RNAI constructs for inhibiting PNPLA3 expression

Abstract

The present invention relates to RNAi constructs for reducing PNPLA3 gene expression. Methods of using such RNAi constructs to treat or prevent liver disease, non-alcoholic fatty liver disease (NAFLD) are also described.

Description

Technical Field

[0001] 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 via RNA interference and methods of using these nucleic acid-based therapeutics to treat or prevent liver diseases, such as non-alcoholic fatty liver disease (NAFLD). Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) constitutes a spectrum of liver pathologies and is the most common chronic liver disease worldwide. Its prevalence has doubled in the past 20 years and is now estimated to affect approximately 20% of the world's 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 as 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) in whom other types of liver disease have been excluded (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) triggers inflammation and hepatocellular damage, leading to a more advanced disease called nonalcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, an estimated 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).

[0003] Phospholipase domain-containing patatin-like 3 (PNPLA3), formerly known as adiponectin (ADPN) and calcium-independent phospholipase A2-ε (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). Originally identified in adipocytes as a membrane-associated lipid-rich protein induced during adipogenesis in mice, it is now well characterized as being expressed in other tissues, including the liver (Wilson et al., supra; Baulande et al. (2001) J Biol Chem 276(36):33336-44; 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 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 on the endoplasmic reticulum and 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). Despite the lack of a secretory signal, data indicate that PNPLA3 is secreted and can be found as disulfide-dependent multimers in human plasma (Winberg et al. (2014) Biochem Biophys Res Commun 446(4):1114-9).Therefore, novel therapeutic agents targeting PNPLA3 function represent a new approach to reduce PNPLA3 levels and treat liver diseases such as nonalcoholic fatty liver disease. Summary of the Invention

[0004] 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 hepatocytes. Sequence-specific inhibition of PNPLA3 expression can be used to treat or prevent conditions associated with PNPLA3 expression, such as liver-related diseases, such as simple fatty liver (steatosis), non-alcoholic 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 a PNPLA3 mRNA sequence. In certain embodiments, the antisense strand comprises a region comprising at least 15 contiguous nucleotides from an antisense sequence listed in Table 1 or Table 2. In some embodiments, the RNAi constructs of the present invention selectively inhibit the PNPLA3-rs738409, PNPLA3-rs738408, and / or PNPLA3-rs738409-rs738408 minor allele relative to a reference allele lacking these alterations.

[0005] In some embodiments, the sense strand of the RNAi constructs described herein comprises a sequence that is fully complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, the sense strand and antisense strand are each about 15 to about 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 nucleotide overhangs 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' end of both the sense strand and the antisense strand. In certain embodiments, the RNAi construct comprises an overhang of two unpaired nucleotides at the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct comprises two unpaired nucleotide overhangs at the 3' end of the antisense strand and a blunt end at the 3' end of the sense strand / 5' end of the antisense strand.

[0006] The RNAi constructs of the present invention may comprise one or more modified nucleotides, including nucleotides having modified ribose rings, core bases or phosphodiester backbones. In some embodiments, the RNAi constructs comprise 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 (BNA), glycol nucleic acids (GNA), reverse bases (e.g., reverse adenosine) or combinations thereof. In a specific embodiment, the RNAi constructs comprise 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 constructs are modified nucleotides.

[0007] 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 constructs described herein comprise at least one phosphorothioate internucleotide bond. In specific embodiments, the phosphorothioate internucleotide bond can be located at the 3' end or the 5' end of the sense strand and / or the antisense strand.

[0008] In some embodiments, the antisense strand and / or sense strand of the RNAi construct of the present invention may comprise or consist of a sequence from the antisense and sense sequences listed in Table 1 or 2. In certain embodiments, the RNAi construct may be any duplex compound listed in any one of Tables 1 to 2. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1D Shown is the screening of five siRNA molecules for dose-dependent mRNA knockdown and functional durability in vivo.

[0010] Figures 2A-2G Shown are the effects of PNPLA3 siRNA molecules on liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation in mice.

[0011] Figures 3A-3G Shown are the effects of PNPLA3 siRNA molecules in vivo on liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation.

[0012] Figures 4A-4D PNPLA3 is shown rs738409-rs738408 Specific siRNA molecules rescue PNPLA3 rs738409 -rs738408The ability to modulate disease-associated phenotypes resulting from overexpression of TNF-α, hepatic triglyceride content, serum TIMP1 levels, and histological signs of steatosis or inflammation.

[0013] Figures 5A-5L PNPLA3 is shown rs738409-rs738408 The ability of specific siRNA molecules to prevent early fibrosis development.

[0014] Figure 6a and 6b The complete sequence of the AAV plasmid containing the PNPLA3 minor allele target sequence is shown. The portion containing the mouse CMV promoter, firefly luciferase reporter gene, and target sequence is underlined.

[0015] Figure 7a and 7b The complete sequence of the AAV plasmid containing the PNPLA3 reference allele target sequence is shown. The portion containing the mouse CMV promoter, firefly luciferase reporter gene, and target sequence is underlined.

[0016] Figure 8 Example images of mice injected with AAV expressing the human PNPLA3 minor allele target sequence (upper row) compared to mice injected with AAV expressing the human PNPLA3 reference allele target sequence (lower row) are shown. After acquiring baseline images (first column), both mice were injected with the same siRNA molecule (D-2878, 3 mpk). Columns 2 to 5 are images captured at weeks 1, 2, 3, and 4, respectively. The software converts the image to grayscale. The lighter areas are areas of low total flux [p / s], while the darker areas are areas of high total flux [p / s]. The insets (rows 2 and 4) show the relative percentage knockdown of siRNA-treated mice at weekly time points, normalized to vehicle-treated mice.

[0017] Figure 9 shows an example of siRNA molecule D-2419, demonstrating dose-dependent and allele-selective mRNA knockdown and functional efficacy in vivo. (A) siRNA molecule D-2419 was injected subcutaneously into the abdomen of mice at 3.0 and 10.0 mg / kg body weight. (B) Data are presented for human PNPLA3 compared to vehicle-treated controls. rs738409-rs738408 Alleles compared to PNPLA3 WTMean relative mRNA knockdown percentages and standard errors of the mean. (C) Livers from the two-week treatment groups were processed for triglyceride content to assess functional efficacy. (D) To control for efficient GalNAc-mediated siRNA delivery, D-2787 (an siRNA cross-reactive to human and mouse HPRT and Hprt, respectively) was delivered at 10 mg / kg and livers were harvested two weeks later. Data represent copies of HPRT mRNA and Hprt mRNA in D-2787-treated mice (N=4) compared to vehicle-treated mice (N=5). (E) Data represent mean relative mRNA knockdown percentages and standard errors of the mean for human HPRT and mouse Hprt mRNA relative to vehicle-treated controls; all normalized to human TBP. (F) To confirm the role of GalNAc receptors in PXB Expression on hepatocytes, mouse Asgr1 mRNA and human ASGR1 mRNA levels were assessed in the absence and presence of D-2419. DETAILED DESCRIPTION

[0018] The present invention relates to compositions and methods for modulating expression of a patatin-like phospholipase domain-containing 3 (PNPLA3) gene. In some embodiments, the gene can be in a cell or subject, such as a mammal (e.g., a human). In some embodiments, the compositions of the present invention comprise RNAi constructs that target PNPLA3 mRNA and reduce PNPLA3 expression in the cell or mammal. Such RNAi constructs can be used to treat or prevent 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.

[0019] In 2008, a genome-wide association study (GWAS) exploring nonsynonymous sequence variations, or single nucleotide polymorphisms (SNPs), associated with NAFLD identified a variant in PNPLA3 (rs738409[G], encoding I148M; also referred to as PNPLA3-rs738409, PNPLA3-ma, or PNPLA3-minor allele) as significantly associated with liver fat content. Since this initial report, subsequent GWAS have confirmed PNPLA3 rs738409 as a major genetic determinant of NAFLD, with significant associations with: 1) elevated levels of alanine aminotransferase (ALT), a serum biomarker of liver injury; 2) NAFLD incidence, progression, and severity; 3) both obese and lean individuals; and 4) the only known SNP to show significant associations with all stages of NAFLD: steatosis, NASH, cirrhosis, and hepatocellular carcinoma. The consensus from numerous GWAS studies suggests that the association of PNPLA3 rs738409 with NAFLD is independent of age, sex, race, metabolic syndrome, body mass index, insulin resistance, and serum lipid profiles. Furthermore, statistical analyses from multiple sources estimate that approximately 50% of NAFLD patients carry the PNPLA3 rs738409 mutation. Patients can be homozygous or heterozygous for the PNPLA3 rs738409 mutation. Additionally, patients with the PNPLA3 rs738409 mutation have been found to also commonly carry the 3-base-pair rs738408 mutation (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 dual minor allele mutation (PNPLA3-dma).

[0020] Researchers have developed mouse models to explore PNPLA3 function in vivo. To date, no detectable metabolic phenotype has been identified as a consequence of Pnpla3 deficiency or Pnpla3 overexpression. I148M Expression in transgenic and knock-in mice resulted in increased hepatic triglyceride levels, similar to NAFLD. Thus, in summary, the in vivo mouse model data suggest that mutant Pnpla3 I148M "The expression of the protein, rather than overexpression of the wild-type protein, is the driver of the disease phenotype. In addition to the high frequency of the minor allele in individuals affected by NAFLD and the major association with disease, these findings highlight PNPLA3 rs738409 as a major therapeutic target for NAFLD."

[0021] RNA interference (RNAi) is a process in which exogenous RNA is introduced into cells, resulting in the specific degradation of the mRNA encoding a targeted protein, leading to reduced protein expression. Advances in RNAi technology and liver delivery, as well as the growing number of positive results from other RNAi-based therapies, suggest that RNAi is a powerful approach for the therapeutic treatment of NAFLD by directly targeting PNPLA3I148M. Numerous GWAS have demonstrated a dose-dependent effect of PNPLA3 rs738409 on the incidence, progression, and severity of NAFLD; the odds ratio tends to be twice as high (if not higher) for homozygous carriers compared with heterozygous carriers, but remains at least twofold higher for heterozygotes compared with wild-type individuals. Therefore, silencing PNPLA3 using allele-discriminating specificity is both a potential approach to lower hepatic triglycerides in PNPLA3I148M carriers and provides a scenario in which heterozygotes can derive benefit without silencing the wild-type allele. Along these lines, we identified SNP-specific short interfering RNAs (siRNAs) for PNPLA3I148M and demonstrated proof of concept in vitro. Using the hepatoma cell lines Hep3B (homozygous for the reference allele PNPLA3I148I) and HEPG2 (homozygous for the minor allele PNPLA3I148M), we identified siRNA sequences capable of specifically inhibiting PNPLA3I148M gene expression. The inhibitory effects of these sequences were confirmed by screening Chinese hamster ovary (CHO) cells overexpressing PNPLA3I148I or PNPLA3I148M. Using an adeno-associated virus (AAV) that overexpresses human PNPLA3I148M in vivo, we then demonstrated that treatment with the minor allele-specific SNP not only specifically reduced human PNPLA3I148M expression in mice, but also significantly reversed hepatic triglyceride accumulation induced by overexpression of human PNPLA3I148M.

[0022] As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, is capable of downregulating the expression of a target gene (e.g., PNPLA3) via an 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., messenger RNA or mRNA) in a sequence-specific manner (e.g., 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 to hybridize to form a duplex region. "Hybridization" refers to the pairing of complementary polynucleotides, typically via hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) between complementary bases in the two polynucleotides. The strand comprising a region having a sequence that is substantially complementary to a target sequence (e.g., a target mRNA) is referred to as the "antisense strand." The "sense strand" refers to the strand comprising a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may comprise a region having a sequence substantially identical to the target sequence.

[0023] In some embodiments, the present invention is an RNAi agent directed against PNPLA3. In some embodiments, the present invention is an RNAi agent that binds at the PNPLA3 rs738409 locus. In some embodiments, the present invention is an RNAi agent that binds at the PNPLA3 rs738408 locus. In some embodiments, the present invention is an RNAi agent that binds at the PNPLA3 rs738409 and rs738408 loci. In some embodiments, the present invention is an RNAi agent that preferentially binds to PNPLA3 rs738409 relative to the native PNPLA3 sequence (PNPLA3-ref). In some embodiments, the present invention is an RNAi agent that preferentially binds to PNPLA3 rs738408 relative to the PNPLA3-ref sequence. In some embodiments, the present invention is an RNAi agent that preferentially binds to PNPLA3-dma relative to PNPLA3-ma. In some embodiments, the present invention is an RNAi molecule comprising any of the sequences found in Tables 1 or 2.

[0024] Double-stranded RNA molecules can include chemical modifications to ribonucleotides, including modifications to the ribose, base, or backbone components of the ribonucleotides, such as those described herein or known in the art. For the purposes of this disclosure, the term "double-stranded RNA" encompasses any such modifications used in double-stranded RNA molecules (e.g., siRNA, shRNA, etc.).

[0025] As used herein, a first sequence is "complementary" to a second sequence if a polynucleotide comprising a first sequence can hybridize to a polynucleotide comprising a second sequence to form a duplex region under certain conditions (e.g., physiological conditions). Other such conditions may include moderate or stringent hybridization conditions known to those of ordinary skill in the art. If a polynucleotide comprising a first sequence is base-paired with a polynucleotide comprising a second sequence over the entire length of one or both nucleotide sequences without any mismatches, the first sequence is considered to be completely complementary to the second sequence (100% complementary). 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 percentage of complementarity can be calculated by dividing the number of bases in the first sequence that are complementary to the bases at corresponding positions in the second or target sequence by the total length of the first sequence. A sequence can also be said to be substantially complementary to another sequence if there are no more than 5, 4, 3, 2 or 1 mismatches in a duplex region of 30 base pairs when the two sequences hybridize. Generally, if any nucleotide overhangs as defined herein are present, the sequences of these overhangs are not taken into account when determining the degree of complementarity between two sequences. For example, a sense strand 21 nucleotides in length and an antisense strand 21 nucleotides in length hybridize to form a 19 base pair duplex region with a 2 nucleotide overhang at the 3' end of each strand, and the two strands would be considered to be fully complementary, as that term is used herein.

[0026] In some embodiments, a region of the antisense strand comprises a sequence that is fully complementary to a region of a target RNA sequence (e.g., PNPLA3 mRNA). In such embodiments, the sense strand may comprise a sequence that is fully 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, e.g., having 1, 2, 3, 4, or 5 mismatches in the duplex region formed by the sense and antisense strands. In certain embodiments, it is preferred that any mismatches occur within the terminal regions (e.g., within 6, 5, 4, 3, 2, or 1 nucleotides of the 5' and / or 3' ends of the strands). In one embodiment, any mismatches in the duplex region formed by the sense and antisense strands occur within 6, 5, 4, 3, 2, or 1 nucleotides of the 5' end of the antisense strand.

[0027] In certain embodiments, the sense and antisense strands of a double-stranded RNA can be two separate molecules that hybridize to form a duplex region but are otherwise unconnected. Such double-stranded RNA molecules formed from two separate strands are referred to as "small interfering RNA" or "short interfering RNA" (siRNA). Thus, in some embodiments, the RNAi constructs of the present invention comprise siRNA.

[0028] When the two substantially complementary chains of dsRNA are composed of separate RNA molecules, those molecules do not need to be but can be covalently linked. When the two chains are covalently linked by means other than an uninterrupted nucleotide chain between the 3' end of one chain and the 5' end of the corresponding other chain forming a duplex structure, the connecting structure is referred to as a "joint". The RNA chains may have the same or different numbers of nucleotides. The maximum number of base pairs in the duplex is the number of nucleotides in the shortest chain of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, RNAi may comprise one or more nucleotide overhangs.

[0029] In other embodiments, the sense strand and antisense strand that hybridize to form a duplex region can be part of a single RNA molecule, that is, the sense strand and antisense strand are part of the self-complementary region of a single RNA molecule. In such cases, a 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 forming a continuous unpaired nucleotide sequence in the loop region. The loop region generally has a sufficient length to allow the RNA molecule to fold back on itself so that the antisense strand can be base-paired with the sense strand to form a duplex or stem region. The loop region can include from about 3 to about 25, from about 5 to about 15, or from about 8 to about 12 unpaired nucleotides. Such RNA molecules with at least a partial self-complementary region are referred to as "short hairpin RNA" (shRNA). In certain embodiments, the loop region can include at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In certain embodiments, the loop region can have 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer unpaired nucleotides. In certain embodiments, the RNAi constructs of the present invention comprise shRNA. The length of a single at least partially self-complementary RNA molecule can be from about 35 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 85 nucleotides, or from about 50 to about 60 nucleotides, and comprises a duplex region and a loop region each having a length as described herein.

[0030] In some embodiments, the RNAi constructs of the present invention comprise a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence that is substantially or completely complementary to a PNPLA3 messenger RNA (mRNA) sequence. As used herein, "PNPLA3 mRNA sequence" refers to any messenger RNA sequence, including splice variants encoding a PNPLA3 protein, including PNPLA3 protein variants or isoforms from any species (e.g., mouse, rat, non-human primate, human). The PNPLA3 protein is also known as adiponectin (ADPN) and calcium-independent phospholipase A2-ε (iPLA(2)ε).

[0031] PNPLA3 mRNA sequences also include transcript sequences expressed as their complementary DNA (cDNA) sequences. A cDNA sequence refers to a 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 the RNAi constructs of the present invention can include a region having a sequence that is substantially or completely complementary to a target PNPLA3 mRNA sequence or PNPLA3 cDNA sequence. PNPLA3 mRNA or cDNA sequences can include, but are not limited to, any PNPLA3 mRNA or cDNA sequence, such as those derived from NCBI Reference Sequence NM_025225.2.

[0032] A region of the antisense strand can be substantially complementary or fully complementary to at least 15 consecutive nucleotides of a PNPLA3 mRNA sequence. In some embodiments, the antisense strand comprises a complementary region to a target region of a PNPLA3 mRNA sequence ranging from about 15 to about 30 consecutive nucleotides, from about 16 to about 28 consecutive nucleotides, from about 18 to about 26 consecutive nucleotides, from about 17 to about 24 consecutive nucleotides, from about 19 to about 25 consecutive nucleotides, from about 19 to about 23 consecutive nucleotides, or from about 19 to about 21 consecutive nucleotides. In certain embodiments, a region of the antisense strand comprising a sequence that is substantially or fully complementary to a PNPLA3 mRNA sequence can, in some embodiments, comprise at least 15 consecutive 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 consecutive 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 2 with no more than 1, 2, or 3 nucleotide mismatches.

[0033] The sense strand of an RNAi construct generally comprises a sequence fully complementary to the antisense strand so that the two chains hybridize under physiological conditions to form a sequence in a duplex region. A "duplex region" refers to a region in two complementary or substantially complementary polynucleotides that interact to form base pairs with each other through Watson-Crick base pairing or other hydrogen bonds to produce a duplex between the two polynucleotides. The duplex region of an RNAi construct should have sufficient length to allow the RNAi construct to enter the RNA interference pathway, such as by using a Dicer enzyme and / or RISC complex. For example, in some embodiments, the length of the duplex region is about 15 to about 30 base pairs. Other lengths of the duplex 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 length of the duplex region is about 17 to about 24 base pairs. In another embodiment, the length of the duplex region is about 19 to about 21 base pairs.

[0034] 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 direct the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells can be broken down into siRNAs by a type III endonuclease called Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer is an RNase-III-like enzyme that processes dsRNA into 19-23 base pair short interfering RNAs with a characteristic two-base 3' overhang (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 direct target recognition (Nykanen et al. (2001) Cell 107:309). Upon binding to the appropriate target 20 mRNA, one or more endonucleases within RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0035] For embodiments in which the sense strand and the antisense strand are two separate molecules (e.g., an RNAi construct comprises an siRNA), the length of the sense strand and the antisense strand need not be the same as the length of the duplex region. For example, one or both chains may be longer than the duplex region and have one or more unpaired nucleotides or mismatches flanking the duplex region. Therefore, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, "nucleotide overhang" refers to one or more unpaired nucleotides or nucleotides extending beyond the end of the chain in the duplex region. When the 3' end of a chain extends beyond the 5' end of another chain or when the 5' end of a chain extends beyond the 3' end of another chain, a nucleotide overhang is generally produced. The length of the 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 specific embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises two nucleotides. The nucleotides in the overhang may 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 may 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.

[0036] The nucleotide overhangs may be located at the 5' end or the 3' end of one or both strands. For example, in one embodiment, the RNAi construct comprises nucleotide overhangs at the 5' end and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises nucleotide overhangs at the 5' end and the 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.

[0037] RNAi constructs can comprise a single nucleotide overhang at one end of a double-stranded RNA molecule and a blunt end at the other end. "Blunt end" means that the sense strand and the antisense strand are completely base-paired at the ends of the molecule, and no unpaired nucleotides extend beyond the duplex region. In certain embodiments, the RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and the 3' end of the sense strand. In certain embodiments, the RNAi construct comprises a blunt end at both ends of the double-stranded RNA molecule. In such embodiments, the sense strand and the antisense strand have the same length, and the length of the duplex region is identical to the sense strand and the antisense strand (that is, the molecule is double-stranded over its entire length).

[0038] The sense strand and the antisense strand can each independently be about 15 to about 30 nucleotides in length, about 18 to about 28 nucleotides in length, about 19 to about 27 nucleotides in length, about 19 to about 25 nucleotides in length, about 19 to about 23 nucleotides in length, about 21 to about 25 nucleotides in length, or about 21 to about 23 nucleotides in length. In certain embodiments, the sense strand and the antisense strand are each about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments, the sense strand and the antisense strand are the same length, but form a duplex region shorter than these strands such that the RNAi construct has two nucleotide overhangs. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each of 21 nucleotides in length, (ii) a duplex region of 19 base pairs in length, and (iii) nucleotide overhangs with two unpaired nucleotides at 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 of 23 nucleotides in length, (ii) a duplex region of 21 base pairs in length, and (iii) nucleotide overhangs with 2 unpaired nucleotides at 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 over their entire length, such that there are no nucleotide overhangs at either end of the double-stranded molecule. In one such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand each of 21 nucleotides in length and (ii) a duplex region of 21 base pairs in length. In another such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand each of 23 nucleotides in length and (ii) a duplex region of 23 base pairs in length.

[0039] In other embodiments, the sense strand or the antisense strand is longer than the other strand, and the two strands form a duplex region with a length equal to the shorter strand length such that the RNAi construct comprises at least one nucleotide overhang. For example, in one embodiment, the RNAi construct comprises (i) a sense strand of 19 nucleotides in length, (ii) an antisense strand of 21 nucleotides in length, (iii) a duplex region of 19 base pairs in length, and (iv) a single nucleotide overhang of 2 unpaired nucleotides at the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand of 21 nucleotides in length, (ii) an antisense strand of 23 nucleotides in length, (iii) a duplex region of 21 base pairs in length, and (iv) a single nucleotide overhang of 2 unpaired nucleotides at the 3' end of the antisense strand.

[0040] The antisense strand of the RNAi construct of the present invention can comprise the sequence of any 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 consecutive nucleotides (counting from the first 19 nucleotides from the 5' end) that is complementary to the PNPLA3 mRNA sequence plus a two-nucleotide overhang. Thus, in some embodiments, the antisense strand comprises the sequence of nucleotides 1-19 of any of SEQ ID NOs: 1-166 or 167-332.

[0041] Modified nucleotides

[0042] The RNAi construct of the present invention may comprise one or more modified nucleotides." modified nucleotides " refer to nucleotides having one or more chemically modified nucleosides, core bases, pentose rings or phosphate groups. As used herein, modified nucleotides do not encompass ribonucleotides containing adenosine monophosphate, guanosine monophosphate, uridine monophosphate and cytidine monophosphate, and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate and deoxycytidine monophosphate. However, the RNAi construct may comprise a combination of modified nucleotides, ribonucleotides and deoxyribonucleotides. Modified nucleotides may be incorporated into one or both chains of a double-stranded RNA molecule to improve the in vivo stability of the RNA molecule, for example, by reducing the susceptibility of the molecule to nucleases and other degradation processes. The effectiveness of RNAi constructs to reduce target gene expression may also be enhanced by incorporating modified nucleotides.

[0043] In certain embodiments, modified nucleotides have the modification of ribose.These sugar modifications can include modification of 2' and / or 5' positions of pentose ring and bicyclic sugar modification.2'-modified nucleotides refer to nucleotides with pentose rings, and the pentose rings have substituents other than H or OH at 2' positions.Such 2' modifications include but are not limited to 2'-O-alkyl (such as alkyl substituted with O-C1-C10 or O-C1-C10), 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 (such as 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.

[0044] " bicyclic sugar modification " refers to the modification of pentose ring, wherein bridging group connects two atoms of ring to form second ring, produces bicyclic sugar structure.In certain embodiments, bicyclic sugar modification comprises the bridging group between 4 ' and 2 ' carbon of pentose ring.The nucleotide comprising the sugar part with bicyclic sugar modification is referred to as bicyclic nucleic acid or BNA in this article.Exemplary bicyclic sugar modification includes but is not limited to α-L-methyleneoxy (4 '-CH2-O-2 ') bicyclic nucleic acid (BNA);β-D-methyleneoxy (4 '-CH2-O-2 ') BNA (also referred to as locked nucleic acid or LNA);Ethyleneoxy (4 '-(CH2)2-O-2 ') BNA;Aminooxy (4 '-CH2-ON (R)-2 ') BNA;Oxyamino (4 '-CH2-N (R)-O-2 ') BNA;Methyl (methyleneoxy) (4 '-CH2-N (R)-O-2 ') BNA; (CH3)-O-2') BNA (also known as constrained ethyl or cEt); methylene-thio (4'-CH2-S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methyl carbocycle (4'-CH2-CH(CH3)-2') BNA; propylene carbocycle (4'-(CH2)3-2') BNA; and methoxy(ethyleneoxy) (4'-CH(CHOMe)-O-2') BNA (also known as constrained MOE or cMOE). These and other sugar-modified nucleotides that can be incorporated into the RNAi constructs of the present invention are described in U.S. Patent No. 9,181,551, U.S. Patent Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, all of which are hereby incorporated by reference in their entirety.

[0045] In some embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-O-allyl modified nucleotides, bicyclic nucleic acids (BNAs), or combinations thereof. In certain embodiments, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, or combinations thereof. In a specific embodiment, the RNAi construct comprises one or more 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or combinations thereof.

[0046] Both the sense and antisense strands of the RNAi construct may comprise one or more modified nucleotides. For example, in some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In certain embodiments, all nucleotides in the sense strand are modified nucleotides. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In other embodiments, all nucleotides in the antisense strand are modified nucleotides. In certain other embodiments, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In these and other embodiments, the modified nucleotides may be 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, or a combination thereof.

[0047] In some embodiments, all pyrimidine nucleotides preceding the 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' occurs in either strand, the cytidine and uridine nucleotides are modified nucleotides, preferably 2'-O-methyl modified nucleotides. In certain embodiments, all pyrimidine nucleotides in the sense strand are modified nucleotides (e.g., 2'-O-methyl modified nucleotides), and the 5' nucleotide in all occurrences of the sequence 5'-CA-3' or 5'-UA-3' in the antisense strand is a modified nucleotide (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.

[0048] In embodiments where the RNAi construct comprises a nucleotide overhang, the nucleotides in the overhang may be ribonucleotides, deoxyribonucleotides, or modified nucleotides. In one embodiment, the nucleotides in the overhang are deoxyribonucleotides, such as deoxythymidine. In another embodiment, the nucleotides in the overhang are modified nucleotides. For example, in some embodiments, the nucleotides in the overhang are 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-methoxyethyl modified nucleotides, or a combination thereof.

[0049] The RNAi construct of the present invention can also include one or more modified internucleotide bonds. As used herein, the term "modified internucleotide bond" refers to a bond between nucleotides except a natural 3' to 5' phosphodiester bond. In certain embodiments, the modified internucleotide bond is a phosphorus-containing internucleotide bond, such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a thiophosphate (P=S), a chiral thiophosphate, a dithiophosphate, a thiophosphoramidate, an alkylthiophosphodiester, and a boranephosphate. In one embodiment, the modified internucleotide bond is a 2' to 5' phosphodiester bond. In other embodiments, the modified internucleotide bond is a bond that does not contain phosphorus internucleotides, and therefore can be referred to as a modified internucleoside bond. Such non-phosphorus-containing bonds include, but are not limited to, morpholino bonds (formed in part by the sugar portion of the nucleoside); siloxane bonds (-O-Si(H)2-O-); sulfide, sulfoxide, and sulfone bonds; formyl and thioformyl bonds; backbones containing olefins; aminosulfonic acid backbones; methylenemethylimino (-CH2-N(CH3)-O-CH2-) and methylenehydrazinyl bonds; sulfonate and sulfonamide bonds; amide bonds; and other bonds with mixed N, O, S, and CH2 components. In one embodiment, the modified internucleoside bond is a peptide-based bond (e.g., aminoethylglycine) that produces peptide nucleic acids or PNAs, such as those described in U.S. Patent 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. No. 6,693,187, U.S. Pat. No. 9,181,551, U.S. Patent Publication No. 2016 / 0122761, and Deleavey and Damha, Chemistry and Biology, Vol. 19: 937-954, 2012, all of which are hereby incorporated by reference in their entirety.

[0050] In certain embodiments, the RNAi construct comprises a bond between one or more phosphorothioate nucleotides. The bond between the phosphorothioate nucleotides can be present in the sense strand, antisense strand or two chains of the RNAi construct. For example, in some embodiments, the sense strand comprises a bond between 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate nucleotides. In other embodiments, the antisense strand comprises a bond between 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate nucleotides. In other embodiments, the two chains comprise a bond between 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate nucleotides. The RNAi construct can comprise a bond between one or more phosphorothioate nucleotides at the 3'-end, 5'-end or 3'-end and 5'-end of the sense strand, antisense strand or two chains. In certain embodiments, the RNAi construct comprises a bond between about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) continuous phosphorothioate nucleotides at the 3'-end of sense strand, antisense strand or two chains. In other embodiments, the RNAi construct comprises a bond between about 1 to about 6 or more (e.g., about 1, 2, 3, 4, 5, 6 or more) continuous phosphorothioate nucleotides at the 5'-end of sense strand, antisense strand or two chains. In one embodiment, the RNAi construct is included in a bond between a single phosphorothioate nucleotide at the 3' end of sense strand and a bond between a single phosphorothioate nucleotide at the 3' end of antisense strand. In another embodiment, the RNAi construct comprises a bond between two continuous phosphorothioate nucleotides at the 3' end of antisense strand (i.e., a bond between the phosphorothioate nucleotides at the first and second nucleotides at the 3' end of antisense strand). In another embodiment, the RNAi construct comprises a bond between two continuous phosphorothioate nucleotides at the 3' end and the 5' end of antisense strand. In another embodiment again, the RNAi construct comprises a bond between two continuous thiophosphate nucleotides at the 3' end and 5' end of the antisense strand and a bond between two continuous thiophosphate nucleotides at the 5' end of the sense strand. In another embodiment, the RNAi construct comprises a bond between two continuous thiophosphate nucleotides at the 3' end and 5' end of the antisense strand and a bond between two continuous thiophosphate nucleotides at the 3' end and 5' end of the sense strand and a bond between two continuous thiophosphate nucleotides at the 3' end and 5' end of the sense strand (that is, a bond between the thiophosphate nucleotides at the first and second nucleotides at the 5' end and 3' end of the antisense strand and a bond between the thiophosphate nucleotides at the first and second nucleotides at the 5' end and 3' end of the sense strand). In any embodiment in which one or both chains comprise a bond between one or more thiophosphate nucleotides, the bond between the remaining nucleotides in the chain can be a natural 3' to 5' phosphodiester bond. For example, in some embodiments, the bond between each nucleotide of the sense strand and the antisense strand is selected from phosphodiester and thiophosphate, and wherein the bond between at least one nucleotide is a thiophosphate.

[0051] In the embodiment that RNAi construct comprises nucleotide overhang, two or more unpaired nucleotides in the overhang can be connected by key between thiophosphate nucleotide.In certain embodiments, all unpaired nucleotides in the nucleotide overhang of 3 ' end of antisense strand and / or sense strand are connected by key between thiophosphate nucleotide.In other embodiments, all unpaired nucleotides in the nucleotide overhang of 5 ' end of antisense strand and / or sense strand are connected by key between thiophosphate nucleotide.In other embodiments, all unpaired nucleotides in any nucleotide overhang are connected by key between thiophosphate nucleotide.

[0052] In certain embodiments, the modified nucleotides incorporated into one or both strands of the RNAi constructs of the invention have modifications to their nucleobases (also referred to herein as "bases"). "Modified nucleobases" or "modified bases" refer to bases other than the naturally occurring purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil. The modified nucleobases may be synthetic or naturally occurring modifications, including but not limited to 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, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen, in particular 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.

[0053] In some embodiments, the modified base is a universal base. "Universal base" refers to a base analog that forms base pairs with all natural bases in RNA and DNA without changing the double helical structure of the resulting duplex region. Universal bases are known to those of ordinary skill 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.

[0054] 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. Chem., Vol. 76: 7295-7300, 2011, all of which are hereby incorporated by reference in their entirety. It is well known to those skilled in the art that guanine, cytosine, adenine, thymine, and uracil can be replaced by other nucleobases, such as the modified nucleobases described above, without substantially changing the base pairing properties of the polynucleotide comprising the nucleotide carrying the replacement nucleobase.

[0055] In some embodiments of the RNAi constructs of the present invention, the sense strand, the antisense strand, or the 5' end of the antisense and sense strands comprises a phosphate moiety. As used herein, the term "phosphate moiety" refers to terminal phosphate groups including unmodified phosphates (-OP=O)(OH)OH) and modified phosphates. Modified phosphates include phosphates in which one or more O and OH groups are substituted with H, O, S, N(R), or alkyl, wherein R is H, an amino protecting group, or an unsubstituted or substituted alkyl group. 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'-monothioate (phosphorothioate); 5'-monodithioate (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.).

[0056] The modified nucleotides that can be incorporated into the RNAi constructs of the present invention may have more than one chemical modification as described herein. For example, the modified nucleotides may have modifications to ribose and modifications to core bases. For example, the modified nucleotides may include 2' sugar modifications (e.g., 2'-fluoro or 2'-methyl) and include modified bases (e.g., 5-methylcytosine or pseudouracil). In other embodiments, the modified nucleotides may include sugar modifications and modifications to 5' phosphate, and when the modified nucleotides are incorporated into polynucleotides, these modifications will produce modified internucleotides or internucleoside bonds. For example, in some embodiments, the modified nucleotides may include sugar modifications, such as 2'-fluoro modifications, 2'-O-methyl modifications, or bicyclic sugar modifications, and 5' thiophosphate groups. Therefore, in some embodiments, one or two chains of the RNAi constructs of the present invention include 2' modified nucleotides or a combination of BNA and thiophosphate internucleotide bonds. In certain embodiments, the sense and antisense strands of the RNAi constructs of the present invention both comprise a combination of 2'-fluoro modified nucleotides, 2'-O-methyl modified nucleotides, and phosphorothioate internucleotide bonds. Exemplary RNAi constructs comprising modified nucleotides and internucleotide bonds are shown in Table 2.

[0057] Function of RNAi constructs

[0058] Preferably, the RNAi constructs of the present invention reduce or inhibit PNPLA3 expression in cells, particularly hepatocytes. Thus, in one embodiment, the present invention provides a method for reducing PNPLA3 expression in a cell by contacting the cell with any of the RNAi constructs described herein. The cell 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. Reduction of PNPLA3 expression in cells or animals treated with the RNAi constructs of the present invention can be determined relative 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, reduction of PNPLA3 expression is assessed by (a) measuring the amount or level of PNPLA3 mRNA in hepatocytes treated with an RNAi construct of the invention, (b) measuring the amount or level of PNPLA3 mRNA in hepatocytes treated with a control RNAi construct (e.g., an RNAi agent directed against an RNA molecule not expressed in hepatocytes or an RNAi construct having a nonsense or scrambled sequence) or treated with no construct, and (c) comparing the measured PNPLA3 mRNA levels from the treated cells in (a) with the measured PNPLA3 mRNA levels from the control cells in (b). Prior to comparison, the PNPLA3 mRNA levels in the treated and control cells can be normalized to the RNA level of a control gene (e.g., 18S ribosomal RNA). PNPLA3 mRNA levels can be measured by a variety of methods, including Northern blot analysis, nuclease protection assays, fluorescence in situ hybridization (FISH), reverse transcriptase (RT)-PCR, real-time RT-PCR, quantitative PCR, and the like.

[0059] In other embodiments, reduction of PNPLA3 expression is assessed by (a) measuring the amount or level of PNPLA3 protein in hepatocytes treated with an RNAi construct of the invention, (b) measuring the amount or level of PNPLA3 protein in hepatocytes treated with a control RNAi construct (e.g., an RNAi agent directed against an RNA molecule not expressed in hepatocytes or an RNAi construct having a nonsense or scrambled sequence) or treated with no construct, and (c) comparing the measured PNPLA3 protein levels from the treated cells in (a) with the measured PNPLA3 protein levels from the control cells in (b). Methods for measuring PNPLA3 protein levels are known to those of ordinary skill in the art and include immunoblotting, immunoassays (e.g., ELISA), and flow cytometry. Exemplary immunoassay-based methods for assessing PNPLA3 protein expression are 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 efficacy of the RNAi constructs of the invention.

[0060] In some embodiments, methods for assessing PNPLA3 expression levels are performed in vitro in cells that naturally express PNPLA3 (e.g., hepatocytes) or cells that have been engineered to express PNPLA3. In certain embodiments, these methods are performed in vitro in hepatocytes. Suitable hepatocytes include, but are not limited to, primary hepatocytes (e.g., human, non-human primate, or rodent hepatocytes), HepAD38 cells, HuH-6 cells, HuH-7 cells, HuH-5-2 cells, BNLCL2 cells, Hep3B cells, or HepG2 cells. In one embodiment, the hepatocytes are Hep3B cells. In another embodiment, the hepatocytes are HepG2 cells.

[0061] In other embodiments, the method of assessing PNPLA3 expression levels is performed in vivo. The RNAi construct and any control RNAi construct can be administered to an animal (e.g., a rodent or non-human primate) and, following treatment, PNPLA3 mRNA or protein levels can be assessed in liver tissue harvested from the animal. Alternatively or additionally, biomarkers or functional phenotypes associated with PNPLA3 expression can be assessed in the treated animal.

[0062] In certain embodiments, the expression of PNPLA3 in hepatocytes is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% by the RNAi constructs of the present invention. In some embodiments, the expression of PNPLA3 in hepatocytes is reduced 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 in hepatocytes is reduced 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, as well as other methods known in the art. For example, in certain embodiments, the RNAi constructs of the invention inhibit PNPLA3 expression by at least 45% at 5 nM in Hep3B cells (containing wild-type PNPLA3) in vitro. In related embodiments, the RNAi constructs of the invention inhibit 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 Hep3B cells in vitro. 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 Hep3B cells in vitro. In certain embodiments, the RNAi constructs of the invention inhibit PNPLA3 expression by at least 45% at 5 nM in HepG2 cells (containing the PNPLA3-rs738409-rs738408 double minor allele). In related embodiments, the RNAi constructs of the invention inhibit PNPLA3 expression in HepG2 cells at 5 nM by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% in vitro. In other embodiments, the RNAi constructs of the invention inhibit PNPLA3 expression in HepG2 cells at 5 nM by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% in vitro. In certain embodiments, the RNAi constructs of the invention inhibit PNPLA3 expression in CHO transfected cells expressing human PNPLA3 I148I or I148M cells at 5 nM by at least 45% in vitro. In related embodiments, the RNAi constructs of the invention inhibit 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 CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro.In other embodiments, the RNAi constructs of the invention inhibit PNPLA3 expression in CHO transfected cells expressing human PNPLA3 I148I or I148M in vitro 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. As described in Examples 2 and 3, reduction of PNPLA3 can be measured using a variety of techniques, including RNA FISH or droplet digital PCR.

[0063] In some embodiments, IC50 values ​​are calculated to assess the efficacy of RNAi constructs of the present invention in inhibiting PNPLA3 expression in hepatocytes. An "IC50 value" is the dose / concentration required to achieve 50% inhibition of a biological or biochemical function. The IC50 value of any particular substance or antagonist can be determined by constructing a dose-response curve and examining the effects of varying concentrations of the substance or antagonist on expression levels or functional activity in any assay. The IC50 value of a given antagonist or substance can be calculated by determining the concentration required to inhibit half of the maximal biological response or native expression level. Thus, the IC50 value of any RNAi construct can be calculated by determining the concentration of RNAi construct required to inhibit half of the native PNPLA3 expression level in hepatocytes (e.g., the PNPLA3 expression level in control hepatocytes) 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 hepatocytes (e.g., Hep3B cells) with an IC50 of less than about 20 nM. For example, the RNAi construct inhibits PNPLA3 expression in hepatocytes 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 hepatocytes (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 hepatocytes (e.g., HepG2 cells) with an IC50 of less than about 20 nM. For example, the RNAi construct inhibits PNPLA3 expression in hepatocytes 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 hepatocytes (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 hepatocytes (e.g., CHO transfected cells expressing human PNPLA3 I148I or I148M) with an IC50 of less than about 20 nM.For example, the RNAi construct inhibits PNPLA3 expression in hepatocytes 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 hepatocytes (e.g., CHO transfected cells expressing human PNPLA3 I148I or I148M) with an IC50 of about 1 nM to about 10 nM.

[0064] The RNAi construct of the present invention can use technology known in the art, for example, using conventional nucleic acid solid phase synthesis to easily make.The polynucleotide of RNAi construct can use standard nucleotide or nucleoside precursor (such as phosphoramidite) to assemble on suitable nucleic acid synthesizer.Automated nucleic acid synthesizer is sold commercially by some suppliers, including DNA / RNA synthesizer from Applied Biosystems (Foster City, California), MerMade synthesizer from BioAutomation (Irving, Texas) and OligoPilot synthesizer from GE Healthcare Life Sciences (Pittsburgh, Pennsylvania).

[0065] The 2' silyl protecting group can be used in conjunction with the acid-labile dimethoxytrityl (DMT) group at the 5' position of ribonucleosides to synthesize oligonucleotides via phosphoramidite chemistry. 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 also be performed in multiple well plates, columns, or on glass slides.

[0066] The 2'-O-silyl group can be removed by exposure to fluoride ions, which can include any fluoride ion source, such as those salts containing fluoride ions paired with inorganic counterions, such as cesium fluoride and potassium fluoride, or those containing fluoride ions paired with organic counterions, such as tetraalkylammonium fluorides. Crown ether catalysts can be used in combination with inorganic fluorides in the deprotection reaction. Preferred fluoride ion sources are tetrabutylammonium fluoride or aminohydrofluoride (e.g., combining aqueous HF with triethylamine in a dipolar aprotic solvent such as dimethylformamide).

[0067] The choice of protecting groups on phosphite triesters and phosphate triesters can alter the stability of the triesters toward fluoride. Methyl protection of phosphate triesters or phosphite triesters can stabilize the bond to fluoride ions and improve process yields.

[0068] Because ribonucleosides have reactive 2' hydroxyl substituents, it may be desirable to protect the reactive 2' position in RNA with a protecting group orthogonal to the 5'-O-dimethoxytrityl protecting group (e.g., a protecting group that is stable to acid treatment). Silyl protecting groups meet this criterion and can be easily removed in the final fluoride deprotection step, which can result in minimal RNA degradation.

[0069] Tetrazolium catalysts can be used in standard phosphoramidite coupling reactions. Preferred catalysts include, for example, tetrazole, S-ethyltetrazole, benzylthiotetrazole, and p-nitrophenyltetrazole.

[0070] As will be appreciated by those of ordinary skill in the art, other methods of synthesizing the RNAi constructs described herein will be readily apparent to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in alternate sequences or orders to obtain the desired compounds. Other synthetic chemical transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on bases), and protecting group methods (protection and deprotection) that can be used to synthesize the RNAi constructs described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. Wuts, Protective Groups in Organic Synthesis, 2nd ed., ed.: John Wiley and Sons, (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1996). Sons (John Wiley & Sons, Inc.) (1995), and subsequent editions. Custom synthesis of RNAi agents is also available from several commercial suppliers, including Dharmacon, Inc. (Lafayette, Colorado), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, California).

[0071] The RNAi constructs of the present invention may comprise a ligand. As used herein, a "ligand" refers to any compound or molecule that is capable of interacting directly or indirectly with another compound or molecule. The interaction of a ligand with another compound or molecule may trigger a biological response (e.g., initiate a signal transduction cascade, induce receptor-mediated endocytosis) or may simply be a physical association. The ligand may modify one or more properties of the attached double-stranded RNA molecule, such as the pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties of the RNA molecule.

[0072] The ligand can 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), cross-linking agents (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-di-O(hexadecyl)glycerol, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., antennary peptides, Tat peptides, RGD peptides), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g., PEG-40K), polyamino acids, and polyamines (such as spermine, spermidine).

[0073] In certain embodiments, the ligand has endosomal lytic properties. The endosomal lytic ligand promotes endosomal lysis and / or transport of the RNAi construct of the present invention or its components from the endosomes of the cell to the cytoplasm. The endosomal lytic ligand can be a polycationic peptide or peptide mimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomal lytic ligand assumes its active conformation at endosomal pH. An "active" conformation is one in which the endosomal lytic ligand promotes endosomal lysis and / or transport of the RNAi construct of the present invention or its components from the endosomes of the cell to the cytoplasm. Exemplary endosomolytic 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 derivatives thereof (Turk et al., Biochem. Biophys. Acta, Vol. 1559: 56-68, 2002). In one embodiment, the endosomolytic component may contain chemical groups (e.g., amino acids) that undergo changes in charge or protonation in response to changes in pH. The endosomolytic component may be linear or branched.

[0074] In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or other steroids. It is reported that oligonucleotides conjugated to cholesterol are more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development [antisense nucleic acid drug development], Vol. 12: 103-228, 2002). Parts comprising a cholesterol moiety and other lipids for conjugation to nucleic acid molecules have also been described in U.S. Patent Nos. 7,851,615, 7,745,608 and 7,833,992, all of which are hereby incorporated by reference in their entirety. In another embodiment, the ligand comprises a folic acid moiety. The polynucleotides conjugated to the folic acid moiety can be taken up by cells via a receptor-mediated endocytosis pathway. Such folic acid-polynucleotide conjugates are described in U.S. Patent No. 8,188,247, which is hereby incorporated by reference in its entirety.

[0075] Given that PNPLA3 is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to deliver RNAi constructs specifically to those liver cells. In some embodiments, RNAi constructs can be specifically targeted to the liver by using ligands that bind to or interact with proteins expressed on the surface of hepatocytes. For example, in certain embodiments, the ligand can comprise an antigen binding protein (e.g., an antibody or binding fragment thereof (e.g., Fab, scFv)) that specifically binds to a receptor expressed on hepatocytes.

[0076] In certain embodiments, the ligand comprises a carbohydrate. "Carbohydrate" refers to a compound consisting of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic) and oxygen, nitrogen, or sulfur atoms bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides, such as starch, glycogen, cellulose, and polysaccharide gum. In certain embodiments, the carbohydrate incorporated into the ligand is a monosaccharide selected from pentoses, hexoses, or heptoses and disaccharides and trisaccharides comprising 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.

[0077] In some embodiments, the ligand comprises a hexose or a 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 a specific embodiment, the ligand comprises N-acetyl-galactosamine. Ligands comprising glucose, galactose and N-acetyl-galactosamine (GalNAc) are particularly effective in targeting compounds to hepatocytes. See, for example, D'Souza and Devarajan, J. Control Release [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. Patent Nos. 7,491,805, 8,106,022, and 8,877,917, U.S. Patent Publication No. 20030130186; and WIPO Publication No. WO 2013166155, all of which are hereby incorporated by reference in their entirety.

[0078] In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, a "multivalent carbohydrate moiety" refers to a moiety comprising two or more carbohydrate units that are capable of independently binding to or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains composed of carbohydrates that can bind to two or more different molecules or 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," "divalent," "trivalent," and "tetravalent" with respect to a carbohydrate moiety refer to carbohydrate moieties having one, two, three, and four binding domains, respectively. The multivalent carbohydrate moiety may 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 divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety can be biantennary or triantennary. In a specific embodiment, the multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent. In another specific embodiment, the multivalent galactose moiety is trivalent or tetravalent. Exemplary trivalent and tetravalent GalNAc-containing ligands for incorporation into the RNAi constructs of the present invention are described in detail below.

[0079] The ligand can be directly or indirectly connected or conjugated to the RNAi construct to the RNA molecule. For example, in some embodiments, the ligand is directly covalently connected to the sense strand or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently connected to the sense strand or antisense strand of the RNAi construct via a joint. The ligand can be connected to the core base, sugar moiety or internucleotide bond of the polynucleotide (such as sense strand or antisense strand) of the RNAi construct of the present invention. Conjugation or connection with a purine core base or its derivative can occur at any position including an endo- and exo-atoms. In certain embodiments, the 2-, 6-, 7- or 8-position of a purine core base is connected to the ligand. Conjugation or connection with a pyrimidine core base or its derivative can also occur at any position. In certain embodiments, the 2-, 5- and 6-position of a pyrimidine core base can be connected to the ligand. Conjugation or connection with the sugar moiety of a nucleotide can occur at any carbon atom. Exemplary carbon atoms that can be connected to the ligand of the sugar moiety include 2', 3' and 5' carbon atoms. In some embodiments, the 1' position of the present invention can be connected to the part, for example, in the basic residue.Between nucleotides, key can also support the part to connect.For example, for phosphorus-containing bond (phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate etc.), the part can be directly connected to phosphorus atom or with O, N or the S atom of phosphorus atom bonding.For key (for example PNA) between the nucleosides containing amine or amides, the part can be connected to the nitrogen-atoms of amine or amides or be connected to the adjacent carbon atom.

[0080] In certain embodiments, the ligand can be connected to the 3' end or 5' end of the sense strand or 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 some 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 to the 2' position of the sugar of the 3' terminal nucleotide of the sense strand.

[0081] In certain embodiments, the ligand is connected to the sense strand or antisense strand via a joint. A "joint" is an atom or atomic group that covalently connects the ligand to the polynucleotide component of the RNAi construct. The joint can be from about 1 to about 30 atomic lengths, from about 2 to about 28 atomic lengths, from about 3 to about 26 atomic lengths, from about 4 to about 24 atomic lengths, from about 6 to about 20 atomic lengths, from about 7 to about 20 atomic lengths, from about 8 to about 20 atomic lengths, from about 8 to about 18 atomic lengths, from about 10 to about 18 atomic lengths, and from about 12 to about 18 atomic lengths. In certain embodiments, the joint can include a bifunctional linking portion, which typically includes an alkyl portion with two functional groups. A functional group is selected to combine a target compound (such as the sense strand or antisense strand of an RNAi construct), and another functional group is selected to substantially combine any selected group, such as a ligand as described herein. In certain embodiments, the joint includes a repeating unit, such as a chain structure or an oligomer of ethylene glycol or an amino acid unit. Examples of functional groups commonly used 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 includes amino, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bonds), etc.

[0082] Linkers that can be used to link the ligand to the sense or antisense strand in the RNAi construct of the present invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, 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, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0083] In certain embodiments, the joint is cleavable. A cleavable joint is sufficiently stable outside the cell, but after entering the target cell, it is cracked to release the joint of the two parts that the joint is kept together. In certain embodiments, the cleavable joint is at least 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least 100 times faster than in the subject's blood or under a second reference condition (which can, for example, be selected to simulate or represent conditions found in blood or serum) cracking in the target cell or under a first reference condition (which can, for example, be selected to simulate or represent conditions found in blood or serum).

[0084] Cleavable joints are susceptible to cleavage agents, such as pH, redox potential, or the presence of degradation molecules. Typically, cleavage agents are more prevalent or found at higher levels or activity in cells than in serum or blood. Examples of such degradation agents include: redox agents selected for specific substrates or without substrate specificity, including, for example, oxidoreductases or reducing agents present in cells, such as thiols, which can degrade redox cleavable joints by reduction; esterases; endosomes or agents that can form an acidic environment, such as those that produce pH 5 or lower; enzymes, peptidases (which can be substrate specific), and phosphatases that hydrolyze or degrade acid cleavable joints by acting as general acids.

[0085] Cleavable linkers may comprise moieties that are sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, ranging from 5.5-6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have a cleavable group that cleaves at a preferred pH, thereby releasing the RNA molecule from the ligand into the cell, or into a desired compartment of the cell.

[0086] 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 cell to be targeted. For example, a liver targeting ligand can be connected to an RNA molecule by a linker that includes an ester group. Hepatocytes are rich in esterases, and therefore the linker will be more effectively cleaved in hepatocytes than in cell types that are not rich in esterases. Other types of cells that are rich in esterases include cells of the lung, renal cortex, and testis. When targeting cells rich in peptidases (e.g., hepatocytes and synovial cells), a linker containing a peptide bond can be used.

[0087] Generally, the suitability of a candidate cleavable joint can be assessed by the ability of a test degradation agent (or condition) cracking candidate joint. It is also desirable to also test the ability of a candidate cleavable joint in blood or when contacted with other non-target tissues to resist cracking. Therefore, the relative susceptibility to cracking between the first condition and the second condition can be determined, wherein the first condition is selected as the cracking in the indication target cell, and the second condition is selected as the cracking in other tissues or biofluids (such as blood or serum) indicated. Assessment can be carried out in a cell-free system, cell, cell culture, organ or tissue culture or whole animal. Preliminary assessment is carried out under cell-free or culture conditions and it may be useful to confirm by further assessing the whole animal. In certain embodiments, useful candidate joints are compared with blood or serum (or under the in vitro conditions selected to simulate intracellular conditions) in cells, and cracking is at least 2, 4, 10, 20, 50, 70 or 100 times faster.

[0088] In other embodiments, redox cleavable joints are used. Redox cleavable joints are cleaved when reduced or oxidized. An example of a reductive cleavable group is a disulfide linking group (-SS-). To determine whether a candidate cleavable joint is a suitable "reducible cleavable joint", or for example, suitable for use with a specific RNAi construct and a specific ligand, one or more methods described herein can be used. For example, candidate joints can be assessed by incubating with dithiothreitol (DTT) or other reducing agents known in the art, the cleavage rate observed in cells (such as target cells) by simulation. Candidate joints can also be assessed under conditions selected to simulate blood or serum conditions. In specific embodiments, candidate joints are cleaved at most 10% in blood. In other embodiments, useful candidate joints are degraded at least 2 times, 4 times, 10 times, 20 times, 50 times, 70 times or 100 times faster in cells (or under in vitro conditions selected to simulate intracellular conditions) compared to blood (or under in vitro conditions selected to simulate extracellular conditions).

[0089] In yet other embodiments, the cleavable linker based on phosphate is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents that hydrolyze the phosphate group in a cell are enzymes, such as phosphatases in a cell. Examples of cleavable groups based on phosphate are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Specific examples 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 specific example is -OP(O)(OH)-O-. These candidate linkers can be evaluated using methods similar to those described above.

[0090] In other embodiments, the linker may include acid cleavable groups, which are groups that crack under acidic conditions. In certain embodiments, the acid cleavable group is cracked in an acidic environment at a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0 or lower), or is cracked by a reagent such as an enzyme that can serve as a general acid. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cracking environment for the acid cleavable group. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. The acid cleavable group can have the general formula -C=NN-, C(O)O, or -OC(O). A specific embodiment is when the carbon of the oxygen connected to the ester (alkoxy) is an aryl, substituted alkyl, or tertiary alkyl such as dimethyl, amyl, or tert-butyl. These candidates can be evaluated using methods similar to those described above.

[0091] In other embodiments, the joint may comprise an ester-based cleavable group that is cleaved by enzymes in the cell, such as esterases and amidases. Examples of ester-based cleavable groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. The ester cleavable group has the general formula -C(O)O- or -OC(O)-. These candidate joints can be evaluated using methods similar to those described above.

[0092] In other embodiments, the joint can include a cleavable group based on a peptide, and these groups are cleaved by enzymes in the cell, such as peptidases and proteases. The cleavable group based on a peptide is the peptide bond formed between amino acids, to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The cleavable group based on a peptide does not include an amide group (-C (O) NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene groups. The peptide bond is the amide bond of a special type formed between amino acids, to produce peptides and proteins. The cleavable group based on a peptide is usually limited to the peptide bond (i.e., amide bond) formed between the amino acids producing peptides and proteins, and does not include a complete amide functional group. The cleavable linking group based on a peptide has the general formula -NHCHRAC (O) NHCHRBC (O) -, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0093] 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 may include linkers 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 hereby incorporated by reference in their entireties.

[0094] In certain embodiments, the ligand covalently attached to the sense strand or antisense strand of the RNAi construct of the present invention comprises a GalNAc moiety, such as 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 other embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

[0095] In certain embodiments, the RNAi construct of the present invention can be delivered to target cells or tissues by administering a vector encoding and controlling the intracellular expression of the RNAi construct." vector" (also referred to herein as "expression vector") is a composition of matter that can be used to deliver a target nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphipathic compounds, plasmids, and viruses. Therefore, 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, etc. Vectors can be replicated in living cells, or can be synthesized.

[0096] Typically, the vector used to express the RNAi construct of the present invention will contain one or more promoters operably linked to the sequence encoding the RNAi construct. As used herein, the phrase "operably linked" or "under transcriptional control" means that the promoter is in the correct position and orientation relative to the polynucleotide sequence to control the transcription initiation of RNA polymerase and the expression of the polynucleotide sequence. "Promoter" refers to a sequence recognized by the cellular synthetic machinery required to initiate specific transcription of a gene sequence or by an introduced synthetic machinery. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI or U6 RNA pol III, and viral promoters (e.g., human cytomegalovirus (CMV) immediate early gene promoter, SV40 early promoter, and Rous sarcoma virus long terminal repeat). In some embodiments, HI or U6 RNA pol III promoters are preferred. The promoter may be tissue-specific or inducible. Of particular interest are liver-specific promoters, such as promoter sequences from 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).

[0097] In some embodiments where the RNAi construct comprises siRNA, two separate chains (sense strand and antisense strand) 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 on a first vector, and the sequence encoding the antisense strand is operably linked to a promoter on a second vector. In this embodiment, the first vector and the second vector are introduced into the target cell together, for example, by infection or transfection, so that the sense strand and the antisense strand, once transcribed, will hybridize in the cell to form an siRNA molecule. In another embodiment, the sense strand and the antisense strand are transcribed by 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, wherein the first promoter and the second promoter 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 synthesis of the sense strand of the siRNA molecule and transcription of the sequence from the second promoter results in synthesis of the antisense strand of the siRNA molecule.

[0098] 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 to produce a single transcript. In some embodiments, the sequence encoding the shRNA comprises an inverted repeat sequence connected by a linker polynucleotide sequence to produce a stem and loop structure of the shRNA after transcription.

[0099] 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 constructs described herein include, but are not limited to, adenoviral vectors, retroviral vectors (e.g., lentiviral vectors, Maloney murine leukemia virus), adeno-associated viral vectors; herpes simplex virus vectors; SV40 vectors; polyoma virus vectors; papilloma virus vectors; picornavirus vectors; and poxvirus vectors (e.g., vaccinia virus). In certain embodiments, the viral vector is a retroviral vector (e.g., a lentiviral vector).

[0100] Various vectors suitable for use in the present invention, methods for inserting nucleic acid sequences encoding siRNA or shRNA molecules into vectors, and methods for delivering vectors to target cells are all within the capabilities of those of ordinary skill in the art. See, e.g., Dornburg, Gene Therap., vol. 2: 301-310, 1995; Eglitis, Biotechniques, vol. 6: 608-614, 1988; Miller, Hum Gene 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. Genet. Biotechnol, vol. 20: 500-505, 2002; Miyagishi et al., Nat Biotechnol, vol. 20: 497-500, 2002; Paddison et al., Genes Dev, 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 hereby incorporated by reference in their entirety.

[0101] The present invention also includes pharmaceutical compositions and formulations comprising the RNAi constructs described herein and a pharmaceutically acceptable carrier, excipient, or diluent. Such compositions and formulations can be used to reduce PNPLA3 expression in a subject in need thereof. When clinical applications are contemplated, pharmaceutical compositions and formulations will be prepared in a form suitable for the intended application. Typically, this will require preparing compositions that are substantially free of pyrogens and other impurities that may be harmful to humans or animals.

[0102] 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, etc. that are acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of these media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the RNAi constructs of the present invention, their use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions, provided that they do not inactivate the carrier or RNAi construct of the composition.

[0103] The compositions and methods for preparing pharmaceutical compositions depend on many standards, including but not limited to route of administration, the type and degree of the disease to be treated or the disorder or the dosage. In certain embodiments, pharmaceutical compositions are prepared based on the expected route of delivery. For example, in certain embodiments, pharmaceutical compositions are prepared for parenteral delivery. Parenteral delivery forms include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal or intramuscular injection or infusion. In one embodiment, pharmaceutical compositions are prepared for intravenous delivery. In this embodiment, pharmaceutical compositions may include a lipid-based delivery vehicle. In another embodiment, pharmaceutical compositions are prepared for subcutaneous delivery. In this embodiment, pharmaceutical compositions may include a targeting ligand (e.g., a ligand containing GalNAc as described herein).

[0104] 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 outcome. In some embodiments, the effective amount is an amount sufficient to reduce PNPLA3 expression in hepatocytes of a subject. In some embodiments, the effective amount may be an amount sufficient to only partially reduce PNPLA3 expression, for example, to a level comparable to that of a wild-type PNPLA3 allele in heterozygotes. Human heterozygote carriers of loss-of-function PNPLA3 variant alleles have been reported to 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, No. 22: 2131-2141, 2016). Thus, without being bound by theory, it is believed that a partial reduction in PNPLA3 expression may be sufficient to achieve a beneficial reduction in serum non-HDL cholesterol and a reduced risk of coronary artery disease and myocardial infarction.

[0105] The effective dose of RNAi construct of the present invention can be from about 0.01mg / kg body weight to about 100mg / kg body weight, about 0.05mg / kg body weight to about 75mg / kg body weight, about 0.1mg / kg body weight to about 50mg / kg body weight, about 1mg / kg to about 30mg / kg body weight, about 2.5mg / kg body weight to about 20mg / kg body weight or about 5mg / kg body weight to about 15mg / kg body weight.In certain embodiments, the single effective dose of RNAi construct of the present invention can be about 0.1mg / kg, about 0.5mg / kg, about 1mg / kg, about 2mg / kg, about 3mg / kg, about 4mg / kg, about 5mg / kg, about 6mg / kg, about 7mg / kg, about 8mg / kg, about 9mg / kg or about 10mg / kg.The pharmaceutical composition that comprises the RNAi construct of effective dose can be weekly, every two weeks, monthly, every quarter or every half a year and use. The exact determination of the effective amount and frequency of administration can be based on several factors, including the patient's size, age, and general condition, the type of disorder to be treated (e.g., myocardial infarction, heart failure, coronary artery disease, hypercholesterolemia), the specific RNAi construct used, and the route of administration. Estimated effective doses and in vivo half-lives for any particular RNAi construct of the invention can be determined using conventional methods and / or by testing in appropriate animal models.

[0106] Administration of the pharmaceutical composition of the present invention can be via any conventional route, as long as the target tissue is accessible via the 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 by direct injection into liver tissue or via the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

[0107] Colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes, can be used as delivery vehicles for the RNAi constructs of the invention or vectors encoding such constructs. Commercially available fat emulsions suitable for delivery of the nucleic acids of the invention include II. III, Nutrilipid, and other similar lipid emulsions. The preferred colloidal system used as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The RNAi construct of the present invention can be encapsulated in a liposome or can form a complex therewith, particularly with a cationic liposome. Alternatively, the RNAi construct of the present invention can be complexed with lipids, particularly with cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC)), distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and positive (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The preparation and use of such colloidal dispersion systems are well known in the art. Exemplary formulations are also disclosed in U.S. Patent No. 5,981,505; U.S. Patent No. 6,217,900; U.S. Patent No. 6,383,512; U.S. Patent No. 5,783,565; U.S. Patent No. 7,202,227; U.S. Patent No. 6,379,965; U.S. Patent No. 6,127,170; U.S. Patent No. 5,837,533; U.S. Patent No. 6,747,014; and WO 03 / 093449.

[0108] In certain embodiments, RNAi constructs of the present invention are fully encapsulated in lipid formulations, for example, to form SPLP, pSPLP, SNALP or other nucleic acid-lipid particles. As used herein, term " SNALP " refers to stable nucleic acid-lipid particles, including SPLP. As used herein, term " SPLP " refers to nucleic acid-lipid particles comprising the plasmid DNA encapsulated in lipid vesicles. SNALP and SPLP generally contain cationic lipids, non-cationic lipids and the lipid (for example, PEG-lipid conjugates) that prevents particle aggregation. SNALP and SPLP are particularly useful for systemic applications because they show the circulation life of extension after intravenous injection, and accumulate at remote sites (for example, the position physically separated from the application site). SPLP includes " pSPLP ", which includes the encapsulated condensing agent-nucleic acid complex described in PCT Publication No. 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 nontoxic. In addition, nucleic acids, when present in nucleic acid-lipid particles, are resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed in, for example, U.S. Patent Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432, and PCT Publication No. WO 96 / 40964.

[0109] Pharmaceutical compositions suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Typically, these preparations are sterile and flow to a degree that is easy to inject. The preparation should remain stable under manufacturing and storage conditions, and should prevent the contamination of microorganisms such as bacteria and fungi. Suitable solvents or dispersion media can include, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), their suitable mixtures, and vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, in the case of a dispersion, by maintaining the desired particle size and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. By using delayed absorption agents in the composition, such as aluminum monostearate and gelatin, the extended absorption of the injectable composition can be achieved.

[0110] Sterile injectable solutions can be prepared by incorporating the active compound into a solvent as needed with an appropriate amount along with any other ingredients (e.g., those listed above), followed by sterilization filtration. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other desired ingredients, e.g., those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods include vacuum drying and freeze drying techniques, which produce a powder of one or more active ingredients plus any other desired ingredients from their previously sterile filtered solutions.

[0111] The compositions of the present invention can generally be formulated in neutral or salt form. Pharmaceutically acceptable salts include, for example, acid addition salts (formed from free amino groups) derived from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or derived from 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 hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) or organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

[0112] For example, for parenteral administration in an aqueous solution, the solution is generally buffered appropriately, and first, for example, the liquid diluent is made isotonic with enough saline or glucose. Such aqueous solutions can be used for, for example, intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Preferably, as known to those of ordinary skill in the art, particularly according to the present disclosure, a sterile aqueous medium is used. For example, a single dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous injection or injected at the proposed infusion site (see, for example, "Remington's Pharmaceutical Sciences" 15th edition, pages 1035-1038 and 1570-1580). For human administration, the preparation should meet the sterility, pyrogenicity, general safety, and purity standards required by FDA standards. In certain embodiments, the pharmaceutical composition of the present invention comprises a sterile saline solution and an RNAi construct as described herein or consists of it. In other embodiments, the pharmaceutical composition of the present invention comprises an RNAi construct as described herein and sterile water (e.g., water for injection, WFI) or consists of it. In other embodiments, the pharmaceutical compositions of the present invention comprise or consist of an RNAi construct described herein and phosphate buffered saline (PBS).

[0113] In some embodiments, the pharmaceutical compositions of the present invention are packaged with or stored within a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, prefilled syringes, autoinjectors, syringe pumps, intracorporeal syringes, and injection pens. Devices for aerosolized or powdered formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes an administration device comprising a pharmaceutical composition of the present invention for treating or preventing one or more disorders described herein.

[0114] Methods for inhibiting PNPLA3 expression

[0115] The present invention also provides methods for inhibiting PNPLA3 gene expression in cells. These methods comprise contacting cells with an RNAi agent (e.g., a double-stranded RNAi agent) in an amount effective to inhibit PNPLA3 expression in the cells, thereby inhibiting PNPLA3 expression in the cells. Contacting cells with the RNAi agent (e.g., a double-stranded RNAi agent) can be performed in vitro or in vivo. Contacting cells with the RNAi agent in vivo includes contacting cells or cell populations in a subject (e.g., a human subject) with the RNAi agent. Combinations of in vitro and in vivo methods of contacting cells are also possible.

[0116] The present invention provides methods for reducing or inhibiting PNPLA3 expression in a subject in need thereof, as well as methods for treating or preventing conditions, diseases, or disorders associated with PNPLA3 expression or activity. A "condition, disease, or disorder associated with PNPLA3 expression" refers to a condition, disease, or disorder in which altered PNPLA3 expression levels or elevated PNPLA3 expression levels are associated with an increased risk of developing the condition, disease, or disorder.

[0117] As described above, contacting the cell can be direct or indirect. In addition, contact with the cell can be achieved via a targeting ligand, including any ligand described herein or known in the art. In a preferred embodiment, the targeting ligand is a carbohydrate moiety, such as a GalNAc3 ligand or any other ligand that guides the RNAi agent to the target site.

[0118] In one embodiment, contacting a cell with an RNAi comprises "introducing" or "delivering the RNAi into a cell" by promoting or enabling uptake or absorption into the cell. The absorption or uptake of the RNAi can occur by unassisted diffusion or active cellular processes or by adjuvants or devices. The introduction of the RNAi into the 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 cells includes methods known in the art, such as electroporation and lipofection. Other methods are described below and / or are known in the art.

[0119] As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "repress," and other similar terms, and includes any level of inhibition.

[0120] The phrase "inhibiting PNPLA3 expression" is intended to refer to inhibiting the expression of any PNPLA3 gene (e.g., a mouse PNPLA3 gene, a rat PNPLA3 gene, a monkey PNPLA3 gene, or a human PNPLA3 gene), as well as variants or mutants of a PNPLA3 gene. Thus, the PNPLA3 gene can be a wild-type PNPLA3 gene, a mutant PNPLA3 gene (e.g., a mutant PNPLA3 gene that produces amyloid deposits), or a transgenic PNPLA3 gene in the context of a genetically manipulated cell, cell population, or organism.

[0121] "Inhibiting the expression of the PNPLA3 gene" includes any level of PNPLA3 gene inhibition, for example, at least partial suppression of PNPLA3 gene expression. PNPLA3 gene expression can be assessed based on the level or change in the level of any variable associated with PNPLA3 gene expression, such as PNPLA3 mRNA levels, PNPLA3 protein levels, or the amount or extent of amyloid deposits. The level can be assessed in a single cell or a population of cells, including, for example, a sample derived from a subject.

[0122] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with PNPLA3 expression compared to a control level. The control level can be any type of control level used in the art, for example, a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that has not been treated or has been 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, the 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%.

[0123] Inhibition of PNPLA3 gene expression can be demonstrated by a decrease in the amount of mRNA expressed by a first cell or cell population (such cells can be present, for example, in a sample from a subject) from which the PNPLA3 gene is transcribed and which has been treated (e.g., by contacting one or more cells with a RNAi agent of the invention, or by administering a RNAi agent of the invention to a subject in which the cells are present) compared to a second cell or cell population (control cells) that is substantially identical to the first cell or cell population but has not been so treated, such that expression of the PNPLA3 gene is inhibited. In a preferred embodiment, inhibition is assessed by expressing the mRNA level in the treated cells as a percentage of the mRNA level in the control cells using the formula:

[0124]

[0125] Alternatively, inhibition of PNPLA3 gene expression can be assessed based on a reduction in a parameter functionally associated with PNPLA3 gene expression, such as PNPLA3 protein expression or Hedgehog pathway protein activity. PNPLA3 gene silencing can be performed constitutively or by genomic engineering in any cell expressing PNPLA3 and determined by any assay known in the art.

[0126] Inhibition of PNPLA3 protein expression can be demonstrated by a decrease in the level of PNPLA3 protein expressed by a cell or cell population (e.g., the level of protein expressed in a sample derived from a subject). As described above, to assess mRNA suppression, inhibition of protein expression levels in treated cells or cell populations can be similarly expressed as a percentage of protein levels in control cells or cell populations.

[0127] Control cells or cell populations that can be used to assess inhibition of PNPLA3 gene expression include cells or cell populations that have not been contacted with the RNAi agents of the invention. For example, control cells or cell populations can be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with the RNAi agent.

[0128] The level of PNPLA3 mRNA expressed by a cell or cell population, or circulating PNPLA3 mRNA, can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of PNPLA3 expression in a sample is determined by detecting mRNA of a transcribed polynucleotide or portion thereof, such as the PNPLA3 gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), the RNeasy RNA prep kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing RNA 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, which is hereby incorporated by reference.

[0129] In one embodiment, PNPLA3 expression levels are determined using nucleic acid probes. As used herein, the term "probe" refers to any molecule capable of selectively binding to a specific PNPLA3. Probes can be synthesized by one of ordinary skill in the art or derived from appropriate biological agents. Probes can be specifically designed for labeling. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0130] The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern blot analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels comprises 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, one or more probes are immobilized on a solid surface and the mRNA is contacted with the one or more probes, for example, in an Affymetrix gene chip array. Known mRNA detection methods suitable for determining PNPLA3 mRNA levels are readily available to those of skill in the art.

[0131] Alternative methods for determining the level of PNPLA3 expression in a sample include nucleic acid amplification and / or reverse transcription (to prepare cDNA) of, for example, mRNA in the sample, such as by RT-PCR (experimental examples are 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-sustaining sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 88:189-193), and reverse transcriptase (Cell Signaling Technology, Inc., 1992). USA 87:1874-1878), transcription amplification system (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., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those of ordinary skill in the art. These detection schemes are particularly useful for detecting nucleic acid molecules if these molecules are present in very low numbers. In a specific aspect of the invention, PNPLA3 expression levels are measured by quantitative fluorescent RT-PCR (i.e., TaqMan TM PNPLA3 mRNA expression levels can be monitored using membrane blotting (e.g., for hybridization analysis, such as Northern blots, Southern blots, dot blots, etc.) 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. Determination of PNPLA3 expression levels can also include the use of nucleic acid probes in solution.

[0132] In preferred embodiments, mRNA expression levels are assessed using branched DNA (bDNA) assays or real-time PCR (qPCR).The use of these methods is described and exemplified in the examples provided herein.

[0133] PNPLA3 protein expression levels can be determined using any method known in the art for measuring protein levels, including, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitin reaction, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assays, electrochemiluminescence assays, and the like.

[0134] In some embodiments, the efficacy of the methods of the present invention can be monitored by detecting or monitoring a reduction in PNPLA3 disease symptoms, such as edema of the extremities, face, throat, upper respiratory tract, abdomen, trunk, and genitals, prodromal symptoms; swelling of the throat; non-pruritic rash; nausea; vomiting; or a reduction in abdominal pain. These symptoms can be assessed in vitro or in vivo using any method known in the art.

[0135] In some embodiments of the methods of the present invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. Inhibition of PNPLA3 expression can be assessed using measurements of PNPLA3 mRNA or PNPLA3 protein levels, or changes in levels, in fluid or tissue samples from specific sites within the subject. In preferred embodiments, these sites are selected from the group consisting of the liver, choroid plexus, retina, and pancreas. The site may also be a small portion or subset of cells from any of the aforementioned sites. The site may also include cells expressing a specific type of receptor.

[0136] Methods for treating or preventing PNPLA3-related diseases

[0137] The present invention provides methods of treatment and prevention 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 suffering from, or susceptible to developing, a PNPLA3-related disease, disorder, and / or condition. Non-limiting examples of PNPLA3-related diseases include, for example, 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). 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.

[0138] In certain embodiments, the present invention provides a method for reducing PNPLA3 expression in a patient in need thereof, comprising administering to the patient any of the RNAi constructs described herein. As used herein, the term "patient" refers to mammals, including humans, and is used interchangeably with the term "subject." Preferably, the level of PNPLA3 expression in the patient's hepatocytes is reduced following administration of the RNAi construct, compared to the level of PNPLA3 expression in a patient who has not received the RNAi construct.

[0139] The methods of the present invention can be used to treat subjects suffering from a PNPLA3-related disease, such as subjects who would benefit from reduced PNPLA3 gene expression and / or PNPLA3 protein production. In one aspect, the present invention provides methods for reducing the expression level of the Patatin-like phospholipase domain-containing 3 (PNPLA3) gene in a subject suffering from non-alcoholic fatty liver disease (NAFLD). In another aspect, the present invention provides methods for reducing PNPLA3 protein levels in a subject suffering from NAFLD. The present invention also provides methods for reducing the activity level of the hedgehog pathway in a subject suffering from NAFLD.

[0140] In another aspect, the present invention provides methods for treating subjects with NAFLD. In one aspect, the present invention provides methods for treating subjects with PNPLA3-related diseases, 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 methods (and uses) of the present invention comprise administering to a subject, such as a human, a therapeutically effective amount of a RNAi agent targeting the PNPLA3 gene of the present invention, or a pharmaceutical composition comprising a RNAi agent targeting the PNPLA3 gene of the present invention, or a vector of the present invention comprising a RNAi agent targeting the PNPLA3 gene.

[0141] In one aspect, the present invention provides methods for preventing at least one symptom in a subject with NAFLD, such as the presence of elevated hedgehog signaling, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider blood vessels, yellowing of the skin and eyes (jaundice), itching, fluid accumulation and swelling in the legs (edema), abdominal swelling (ascites), and confusion. These methods comprise administering to the subject a therapeutically effective amount of an RNAi agent, such as a dsRNA, pharmaceutical composition, or vector of the present invention, thereby preventing at least one symptom in a subject with a disorder that would benefit from reduced PNPLA3 gene expression.

[0142] In another aspect, the present invention provides use of a therapeutically effective amount of a RNAi agent of the present invention for treating a subject, e.g., a subject who would benefit from reduction and / or inhibition of PNPLA3 gene expression. In another aspect, the present invention provides use of a RNAi agent, e.g., a dsRNA, or a pharmaceutical composition comprising a RNAi agent targeting the PNPLA3 gene, of the present invention for preparing a medicament for treating a subject, e.g., a subject who would benefit from reduction and / or inhibition of PNPLA3 gene expression and / or PNPLA3 protein production, e.g., a subject suffering from a disorder that would benefit from reduction of PNPLA3 gene expression, e.g., a PNPLA3-related disease.

[0143] In another aspect, the present invention provides use of an RNAi (e.g., dsRNA) of the invention for preventing at least one symptom in a subject having a disorder that would benefit from reduction and / or inhibition of PNPLA3 gene expression and / or PNPLA3 protein production.

[0144] In another aspect, the present invention provides use of a RNAi agent of the invention in the preparation of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduction and / or inhibition of PNPLA3 gene expression and / or PNPLA3 protein production, such as a PNPLA3-associated disease.

[0145] In one embodiment, a RNAi agent targeting PNPLA3 is administered to a subject having a PNPLA3-related disease, such as non-alcoholic fatty liver disease (NAFLD), such that expression of the PNPLA3 gene, for example, in cells, tissues, blood, or other tissues or fluids of the subject is reduced 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%, 112%, 113%, 14 , 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 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.

[0146] The methods and uses of the present invention comprise administering a composition as described herein such that expression of the target PNPLA3 gene is reduced, for example, for 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 two, three, four, five, six, seven, or more days, for example, about one, two, three, or about four weeks or longer.

[0147] 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 a PNPLA3-associated disease (e.g., non-alcoholic fatty liver disease (NAFLD)) in a patient suffering from such a disease or disorder. In this context, "reduction" refers to a statistically significant decrease 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 efficacy of a treatment or prevention of a disease can be assessed, for example, by measuring the level of disease progression, disease remission, symptom severity, pain relief, quality of life, drug dosage required to maintain a therapeutic effect, disease markers, or any other measurable parameter applicable to the specific disease being treated or targeted for prevention. Monitoring the efficacy of a treatment or prevention by measuring any one or any combination of these parameters is within the capabilities of one of ordinary skill in the art. For example, the efficacy of NAFLD treatment can be assessed, for example, by periodically monitoring NAFLD symptoms, liver fat levels, or expression of downstream genes. Comparison of subsequent readings with initial readings provides the physician with an indication of whether the treatment is effective. Monitoring therapeutic or preventive efficacy by measuring any one or any combination of these parameters is within the capabilities of one of ordinary skill in the art. In conjunction with administration of a PNPLA3-targeting RNAi or pharmaceutical composition thereof, "effective against" a PNPLA3-related disease means that administration in a clinically appropriate manner produces a beneficial effect in at least a statistically significant portion of patients, such as amelioration of symptoms, cure, disease alleviation, life extension, improved quality of life, or other effects generally considered positive by physicians familiar with treating NAFLD and / or PNPLA3-related diseases and associated etiologies.

[0148] A therapeutic or preventive effect is evident when one or more parameters of the disease state are statistically significantly improved, or when symptoms fail to worsen or otherwise be expected to appear. For example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50%, or more, in a measurable disease parameter can indicate effective treatment. The efficacy of a given RNAi drug or formulation of the drug can also be judged using experimental animal models for a given disease known in the art. When using an experimental animal model, therapeutic efficacy is demonstrated when a statistically significant reduction in a marker or symptom is observed.

[0149] A therapeutically effective amount of RNAi can be administered to a subject, such as 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, 6.6mg / 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 dsRNA, 8.9mg / kg dsRNA, 9.0mg / kg dsRNA, 9.1mg / kg dsRNA, 9.2mg / kg dsRNA, 9.3mg / kg dsRNA, 9.4mg / kg dsRNA, 9.5mg / kg dsRNA, 9.6mg / kg dsRNA, 9.7mg / kg dsRNA, 9.8mg / kg dsRNA, 9.9mg / kg dsRNA, 9.0mg / kg dsRNA, 10mg / kg dsRNA, 15mg / kg dsRNA, 20mg / kg dsRNA, 25mg / kg dsRNA, 30mg / kg dsRNA, 35mg / kg dsRNA, 40mg / kg dsRNA, 45 mg / kg dsRNA or approximately 50 mg / kg dsRNA. In one embodiment, 0.5 mg / kg of dsRNA may be administered to a subject. Values ​​and ranges intermediate to these values ​​are also intended to be part of the present invention.

[0150] Administration of RNAi can reduce the presence of PNPLA3 protein levels in, for example, cells, tissues, blood, urine, or other compartments of a patient by at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111 %, 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.

[0151] Before administering the full dose of RNAi, a smaller dose, such as a 5% infusion, can be administered to the patient and adverse reactions, such as allergic reactions, can be monitored. In another example, the patient can be monitored for unwanted immune stimulation, such as increased cytokine (e.g., TNF-α or INF-α) levels.

[0152] Due to the inhibitory effect on PNPLA3 expression, the composition according to the present invention or the pharmaceutical composition prepared therefrom can improve the quality of life.

[0153] The RNAi of the present invention can be administered in a "naked" form, where the modified or unmodified RNAi agent is directly suspended in an aqueous or suitable buffered solvent as "free RNAi". Free RNAi is administered in the absence of a pharmaceutical composition. Free RNAi can be in a suitable buffer solution. The buffer solution can 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 weight molar osmotic pressure concentration of the buffer solution containing RNAi can be adjusted to make it suitable for administration to a subject.

[0154] Alternatively, the RNAi of the invention can be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

[0155] A subject who would benefit from reduction and / or inhibition of PNPLA3 gene expression is a subject suffering from non-alcoholic fatty liver disease (NAFLD) and / or a PNPLA3-related disease or disorder as described herein.

[0156] Treatment of subjects who would benefit from reduction and / or inhibition of PNPLA3 gene expression includes both therapeutic and prophylactic treatments.

[0157] The present invention further provides methods for treating subjects who would benefit from reduction and / or inhibition of PNPLA3 gene expression (e.g., subjects suffering from a PNPLA3-associated disease) and uses of RNAi agents or pharmaceutical compositions thereof in combination with other drugs and / or other treatments (e.g., with known drugs and / or known treatments, such as those currently used to treat these disorders) for treating subjects who would benefit from reduction and / or inhibition of PNPLA3 gene expression.

[0158] For example, in certain embodiments, RNAi agents targeting the PNPLA3 gene are administered in combination with agents useful for treating PNPLA3-related diseases, such as those described elsewhere herein. For example, other therapeutic agents and treatment methods suitable for treating subjects who would benefit from reduced PNPLA3 expression, such as subjects with a PNPLA3-related disease, include RNAi agents targeting different portions of the PNPLA3 gene, therapeutic agents, and / or procedures for treating a PNPLA3-related disease, or any combination of the foregoing.

[0159] In certain embodiments, a first RNAi agent targeting the PNPLA3 gene is administered in combination with a second RNAi agent targeting a different portion of the PNPLA3 gene. For example, the first RNAi agent comprises a first sense strand and a first antisense strand forming a double-stranded region, wherein substantially all nucleotides of the first sense strand and substantially all nucleotides of the first antisense strand are modified nucleotides, wherein the first sense strand is conjugated to a ligand linked to the 3' end, and wherein the ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker; and the second RNAi agent comprises a second sense strand and a second antisense strand forming a double-stranded region, wherein substantially all nucleotides of the second sense strand and substantially all nucleotides of the second antisense strand are modified nucleotides, wherein the second sense strand is conjugated to a ligand linked to the 3' end, and wherein the ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker.

[0160] In one embodiment, all nucleotides of the first and second sense strands and / or all nucleotides of the first and second antisense strands comprise a modification.

[0161] In one embodiment, at least one modified nucleotide is selected from the group consisting of: 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing non-natural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0162] In certain embodiments, a first RNAi agent targeting the PNPLA3 gene is administered in combination with a second RNAi agent targeting a gene other than the PNPLA3 gene. For example, a RNAi agent targeting the PNPLA3 gene can be administered in combination with a RNAi agent targeting the SCAP gene. The first RNAi agent targeting the PNPLA3 gene and the second RNAi agent targeting a gene other than the PNPLA3 gene (e.g., the SCAP gene) can be administered as part of the same pharmaceutical composition. Alternatively, the first RNAi agent targeting the PNPLA3 gene and the second RNAi agent targeting a gene other than the PNPLA3 gene (e.g., the SCAP gene) can be administered as part of different pharmaceutical compositions.

[0163] The RNAi agent and the additional therapeutic agent and / or treatment can be administered simultaneously and / or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at a different time and / or by another method known in the art or described herein.

[0164] The present invention also provides methods for reducing and / or inhibiting PNPLA3 expression in cells using RNAi agents of the present invention and / or compositions containing the same. In other aspects, the present invention provides RNAi agents of the present invention and / or compositions containing the same for reducing and / or inhibiting PNPLA3 gene expression in cells. In yet other aspects, the present invention provides uses of RNAi agents of the present invention and / or compositions containing the same for preparing a medicament for reducing and / or inhibiting PNPLA3 gene expression in cells. In other aspects, the present invention provides RNAi agents of the present invention and / or compositions containing the same for reducing and / or inhibiting PNPLA3 protein production in cells. In yet other aspects, the present invention provides uses of RNAi agents of the present invention and / or compositions containing the same for preparing a medicament for reducing and / or inhibiting PNPLA3 protein production in cells. These methods and uses comprise contacting cells with an RNAi agent of the present invention (e.g., dsRNA) and maintaining the cells for a sufficient time to achieve degradation of PNPLA3 gene mRNA transcripts, thereby inhibiting PNPLA3 gene expression or inhibiting PNPLA3 protein production in the cells.

[0165] Reduction in gene expression can be assessed by any method known in the art. For example, reduction in PNPLA3 expression can be determined by determining the level of PNPLA3 mRNA expression using methods routinely available to those skilled in the art, such as Northern blotting, qRT-PCR; by determining the level of PNPLA3 protein using methods routinely available to those skilled in the art, such as immunoblotting, immunological techniques, flow cytometry, ELISA; and / or by determining the biological activity of PNPLA3.

[0166] In the methods and uses of the present invention, the cells may be contacted in vitro or in vivo, ie, the cells may be within the body of a subject.

[0167] 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 containing an expression vector containing the PNPLA3 gene or a portion of the PNPLA3 gene. Cells suitable for the methods and uses of the present invention can be mammalian cells, such as primate cells (e.g., 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), avian cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell.

[0168] PNPLA3 gene expression can be inhibited in a cell 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%, 119%, 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%.

[0169] PNPLA3 protein production can be inhibited in a cell 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%, 119%, 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%.

[0170] The in vivo methods and uses of the present invention may include administering to a subject a composition comprising an RNAi agent, wherein the RNAi agent comprises a nucleotide sequence complementary to at least a portion of an RNA transcript of a PNPLA3 gene of a mammal to be treated. When the organism to be treated is a human, the composition may be administered by any means known in the art, including but not limited to subcutaneous, intravenous, oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, airway (aerosol), nasal, rectal, and topical (including oral and sublingual) administration. In certain embodiments, the composition is administered by subcutaneous or intravenous infusion or injection. In one embodiment, the composition is administered by subcutaneous injection.

[0171] In some embodiments, administration is via a long-acting injection. Long-acting injections can release the RNAi in a consistent manner over an extended period of time. Thus, long-acting injections can reduce the frequency of dosing required to achieve a desired effect, such as a desired PNPLA3 inhibition or therapeutic or preventive effect. Long-acting injections can also provide more consistent serum concentrations. Long-acting injections can include subcutaneous or intramuscular injections. In preferred embodiments, the long-acting injection is a subcutaneous injection.

[0172] In some embodiments, administration is via a pump. The pump can 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. Infusion pumps can be used for intravenous, subcutaneous, intraarterial, or epidural infusion. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi to the subject.

[0173] 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 of administration and site of administration can be selected to enhance targeting.

[0174] In one aspect, the present invention also provides a method for inhibiting the expression of the PNPLA3 gene in a mammal (e.g., a human). The present invention also provides a composition comprising an RNAi (e.g., dsRNA) targeting the PNPLA3 gene in a mammalian cell, for inhibiting the expression of the PNPLA3 gene in a mammal. In another aspect, the present invention provides the use of an RNAi (e.g., dsRNA) targeting the PNPLA3 gene in a mammalian cell in the preparation of a medicament for inhibiting the expression of the PNPLA3 gene in a mammal.

[0175] These methods and uses include administering to a mammal (e.g., a human) a composition comprising an RNAi (e.g., dsRNA) targeted to a PNPLA3 gene in a cell of the mammal and maintaining the mammal for a sufficient time to achieve degradation of the mRNA transcript of the PNPLA3 gene, thereby inhibiting expression of the PNPLA3 gene in the mammal.

[0176] Reduction of gene expression can be assessed in peripheral blood samples of subjects administered RNAi by any method known in the art (e.g., qRT-PCR as described herein). Reduction of protein production can be assessed by any method known in the art and by methods described herein (e.g., ELISA or immunoblotting). In one embodiment, tissue samples are used as tissue material to monitor reduction of PNPLA3 gene and / or protein expression. In another embodiment, blood samples are used as tissue material to monitor reduction of PNPLA3 gene and / or protein expression.

[0177] In one embodiment, verification of RISC-mediated cleavage of the target in vivo following administration of the RNAi agent is accomplished by performing 5'-RACE or modifications of protocols known in the art (Lasham A et al. (2010) Nucleic Acid Res., 38(3) p-el9) (Zimmermann et al. (2006) Nature 441: 111-4).

[0178] It should be understood that all RNA 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 uracil bases for thymine bases in the sequence. The present invention includes sequences of deoxyribonucleic acid sequences, ribonucleic acid sequences, and mixtures of deoxyribonucleotides and ribonucleotides containing all sequences disclosed herein.

[0179] Additionally, any nucleic acid sequence disclosed herein can be modified with any combination of chemical modifications. It will be readily understood by those skilled in the art that, in some cases, nomenclature describing modified polynucleotides such as "RNA" or "DNA" is arbitrary. For example, a polynucleotide comprising a nucleotide having a 2'-OH substituent on a ribose sugar and a thymine base can be described as a DNA molecule having a modified sugar (2'-OH replacing the natural 2'-H of DNA) or as an RNA molecule having a modified base (thymine (methylated uracil) replacing the natural uracil of RNA).

[0180] 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 those with modified nucleoside bases. As another example and without limitation, a polynucleotide having the sequence "ATCGATCG" encompasses any polynucleotide having this sequence, modified or unmodified, including but not limited to compounds comprising RNA bases, such as those having the sequence "AUCGAUCG," and those having some DNA bases and some RNA bases (e.g., "AUCGATCG"), and polynucleotides having other modified bases (e.g., "ATmeCGAUCG"), wherein meC represents a cytosine base containing a methyl group at the 5-position.

[0181] The following examples, including experiments performed and results achieved, are for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

[0182] Incorporated by Reference

[0183] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, citation of a reference herein should not be construed as an admission that such reference is prior art to the present invention. In the event that any definition or term provided in an incorporated reference differs from the terminology and discussion provided herein, the present terminology and definitions shall prevail.

[0184] equivalent

[0185] The foregoing written description is believed to be sufficient to enable one of ordinary skill in the art to practice the invention. The foregoing description and examples detail certain preferred embodiments of the invention and describe the best mode contemplated by the inventors. However, it will be understood that no matter how detailed the foregoing appears in text, the invention can be implemented in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0186] The following examples, including the experiments performed and the results achieved, are provided for illustrative purposes only and are not intended to be construed as limiting the present invention.

[0187] All animal experiments described herein are approved by the Amgen Research Animal Care and Use Committee (IACUC) of American business Amgen, Inc., and are nursed according to the 8th edition of the Guide for the Care and Use of Laboratory Animals (National Research Council (U.S.)), the Update Committee on the Care and Use of Laboratory Animals, the Institute of Laboratory Animals (U.S.), and the National Academy of Sciences Press (U.S.) (2011) Guide for the Care and Use of Laboratory Animals, 8th edition (National Academy of Sciences Press (Washington, D.C.)). Mice are housed individually in an air-conditioned room at 22°C ± 2°C with 12 hours of light; 12 hours of dark cycle (0600-1800 hours). Unless otherwise stated, animals can arbitrarily obtain regular food (Envigo, 2920X; or food otherwise specified) and water (reverse osmosis purification) is obtained by an automatic watering system. At the end, blood is collected by cardiac puncture under deep anesthesia, and then euthanasia is implemented by secondary physical methods according to the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines.

[0188] Example 1: Selection, design, and synthesis of modified PNPLA3 siRNA molecules

[0189] Bioinformatics analysis of the human PNPLA3 transcript (NM_025225.2) was used to identify and select optimal sequences for therapeutic siRNA molecules targeting patatin-like phospholipase domain 3 (PNPLA3). Table 1 shows the sequences identified as having therapeutic properties. Throughout the various sequences, INVAB is inverted A basic, INVDA is inverted deoxythymidine, GNA is glycol nucleic acid, dT is deoxythymidine, and dC is deoxycytosine.

[0190] Table 1. siRNA sequences targeting PNPLA3

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205] To improve the efficacy and in vivo stability of PNPLA3 siRNA sequences, chemical modifications were incorporated into the PNPLA3 siRNA molecules. Specifically, 2'-O-methyl and 2'-fluoro modifications of the ribose sugar were incorporated into specific positions within the PNPLA3 siRNA. Phosphorothioate internucleotide linkages were also incorporated at the termini of the antisense and / or sense sequences. Table 2 below describes the modifications to the sense and antisense sequences of each modified PNPLA3 siRNA. The nucleotide sequences in Table 2 and elsewhere in this application are listed according to the following symbols: A, U, G, and C = corresponding ribonucleotides; dT = deoxythymidine; dA = deoxyadenosine; dC = deoxycytidine; dG = deoxyguanosine; invDT = inverted deoxythymidine; invDA = inverted deoxyadenosine; invDC = inverted deoxycytidine; invDG = inverted deoxyguanosine; a, u, g, and c = corresponding 2'-O-methyl ribonucleotides; Af, Uf, Gf, and Cf = corresponding 2'-deoxy-2'-fluoro ("2'-fluoro") ribonucleotides; Ab = abasic; MeO-I = 2'methoxyinosine; GNA = glycol nucleic acid; sGNA = glycol nucleic acid with a 3' phosphorothioate; LNA = locked nucleic acid. The insertion of an "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothioate diester group (e.g., a phosphorothioate internucleotide bond). Unless otherwise noted, all other nucleotides are linked via 3'-5' phosphodiester groups. Each siRNA compound in Table 2 contains a 19 base pair duplex region with 2 nucleotide overhangs at the 3' end of both strands or a blunt end at one or both ends. GalNAc3K2AhxC6 is:

[0206]

[0207] Table 2. siRNA sequences targeting PNPLA3 with modifications

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] Example 2: Efficacy of Selected PNPLA3 siRNA Molecules in RNA FISH Assay

[0230] A panel of fully chemically modified siRNAs, including siRNAs spanning the rs738409 and / or rs738408 SNPs in PNPLA3, was prepared and tested for efficacy and selectivity in vitro for mRNA knockdown. Each siRNA duplex consisted of two strands: a sense or "follower" strand and an antisense or "guide" strand. The strands were 21 or 23 nucleotides long, with 19 complementary base pairs. In some cases, a two-base-pair 3' overhang was present. siRNAs were prepared in which the native 2'-OH group in the ribose sugar of each nucleotide was replaced with a 2'-OMe or 2'-F group. Optionally, the phosphodiester internucleotide linkage on one or both strands was replaced with a phosphorothioate to reduce exonuclease degradation.

[0231] The efficacy of each siRNA molecule in reducing PNPLA3 expression was evaluated using a 384-well format in vitro siRNA transfection assay followed by a fluorescence in situ hybridization (RNA FISH) assay to determine IC50 and maximum activity values. The assay was performed on the 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 in EMEM medium (ATCC 30-2003) supplemented with 10% fetal bovine serum and 1% antibiotic / antimycotic at 37°C and 5% CO2. CHO cells expressing human PNPLA3 I148I were maintained in culture medium containing 50% CD-CHO (Life Technologies), 50% Ex-Cell CHO 5 medium (Sigma), 8 mM L-glutamine, 1xHT, 1% antibiotic / antimycotic, and 10 μg / mL puromycin at 37°C and 5% CO2.

[0232] 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 / 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 / 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, with a final density of 2000 cells / well in 40 μl of medium. After incubation at room temperature for 20 minutes, the plates were transferred to a 37°C and 5% CO2 incubator. Hep3B cell transfection assays were incubated for 72 hours, and CHO human PNPLA3 I148I transfection assays were incubated for 48 hours.

[0233] At harvest, cells were fixed in 8% formaldehyde fixative solution (Thermo Scientific) at room temperature for 15 minutes. The plates were then dehydrated by sequential 50%, 70%, and 100% ethanol washes. The plates were then sealed and stored at -20°C.

[0234] Using Affymetrix RNA FISH assays were performed using the View RNA HC Screening Assay Kit (QVP0011), the Affymetrix View HC Signal Amplification Kit 3-plex (QVP0213), and the following Affymetrix gene-specific probes: PNPLA3 Human 0.33 mL View RNA Type 6 (650 markers) VA6-20279-01 and PPIB Human 0.44 mL View RNA Type 1 (488 markers) VA1-10148-01.

[0235] The plate was first rehydrated by continuous 100%, 70% and 50% ethanol washing. The cells were then washed with PBS and permeabilized and digested with proteases according to the kit instructions. The target working probe group was prepared according to the manufacturer's protocol, added to the wells, and incubated for 3 hours at 40°C. The manufacturer's protocol was followed to perform continuous hybridization using the working probe group, working preamplifier, working amplifier, and working LP. Finally, nuclear counterstaining (Hoechst 33342 and Cell Mask Blue; Molecular Probes) was applied. The plate was incubated at room temperature for 30 minutes, washed with PBS, covered with 80 μl of PBS, and then the plate was sealed for imaging.

[0236] 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.

[0237] RNA FISH data were analyzed using Columbus software, and images were generated using Genedata Screener. The results for PNPLA3 I148I assays in CHO-transfected cells are shown in Table 3. The results for PNPLA3 I148M assays in CHO-transfected cells are shown in Table 4. PNPLA3 knockdown is provided as a percentage of knockdown compared to the control. Negative values ​​indicate decreased PNPLA3 levels.

[0238] Table 3. RNA FISH assay of PNPLA3 I148I transfected with CHO cells

[0239]

[0240]

[0241]

[0242] Table 4. RNA FISH assay of PNPLA3 I148M transfected with CHO

[0243]

[0244]

[0245] RNA FISH is also run on a hepatocyte line containing double mutant PNPLA3-rs738408-rs738409 and a control wild-type cell line Hep3B. Hep3B and HepG2 cells (purchased from ATCC) are 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 is performed as follows: depending on the cell line, 1 μL test siRNA and 4 μL ordinary MEM or EMEM are added to a PDL-coated CellCarrier-384Ultra assay plate (PerkinElmer) by BioMek FX (Beckman Coulter). 5 μL of Lipofectamine RNAiMAX (Thermo Fisher Scientific) was pre-diluted in plain MEM or EMEM (specifically, for Hep3B, 0.035 μL RNAiMAX in 5 μL MEM, and for HepG2, 0.06 μL RNAiMAX in 5 μL EMEM) and then dispensed into the assay plate via a Multidrop Combi reagent dispenser (Thermo Fisher Scientific). After incubating the siRNA / RNAiMAX mixture for 20 minutes at room temperature (RT), 30 μL Hep3B or HepG2 cells (2000 cells per well) in MEM or EMEM supplemented with 10% FBS and 1% PS were added to the transfection complex using a Multidrop Combi reagent dispenser. The assay plate was incubated at room temperature for 20 minutes and then placed in an incubator. The cells were then incubated at 37°C and 5% CO2 for 72 hours. ViewRNA ISH cell assays were performed using an in-house assembled automated FISH assay platform for liquid handling following the manufacturer's protocol (Thermo Fisher Scientific). 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.The target specific probe pairs (Thermo Fisher Scientific) were incubated for 3 hours, while the preamplifier, amplifier and label probe (Thermo Fisher Scientific) were incubated for 1 hour each. All hybridization steps were performed at 40°C in a Cytomat 2C-LIN automatic incubator (Thermo Fisher Scientific). After the hybridization reaction, the cells were stained with Hoechst and CellMask Blue (Thermo Fisher Scientific) for 30 minutes and then imaged on an Opera Phenix (Perkin Elmer). Images were analyzed using a Columbus imaging data storage and analysis system (Perkin Elmer) to obtain the average spot count for each cell. High (containing phosphate buffered saline, Corning) and low (no target probe pair) control wells were used to normalize spot counts. Normalized values ​​were plotted relative to total siRNA concentration, and the data were fitted to a four-parameter sigmoidal model in Genedata Screener (Genedata) to obtain IC50 and maximum activity. The results for HepG2 cells are shown in Table 5, and the results for Hep3B cells are shown in Table 6. PNPLA3 knockdown provides the percentage of knockdown compared to the control. Negative values ​​indicate a decrease in PNPLA3 levels. In cases where duplexes were run more than once, the average IC50 is shown, along with the standard deviation.

[0246] Table 5. RNA FISH assay of HepG2 liver cells

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] Table 6. RNA FISH assay of Hep3B cells

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] Example 3: Droplet Digital PCR Assay for siRNAs to PNPLA3-rs738409 and PNPLA3-rs738409-rs738408

[0269] Following the manufacturer's protocol, human primary hepatocytes (Xenotech / Sekisui donor lot #HC3-38) were thawed in OptiThaw medium (Xenotech catalog number K8000), centrifuged, and after aspiration of the medium, resuspended in OptiPlate Hepatocyte Medium (Xenotech catalog number K8200) and plated into 96-well collagen-coated plates (Greiner catalog number 655950). After incubation for 2-4 hours, the medium was removed and replaced with OptiCulture Hepatocyte Medium (Xenotech catalog number K8300). 2-4 hours after adding OptiCulture medium, GalNAc-conjugated siRNA was delivered to the cells by free uptake (without transfection reagent). The cells were incubated at 37°C and 5% CO2 for 24-72 hours. The cells were then lysed with Qiagen RLT buffer (79216) + 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 a Qiagen RNeasy 96QIACube HT kit (74171) according to the manufacturer's instructions. Samples were analyzed using the QIAxpert system (9002340). cDNA was synthesized from the RNA samples using an Applied Biosystems HighCapacity cDNA Reverse Transcription kit (4368813). Reactions were assembled according to the manufacturer's instructions, with input RNA concentrations varying with the sample. Reverse transcription was performed on a BioRad Tetrad Thermal Cycler (Model #PTC-0240G) under the following conditions: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 minutes, and then (optionally) 4°C indefinite hold.

[0270] Droplet digital PCR (ddPCR) was performed using the QX200 AutoDG Droplet Digital PCR System from BioRad according to the manufacturer's instructions. The reaction was assembled into an Eppendorf clear 96-well PCR plate (951020303) using BioRad ddPCR Supermix for Probes (1863010) and qPCR assays were performed using fluorescently labeled PNPLA3 (IDTHs.PT.58.21464637, primer to probe ratio 3.6:1 and TBP (IDT) Hs.PT.53a.20105486, probe ratio 3.6:1) and RNase-free water (Ambion, AM9937). The final primer / probe concentrations were 900 nM / 250 nM, respectively, with different cDNA concentrations input into the wells. Droplets were formed using a BioRad Auto DG droplet generator (1864101) equipped with the manufacturer's recommended consumables (BioRad DG32 cartridge 1864108, BioRad tips 1864121, Eppendorf blue 96-well PCR plate 951020362, BioRad droplet generation oil for probes 1864110, and BioRad droplet plate assembly). Droplets were amplified on a BioRad C1000 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 using a 2°C / second ramp rate, enzyme inactivation at 98°C for 10 minutes, and then (optionally) an unlimited hold at 4°C. The samples were then read on a BioRad QX200 microdroplet reader, which measures the FAM / HEX signal associated with the concentration of PNPLA3 or TBP. Data were analyzed using BioRad's QuantaSoft software package. Samples were gated by channel (fluorescent marker) to determine the concentration of each sample. Each sample was then expressed as a ratio of target gene (PNPLA3) concentration / housekeeping gene (TBP) concentration to control for sample loading differences. The data were then imported into Genedata Screener, where each test siRNA was normalized to the median value of the neutral control well (buffer only). IC50 values ​​are reported in Table 7.

[0271] Table 7. ddPCR assays on primary hepatocytes

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[0273]

[0274]

[0275]

[0276]

[0277]

[0278] Example 4: Efficacy screening of selected PNPLA3 siRNA molecules in a humanized mouse model

[0279] Human PNPLA3 was driven by intravenous injection of relevant adenovirus (AAV; serotype AAV8 or AAV7; endotoxin-free, prepared in-house by Amgen) diluted to 4e11 to 1e12 viral particles per animal in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) into the tail vein of C57BL / 6NCrl male mice (Charles River Laboratories Inc.). WT (PNPLA3-WT), PNPLA3 rs738409 (PNPLA3-I148M), or PNPLA3 rs738409-rs738408 (PNPLA3-I148M DM) expression in the liver. Mice were typically 10-12 weeks old, and each group included n=4-6 animals. Each round of screening included at least two vehicle-treated control groups: AAV-empty vector and AAV-PNPLA3 treated with vehicle. WT or PNPLA3 rs738409 and PNPLA3 rs738409-rs738408 Targeting AAV-PNPLA3 WT 、PNPLA3 rs738409 and / or PNPLA3 rs738409-rs738408All siRNAs were tested. Two weeks after AAV injection, mice were treated with a single dose of siRNA D-2324 (0.5 mM) at 0.5, 1.0, 3.0, or 5.0 mg / kg animal diluted in phosphate buffered saline (Thermo Fisher Scientific, 14190-136) by subcutaneous injection. 8, 15, 22, 28, or 42 days after siRNA injection, livers were collected from animals, snap-frozen in liquid nitrogen, and purified RNA was processed using a QIACube HT instrument (Qiagen, 9001793) and RNeasy 96QIACube HT kit (Qiagen, 74171) according to manufacturer's instructions. Samples were analyzed using the QIAxpert system (Qiagen, 9002340). RNA was treated with RQ1 RNase-free DNase (Promega, M6101) and quantified using TaqMan TM RNA-to-C T TM Real-time qPCR was performed using the 1-Step kit (Applied Biosystems, 4392653). Real-time qPCR was performed on a QuantStudio Real-Time PCR machine. Results were based on gene expression of human PNPLA3 normalized to mouse Gapdh (TaqMan® from Invitrogen). TM The results were assayed for hs00228747_ml and 4352932E, respectively, and are presented as relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated control animals. Endogenous mouse Pnpla3 expression was determined for comparison (Invitrogen, Mm00504420_ml).

[0280] For liver triglyceride content analysis, by homogenization in one milliliter of isopropyl alcohol from the approximately 0.05-0.1 milligram of frozen liver of animal.After incubation on ice one hour, sample is rotated with 10,000rpm in microcentrifuge, and supernatant is transferred in the deep well 96 orifice plates of cleaning.According to manufacturer specification, use colorimetric Infinity triglyceride reagent (Sai ​​Mo Fei Shi Er Science and Technology Co., Ltd. (Thermo Fisher Scientific), TR22421) and triglyceride standard substance (Pointe Scientific Co., Ltd. (Pointe Scientific, T7531-STD) to determine triglyceride content.Result is expressed as the triglyceride milligram number of every milligram of tissue.

[0281] Figures 1A-1D Examples of 5 siRNA molecules screened for dose-dependent mRNA knockdown and functional durability in vivo. Two weeks after intravenous AAV injection, cells expressing human PNPLA3 were treated with siRNA. rs738409-rs738408 Mice. N = 6 mice per group; data are expressed 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 mg / kg body weight. After four weeks of siRNA treatment, mice were sacrificed, and livers were harvested and processed for gene expression analysis. Data represent the expression of human PNPLA3 in each group relative to a vehicle-treated control group. rs738409 -rs738408 (B) Livers from the same four-week treatment groups were also treated for triglyceride content to determine functional efficacy. Data represent the average number of milligrams of triglyceride per gram of treated tissue. (C) siRNA was injected subcutaneously into the abdomen of parallel groups of animals at 1.0 and 3.0 mg / kg body weight. Mice were harvested six weeks after siRNA treatment to compare the durability of the siRNA molecules in vivo. Livers were collected and processed for gene expression analysis. Data represent the relative expression of human PNPLA3 in each group relative to a vehicle-treated control group. rs738409-rs738408 (D) Livers from the same six-week treatment groups were also treated for triglyceride content to gain functional efficacy over time. Data represent the average milligrams of triglyceride per gram of treated tissue.

[0282] The relative knockdown data are shown in Tables 8-12, which show the relative knockdown and various doses at days 8, 15, 22, 28, and 42. PNPLA3 knockdown is expressed as a percentage, where negative values ​​indicate a decrease in PNPLA3 levels.

[0283] Table 8. PNPLA3 knockdown assay on day 8

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[0285]

[0286]

[0287] Table 9. Day 15 PNPLA3 knockdown assay

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[0289]

[0290]

[0291]

[0292] Table 10. PNPLA3 knockdown assay on day 22

[0293]

[0294] Table 11. PNPLA3 knockdown assay on day 28

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[0296]

[0297]

[0298] Table 12. PNPLA3 knockdown assay on day 42

[0299]

[0300]

[0301] Example 5: Humanized PNPLA3 rs738409-rs738408 Preventing and rescuing NAFLD with siRNA in a mouse model

[0302] The "American Lifestyle-Induced Obesity Syndrome" or ALIOS mouse model for NAFLD / NASH was developed by feeding mice a diet high in trans fat (45% total fat) and sugar (Tetri 2008). For these studies, eight- to ten-week-old C57BL / 6NCrl male mice (Charles River Laboratories Inc.) were injected with either AAV empty vector or AAV8-PNPLA3 as previously described. rs738409-rs738408 At the time of AAV injection, mice were maintained on normal chow or received ALIOS diet (Envigo, TD.06303) with drinking water consisting of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively) until harvest. rs738409-rs738408 Overexpression of α-glucose in this setting accelerated and exacerbated the NAFLD phenotype (data not shown).

[0303] Two weeks after AAV injection and diet initiation, mice were treated with a single dose of siRNA D-2324 (0.5 mM) or vehicle control at 5.0 mg / kg animal diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) via subcutaneous injection. Dosing was repeated every two weeks until harvest. At harvest, body weights were collected, serum was collected by cardiac puncture under isoflurane anesthesia, and liver weights were obtained. The median lobe was fixed with 10% neutral buffered formalin, then paraffinized and embedded. The remainder of the liver was snap-frozen for content and gene expression analysis as described previously.

[0304] As previously described, quick-frozen liver tissue was processed for RNA and gene expression analysis. Results are shown as raw Ct values ​​and relative mRNA expression of the indicated genes normalized to mouse Gapdh. (TaqMan TM Assay: human PNPLA3, hs00228747_m1; mouse Pnpla3, Mm00504420_m1; mouse Gapdh, 4352932E).

[0305] Formalin-fixed tissues were processed for hematoxylin and eosin staining according to the manufacturer's instructions (Dako, CS70030-2, CS70130-2, respectively).Steatosis and inflammation were scored by a board-certified pathologist.

[0306] 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.

[0307] Figures 2A-2G To evaluate PNPLA3 rs738409-rs738408 The specific siRNA molecule D-2324 prevents the development of NAFLD-related phenotypes and PNPLA3 rs738409-rs738408 To overexpress the ability of AAV8-empty vector (EV) or AAV8-PNPLA3 rs738409-rs738408 or vehicle and maintained a normal diet or transitioned to the ALIOS diet. Two weeks after AAV injection, mice were treated with siRNA or vehicle every other week for six weeks; there were three rounds of injections. Mice were harvested at the eight-week time point. Results are presented as group means and standard errors, N = 8 per group. Asterisks represent AAV8-PNPLA3 rs738409-rs738408Statistical significance of the groups was determined by one-way ANOVA using Dunnett's multiple comparisons test. (A) Ratio of liver weight (g) to body weight (g) 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; PNPLA3 in ALIOS-fed mice rs738409-rs738408 +Vehicle and PNPLA3 rs738409-rs738408 (C) Analysis of mouse Pnpla3 mRNA expression in the liver by qPCR showed that endogenous Pnpla3 was not significantly altered by AAV-mediated overexpression or siRNA silencing. (Left) Raw Ct values ​​and (right) relative fold mRNA expression normalized to mouse Gapdh; PNPLA3 in the chow-fed AAV-free group was compared with that in the ALIOS-fed group. rs738409-rs738408 +Vehicle and PNPLA3 rs738409-rs738408 (D) Liver triglyceride content is expressed as mg triglyceride per gram of liver tissue. Adjusted P values: No AAV + vehicle group, **** < 0.0001, AAV-EV + vehicle group, * = 0.0393, AAV-PNPLA3 rs738409-rs738408 +siRNA, **=0.0063. (E) Serum TIMP1 is expressed as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, ****<0.0001, AAV-EV + vehicle group, ****<0.0001, AAV-PNPLA3 rs738409-rs738408 +siRNA, ****<0.0001. (F) Histological signs of steatosis based on H&E staining, scored as: within normal range (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 range (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.

[0308] Figures 3A-3G To evaluate PNPLA3 rs738409-rs738408 Specific siRNA molecules prevent PNPLA3 from inducing inflammatory cytokines after disease onset. rs738409-rs738408 To determine the ability of mice to further develop induced EV-mediated disease, AAV8-empty vector (EV) or AAV8-PNPLA3 was administered to mice. rs738409-rs738408 or vehicle and maintained a regular diet or transitioned to the ALIOS diet. Eight weeks after AAV injection and dietary changes, mice were treated with siRNA or vehicle every other week for an additional eight weeks; a total of four rounds of injections. Mice were harvested at the sixteen-week time point. Although no changes were observed in steatosis, several other disease-related endpoints were significantly reduced if siRNA treatment was initiated after disease induction. Results are expressed as mean and standard error, N = 8 per group. Asterisks represent AAV8-PNPLA3 rs738409-rs738408 Statistical significance of the groups was determined by one-way ANOVA using Dunnett's multiple comparisons test. (A) Ratio of liver weight (g) to body weight (g) 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; PNPLA3 in ALIOS-fed mice rs738409-rs738408 +Vehicle and PNPLA3 rs738409-rs738408 (C) Analysis of mouse Pnpla3 mRNA expression in the liver by qPCR showed that endogenous Pnpla3 was not significantly altered by AAV-mediated overexpression or siRNA silencing. (Left) Raw Ct values ​​and (right) relative fold mRNA expression normalized to mouse Gapdh; PNPLA3 in the chow-fed AAV-free group was compared with that in the ALIOS-fed group. rs738409-rs738408 +Vehicle and PNPLA3 rs738409-rs738408(D) Liver triglyceride content is expressed as mg triglyceride per gram of liver tissue. Adjusted P values: AAV-EV+vehicle, not significant, AAV-PNPLA3 rs738409-rs738408 +siRNA, *=0.0403. (E) Serum TIMP1 is expressed as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, ****<0.0001, AAV-EV + vehicle group, **=0.0027, AAV-PNPLA3 rs738409-rs738408 +siRNA, **=0.002. (F) Histological signs of steatosis based on H&E staining, scored as: within normal range (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 range (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.

[0309] Figures 4A-4D To evaluate PNPLA3 rs738409-rs738408 Specific siRNA molecules rescue the overexpression of PNPLA3 rs738409-rs738408 The ability to induce disease-relevant phenotypes derived from eight-week AAV8-PNPLA3 fed ALIOS rs738409-rs738408 - Liver and serum from vehicle-treated mice were compared with ALIOS-fed AAV8-PNPLA3 mice treated with vehicle or siRNA for sixteen weeks. rs738409-rs738408 Serum and liver of mice were compared. Although no changes in steatosis were observed with siRNA treatment at this time point, liver triglycerides, serum TIMP1, and inflammation were statistically lower at 16 weeks compared to vehicle controls at 8 weeks. Results are presented as mean ± standard error, N = 8 per group. Asterisks represent the difference between the two groups after 8 weeks of AAV8-PNPLA3 therapy. rs738409-rs738408 Statistical significance of the vehicle-treated group was determined by one-way ANOVA using Dunnett's multiple comparison test. (A) Liver triglyceride content is expressed as mg triglyceride per gram of liver tissue. Adjusted P value: 16WK AAV-PNPLA3 rs738409-rs738408 +Vehicle, not significant; 16WK AAV-PNPLA3 rs738409-rs738408+siRNA, **=0.0011. (B) Serum Timp1 expressed as picograms per milliliter of serum. Adjusted P value: 16WK AAV-PNPLA3 rs738409-rs738408 +Vehicle, not significant; 16WK AAV-PNPLA3 rs738409-rs738408 +siRNA, *=0.0134. (C) Histological signs of steatosis based on H&E staining, scored as: within normal range (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P value: 16WK AAV-PNPLA3 rs738409-rs738408 +Vehicle, not significant; 16WK AAV-PNPLA3 rs738409-rs738408 +siRNA, not significant. (D) Histological signs of inflammation based on H&E staining, scored as: within normal range (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P value: 16WK AAV-PNPLA3 rs738409-rs738408 +Vehicle, not significant; 16WK AAV-PNPLA3 rs738409-rs738408 +siRNA, *=0.0112.

[0310] Example 6: Humanized PNPLA3 rs738409-rs738408 Preventing liver fibrosis with siRNA in a mouse model

[0311] The "AMLN" diet (Clapper 2013), developed by Amylin Pharmaceuticals, is a modified version of the ALIOS diet. The diet includes a tenfold increase in cholesterol (2%) and additional sucrose. Mice receiving the "AMLN" diet develop mild to moderate fibrosis after 20-30 weeks (Clapper, Mells, and Kristiansen paper). For this study, eight to ten-week-old C57BL / 6NCrl male mice (Charles River Laboratories Inc.) were injected with either AAV-empty vector or AAV-PNPLA3 as described above. rs738409-rs738408 At the time of AAV injection, mice continued their normal diet or received Envigo diet TD.170748 with drinking water consisting of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively) until harvest.

[0312] Two weeks after AAV injection and diet initiation, mice were treated subcutaneously with a single dose of siRNA D-2324 (0.5 mM) at 5.0 mg / kg animal diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) or vehicle control. Dosing was repeated every two weeks until harvest. At harvest, body weights were collected, serum was collected by cardiac puncture under isoflurane anesthesia, and liver weights were obtained. The median lobe was fixed with 10% neutral buffered formalin, then paraffinized and embedded. The remainder of the liver was quickly frozen for gene expression analysis.

[0313] As previously described, quick-frozen liver tissue was processed for RNA and gene expression analysis. Results are shown as raw Ct values ​​and relative mRNA expression of the indicated genes normalized to mouse Gapdh. (TaqMan TM Assay: 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).

[0314] Formalin-fixed tissue was processed according to the manufacturer's instructions for use in hematoxylin and eosin and Masson's Trichrome staining (Dako, CS70030-2, CS70130-2, AR17311-2). Anti-smooth muscle actin staining was performed using a DAKO automated stainer without antigen retrieval. 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 hematoxylin staining. Steatosis, inflammation, oval cell / biliary proliferation, and the amount of aSMA-positive cells were scored by a board-certified pathologist.

[0315] 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, cytokeratin 18-M30 has been identified as a potential biomarker for NAFLD / NASH, including early fibrosis (Neuman 2014 and Yang 2015). Figures 5A-5L To evaluate PNPLA3 rs738409-rs738408 The ability of specific siRNA molecules to prevent early fibrosis development in mice receiving AAV8-empty vector (EV) or AAV8-PNPLA3 rs738409-rs738408 Two weeks after AAV injection, mice were treated with siRNA, D-2324, or vehicle every other week for an additional ten weeks; a total of six rounds of injections. Mice were harvested at the ten-week time point. Results are presented as mean and standard error for AAV+vehicle-free diets fed with chow and AAV8-PNPLA3-fed with AMLN. rs738409-rs738408 + vehicle, N=8 per group; AAV8-PNPLA3 fed by AMLN rs738409-rs738408 +Vehicle and AAV8-PNPLA3 rs738409-rs738408 +siRNA, N=12 per group. Asterisks represent AAV8-PNPLA3 rs738409-rs738408 - Statistical significance of vehicle-treated groups by one-way ANOVA with Dunnett's multiple comparison test. (A) Ratio of liver weight (g) to body weight (g) 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 +Vehicle and PNPLA3 rs738409-rs738408 (C) Analysis of mouse Pnpla3 mRNA expression in the liver by qPCR showed that endogenous Pnpla3 was not significantly altered by AAV-mediated overexpression or siRNA silencing. (Left) Raw Ct values ​​and (right) relative fold mRNA expression normalized to mouse Gapdh; PNPLA3 in the chow-fed AAV-free group was compared with that in the AMLN-fed group. rs738409-rs738408 +Vehicle and PNPLA3 rs738409-rs738408(D) Serum Timp1 is expressed as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, **** < 0.0001, AAV-EV + vehicle group, **** < 0.0001, AAV-PNPLA3 rs738409-rs738408 +siRNA, ****<0.0001. (E) Serum CK18m30 expressed as picograms per milliliter of serum. Adjusted P values: No AAV + vehicle group, ****<0.0001, AAV-EV + vehicle group, ****<0.0001, AAV-PNPLA3 rs738409-rs738408 +siRNA, ****<0.0001. (F) Histological signs of inflammation based on H&E staining, scored as: within normal range (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 range (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 of anti-smooth muscle actin, scored as: within normal range (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 of fibrosis, scored as: within normal range (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P values: no AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3 rs738409-rs738408 +siRNA, not significant. (J) Mouse Col1a1 mRNA expression in the liver was obtained by qPCR. Normalized to the relative fold mRNA expression of mouse Gapdh. Adjusted P values: No AAV + vehicle group, **** < 0.0001, AAV-EV + vehicle group, **** < 0.0001, AAV-PNPLA3 rs738409-rs738408+siRNA, ****<0.0001. (K) Mouse Col3a1 mRNA expression in the liver was obtained by qPCR. Normalized to the relative fold mRNA expression of mouse Gapdh. Adjusted P values: No AAV + vehicle group, ****<0.0001, AAV-EV + vehicle group, ****<0.0001, AAV-PNPLA3 rs738409-rs738408 +siRNA, ****<0.0001. (L) Mouse Col4a1 mRNA expression in liver was obtained by qPCR. Normalized to the relative fold mRNA expression of mouse Gapdh. Adjusted P values: No AAV + vehicle group, ****<0.0001, AAV-EV + vehicle group, ***<0.0005, AAV-PNPLA3 rs738409-rs738408 +siRNA, **<0.0041.

[0316] Example 7: Screening PNPLA3 siRNA molecules using a bioluminescent imaging mouse model

[0317] BALB / c male mice (Charles River Laboratories Inc.), typically 10-12 weeks old, were injected with adenovirus of interest (AAV; serotype AAVDJ8; endotoxin-free, produced in-house by Amgen, Inc.) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). AAV was administered to each animal at a dose ranging from 5e11 to 7.5e11 viral particles and injected intravenously into the tail vein. AAV constructs were designed using the murine cytomegalovirus (CMV) promoter and the firefly luciferase reporter gene, with a string of nucleotides at the 3' end containing human PNPLA3. WT (reference allele) or human PNPLA3 rs738409-rs738408 The segments of the (minor allele) siRNA target sequence, as well as other non-SNPs spanning the human PNPLA3 target sequence of interest, are shown in Figures 6 and 7.

[0318] Two weeks after AAV injection, mice were injected with RediJect D-Luciferin (PerkinElmer, 770504) according to the manufacturer's instructions. Bioluminescent signals in mice were captured using the IVIS Spectrum In VivoImaging System (PerkinElmer) and analyzed using Living Image software (PerkinElmer). Mice were then randomly divided into n=5 groups based on the total flux [photons / second] signal in the liver region. After randomization to treatment groups, mice were administered a single dose of siRNA (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). siRNA was administered via subcutaneous injection at the specified mg / kg animal dose. A vehicle-treated control group was included for each round of screening and each AAV type.

[0319] At 1, 2, 3, and 4 weeks after siRNA injection, mice were re-imaged and total flux [p / s] measurements were collected using the same defined target area established for the baseline readings. For each animal, the relative knockdown percentage was determined by calculating the percentage change in total flux at week 1, 2, 3, or 4 relative to the animal's baseline total flux, normalized to the average change in total flux at the vehicle control group baseline at the same time point. For example, the relative knockdown of animals treated with siRNA was calculated as follows: (total flux of animals at week 2) / (total flux of animals at baseline), normalized to the average (total flux of vehicle animals at week 2 / total flux of vehicle animals at baseline). Figure 8 Depicted are representative images of animals injected with AAV expressing either the human PNPLA3 minor or reference allele target sequences before and after siRNA treatment, along with the change in total flux over time.

[0320] Table 13. PNPLA3 knockdown assay

[0321]

[0322]

[0323]

[0324]

[0325] Example 8: PNPLA3 in a Chimeric Humanized Liver Mouse Model rs738409-rs738408 Confirmation of efficacy of selective siRNA molecules

[0326] To evaluate the efficacy of PNPLA3 minor allele-selective siRNA in human hepatocytes in vivo, chimeric humanized hepatic PXB mice from PhoenixBio Co., Ltd (Japan) were used (Miyamoto et al. (2017) Xenobiotica 47(12):1052-1063; Tateno et al. (2015) PLoS One 10(11):e0142145. doi:10.1371 / journal.pone.0142145). Male mice were approximately four months old at the start of the study; at least three months after transplantation. The mice had a human hepatocyte replacement index of 90%-95% as determined by PhoenixBio and were resistant to PNPLA3 based on previous genotyping. rs738409 (Lot BD195) is heterozygous. Upon arrival, mice were placed on ProLab RMH 3000 chow as recommended by PhoenixBio. After a one-week acclimation period, the diet was switched to a high-fat, fructose, NASH-inducing diet (Research Diets, D19021301). After one week on the NASH diet, mice were randomized into groups based on body weight measurements. Mice were treated subcutaneously with a single dose of siRNA (0.5 mM) at 3.0 or 10.0 mg / kg animal diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) or received vehicle alone. Two or four weeks after siRNA injection, livers were collected from animals, snap-frozen in liquid nitrogen, and purified RNA was processed using the QIAcube Automated DNA / RNA Isolation Purification System (Qiagen) and the RNeasy Mini QIAcube Kit (Qiagen, 74116) according to the manufacturer's instructions. TMSamples were analyzed using a 8000 spectrophotometer (Thermo Scientific, ND-8000-GL). RNA was treated with RQ1 RNase-free DNase (Promega, M6101) and prepared for digital droplet PCR (ddPCR) according to the manufacturer's instructions. AccuScript High-Fidelity 1st Strand cDNA Synthesis Kit (Thermo Fisher, 200820) was used for reverse transcription reactions, and ddPCR Supermix for Probes (BioRad, 1863010) was used to assemble the PCR reactions. ddPCR was performed using the AutoDG Droplet Digital PCR System (BioRad, QX200). The following TaqMan TM Assays were purchased from Invitrogen: human PNPLA3 (Hs00228747_m1), human ASGR1 (Hs1005019_m1), mouse Asgr1 (Mm01245581_m1), and human PNPLA3 rs738409 minor / reference allele discrimination assay (C______7241_10). The following assays were purchased from Integrated DNA Technologies Inc.: human TBP (Hs.PT 53a.20105486; primer to probe ratio of 3.6:1), human HPRT1 (Hs.PT.39a.22214821; primer to probe ratio of 3.6:1), and mouse Hprt (Mm.PT.39a.22214828; primer to probe ratio of 3.6:1). Results for human PNPLA3, HPRT, and ASGR1, and mouse Hprt and Asgr1 are expressed as copies / 20 μl reaction, normalized to human TBP. Data for human PNPLA3, human HPRT, and mouse Hprt are also expressed as relative percent knockdown of mRNA expression compared to vehicle-treated control animals.

[0327] For liver triglyceride content analysis, by homogenization in one milliliter of isopropyl alcohol from the approximately 0.05-0.1 milligram of frozen liver of mice.After incubation on ice for one hour, sample is rotated with 10,000rpm in microcentrifuge, and supernatant is transferred in the deep well 96 orifice plates of cleaning.According to manufacturer's specification, use colorimetric Infinity triglyceride reagent (Sai ​​Mo Fei Shi Er science and technology company (Thermo Fisher Scientific), TR22421) and triglyceride standard substance (Pointe science and technology company (Pointe Scientific, T7531-STD) and the SpectraMax Plus microplate reader (molecular device company (Molecular Devices)) with SoftMax Pro6 software to determine triglyceride content.Result is expressed as the triglyceride milligram number of every gram of liver tissue.

[0328] Figure 9 shows an example of siRNA molecule D-2419 demonstrating dose-dependent and allele-selective mRNA knockdown and functional efficacy in vivo. rs738409-rs738408 Heterozygous mice. (A) siRNA molecule D-2419 was subcutaneously injected into the abdomen of mice at 3.0 and 10.0 mg / kg body weight. After two and four weeks of siRNA treatment, mice were sacrificed and livers were collected and processed for analysis. ddPCR and allele-specific Two dye reagents were used to distinguish the minor allele of PNPLA3 from the reference allele. The data showed that PNPLA3 rs738409-rs738408 Dose-dependent and allele-selective knockdown of PNPLA3 WT No measurable changes. N = 5 mice per group; data are presented as mean and standard error of the mean. Two-way ANOVA, ** < 0.001, *** < 0.001, **** < 0.0001, NS = not significant. (B) Data represent human PNPLA3 relative to vehicle-treated controls. rs738409-rs738408 Alleles compared to PNPLA3 WTMean relative mRNA knockdown percentage and standard error of the mean. Values ​​relative to the two-week vehicle control mean are normalized to human TBP. (C) Livers from the two-week treatment groups were processed for triglyceride content to assess functional efficacy. Data represent milligrams of triglyceride per gram of liver. N = 5 mice per group; data are expressed as mean and standard error of the mean. One-way ANOVA, **0.01, NS = not significant. (D) To control for efficient GalNAc-mediated siRNA delivery, D-2787 (an siRNA cross-reactive to human and mouse HPRT and Hprt, respectively) was delivered at 10 mg / kg, and livers were harvested two weeks later. Data represent copies of HPRT mRNA and Hprt mRNA in D-2787-treated mice (N = 4) compared to vehicle-treated mice (N = 5). Data are expressed as mean and standard error of the mean. One-way ANOVA, *0.01. (E) Data represent the mean relative mRNA knockdown percentage of human HPRT and mouse Hprt mRNA relative to vehicle-treated controls and standard error of the mean; all normalized to human TBP. (F) To confirm the role of GalNAc receptors in PXB mice Expression on hepatocytes, mouse Asgr1 mRNA and human ASGR1 mRNA levels were assessed in the absence and presence of D-2419 at two and four weeks after siRNA injection. N = 5 mice per group; data are presented as mean and standard error of the mean.

Claims

1. An RNAi construct selected from the following duplexes: Where A, U, G, and C = the corresponding ribonucleotides; a, u, g, and c = the corresponding 2'-O-methyl ribonucleotides; Af, Uf, Gf, and Cf = the corresponding 2'-deoxy-2'-fluororibonucleotides; Ab = abasic; invAb = inverted abasic; LNA = locked nucleic acid; GalNAc3K2AhxC6 is: And the insertion of an "s" in the sequence indicates that two adjacent nucleotides are linked by a phosphorothioate diester group.

2. A pharmaceutical composition comprising the RNAi construct according to claim 1 and a pharmaceutically acceptable carrier, excipient or diluent.

3. Use of the RNAi construct according to claim 1 in the preparation of a medicament for treating non-alcoholic fatty liver disease or liver fibrosis.

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