the ligand can 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.

[0075] 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. Linkers can be about 1 to about 30 atoms in length, about 2 to about 28 atoms in length, about 3 to about 26 atoms in length, about 4 to about 24 atoms in length, about 6 to about 20 atoms in length, about 7 to about 20 atoms in length, about 8 to about 20 atoms in length, about 8 to about 18 atoms in length, about 10 to about 18 atoms in length, and about 12 to about 18 atoms in length. In some embodiments, the linker may comprise a bifunctional linking moiety, typically comprising an alkyl moiety bearing 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 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 moiety comprises amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bond), etc.

[0076] Linkers that can be used to attach a ligand to the sense or antisense strand in an RNAi construct of the present 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.

[0077] In certain embodiments, the linker is cleavable. A cleavable linker is one that is sufficiently stable outside the cell but is cleaved after entry into the target cell to release the two moieties that the linker holds together. In some embodiments, the cleavable linker is cleaved at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, or 90-fold or more, or at least 100-fold faster in the target cell or under a first reference condition (e.g., which may be selected to mimic or correspond to intracellular conditions) than in the subject's blood or under a second reference condition (e.g., which may be selected to mimic or correspond to conditions found in blood or serum).

[0078] Cleavable linkers are sensitive to cleaving agents, such as pH, redox potential, or the presence of degradative 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 degradative agents include oxidizing or reducing agents, such as mercaptans, that are present inside cells and can degrade redox-cleavable linkers by reduction, which are selective for specific substrates or have no substrate specificity; 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.

[0079] 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.

[0080] The linker can include a cleavable group that can be cleaved by a specific enzyme. The type of cleavable group incorporated into the linker can depend on the target cell. For example, a liver targeting ligand can be linked to an RNA molecule via a linker containing an ester group. Liver cells are rich in esterase, so the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterase. Other types of cells that are rich in esterase include lung, renal cortex, and testicular cells. Linkers containing peptide bonds can be used when targeting peptidase-rich cells such as liver cells and synovial cells.

[0081] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage when in contact with blood or other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between first conditions selected to be indicative of cleavage in target cells and second conditions selected to be indicative of cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, cells, cell culture, organ or tissue culture, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions and confirm with further evaluations in whole animals. In some embodiments, useful candidate linkers are 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) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0082] In other embodiments, a redox-cleavable linker is utilized. A redox-cleavable linker is cleaved upon reduction or oxidation. One example of a reductively cleavable group is a disulfide linking group (-SS-). One or more methods described herein can be used to determine whether a candidate cleavable linker is a suitable "reductively cleavable linker," or whether it is suitable for use with, for example, a particular RNAi construct and a particular ligand. For example, a candidate linker can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents known in the art that mimic the cleavage rate that would be observed in cells, such as target cells. The candidate linker can also be evaluated under conditions selected to mimic blood or serum conditions. In certain embodiments, the candidate linker is cleaved at a maximum of 10% in blood. In other embodiments, useful linker candidates are degraded 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 under in vitro conditions selected to mimic extracellular conditions).

[0083] In yet other embodiments, 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 within a cell is an enzyme such as an intracellular phosphatase. 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-, and -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.

[0084] 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 below (e.g., about 6.0, 5.5, 5.0 or below) 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 bonded 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.

[0085] 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 linker candidates can be evaluated using methods similar to those described above.

[0086] 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, but do not include the entire amide functionality. Peptidic cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0087] Other types of linkers suitable for attaching a ligand to the sense or antisense strand in the RNAi constructs 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.

[0088] 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.

[0089] In some embodiments, the RNAi constructs of the present invention can be delivered to cells or tissues 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 into 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. Vectors can be replicated within living cells or can be synthetically produced.

[0090] Generally, vectors for expressing RNAi constructs of the present invention will contain one or more promoters operably linked to a sequence encoding the RNAi construct. As used herein, the phrases "operably linked" or "under transcriptional control" mean that the promoter is appropriately positioned and oriented relative to the polynucleotide sequence to control initiation of transcription by RNA polymerase and expression of the polynucleotide sequence. A "promoter" refers to a sequence recognized by or introduced into the synthetic machinery of a cell and required to initiate transcription of a specific gene sequence. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI, or U6 RNA pol III, and viral promoters (e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In some embodiments, the HI or U6 RNA pol III promoter is preferred. The promoter may be a tissue-specific or inducible promoter. Of particular interest are liver-specific promoters, such as promoter sequences derived 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).

[0091] In some embodiments in which 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 embodiments, the first and second vectors are simultaneously introduced into a target cell, for example, by infection or transfection, so that the sense and antisense strands are transcribed and hybridize to form an siRNA molecule within the cell. 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, and 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 opposite orientation, such that transcription of the sequence from the first promoter results in the synthesis of the sense strand of the siRNA molecule, and transcription of the sequence from the second promoter results in the synthesis of the antisense strand of the siRNA molecule.

[0092] 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.

[0093] 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 (for example, 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 (for example, vaccinia virus).In some embodiments, the viral vector is a retrovirus vector (for example, lentivirus vector).

[0094] 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. For example, 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. al., Nat Biotechnol, Vol. 20:497-500, 2002; Paddison et al., GenesDev, Vol. 16:948-958, 2002; Paul et al., Nat 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 (all of which are incorporated by reference in their entireties).

[0095] The present invention also includes pharmaceutical compositions and preparations comprising the RNAi constructs described herein and pharmaceutically acceptable carriers, excipients or diluents.Such compositions and preparations are useful for reducing the expression of PNPLA3 in subjects in need.When considering clinical use, pharmaceutical compositions and preparations will be prepared in a form suitable for the intended use.Generally, this will entail preparing compositions that are essentially free of pyrogens and other impurities that may be harmful to humans or animals.

[0096] The phrases "pharmaceutically 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, "pharmaceutically acceptable carriers, excipients, or diluents" include solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for human administration. 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 compositions, provided that they do not inactivate the vector or RNAi construct of the composition.

[0097] The composition and method of pharmaceutical composition formulation depend on several factors, including, but not limited to, the route of administration, the type and severity of the disease or disorder being 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 modes include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such embodiments, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such embodiments, the pharmaceutical composition may include a targeting ligand (e.g., a GalNAc-containing ligand described herein).

[0098] In some embodiments, the pharmaceutical composition comprises an effective amount of an RNAi construct described herein. An "effective amount" is an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, an effective amount is an amount sufficient to reduce PNPLA3 expression in the subject's liver cells. 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). Therefore, without being bound by theory, it is believed that partial reduction of PNPLA3 expression may be sufficient to achieve beneficial reductions in serum non-HDL cholesterol and reduced risks of coronary artery disease and myocardial infarction.

[0099] An effective amount of an RNAi construct of the present invention can be about 0.01 mg / kg to about 100 mg / kg body weight, about 0.05 mg / kg to about 75 mg / kg body weight, about 0.1 mg / kg to about 50 mg / kg body weight, about 1 mg / kg to about 30 mg / kg body weight, about 2.5 mg / kg to about 20 mg / kg body weight, or about 5 mg / kg to about 15 mg / kg body weight. In certain embodiments, an effective single dose of an RNAi construct of the present invention can 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 an RNAi construct can 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.

[0100] Administration of the pharmaceutical compositions of the present invention can be via any common route, as 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 compositions are administered parenterally. For example, in certain embodiments, the pharmaceutical compositions are administered intravenously. In other embodiments, the pharmaceutical compositions are administered subcutaneously.

[0101] 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 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 an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the present invention can be encapsulated within 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 03 / 093449.

[0102] 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 stable nucleic acid-lipid particles, including SPLPs. As used herein, the term "SPLP" refers to nucleic acid-lipid particles containing plasmid DNA encapsulated in lipid vesicles. SNALPs and SPLPs typically 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 circulatory lifetimes after intravenous injection and accumulate at distal sites (e.g., sites physically distant from the administration site). SPLPs include "pSPLPs" containing encapsulated condensing agent-nucleic acid complexes, as described in WO 00 / 03683. Nucleic acid-lipid particles typically 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. Additionally, 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. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and WO 96 / 40964.

[0103] Pharmaceutical compositions suitable for injectable 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 easy syringability exists. Preparations should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. Suitable solvents or dispersion media may contain, for example, water, ethanol, polyol (e.g., 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 achieved 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, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0104] Sterile solution for injection can be prepared by incorporating active compound in appropriate amount with any other desired components (for example, as listed above) into solvent, followed by filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other desired components, for example, as listed above.For sterile powder for preparing sterile solution for injection, preferred preparation method includes vacuum drying and freeze-drying, which produces powder of active ingredient and any other desired components from its solution that has been previously sterilized and filtered.

[0105] The compositions of the present invention can generally be formulated in a neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed with 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 with 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.).

[0106] For parenteral administration in an aqueous solution, for example, the solution is usually suitably buffered and the liquid diluent 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, e.g., "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 required by FDA standards. In certain embodiments, the pharmaceutical compositions of the invention comprise or consist of sterile saline and the RNAi constructs described herein. In other embodiments, 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, pharmaceutical compositions of the invention comprise or consist of an RNAi construct described herein and phosphate buffered saline (PBS).

[0107] In some embodiments, the pharmaceutical composition of the present invention is packaged in or stored in an administration device.Devices for injectable formulations include, but are not limited to, injection ports, pre-filled syringes, automatic injection devices, injection pumps, wearable syringes, and injection pens.Devices for aerosolized or powdered formulations include, but are not limited to, inhalers, inhalers, inhalers, etc.Therefore, the present invention includes an administration device comprising the pharmaceutical composition of the present invention for treating or preventing one or more of the disorders described herein.

[0108] Methods for inhibiting PNPLA3 expression The present invention also provides a method for inhibiting the expression of PNPLA3 gene in cells.The method comprises contacting cells with an RNAi agent, for example, a double-stranded RNAi agent, in an amount effective to inhibit the expression of PNPLA3 in cells, thereby inhibiting the expression of PNPLA3 in cells.The contact of cells with an RNAi agent, for example, a double-stranded RNAi agent, can be carried out in vitro or in vivo.Contacting cells with an RNAi agent in vivo includes contacting a cell or a group of cells in a subject, for example, a human subject, with an RNAi agent.A combination of in vitro and in vivo methods of contacting cells is also possible.

[0109] The present invention provides methods for reducing or inhibiting the expression of PNPLA3 in a subject in need thereof, and methods for treating or preventing conditions, diseases, or disorders associated with PNPLA3 expression or activity. "Conditions, diseases, or disorders associated with PNPLA3 expression" refers to conditions, diseases, or disorders in which altered expression levels of PNPLA3 or elevated expression levels of PNPLA3 are associated with an increased risk of developing the condition, disease, or disorder.

[0110] The contact of cells can be direct or indirect as described above.In addition, 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 can guide RNAi agent to the target site.

[0111] In one embodiment, contacting a cell with an RNAi includes "introducing" or "delivering the RNAi to a cell" by facilitating or causing cellular uptake or absorption. Absorption or uptake of the RNAi can occur by unassisted diffusive or active cellular processes, or by auxiliary agents or devices. 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. Additional techniques are described herein below and / or known in the art.

[0112] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes all levels of inhibition.

[0113] The phrase "inhibiting the 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.

[0114] "Inhibiting the expression of the PNPLA3 gene" includes any level of inhibition of the PNPLA3 gene, for example, at least partial suppression of the expression of the PNPLA3 gene. The 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, the PNPLA3 mRNA level, the PNPLA3 protein level, or the number or extent of amyloid deposits. This level can be evaluated, for example, in individual cells or cell groups, including samples derived from a subject.

[0115] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables related to PNPLA3 expression compared to a control level, which can be any type of control level utilized in the art, such as a baseline level before 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 agent 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%.

[0116] Inhibition of expression of the PNPLA3 gene can be evidenced by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can 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 cells with an RNAi agent of the present invention or by administering an RNAi agent of the present invention to a subject in which the cells are or were present) such that expression of the PNPLA3 gene is inhibited, 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 can 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 cells with an RNAi agent of the present invention or by administering an RNAi agent of the present invention to a subject in which the cells are or were present) such that expression of the PNPLA3 gene is inhibited.

number

[0117] Alternatively, inhibition of PNPLA3 gene expression may be assessed in terms of a decrease in a parameter functionally related to PNPLA3 gene expression, such as PNPLA3 protein expression or Hedgehog pathway protein activity. PNPLA3 gene silencing can be determined by any assay known in the art in any cell that constitutively or by genome manipulation expresses PNPLA3.

[0118] Inhibition of PNPLA3 protein expression 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, the inhibition of protein expression levels in treated cells or cell groups can also be expressed as a percentage of the protein levels in control cells or cell groups.

[0119] 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 (for example, human or animal subject) before the subject is treated with the RNAi agent.

[0120] 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 the PNPLA3 gene mRNA, 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), the RNeasy RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing 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.

[0121] In one embodiment, the expression level of PNPLA3 is determined using a nucleic acid probe. As used herein, the term "probe" 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 appropriate 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, protein, antibody, and organic molecules.

[0122] 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 for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PNPLA3 mRNA. 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 PNPLA3 mRNA levels.

[0123] 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. (1988) Bio / Technology 6:1197), and the like.

[0013] Methods for detecting PNPLA3 mRNA include, for example, nucleic acid amplification of mRNA in a sample and / or reverse transcriptase (to prepare cDNA) using a method well known to those skilled in the art, 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, particularly when they are present in very low numbers. In a specific embodiment of the present invention, the level of PNPLA3 expression 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, and dot), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acids). 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 involve the use of nucleic acid probes in solution.

[0124] In preferred embodiments, the level of mRNA expression 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.

[0125] The level of PNPLA3 protein expression can be determined using any method known in the art for measuring protein levels, such as 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 (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.

[0126] 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 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.

[0127] In some embodiments of the method of the present invention, the RNAi agent is administered to a subject so that the RNAi agent is delivered to a specific site within the subject. Inhibition of PNPLA3 expression can be evaluated by measuring the level or change in the level of PNPLA3 mRNA or PNPLA3 protein in a sample derived from body fluid or tissue from a specific site within the subject. In a preferred embodiment, the site is selected from the group consisting of the liver, choroid plexus, retina, and pancreas. The site can also 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.

[0128] Methods for treating or preventing PNPLA3-related diseases The present invention provides therapeutic and preventive methods for a subject suffering from or susceptible to a PNPLA3-related disease, disorder, and / or condition, comprising administering a composition comprising an RNAi agent, a pharmaceutical composition comprising an RNAi agent, or a vector comprising an RNAi agent of the present invention to a subject. Non-limiting examples of PNPLA3-related diseases include fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis, accumulation of fat in the liver, liver inflammation, hepatocellular 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.

[0129] In some 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 mammals, including humans, 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 administering the RNAi construct, compared with the expression level of PNPLA3 in the patient who has not received the RNAi construct.

[0130] The method of the present invention is useful for treating subjects with PNPLA3-related diseases, for example, 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 activity level of Hedgehog pathway in subjects with NAFLD.

[0131] 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), non-alcoholic steatohepatitis (NASH), cirrhosis, fat accumulation in the liver, liver inflammation, hepatocellular necrosis, liver fibrosis, obesity, or non-alcoholic 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 the PNPLA3 gene, or a pharmaceutical composition comprising the RNAi agent of the present invention that targets the PNPLA3 gene, or the vector of the present invention that comprises the RNAi agent of the present invention that targets the PNPLA3 gene.

[0132] 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, pharmaceutical composition or vector of the present invention, to the subject, thereby preventing at least one symptom of the subject with the disorder that will benefit from reducing PNPLA3 gene expression.

[0133] In another aspect, the present invention provides the use of a therapeutically effective amount of an 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 an RNAi agent of the present invention that targets the PNPLA3 gene, for example, a dsRNA, or a pharmaceutical composition comprising an RNAi agent that targets 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 subject with a PNPLA3-related disease, for example, a subject that will benefit from reducing and / or inhibiting PNPLA3 gene expression and / or PNPLA3 protein production.

[0134] In another aspect, the present invention provides the 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.

[0135] 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-associated disease.

[0136] In one embodiment, the RNAi agent targeting PNPLA3 is a dsRNA agent that, when administered to a subject, e.g., reduces or at least reduces the expression of the PNPLA3 gene in the 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%, 110%, 111%, 112%, 113%, 114%, 115 The compound is administered to a subject having 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.

[0137] The methods and uses of the present invention include administering a composition 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.

[0138] The administration of dsRNA according to the methods and uses 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.In this context, "reduction" refers to a statistically significant reduction in such levels.The 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 those skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. For example, the effectiveness of treating NAFLD can be evaluated by, for example, regularly monitoring NAFLD symptoms, liver fat levels, or the expression of downstream genes. Comparing the initial reading with the subsequent reading provides the physician with an indication of whether the treatment is effective. It is well within the capabilities of those skilled in the art to monitor the effectiveness of treatment or prevention by measuring any one or any combination of such parameters. In relation to the administration of an RNAi targeting PNPLA3 or a pharmaceutical composition thereof, "effective against" PNPLA3-related diseases means that administration in a clinically relevant manner will result in beneficial effects, such as symptom improvement, cure, disease reduction, prolongation of life, improvement in quality of life, or other effects generally recognized as favorable by physicians familiar with the treatment of NAFLD and / or PNPLA3-related diseases and related causes, in at least a statistically significant proportion of patients.

[0139] The therapeutic or preventive effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or when there is no worsening 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 effectiveness 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 effectiveness of treatment is proven when a statistically significant reduction in markers or symptoms is observed.

[0140] The subjects included dsRNA at about 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.

[0141] Administration of an RNAi may, for example, reduce the presence of PNPLA3 protein levels in the patient's cells, tissues, blood, urine, or other compartment 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%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, %, 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.

[0142] 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. In another example, the patient may be monitored for unwanted immunostimulatory effects, such as elevated cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0143] 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.

[0144] The RNAi of the present invention may be administered in a "naked" form, in which the modified or unmodified RNAi agent is directly suspended in an aqueous or suitable buffer solvent as a "free RNAi." The free RNAi is administered in the absence of a pharmaceutical composition. The free RNAi may be in a suitable buffer solution. The buffer solution may contain 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 the RNAi may be adjusted to be suitable for administration to a subject.

[0145] Alternatively, the RNAi of the present invention can be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

[0146] Subjects who 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.

[0147] Treatment of subjects that would benefit from reducing and / or inhibiting PNPLA3 gene expression includes therapeutic and prophylactic treatment.

[0148] The present invention further provides methods for treating subjects who would benefit from reducing and / or inhibiting PNPLA3 gene expression, e.g., subjects with PNPLA3-related diseases, 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.

[0149] For example, in some embodiments, the RNAi that targets PNPLA3 gene is administered in combination with, for example, the agent that is 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 reducing 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 that is used to treat PNPLA3-related disease, or any combination of the above.

[0150] In some embodiments, the first RNAi agent that targets PNPLA3 gene is administered in combination with the second RNAi agent that targets different parts of PNPLA3 gene.For example, the first RNAi agent comprises a first sense strand and a first antisense strand that form a duplex 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 with the 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 duplex 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 with the 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.

[0151] 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 a modification.

[0152] 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 containing a non-natural base, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide containing a phosphorothioate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimic.

[0153] In certain embodiments, the first RNAi agent that targets PNPLA3 gene is administered in combination with the second RNAi agent that targets a gene other than PNPLA3 gene.For example, the RNAi agent that targets PNPLA3 gene can be administered in combination with the RNAi agent that targets SCAP gene.The first RNAi agent that targets PNPLA3 gene and the second RNAi agent that targets a gene other than PNPLA3 gene, for example, SCAP gene, can be administered as part of the same pharmaceutical composition.Alternatively, the first RNAi agent that targets PNPLA3 gene and the second RNAi agent that targets a gene other than PNPLA3 gene, for example, SCAP gene, can be administered as part of different pharmaceutical compositions.

[0154] 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.

[0155] The present invention also provides a method for using an RNAi agent of the present invention and / or a composition containing an RNAi agent of the present invention to reduce and / or inhibit PNPLA3 expression in a cell. In another aspect, the present invention provides an RNAi of the present invention and / or a composition containing 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 a use of an RNAi of the present invention and / or a composition containing the RNAi of the present invention 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 an RNAi of the present invention and / or a composition containing 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 a use of an RNAi of the present invention and / or a composition containing the RNAi of the present invention 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 an RNAi of the present invention, for example, 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.

[0156] The reduction of gene expression can be evaluated by any method known in the art.For example, the reduction of PNPLA3 expression can be determined by using methods familiar to those skilled in the art, such as Northern blotting, qRT-PCR to determine the mRNA expression level of PNPLA3, by using methods familiar to those skilled in the art such as Western blotting, immunological method, flow cytometry, ELISA, etc. to determine the protein level of PNPLA3, and / or by determining the biological activity of PNPLA3.

[0157] 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.

[0158] Cells suitable for treatment using the methods of the present invention can be any cells that express the PNPLA3 gene, such as cells from a subject with NAFLD, or cells that contain an expression vector containing the PNPLA3 gene or a portion of the PNPLA3 gene.Cells suitable for use in the methods and uses of the present invention can be mammalian cells, such as primate cells (human cells or non-human primate cells, such as monkey cells or chimpanzee cells), non-primate cells (such as cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (such as duck cells or goose cells), or whale cells.In one embodiment, the cells are human cells.

[0159] 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%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124 %, 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.

[0160] 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%, 1109%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 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%.

[0161] The in vivo method and use of the present invention may comprise administering to a subject a composition containing RNAi, wherein the RNAi comprises 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 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 (aerosol), nasal, parenteral routes including rectal, and topical (including buccal and sublingual) administration.In some embodiments, the composition is administered by subcutaneous or intravenous infusion or injection.In one embodiment, the composition is administered by subcutaneous injection.

[0162] In some embodiments, administration is via depot injection. Depot injection can release RNAi consistently for a long period of time. Therefore, depot injection can reduce the frequency of administration required to achieve desired effect, for example, desired PNPLA3 inhibition, or therapeutic or preventive 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.

[0163] In some embodiments, administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump implanted subcutaneously. 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.

[0164] 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.

[0165] 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, for example, the 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, for example, the dsRNA that targets PNPLA3 gene in mammalian cells, in the manufacture of the medicine that inhibits the expression of PNPLA3 gene in mammals.

[0166] The methods and uses include administering to a mammal, e.g., a human, a composition comprising RNAi, e.g., dsRNA, that targets the PNPLA3 gene in the cells of the mammal, and maintaining the mammal for a sufficient time to obtain degradation of mRNA transcripts of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the mammal.

[0167] The reduction of gene expression can be evaluated in the peripheral blood sample of the subject receiving RNAi by any method known in the art, for example, by qRT-PCR as described herein.The reduction of protein production can be evaluated by any method and method known in the art, for example, by ELISA or Western blotting as described herein.In one embodiment, tissue sample serves as the tissue material for monitoring the reduction of PNPLA3 gene and / or protein expression.In another embodiment, blood sample serves as the tissue material for monitoring the reduction of PNPLA3 gene and / or protein expression.

[0168] In one embodiment, verification of RISC-mediated cleavage of the target in vivo after administration of the 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-e19) (Zimmermann et al. (2006) Nature 441:111-4).

[0169] It is understood that all ribonucleic acid sequences disclosed herein can be converted to deoxyribonucleic acid sequences by substituting thymine bases for uracil bases in the sequence. Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted to ribonucleic acid sequences by substituting thymine bases for uracil bases in the sequence. Deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing mixtures of deoxyribonucleotides and ribonucleotides of all sequences disclosed herein are encompassed by the present invention.

[0170] In addition, any nucleic acid sequence disclosed herein can 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 comprising nucleotides with 2'-OH substituents on the ribose sugar and thymine bases can be described as a DNA molecule with modified sugars (2'-OH for the natural 2'-H in DNA) or an RNA molecule with modified bases (thymine (methylated uracil) for the natural uracil in RNA).

[0171] 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 indicates a cytosine base with a methyl group at the 5-position.

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

[0173] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are incorporated by reference herein 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.

[0174] equivalent The foregoing specification is considered sufficient to enable one skilled in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and set forth the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears, it will be understood that the invention can be practiced in many ways and should be construed in accordance with the appended claims and their equivalents.

[0175] 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. [Example]

[0176] 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.

[0177] Table 1

[0178] Table 2

[0179] Table 3

[0180] Table 4

[0181] Table 5

[0182] Table 6

[0183] Table 7

[0184] Table 8

[0185] Table 9

[0186] Table 10

[0187] Table 11

[0188] Table 12

[0189] Table 13

[0190] Table 14

[0191] Table 15

[0192] Table 16

[0193] Table 17

[0194] Table 18

[0195] Table 19

[0196] Table 20

[0197] Table 21

[0198] [Table 22]

[0199] [Table 23]

[0200] [Table 24]

[0201] [Table 25]

[0202] To improve the potency and in vivo stability of PNPLA3 siRNA sequences, chemical modifications were incorporated into PNPLA3 siRNA molecules. Specifically, 2'-O-methyl and 2'-fluoro modifications of the ribose sugar were incorporated at specific positions within PNPLA3 siRNA. Phosphorothioate internucleotide linkages were also incorporated at the ends of the antisense and / or sense sequences. Table 2 below shows the modifications in each sense and antisense sequence of modified PNPLA3 siRNA. The nucleotide sequences in Table X are listed according to the following notation: A, U, G, and C = corresponding ribonucleotides; dT = deoxythymidine; a, u, g, and c = corresponding 2'-O-methyl ribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides. The insertion of "s" in a 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 a 3'-5' phosphodiester group. 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] is.

[0203] [Table 26]

[0204] [Table 27]

[0205] [Table 28]

[0206] [Table 29]

[0207] [Table 30]

[0208] [Table 31]

[0209] [Table 32]

[0210] [Table 33]

[0211] [Table 34]

[0212] [Table 35]

[0213] Table 36

[0214] Table 37

[0215] Table 38

[0216] Table 39

[0217] Table 40

[0218] Table 41

[0219] Table 42

[0220] Table 43

[0221] Table 44

[0222] Table 45

[0223] 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 in vitro mRNA knockdown potency and selectivity. 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 contained 19 complementary base pairs. In some cases, there was a two-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 linkage in one or both strands was replaced with a phosphorothioate to reduce exonucleolytic degradation.

[0224] The effectiveness of each siRNA molecule 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 cells were 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.

[0225] 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 2,000 cells / well in 40 μl medium. After 20 minutes 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.

[0226] 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.

[0227] 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 labels) VA6-20279-01 and PPIB Human 0.44 mL View RNA Type 1 (488 labels) VA1-10148-01.

[0228] First, the plate was rehydrated by successive 100%, 70%, and 50% ethanol washes. Next, the cells were washed with PBS and then permeabilized and protease digested according to the kit instructions. The desired Working Probe Set was prepared according to the manufacturer's protocol, added to the wells, and incubated at 40°C for 3 hours. The manufacturer's protocol was adopted for sequential hybridization with the Working Probe Set, Working PreAmp, Working Amp, and Working LP. Finally, a nuclear counterstain was applied (Hoechst 33342 and Cell Mask Blue; Molecular Probes). The plate was incubated at room temperature for 30 minutes, washed with PBS, and overlaid with 80 μl of PBS. The plate was then sealed for imaging.

[0229] 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.

[0230] RNA FISH data was analyzed using Columbus software, and images were generated using Genedata Screener. The results of the assay for CHO transfected with PNPLA3 I148I are shown in Table 3. The results of the assay for CHO transfected with PNPLA3 I148M are shown in Table 4. PNPLA3 knockdown provides the percentage of knockdown compared to the control. Negative values ​​indicate a reduction in PNPLA3 levels.

[0231] [Table 46]

[0232] Table 47

[0233] Table 48

[0234] 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 minimal 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) using a BioMek FX (Beckman Coulter). Next, 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) pre-diluted in simple MEM or EMEM (specifically, 0.035 μL of RNAiMAX in 5 μL for Hep3B and 0.06 μL of RNAiMAX in 5 μL of EMEM for HepG2) was dispensed into the assay plate using a Multidrop Combi reagent dispenser (Thermo Fisher Scientific). After a 20-minute incubation of the siRNA / RNAiMAX mixture at room temperature (RT), 30 μL of either Hep3B or HepG2 cells (2,000 cells per well) in MEM or EMEM supplemented with 10% FBS and 1% PS was added to the transfection complex using the Multidrop Combi reagent dispenser. The assay plate was incubated for 20 minutes at RT before being placed in an incubator. The cells were then incubated at 37°C and 5% CO for 72 hours. 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 minutes at room temperature, permeabilized with detergent for 3 minutes at room temperature, and then treated with protease solution for 10 minutes at room temperature. Incubation with the target-specific probe pair (Thermo Fisher Scientific) was performed for 3 hours, except for the preamplifier, amplifier, and label probe (Thermo Fisher Scientific), which were each incubated for 1 hour. 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 minutes and then imaged on an Opera Phenix (PerkinElmer). Images were analyzed using a Columbus Image Data Archiving and Analysis System (PerkinElmer) to obtain average spot counts 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 the data were fitted to a four-parameter sigmoidal model in Genedata Screener (Genedata) to obtain IC50 and maximal activity. Assay results for HepG2 cells are shown in Table 5, and assay results for Hep3B cells are shown in Table 6. PNPLA3 knockdown provides the percentage of knockdown compared to the control. Negative values ​​indicate reduced PNPLA3 levels. Where duplexes were performed more than once, the average IC50 is shown with the standard deviation.

[0235] [Table 49]

[0236] [Table 50]

[0237] [Table 51]

[0238] [Table 52]

[0239] Example 3: Droplet digital PCR assay of siRNA for PNPLA3-rs738409 and PNPLA3-rs738409-rs738408 According to the manufacturer's protocol, human primary hepatocytes (Xenotech / Sekisui donor lot #HC3-38) were thawed in OptiThaw medium (Xenotech cat#K8000). The cells were centrifuged, the medium was aspirated, and the cells were resuspended in OptiPlate Hepatocyte Medium (Xenotech cat#K8200) and plated onto 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). Two to four hours after the addition of OptiCulture medium, GalNAc-conjugated siRNA was delivered into the cells by free uptake (no transfection reagent). The cells were incubated at 37°C and 5% CO2 for 24-72 hours. Cells were then lysed in Qiagen's RLT buffer (79216) supplemented with 1% 2-mercaptoethanol (Sigma, M-3148), and the 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 varied depending on the sample. Reverse transcription was carried out on a BioRad quadruplicate thermal cycler (model number 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 4°C hold.

[0240] Droplet digital PCR (ddPCR) was performed using BioRad's QX200 AutoDG droplet digital PCR system according to the manufacturer's instructions. 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 varied between wells. Droplets were generated using a BioRad Auto DG droplet generator (1864101) set up with the manufacturer's recommended consumables (BioRad DG32 cartridge 1864108, BioRad tip 864121, 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 at 95°C for 10 minutes, denaturation at 94°C for 30 seconds, followed by annealing / extension at 60°C for 1 minute, 40 cycles with a ramp rate of 2°C / second, enzyme deactivation at 98°C for 10 minutes, followed by an optional 4°C hold. Samples were then read on a BioRad QX200 droplet reader, which measures the FAM / HEX signal, which correlates with PNPLA3 or TBP concentration. Data were analyzed using BioRad's QuantaSoft software package. Samples were gated by channel (fluorescent label) to determine the concentration per sample. Each sample is then expressed as a ratio of the concentration of the gene of interest (PNPLA3) to the concentration of the housekeeping gene (TBP) relative to the control for different sample loadings.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.

[0241] [Table 53]

[0242] [Table 54]

[0243] [Table 55]

[0244] [Table 56]

[0245] [Table 57]

[0246] Example 4: Efficacy screening of selected PNPLA3 siRNA molecules in a humanized mouse model Adeno-associated virus (AAV; serotype 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 the expression of human PNPLA3 in the liver. WT (PNPLA3-WT), PNPLA3 rs738409 (PNPLA3-I148M), or PNPLA3 rs738409-rs738408Mice were generally 10-12 weeks old, with 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 subcutaneously with a single dose of 0.5 mM siRNA D-2324 diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) 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, snap-frozen in liquid nitrogen, and processed for purified RNA using a QIACube HT instrument (Qiagen, 9001793) and an 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 prepared for real-time qPCR using the TaqMan™ RNA-to-CT™ 1-Step Kit (Applied Biosystems, 4392653). Real-time qPCR was performed on a QuantStudio real-time PCR instrument. Results are expressed as the relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated control animals, based on human PNPLA3 gene expression normalized to mouse Gapdh (TaqMan™ assays from Invitrogen, hs00228747_m1 and 4352932E, respectively). Endogenous mouse Pnpla3 expression was determined for comparison (Invitrogen, Mm00504420_m1).

[0247] For liver triglyceride content analysis, approximately 0.05–0.1 milligrams of frozen liver from each animal was homogenized in 1 milliliter of isopropanol. After 1 hour of incubation on ice, the sample was 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 determined using the colorimetric Infinity Triglyceride Reagent (Thermo Fisher Scientific, TR22421) and triglyceride standard (Pointe Scientific, T7531-STD) according to the manufacturer's instructions. Results are presented as milligrams of triglyceride per milligram of tissue.

[0248] All animal experiments described herein were approved by Amgen's Institutional Animal Care and Use Committee (IACUC) and in accordance with the Guide for the Care and Use of Laboratory Animals, 8 thMice were cared for in accordance with the Committee for Update of the National Research Council (US) 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-hour light:12-hour dark cycle (0600-1800 h). Animals had access to a regular chow diet (Envigo, 2920X) ad libitum and water (reverse osmosis purified) via an automated watering system unless otherwise indicated. At termination, blood was collected by cardiac puncture under deep anesthesia and subsequently approved by the Association for Assessment and Accreditation of Laboratory Animals. The animals were euthanized by secondary physical methods in accordance with the AAALAC guidelines. Figure 1A-D. An example of five siRNA molecules screened for both in vivo dose-dependent mRNA knockdown and functional durability. Human PNPLA3 rs738409-rs73840 Mice expressing human PNPLA3 were treated with siRNA two 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 collected and processed for gene expression analysis. Data are shown 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 parallel cohorts 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 collected 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 group were also processed for triglyceride content to obtain functional efficacy over time. Data represent the average milligrams of triglyceride per gram of processed tissue.

[0249] Data on relative knockdown are shown in Tables 8-12, which show the relative knockdown at various doses and on days 8, 15, 22, 28, and 42, respectively. PNPLA3 knockdown is a percentage with negative values ​​indicating reduced PNPLA3 levels.

[0250] [Table 58]

[0251] [Table 59]

[0252] [Table 60]

[0253] [Table 61]

[0254] [Table 62]

[0255] [Table 63]

[0256] [Table 64]

[0257] [Table 65]

[0258] [Table 66]

[0259] [Table 67]

[0260] [Table 68]

[0261] [Table 69]

[0262] 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 fat (45% of total fat) and sugar (Tetri 2008). For these studies, 8-10 week-old C57BL / 6NCrl male mice (Charles River Laboratories Inc.) were transfected with either an AAV empty vector or AAV8-PNPLA3 vector as previously described. rs738409-rs738408 At the time of AAV injection, mice were maintained on a regular chow diet until harvest or placed on an ALIOS diet (Envigo, TD.06303) with drinking water consisting of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively). rs738409-rs738408 We established that overexpression of α-glucan promotes and exacerbates the NAFLD phenotype in this context (data not shown).

[0263] Two weeks after AAV injection and initiation of the diet, mice were treated with a single dose of 5.0 milligrams per kilogram of animal of siRNA D-2324 (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 4190-136) or vehicle 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 in 10% neutral-buffered formalin and subsequently processed and embedded in paraffin. The remainder of the liver was snap-frozen for content and gene expression analysis as previously described.

[0264] 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_m1; mouse Pnpla3, Mm00504420_m1; mouse Gapdh, 4352932E).

[0265] 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.

[0266] Serum analysis 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.

[0267] Figure 2A-G. NAFLD and PNPLA3 rs738409-rs738408 PNPLA3 prevents the development of phenotypes associated with its overexpression rs738409-rs738408 To evaluate the efficacy of the specific siRNA molecule D-2324, mice were transfected with either AAV8 empty vector (EV) or AAV8-PNPLA3. rs738409-rs738408 Mice received either siRNA or vehicle and were 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 (a total of three 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 using Dunnett's multiple comparison test. rs738409-rs738408 Statistical significance for each 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, **=0.0018; AAV-PNPLA3 rs738409-rs738408 + siRNA, ****<0.0001. (B) Confirmation of human PNPLA3 mRNA expression and silencing in the 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(C) Analysis of mouse Pnpla3 mRNA expression in the 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 ALIOS-fed PNPLA3 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.

[0268] 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-rs738408Mice received either siRNA or vehicle and were maintained on a normal chow diet or transitioned to the ALIOS diet. Eight weeks after AAV injection and dietary change, mice were treated with siRNA or vehicle every other week for eight weeks (four injection rounds in total). Mice were harvested at the 16-week time point. No changes in adiposity were observed, 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 difference between AAV8-PNPLA3 and AAV8-PNPLA3, generated by one-way ANOVA using Dunnett's multiple comparison test. rs738409-rs738408 Statistical significance for each 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, ***=0.0006. (B) Confirmation of human PNPLA3 mRNA expression and silencing in the 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 (C) Analysis of mouse Pnpla3 mRNA expression in the 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 ALIOS-fed PNPLA3 rs738409-rs738408 + Solvent and PNPLA3 rs738409-rs738408 + siRNA groups. (D) Liver triglyceride content, expressed 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.

[0269] Figure 4A-D. PNPLA3 rs738409-rs738408 PNPLA3 rescues disease-associated phenotypes caused by overexpression of PNPLA3 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 by ALIOS ingestion for 16 weeks using vehicle- or siRNA-treated AAV8-PNPLA3 rs738409-rs738408 The liver and serum levels from 8-week AAV8-PNPLA3 mice were compared. No changes in steatosis were observed with siRNA treatment at this time point, but liver glycerides, serum TIMP1, and inflammation were all statistically lower at week 16 compared to the vehicle control at week 8. Results are shown as mean and standard error, N=8 per group. Asterisks indicate the mean values ​​generated by one-way ANOVA using Dunnett's multiple comparison test. rs738409-rs738408 Statistical significance is expressed relative to the vehicle-treated cohort. (A) Liver triglyceride content shown as milligrams of triglyceride per gram of liver tissue. Adjusted P-value: 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 value: 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.

[0270] 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 placed on 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 an 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 placed on Envigo diet, TD.170748, with drinking water composed entirely of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively).

[0271] Two weeks after AAV injection and initiation of the diet, mice were treated with a single dose of 5.0 milligrams per kilogram of animal of siRNA D-2324 (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) or vehicle 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 in 10% neutral-buffered formalin and subsequently processed and embedded in paraffin. The remainder of the liver was snap-frozen for gene expression analysis.

[0272] 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).

[0273] Formalin-fixed tissues were processed for hemotoxylin, eosin, and Masson's trichrome staining (Dako, CS70030-2, CS70130-2, and AR17311-2, respectively) according to the manufacturer's instructions. Anti-smooth muscle actin staining was performed without antigen retrieval 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.

[0274] 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 received either siRNA D-2324 or vehicle and were maintained 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 (a total of six injection rounds). Mice were harvested at the 10-week time point. Results are presented as mean and standard error. Chow-fed mice (no AAV + vehicle) and AMLN-fed mice (AAV8-PNPLA3) were treated with siRNA D-2324 or vehicle. 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 using Dunnett's multiple comparison test. AAV8-PNPLA3 rs738409-rs738408 Statistical significance is shown relative to the vehicle-treated cohort. (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 the 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 the 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 levels 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: AAV-free + vehicle group, ****<0.0001; AAV-EV + vehicle, not significant; AAV-PNPLA3 rs738409-rs738408 + siRNA, not significant. (J) Mouse Col1a1 mRNA expression in the 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 the 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 the 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.

Claims

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises the antisense sequence of SEQ ID NO: 306, SEQ ID NO: 308, or SEQ ID NO: 324, and the RNAi construct inhibits the expression of a patatin-like phospholipase domain-containing 3(PNPLA3)-rs738409 minor allele, a PNPLA3-rs738408 minor allele, or a PNPLA3-rs738409-rs738408 biminor allele.

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

3. The RNAi construct according to claim 1, wherein the sense strand comprises the sense sequence of SEQ ID NO: 305, SEQ ID NO: 307, or SEQ ID NO:

323.

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

5. The RNAi construct according to claim 1, wherein the construct inhibits the PNPLA3-rs738409-rs738408 biminor allele.

6. The RNAi construct according to claim 1, wherein the RNAi construct comprises at least one blunt end.

7. The RNAi construct according to claim 1, wherein the RNAi construct comprises at least one nucleotide overhang of 1 to 4 unpaired nucleotides.

8. The RNAi construct according to claim 7, wherein the RNAi construct includes a nucleotide overhang at the 3' end of the sense strand, the 3' end of the antisense strand, or at the 3' ends of both the sense strand and the antisense strand.

9. The RNAi construct according to claim 8, wherein the nucleotide overhang comprises a 5'-UU-3' dinucleotide or a 5'-dTdT-3' dinucleotide.

10. The RNAi construct according to claim 1, wherein the RNAi construct reduces the expression level of PNPLA3 in liver cells incubated with the RNAi construct compared to the expression level of PNPLA3 in liver cells incubated with a control RNAi construct.

11. The RNAi construct according to claim 10, wherein the liver cells are Hep3B or HepG2 cells.

12. The RNAi construct is (a) In vitro inhibition of at least 10% of PNPLA3 expression in Hep3B cells at 5 nM; (b) In vitro inhibition of at least 10% of PNPLA3 expression in HepG2 cells at 5 nM; (c) Inhibit PNPLA3 expression in Hep3B cells with an IC50 of less than 1 nM; or (d) The RNAi construct according to claim 10, which inhibits PNPLA3 expression in HepG2 cells with an IC50 of less than 1 nM.

13. (a) The antisense strand includes the antisense sequence of Sequence ID No. 306, and the sense strand includes the sense sequence of Sequence ID No. 305; (b) The antisense chain includes the antisense sequence of Sequence ID No. 308, and the sense chain includes the sense sequence of Sequence ID No. 307; or (c) The RNAi construct according to claim 1, wherein the antisense strand comprises the antisense sequence of SEQ ID NO: 324, and the sense strand comprises the sense sequence of SEQ ID NO:

323.

14. A pharmaceutical composition comprising the RNAi construct described in Claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

15. A pharmaceutical composition comprising the RNAi construct according to claim 1 for use in the treatment of non-alcoholic steatohepatitis (NASH).

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