EA047656B1 - RNAi CONSTRUCTS DESIGNED TO SUPPRESS PNPLA3 EXPRESSION - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to compositions and methods for modulating the expression of patatin-like phospholipase domain 3 (PNPLA3) in the liver. In particular, the present invention relates to nucleic acid therapeutics for reducing the expression of PNPLA3 via RNA interference and to methods of using such nucleic acid therapeutics to treat or prevent a liver disease such as non-alcoholic fatty liver disease (NAFLD).

Prerequisites for the creation of an invention

As part of a spectrum of liver pathologies, non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, the incidence of which has doubled over the past 20 years and is currently 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 by the presence of cytoplasmic lipid droplets in more than 5% of hepatocytes in an individual 1) with no history of excess 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, causes inflammation and liver cell damage, leading to a later stage of the disease called nonalcoholic steatohepatitis (NASH) (Rinella, supra). As of 2015, 75–100 million Americans are projected to develop NAFLD, with NASH accounting for approximately 10–30% of NAFLD diagnoses (Rinella, supra; Younossi et al. (2016) Hepatology, 64(5):1577–1586).

Patatin-like phospholipase domain 3 (PNPLA3), previously known as adiponutrin (ADPN) and calcium-independent phospholipase A2-epsilon (iPLA(2^), 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). Initially identified in lipocytes as a membrane-associated protein enriched in adipose tissue that is 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 either triacylglycerol lipase activity or transacylating activity (Jenkins et al., above; Kumari et al. (2012), Cell Metab. 15(5):691–702; He et al. (2010), J. Biol. Chem. 285(9):6706–15). In hepatocytes, PNPLA3 is expressed in the endoplasmic reticulum and on lipid membranes and predominantly exhibits triacylglycerol hydrolase activity (He et al., above; 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 detected in human plasma as disulfide-linked multimers (Winberg et al. (2014) Biochem. Biophys. Res. Commun, 446(4):1114-9). Accordingly, a new therapeutic agent that specifically targets PNPLA3 function represents a new approach to reduce PNPLA3 levels and treat liver diseases such as non-alcoholic fatty liver disease.

Brief description of the invention

The present invention is based, in part, on the design and production of RNAi constructs that target the PNPLA3 gene and reduce PNPLA3 expression in liver cells. Inhibition of PNPLA3 expression at a specific sequence is useful for treating or preventing conditions associated with PNPLA3 expression, such as liver diseases such as, for example, fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), cirrhosis (irreversible progressive scarring of the liver), or PNPLA3-associated obesity. Accordingly, 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 that is complementary to a PNPLA3 mRNA sequence. In certain embodiments, the antisense strand comprises a region having at least 15 contiguous nucleotides of the antisense sequence set forth in Table 1 or 2. In some embodiments, the RNAi of the present invention selectively suppresses the minor alleles of PNPLA3-rs738409, PNPLA3-rs738408 and/or PNPLA3-rs738409rs738408 compared to a reference allele that does not contain these changes.

In some embodiments, the sense strand of the RNAi constructs described herein comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length. In these and other embodiments, each sense and

- 1 047 656 antisense strands have a length of from about 15 to about 30 nucleotides. In some embodiments, the RNAi constructs comprise at least one blunt end. In other embodiments, the RNAi constructs comprise 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 and antisense strands. In certain embodiments, the RNAi constructs comprise an overhang of two unpaired nucleotides at the 3' end of the sense strand and at the 3' end of the antisense strand. In other embodiments, RNAi constructs comprise a sticky end of two unpaired nucleotides on the 3' end of the antisense strand and a blunt end on the 3' end of the sense strand/5' end of the antisense strand.

The RNAi constructs of the present invention may comprise one or more modified nucleotides, including nucleotides having modifications to the ribose ring, nucleobase, or phosphodiester backbone. 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), inverted bases (e.g., inverted adenosine), or combinations thereof. In one 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 construct are modified nucleotides.

In some embodiments, the RNAi constructs comprise at least one backbone modification, such as a modified internucleotide or internucleoside linkage. In certain embodiments, the RNAi constructs described herein comprise at least one phosphorothioate internucleotide linkage. In particular embodiments, the phosphorothioate internucleotide linkages can be located at the 3' or 5' ends of the sense and/or antisense strands.

In some embodiments, the antisense strand and/or sense strand of the RNAi constructs of the present invention may comprise or consist of a sequence from the antisense and sense sequences set forth in Table 1 or 2. In certain embodiments, the RNAi constructs may be any of the duplex compounds listed in either Table 1 or 2.

Brief description of graphic materials

Figures 1A–1D show the screening of five siRNA molecules for both dose-dependent mRNA knockdown and functional stability in vivo.

Figures 2A-2G show the effect of siRNA molecules on PNPLA3 in mice in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological evidence of steatosis or inflammation.

Figures 3A-3G show the effect of siRNA molecules on PNPLA3 in vivo, liver weight, confirmation of human PNPLA3 expression, liver triglyceride content, serum TIMP1 levels, and histological evidence of steatosis or inflammation.

Figures 4A-4D show the ability of the PNPLA3 ^{rs738409-rs738408} specific siRNA molecule to rescue disease-associated phenotypes by overexpressing PNPLA3 ^{rs738409-rs738408} , liver triglyceride content, serum TIMP1 levels, and histological evidence of steatosis or inflammation.

Figures 5A-5L show the ability of siRNA molecules specific for PNPLA3 ^{rs738409-rs738408} to prevent the development of early fibrosis.

Figures 6A and 6B show the complete sequence of an AAV-based plasmid containing the target sequences of the minor allele of PNPLA3. The portion containing the mouse CMV promoter, firefly luciferase reporter gene, and target sequences is underlined.

Figures 7A and 7B show the complete sequence of an AAV-based plasmid containing the target sequences of the PNPLA3 reference allele. The portion containing the mouse CMV promoter, firefly luciferase reporter gene, and target sequences is underlined.

Figure 8 shows illustrative images of a mouse injected with AAV expressing target sequences of the human PNPLA3 minor allele (top row) compared to a mouse injected with AAV expressing target sequences of the human PNPLA3 reference allele (bottom row). After baseline images were obtained (first column), the same siRNA molecule was injected into both mice (D-2878 at 3 mg/kg). Columns 2–5 represent images acquired at weeks 1, 2, 3, and 4, respectively. Colors in the images were converted to grayscale using Living Image® software. Lighter areas represent areas of low overall flux intensity [f/s], compared to darker areas with

- 2 047656 high flux [f/s]. Relative percentage of knockdown in mice treated with siRNA at each time point weekly, normalized to vehicle-treated mice, is shown in the inset (rows 2 and 4).

Figure 9 shows an example of an siRNA molecule, D-2419, demonstrating both dose-dependent and allele-selective mRNA knockdown and functional efficacy in vivo.

(A) The siRNA molecule, D-2419, was administered at 3.0 and 10.0 milligrams per kilogram of body weight subcutaneously into the abdomen of mice.

(B) Data represent the mean relative percentage of mRNA knockdown and standard error of the mean for the human PNPLA3 ^{rs738409-rs738408} allele compared to PNPLA3 ^WT , determined in the vehicle-treated control group.

(C) Liver tissues isolated from the two-week treatment group were processed for triglyceride content to assess functional efficacy.

(D) To control for efficient GalNAc-mediated delivery of D-2787 siRNA, siRNA cross-reacting with human and mouse HPRT and Hprt, respectively, was delivered at 10 mg/kg and liver tissues were harvested two weeks later. Data represent HPRT mRNA and Hprt mRNA copies in D-2787-treated mice (N=4) compared to vehicle-treated mice (N=5).

(E) Data represent the mean relative percentage of mRNA knockdown and standard error of the mean for human HPRT and mouse Hprt mRNA, respectively, determined from the vehicle-treated control group; all data are normalized to human TBP.

(F) To confirm GalNAc receptor expression on PXB® mouse hepatocytes, mouse Asgr1 mRNA and human ASGR1 mRNA levels were assessed in the absence and presence of D-2419.

Detailed description of the invention

The present invention is directed to compositions and methods for regulating the expression of a gene encoding patatin-like phospholipase domain-containing protein 3 (PNPLA3). In some embodiments, the gene may be within 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 a cell or mammal. Such RNAi constructs are useful for treating or preventing various forms of liver disease, such as, for example, fatty liver disease (steatosis), nonalcoholic steatohepatitis (NASH), liver cirrhosis (irreversible progressive scarring of the liver), or PNPLA3-associated obesity.

In 2008, genome-wide association studies (GWAS) looking for nonsynonymous sequence variations, or single nucleotide polymorphisms (SNPs), associated with NAFLD identified a PNPLA3 variant (rs738409[G], encoding I148M; referred to as PNPLA3-rs738409, PNPLA3-ma, or the minor allele of PNPLA3) that was significantly associated with liver fat content. Following this initial report, subsequent GWAS confirmed PNPLA3-rs738409 as a major genetic determinant of NAFLD, significantly associated with 1) elevated levels of the serum liver injury biomarker alanine aminotransferase (ALT), 2) the incidence, progression, and severity of NAFLD, 3) detectable in both obese and lean individuals, and 4) with the only known SNP found to be significantly associated with all stages of NAFLD: steatosis, NASH, cirrhosis, and hepatocellular carcinoma. Consensus among multiple GWAS indicates that the association of PNPLA3 rs738409 with NAFLD is independent of age, sex, ethnicity, metabolic syndrome, body mass index, insulin resistance, and serum lipids. In addition, according to statistical analyses from several sources, approximately 50% of patients with NAFLD are carriers of the PNPLA3 rs738409 mutation. Patients can be carriers of either homozygous or heterozygous PNPLA3 rs738409 mutation. Furthermore, it has been found that patients carrying the PNPLA3 rs738409 mutation also frequently carry a mutation three base pairs away from rs738408 (Tian et al. (2010) Nature Genetics, 42:21-23). Thus, a patient can have a minor allele of PNPLA3-rs738409, a minor allele of PNPLA3-rs738408, or a mutation in two minor alleles (PNPLA3-dma) of PNPLA3-rs738409-rs738408.

The researchers developed mouse models to study PNPLA3 function in vivo. To date, no detectable metabolic phenotype has been identified associated with Pnpla3 deficiency or Pnpla3 overexpression. In contrast, Pnpla3 ^I148M expression in both transgenic and knockin mice resulted in elevated hepatic triglyceride levels similar to that observed in NAFLD. Thus, taken together, the in vivo mouse model data implicate expression of the mutant form of the Pnpla3 ^I148M protein, rather than overexpression of the wild-type protein, as a contributing factor to the disease phenotype. These observations, in addition to the high frequency of minor alleles in individuals with NAFLD and the predominant association with the disease, highlight the importance of PNPLA3-rs738409 as a major therapeutic target in NAFLD.

- 3 047656

RNA interference (RNAi) is the process of introducing exogenous RNA into a cell, which results in specific degradation of the mRNA encoding the target protein, followed by downregulation of the protein expression. Advances in both RNAi technology and liver delivery, as well as the increasing positive results with RNAi-based therapeutics, suggest that RNAi represents a compelling therapeutic option for targeting NAFLD by directly targeting PNPLA3I148M. Multiple GWAS indicate a dose-response effect of PNPLA3 rs738409 on NAFLD incidence, progression, and severity (GWAS); a trend toward a doubling, if not greater, increase in the odds ratio was observed for carriers of the homozygous genotype compared to carriers of the heterozygous genotype, but the odds ratio was at least twice as great for individuals with the heterozygous genotype compared to individuals with the wild-type allele. Thus, silencing of PNPLA3 using allele recognition specificity may both be a potential means of reducing hepatic triglyceride levels in PNPLA3I148M carriers and represent an option in which heterozygous carriers can benefit without silencing the wild-type allele. Accordingly, the present inventors identified short interfering RNAs (siRNAs) specific for the PNPLA3I148M SNP and demonstrated proof of concept in vitro. Using both 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 suppressing the expression of the PNPLA3I148M gene. The inhibitory effect of these sequences was confirmed by screening in Chinese hamster ovary (CHO) cells overexpressing either PNPLA3I148I or PNPLA3I148M. Using adeno-associated virus (AAV) to achieve in vivo overexpression of human PNPLA3I148M, the present inventors then showed that treatment with minor allele-specific SNPs not only caused a specific reduction in human PNPLA3I148M expression in mice, but also significantly reversed the accumulation of hepatic triglycerides induced by human PNPLA3I148M overexpression.

As used herein, the term "RNAi construct" refers to an agent comprising an RNA molecule that, when introduced into a cell, is capable of reducing the expression of a target gene (e.g., PNPLA3) via an RNA interference mechanism. RNA interference is a process by which a nucleic acid molecule causes the cleavage and degradation of a target RNA molecule (e.g., messenger RNA or mRNA) in a sequence-specific manner, such as 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 adjacent nucleotides that are sufficiently complementary to each other to hybridize to form a duplex region. The terms hybridize or hybridization refer to the pairing of complementary polynucleotides, usually via hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds) between complementary bases in the two polynucleotides. The strand containing the region having a sequence that is essentially complementary to the target sequence (e.g., the target mRNA) is called the antisense strand.

The term sense strand refers to a strand that includes a region that is substantially complementary to a region of the antisense strand. In some embodiments, the sense strand may include a region that has a sequence that is substantially identical to a target sequence.

In some embodiments, the invention provides an RNAi agent that targets PNPLA3. In some embodiments, the invention provides an RNAi agent that binds to the rs738409 site of PNPLA3. In some embodiments, the invention provides an RNAi agent that binds to the rs738408 site of PNPLA3. In some embodiments, the invention provides an RNAi agent that binds to both the rs738409 site and the rs738408 site of PNPLA3. In some embodiments, the invention provides an RNAi agent that preferentially binds to rs738409 of PNPLA3 over the native sequence of PNPLA3 (PNPLA3-ref). In some embodiments, the invention provides an RNAi agent that preferentially binds to rs738408 of PNPLA3 over the PNPLA3-ref sequence. In some embodiments, the present invention provides an RNAi agent that preferentially binds to PNPLA3-dma over PNPLA3-ma. In some embodiments, the present invention provides an RNAi molecule that comprises any of the sequences listed in Table 1 or 2.

The double-stranded RNA molecule may include chemical modifications of the ribonucleotides, including modifications of the ribose sugar, base, or backbone components of the ribonucleotides, such as those described herein or known in the art. Any such modifications that are used in a double-stranded RNA molecule (e.g., siRNA, shRNA, or

- 4 047656 and the like), are encompassed by the term double-stranded RNA for the purposes of the present invention.

According to the terminology used herein, a first sequence is complementary to a second sequence if a polynucleotide comprising the first sequence can hybridize to a polynucleotide comprising the second sequence to form a duplex region under certain conditions, such as physiological conditions. Other such conditions may include moderate or stringent hybridization conditions, which are known to those skilled in the art. A first sequence is considered to be fully complementary (100% complementary) to a second sequence if a polynucleotide comprising the first base sequence binds to a polynucleotide comprising the second sequence along the entire length of one or both nucleotide sequences without any mismatched bases. 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 may be calculated by dividing the number of bases in the first sequence that are complementary to bases at corresponding positions in the second or target sequence by the total length of the first sequence. A sequence may also be said to be substantially complementary to another sequence when the two sequences hybridize to form no more than 5, 4, 3, 2, or 1 mismatch within a 30 base pair duplex region. Generally, if any nucleotide overhangs are present as defined herein, the sequence of such overhangs is not taken into account in determining the degree of complementarity between two sequences. For example, a 21 nucleotide long sense strand and a 21 nucleotide long antisense strand that hybridize to form a 19 base pair duplex region with 2 nucleotide overhangs at the 3' end of each strand would be considered to be fully complementary within the meaning of the term as used herein.

In some embodiments, a region of the antisense strand comprises a sequence that is completely 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 completely complementary to a sequence of the antisense strand. In other such embodiments, the sense strand may comprise a sequence that is substantially complementary to a sequence of the antisense strand, such as having 1, 2, 3, 4, or 5 mismatches in a duplex region formed by the sense and antisense strands. In certain embodiments, it is preferred that any mismatches be in 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 mismatched bases in a duplex region formed from the sense and antisense strands are within 6, 5, 4, 3, 2, or 1 nucleotides of the 5' end of the antisense strand.

In certain embodiments, the sense strand and the antisense strand of a double-stranded RNA may be two separate molecules that hybridize to form a duplex region but are otherwise unrelated. Such double-stranded RNA molecules formed by two separate strands are called small interfering RNAs or short interfering RNAs (siRNAs). Thus, in some embodiments, the RNAi constructs of the present invention comprise siRNA.

If two essentially complementary strands of dsRNA are formed by separate RNA molecules, the molecules need not be, but may be, covalently linked. Where two strands are covalently joined by other means than by forming a continuous chain of nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand, forming a duplex structure, the connecting structure is called a linker. The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs in a duplex is equal to the number of nucleotides in the shortest strand of dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, RNAi may contain one or more nucleotide overhangs.

In other embodiments, the sense strand and the antisense strand that hybridize to form a duplex region may be part of a single RNA molecule, i.e., the sense and antisense strands may be part of a self-complementary region of a single RNA molecule. In such cases, the single RNA molecule comprises a duplex region (also referred to as a stem region) and a loop region. The 3' end of the sense strand is connected to the 5' end of the antisense strand by a continuous sequence of unpaired nucleotides that will form a loop region. The loop region is typically long enough to allow the RNA molecule to fold back so that the antisense strand can pair with the sense strand to form a duplex or stem region. The loop region may comprise 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 partially self-complementary regions are referred to as short hairpin RNAs (shRNAs). In some embodiments, the loop region may comprise

- 5,047,656 at least 1, 2, 3, 4, 5, 10, 20, or 25 unpaired nucleotides. In some embodiments, the loop region may comprise 10, 9, 8, 7, 6, 5, 4, 3, 2, or less unpaired nucleotides. In certain embodiments, the RNAi constructs of the present invention comprise shRNA. The length of one at least partially self-complementary RNA molecule may be from about 35 to about 100 nucleotides, from about 45 to about 85 nucleotides, or from about 50 to about 60 nucleotides and comprise a duplex region and a loop region, each of which has a length as specified herein.

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, the term 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). PNPLA 3 protein is also known as adiponutrin (ADPN) and calcium-independent phospholipase A2-epsilon (iPLA(2^)).

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

The region of the antisense strand may be substantially complementary or fully complementary to at least 15 consecutive nucleotides of the PNPLA3 mRNA sequence. In some embodiments, the target region of the PNPLA3 mRNA sequence whose antisense strand comprises the complementary region may be 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, the portion of the antisense strand comprising a sequence that is substantially or completely complementary to a sequence of the PNPLA3 mRNA may, in some embodiments, comprise at least 15 contiguous nucleotides of the antisense sequence shown in Table 1 or 2. In other embodiments, the antisense sequence comprises at least 16, at least 17, at least 18, or at least 19 contiguous nucleotides of the antisense sequence shown in Table 1 or 2. In some embodiments, the sense and/or antisense sequence comprises at least 15 nucleotides of the sequence shown in Table 1 or 2, with no more than 1, 2, or 3 mismatched nucleotides.

The sense strand of an RNAi construct typically contains a sequence that is so complementary to the sequence of the antisense strand that the two strands hybridize under physiological conditions to form a duplex region.

The term "duplex region" refers to a region in two complementary or substantially complementary polynucleotides that base pair with each other either by Watson-Crick base pairing or other hydrogen bonding interactions, creating a duplex between the two polynucleotides. The duplex region of an RNAi construct should be of sufficient length to allow the RNAi construct to integrate into the RNA interference machinery, such as by interacting with the Dicer enzyme and/or the RISC complex. For example, in some embodiments, the duplex region is from about 15 to about 30 base pairs in length. Other values of the length of the duplex region within this range are also suitable, such as from about 15 to about 28 base pairs, from about 15 to about 26 base pairs, from about 15 to about 24 base pairs, from about 15 to about 22 base pairs, from about 17 to about 28 base pairs, from about 17 to about 26 base pairs, from about 17 to about 24 base pairs, from about 17 to about 23 base pairs, from about 17 to about 21 base pairs, from about 19 to about 25 base pairs, from about 19 to about 23 base pairs or

- 6,047,656 from about 19 to about 21 base pairs. In one embodiment, the duplex region is from about 17 to about 24 base pairs in length. In another embodiment, the duplex region is from about 19 to about 21 base pairs in length.

In some embodiments, the RNAi agent of the present invention comprises a duplex region of about 24 to about 30 nucleotides that interacts with a target RNA sequence, such as a target sequence of PNPLA3 mRNA, to direct cleavage of the target RNA. Without being limited by theory, the present inventors recall that long double-stranded RNA introduced into cells can be cleaved into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, 15 processes dsRNA to form short interfering RNAs of 19-23 bp in length with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature, 409:363). The siRNAs are then incorporated into the RNA-induced silencing complex (RISC), in which 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 mRNA, one or more endonucleases in the RISC complex cleave the target, inducing silencing (Elbashir, et al., (2001), Genes Dev. 15:188).

For embodiments in which the sense strand and the antisense strand are two separate molecules (e.g., when the RNAi construct comprises siRNA), the sense strand and the antisense strand need not be the same length as the duplex region. For example, one or both strands may be longer than the duplex region and contain one or more unpaired nucleotides or mismatched nucleotides flanking the duplex region. Thus, in some embodiments, the RNAi construct comprises at least one nucleotide overhang. As used herein, the term nucleotide overhang refers to an unpaired nucleotide or nucleotides that extend beyond the duplex region at the terminal ends of the strands. Nucleotide overhangs are typically formed when the 3' end of one strand extends beyond the 5' end of the other strand, or when the 5' end of one strand extends beyond the 3' end of the other strand. The length of a nucleotide overhang is typically from 1 to 6 nucleotides, from 1 to 5 nucleotides, from 1 to 4 nucleotides, from 1 to 3 nucleotides, from 2 to 6 nucleotides, from 2 to 5 nucleotides, or from 2 to 4 nucleotides. In some embodiments, the nucleotide overhang comprises 1, 2, 3, 4, 5, or 6 nucleotides. In one particular embodiment, the nucleotide overhang comprises 1 to 4 nucleotides. In certain embodiments, the nucleotide overhang comprises 2 nucleotides. The nucleotides in the overhang may be ribonucleotides, deoxyribonucleotides, or modified nucleotides as described herein. In some embodiments, the overhang comprises a dinucleotide that is 5'-uridine-uridine-3' (5'-UU-3'). In such embodiments, the UU dinucleotide may comprise ribonucleotides or modified nucleotides, such as 2'-modified nucleotides. In other embodiments, the overhang comprises a dinucleotide that is 5'-deoxythymidine-deoxythymidine-3' (5'-dTdT-3').

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

RNAi constructs may comprise a single nucleotide overhang at one end of a double-stranded RNA molecule and a blunt end at the other. The term blunt end means that the sense strand and the antisense strand are completely base-paired at the end of the molecule and that there are no unpaired nucleotides that protrude beyond the duplex region. In some embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the sense strand and a blunt end at the 5' end of the sense strand and at the 3' end of the antisense strand. In other embodiments, an RNAi construct comprises a nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand and at the 3' end of the sense strand. In certain embodiments, an 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 are the same length, and the duplex region is the same length as the sense and antisense strands (i.e., the molecule is double-stranded along its entire length).

The length of each sense strand and antisense strand can independently be from about 15 to about 30 nucleotides, from about 18 to about

- 7,047,656 nucleotides, from about 19 to about 27 nucleotides, from about 19 to about 25 nucleotides, from about 19 to about 23 nucleotides, from about 21 to about 25 nucleotides, or from about 21 to about 23 nucleotides. In certain embodiments, the length of each sense strand and antisense strand is about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides. In some embodiments, the sense strand and antisense strand are the same length but form a duplex region that is shorter than these strands, such that the RNAi construct comprises two nucleotide ends. For example, in one embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand each being 21 nucleotides in length; (ii) a duplex region that is 19 base pairs in length; and (iii) nucleotide overhangs of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the RNAi construct comprises (i) a sense strand and an antisense strand that are each 23 nucleotides in length; (ii) a duplex region that is 21 base pairs in length; and (iii) nucleotide overhangs of two unpaired nucleotides at both the 3' end of the sense strand and the 3' end of the antisense strand. In other embodiments, the sense strand and antisense strand are the same length and form a duplex region along 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 which is 21 nucleotides in length; and (ii) a duplex region having a length of 21 base pairs. In another such embodiment, the RNAi construct is blunt-ended and comprises (i) a sense strand and an antisense strand, each of which is 23 nucleotides in length; and (ii) a duplex region having a length of 23 base pairs.

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

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

Modified nucleotides.

The RNAi constructs of the present invention may comprise one or more modified nucleotides. The term modified nucleotide refers to a nucleotide that has one or more chemical modifications to a nucleoside, a nucleobase, a pentose ring, or a phosphate group. As used herein, modified nucleotides do not include 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 constructs may comprise combinations of modified nucleotides, ribonucleotides, and deoxyribonucleotides. The incorporation of modified nucleotides into one or both strands of double-stranded RNA molecules can improve the stability of RNA molecules in vivo, for example by reducing the susceptibility of the molecules to nucleases and other degradation processes. The efficiency of RNAi constructs in reducing target gene expression can also be increased by incorporating modified nucleotides.

In certain embodiments, the modified nucleotides have a modification of the ribose sugar. These sugar modifications can include modifications at the 2' and/or 5' position of the pentose ring, as well as modifications of the bicyclic sugar. The term "N'-modified nucleotide" refers to a nucleotide having a pentose ring with a substituent at the 2' position other than H or OH. Such 2'-modifications include, but are not limited to, 2'-O-alkyl (e.g., O-C1-C10 or O-Ci-Cio substituted alkyl), 2'-O-allyl (O- _CH2CH = _Ch2 ), 2'-C-allyl, 2'-fluoro, 2'-0-methyl (OCH), 2'-O-methoxyethyl (O-( _CH2 ) _2OCH ), ₃ , 2'-O(CH ₂ ) ₂ SCH, 2'-O-aminoαalkyl, 2'-amino (for example, NH ₂ ),

- 8 047656

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.

The term bicyclic sugar modification refers to a modification of a pentose ring in which a bridge connects two atoms of the ring to form a second ring, resulting in a bicyclic sugar structure. In some embodiments, the bicyclic sugar modification comprises a bridge between the carbon atoms of the pentose ring at the 4' and 2' positions. Nucleotides comprising a sugar moiety with a bicyclic sugar modification are used herein as the term bicyclic nucleic acids or BNAs. Exemplary bicyclic sugar modifications include, but are not limited to, aL-methyleneoxy (4'-CH ₂ -O-2') bicyclic nucleic acid (BNA); eD-methyleneoxy (4'-CH ₂ -O-2') BNA (also referred to as a locked nucleic acid or LNA); ethyleneoxy (4'-(CH ₂ ) ₂ -O-2') BNA; aminooxy (4'-CH ₂ -ON(R)-2') BNA; oxyamino (4'-CH2-N(R)-O-2') BNA; methyl (methyleneoxy) (4'-CH(CH)-O-2') BNA (also called ethyl constrained or cEt); methylene-thio (4'-CH ₂ -S-2') BNA; methylene-amino (4'-CH2-N(R)-2') BNA; methylcarbocyclic (4'-CH2-CH(CH)-2') BNA; propylenecarbocyclic (4'-(CH ₂ ) ₃ -2') BNA and methoxy (ethyleneoxy) (4'-CH(CH ₂ OMe)-O-2') BNA (also called conformationally 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 incorporated herein by reference in their entireties.

In some embodiments, the RNAi constructs comprise 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 constructs comprise one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, or combinations thereof. In one specific embodiment, the RNAi constructs comprise one or more 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, or combinations thereof.

Both the sense and antisense strands of the RNAi constructs 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 some 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 combinations thereof.

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

In some embodiments in which 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 combinations thereof.

The RNAi constructs of the present invention may also comprise one or more

- 9,047,656 modified internucleotide linkages. As used herein, the term modified internucleotide linkage refers to an internucleotide linkage other than a natural 3'-5' phosphodiester linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing internucleotide linkage such as a phosphotriester, an aminoalkylphosphotriester, an alkylphosphonate (e.g., methylphosphonate, 3'-alkylenephosphonate), a phosphinate, a phosphoramidate (e.g., 3'-aminophosphoramidate and aminoalkylphosphoramidate), a phosphorothioate (P=S), a chiralphosphorothioate, a phosphorodithioate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, and a boranophosphate. In one embodiment, the modified internucleotide linkage is a 2'-5' phosphodiester linkage. In other embodiments, the modified internucleotide linkage is a non-phosphorus internucleotide linkage and thus may be referred to as a modified internucleoside linkage. Such non-phosphorus-containing linkages include, but are not limited to, morpholine linkages (formed in part from the sugar moiety of a nucleoside); siloxane linkages (-O-Si(H) ₂ -O-); sulfide, sulfoxide, and sulfone linkages; formacetyl and thioformacetyl linkages; alkene-containing backbones; sulfamate backbones; methylenemethylimine ( _-CH2 -N(CH)-O- _CH2- ) and methylenehydrazine linkages; sulfonate and sulfonamide linkages; amide linkages and others having mixed moieties containing N, O, S, and CH2. In one embodiment, the modified internucleoside linkage is a peptide linkage (e.g., aminoethylglycine) to create a peptide nucleic acid or PNA, 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 construct of the present invention are described in U.S. Patent Nos. 6,693,187, 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 herein incorporated by reference in their entireties.

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

In some embodiments in which the RNAi construct comprises a nucleotide overhang, two or more unpaired nucleotides within the overhang may be joined at

- 10 047656 by means of a phosphorothioate internucleotide linkage. In certain embodiments, all unpaired nucleotides in the nucleotide overhang at the 3' end of the antisense strand and/or the sense strand are linked by phosphorothioate internucleotide linkages. In other embodiments, all unpaired nucleotides in the nucleotide overhang at the 5' end of the antisense strand and/or the sense strand are linked by phosphorothioate internucleotide linkages. In yet further embodiments, all unpaired nucleotides in either nucleotide overhang are linked by phosphorothioate internucleotide linkages.

In certain embodiments, modified nucleotides incorporated into one or both strands of an RNAi construct of the present invention have a modification of the nucleobase (also referred to herein as a base). The term modified nucleobase or modified base refers to a base other than the naturally occurring purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). The modified nucleobases may be synthetic or naturally occurring modifications and include, but are 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-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, 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, 3-deazaguanine and 3-deazaadenine.

In some embodiments, the modified base is a universal base. The term universal base refers to a base analog that non-selectively base pairs with all natural bases in RNA and DNA without altering the double helical structure of the resulting duplex region. Universal bases are known to those skilled in the art and include, but are not limited to, inosine, C-phenyl, C-naphthyl and other aromatic derivatives, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole.

Other suitable modified bases that can be incorporated into the RNAi constructs of the present invention include those described in Herdewijn, Antisense Nucleic Acid Drug Dev., Vol. 10, 297-310, 2000 and Peacock et al., J. Org. Chern., Vol. 76, 7295-7300, 2011, all of which are incorporated herein by reference in their entireties. It is well known to one of skill in the art that guanine, cytosine, adenine, thymine, and uracil can be replaced with other nucleobases, such as the modified nucleobases described above, without substantially altering the base pairing properties of a polynucleotide comprising a nucleotide with such a substituted nucleobase.

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

Modified nucleotides that can be incorporated into the RNAi constructs of the present invention can have more than one chemical modification described herein. For example, a modified nucleotide can have a modification of the ribose sugar as well as a modification of the nucleobase. As an example, a modified nucleotide can comprise a 2' sugar modification (e.g., 2'-fluoro and 2'-methyl) and comprise a modified base (e.g., 5-methylcytosine or pseudouracil). In other embodiments, a modified nucleotide can comprise a sugar modification in combination with a 5'-phosphate modification, wherein when the modified nucleotide is incorporated into a polynucleotide, a modified internucleotide or internucleoside linkage is formed. For example, in some embodiments, a modified nucleotide can comprise a sugar modification, such as a 2'-fluoro modification, a 2'-O-methyl modification, or a bicyclic sugar modification, and a 5'-phosphorothioate group. Accordingly, in some embodiments, one or both strands of the RNAi construct are

- 11 047 656 of the present invention comprise a combination of 2'-modified nucleotides or BNAs and phosphorothioate internucleotide linkages. In certain embodiments, both the sense and antisense strands of an RNAi construct of the present invention comprise a combination of 2'-fluoro-modified nucleotides, 2'-O-methyl-modified nucleotides, and phosphorothioate internucleotide linkages. Exemplary RNAi constructs comprising modified nucleotides and internucleotide linkages are shown in Table 2.

Function of the construct for RNAi.

Preferably, the RNAi constructs of the present invention reduce or suppress the expression of PNPLA3 in cells, particularly liver cells. Accordingly, in one embodiment, the present invention provides a method of reducing the expression of PNPLA3 i in a cell by contacting the cell with any of the RNAi constructs described herein. The cell may be in vitro or in vivo. The expression of PNPLA3 can be assessed by measuring the amount or level of PNPLA3 mRNA, PNPLA3 protein, or another biomarker associated with the expression of PNPLA3. A reduction in the expression of PNPLA3 in cells or animals treated with an RNAi construct of the present invention can be determined in comparison to the expression of PNPLA3 in cells or animals not treated with the RNAi construct or treated with a control RNAi construct. For example, in some embodiments, a decrease in PNPLA3 expression is assessed by (a) measuring the amount or level of PNPLA3 mRNA in liver cells treated with an RNAi construct of the invention; (b) measuring the amount or level of PNPLA3 mRNA in liver cells treated with a control RNAi construct (e.g., an RNAi agent targeted to an RNA molecule not expressed in liver cells, or an RNAi construct having a nonsense or scrambled sequence) or treated without the construct; and (c) comparing the measured PNPLA3 mRNA levels in the treated cells of (a) with the measured PNPLA3 mRNA levels in the control cells of (b). The PNPLA3 mRNA levels in the treated cells and in the control cells can be normalized relative to the RNA levels of a control gene (e.g., 18S ribosomal RNA) prior to comparison. PNPLA3 mRNA levels can be measured using a variety of techniques including Northern blot, nuclease protection assay, in situ hybridization (FISH), reverse transcription-PCR ((RT)-PCR), real-time PCR (RT-PCR), quantitative PCR, and the like.

In other embodiments, the reduction in PNPLA3 expression is assessed by (a) measuring the amount or level of PNPLA3 protein in liver cells treated with an RNAi construct of the invention; (b) measuring the amount or level of PNPLA3 protein in liver cells treated with a control RNAi construct (e.g., an RNAi agent directed to an RNA molecule not expressed in liver cells, or an RNAi construct having a nonsense or scrambled sequence) or treated without the construct; and (c) comparing the measured PNPLA3 protein levels in the treated cells of (a) with the measured PNPLA3 protein levels in the control cells of (b). Techniques for measuring PNPLA3 protein levels are known to those of skill in the art and include Western blotting, immunoassays (e.g., ELISA), and flow cytometry. An exemplary immunoassay-based assay for assessing PNPLA3 protein expression is described in Example 2. An exemplary assay for measuring PNPLA3 mRNA using RNA FISH is described in Example 3. Any assay capable of measuring PNPLA3 mRNA or protein can be used to assess the efficacy of the RNAi construct of the present invention.

In some embodiments, the methods for assessing PNPLA3 expression levels are performed in vitro in cells that natively express PNPLA3 (e.g., liver cells) or in cells that have been engineered to express PNPLA3. In certain embodiments, the methods are performed in vitro in liver cells. Suitable liver cells 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 liver cells are Hep3B cells. In another embodiment, the liver cells are HepG2 cells.

In other embodiments, the methods for assessing PNPLA3 expression levels are performed in vivo. The RNAi constructs and any control RNAi constructs can be administered to an animal (e.g., a rodent or non-human primate) and the levels of PNPLA3 mRNA or protein can be assessed in a liver tissue sample collected from the animal after treatment. Alternatively or additionally, a biomarker or functional phenotype associated with PNPLA3 expression can be assessed in animals treated with the agent.

In certain embodiments, PNPLA3 expression is reduced in liver cells by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% due to exposure to an RNAi construct of the invention. In some embodiments, PNPLA3 expression is reduced in liver cells by at least 60%, at least 65%, at least 70%, at least 75%, at least

- 12 047656

80% or at least 85% due to exposure to an RNAi construct of the invention. In other embodiments, the expression of PNPLA3 is reduced in liver cells by about 90% or more, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% or more due to exposure to an RNAi construct of the invention. The percentage reduction in PNPLA3 expression can be measured using 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 suppress the expression of PNPLA3 by at least 45% at a concentration of 5 nM in Hep3B cells (containing wild-type PNPLA3) in vitro. In related embodiments, RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% at a concentration of 5 nM in Hep3B cells in vitro. In other embodiments, RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% at a concentration of 5 nM in Hep3B cells in vitro. In certain embodiments, RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 45% at a concentration of 5 nM in HepG2 cells (containing two minor alleles of PNPLA3-rs738409-rs738408) in vitro. In related embodiments, RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% at a concentration of 5 nM in HepG2 cells in vitro. In other embodiments, the RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% at a concentration of 5 nM in HepG2 cells in vitro. In certain embodiments, the RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 45% at a concentration of 5 nM in transfected CHO cells expressing human PNPLA3 I148I or I148M in vitro. In related embodiments, RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% at a concentration of 5 nM in transfected CHO cells expressing human PNPLA3 I148I or I148M in vitro. In other embodiments, RNAi constructs of the present invention suppress the expression of PNPLA3 by at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% at a concentration of 5 nM in transfected CHO cells expressing human PNPLA3 I148I or I148M in vitro. PNPLA3 reduction can be measured using a variety of techniques, including RNA FISH or droplet digital PCR, as described in Examples 2 and 3.

In some embodiments, an _IC50 value is calculated to assess the ability of an RNAi construct of the present invention to suppress PNPLA3 expression in liver cells. The term _IC50 value means the dose/concentration required to achieve 50% suppression of a biological or biochemical function. The _IC50 value for any particular substance or antagonist can be determined by constructing a dose-response curve and examining the effect of different concentrations of the substance or antagonist on the expression levels or functional activity measured in any assay. _IC50 values can be calculated for a given antagonist or substance by determining the concentration required to suppress half the maximal biological response or half the native expression levels. Thus, the IC50 value for any RNAi construct can be calculated by determining the concentration of the RNAi construct required to suppress half the level of native expression of PNPLA3 in liver cells (e.g., the level of expression of PNPLA3 in control liver cells) in any assay, such as an immunoassay, or an RNA FISH assay, or a droplet digital PCR assay, which are described in the examples. The RNAi constructs of the present invention can suppress the expression of PNPLA3 in liver cells (e.g., Hep3B cells) with an _IC50 value of less than about 20 nM. For example, the RNAi constructs downregulate PNPLA3 expression in liver cells with an _IC50 of about 0.001 to about 20 nM, about 0.001 to about 10 nM, about 0.001 to about 5 nM, about 0.001 to about 1 nM, about 0.1 to about 10 nM, about 0.1 to about 5 nM, or about 0.1 to about 1 nM. In certain embodiments, the RNAi construct downregulates PNPLA3 expression in liver cells (e.g., Hep3B cells) with an IC50 of about 1 to about 10 nM. The RNAi constructs of the present invention can downregulate PNPLA3 expression in liver cells (e.g., HepG2 cells) with an _IC50 of less than about 20 nM. For example, RNAi constructs suppress PNPLA3 expression in liver cells with an IC ₅₀ value of

- 13 047656 from about 0.001 to about 20 nM, from about 0.001 to about 10 nM, from about 0.001 to about 5 nM, from about 0.001 to about 1 nM, from about 0.1 to about 10 nM, from about 0.1 to about 5 nM, or from about 0.1 to about 1 nM. In certain embodiments, the RNAi construct suppresses PNPLA3 expression in liver cells (e.g., HepG2 cells) with an IC ₅₀ of about 1 to about 10 nM. The RNAi constructs of the present invention can downregulate PNPLA3 expression in liver cells (e.g., transfected CHO cells expressing human PNPLA3 I148I or I148M) with an _IC50 of less than about 20 nM. For example, the RNAi constructs downregulate PNPLA3 expression in liver cells with an IC50 of from about 0.001 to about 20 nM, from about 0.001 to about 10 nM, from about 0.001 to about 5 nM, from about 0.001 to about 1 nM, from about 0.1 to about 10 nM, from about 0.1 to about 5 nM, or from about 0 to about 1 nM. In certain embodiments, the RNAi construct suppresses PNPLA3 expression in liver cells (e.g., transfected CHO cells expressing human PNPLA3 I148I or I148M) with an IC ₅₀ of about 1 to about 10 nM.

The RNAi constructs of the present invention can be readily prepared using techniques known in the art, such as using conventional solid-phase nucleic acid synthesis. The polynucleotides of the RNAi construct can be assembled on a suitable nucleic acid synthesizer using standard nucleotide or nucleoside precursors (e.g., phosphoramidites). Automated nucleic acid synthesizers are commercially available from several suppliers, including DNA/RNA synthesizers from Applied Biosystems (Foster City, Calif.), MerMade synthesizers from BioAutomation (Irving, Texas), and OligoPilot synthesizers from GE Healthcare Life Sciences (Pittsburgh, Pa.).

For the synthesis of oligonucleotides via phosphoramidite chemistry, a 2'-silyl protecting group can be used in combination with acid-labile dimethoxytrityl (DMT) at the 5' position of ribonucleosides. The final deprotection conditions are known to result in no significant degradation of the RNA products. All synthesis processes can be performed in any automated or manual synthesizer in large, medium or small scale. Synthesis can also be performed in multiwell plates, columns or glass slides.

The 2'-O-silyl group can be removed by the action of fluoride ions, which can be any source of fluoride ion, for example, those salts which contain fluoride ion in combination with inorganic counterions, for example, cesium fluoride and potassium fluoride, or those salts which contain fluoride ion paired with an organic counterion, for example, tetraalkylammonium fluoride. A crown ether catalyst in combination with an inorganic fluoride can be used in the deprotection reaction. Preferred sources of fluoride ion are tetrabutylammonium fluoride or aminohydrofluorides (for example, combining aqueous HF with triethylamine in a dipolar aprotic solvent, for example, dimethylformamide).

The choice of protecting groups for use on phosphite triesters and phosphotriesters can alter the stability of the triesters toward fluorine. Methyl protection of a phosphotriester or phosphitetriester can stabilize the bond to fluorine ions and improve process yield.

Since ribonucleosides have a reactive 2'-hydroxyl substituent, it may be desirable to protect the reactive 2' position in RNA with a protecting group that is orthogonal to the 5'-O-dimethoxytrityl protecting group, such as one that is stable to acid treatment. Silyl protecting groups meet this criterion and can be easily removed in the final fluorine deprotection step, which may result in minimal degradation of the RNA.

In the standard phosphoramidite coupling reaction, tetrazole catalysts can be used. Preferred catalysts include, for example, tetrazole, S-ethyltetrazole, benzylthiotetrazole, nitrophenyltetrazole.

One skilled in the art will appreciate that additional methods for synthesizing the RNAi constructs described herein will be apparent to those skilled in the art. Furthermore, the various synthetic steps can be performed in an alternative sequence or to provide the desired compounds. Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc., present on bases), and protecting group methodology (protection and deprotection) useful in synthesizing 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); TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, ^2nd Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994) and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and their subsequent editions. Custom RNAi synthesis reagents are also available from several commercial suppliers, including Dharmacon,

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Inc. (Lafayette, Colorado), AxoLabs GmbH (Kulmbach, Germany), and Ambion, Inc. (Foster City, California).

The RNAi constructs of the present invention may comprise a ligand. As used herein, the term ligand refers to any compound or molecule that is directly or indirectly capable of interacting with another compound or molecule. The interaction of the ligand with another compound or molecule may elicit 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 double-stranded RNA molecule to which it is attached, such as pharmacodynamics, pharmacokinetic, binding, absorption properties, cellular distribution, cellular uptake, charge, and/or clearance of the RNA molecule.

The ligand may include 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 liver cells). Other examples of ligands include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphirin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine), peptides (e.g., peptide antennapedia), Tat peptide, RGD peptides), alkylating agents, polymers such as polyethylene glycol (PEG) (e.g. PEG-40K), polyamino acids and polyamines (e.g. spermine, spermidine).

In certain embodiments, the ligands have endosomolytic properties. Endosomolytic ligands promote lysis of the endosome and/or transport of the RNAi construct of the present invention or components thereof from the endosome to the cytoplasm of the cell. The endosomolytic ligand can be a polycationic peptide or a peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand is believed to have a functionally active conformation at the pH of the endosome. By active conformation is meant a conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the RNAi construct of the present invention or components thereof from the endosome to the cytoplasm of the cell. 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 can comprise a chemical group (e.g., an amino acid) that undergoes a charge change or protonation in response to a change in pH. The endosomolytic component can be linear or branched.

In some embodiments, the ligand comprises a lipid or other hydrophobic molecule. In one embodiment, the ligand comprises a cholesterol moiety or another steroid. Cholesterol-conjugated oligonucleotides have been reported to be more active than their unconjugated counterparts (Manoharan, Antisense Nucleic Acid Drug Development, Vol. 12, 103-228, 2002). Ligands comprising cholesterol moieties and other lipids for conjugation to nucleic acid molecules have also been described in U.S. Pat. Nos. 7,851,615; and 7,833,992, all of which are incorporated herein by reference in their entireties. In another embodiment, the ligand comprises a folate moiety. Polynucleotides conjugated to folate moieties can be taken up by cells via the receptor-mediated endocytosis pathway. Such folate polynucleotide conjugates are described in U.S. Patent No. 8,188,247, which is incorporated herein by reference in its entirety.

Given that PNPLA3 is expressed in liver cells (e.g., hepatocytes), in certain embodiments, it is desirable to specifically deliver the RNAi construct to these liver cells. In some embodiments, the RNAi constructs can be specifically targeted to the liver by using ligands that bind or interact with proteins expressed on the surface of liver cells. For example, in certain embodiments, the ligands can comprise antigen-binding proteins (e.g., antibodies or binding fragments thereof (e.g., Fab, scFv)) that specifically bind to a receptor expressed on hepatocytes.

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In certain embodiments, the ligand comprises a carbohydrate. The term 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) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Carbohydrates include, but are not limited to, sugars (e.g., monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starches, glycogen, cellulose, and polysaccharide gums. In some embodiments, the carbohydrate included in the ligand is a monosaccharide selected from pentose, hexose, or heptose, and di- and trisaccharides including 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.

In some embodiments, the ligand comprises a hexose or a hexosamine. The hexose can be selected from glucose, galactose, mannose, fucose, or fructose. The hexosamine can 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-acetylgalactosamine. In specific embodiments, the ligand comprises N-acetylgalactosamine. Ligands comprising glucose, galactose, and N-acetylgalactosamine (GalNAc) are particularly effective in targeting compounds to liver cells. See, for example, D'Souza and Devarajan, J. Control Release, Vol. 203, 126-139, 2015. Examples of GalNAc or galactose-containing ligands that can be incorporated into the RNAi constructs of the present invention are described in U.S. Patent Nos. 7,491,805; 8,106,022; and 8,877,917; U.S. Patent Publication No. 2003/0130186; and WIPO Publication No. WO 2013/166155, all of which are incorporated herein by reference in their entireties.

In certain embodiments, the ligand comprises a multivalent carbohydrate moiety. As used herein, the term multivalent carbohydrate moiety refers to a moiety comprising two or more carbohydrate units that are capable of independently binding or interacting with other molecules. For example, a multivalent carbohydrate moiety comprises two or more binding domains consisting 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 in the carbohydrate moiety. For example, the terms monovalent, divalent, trivalent, and tetravalent, when referring to a carbohydrate moiety, refer to carbohydrate moieties with 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-acetylgalactosamine 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-acetylgalactosamine moiety. In these and other embodiments, the multivalent carbohydrate moiety is divalent, trivalent, or tetravalent. In such embodiments, the multivalent carbohydrate moiety may be biantennary or triantennary. In one particular embodiment, the multivalent N-acetylgalactosamine moiety is trivalent or tetravalent. In another particular embodiment, the multivalent galactose moiety is trivalent or tetravalent. Illustrative trivalent and tetravalent GalNAc-containing ligands for incorporation into RNAi constructs of the present invention are described in detail below.

The ligand may be attached or conjugated to the RNA molecule of the RNAi construct directly or indirectly. For example, in some embodiments, the ligand is covalently attached directly to the sense or antisense strand of the RNAi construct. In other embodiments, the ligand is covalently attached through a linker to the sense or antisense strand of the RNAi construct. The ligand may be attached to nucleobases, sugar moieties, or internucleotide linkages of polynucleotides (e.g., the sense strand or the antisense strand) of the RNAi construct of the present invention. Conjugation or attachment to purine nucleobases or derivatives thereof may occur at any position, including endocyclic and exocyclic atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of the purine nucleobase is attached to the ligand. Conjugation with or attachment to pyrimidine nucleobases or derivatives thereof may also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase may be attached to the ligand. Conjugation with or attachment to sugar moieties of nucleotides may occur at any carbon atom. Exemplary carbon atoms of a sugar moiety that may be attached to a ligand include

- 16 047656 carbon atoms at positions 2'-, 3'- and 5'. The atom at position 1' may also be attached to a ligand, such as a base residue. Internucleotide linkages may also support ligand attachment. In the case of phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithioate, phosphoramidate, etc.), the ligand may be attached directly to the phosphorus atom or to an O, N, or S atom bonded to the phosphorus atom. In the case of amine-containing or amide-containing internucleoside linkages (e.g., PNA), the ligand may be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

In certain embodiments, the ligand can be attached to the 3' or 5' end of the sense 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 (e.g., before 1, 2, 3, or 4 terminal nucleotides). In some embodiments, the ligand is attached at the 2' position of a sugar included in the 3' terminal nucleotide of the sense strand.

In certain embodiments, the ligand is attached to the sense or antisense strand by a linker. The term "linker" means an atom or group of atoms that covalently connect the ligand to the polynucleotide component of the RNAi construct. The length of the linker can be from about 1 to about 30 atoms, from about 2 to about 28 atoms, from about 3 to about 26 atoms, from about 4 to about 6 atoms, from about 6 to about 20 atoms, from about 7 to about 8 atoms, from about 8 to about 20 atoms, from about 8 to about 10 atoms, from about 10 to about 18 atoms, and from about 12 to about 18 atoms. In some embodiments, the linker can comprise a bifunctional linking moiety, which typically comprises an alkyl moiety with two functional groups. One of the functional groups is selected to link to a compound of interest (e.g., a sense or antisense strand of an RNAi construct), and the other is selected to link to essentially any selected group, such as a ligand, as described herein. In certain embodiments, the linker comprises a chain structure or oligomer that is composed of repeating units, such as ethylene glycol or amino acid units. Examples of functional groups that are commonly used in a bifunctional linking moiety include, but are not limited to, electrophiles for reaction with nucleophilic groups and nucleophiles for reaction with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturated groups (e.g., double or triple bonds), and the like.

Linkers that can be used to attach a ligand to the sense or antisense strand in an RNAi construct of the present invention include, but are not limited to, pyrrolidine, 8-amino-3,6-dioxaoctanoic acid, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, 6-aminohexanoic acid, substituted C ₁ -C ₁₀ alkyl, substituted or unsubstituted C ₂ -C ₁₀ alkenyl, or substituted or unsubstituted C 2 -C 10 alkylyl. Preferred substituent groups for such linkers include, but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In certain embodiments, the linkers are cleavable. A cleavable linker is a linker that is sufficiently stable outside the cell, but which, upon entering the target cell, cleaves, releasing the two portions that the linker holds together. In some embodiments, the cleavable linker cleaves at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least 100-fold faster in the target cell or under a first control condition (which may be selected, for example, to mimic or represent intracellular conditions) than in the subject's blood or under a second control condition (which may be selected, for example, to mimic or represent conditions observed in blood or blood serum).

Cleavable linkers are sensitive to cleavage factors such as pH, redox potential, or the presence of degradative molecules. Typically, cleavable agents are more abundant or found at higher levels or activity inside cells than in serum or whole blood. Examples of such degradative agents include: redox agents that are substrate-selective or that lack substrate specificity, including, for example, oxidative or reductive enzymes or reducing agents such as mercaptans present in cells that can degrade a redox cleavable linker by reduction; esterases; endosomes; or agents that can create an acidic environment, such as those that

- 17 047656 result in a pH of five or lower; enzymes that can hydrolyze or degrade the acid-cleavable linker by acting as a general acid, peptidases (which may be substrate specific) and phosphatases.

The cleavable linker may contain a moiety that is sensitive to pH. The pH of blood serum is 7.4, while the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers will have a cleavable group that cleaves at a preferred pH, thereby releasing the RNA molecule from the ligand within the cell or into a desired compartment of the cell.

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

In general, the suitability of a cleavable candidate linker can be assessed by testing the ability of a degradative agent (or condition) to cleave the candidate linker. It will also be desirable to test the candidate cleavable linker for its ability to resist cleavage in blood or upon contact with other non-target tissue. In this way, the relative susceptibility to cleavage can be determined and selected between the first and second conditions, where the first is selected to indicate cleavage in the target cell and the second is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Such evaluation can be performed in cell-free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform an initial evaluation in cell-free conditions or culture conditions and confirm the results by further evaluation in whole animals. In some embodiments, useful candidate linkers are cleaved at least 2, 4, 10, 20, 50, 70, or 100 times faster inside a cell (or under in vitro conditions selected to mimic intracellular conditions) than in blood or blood serum (or under in vitro conditions selected to mimic extracellular conditions).

In other embodiments, redox-cleavable linkers are used. Redox-cleavable linkers are cleavable upon oxidation or reduction. An example of a reductively cleavable group is a disulfide linking group (-S-S-). To determine whether a candidate cleavable linker is a suitable reductively cleavable linker or, for example, whether it is suitable for use with a particular RNAi construct and a particular ligand, one or more of the techniques described herein can be used. For example, a candidate linker can be evaluated by incubation with dithiothreitol (DTT) or another reducing agent known in the art that mimics the rate of cleavage that occurs in a cell, such as a target cell. Candidate linkers can also be evaluated under conditions selected to mimic conditions that occur in blood or serum. In a particular embodiment, candidate linkers are cleaved in blood by no more than 10%. In other embodiments, useful candidate linkers are cleaved at least 2, 4, 10, 20, 50, 70, or 100 times faster inside a cell (or under in vitro conditions selected to mimic intracellular conditions) than in blood (or under in vitro conditions selected to mimic extracellular conditions).

In still further embodiments, cleavable phosphate-based linkers are cleaved by agents that destroy or hydrolyze the phosphate group. An example of an agent that hydrolyzes phosphate groups in cells are enzymes such as intracellular phosphatases.

Examples of phosphate-based cleavable groups are -OP(O)(OR _k )-O-, -OP(S)(OR _k )-O-, -OP(S)(SRk)-O-, -SP(O) (OR _k )-O-, -OP(O)(OR _k )-S-, -SP(O)(OR _k )-S-, -OP(S)(OR _k )-S-, -SP( S)(OR _k )-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 embodiments include -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -SP(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-, -S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-.

Another specific embodiment is -O-P(O)(OH)-O-. These candidate linkers can be evaluated using methods similar to those described above.

In other embodiments, the linkers may contain acid-cleavable groups, which are groups that are cleavable under acidic conditions. In some embodiments, the acid-cleavable groups are cleavable in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower) or by means such as enzymes that can act as a normal acid. In a cell, specific organelles with

- 18 047656 low pH, such as endosomes and lysosomes, can provide an environment for the cleavage of acid-cleavable groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A particular embodiment is the case where the carbon attached to the oxygen of the ester (an alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethyl, pentyl, or tert-butyl. These candidate groups can be evaluated using techniques similar to those described above.

In other embodiments, the linkers may comprise ester-based cleavable groups that are cleavable by enzymes such as intracellular esterases and amidases. Examples of cleavable ester groups include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable groups have the general formula -C(O)O- or -OC(O)-. These candidate linkers may be evaluated using methods similar to those described above.

In additional embodiments, the linkers may comprise peptide-based cleavable groups that are cleavable by enzymes such as intracellular peptidases and proteases. Peptide-based cleavable groups are peptide bonds formed between amino acids to form oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond between amino acids necessary for the formation of peptides and proteins. A peptide-based cleavable group is typically limited to a peptide bond (i.e., an amide bond) between amino acids forming peptides and proteins and does not include the entire amide functionality. Peptide-based cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of two adjacent amino acids. These candidate groups can be evaluated using methods similar to those described above.

Other types of linkers suitable for attaching ligands to the sense or antisense strands in the RNAi construct of the present invention are known in the art and may include those described in U.S. Patent Nos. 7,723,509; 8,017,762; 8,828,956; 8,877,917; and 9,181,551, all of which are incorporated herein by reference in their entireties.

In certain embodiments, a ligand covalently attached to the sense or antisense strand of an 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 further embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 3' end of the sense strand. In yet further embodiments, the multivalent GalNAc moiety is a tetravalent GalNAc moiety and is attached to the 5' end of the sense strand.

In some embodiments, the RNAi constructs of the present invention can be delivered to a cell or tissue of interest by introducing a vector that encodes and controls intracellular expression of the RNAi construct. The term vector (also referred to herein as an expression vector) refers to a composition of matter that can be used to deliver a nucleic acid of interest into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, and plasmids and viruses. Thus, the term vector includes an autonomously replicating plasmid or virus. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated viral vectors, retrovirus vectors, and the like. The vector can replicate in a living cell or can be produced synthetically.

In general, a vector for expressing an RNAi construct of the present invention will comprise one or more promoters operably linked to sequences encoding the RNAi construct. As used herein, the terms operably linked or under transcriptional control mean that the promoter is in the correct position and orientation relative to the polynucleotide sequence to control the initiation of transcription by RNA polymerase and the expression of the polynucleotide sequence. The term promoter refers to a sequence recognized by the synthetic machinery of a cell, or to an introduced synthetic machinery, necessary for the initiation of specific transcription of a gene sequence. Suitable promoters include, but are not limited to, RNA pol I, pol II, HI, or U6 RNA pol III, as well as viral promoters (e.g., the cytomegalovirus immediate early gene promoter).

- 19 047656 human lovirus (CMV), SV40 early promoter and Rous sarcoma virus long circular repeat). In some embodiments, the HI or U6RNA pol III promoter is preferred. The promoter may be tissue-specific or an inducible promoter. Of particular interest are liver-specific promoters such as the promoter sequences of the human alpha-1 antitrypsin gene, the albumin gene, the hemopexin gene and the liver lipase gene. Inducible promoters include those regulated by ecdysone, estrogen, progesterone, tetracycline and isopropyl-PD 1 -thiogalactopyranoside (IPTG).

In some embodiments in which the RNAi construct comprises siRNA, two separate strands (sense and antisense strands) may be expressed from a single vector or from two separate vectors. For example, in one embodiment, a sequence encoding the sense strand is operably linked to a promoter on a first vector and a sequence encoding the antisense strand is operably linked to a promoter on a second vector. In such an embodiment, the first and second vectors are introduced together, such as by infection or transfection, into a target cell such that the sense and antisense strands, after transcription, will hybridize within the cell to form an siRNA molecule. In another embodiment, the sense and antisense strands are transcribed from two different promoters located on a single vector. In some such embodiments, the sequence encoding the sense strand is operably linked to the first promoter and the sequence encoding the antisense strand is operably linked to the second promoter, wherein the first and second promoters are located on 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 direction, 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.

In other embodiments, in which the RNAi construct comprises an shRNA, a sequence encoding one at least partially self-complementary RNA molecule is operably linked to a promoter, resulting in the production of one transcript. In some embodiments, the shRNA encoding sequence comprises an inverted repeat linked by a linker polynucleotide sequence, resulting in the production of an shRNA stem and loop structure after transcription.

In some embodiments, a vector encoding an 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 pox virus (e.g., vaccinia virus) vectors. In certain embodiments, the viral vector is a retroviral vector (e.g., a lentiviral vector).

Various vectors suitable for use in the present invention, methods of inserting nucleic acid sequences encoding siRNA or shRNA molecules into vectors, and methods of delivering the vectors to cells of interest are known to those skilled in the art. See, for example, Dornburg, Gene Therap., Vol. 2, 301-310, 1995; Eglitis, Biotechniques, Vol. 6, 608-614, 1988; Miller, HumGene Therap., Vol. 1, 5-14, 1990; Anderson, Nature, Vol. 392, 25-30, 1998; Rubinson D.A. et al., Nat. Genet., Vol. 33, 401-406, 2003; Brummelkamp et al., Science, Vol. 296, 550-553, 2002; Brummelkamp et al., Cancer Cell, Vol. 2, 243-247, 2002; Lee et al., Nat. Biotechnol., Vol. 20, 500-505, 2002; Miyagishi et al., Nat. Biotechnol., Vol. 20, 497-500, 2002; Paddison et al., GenesDev, Vol. 16, 948-958, 2002; Paul et al., Nat. Biotechnol, Vol. 20, 505-508, 2002; Sui et al., Proc. Natl. Acad. Sci. USA, Vol. 99, 5515-5520, 2002 and Yu et al., Proc. Natl. Acad. Sci. USA, Vol. 99, 6047-6052, 2002, all of which are incorporated herein by reference in their entireties.

Also included in the present invention are pharmaceutical compositions and formulations comprising the RNAi constructs described herein and pharmaceutically acceptable carriers, excipients or diluents. Such compositions and formulations are useful in reducing the expression of PNPLA3 in a subject in need thereof. When intended for clinical use, the pharmaceutical compositions and formulations will be manufactured in a form suitable for the intended use. In general, this includes compositions that are substantially free of pyrogens as well as other impurities that may be harmful to humans or animals.

The phrases pharmaceutically acceptable or pharmacologically acceptable refer to molecular entities and compositions that do not produce undesirable, allergic or other adverse reactions when administered to an animal or human. As used herein, the term pharmaceutically acceptable carrier, excipient or diluent includes solvents,

- 20 047656 buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, suitable for use in formulating pharmaceutical preparations, such as pharmaceutical preparations suitable for administration to humans. The use of such media and vehicles for pharmaceutically active substances is well known in the art. Except where any conventional media or vehicle is incompatible with the RNAi constructs of the present invention, their use in therapeutic compositions is contemplated. Additional active ingredients may also be included in the compositions, provided that they do not inactivate the vectors or RNAi constructs in the compositions.

Compositions and methods for formulating pharmaceutical compositions depend on a number of criteria, including, but not limited to, the route of administration, the type and extent of the disease or disorder being treated, or the dose being administered. In some embodiments, pharmaceutical compositions are formulated based on the intended route of delivery. For example, in certain embodiments, pharmaceutical compositions are formulated for parenteral delivery. Parenteral forms of delivery include intravenous, intraarterial, subcutaneous, intrathecal, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the pharmaceutical composition is formulated for intravenous delivery. In such an embodiment, the pharmaceutical composition may include a lipid-based delivery vehicle. In another embodiment, the pharmaceutical composition is formulated for subcutaneous delivery. In such an embodiment, the pharmaceutical composition may include a targeting ligand (e.g., the GalNAc-containing ligands described herein).

In some embodiments, the pharmaceutical compositions comprise an effective amount of an RNAi construct described herein. The term "effective amount" means an amount sufficient to produce a beneficial or desired clinical result. In some embodiments, an effective amount means an amount sufficient to reduce the expression of PNPLA3 in hepatocytes of a subject. In some embodiments, an effective amount may be an amount sufficient to only partially reduce the expression of PNPLA3, such as to a level comparable to the expression of the wild-type PNPLA3 allele in human heterozygotes. Humans heterozygous for loss-of-function allelic variants of PNPLA3 have been reported to have lower serum non-HDL cholesterol levels and a lower risk of coronary heart disease and myocardial infarction compared to individuals without the corresponding allelic variants (Nioi et al., New England Journal of Medicine, Vol. 374(22):2131-2141, 2016). Thus, without being limited by theory, it is believed that partial reduction of PNPLA3 expression may be sufficient to achieve beneficial reductions in serum non-HDL cholesterol levels and a reduction in the risk of coronary heart disease and myocardial infarction.

An effective amount of an RNAi construct of the present invention can be from about 0.01 to about 100 mg/kg body weight, from about 0.05 to about 75 mg/kg body weight, from about 0.1 to about 50 mg/kg body weight, from about 1 to about 30 mg/kg body weight, from about 2.5 to about 20 mg/kg body weight, or from about 5 to about 15 mg/kg body weight. In certain embodiments, a single effective dose of an RNAi construct of the present invention can be about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg. A pharmaceutical composition comprising an effective amount of an RNAi construct may be administered weekly, biweekly, monthly, quarterly, or biennially. The precise determination of what is considered an effective amount and frequency of administration may be based on several factors, including the patient's weight, age, and general condition, the type of disorder being treated (e.g., myocardial infarction, heart failure, ischemic heart disease, hypercholesterolemia), the particular RNAi construct used, and the route of administration. Estimates of effective dosages and in vivo half-lives for any particular RNAi construct of the present invention may be determined using routine techniques and/or testing in appropriate animal models.

Administration of the pharmaceutical compositions of the present invention can be accomplished by any conventional route, as long as the target tissue is accessible by that route of administration. Such routes of administration 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 delivery via the hepatic portal vein. In some embodiments, the pharmaceutical composition is administered parenterally. For example, in certain embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, the pharmaceutical composition is administered subcutaneously.

- 21 047656

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 present invention or vectors encoding such constructs. Commercially available lipid emulsions that are suitable for delivering the nucleic acids of the invention include Intralipid®, Liposyn®, Liposyn®II, Liposyn®III, Nutrilipid and other similar lipid emulsions. A preferred colloidal system for use as an in vivo delivery vehicle is a liposome (i.e., an artificial membrane vesicle). The RNAi constructs of the present invention can be encapsulated in or complexed with liposomes, in particular cationic liposomes. Alternatively, the RNAi constructs of the present invention can be complexed with lipids, particularly cationic lipids. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidylethanolamine) (DOPE), dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC)), distearoylphosphatidylcholine), negatively charged (e.g., dimyristoylphosphatidylglycerol (DMPG)) and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DO™A)) lipids and liposomes. The preparation and use of such colloidal dispersion systems are well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505; 6,217,900; 6,383,512; 5783565; 7202227; 6379965; 6127170; 5837533; 6747014 and PCT Application Publication No. WO 03/093449.

In some embodiments, the RNAi constructs of the present invention are fully encapsulated in a lipid formulation, such as to form SPLPs, pSPLPs, SNALPs, or other nucleic acid-lipid particles. As used herein, the term SNALPs refers to a stable nucleic acid-lipid particle, including SPLPs. As used herein, the term SPLPs refers to a nucleic acid-lipid particle that contains plasmid DNA encapsulated in a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents particle aggregation (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are particularly useful for systemic applications because they exhibit extended circulation times in the bloodstream after intravenous administration and accumulate at remote sites (e.g., physically separated from the site of administration). SPLPs include pSPLPs that include an encapsulated complex of a condensing agent and a nucleic acid as disclosed in PCT Publication No. WO 00/03683. The nucleic acid-lipid particles typically have an average diameter of from about 50 to about 150 nM, from about 60 to about 130 nM, from about 70 to about 110 nM, or from about 70 to about 90 nM, and are substantially non-toxic. Furthermore, when nucleic acids are present in the nucleic acid-lipid particles, they are resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and a method for making them are disclosed, for example, in U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6815432 and publication of PCT application No. WO 96/40964.

Pharmaceutical compositions suitable for injection use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. As a rule, these preparations are sterile and fluid to such an extent that they are able to pass easily through a needle during administration. The preparations must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. Suitable solvents or dispersion vehicles may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be caused by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using absorption-retarding agents in the compositions, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by adding the active compounds in the appropriate amount to the solvent with any other ingredients (e.g., those enumerated above) as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier medium which contains the basic dispersion medium and the required other ingredients from, for example, those enumerated above. In the case of sterile powders intended for the preparation of sterile injectable solutions, preferred processes for their preparation include the method

- 22 047656 vacuum drying and freeze drying machines which provide a powder of the active ingredient(s) plus any additional required ingredient from its previously sterile filtered solution.

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 with free amino groups) derived from inorganic acids (e.g., hydrochloric or phosphoric acids) or derived from organic acids (e.g., acetic, oxalic, tartaric, mandelic, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or iron hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.).

For example, for parenteral administration in an aqueous solution, the solution is, for example, sufficiently buffered and the liquid diluent is first rendered isotonic, for example, by using a sufficient amount of saline or glucose. Such aqueous solutions can be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are used, as are known to those skilled in the art, especially in light of the disclosure of the present invention. By way of illustration, a single dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or administered at the proposed infusion site (see, for example, Remington's Pharmaceutical Sciences 15 ^th Edition, pp. 1035-1038 and 1570-1580). For human administration, the preparations must meet the standards of sterility, pyrogenicity, general safety and purity as required by FDA standards. In certain embodiments, a pharmaceutical composition of the present invention comprises or consists of sterile saline and an RNAi construct described herein. In other embodiments, a pharmaceutical composition of the present invention comprises or consists of an RNAi construct described herein and sterile water (e.g., water for injection, WFI). In still other embodiments, a pharmaceutical composition of the present invention comprises or consists of an RNAi construct described herein and phosphate buffered saline (PBS).

In some embodiments, the pharmaceutical compositions of the present invention are packaged or stored in an administration device. Devices for administering injectable formulations include, but are not limited to, injection port systems, prefilled syringes, automatic injectors, injection pumps, body injectors, and injection pens. Devices for administering aerosol or powder formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes administration devices containing a pharmaceutical composition of the present invention for the treatment or prevention of one or more disorders described herein.

Methods for suppressing PNPLA3 expression

The present invention also provides methods for suppressing the expression of the PNPLA3 gene in a cell. The methods include contacting the cell with an RNAi agent, such as a double-stranded RNAi agent, in an amount that is effective to suppress the expression of PNPLA3 within the cell, thereby suppressing the expression of PNPLA3 within the cell. Contacting the cell with an RNAi agent, such as a double-stranded RNAi agent, can be performed in vitro or in vivo. Contacting the cell with an RNAi agent in vivo includes contacting a cell or a group of cells within a subject, such as a human subject, with the RNAi agent. Combinations of in vitro and in vivo cell contact methods are also possible.

The present invention provides methods for reducing or suppressing the expression of PNPLA3 in a subject in need thereof, as well as methods for treating or preventing conditions, diseases or disorders associated with the expression or activity of PNPLA3. The term condition, disease or disorder associated with the expression of PNPLA3 refers to conditions, diseases or disorders in which changes in the expression levels of PNPLA3 are observed or in which increased levels of PNPLA3 expression are associated with an increased risk of developing the condition, disease or disorder.

Contacting the cell may be direct or indirect, as discussed above. In addition, contacting the cell may be via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, such as a GalNAc3 ligand or any other ligand that directs the RNAi agent to the site of interest.

In one embodiment, contacting the cell with the RNAi comprises introducing or delivering the RNAi agent into the cell by facilitating or effecting uptake by the cell or by absorption into the cell. The uptake or uptake of the RNAi agent may occur through uncontrolled diffusion or active cellular processes or through assisted

- 23 047656 means or devices. The introduction of the RNAi agent into the cell can be carried out in vitro and/or in vivo. For example, for in vivo administration, RNAi can be administered into a tissue site or administered systemically. In vitro introduction into the cell includes methods known in the art, such as electroporation and lipofection. Additional approaches are described herein below and/or are known in the art.

The term suppression as used in this document is used interchangeably with the terms reduction, silencing, down-regulation, suppression and other similar terms and includes any level of suppression.

The phrase suppressing the expression of PNPLA3 means suppressing the expression of any PNPLA3 gene (such as a mouse PNPLA3 gene, a rat PNPLA3 gene, a monkey PNPLA3 gene, or a human PNPLA3 gene), as well as variants or mutant variants of the PNPLA3 gene. Thus, the PNPLA3 gene may be a wild-type PNPLA3 gene, a mutant PNPLA3 gene (such as a mutant PNPLA3 gene that causes amyloid deposition), or a genetically modified PNPLA3 gene in the context of a genetically modified cell, group of cells, or organism.

The term suppression of PNPLA3 gene expression includes any level of suppression of PNPLA3 gene, such as 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 parameter associated with PNPLA3 gene expression, such as the level of PNPLA3 mRNA, PNPLA3 protein, or the amount or extent of amyloid deposits. This level can be assessed in a single cell or in a group of cells, including, for example, a sample obtained from a subject.

Suppression can be assessed by a decrease in the absolute or relative level of one or more parameters associated with PNPLA3 expression compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined for a similar subject, cell, or sample that is untreated or treated with a control solution (such as, for example, a buffer-only control or an inactive agent control). In some embodiments of the methods of the present invention, PNPLA3 gene expression is suppressed 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%,

By smaller mere approximately 35%, By smaller mere approximately 40%, By smaller mere approximately 45%, By smaller mere approximately 50%, By smaller mere approximately 55%, By smaller mere approximately 60%, By smaller mere approximately 65%, By smaller mere approximately 70%, By smaller mere approximately 75%, By smaller mere approximately 80%, By smaller mere approximately 85%, By smaller mere approximately 90%, By smaller mere approximately 91%, By smaller mere approximately 92%, By smaller mere approximately 93%, By smaller mere approximately 94%, By smaller mere approximately 95%, By smaller mere approximately 96%, By smaller mere approximately 97%, By smaller mere approximately 98%

or at least approximately 99%.

The downregulation of the expression of the PNPLA3 gene may be exhibited by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample obtained from a subject) in which the PNPLA3 gene is transcribed and which has been or has been treated (for example, by contacting the cell or cells with an RNAi agent of the invention or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the expression of the PNPLA3 gene is downregulated compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but which has not been or has not been so treated (control cell(s)). In preferred embodiments, the downregulation is assessed by expressing the mRNA level in the treated cells as a percentage of the mRNA level in the control cells using the following formula:

|(mRNA in control cells) - (mRNA in treated cells) ।qqo/ (mRNA in control cells)

Alternatively, suppression of PNPLA3 gene expression can be assessed in terms of a decrease in a parameter that is functionally related to PNPLA3 gene expression, such as PNPLA3 protein expression or Hedgehog signaling pathway protein activity. PNPLA3 gene silencing can be determined in any cell that expresses PNPLA3 either constitutively or as a result of genetic engineering and by any assay known in the art.

Suppression of PNPLA3 protein expression may be demonstrated by a decrease in the level of PNPLA3 protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample obtained from a subject). As explained above, to assess the degree of mRNA suppression, the suppression of protein expression levels in a treated cell or group of cells may be similarly expressed as

- 24 047656 as a percentage of the protein level in a control cell or group of cells.

A control cell or group of cells that can be used to assess the degree of suppression of PNPLA3 gene expression includes a cell or group of cells that have not yet been contacted with the RNAi agent of the present invention. For example, a control cell or group of cells can be obtained from an individual subject (e.g., a human subject or an animal subject) prior to treating the subject with the RNAi agent.

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

In one embodiment, the level of PNPLA3 expression is determined using a nucleic acid probe. The term probe as used herein refers to any molecule that is capable of selectively binding to a particular PNPLA3. Probes can be synthesized by one skilled in the art or obtained from appropriate biological preparations. Probes can be specially designed to introduce a label. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

The isolated mRNA can be used in hybridization or amplification assays, which include, but are not limited to, Southern and Northern blotting, polymerase chain reaction (PCR)-based assays, and probe arrays. One technique for detecting mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PNPLA3 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, such as by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as a nitrocellulose membrane. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), such as on an Affymetrix DNA chip. One skilled in the art can readily adapt known mRNA detection methods for use in determining PNPLA3 mRNA levels.

An alternative method for determining PNPLA3 expression in a sample involves a nucleic acid amplification process and/or reverse transcriptase action (to produce cDNA) on, for example, mRNA in the sample, such as by reverse transcription-PCR (an experimental embodiment is described in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991), Proc. Natl. Acad. Sci. USA, 88:189-193), self-sustained sequence replication (Guatelli et al. (1990), Proc. Natl. Acad. Sci. USA, 87:1874-1878), transcription amplification 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. Patent No. 5,854,033), or any other method of amplifying nucleic acids, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very low amounts. In particular aspects of the invention, the level of PNPLA3 expression is determined using quantitative fluorogenic reverse transcription-PCR (i.e., the TaqMan™ system). Monitoring PNPLA3 mRNA expression levels can be performed using membrane blots (such as those used in hybridization assays, e.g., 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. Determining PNPLA3 expression levels can also include the use of nucleic acid probes in solution.

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

The expression level of the PNPLA3 protein can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis.

- 25 047656 phoresis, capillary electrophoresis, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), hyperdiffusion chromatography, liquid or gel precipitation reactions in liquid or in gel, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays, electrochemiluminescence assays, etc.

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

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. Suppression of PNPLA3 expression can be assessed using measurements of the level or changes in the level of PNPLA3 mRNA or PNPLA3 protein in a sample obtained from a fluid or tissue in a specific site of the subject. In preferred embodiments, the site is selected from the group consisting of the liver, choroid plexus, retina, and pancreas. The site can also be a portion or subset of cells from any of the aforementioned sites. The site can also include cells that express a specific type of receptor.

Methods for treating or preventing diseases associated with PNPLA3.

The present invention provides therapeutic and prophylactic methods that comprise administering to a subject with a disease, disorder and/or condition associated with PNPLA3 or prone to developing a disease, disorder and/or condition associated with PNPLA3 a composition comprising an RNAi agent or pharmaceutical compositions comprising an RNAi agent or vectors expressing an RNAi agent of the present invention. Non-limiting examples of diseases associated with PNPLA3 include, for example, fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), liver cirrhosis, liver fat accumulation, liver inflammation, hepatocellular necrosis, liver fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). In one embodiment, the disease associated with PNPLA3 is NAFLD. In another embodiment, the disease associated with PNPLA3 is NASH. In another embodiment, the disease associated with PNPLA3 is fatty liver disease (steatosis). In another embodiment, the disease associated with PNPLA3 is insulin resistance. In another embodiment, the disease associated with PNPLA3 is not insulin resistance.

In certain embodiments, the present invention provides a method for reducing the expression of PNPLA3 in a patient in need thereof, comprising administering to the patient any of the RNAi constructs described herein. The term patient, as used herein, refers to a mammal, including a human, and may be used interchangeably with the term subject. Preferably, the level of PNPLA3 expression in hepatocytes in the patient is reduced following administration of the RNAi construct, compared to the level of PNPLA3 expression in a patient that has not received the RNAi construct.

The methods of the present invention are useful for treating a subject with a disease associated with PNPLA3, such as a subject that can benefit from reducing the expression of the PNPLA3 gene and/or the production of PNPLA3 protein. In one aspect, the present invention provides methods for reducing the expression level of the gene encoding the patatin-like phospholipase domain-containing protein 3 (PNPLA3) in a subject with non-alcoholic fatty liver disease (NAFLD). In another aspect, the present invention provides methods for reducing the level of PNPLA3 protein in a subject with NAFLD. The present invention also provides methods for reducing the level of hedgehog signaling pathway activity in a subject with NAFLD.

In another aspect, the present invention provides methods of treating a subject with NAFLD. In one aspect, the present invention provides methods of treating a subject with a disease associated with PNPLA3, such as fatty liver disease (steatosis), non-alcoholic steatohepatitis (NASH), liver cirrhosis, liver fat accumulation, liver inflammation, hepatocellular necrosis, liver fibrosis, obesity, or non-alcoholic fatty liver disease (NAFLD). The methods of treatment (and uses) of the present invention comprise administering to a subject, such as a human, a therapeutically effective amount of an RNAi agent of the present invention that targets a PNPLA3 gene, or a pharmaceutical composition comprising an RNAi agent of the present invention that targets a PNPLA3 gene, or a vector of the present invention expressing an RNAi agent that targets a PNPLA3 gene.

- 26 047656

In one aspect, the present invention provides methods for preventing at least one symptom in a subject with NAFLD, such as the presence of increased activity of hedgehog signaling pathways, fatigue, weakness, weight loss, loss of appetite, nausea, abdominal pain, spider veins, yellowing of the skin and eyes (jaundice), itching, fluid accumulation and swelling of the legs (edema), swelling of the abdomen (ascites), and confusion. The methods comprise administering to the subject a therapeutically effective amount of an RNAi agent, such as dsRNA, pharmaceutical compositions, or vectors of the present invention, thereby preventing the occurrence of at least one symptom in a subject with a disorder that can be improved by reducing the expression of the PNPLA3 gene.

In another aspect, the present invention provides embodiments of using a therapeutically effective amount of an RNAi agent of the present invention to treat a subject, such as a subject that can benefit from reducing and/or suppressing the expression of a PNPLA3 gene. In a further aspect, the present invention provides embodiments of using an RNAi agent, such as a dsRNA, of the present invention that targets a PNPLA3 gene, or a pharmaceutical composition comprising an RNAi agent that targets a PNPLA3 gene, in the manufacture of a medicament for treating a subject, such as a subject that can benefit from reducing and/or suppressing the expression of a PNPLA3 gene and/or the production of a PNPLA3 protein, such as a subject with a disorder that can be ameliorated by reducing the expression of a PNPLA3 gene, such as a disease associated with PNPLA3.

In another aspect of the present invention, embodiments are provided of the use of an RNAi agent, such as a dsRNA, of the present invention to prevent the occurrence of at least one symptom in a subject suffering from a disorder that can be ameliorated by reducing and/or suppressing PNPLA3 gene expression and/or PNPLA3 protein production.

In a further aspect of the present invention, embodiments are provided for using the RNAi agent of the present invention in the manufacture of a medicament intended to prevent the occurrence of at least one symptom in a subject suffering from a disorder that can be ameliorated by reducing and/or suppressing PNPLA3 gene expression and/or PNPLA3 protein production, such as a PNPLA3-associated disease.

In one embodiment, an RNAi agent that targets PNPLA3 is administered to a subject with a PNPLA3-associated disease, such as. non-alcoholic fatty liver disease (NAFLD), whereby expression of the PNPLA3 gene, for example, in a cell, tissue, blood or other tissue or fluid in 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, 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 if a dsRNA-based agent is administered to the subject.

The methods and uses of the present invention comprise administering a composition as described herein, whereby the expression of a PNPLA3 target gene is reduced, such as 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, the expression of a PNPLA3 target gene is reduced over an extended period, such as for at least about two, three, four, five, six, seven days or more, such as for about one week, two weeks, three weeks, or about four weeks or longer.

Administration of dsRNA according to the methods and uses of the present invention may result in a reduction in the severity, signs, symptoms and/or level of markers of diseases or disorders in a patient with a disease associated with PNPLA3, such as non-alcoholic fatty liver disease (NAFLD). By reduction in this context is meant a statistically significant reduction in such level. The reduction may be, for example, at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or about 100%. The effectiveness of treating or preventing a disease may be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain relief, quality of life, the dose of drug needed to maintain the treatment effect, the level of a disease marker, or any other measurable parameter relevant to the disease being treated or targeted for prevention. One of skill in the art is well placed to monitor the effectiveness of the treatment or prevention by measuring any one or a combination of such parameters. For example, the effectiveness of treatment for NAFLD may be assessed, for example, by periodically monitoring NAFLD symptoms, liver fat levels, or the expression of genes that regulate downstream signaling cascades. Comparison of the later readings with the initial readings provides the physician with an indication of whether the treatment is effective. One of skill in the art is well placed to monitor the effectiveness of the treatment or prevention by measuring any one or a combination of such parameters. In connection with the administration of an RNAi agent targeting PNPLA3 or its pharma

- 27 047656 ceutical composition efficacy against a PNPLA3-associated disease indicates that administration in a clinically acceptable manner results in a beneficial effect in at least a statistically significant proportion of patients, such as improvement in symptoms, cure, reduction in disease severity, prolongation of life, improvement in quality of life, or other effect that is generally recognized as beneficial by physicians familiar with the treatment of NAFLD and/or PNPLA3-associated disease and related causes.

A therapeutic or preventive effect is demonstrated when a statistically significant improvement in one or more disease status parameters is observed, or when symptoms do not worsen or develop where they would otherwise be expected. As an example, a favorable change of at least 10% in a measurable disease parameter, and preferably at least 20, 30, 40, 50% or more, may indicate an effective treatment. The efficacy of a given RNAi-based medicinal product or formulation of the medicinal product may also be assessed using an animal model known in the art for the disease. When using an animal model, the efficacy of the treatment is confirmed when a statistically significant reduction in a marker or symptom is observed.

The subjects may be administered a therapeutic amount of the RNAi agent, such as about 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,

3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4, 2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5,

5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6, 8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1,

8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 9.0, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 mg/kg dsRNA. In one embodiment, 0.5 mg/kg dsRNA can be administered to subjects. Values and ranges intermediate to these values are also contemplated as part of this invention.

Administration of the RNAi agent can reduce the levels of PNPLA3 protein present, for example, in a cell, tissue, blood, urine, or other compartment of the patient's body by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1, 16, 17, 18, 19, 20, 21, 22, 23, 24, 2, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3, 36, 37, 38, 39, 40, 41, 42, 43, 44, 4, 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 or at least about 99% or more.

Before administering the full dose of RNAi agent, patients may be given a smaller dose, such as a 5% infusion, and monitored for side effects such as an allergic reaction. In another example, the patient may be monitored for unwanted immune-stimulatory effects such as increased cytokine levels (e.g., TNF-alpha or INF-alpha).

Due to the inhibitory effects on the expression of PNPLA3, the composition of the present invention or a pharmaceutical composition obtained therefrom can improve the quality of life.

The RNAi agent of the present invention can be administered in a naked form, wherein the modified or unmodified RNAi agent is directly suspended in an aqueous or suitable buffer solvent as a free RNAi agent. The free RNAi agent is administered in the absence of a pharmaceutical composition. The free RNAi agent can be in a suitable buffer solution. The buffer solution can contain acetate, citrate, prolamine, carbonate or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate-buffered saline (PBS). The pH and osmolality of the buffer solution containing the RNAi agent can be adjusted so that it is suitable for administration to a subject.

Alternatively, the RNAi agent of the present invention may be administered as a pharmaceutical composition, such as a liposomal formulation of dsRNA.

Subjects who may benefit from reduction and/or suppression of PNPLA3 gene expression include subjects with non-alcoholic fatty liver disease (NAFLD) and/or a PNPLA3-associated disease or disorder described herein.

Treatment of a subject who may benefit from reduction and/or suppression of PNPLA3 gene expression includes therapeutic and prophylactic treatment.

The present invention further provides methods and uses of an RNAi agent or a pharmaceutical composition thereof for treating a subject that may benefit from reducing and/or suppressing the expression of a PNPLA3 gene, such as a subject with a disease associated with PNPLA3, in combination with other pharmaceuticals and/or other therapeutic methods, such as known pharmaceuticals and/or known therapeutic methods, such as, for example, those currently used to treat these disorders.

For example, in certain embodiments, an RNAi agent that specifically targets

- 28 047656 acting on the PNPLA3 gene, is administered in combination with, for example, an agent that is useful in the treatment of a disease associated with PNPLA3, described elsewhere herein. For example, additional therapeutic agents and therapeutic methods suitable for treating a subject that may benefit from a decrease in PNPLA3 expression, such as a subject with a disease associated with PNPLA3, include an RNAi agent that targets another portion of the PNPLA3 gene, a therapeutic agent and/or procedures for treating a disease associated with PNPLA3, or a combination of any of the foregoing.

In certain embodiments, a first RNAi agent that targets a PNPLA3 gene is administered in combination with a second RNAi agent that targets another portion of the PNPLA3 gene. For example, the first RNAi agent comprises a first sense strand and a first antisense strand that form a double-stranded region, wherein substantially all of the nucleotides of said first sense strand and substantially all of the nucleotides of the first antisense strand are modified nucleotides, wherein said first sense strand is conjugated to a ligand attached at the 3' end, and wherein the ligand is one or more GalNAc derivatives attached via a divalent 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 of the nucleotides of the second sense strand and substantially all of the nucleotides of the second antisense strand are modified nucleotides, wherein the second sense strand is conjugated to a ligand attached at the 3' end, and wherein the ligand is one or more GalNAc derivatives attached via a divalent or trivalent branched linker.

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.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 3'-terminal deoxythymine (dT) nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a closed nucleotide, an open nucleotide, a conformationally constrained nucleotide, an ethyl-constrained nucleotide, a base-deleted nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholine nucleotide, a phosphoramidate, a nucleotide containing non-natural base, tetrahydropyran-modified nucleotide, 1,5-anhydroxyhexitol-modified nucleotide, cyclohexenyl-modified nucleotide, nucleotide containing a phosphorothioate group, nucleotide containing a methylphosphonate group, nucleotide containing a 5'-phosphate, and nucleotide containing a 5'-phosphate mimetic.

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

The RNAi agent and the additional therapeutic agent and/or drug may be administered simultaneously and/or in the same combination, such as parenterally, or the additional therapeutic agent may be administered as part of a separate composition, or at different times, and/or in another manner known in the art or described herein.

The present invention also provides methods of using an RNAi agent of the present invention and/or a composition comprising an RNAi agent of the present invention to reduce and/or suppress the expression of PNPLA3 in a cell. In other aspects, the present invention provides an RNAi agent of the present invention and/or a composition comprising an RNAi agent of the present invention for use in reducing and/or suppressing the expression of a PNPLA3 gene in a cell. In yet other aspects, the present invention provides a use of an RNAi agent of the present invention and/or a composition comprising an RNAi agent of the present invention or the manufacture of a medicament for reducing and/or suppressing the expression of a PNPLA3 gene in a cell. In other aspects, the present invention provides an RNAi agent of the present invention and/or a composition comprising an RNAi agent of the present invention for use in reducing and/or suppressing the production of a PNPLA3 protein in a cell. In yet other aspects, the present invention provides a use of an RNAi agent of the present invention and/or a composition comprising an RNAi agent of the present invention

- 29 047656 of the present invention, for the manufacture of a medicament for reducing and/or suppressing the production of PNPLA3 protein in a cell. The methods and uses include contacting the cell with an RNAi agent, such as dsRNA, of the present invention and maintaining the cell for a time sufficient to achieve degradation of the mRNA transcript of the PNPLA3 gene, thereby suppressing the expression of the PNPLA3 gene or suppressing the production of PNPLA3 protein in the cell.

The decrease in gene expression can be assessed by any methods known in the art. For example, a decrease in PNPLA3 expression can be determined by determining the level of PNPLA3 mRNA expression using methods routinely practiced by a person skilled in the art, such as Northern blotting, qRT-PCR, by determining the level of PNPLA3 protein using methods routinely practiced by a person skilled in the art, such as Western blotting, immunological techniques, flow cytometric techniques, ELISA, and/or by determining the biological activity of PNPLA3.

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

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

The expression of the PNPLA3 gene in the cell can be reduced by at least approximately 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 or approximately 100%.

The production of PNPLA3 protein in a cell can be reduced 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 or approximately 100%.

The in vivo methods and uses of the present invention may comprise administering to a subject a composition comprising an RNAi agent, wherein the RNAi agent comprises a nucleotide sequence that is 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 organism, the composition may be administered by any route 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, respiratory (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection. In one embodiment, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is performed by injection of a depot agent. When injecting a depot agent, the RNAi agent can be released in a consistent manner over a long period of time. Thus, injection of a depot agent can reduce the frequency of dosing required to achieve the desired effect, such as the desired suppression of PNPLA3 or a therapeutic or prophylactic effect. Injection of a depot agent can also provide more constant serum concentrations. Injections of depot agents can include subcutaneous injections or intramuscular injections. In preferred embodiments, the injection of a depot agent is a subcutaneous injection.

In some embodiments, the administration is performed via an injection pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. The infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a

- 30 047656 skin infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers an RNAi agent to a subject.

The route of administration may be selected depending on whether local or systemic treatment is required and the area to be treated. The route and site of administration may be selected to enhance the targeted effect.

In one aspect, the present invention also provides methods for suppressing the expression of a PNPLA3 gene in a mammal, such as a human. The present invention also provides a composition comprising an RNAi agent, such as a dsRNA, that targets a PNPLA3 gene in a mammalian cell for use in suppressing the expression of a PNPLA3 gene in a mammal. In another aspect, the present invention provides uses of an RNAi agent, such as a dsRNA, that targets a PNPLA3 gene in a mammalian cell, in the manufacture of a medicament for suppressing the expression of a PNPLA3 gene in a mammal.

Methods and uses include administering to a mammal, such as a human, a composition comprising an RNAi agent, such as dsRNA, that specifically targets a PNPLA3 gene in a cell of the mammal, and maintaining the mammal for a time sufficient to achieve degradation of the mRNA transcript of the PNPLA3 gene, thereby suppressing expression of the PNPLA3 gene in the mammal.

A decrease in gene expression can be assessed in a peripheral blood sample from a subject who has been administered an RNAi agent using any method known in the art, such as qRTPCR described herein. A decrease in protein production can be assessed using any method known in the art, such as ELISA or Western blotting described herein. In one embodiment, the tissue sample serves as a tissue material for monitoring a decrease in PNPLA3 gene and/or protein expression. In another embodiment, the blood sample serves as a tissue material for monitoring a decrease in PNPLA3 gene and/or protein expression.

In one embodiment, verification of RISC-mediated target cleavage in vivo following administration of the RNAi agent is performed using 5'-RACE or modifications of the protocol known in the art (Lasham A et al., (2010), Nucleic Acid Res., 38(3), p-e19) (Zimmermann et al. (2006), Nature, 441:111-4).

It is understood that all ribonucleic acid sequences disclosed herein can be converted into deoxyribonucleic acid sequences by replacing the thymine base in the sequence with a uracil base. Similarly, all deoxyribonucleic acid sequences disclosed herein can be converted into ribonucleic acid sequences by replacing the uracil base in the sequence with a thymine base. The present invention includes deoxyribonucleic acid sequences, ribonucleic acid sequences, and sequences containing mixtures of deoxyribonucleotides and ribonucleotides in all sequences disclosed herein.

In addition, any nucleic acid sequences disclosed herein may be modified by any combination of chemical modifications. One skilled in the art will readily appreciate that the designation RNA or DNA to describe modified polynucleotides is arbitrary in some instances. For example, a polynucleotide comprising a nucleotide having a 2'-OH substituent on the ribose sugar and a thymine base may be described as a DNA molecule having a modified sugar (2'-OH instead of the natural 2'-H atom of DNA) or as an RNA molecule having a modified base (thymine (methylated uracil) instead of the natural uracil of RNA).

Accordingly, the nucleic acid sequences provided herein, including but not limited to those recited in the sequence listing, are intended to encompass nucleic acids comprising any combination of natural or modified RNA and/or DNA, including but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, a polynucleotide having the sequence ATCGATCG encompasses any polynucleotides having such a sequence, whether modified or unmodified, including but not limited to those compounds comprising RNA bases, such as those having the sequence AUCGAUCG, and those compounds having some DNA bases and some RNA bases, such as AUCGATCG, as well as polynucleotides having other modified bases, such as ATmeCGAUCG, where meC is a cytosine base containing a methyl group at the 5 position.

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

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Inclusion via link

All publications, patents, and patent applications mentioned in this specification are herein incorporated 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. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussions provided herein, the terms and definitions in this specification control.

Equivalents

The foregoing written description is considered sufficient to enable one skilled in the art to practice the present invention. The foregoing description and examples have described in detail certain preferred embodiments of the present invention and have described the best mode contemplated by the inventors of the present invention. However, it should be understood that no matter how detailed the foregoing may appear in the text, the present invention may be practiced in many ways, and therefore the present invention should be construed in accordance with the appended claims and any equivalents thereof.

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

All animal experiments described in this paper were approved by the Amgen Institutional Animal Care and Use Committee (IACUC) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals, ^8th Edition (National Research Council (US), Committee for the Update Guide for the Care and Use of Laboratory Animals., Institute for Laboratory Animal Research (US) and National Academies Press (US) (2011), Guide for the care and use of laboratory animals. 8th Ed., National Academies Press, Washington, DC Mice were housed indoors in an air-conditioned room maintained at 22 ± 2°C and maintained on a 12-hour light/12-hour dark cycle (0600–1800 h). Animals had free access to standard chow (Envigo, 2920X, or chow as otherwise indicated) and water (reverse osmosis purified) via an automatic waterer unless otherwise indicated. otherwise specified. At the end of the specified period, blood was collected by cardiac puncture under deep anesthesia, and then, following the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), the animals were euthanized by a secondary physical method.

Example 1. Selection, design and synthesis of modified PNPLA3 siRNA molecules.

Identification and selection of optimal sequences of therapeutic siRNA molecules targeting patatin-like phospholipase domain 3 (PNPLA3) were identified using bioinformatic analysis of the human PNPLA3 transcript (NM_025225.2). Table 1 shows the sequences identified as having therapeutic properties. Throughout the various sequences, {INVAB} denotes inverted base A, {INVDA} denotes inverted deoxythymidine, GNA denotes glycolic nucleic acid, dT denotes deoxythymidine, and dC denotes deoxycytosine.

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Table 1

siRNA sequences targeting PNPLA3

Duplex No. Semantic consecutive ть (5'-3') SEQ Ю NO: (semantic) Antisense sequence (5'-3') SEQ ID NO: (antisense) D-1000 GGGCAAUAAAG UACCUGCUUU 1 AGCAGGUACUUU AUUGCCCUU 2 D-1001 CGGCCAAUGUC CACCAGCUUU 3 AGCUGGUGGACA UUGGCCGUU 4 D-1002 GGUCCAGCCUG AACUUCUUUU 5 AAGAAGUUCAGG CUGGACCUU 6 D-1003 GCUUCAUCCCC UUCUACAGUU 7 CUGUAGAAGGGG AUGAAGCUU 8 D-1004 GCGGCUUCCUG GGCUUCUAUU 9 UAGAAGCCCAGG AAGCCGCUU 10 D-1005 GCCUCUGAGCU GAGUUGGUUU 11 ACCAACUCAGCU CAGAGGCUU 12 D-1006 GUGACAACGUA CCCUUCAUUU 13 AUGAAGGGUACG UUGUCACUU 14 D-1007 CCCGCCUCCAG GUCCCAAAUU 15 UUUGGGACCUGG AGGCGGGUU 16 D-1008 CUUCAUCCCCU UCUACAGUUU 17 ACUGUAGAAGGG GAUGAAGUU 18

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D-1009 GGUAUGUUCCU GCUUCAUGUU 19 CAUGAAGCAGGA ACAUACCUU 20 D-1010 GUAUGUUCCUG CUUCAUGCUU 21 GCAUGAAGCAGG AACAUACUU 22 D-1011 UAUGUUCCUGC UUCAUGCCUU 23 GGCAUGAAGCAG GAACAUAUU 24 D-1012 AUGUUCCUGCU UCAUGCCCUU 25 GGGCAUGAAGCA GGAACAUUU 26 D-1013 UGUUCCUGCUU CAUGCCCUUU 27 AGGGCAUGAAGC AGGAACAUU 28 D-1014 GUUCCUGCUUC AUGCCCUUUU 29 AAGGGCAUGAAG CAGGAACUU 30 D-1015 UUCCUGCUUCA UGCCCUUCUU 31 GAAGGGCAUGAA GCAGGAAUU 32 D-1016 UCCUGCUUCAU GCCCUUCUUU 33 AGAAGGGCAUGA AGCAGGAUU 34 D-1017 CCUGCUUCAUG CCCUUCUAUU 35 UAGAAGGGCAUG AAGCAGGUU 36 D-1018 CUGCUUCAUGC CCUUCUACUU 37 GUAGAGGGCAU GAAGCAGUU 38 D-1019 UGCUUCAUGCC CUUCUACAUU 39 UGUAGAAGGGCA UGAAGCAUU 40 D-1020 GCUUCAUGCCC UUCUACAGUU 41 CUGUAGAAGGGC AUGAAGCUU 42 D-1021 CUUCAUGCCCU UCUACAGUUU 43 ACUGUAGAAGGG CAUGAAGUU 44 D-1022 UUCAUGCCCUU CUACAGUGUU 45 CACUGUAGAGG GCAUGAAUU 46 D-1023 UCAUGCCCUUC UACAGUGGUU 47 CCACUGUAGAAG GGCAUGAUU 48

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D-1024 CAUGCCCUUCU ACAGUGGCUU 49 GCCACUGUAGAA GGGCAUGUU 50 D-1025 AUGCCCUUCUA CAGUGGCCUU 51 GGCCACUGUAGA AGGGCAUUU 52 D-1026 UGCCCUUCUAC AGUGGCCUUU 53 AGGCCACUGUAG AAGGGCAUU 54 D-1027 GCCCUUCUACA GUGGCCUUUU 55 AAGGCCACUGUA GAAGGGCUU 56 D-1028 GGUAUGUUCCU GCUUCAUCUU 57 GAUGAAGCAGGA ACAUACCUU 58 D-1029 GUAUGUUCCUG CUUCAUCCUU 59 GGAUGAAGCAGG AACAUACUU 60 D-1030 UAUGUUCCUGC UUCAUCCCUU 61 GGGAUGAAGCAG GAACAUAUU 62 D-1031 AUGUUCCUGCU UCAUCCCCUU 63 GGGGAUGAAGCA GGAACAUUU 64 D-1032 UGUUCCUGCUU CAUCCCCUUU 65 AGGGGAUGAAGC AGGAACAUU 66 D-1033 GUUCCUGCUUC AUCCCCUUUU 67 AAGGGGAUGAAG CAGGAACUU 68 D-1034 UUCCUGCUUCA uccccuucuu 69 GAAGGGGAUGAA GCAGGAAUU 70 D-1035 UCCUGCUUCAU ccccuucuuu 71 AGAAGGGGAUGA AGCAGGAUU 72 D-1036 CCUGCUUCAUC CCCUUCUAUU 73 UAGAAGGGGAUG AAGCAGGUU 74 D-1037 CUGCUUCAUCC CCUUCUACUU 75 GUAGAGGGGAU GAAGCAGUU 76 D-1038 UGCUUCAUCCC CUUCUACAUU 77 UGUAGAAGGGGA UGAAGCAUU 78

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D-1039 UUCAUCCCCUU CUACAGUGUU 79 CACUGUAGAGG GGAUGAAUU 80 D-1040 UCAUCCCCUUC UACAGUGGUU 81 CCACUGUAGAAG GGGAUGAUU 82 D-1041 CAUCCCCUUCU ACAGUGGCUU 83 GCCACUGUAGAA GGGGAUGUU 84 D-1042 UCCCCUUCUAC AGUGGCCUUU 85 AGGCCACUGUAG AAGGGGAUU 86 D-1043 GAUCAGGACCC GAGCCGAUUU 87 AUCGGCUCGGGU CCUGAUCUU 88 D-1044 UGGGCUUCUAC CACGUCGUUU 89 ACGACGUGGUAG AAGCCCAUU 90 D-1045 GAGCGAGCACG CCCCGCAUUU 91 AUGCGGGGCGUG CUCGCUCUU 92 D-1046 UGCACUGCGUC GGCGUCCUUU 93 AGGACGCCGACG CAGUGCAUU 94 D-1047 UGGAGCAGACU CUGCAGGUUU 95 ACCUGCAGAGUC UGCUCCAUU 96 D-1048 UGCAGGUCCUC UCAGAUCUUU 97 AGAUCUGAGAGG ACCUGCAUU 98 D-1049 CCCGGCCAAUG UCCACCAUUU 99 AUGGUGGACAUU GGCCGGGUU 100 D-1050 UUCUACAGUGG CCUUAUCUUU 101 AGAUAAGGCCAC UGUAGAAUU 102 D-1051 UCUACAGUGGC CUUAUCCUUU 103 AGGAUAAGGCCA CUGUAGAUU 104 D-1052 CUUCCUUCAGA GGCGUGCUUU 105 AGCACGCCUCUG AAGGAAGUU 106 D-1053 UUCCUUCAGAG GCGUGCGAUU 107 UCGCACGCCUCU GAAGGAAUU 108

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D-1054 GCGUGCGAUAU GUGGAUGUUU 109 ACAUCCACAUAU CGCACGCUU 110 D-1055 CGUGCGAUAUG UGGAUGGAUU 111 UCCAUCCACAUA UCGCACGUU 112 D-1056 UGGAUGGAGGA GUGAGUGAUU 113 UCACUCACUCCCUC CAUCCAUU 114 D-1057 ACGUACCCUUC AUUGAUGUUU 115 ACAUCAAUGAAG GGUACGUUU 116 D-1058 UGGACAUCACC AAGCUCAUUU 117 AUGAGCUUGGUG AUGUCCAUU 118 D-1059 CACCUGCGUCU CAGCAUCUUU 119 AGAUGCUGAGAC GCAGGUGUU 120 D-1060 ACCUGCGUCUC AGCAUCCUUU 121 AGGAUGCUGAGA CGCAGGUUU 122 D-1061 CCAGAGACUGG UGACAUGUUU 123 ACAUGUCACCAG UCUCUGGUU 124 D-1062 AUGGCUUCCAG AUAUGCCUUU 125 AGGCAUAUCUGG AAGCCAUUU 126 D-1063 CCGCCUCCAGG UCCCAAAUUU 127 AUUUGGGACCUG GAGGCGGUU 128 D-1064 UACCUGCUGGU GCUGAGGUUU 129 ACCUCAGCACCA GCAGGUAUU 130 D-1065 ACCUGCUGGUG CUGAGGGUUU 131 ACCCUCAGCACCA GCAGGUUU 132 D-1066 CUCUCCACCUU UCCCAGUUUU 133 AACUGGGAAAGG UGGAGAGUU 134 D-1067 UUUUUCACCUA ACUAAAAUUU 135 AUUUUAGUUAGG UGAAAAAUU 136 D-1068 CGGCCAAUGUC CACCAGCUUU 137 AGCUGGUGGACA UUGGCCGUU 138

- 37 047656

D-1069 GGUCCAGCCUG AACUUCUUUU 139 AAGAAGUUCAGG CUGGACCUU 140 D-1070 GCGGCUUCCUG GGCUUCUAUU 141 UAGAAGCCCAGG AAGCCGCUU 142 D-1071 GUGACAACGUA CCCUUCAUUU 143 AUGAAGGGUACG UUGUCACUU 144 D-1072 GGUAUGUUCCU GCUUCAUGUU 145 CAUGAAGCAGGA ACAUACCUU 146 D-1073 GUAUGUUCCUG CUUCAUGCUU 147 GCAUGAAGCAGG AACAUACUU 148 D-1074 UGUUCCUGCUU CAUGCCCUUU 149 AGGGCAUGAAGC AGGAACAUU 150 D-1075 GUUCCUGCUUC AUGCCCUUUU 151 AAGGGCAUGAAG CAGGAACUU 152 D-1076 CCUGCUUCAUG CCCUUCUAUU 153 UAGAAGGGCAUG AAGCAGGUU 154 D-1077 GCUUCAUGCCC UUCUACAGUU 155 CUGUAGAAGGGC AUGAAGCUU 156 D-1078 CUUCAUGCCCU UCUACAGUUU 157 ACUGUAGAAGGG CAUGAAGUU 158 D-1079 UUCAUGCCCUU CUACAGUGUU 159 CACUGUAGAGG GCAUGAAUU 160 D-1080 AUGGCUUCCAG AUAUGCCUUU 161 AGGCAUAUCUGG AAGCCAUUU 162 D-1081 AUGCCCUUCUA CAGUGGCCUU 163 GGCCACUGUAGA AGGGCAUUU 164 D-1082 GCUUCAUGCCC UUCUACAUUU 165 AUGUAGAGGGC AUGAAGCUU 166 D-1083 GGAAAGACUGU UCCAAAAAUU 333 UUUUUGGAACAG UCUUUCCUU 334

- 38 047656

D-1084 GGUAUGUUCCU GCUUCAUGUU 335 CAUGAAGCAGGA ACAUACCUU 336 D-1085 GUAUGUUCCUG CUUCAUGCUU 337 GCAUGAAGCAGG AACAUACUU 338 D-1086 UGUUCCUGCUU CAUGCCCUUU 339 AGGGCAUGAAGC AGGAACAUU 340 D-1087 GCUUCAUGCCC UUCUACAGUU 341 CUGUAGAAGGGC AUGAAGCUU 342 D-1088 CUUCAUGCCCU UCUACAGUUU 343 ACUGUAGAAGGG CAUGAAGUU 344 D-1089 GCGGCUUCCUG GGCUUCUAUU 345 UAGAAGCCCAGG AAGCCGCUU 346 D-1090 GUUCCUGCUUC AUGCCCUUUU 347 AAGGGCAUGAAG CAGGAACUU 348 D-1091 AUGGCUUCCAG AUAUGCCUUU 349 AGGCAUAUCUGG AAGCCAUUU 350 D-1092 CCUGCUUCAUG CCCUUCUAUU 351 UAGAAGGGCAUG AAGCAGGUU 352 D-1093 UUCAUGCCCUU CUACAGUUUU 353 AACUGUAGAGG GCAUGAAUU 354 D-1094 UUCAUGCCCUU CUACAGUGUU 355 CACUGUAGAGG GCAUGAAUU 356 D-1095 GCUUCAUGCCC UUCUACAUUU 357 AUGUAGAGGGC AUGAAGCUU 358 D-1096 GGUCCAGCCUG AACUUCUUUU 359 AAGAAGUUCAGG CUGGACCUU 360 D-1097 GCGGCUUCCUG GGCUUCUAUU 361 UAGAAGCCCAGG AAGCCGCUU 362 D-1098 GCGGCUUCCUG GGCUUCUAUU 363 UAGAAGCCCAGG AAGCCGCUU 364

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D-1099 GUUCCUGCUUC AUGCCCUUUU 365 AAGGGCAUGAAG CAGGAACUU 366 D-1100 GUUCCUGCUUC AUGCCCUUUU 367 AAGGGCAUGAAG CAGGAACUU 368 D-1101 CCUGCUUCAUG CCCUUCUAUU 369 UAGAAGGGCAUG AAGCAGGUU 370 D-1102 CCUGCUUCAUG CCCUUCUAUU 371 UAGAAGGGCAUG AAGCAGGUU 372 D-1103 AUGCCCUUCUA CAGUGGCCUU 373 GGCCACUGUAGA AGGGCAUUU 374 D-1104 AUGGCUUCCAG AUAUGCCUUU 375 AGGCAUAUCUGG AAGCCAUUU 376 D-1105 GUGACAACGUA CCCUUCAUUU 377 AUGAAGGGUACG UUGUCACUU 378 D-1106 GUAUGUUCCUG CUUCAUGCUU 379 GCAUGAAGCAGG AACAUACUU 380 D-1107 GUAUGUUCCUG CUUCAUGCUU 381 GCAUGAAGCAGG AACAUACUU 382 D-1108 GUAUGUUCCUG CUUCAUGCUU 383 GCAUGAAGCAGG AACAUACUU 384 D-1109 GUAUGUUCCUG CUUCAUGCCU 385 AGGCAUGAAGCA GGAACAUACUU 386 D-1110 UGGUAUGUUCC UGCUUCAUGU 387 GCAUGAAGCAGG AACAUACCAUU 388 D-llll GUAUGUUCCUG CUUCAUGU 389 GCAUGAAGCAGG AACAUACUU 390 D-1112 GUAUGUUCCUG CUUCAUGC{INV AB] 391 GCAUGAAGCAGG AACAUACUU 392 D-1113 GCUUCAUGCCC 393 AUGUAGAGGGC 394

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UUCUACAUUU AUGAAGCUU D-1114 GCUUCAUGCCC UUCUACAUUU 395 AUGUAGAGGGC AUGAAGCUU 396 D-1115 GCUUCAUGCCC UUCUACAUUU 397 AUGUAGAGGGC AUGAAGCUU 398 D-1116 GCUUCAUGCCC UUCUACAUUU 399 AUGUAGAGGGC AUGAAGCUU 400 D-1117 GCUUCAUGCCC UUCUACAUUU 401 AUGUAGAGGGC AUGAAGCUU 402 D-1118 GCUUCAUGCCC UUCUACAUUU 403 AUGUAGAGGGC AUGAAGCUU 404 D-1119 GCUUCAUGCCC UUCUACAUUU 405 AUGUAGAGGGC AUGAAGCUU 406 D-1120 GCUUCAUGCCC UUCUACAUUU 407 AUGUAGAGGGC AUGAAGCUU 408 D-1121 GCUUCAUGCCC UUCUACAUUU 409 AUGUAGAGGGC AUGAAGCUU 410 D-1122 GCUUCAUGCCC UUCUACAUUU 411 AUGUAGAGGGC AUGAAGCUU 412 D-1123 GCUUCAUGCCC UUCUACAUUU 413 AUGUAGAGGGC AUGAAGCUU 414 D-1124 GCUUCAUGCCC UUCUACAUUU 415 AUGUAGAGGGC AUGAAGCUU 416 D-1125 GCUUCAUGCCC UUCUACAUUU 417 AUGUAGAGGGC AUGAAGCUU 418 D-1126 GCUUCAUGCCC UUCUACAUUU 419 AUGUAGAGGGC AUGAAGCUU 420 D-1127 GCUUCAUGCCC UUCUACAUUU 421 AUGUAGAGGGC AUGAAGCUU 422 D-1128 GCUUCAUGCCC 423 AUGUAGAGGGC 424

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UUCUACAUUU AUGAAGCUU D-1129 GCUUCAUG[DC] CCUUCUACAUU u 425 AUGUAGAGGGC AUGAAGCUU 426 D-1130 GCUUCAUGCCC DC]UUCUACAU uu 427 AUGUAGAGGGC AUGAAGCUU 428 D-1131 GCUUCAUGC[D C]CUUCUACAU UU 429 AUGUAGAGGGC AUGAAGCUU 430 D-1132 GCUUCAUGCCC UUCUACAUUU 431 AUGUAGAGGGC AUGAAGCUU 432 D-1133 GCUUCAUGCCC UUCUACAUUU 433 AUGUAGAGGGC AUGAAGCUU 434 D-1134 GCUUCAUGCCC UUCUACAUUU 435 AUGUAGAGGGC AUGAAGCUU 436 D-1135 GCUUCAUGCCC UUCUACAUUU 437 AUGUAGAGGGC AUGAAGCUU 438 D-1136 GCUUCAUGCCC UUCUACAUUU 439 AUGUAGAGGGC AUGAAGCUU 440 D-1137 GCUUCAUGCCC UUCUACAGUU 441 AACUGUAGAAGG GCAUGAAGCUU 442 D-1138 CUGCUUCAUGC CCUUCUACAU 443 AUGUAGAGGGC AUGAAGCAGUU 444 D-1139 GCUUCAUGCCC UUCUACAU 445 AUGUAGAGGGC AUGAAGCUU 446 D-1140 GCUUCAUGCCC UUCUACAU{INV AB] 447 AUGUAGAGGGC AUGAAGCUU 448 D-1141 GCUUCAUGCCC UUCUACAUUU{I 449 AUGUAGAGGGC AUGAAGCUU 450

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NV AB} D-1142 GCUUCAUGCCC UUCUACAUUU 451 AUGUAGAGGGC AUGAAGCUU 452 D-1143 GCUUCAUGCCC UUCUACAUUU 453 AUGUAGAGGGC AUGAAGCUU 454 D-1144 GCUUCAUGCCC UUCUACAUUU 455 AUGUAGAGGGC AUGAAGCUU 456 D-1145 GCUUCAUGCCC UUCUACAUUU 457 AUGUAGAGGGC AUGAAGCUU 458 D-1146 GCUUCAUGCCC UUCUACAUUU 459 AUGUAGAGGGC AUGAAGCUU 460 D-1147 GCUUCAUGCCC UUCUACAUUU 461 AUGUAGAGGGC AUGAAGCUU 462 D-1148 GCUUCAUGCCC UUCUACAUUU 463 AUGUAGAGGGC AUGAAGCUU 464 D-1149 GCUUCAUGCCC UUCUACAUUU 465 AUGUAGAGGGC AUGAAGCUU 466 D-1150 GCUUCAUGCCC UUCUACAUUU 467 AUGUAGAGGGC AUGAAGCUU 468 D-1151 GUAUGUUCCUG CUUCAUGCUU{I NV AB} 469 GCAUGAAGCAGG AACAUACUU 470 D-1152 GGUAUGUUCCU GCUUCAUUUU 471 AAUGAAGCAGGA ACAUACCUU 472 D-1153 GUAUGUUCCUG CUUCAUGUUU 473 ACAUGAAGCAGG AACAUACUU 474 D-1154 CGGCCAAUGUC CACCAGCUUU 475 AGCUGGUGGACA UUGGCCGUU 476 D-1155 UGGAGCAGACU CUGCAGGUUU 477 ACCUGCAGAGUC UGCUCCAUU 478

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D-1156 ACGUACCCUUC AUUGAUGUUU 479 ACAUCAAUGAAG GGUACGUUU 480 D-1157 CCAGAGACUGG UGACAUGUUU 481 ACAUGUCACCAG UCUCUGGUU 482 D-1158 [INVAB]GCUUC AUGCCUUUCUA CAUUU 483 AUGUAGAAAGGC AUGAAGCUU 484 D-1159 [INVAB]GCUUC AUGCCUUUCUA CAUUU 485 AAAUGUAGAAAG GCAUGAAGCUU 486 D-1160 [INVAB]GCUUC AUGCCUUUCUA CAUUU 487 AAAUGUAGAAAG GCAUGAAGCUU 488 D-1161 UGCUUCAUGCC UUUCUACAUU 489 UGUAGAAAGGCA UGAAGCAUU 490 D-1162 UAUGUUCCUGC UUCAUGCUUU 491 AGCAUGAAGCAG GAACAUAUU 492 D-1163 UUCCUGCUUCA UGCCUUUUUU 493 AAAAGGCAUGAA GCAGGAAUU 494 D-1164 UCAUGCCUUUC UACAGUGUUU 495 ACACUGUAGAAA GGCAUGAUU 496 D-1165 CAUGCCUUUCU ACAGUGGUUU 497 ACCACUGUAGAA AGGCAUGUU 498 D-1166 AUGCCUUUCUA CAGUGGCUUU 499 AGCCACUGUAGA AAGGCAUUU 500 D-1167 GGUAUGUUCCU GCUUCAUAUU 501 UAUGAAGCAGGA ACAUACCUU 502 D-1168 GUAUGUUCCUG CUUCAUGAUU 503 UCAUGAAGCAGG AACAUACUU 504 D-1169 UAUGUUCCUGC 505 UGCAUGAAGCAG 506

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UUCAUGCAUU GAACAUAUU D-1170 UUCCUGCUUCA UGCCUUUAUU 507 UAAAAGGCAUGAA GCAGGAAUU 508 D-1171 CUGCUUCAUGC CUUUCUAAUU 509 UUAGAAAGGCAU GAAGCAGUU 510 D-1172 GCUUCAUGCCU UUCUACAAUU 511 UUGUAGAAAGGC AUGAAGCUU 512 D-1173 UUCAUGCCUUU CUACAGUAUU 513 UACUGUAGAAAG GCAUGAAUU 514 D-1174 UCAUGCCUUUC UACAGUGAUU 515 UCACUGUAGAAA GGCAUGAUU 516 D-1175 CAUGCCUUUCU ACAGUGGAUU 517 UCCACUGUAGAA AGGCAUGUU 518 D-1176 AUGCCUUUCUA CAGUGGCAUU 519 UGCCACUGUAGA AAGGCAUUU 520 D-1177 ACGUACCCUUC AUUGAUGAUU 521 UCAUCAAUGAAG GGUACGUUU 522 D-1178 CCAGAGACUGG UGACAUGAUU 523 UCAUGUCACCAG UCUCUGGUU 524 D-1179 AUGGCUUCCAG AUAUGCCAUU 525 UGGCAUAUCUGG AAGCCAUUU 526 D-1180 GUUCCUGCUUC AUGCCUUUUU 527 AAAGGCAUGAAG CAGGAACUU 528 D-1181 CCUGCUUCAUG CCUUUCUAUU 529 UAGAAAGGCAUG AAGCAGGUU 530 D-1182 GCUUCAUGCCU UUCUACAUUU 531 AUGUAGAAAGGC AUGAAGCUU 532 D-1183 CUUCAUGCCUU UCUACAGUUU 533 ACUGUAGAAAGG CAUGAAGUU 534 D-1184 UUCAUGCCUUU 535 AACUGUAGAAAG 536

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CUACAGUUUU GCAUGAAUU D-1185 GCUUCAUCCCU UUCUACAUUU 537 AUGUAGAAAGGG AUGAAGCUU 538 D-1186 [INVABJGCUUC AUGCCCUUCUA CAUUU 539 AUGUAGAGGGC AUGAAGCUU 540 D-1187 [INVABJGCUUC AUGCCCUUCUA CAUUU 541 AAAUGUAGAGG GCAUGAAGCUU 542 D-1188 [INVABJGCUUC AUGCCCUUCUA CAUUU 543 AAAUGUAGAGG GCAUGAAGCUU 544 D-1189 [INVABJGCGGC UUCCUGGGCUU CUAUU 545 UAGAAGCCCAGG AAGCCGCUU 546 D-1190 [INVABJCUGCG GCUUCCUGGGC UUCUA 547 UAGAAGCCCAGG AAGCCGCAGUU 548 D-1191 CUGCGGCUUCC UGGGCUUCU{IN VABJ 549 UAGAAGCCCAGG AAGCCGCAGUU 550 D-1192 [INVABJCUGCG GCUUCCUGGGC UUCUA 551 UAGAAGCCCAGG AAGCCGCAGUU 552 D-1193 [INVABJGCGGC UUCCUGGGCUU CUAUU 553 UAGAAGCCCAGG AAGCCGCUU 554 D-1194 CUGCGGCUUCC UGGGCUUCU{IN VABJ 555 UAGAAGCCCAGG AAGCCGCAGUU 556 D-1195 [INVABJCUGCG 557 UAGAAGCCCAGG 558

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GCUUCCUGGGC UUCUA AAGCCGCAGUU D-1196 [INVAB] GCGGC UUCCUGGGCUU CUAUU 559 UAGAAGCCCAGG AAGCCGCUU 560 D-1197 CUGCGGCUUCC UGGGCUUCU{IN VAB] 561 UAGAAGCCCAGG AAGCCGCAGUU 562 D-1198 [INVAB]CUGCG GCUUCCUGGGC UUCUA 563 UAGAAGCCCAGG AAGCCGCAGUU 564 D-1199 [INVAB] GCGGC UUCCUGGGCUU CUAUU 565 UAGAAGCCCAGG AAGCCGCUU 566 D-1200 [INVAB]CUGCG GCUUCCUGGGC UUCUA 567 UAGAAGCCCAGG AAGCCGCAGUU 568 D-1201 [INVAB]CUGCG GCUUCCUGGGC UUCUA 569 UAGAAGCCCAGG AAGCCGCAGUU 570 D-1202 [INVAB]CUGCG GCUUCCUGGGC UUCUA 571 UAGAAGCCCAGG AAGCCGCAGUU 572 D-1203 [INVAB]AUGGC UUCCAGAUAUG CCUUU 573 AGGCAUAUCUGG AAGCCAUUU 574 D-1204 [INVAB]ACAUG GCUUCCAGAUA UGCCU 575 AGGCAUAUCUGG AAGCCAUGUUU 576 D-1205 ACAUGGCUUCC AGAUAUGCC{IN VAB} 577 AGGCAUAUCUGG AAGCCAUGUUU 578

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D-1206 [INVAB]ACAUG GCUUCCAGAUA UGCCU 579 AGGCAUAUCUGG AAGCCAUGUUU 580 D-1207 [INVAB]AUGGC UUCCAGAUAUG CCUUU 581 AGGCAUAUCUGG AAGCCAUUU 582 D-1208 ACAUGGCUUCC AGAUAUGCC{IN VAB} 583 AGGCAUAUCUGG AAGCCAUGUUU 584 D-1209 [INVABJACAUG GCUUCCAGAUA UGCCU 585 AGGCAUAUCUGG AAGCCAUGUUU 586 D-1210 [TNVAB]AUGGC UUCCAGAUAUG CCUUU 587 AGGCAUAUCUGG AAGCCAUUU 588 D-1211 ACAUGGCUUCC AGAUAUGCC{IN VAB} 589 AGGCAUAUCUGG AAGCCAUGUUU 590 D-1212 [INVAB]ACAUG GCUUCCAGAUA UGCCU 591 AGGCAUAUCUGG AAGCCAUGUUU 592 D-1213 [INVAB]AUGGC UUCCAGAUAUG CCUUU 593 AGGCAUAUCUGG AAGCCAUUU 594 D-1214 ACAUGGCUUCC AGAUAUGCC{IN VAB} 595 AGGCAUAUCUGG AAGCCAUGUUU 596 D-1215 [INVAB]ACAUG GCUUCCAGAUA UGCCU 597 AGGCAUAUCUGG AAGCCAUGUUU 598 D-1216 [INVAB]ACAUG 599 AGGCAUAUCUGG 600

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GCUUCCAGAUA UGCCU AAGCCAUGUUU D-1217 [INVABJACAUG GCUUCCAGAUA UGCCU 601 AGGCAUAUCUGG AAGCCAUGUUU 602 D-1218 [INVABJACAUG GCUUCCAGAUA UGCCU 603 AGGCAUAUCUGG AAGCCAUGUUU 604 D-1219 [INVABJACGUA CCCUUCAUUGA UGUUU 605 ACAUCAAUGAAG GGUACGUUU 606 D-1220 [INVABJCAACG UACCCUUCAUU GAUGU 607 ACAUCAAUGAAG GGUACGUUGUU 608 D-1221 [INVABJCAACG UACCCUUCAUU GAUGU 609 ACAUCAAUGAAG GGUACGUUGUU 610 D-1222 [INVABJACGUA CCCUUCAUUGA UGUUU 611 ACAUCAAUGAAG GGUACGUUU 612 D-1223 CAACGUACCCCU UCAUUGAUGJI NVABJ 613 ACAUCAAUGAAG GGUACGUUGUU 614 D-1224 [INVABJACGUA CCCUUCAUUGA UGUUU 615 ACAUCAAUGAAG GGUACGUUU 616 D-1225 CAACGUACCCCU UCAUUGAUGJI NVAB} 617 ACAUCAAUGAAG GGUACGUUGUU 618 D-1226 [INVABJCAACG UACCCUUCAUU GAUGU 619 ACAUCAAUGAAG GGUACGUUGUU 620

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D-1227 [INVABJACGUA CCCUUCAUUGA UGUUU 621 ACAUCAAUGAAG GGUACGUUU 622 D-1228 CAACGUACCCCU UCAUUGAUGJI NVABJ 623 ACAUCAAUGAAG GGUACGUUGUU 624 D-1229 [INVABJCAACG UACCCUUCAUU GAUGU 625 ACAUCAAUGAAG GGUACGUUGUU 626 D-1230 [INVABJCAACG UACCCUUCAUU GAUGU 627 ACAUCAAUGAAG GGUACGUUGUU 628 D-1231 [INVABJCAACG UACCCUUCAUU GAUGU 629 ACAUCAAUGAAG GGUACGUUGUU 630 D-1232 [INVABJCAACG UACCCUUCAUU GAUGU 631 ACAUCAAUGAAG GGUACGUUGUU 632 D-1233 CUGCUUCAUGC CUUUCUACAU 633 AUGUAGAAAGGC AUGAAGCAGUU 634 D-1234 CUGCUUCAUGC CUUUCUACAU 635 AUGUAGAAAGGC AUGAAGCAGUU 636 D-1235 [INVABJGCUUC AUGCCUUUCUA CAUUU 637 AUGUAGAAAGGC AUGAAGCUU 638 D-1236 [INVABJGCUUC AUGCCUUUCUA CAUUU 639 AUGUAGAAAGGC AUGAAGCUU 640 D-1237 CUGCUUCAUGC CUUUCUACAU 641 AUGUAGAAAGGC AUGAAGCAGUU 642 D-1238 CUGCUUCAUGC 643 AUGUAGAAAGGC 644

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CUUUCUACAU AUGAAGCAGUU D-1239 [INVABJCUGCU UCAUGCCUUUC UACAU 645 AUGUAGAAAGGC AUGAAGCAGUU 646 D-1240 CUGCUUCAUGC CUUUCUACA{IN VAB} 647 AUGUAGAAAGGC AUGAAGCAGUU 648 D-1241 [INVAB]CUGCU UCAUGCCUUUC UACAU 649 AUGUAGAAAGGC AUGAAGCAGUU 650 D-1242 [INVAB]CUGCU UCAUGC[DC]UU UCUACAU 651 AUGUAGAAAGGC AUGAAGCAGUU 652 D-1243 CUGCUUCAUGC CUUUCUACAU 653 AUGUAGAAAGGC AUGAAGCAGUU 654 D-1244 [INVAB]CUGCU UCAUGCCUUUC UACAU 655 AUGUAGAAAGGC AUGAAGCAGUU 656 D-1245 CUGCUUCAUGC CUUUCUACAU 657 AUGUAGAAAGGC AUGAAGCAGUU 658 D-1246 CUGCUUCAUGC CUUUCUACA{IN VAB} 659 AUGUAGAAAGGC AUGAAGCAGUU 660 D-1247 [INVAB]CUGCU UCAUGCCUUUC UACAU 661 AUGUAGAAAGGC AUGAAGCAGUU 662 D-1248 CUGCUUCAUGC CUUUCUACAU 663 AUGUAGAAAGGC AUGAAGCAGUU 664 D-1249 CUGCUUCAUGC CUUUCUACA{IN VAB} 665 AUGUAGAAAGGC AUGAAGCAGUU 666

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D-1250 [INVAB]CUGCU UCAUGCCUUUC UACAU 667 AUGUAGAAAGGC AUGAAGCAGUU 668 D-1251 CUGCUUCAUGC CUUUCUACA{IN VAB} 669 AUGUAGAAAGGC AUGAAGCAGUU 670 D-1252 [INVABJCUGCU UCAUGCCUUUC UACAU 671 AUGUAGAAAGGC AUGAAGCAGUU 672 D-1253 [INVABJGCUUC AUGCCUUUCUA CAUUU 673 AUGUAGAAAGGC AUGAAGCUU 674 D-1254 [INVABJGCUUC AUGCCUUUCUA CAUUU 675 AUGUAGAAAGGC AUGAAGCUU 676 D-1255 [INVAB]GCUUC AUGCCUUUCUA CAUUU 677 AUGUAGAAAGGC AUGAAGCUU 678 D-1256 [INVAB]GCUUC AUGCCUUUCUA CAUUU 679 AUGUAGAAAGGC AUGAAGCUU 680 D-1257 [INVAB]GCUUC AUGCCUUUCUA CAUUU 681 AUGUAGAAAGGC AUGAAGCUU 682 D-1258 CUGCUUCAUGC CCUUCUACAU 683 AUGUAGAGGGC AUGAAGCAGUU 684 D-1259 [INVAB]GCUUC AUGCCCUUCUA CAUUU 685 AUGUAGAGGGC AUGAAGCUU 686 D-1260 [INVAB]GCUUC AUGCCCUUCUA 687 AUGUAGAGGGC AUGAAGCUU 688

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CAUUU D-1261 [INVABJGCUUC AUGCCCUUCUA CAUUU 689 AUGUAGAGGGC AUGAAGCUU 690 D-1262 CUGCUUCAUGC CCUUCUACA{IN VAB} 691 AUGUAGAGGGC AUGAAGCAGUU 692 D-1263 [INVAB]CUGCU UCAUGCCCUUC UACAU 693 AUGUAGAGGGC AUGAAGCAGUU 694 D-1264 [INVAB]GCUUC AUGCCCUUCUA CAUUU 695 AUGUAGAGGGC AUGAAGCUU 696 D-1265 AUGUUCCUGCU UCAUGCCUUU 697 AGGCAUGAAGCA GGAACAUUU 698 D-1266 UGUUCCUGCUU CAUGCCUUUU 699 AAGGCAUGAAGC AGGAACAUU 700 D-1267 UGCCUUUCUAC AGUGGCCUUU 701 AGGCCACUGUAG AAAGGCAUU 702 D-1268 CGUACUUCGUC CUUGUAUGUU 703 CAUACAAGGACG AAGUACGUU 704 D-1269 [TNVAB]GCUUC AUGC[DC]CUUC UACAUUU 705 AUGUAGAGGGC AUGAAGCUU 706 D-1270 [INVAB]GCUUC AUGCCCUUCUA CAUUU 707 AUGUAGAGGGC AUGAAGCUU 708 D-1271 [INVAB]GCUUC AUGCCCUUCUA CAUUU 709 AUGUAGAGGGC AUGAAGCUU 710 D-1272 [INVAB]GCUUC 711 AUGUAGAGGGC 712

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AUGCCCUUCUA CAUUU AUGAAGCUU D-1273 [TNVAB]GCUUC AUGCCCUUCUA CAUUU 713 AUGUAGAGGGC AUGAAGCUU 714 D-1274 [INVAB]GCUUC AUGCCCUUCUA CAUUU 715 AUGUAGAGGGC AUGAAGCUU 716 D-1275 [INVAB]CCUGC UUCAUGCCUUU CUAUU 717 UAGAAAGGCAUG AAGCAGGUU 718 D-1276 [INVAB]CCUGC UUCAUGCCUUU CUAUU 719 UAGAAAGGCAUG AAGCAGGUU 720 D-1277 UUCCUGCUUCA UGCCUUUCU{IN VAB} 721 UAGAAAGGCAUG AAGCAGGAAUU 722 D-1278 [INVAB]CCUGC UUCAUGCCUUU CUAUU 723 UAGAAAGGCAUG AAGCAGGUU 724 D-1279 UUCCUGCUUCA UGCCUUUCU{IN VAB} 725 UAGAAAGGCAUG AAGCAGGAAUU 726 D-1280 [INVAB]AUGCC UUUCUACAGUG GCUUU 727 AGCCACUGUAGA AAGGCAUUU 728 D-1281 [INVAB]AUGCC UUUCUACAGUG GCUUU 729 AGCCACUGUAGA AAGGCAUUU 730 D-1282 [INVAB]AUGCC UUUCUACAGUG GCUUU 731 AGCCACUGUAGA AAGGCAUUU 732

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D-1283 UCAUGCCUUUCUACAGUGGC{IN VAB} 733 AGCCACUGUAGA AAGGCAUGAUU 734 D-1284 [INVAB]AUGCC UUUCUACAGUG GCUUU 735 AGCCACUGUAGA AAGGCAUUU 736 D-1285 [INVABJUCAUG CCUUUCUACAG UGGCU 737 AGCCACUGUAGA AAGGCAUGAUU 738 D-1286 [INVABJUGCUU CAUGCCUUUCU ACAGU 739 ACUGUAGAAAGG CAUGAAGCAUU 740 D-1287 [TNVAB]CUUCA UGCCUUUCUAC AGUUU 741 ACUGUAGAAAGG CAUGAAGUU 742 D-1289 UGCUUCAUGCC UUUCUACAG{IN VAB} 743 ACUGUAGAAAGG CAUGAAGCAUU 744 D-1290 [INVAB]CUUCA UGCCUUUCUAC AGUUU 745 ACUGUAGAAAGG CAUGAAGUU 746 D-1291 UGCUUCAUGCC UUUCUACAG{IN VAB} 747 ACUGUAGAAAGG CAUGAAGCAUU 748 D-1292 [INVAB]UGCUU CAUGCCUUUCU ACAGU 749 ACUGUAGAAAGG CAUGAAGCAUU 750 D-1293 [INVAB]UGCUU CAUGCCUUUCU ACAGU 751 ACUGUAGAAAGG CAUGAAGCAUU 752 D-1294 [INVAB]UGCUU 753 ACUGUAGAAAGG 754

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CAUGCCUUUCU ACAGU CAUGAAGCAUU D-1295 [TNVAB]UGCUU CAUGCCUUUCU ACAGU 755 ACUGUAGAAAGG CAUGAAGCAUU 756 D-1296 [INVAB]GUUCC UGCUUCAUGCC UUUUU 757 AAAGGCAUGAAG CAGGAACUU 758 D-1297 [INVAB]CCUGC UUCAUGCCUUU CUAUU 759 UAGAAAGGCAUG AAGCAGGUU 760 D-1298 [INVAB]CUUCA UGCCUUUCUAC AGUUU 761 ACUGUAGAAAGG CAUGAAGUU 762 D-1299 [INVAB]UUCCU GCUUCAUGCCU UUCUA 763 UAGAAAGGCAUG AAGCAGGAAUU 764 D-1300 UUCCUGCUUCA UGCCUUUCU{IN VAB} 765 UAGAAAGGCAUG AAGCAGGAAUU 766 D-1301 [INVAB]UUCCU GCUUCAUGCCU UUCUA 767 UAGAAAGGCAUG AAGCAGGAAUU 768 D-1302 UUCCUGCUUCA UGCCUUUCU{IN VAB} 769 UAGAAAGGCAUG AAGCAGGAAUU 770 D-1303 [INVAB]UUCCU GCUUCAUGCCU UUCUA 771 UAGAAAGGCAUG AAGCAGGAAUU 772 D-1304 [INVAB]UUCCU GCUUCAUGCCU UUCUA 773 UAGAAAGGCAUG AAGCAGGAAUU 774

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D-1305 [INVABJUUCCU GCUUCAUGCCU UUCUA 775 UAGAAAGGCAUG AAGCAGGAAUU 776 D-1306 [INVABJUUCCU GCUUCAUGCCU UUCUA 777 UAGAAAGGCAUG AAGCAGGAAUU 778 D-1307 [INVABJUUCCU GCUUCAUGCCU UUCUA 779 UAGAAAGGCAUG AAGCAGGAAUU 780 D-1308 [INVABJUUCCU GCUUCAUGCCU UUCUA 781 UAGAAAGGCAUG AAGCAGGAAUU 782 D-1309 [INVABJUCAUG CCUUUCUACAG UGGCU 783 AGCCACUGUAGA AAGGCAUGAUU 784 D-1310 UCAUGCCUUUCUACAGUGGC{IN VAB} 785 AGCCACUGUAGA AAGGCAUGAUU 786 D-1311 [INVABJUCAUG CCUUUCUACAG UGGCU 787 AGCCACUGUAGA AAGGCAUGAUU 788 D-1312 UCAUGCCUUUCUACAGUGGC{IN VAB} 789 AGCCACUGUAGA AAGGCAUGAUU 790 D-1313 [INVABJUCAUG CCUUUCUACAG UGGCU 791 AGCCACUGUAGA AAGGCAUGAUU 792 D-1314 [INVABJUCAUG CCUUUCUACAG UGGCU 793 AGCCACUGUAGA AAGGCAUGAUU 794 D-1315 UCAUGCCUUUC 795 AGCCACUGUAGA 796

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UACAGUGGC{IN VAB} AAGGCAUGAUU D-1316 [TNVAB]UCAUG CCUUUCUACAG UGGCU 797 AGCCACUGUAGA AAGGCAUGAUU 798 D-1317 [INVAB]UCAUG CCUUUCUACAG UGGCU 799 AGCCACUGUAGA AAGGCAUGAUU 800 D-1318 [INVAB]UCAUG CCUUUCUACAG UGGCU 801 AGCCACUGUAGA AAGGCAUGAUU 802 D-1319 [INVAB]UGCUU CAUGCCUUUCU ACAGU 803 ACUGUAGAAAGG CAUGAAGCAUU 804 D-1320 UGCUUCAUGCC UUUCUACAG{IN VAB} 805 ACUGUAGAAAGG CAUGAAGCAUU 806 D-1321 UGCUUCAUGCC UUUCUACAG{IN VAB} 807 ACUGUAGAAAGG CAUGAAGCAUU 808 D-1322 [INVAB]UGCUU CAUGCCUUUCU ACAGU 809 ACUGUAGAAAGG CAUGAAGCAUU 810 D-1323 [INVAB]CUUCA UGCCUUUCUAC AGUUU 811 ACUGUAGAAAGG CAUGAAGUU 812 D-1324 [INVAB]UGCUU CAUGCCUUUCU ACAGU 813 ACUGUAGAAAGG CAUGAAGCAUU 814 D-1325 [INVAB]GCUUC AUGCCUUUCUA CAUUU 815 AUGUAGAAAGGC AUGAAGCUU 816

- 58 047656

D-1326 [INVAB]CAUGC CUUUCUACAGU GGUUU 817 ACCACUGUAGAA AGGCAUGUU 818 D-1327 [INVAB]CUGCU UCAUGCCUUUC UAAUU 819 UUAGAAAGGCAU GAAGCAGUU 820 D-1328 [INVABJGCUUC AUGCCUUUCUA CAAUU 821 UUGUAGAAAGGC AUGAAGCUU 822 D-1329 [INVAB]UUCAU GCCUUUCUACA GUAUU 823 UACUGUAGAAAG GCAUGAAUU 824 D-1330 [TNVAB]GUUCC UGCUUCAUGCC UUUUU 825 AAAGGCAUGAAG CAGGAACUU 826 D-1331 AUGUUCCUGCU UCAUGCCUU{IN VAB} 827 AAAGGCAUGAAG CAGGAACAUUU 828 D-1332 [INVAB]AUGUU CCUGCUUCAUG CCUUU 829 AAAGGCAUGAAG CAGGAACAUUU 830 D-1333 [INVAB]GUUCC UGCUUCAUGCC UUUUU 831 AAAGGCAUGAAG CAGGAACUU 832 D-1334 AUGUUCCUGCU UCAUGCCUU{IN VAB} 833 AAAGGCAUGAAG CAGGAACAUUU 834 D-1335 [INVAB]AUGUU CCUGCUUCAUG CCUUU 835 AAAGGCAUGAAG CAGGAACAUUU 836 D-1336 [INVAB]GUUCC 837 AAAGGCAUGAAG 838

- 59 047656

UGCUUCAUGCC UUUUU CAGGAACUU D-1337 AUGUUCCUGCU UCAUGCCUU{IN VABJ 839 AAAGGCAUGAAG CAGGAACAUUU 840 D-1338 [INVABJAUGUU CCUGCUUCAUG CCUUU 841 AAAGGCAUGAAG CAGGAACAUUU 842 D-1339 [INVABJAUGUU CCUGCUUCAUG CCUUU 843 AAAGGCAUGAAG CAGGAACAUUU 844 D-1340 [INVABJAUGUU CCUGCUUCAUG CCUUU 845 AAAGGCAUGAAG CAGGAACAUUU 846 D-1341 [INVABJGCUUC AUGCCUUUCUA CAGUA 847 UACUGUAGAAAG GCAUGAAGCUU 848 D-1342 [INVABJGCUUC AUGCCUUUCUA CAGUA 849 UACUGUAGAAAG GCAUGAAGCUU 850 D-1343 [INVABJUUCAU GCCUUUCUACA GUAUU 851 UACUGUAGAAAG GCAUGAAUU 852 D-1344 [INVABJGCUUC AUGCCUUUCUA CAGUA 853 UACUGUAGAAAG GCAUGAAGCUU 854 D-1345 GCUUCAUGCCU UUCUACAGUJIN VABJ 855 UACUGUAGAAAG GCAUGAAGCUU 856 D-1346 [INVABJGCGGC UUCCUGGGCUU CUAUU 857 UAGAAGCCCAGG AAGCCGCUU 858

- 60 047656

D-1347 [INVAB]AUGGC UUCCAGAUAUG CCUUU 859 AGGCAUAUCUGG AAGCCAUUU 860 D-1348 [INVAB]ACAUG GCUUCCAGAUA UGCCU 861 AGGCAUAUCUGG AAGCCAUGUUU 862 D-1349 [INVABJACAUG GCUUCCAGAUA UGCCU 863 AGGCAUAUCUGG AAGCCAUGUUU 864 D-1350 [INVABJCAACG UACCCUUCAUU GAUGU 865 ACAUCAAUGAAG GGUACGUUGUU 866 D-1351 [TNVAB]CAACG UACCCUUCAUU GAUGU 867 ACAUCAAUGAAG GGUACGUUGUU 868 D-1352 [INVAB]ACGUA CCCUUCAUUGA UGUUU 869 ACAUCAAUGAAG GGUACGUUU 870 D-1353 CUGCUUCAUGC CUUUCUACA{IN VAB} 871 AUGUAGAAAGGC AUGAAGCAGUU 872 D-1354 [INVAB]CUGCU UCAUGCCUUUC UACAU 873 AUGUAGAAAGGC AUGAAGCAGUU 874 D-1355 [INVAB]GCUUC AUGCCUUUCUA CAUUU 875 AUGUAGAAAGGC AUGAAGCUU 876 D-1356 CUGCUUCAUGC CUUUCUACA{IN VAB} 877 AUGUAGAAAGGC AUGAAGCAGUU 878 D-1357 [INVAB]CUGCU 879 AUGUAGAAAGGC 880

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UCAUGCCUUUC UACAU AUGAAGCAGUU D-1358 CUGCUUCAUGC CUUUCUACAJIN VAB} 881 AUGUAGAAAGGC AUGAAGCAGUU 882 D-1359 [INVABJCUGCUUCAUGCCUUUCUACAJINVAB} 883 AUGUAGAAAGGC AUGAAGCAGUU 884 D-1360 [INVABJGCUUC AUGCCUUUCUA CAUUU 885 AUGUAGAAAGGC AUGAAGCUU 886 D-1361 [INVABJCUUCA UGCCUUUCUAC AGUUU 887 ACUGUAGAAAGG CAUGAAGUU 888 D-1362 UGCUUCAUGCC UUUCUACAGJIN VAB} 889 ACUGUAGAAAGG CAUGAAGCAUU 890 D-1363 [INVABJCUGCU UCAUGCCUUUC UACAU 891 AUGUAGAAAGGC AUGAAGCAGUU 892 D-1364 CUGCUUCAUGC CUUUCUACAJIN VAB} 893 AUGUAGAAAGGC AUGAAGCAGUU 894 D-1365 CUGCUUCAUGC CUUUCUACAJIN VAB} 895 AUGUAGAAAGGC AUGAAGCAGUU 896 D-1366 [INVABJCUUCA UCCCUUUCUAC AGUUU 897 ACUGUAGAAAGG GAUGAAGUU 898 D-1367 [INVABJGCUUC AUCCCUUUCUA CAUUU 899 AUGUAGAAAGGG AUGAAGCUU 900

- 62 047656

D-1368 UGCUUCAUCCC UUUCUACAG{IN VAB} 901 ACUGUAGAAAGG GAUGAAGCAUU 902 D-1369 CUGCUUCAUCC CUUUCUACA{IN VAB} 903 AUGUAGAAAGGG AUGAAGCAGUU 904 D-1370 [INVAB]ACAUU GCUCUUUCACC UGAUU 905 UCAGGUGAAAGA GCAAUGUUU 906 D-1371 CUGCUUCAUGC CUUUCUACA{IN VAB} 907 AUGUAGAAAGGC AUGAAGCAGUU 908 D-1372 CUGCUUCAUGC CUUUCUACA{IN VAB} 909 AUGUAGAAAGGC AUGAAGCAGUU 910 D-1373 CUGCUUCAUGC CUUUCUACA{IN VAB} 911 AUGUAGAAAGGC AUGAAGCAGUU 912 D-1374 CUGCUUCAUGC CUUUCUACA{IN VAB} 913 AUGUAGAAAGGC AUGAAGCAGUU 914 D-1375 CUGCUUCAUGC CUUUCUACA{IN VAB} 915 AUGUAGAAAGGC AUGAAGCAGUU 916 D-1376 CUGCUUCAUGC CUUUCUACA{IN VAB} 917 AUGUAGAAAGGC AUGAAGCAGUU 918 D-1377 CUGCUUCAUGC CUUUCUACA{IN VAB} 919 AUGUAGAAAGGC AUGAAGCAGUU 920 D-1378 CUGCUUCAUGC 921 AUGUAGAAAGGC 922

- 63 047656

CUUUCUACA{IN VAB} AUGAAGCAGUU D-1379 CUGCUUCAUGC CUUUCUACA{IN VAB} 923 AUGUAGAAAGGC AUGAAGCAGUU 924 D-1380 CUGCUUCAUGC CUUUCUACA{IN VAB} 925 AUGUAGAAAGGC AUGAAGCAGUU 926 D-1381 CUGCUUCAUGC CUUUCUACA{IN VAB} 927 AUGUAGAAAGGC AUGAAGCAGUU 928 D-1381 [INVAB]CUUCA UGCC[DT]UUCU ACAGUUU 929 ACUGUAGAAAGG CAUGAAGUU 930 D-1382 [INVAB]CUUCA UGCCUUUCUAC AGUUU 931 ACUGUAGAAAGG CAUGAAGUU 932 D-1383 [INVAB]CUUCA UGCCUUUCUAC AGUUU 933 ACUGUAGAAAGG CAUGAAGUU 934 D-1384 [INVAB]CUUCA UGCCUUUCUAC AGUUU 935 ACUGUAGAAAGG CAUGAAGUU 936 D-1385 [INVAB]CUUCA UGCCUUUCUAC AGUUU 937 ACUGUAGAAAGG CAUGAAGUU 938 D-1386 [INVAB]CUUCA UGC[DC]UUUCU ACAGUUU 939 ACUGUAGAAAGG CAUGAAGUU 940 D-1387 [INVAB]CUUCA UGCCU[DT]UCU ACAGUUU 941 ACUGUAGAAAGG CAUGAAGUU 942

- 64 047656

D-1388 [INVAB]GCUUC AUGGGAUUCUA CAUUU 943 AUGUAGAAUCCC AUGAAGCUU 944 D-1389 [INVAB]CUUCA UGCGAAUCUAC AGUUU 945 ACUGUAGAUUCG CAUGAAGUU 946 D-1390 CUGCUUCAUGC CUUUCUACA{IN VAB} 947 UUGUAGAAAGGC AUGAAGCAGUU 948 D-1391 [INVAB]CUGCU UCAUGCCUUUC UACAA 949 UUGUAGAAAGGC AUGAAGCAGUU 950 D-1392 CUGCUUCAUGC CUUUCUACA{IN VDA} 951 UUGUAGAAAGGC AUGAAGCAGUU 952 D-1393 CUGCUUCAUGG GAUUCUACA{IN VAB} 953 AUGUAGAAUCCC AUGAAGCAGUU 954 D-1394 [INVAB]CUGCU UCAUGGGAUUC UACAU 955 AUGUAGAAUCCC AUGAAGCAGUU 956 D-1395 [INVAB]CUUCA UGCCUUUCUAC AGUUU 957 ACUGUAGAAAGG CAUGAAGUU 958 D-1396 [INVAB]CUUCA UGCCUUUCUAC AGUUU 959 ACUGUAGAAAGG CAUGAAGUU 960 D-1397 [INVAB]GCUUC AUGCCUUUCUA CAUUU 961 AUGUAGAAAGGC AUGAAGCUU 962 D-1398 [INVAB]UGCUU 963 ACUGUAGAAAGG 964

- 65 047656

CAUGCCUUUCU ACAG{INVAB} CAUGAAGCAUU D-1399 [TNVAB]CUUCA UGCCUUUCUAC AGUUU 965 ACUGUAGAAAGG CAUGAAGUU 966 D-1400 UGCUUCAUGCC UUUCUACAG{IN VAB} 967 ACUGUAGAAAGG CAUGAAGCAUU 968 D-1401 UGCUUCAUGCC UUUCUACAG{IN VAB} 969 ACUGUAGAAAGG CAUGAAGCAUU 970 D-1402 UGCUUCAUGCC UUUCUACAG{IN VAB} 971 ACUGUAGAAAGG CAUGAAGCAUU 972 D-1403 [INVAB]CUUCA UGCCUUUCUAC AGUUU 973 ACUGUAGAAAGG CAUGAAGUU 974 D-1404 UGCUUCAUGCC UUUCUACAG{IN VAB} 975 ACUGUAGAAAGG CAUGAAGCAUU 976 D-1405 AUGCCUUUCUA CAGUGGCUU{IN VAB} 977 AGCCACUGUAGA AAGGCAUGAUU 978 D-1406 UCAUGCCUUUCUACAGUGGC{IN VAB} 979 AGCCACUGUAGA AAGGCAUGAUU 980 D-1407 [INVAB]AUGCC UUUCUACAGUG GCUUU 981 AGCCACUGUAGA AAGGCAUUU 982 D-1408 [INVAB]AUGCC UUUCUACAGUG GCUUU 983 AGCCACUGUAGA AAGGCAUUU 984

- 66 047656

D-1409 [INVAB]UCAUG CCUUUCUACAG UGGCU 985 AGCCACUGUAGA AAGGCAUGAUU 986 D-1410 UCAUGCCUUUCUACAGUGGC{IN VAB} 987 AGCCACUGUAGA AAGGCAUGAUU 988 D-1411 [INVABJAUGCC UUUCUACAGUG GCUUU 989 AGCCACUGUAGA AAGGCAUUU 990 D-1412 UCAUGCCUUUCUACAGUGGC{IN VAB} 991 AGCCACUGUAGA AAGGCAUGAUU 992 D-1413 UCAUGCCUUUCUACAGUGGC{IN VAB] 993 AGCCACUGUAGA AAGGCAUGAUU 994 D-1414 [INVAB]UCAUG CCUUUCUACAG UGGC{INVAB} 995 AGCCACUGUAGA AAGGCAUGAUU 996 D-1415 [INVAB]AUGCC UUUCUACAGUG GCAUU 997 UGCCACUGUAGA AAGGCAUUU 998 D-1416 UCAUGCCUUUCUACAGUGGC{IN VAB} 999 UGCCACUGUAGA AAGGCAUGAUU 1000 D-1417 [INVAB]UCAUG CCUUUCUACAG UGGCA 1001 UGCCACUGUAGA AAGGCAUGAUU 1002 D-1418 UCAUGCCUUUCUACAGUGGC{IN VDA} 1003 UGCCACUGUAGA AAGGCAUGAUU 1004 D-1419 CUGCUUCAUGC 1005 AUGUAGAAAGGC 1006

- 67 047656

CUUUCUACA{IN VAB} AUGAAGCAGUU D-1420 CUGCUUCAUGC CUUUCUACA{IN VAB} 1007 UUGUAGAAAGGC AUGAAGCAGUU 1008 D-1421 CUGCUUCAUGC CUUUCUACA{IN VDA} 1009 UUGUAGAAAGGC AUGAAGCAGUU 1010 D-1422 UCAUGCCUUUCUACAGUGGC{IN VAB} 1011 UGCCACUGUAGA AAGGCAUGAUU 1012 D-1423 UCAUGCCUUUCUACAGUGGC{IN VDA} 1013 UGCCACUGUAGA AAGGCAUGAUU 1014 D-1424 [INVAB]CUUCA UGCCUUUCUAC AGUUU 1015 ACUGUAGAAAGG CAUGAAGUU 1016 D-1425 CUGCUUCAUGC CUUUCUACA{IN VAB} 1017 AUGUA[AB]AAAG GCAUGAAGCAGU U 1018 D-1426 CUGCUUCAUGC CUUUCUACA{IN VAB} 1019 AUGUA[AB]AAAG GCAUGAAGCAGU U 1020 D-1427 UGCUUCAUGCC UUUCUACAG{IN VAB} 1021 ACUGU[AB]GAAA GGCAUGAAGCAU u 1022 D-1428 UGCUUCAUGCC UUUCUACAG{IN VAB} 1023 ACUGU[AB]GAAA GGCAUGAAGCAU U 1024 D-1429 UCAUGCCUUUCUACAGUGGC{IN VAB} 1025 AGCCA[AB]UGUA GAAAGGCAUGAU U 1026

- 68 047656

D-1430 UCAUGCCUUUCUACAGUGGC{IN VAB} 1027 AGCCA[AB]UGUA GAAAGGCAUGAU U 1028 D-1431 CUGCUUCAUGC CUUUCUACA{IN VAB} 1029 AU[GNAG]UAGAAAGGCAU GAAGCAGUU 1030 D-1432 CUGCUUCAUGC CUUUCUACA{IN VAB} 1031 AUG[GNA- U]AGAAAGGCAUG AAGCAGUU 1032 D-1433 CUGCUUCAUGC CUUUCUACA{IN VAB} 1033 AUGU[GNAA]GAAAGGCAUGA AGCAGUU 1034 D-1434 CUGCUUCAUGC CUUUCUACA{IN VAB} 1035 AUGUA[GNA- G]AAAGGCAUGAA GCAGUU 1036 D-1435 CUGCUUCAUGC CUUUCUACA{IN VAB} 1037 AUGUAG[GNAA]AAGGCAUGAAG CAGUU 1038 D-1436 CUGCUUCAUGC CUUUCUACA{IN VAB} 1039 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1040 D-1437 CUGCUUCAUGC CUUUCUACA{IN VAB} 1041 AUGUAGAAAGGC AUGAAGCAGUU 1042 D-1438 [INVAB]CUGCU UCAUGCCUUUC UACAU 1043 AUGUAGAAAGGC AUGAAGCAGUU 1044 D-1439 [INVAB]CUGCU UCAUGCCUUUC UACA{INVAB} 1045 AUGUAGAAAGGC AUGAAGCAGUU 1046 D-1440 CUGCUUCAUGC 1047 AUGUAGAAAGGC 1048

- 69 047656

CUUUCUACA{IN VAB} AUGAAGCAGUU D-1441 CUGCUUCAUGC CUUUCUACA{IN VAB} 1049 AUGUAGAAAGGC AUGAAGCAGUU 1050 D-1442 [INVAB]CUGCU UCAUGCCUUUC UACAU 1051 AUGUAGAAAGGC AUGAAGCAGUU 1052 D-1443 CUGCUUCAUGC CUUUCUACA{IN VAB} 1053 AUGUAGAAAGGC AUGAAGCAGUU 1054 D-1444 CUGCUUCAUGC CUUUCUACA{IN VAB} 1055 AUGUAGAAAGGC AUGAAGCAGUU 1056 D-1445 [INVAB]CUGCU UCAUGCCUUUC UACAU 1057 AUGUAGAAAGGC AUGAAGCAGUU 1058 D-1446 [INVAB]CUGCU UCAUGCCUUUC UACA{INVAB} 1059 AUGUAGAAAGGC AUGAAGCAGUU 1060 D-1447 CUGCUUCAUGC CUUUCUACA{IN VAB} 1061 A[AB]GUAGAAAG GCAUGAAGCAGU U 1062 D-1448 CUGCUUCAUGC CUUUCUACA{IN VAB} 1063 AU[AB]UAGAAAG GCAUGAAGCAGU u 1064 D-1449 CUGCUUCAUGC CUUUCUACA{IN VAB} 1065 AUG[AB]AGAAAG GCAUGAAGCAGU U 1066 D-1450 CUGCUUCAUGC CUUUCUACA{IN VAB} 1067 AUGU[AB]GAAAG GCAUGAAGCAGU U 1068

- 70 047656

D-1451 CUGCUUCAUGC CUUUCUACA{IN VAB} 1069 AUGUAG[AB]AAG GCAUGAAGCAGU U 1070 D-1452 CUGCUUCAUGC CUUUCUACA{IN VAB} 1071 AUGUAGA[AB]AG GCAUGAAGCAGU U 1072 D-1453 CAACGUACCCCU UCAUUGAUG{I NV AB} 1073 ACAUCAAUGAAG GGUACGUUGUU 1074 D-1454 CAACGUACCCCU UCAUUGAUG{I NV AB} 1075 ACAUCAAUGAAG GGUACGUUGUU 1076 D-1455 ACAUGGCUUCC AGAUAUGCC{IN VAB} 1077 AGGCAUAUCUGG AAGCCAUGUUU 1078 D-1456 ACAUGGCUUCC AGAUAUGCC{IN VAB} 1079 AGGCAUAUCUGG AAGCCAUGUUU 1080 D-1457 CUGCUUCAUGC CUUUCUACA{IN VAB} 1081 AUGUAGAAAGGC AUGAAGCAGUU 1082 D-1458 CUGCUUCAUGC CUUUCUACA{IN VAB} 1083 AUGUAGAAAGGC AUGAAGCAGUU 1084 D-1459 CUGCUUCAUGC CUUUCUACA{IN VAB} 1085 AUGUAGAAAGGC AUGAAGCAGUU 1086 D-1460 CUGCGGCUUCC UGGGCUUCU{IN VAB} 1087 UAGAAGCCCAGG AAGCCGCAGUU 1088 D-1461 CUGCGGCUUCC 1089 UAGAAGCCCAGG 1090

- 71 047656

UGGGCUUCU{IN VAB} AAGCCGCAGUU D-1462 UGCUUCAUGCC UUUCUACAG{IN VAB} 1091 ACUGUAGAAAGG CAUGAAGCAUU 1092 D-1463 [INVAB]UGCUU CAUGCCUUUCU ACAGU 1093 ACUGUAGAAAGG CAUGAAGCAUU 1094 D-1464 [INVAB]UGCUU CAUGCCUUUCU ACAG{INVAB} 1095 ACUGUAGAAAGG CAUGAAGCAUU 1096 D-1465 UGCUUCAUGCC UUUCUACAG{IN VAB} 1097 ACUGUAGAAAGG CAUGAAGCAUU 1098 D-1466 UGCUUCAUGCC UUUCUACAG{IN VAB} 1099 ACUGUAGAAAGG CAUGAAGCAUU 1100 D-1467 UGCUUCAUGCC UUUCUACAG{IN VAB} 1101 ACUGUAGAAAGG CAUGAAGCAUU 1102 D-1468 CUGCUUCAUGC CUUUCUACAU 1103 AUGUAGAAAGGC AUGAAGCAGUU 1104 D-1469 CUGCUUCAUGC CUUUCUACAU 1105 AUGUAGAAAGGC AUGAAGCAGUU 1106 D-1470 CUGCUUCAUGC CUUUCUACAU 1107 AUGUAGAAAGGC AUGAAGCAGUU 1108 D-1471 UGCUUCAUGCC UUUCUACAG{IN VAB} 1109 UCUGUAGAAAGG CAUGAAGCAUU 1110 D-1472 UGCUUCAUGCC UUUCUACAG{IN 1111 UCUGUAGAAAGG CAUGAAGCAUU 1112

- 72 047656

VDA} D-1473 CUGCUUCAUGC CUUUCUACA{TN VDT} 1113 AUGUAGAAAGGC AUGAAGCAGUU 1114 D-1474 CUGCUUCAUGC CUUUCUACA{IN VAB} 1115 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1116 D-1475 CUGCUUCAUGC CUUUCUACA{IN VAB} 1117 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1118 D-1476 CUGCUUCAUGC CUUUCUACA{IN VAB} 1119 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1120 D-1477 CUGCUUCAUGC CUUUCUACA{IN VAB} 1121 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1122 D-1478 CUGCUUCAUGC CUUUCUACA{IN VAB} 1123 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1124 D-1479 CUGCUUCAUGC CUUUCUACA{IN VAB} 1125 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1126 D-1480 CUGCUUCAUGC CUUUCUACA{IN VAB} 1127 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1128 D-1481 CUGCUUCAUGC CUUUCUACA{IN VAB} 1129 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1130 D-1482 CUGCUUCAUGC CUUUCUACA{IN VAB} 1131 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1132

- 73 047656

D-1483 CUGCUUCAUGC CUUUCUACA{1N VAB} 1133 AUGUAGA[GNAAJAGGCAUGAAGC AGUU 1134 D-1484 CUGCUUCAUGC CUUUCUACA{IN VAB} 1135 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1136 D-1485 CUGCUUCAUGC CUUUCUACA{IN VAB} 1137 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1138 D-1486 CUGCUUCAUGC CUUUCUACA{IN VAB} 1139 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1140 D-1487 CUGCUUCAUGC CUUUCUACA{IN VAB} 1141 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1142 D-1488 CUGCUUCAUGC CUUUCUACA{IN VAB} 1143 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1144 D-1489 CUGCUUCAUGC CUUUCUACA{IN VAB} 1145 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1146 D-1490 CUGCUUCAUGC CUUUCUACA{IN VAB} 1147 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1148 D-1491 CUGCUUCAUGC CUUUCUACA{IN VAB} 1149 AUGUAGA[GNA- A]A[DG]GCAUGAA GCAGUU 1150 D-1492 CUGCUUCAUGC CUUUCUACA{IN VAB} 1151 AUGUAGA[GNA- A][DA]GGCAUGAA GCAGUU 1152 D-1493 CUGCUUCAUGC 1153 AUGUAGA[GNA- 1154

- 74 047656

CUUUCUACAJIN VAB} A]AG[DG]CAUGAA GCAGUU D-1494 CUGCUUCAUGC CUUUCUACAJIN VAB} 1155 AUGUAGAJGNA- A]AGGCAUGAAGC AGUU 1156 D-1495 CUGCUUCAUGC CUUUCUACAJIN VAB} 1157 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1158 D-1496 CUGCUUCAUGC CUUUCUACAJIN VAB} 1159 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1160 D-1497 CUGCUUCAUGC CUUUCUACAJIN VAB} 1161 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1162 D-1498 CUGCUUCAUGC CUUUCUACAJIN VAB} 1163 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1164 D-1499 CUGCUUCAUGC CUUUCUACAJIN VAB} 1165 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1166 D-1500 CUGCUUCAUGC CUUUCUACAJIN VAB} 1167 AUGUAGAAAGGC AUGAAGCAGUU 1168 D-1501 UGCUUCAUGCC UUUCUACAGJIN VDA} 1169 U[GNA- C]UGUAGAAAGGC AUGAAGCAUU 1170 D-1502 UGCUUCAUGCC UUUCUACAGJIN VDA} 1171 UC[GNAU]GUAGAAAGGCA UGAAGCAUU 1172 D-1503 UGCUUCAUGCC UUUCUACAGJIN VDA} 1173 UCUG[GNAU]AGAAAGGCAUG AAGCAUU 1174

- 75 047656

D-1504 UGCUUCAUGCC UUUCUACAG{IN VDA} 1175 UCUGU[GNAAJGAAAGGCAUGA AGCAUU 1176 D-1505 UGCUUCAUGCC UUUCUACAG{IN VDA} 1177 UCUGUAG[GNAA]AAGGCAUGAAG CAUU 1178 D-1506 UGCUUCAUGCC UUUCUACAG{IN VDA} 1179 U[AB]UGUAGAAA GGCAUGAAGCAU U 1180 D-1507 UGCUUCAUGCC UUUCUACAG{IN VDA} 1181 UC[AB]GUAGAAA GGCAUGAAGCAU U 1182 D-1508 UGCUUCAUGCC UUUCUACAG{IN VDA} 1183 UCU[AB]UAGAAA GGCAUGAAGCAU u 1184 D-1509 UGCUUCAUGCC UUUCUACAG{IN VDA} 1185 UCUG[AB]AGAAA GGCAUGAAGCAU U 1186 D-1510 UGCUUCAUGCC UUUCUACAG{IN VDA} 1187 UCUGU[AB]GAAA GGCAUGAAGCAU U 1188 D-1511 UGCUUCAUGCC UUUCUACAG{IN VDA} 1189 UCUGUA[AB]AAA GGCAUGAAGCAU U 1190 D-1512 UGCUUCAUGCC UUUCUACAG{IN VDA} 1191 UCUGUAG[AB]AA GGCAUGAAGCAU U 1192 D-1513 UCAUGCCUUUCUACAGUGGC{IN VDT} 1193 AGCCACUGUAGA AAGGCAUGAUU 1194 D-1514 UCAUGCCUUUC 1195 UGCCACUGUAGA 1196

- 76 047656

UACAGUGGC{IN VAB} AAGGCAUGAUU D-1515 UCAUGCCUUUCUACAGUGGC{IN VDA} 1197 UGCCACUGUAGA AAGGCAUGAUU 1198 D-1516 UCCUGCUUCAU GCCUUUCUA{IN VDT} 1199 AUAGAAAGGCAU GAAGCAGGAUU 1200 D-1517 UCCUGCUUCAU GCCUUUCUA{IN VAB} 1201 UUAGAAAGGCAU GAAGCAGGAUU 1202 D-1518 UCCUGCUUCAU GCCUUUCUA{IN VDA} 1203 UUAGAAAGGCAU GAAGCAGGAUU 1204 D-1519 UAUGUUCCUGC UUCAUGCCU{IN VDT} 1205 AAGGCAUGAAGC AGGAACAUAUU 1206 D-1520 UAUGUUCCUGC UUCAUGCCU{IN VAB} 1207 UAGGCAUGAAGC AGGAACAUAUU 1208 D-1521 UAUGUUCCUGC UUCAUGCCU{IN VDA} 1209 UAGGCAUGAAGC AGGAACAUAUU 1210 D-1522 UCCUGCUUCAU GCCUUUCUA{IN VAB} 1211 AUAGAAAGGCAU GAAGCAGGAUU 1212 D-1523 UAUGUUCCUGC UUCAUGCCU{IN VAB} 1213 AAGGCAUGAAGC AGGAACAUAUU 1214 D-1524 UCAUGCCUUUCUACAGUGGC{IN VDT} 1215 AGCCACUGUAGA AAGGCAUGAUU 1216

- 77 047656

D-1525 UCCUGCUUCAU GCCUUUCUA{1N VDT} 1217 AUAGAAAGGCAU GAAGCAGGAUU 1218 D-1526 UCCUGCUUCAU GCCUUUCUA{IN VDA} 1219 UUAGAAAGGCAU GAAGCAGGAUU 1220 D-1527 UAUGUUCCUGC UUCAUGCCU{IN VDA} 1221 UAGGCAUGAAGC AGGAACAUAUU 1222 D-1528 UGCUUCAUGCC UUUCUACAG{IN VDA} 1223 UCUGUA[GNAG]AAAGGCAUGAA GCAUU 1224 D-1529 CUGCUUCAUGC CUUUCUACA{IN VAB} 1225 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1226 D-1530 CUGCUUCAUGC CUUUCUACA{IN VAB} 1227 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1228 D-1531 CUGCUUCAUGC CUUUCUACA{IN VAB} 1229 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1230 D-1532 CUGCUUCAUGC CUUUCUACA{IN VAB} 1231 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1232 D-1533 CUGCUUCAUGC CUUUCUACA{IN VAB} 1233 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1234 D-1534 CUGCUUCAUGC CUUUCUACA{IN VAB} 1235 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1236 D-1535 CUGCUUCAUGC 1237 AUGUAGA[GNA- 1238

- 78 047656

CUUUCUACA{IN VAB} A]AGGCAUGAAGC AGUU D-1536 CUGCUUCAUGC CUUUCUACA{IN VAB} 1239 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1240 D-1537 CUGCUUCAUGC CUUUCUACA{IN VAB} 1241 AUGUAGAFGNAA]AGGCAUGAAGC AGUU 1242 D-1538 CUGCUUCAUGC CU[LNAT]UCUACA{INV AB} 1243 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1244 D-1539 CUGCUUCAUGC C[LNAT]UUCUACA{IN VAB} 1245 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1246 D-1540 CUGCUUCAUGC CUU[LNAT]CUACA{INVA B} 1247 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1248 D-1541 CUGCUUCAUGC CUUUC[LNAT]ACA{INVAB} 1249 AUGU[GNAA]GAAAGGCAUGA AGCAGUU 1250 D-1542 CUGCUUCAUGC CUUUCU[LNAA]CA{INVAB} 1251 AUG[GNA- U]AGAAAGGCAUG AAGCAGUU 1252 D-1543 CUGCUUCAUGC CUUUCUAC[LN A-A]{INVAB} 1253 A[GNA- U]GUAGAAAGGCA UGAAGCAGUU 1254 D-1544 CUGCUUCAUGC CUU[LNAT]CUACA{INVA 1255 AUGUAG[AB]AAG GCAUGAAGCAGU U 1256

- 79 047656

B} D-1545 CUGCUUCAUGC CUUUQLNAT]ACA{INVAB} 1257 AUGU[AB]GAAAG GCAUGAAGCAGU U 1258 D-1546 CUGCUUCAUGC CUUUCU[LNAA]CA{INVAB} 1259 AUG[AB]AGAAAG GCAUGAAGCAGU U 1260 D-1547 CUGCUUCAUGC CUUUCUACA{IN VAB} 1261 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1262 D-1548 CUGCUUCAUGC CUUUCUACA{IN VAB} 1263 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1264 D-1549 CUGCUUCAUGC CU[LNAT]UCUACA{INV AB} 1265 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1266 D-1550 CUGCUUCAUGC CU[LNAT]UCUACA{INV AB} 1267 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1268 D-1551 CUGCUUCAUGC CU[LNAT]UCUACA{INV AB} 1269 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1270 D-1552 CUGCUUCAUGC CU[LNAT]UCUACA{INV AB} 1271 AUGUAGA[GNAA]AGGCAUGAAGC AGUU 1272 D-1553 UGCUUCAUGCC UUUCU[LNAA]CAG{INVDA} 1273 UCUG[AB]AGAAA GGCAUGAAGCAU U 1274

- 80 047656

D-1554 UGCUUCAUGCC UUUCILNAT]ACAG{INVDA} 1275 UCUGU[AB]GAAA GGCAUGAAGCAU U 1276 D-1555 UGCUUCAUGCC UU[LNA- T]CUACAG{INV DA} 1277 UCUGUAG[AB]AA GGCAUGAAGCAU U 1278 D-1556 UGCUUCAUGCC UUUCUAC[LNAA]G{INVDA} 1279 UC[GNAU]GUAGAAAGGCA UGAAGCAUU 1280 D-1557 UGCUUCAUGCC UUUCU[LNAA]CAG{INVDA} 1281 UCUG[GNAU]AGAAAGGCAUG AAGCAUU 1282 D-1558 UGCUUCAUGCC UUUC[LNAT]ACAG{INVDA} 1283 UCUGU[GNAA]GAAAGGCAUGA AGCAUU 1284 D-1559 UGCUUCAUGCC UUUCUACA[LN A-G]{INVDA} 1285 U[AB]UGUAGAAA GGCAUGAAGCAU U 1286 D-1560 UGCUUCAUGCC UUUCUAQLNAA]G{INVDA} 1287 UC[AB]GUAGAAA GGCAUGAAGCAU U 1288 D-1561 UCAUGCCUUUC UACA[LNAG]UGGC{INVAB } 1289 AGCCA[AB]UGUA GAAAGGCAUGAU U 1290 D-1562 UCAUGCCUUUC UACAG[LNAT]GGC{INVAB} 1291 AGCCA[AB]UGUA GAAAGGCAUGAU U 1292 D-1563 UCAUGCCUUUCUACAGUGGC{IN VAB} 1293 AGCCA[AB]UGUA GAAAGGCAUGAU U 1294

- 81 047656

D-1564 GGUAUGUUCCU GCUUCAUUUU 2257 AAUGAAGCAGGA ACAUACCUU 2258 D-1565 GGUAUGUUCCU GCUUCAUAUU 2259 UAUGAAGCAGGA ACAUACCUU 2260 D-1566 GUAUGUUCCUG CUUCAUGUUU 2261 ACAUGAAGCAGG AACAUACUU 2262 D-1567 UAUGUUCCUGC UUCAUGCAUU 2263 UGCAUGAAGCAG GAACAUAUU 2264 D-1568 AUGUUCCUGCU UCAUGCCUUU 2265 AGGCAUGAAGCA GGAACAUUU 2266 D-1569 UGUUCCUGCUU CAUGCCUUUU 2267 AAGGCAUGAAGC AGGAACAUU 2268 D-1570 GUUCCUGCUUC AUGCCUUUUU 2269 AAAGGCAUGAAG CAGGAACUU 2270 D-1571 UUCCUGCUUCA UGCCUUUUUU 2271 AAAAGGCAUGAA GCAGGAAUU 2272 D-1572 UUCCUGCUUCA UGCCUUUAUU 2273 UAAAAGGCAUGAA GCAGGAAUU 2274 D-1573 UCCUGCUUCAU GCCUUUCUUU 2275 AGAAAGGCAUGA AGCAGGAUU 2276 D-1574 CCUGCUUCAUG CCUUUCUAUU 2277 UAGAAAGGCAUG AAGCAGGUU 2278 D-1575 CUGCUUCAUGC CUUUCUAUUU 2279 AUAGAAAGGCAU GAAGCAGUU 2280 D-1576 CUGCUUCAUGC CUUUCUAAUU 2281 UUAGAAAGGCAU GAAGCAGUU 2282 D-1577 UGCUUCAUGCC UUUCUACAUU 2283 UGUAGAAAGGCA UGAAGCAUU 2284 D-1578 GCUUCAUGCCU UUCUACAUUU 2285 AUGUAGAAAGGC AUGAAGCUU 2286

- 82 047656

D-1579 GCUUCAUGCCU UUCUACAAUU 2287 UUGUAGAAAGGC AUGAAGCUU 2288 D-1580 CUUCAUGCCUU UCUACAGUUU 2289 ACUGUAGAAAGG CAUGAAGUU 2290 D-1581 UUCAUGCCUUU CUACAGUUUU 2291 AACUGUAGAAAG GCAUGAAUU 2292 D-1582 UUCAUGCCUUU CUACAGUAUU 2293 UACUGUAGAAAG GCAUGAAUU 2294 D-1583 UCAUGCCUUUC UACAGUGUUU 2295 ACACUGUAGAAA GGCAUGAUU 2296 D-1584 UCAUGCCUUUC UACAGUGAUU 2297 UCACUGUAGAAA GGCAUGAUU 2298 D-1585 CAUGCCUUUCU ACAGUGGUUU 2299 ACCACUGUAGAA AGGCAUGUU 2300 D-1586 CAUGCCUUUCU ACAGUGGAUU 2301 UCCACUGUAGAA AGGCAUGUU 2302 D-1587 AUGCCUUUCUA CAGUGGCUUU 2303 AGCCACUGUAGA AAGGCAUUU 2304 D-1588 AUGCCUUUCUA CAGUGGCAUU 2305 UGCCACUGUAGA AAGGCAUUU 2306 D-1589 UGCCUUUCUAC AGUGGCCUUU 2307 AGGCCACUGUAG AAAGGCAUU 2308 D-1590 GCCUUUCUACA GUGGCCUUUU 2309 AAGGCCACUGUA GAAAGGCUU 2310 D-1591 GGUAUGUUCCU GCUUCAUCUU 2311 GAUGAAGCAGGA ACAUACCUU 2312 D-1592 GUAUGUUCCUG CUUCAUCCUU 2313 GGAUGAAGCAGG AACAUACUU 2314 D-1593 UAUGUUCCUGC UUCAUCCCUU 2315 GGGAUGAAGCAG GAACAUAUU 2316

- 83 047656

D-1594 AUGUUCCUGCU UCAUCCCCUU 2317 GGGGAUGAAGCA GGAACAUUU 2318 D-1595 UGUUCCUGCUU CAUCCCCUUU 2319 AGGGGAUGAAGC AGGAACAUU 2320 D-1596 GUUCCUGCUUC AUCCCCUUUU 2321 AAGGGGAUGAAG CAGGAACUU 2322 D-1597 UUCCUGCUUCA uccccuucuu 2323 GAAGGGGAUGAA GCAGGAAUU 2324 D-1598 UCCUGCUUCAU ccccuucuuu 2325 AGAAGGGGAUGA AGCAGGAUU 2326 D-1599 CCUGCUUCAUC CCCUUCUAUU 2327 UAGAAGGGGAUG AAGCAGGUU 2328 D-1600 CUGCUUCAUCC CCUUCUACUU 2329 GUAGAGGGGAU GAAGCAGUU 2330 D-1601 UGCUUCAUCCC CUUCUACAUU 2331 UGUAGAAGGGGA UGAAGCAUU 2332 D-1602 GCUUCAUCCCC UUCUACAGUU 2333 CUGUAGAAGGGG AUGAAGCUU 2334 D-1603 CUUCAUCCCCU UCUACAGUUU 2335 ACUGUAGAAGGG GAUGAAGUU 2336 D-1604 UUCAUCCCCUU CUACAGUGUU 2337 CACUGUAGAGG GGAUGAAUU 2338 D-1605 UCAUCCCCUUC UACAGUGGUU 2339 CCACUGUAGAAG GGGAUGAUU 2340 D-1606 CAUCCCCUUCU ACAGUGGCUU 2341 GCCACUGUAGAA GGGGAUGUU 2342 D-1607 AUGCCCUUCUA CAGUGGCCUU 2343 GGCCACUGUAGA AGGGGAUUU 2344 D-1608 UCCCCUUCUAC AGUGGCCUUU 2345 AGGCCACUGUAG AAGGGGAUU 2346

- 84 047656

D-1609 CCCCUUCUACA GUGGCCUUUU 2347 AAGGCCACUGUA GAAGGGGUU 2348 D-1610 CUGCUUCAUGC CUUUCUACAA 2441 AUGUAGAAAGGCAUGAAG CAGUU 2442 D-1611 CUGCUUCAUGC CUUUCUACAA 2443 AUGUAGAAAGGCAUGAAG CAGUU 2444 D-1612 CUGCUUCAUGC CUUUCUACAA 2445 AUGUAGAAAGGCAUGAAG CAGUU 2446 D-1613 CUGCUUCAUGC CUUUCUACAA 2447 AUGUAGAAAGGCAUGAAG CAGUU 2448 D-1614 CUGCUUCAUGC CUUUCUACAA 2449 AUGUAGAAAGGCAUGAAG CAGUU 2450 D-1615 CUGCUUCAUGC CUUUCUACAA 2451 AUGUAGAAAGGCAUGAAG CAGUU 2452 D-1616 CUGCUUCAUGC CUUUCUACAA 2453 AUGUAGAAAGGCAUGAAG CAGUU 2454 D-1617 CUGCUUCAUGC CUUUCUACAA 2455 AUGUAGAAAGGCAUGAAG CAGUU 2456 D-1618 CUGCUUCAUGC CUUUCUACAA 2457 AUGUAGAAAGGCAUGAAG CAGUU 2458 D-1619 CUGCUUCAUGC CUUUCUACAA 2459 AUGUAGAAAGGCAUGAAG CAGUU 2460 D-1620 CUGCUUCAUGC CUUUCUACAA 2461 AUGUAGAAAGGCAUGAAG CAGUU 2462 D-1621 CUGCUUCAUGC CUUUCUACAA 2463 AUGUAGAAAGGCAUGAAG CAGUU 2464 D-1622 CUGCUUCAUGC CUUUCUACAA 2465 AUGUAGAAAGGCAUGAAG CAGUU 2466 D-1623 CUGCUUCAUGC CUUUCUACAA 2467 AUGUAGAAAGGCAUGAAG CAGUU 2468 D-1624 CUGCUUCAUGC CUUUCUACAA 2469 AUGUAGAAAGGCAUGAAG CAGUU 2470 D-1625 CUGCUUCAUGC CUUUCUACAA 2471 AUGUAGAAAGGCAUGAAG CAGUU 2472

- 85 047656

D-1626 CUGCUUCAUGC CUUUCUACAA 2473 AUGUAGAAAGGCAUGAAG CAGUU 2474 D-1627 CUGCUUCAUGC CUUUCUACAA 2475 AUGUAGAAAGGCAUGAAG CAGUU 2476 D-1628 CUGCUUCAUGC CUUUCUACAA 2477 AUGUAGAAAGGCAUGAAG CAGUU 2478 D-1629 CUGCUUCAUGC CUUUCUACAA 2479 AUGUAGAAAGGCAUGAAG CAGUU 2480 D-1630 CUGCUUCAUGC CUUUCUACAA 2481 AUGUAGAAAGGCAUGAAG CAGUU 2482 D-1631 CUGCUUCAUGC CUUUCUACAA 2483 AUGUAGAAAGGCAUGAAG CAGUU 2484 D-1632 CUGCUUCAUGC CUUUCUACAA 2485 AUGUAGAAAGGCAUGAAG CAGUU 2486 D-1633 CUGCUUCAUGC CUUUCUACAA 2487 AUGUAGAAAGGCAUGAAG CAGUU 2488 D-1634 CUGCUUCAUGC CUUUCUACAA 2489 AUGUAGAAAGGCAUGAAG CAGUU 2490 D-1635 CUGCUUCAUGC CUUUCUACAA 2491 AUGUAGAAAGGCAUGAAG CAGUU 2492 D-1636 CUGCUUCAUGC CUUUCUACAA 2493 AUGUAGAAAGGCAUGAAG CAGUU 2494 D-1637 UGCUUCAUGCC UUUCUACAGA 2495 UCUGUAGAAAGGCAUGAA GCAUU 2496 D-1638 UGCUUCAUGCC UUUCUACAGA 2497 UCUGUAGAAAGGCAUGAA GCAUU 2498 D-1639 UGCUUCAUGCC UUUCUACAGA 2499 UCUGUAGAAAGGCAUGAA GCAUU 2500 D-1640 UGCUUCAUGCC UUUCUACAGA 2501 UCUGUAGAAAGGCAUGAA GCAUU 2502 D-1641 UGCUUCAUGCC UUUCUACAGA 2503 UCUGUAGAAAGGCAUGAA GCAUU 2504 D-1642 UGCUUCAUGCC UUUCUACAGA 2505 UAUGUAGAAAGGCAUGAA GCAUU 2506

- 86 047656

D-1643 UGCUUCAUGCC UUUCUACAGA 2507 UCAGUAGAAAGGCAUGAA GCAUU 2508 D-1644 UGCUUCAUGCC UUUCUACAGA 2509 UCUAUAGAAAGGCAUGAA GCAUU 2510 D-1645 UGCUUCAUGCC UUUCUACAGA 2511 UCUGAAGAAAGGCAUGAA GCAUU 2512 D-1646 UGCUUCAUGCC UUUCUACAGA 2513 UCUGUAGAAAGGCAUGAA GCAUU 2514 D-1647 UGCUUCAUGCC UUUCUACAGA 2515 UCUGUAAAAAGGCAUGAA GCAUU 2516 D-1648 UGCUUCAUGCC UUUCUACAGA 2517 UCUGUAGAAAGGCAUGAA GCAUU 2518 D-1649 UCAUGCCUUUC UACAGUGGCT 2519 AGCCACUGUAGAAAGGCA UGAUU 2520 D-1650 UCAUGCCUUUC UACAGUGGCA 2521 UGCCACUGUAGAAAGGCA UGAUU 2522 D-1651 UCAUGCCUUUC UACAGUGGCA 2523 UGCCACUGUAGAAAGGCA UGAUU 2524 D-1652 UCCUGCUUCAU GCCUUUCUAT 2525 AUAGAAAGGCAUGAAGCA GGAUU 2526 D-1653 UCCUGCUUCAU GCCUUUCUAA 2527 UUAGAAAGGCAUGAAGCA GGAUU 2528 D-1654 UCCUGCUUCAU GCCUUUCUAA 2529 UUAGAAAGGCAUGAAGCA GGAUU 2530 D-1655 UAUGUUCCUGC UUCAUGCCUT 2531 A AGGC AUGA AGC AGGA AC AUAUU 2532 D-1656 UAUGUUCCUGC UUCAUGCCUA 2533 UAGGCAUGAAGCAGGAAC AUAUU 2534 D-1657 UAUGUUCCUGC UUCAUGCCUA 2535 UAGGCAUGAAGCAGGAAC AUAUU 2536 D-1658 UCCUGCUUCAU GCCUUUCUAA 2537 AUAGAAAGGCAUGAAGCA GGAUU 2538 D-1659 UAUGUUCCUGC UUCAUGCCUA 2539 AAGGCAUGAAGCAGGAAC AUAUU 2540

- 87 047656

D-1660 UAUGUUCCUGC UUCAUGCCUA 2541 AAGGCAUGAAGCAGGAAC AUAUU 2542 D-1661 UCAUGCCUUUC UACAGUGGCT 2543 AGCCACUGUAGAAAGGCA UGAUU 2544 D-1662 UCCUGCUUCAU GCCUUUCUAT 2545 AUAGAAAGGCAUGAAGCA GGAUU 2546 D-1663 UCCUGCUUCAU GCCUUUCUAA 2547 UUAGAAAGGCAUGAAGCA GGAUU 2548 D-1664 UAUGUUCCUGC UUCAUGCCUA 2549 UAGGCAUGAAGCAGGAAC AUAUU 2550 D-1665 UGCUUCAUGCC UUUCUACAGA 2551 UCUGUAGAAAGGCAUGAA GCAUU 2552 D-1666 CUGCUUCAUGC CUUUCUACAA 2553 AUGUAGAAAGGCAUGAAG CAGUU 2554 D-1667 CUGCUUCAUGC CUUUCUACAA 2555 AUGUAGAAAGGCAUGAAG CAGUU 2556 D-1668 CUGCUUCAUGC CUUUCUACAA 2557 AUGUAGAAAGGCAUGAAG CAGUU 2558 D-1669 CUGCUUCAUGC CUUUCUACAA 2559 AUGUAGAAAGGCAUGAAG CAGUU 2560 D-1670 CUGCUUCAUGC CUUUCUACAA 2561 AUGUAGAAAGGCAUGAAG CAGUU 2562 D-1671 CUGCUUCAUGC CUUUCUACAA 2563 AUGUAGAAAGGCAUGAAG CAGUU 2564 D-1672 CUGCUUCAUGC CUUUCUACAA 2565 AUGUAGAAAGGCAUGAAG CAGUU 2566 D-1673 CUGCUUCAUGC CUUUCUACAA 2567 AUGUAGAAAGGCAUGAAG CAGUU 2568 D-1674 CUGCUUCAUGC CUUUCUACAA 2569 AUGUAGAAAGGCAUGAAG CAGUU 2570 D-1675 CUGCUUCAUGC CUTUCUACAA 2571 AUGUAGAAAGGCAUGAAG CAGUU 2572 D-1676 CUGCUUCAUGC CTUUCUACAA 2573 AUGUAGAAAGGCAUGAAG CAGUU 2574

- 88 047656

D-1677 CUGCUUCAUGC CUUTCUACAA 2575 AUGUAGAAAGGCAUGAAG CAGUU 2576 D-1678 CUGCUUCAUGC CUUUCTACAA 2577 AUGUAGAAAGGCAUGAAG CAGUU 2578 D-1679 CUGCUUCAUGC CUUUCUACAA 2579 AUGUAGAAAGGCAUGAAG CAGUU 2580 D-1680 CUGCUUCAUGC CUUUCUACAA 2581 AUGUAGAAAGGCAUGAAG CAGUU 2582 D-1681 CUGCUUCAUGC CUUTCUACAA 2583 AUGUAGAAAGGCAUGAAG CAGUU 2584 D-1682 CUGCUUCAUGC CUUUCTACAA 2585 AUGUAGAAAGGCAUGAAG CAGUU 2586 D-1683 CUGCUUCAUGC CUUUCUACAA 2587 AUGAAGAAAGGCAUGAAG CAGUU 2588 D-1684 CUGCUUCAUGC CUUUCUACAA 2589 AUGUAGAAAGGCAUGAAG CAGUU 2590 D-1685 CUGCUUCAUGC CUUUCUACAA 2591 AUGUAGAAAGGCAUGAAG CAGUU 2592 D-1686 CUGCUUCAUGC CUTUCUACAA 2593 AUGUAGAAAGGCAUGAAG CAGUU 2594 D-1687 CUGCUUCAUGC CUTUCUACAA 2595 AUGUAGAAAGGCAUGAAG CAGUU 2596 D-1688 CUGCUUCAUGC CUTUCUACAA 2597 AUGUAGAAAGGCAUGAAG CAGUU 2598 D-1689 CUGCUUCAUGC CUTUCUACAA 2599 AUGUAGAAAGGCAUGAAG CAGUU 2600 D-1690 UGCUUCAUGCC UUUCUACAGA 2601 UCUGAAGAAAGGCAUGAA GCAUU 2602 D-1691 UGCUUCAUGCC UUUCTACAGA 2603 UCUGUAGAAAGGCAUGAA GCAUU 2604 D-1692 UGCUUCAUGCC UUTCUACAGA 2605 UCUGUAGAAAGGCAUGAA GCAUU 2606 D-1693 UGCUUCAUGCC UUUCUACAGA 2607 UCUGUAGAAAGGCAUGAA GCAUU 2608

- 89 047656

D-1694 UGCUUCAUGCC UUUCUACAGA 2609 UCUGUAGAAAGGCAUGAA GCAUU 2610 D-1695 UGCUUCAUGCC UUUCTACAGA 2611 UCUGUAGAAAGGCAUGAA GCAUU 2612 D-1696 UGCUUCAUGCC UUUCUACAGA 2613 UAUGUAGAAAGGCAUGAA GCAUU 2614 D-1697 UGCUUCAUGCC UUUCUACAGA 2615 UCAGUAGAAAGGCAUGAA GCAUU 2616 D-1698 UCAUGCCUUUC UACAGUGGCA 2617 AGCCAAUGUAGAAAGGCA UGAUU 2618 D-1699 UCAUGCCUUUC UACAGTGGCA 2619 AGCCAAUGUAGAAAGGCA UGAUU 2620 D-1700 UCAUGCCUUUC UACAGUGGCA 2621 AGCCAAUGUAGAAAGGCA UGAUU 2622 D-1701 CUGCUUCAUGC CUUUCUACAA 2623 AUGUAGAAAGGCAUGAAG CAGUU 2624 D-1702 CUGCUUCAUGC CUUUCUACAA 2625 AUAUAGAAAGGCAUGAAG CAGUU 2626 D-1703 UGCUUCAUGCC UUUCUACAGA 2627 UCUAUAGAAAGGCAUGAA GCAUU 2628 D-1704 UGCUUCAUGCC UUUCUACAGA 2629 UCUGUAGAAAGGCAUGAA GCAUU 2630 D-1705 UCAUGCCUUUC UACAGUGGCA 2631 AGCCAAUGUAGAAAGGCA UGAUU 2632 D-1706 UCAUGCCUUUC UACAGUGGCA 2633 AGCCAAUGUAGAAAGGCA UGAUU 2634 D-1707 UCAUGCCUUUC UACAGUGGCA 2635 AGCCAAUGUAGAAAGGCA UGAUU 2636 D-1708 UCAUGCCUUUC UACAGUGGCA 2637 AGCCAAUGUAGAAAGGCA UGAUU 2638 D-1709 CUGCUUCAUGC CUUUCUACAA 2639 A[MEO- I] GUAG AAAGGC AUGAAGC AGUU 2640 D-1710 CUGCUUCAUGC 2641 AUG[MEO- 2642

- 90 047656

CUUUCUACAA I]AGAAAGGCAUGAAGCAG UU D-1711 CUGCUUCAUGC CUUUCUACAA 2643 AUGU[MEO- I] GAAAGGC AUG AAGC AGU U 2644 D-1712 CUGCUUCAUGC CUUUCUACAA 2645 AUGUA[MEO- I]AAAGGCAUGAAGCAGUU 2646 D-1713 CUGCUUCAUGC CUUUCUACAA 2647 AUGUAGA[MEO- I]AGGC AUGAAGCAGUU 2648 D-1714 CUGCUUCAUGC CUUUCUACCA 2649 A[MEO- I]GUAGAAAGGCAUGAAGC AGUU 2650 D-1715 CUGCUUCAUGC CUUUCUCCAA 2651 AUG[MEO- I]AGAAAGGCAUGAAGCAG UU 2652 D-1716 CUGCUUCAUGC CUUCCUACAA 2653 AUGUAG[MEO- I]AAGGCAUGAAGCAGUU 2654 D-1717 CUGCUUCAUGC CUCUCUACAA 2655 AUGUAGA[MEO- HAGGCAUGAAGCAGUU 2656 D-1718 CUGCUUCAUGC CUUUCUACUA 2657 AAGUAGAAAGGCAUGAAG CAGUU 2658 D-1719 CUGCUUCAUGC CUUUCUUCAA 2659 AUGAAGAAAGGCAUGAAG CAGUU 2660 D-1720 CUGCUUCAUGC CUUUCAACAA 2661 AUGUUGAAAGGCAUGAAG CAGUU 2662 D-1721 CUGCUUCAUGC CUAUCUACAA 2663 AUGUAGAUAGGCAUGAAG CAGUU 2664 D-1722 UGCUUCAUGCC UUUCUACACA 2665 A[MEO- I]UGUAGAAAGGCAUGAAG CAUU 2666 D-1723 UGCUUCAUGCC UUUCUACAGA 2667 AC[MEO- I]GUAGAAAGGCAUGAAGC AUU 2668 D-1724 UGCUUCAUGCC 2669 ACUG[MEO- 2670

- 91 047656

UUUCUACAGA I]AGAAAGGCAUGAAGCAU U D-1725 UGCUUCAUGCC UUUCUACAGA 2671 ACUGU[MEO- I] GAAAGGC AUG AAGC AUU 2672 D-1726 UGCUUCAUGCC UUUCUACAGA 2673 ACUGUA[MEO- I]AAAGGCAUGAAGCAUU 2674 D-1727 UGCUUCAUGCC UUUCUACCGA 2675 AC[MEO- I] GUAG AAAGGC AUGAAGC AUU 2676 D-1728 UGCUUCAUGCC UUUCUCCAGA 2677 ACUG[MEO- I]AGAAAGGCAUGAAGCAU u 2678 D-1729 UGCUUCAUGCC UUUCCACAGA 2679 ACUGU[MEO- I] GAAAGGC AUG AAGC AUU 2680 D-1730 CUGCUUCAUGC CUUUCUAAAA 2681 AUUUAGAAAGGCAUGAAG CAGUU 2682 D-1731 CUGCUUCAUGC CUUUAUACAA 2683 AUGUAUAAAGGCAUGAAG CAGUU 2684 D-1732 CUGCUUCAUGC CUUUCUACAA 2685 AUGUAGAAAGGCAUGAAG CAGUU 2686 D-1733 CUGCUUCAUGC CUTUCUACAA 2687 AUGUAGAAAGGCAUGAAG CAGUU 2688 D-1734 UGCUUCAUGCC UUUCUACAGA 2689 UCUGUAGAAAGGCAUGAA GCAUU 2690 D-1735 UGCUUCAUGCC UUTCUACAGA 2691 UCUGUAGAAAGGCAUGAA GCAUU 2692 D-1736 UCAUGCCUUUC UACAGUGGCA 2693 AGCCAAUGUAGAAAGGCA UGAUU 2694 D-1737 CUGCUUCAUGC CUUUCUACAA 2695 AUGUAAAAAGGCAUGAAG CAGUU 2696 D-1738 UCAUGCCUUUC UACAGUGGCA 2697 AGCCAAUGUAGAAAGGCA UGAUU 2698 D-1739 CUGCUUCAUGC CUUUCUACAA 2699 AU[MEO- I]UAGAAAGGCAUGAAGCA 2700

- 92 047656

GUU D-1740 CUGCUUCAUGC CUUUCUAGAA 2701 AUGUAGAAAGGCAUGAAG CAGUU 2702 D-1741 CUGCUUCAUGC CUUACUACAA 2703 AUGUAGUAAGGCAUGAAG CAGUU 2704 D-1742 UGCUUCAUGCC UUUCUACAGA 2705 ACU[MEO- IJUAGAAAGGCAUGAAGCA UU 2706 D-1743 UGCUUCAUGCC UUUCUACAGA 2707 ACUGUAG[MEO- IJAAGGCAUGAAGCAUU 2708 D-1744 UGCUUCAUGCC UUCCUACAGA 2709 ACUGUAG[MEO- IJAAGGCAUGAAGCAUU 2710 D-1745 CUGCUUCAUGC CUUUCUAUAA 2711 AUAUAGAAAGGCAUGAAG CAGUU 2712 D-1746 CUGCUUCAUGC CUUUUUACAA 2713 AUGUAAAAAGGCAUGAAG CAGUU 2714 D-1747 CUGCUUCAUGC CUUUCUACAA 2715 AUGUAGAAAGGCAUGAAG CAGUU 2716 D-1748 CUGCUUCAUGC CUUUCUACAA 2717 AUGUAGAAAGGCAUGAAG CAGUU 2718 D-1749 CUGCUUCAUGC CUUTCUACAA 2719 AUGUAGAAAGGCAUGAAG CAGUU 2720 D-1750 CUGCUUCAUGC CUTUCUACAA 2721 AUGUAGAAAGGCAUGAAG CAGUU 2722 D-1751 CUGCUUCAUGC CUUUCUACAA 2723 AAGUAGAAAGGCAUGAAG CAGUU 2724 D-1752 CUGCUUCAUGC CUUUCUACAA 2725 AUGUAGAAAGGCAUGAAG CAGUU 2726 D-1753 UGCUUCAUGCC UUUCUACAGA 2727 UCUGUAAAAAGGCAUGAA GCAUU 2728 D-1754 CUGCUUCAUGC CUUUCUACAA 2729 AUGUAG[MEO- IJAAGGCAUGAAGCAGUU 2730 D-1755 CUGCUUCAUGC CUUUCUACAA 2731 AUGUAGAAAGGCAUGAAG CAGUU 2732

- 93 047656

D-1756 CUGCUUCAUGC CUTUCUACAA 2733 AUGUAGAAAGGCAUGAAG CAGUU 2734 D-1757 CUGCUUCAUGC CUUUCUACAA 2735 AUGUAGAAAGGCAUGAAG CAGUU 2736 D-1758 CUGCUUCAUGC CUUUCUACAA 2737 AUGUAGAAAGGCAUGAAG CAGUU 2738 D-1759 CUGCUUCAUGC CUUUCUACAA 2739 AUGUAGAAAGGCAUGAAG CAGUU 2740 D-1760 CUGCUUCAUGC CUUUCUACAA 2741 AUGUAGAAAGGCAUGAAG CAGUU 2742 D-1761 CUGCUUCAUGC CUUUCUACAA 2743 AUGUAGAAAGGCAUGAAG CAGUU 2744 D-1762 CUGCUUCAUGC CUUUCUACAA 2745 AUGUAGAAAGGCAUGAAG CAGUU 2746 D-1763 CUGCUUCAUGC CUTUCUACAA 2747 AUGUAGAAAGGCAUGAAG CAGUU 2748 D-1764 CUGCUUCAUGC CUUUCUACAA 2749 AUGUAGAAAGGCAUGAAG CAGUU 2750 D-1765 UCAUGCCUUUC UACAGUGGCA 2751 AGCCAAUGUAGAAAGGCA UGAUU 2752 D-1766 UCAUGCCUUUC UACAGUGGCA 2753 AGCCAAUGUAGAAAGGCA UGAUU 2754 D-1767 CUGCUUCAUGG GAUUCUACAA 2755 AUGUAGAAUCCCAUGAAG CAGUU 2756 D-1768 CUGCUUCAUGC CUUUCUACAA 2757 AUGUAGAAAGGCAUGAAG CAGUU 2758 D-1769 GUAUGUUCCUG CUUCAUGCCA 2759 AGGCAUGAAGCAGGAACA UACUU 2760 D-1770 UGUCCUGCUUC AUGCCUUUCA 2761 AGAAAGGCAUGAAGCAGG ACAUU 2762 D-1771 GCUUCAUGCCU UUCUACAGUA 2763 AACUGUAGAAAGGCAUGA AGCUU 2764 D-1772 CUUCAUGCCUU UCUACAGUGA 2765 ACACUGUAGAAAGGCAUG AAGUU 2766

- 94 047656

D-1773 UUCAUGCCUUU CUACAGUGGA 2767 ACCACUGUAGAAAGGCAU GAAUU 2768 D-1774 CUGCUUCAUGC CUUUCAACAA 2769 AUGUUGAAAGGCAUGAAG CAGUU 2770 D-1775 CUGCUUCAUGC CUUUCUCCAA 2771 AUG[MEO- IJAGAAAGGCAUGAAGCAG UU 2772 D-1776 UGCUUCAUGCC UUUCUCCAGA 2773 ACUGJMEO- IJAGAAAGGCAUGAAGCAU U 2774 D-1777 CUGCUUCAUGC CUUUCUACAA 2775 AUGU[MEO- I]GAAAGGCAUGAAGCAGU U 2776 D-1778 UGCUUCAUGCC UUUCUACAGA 2777 ACUGU[MEO- I]GAAAGGCAUGAAGCAUU 2778 D-1779 CUGCUUCAUGC CUUUCUACAA 2779 AUGUA[MEO- I]AAAGGCAUGAAGCAGUU 2780 D-1780 UGCUUCAUGCC UUUCUACAGA 2781 ACUGUA[MEO- IJAAAGGCAUGAAGCAUU 2782 D-1781 CUGCUUCAUGC CUUCCUACAA 2783 AUGUAG[MEO- IJAAGGCAUGAAGCAGUU 2784 D-1782 CUGCUUCAUGC CUCUCUACAA 2785 AUGUAGA[MEO- IJAGGCAUGAAGCAGUU 2786 D-1783 UGCUUCAUGCC UUUCUACAGA 2787 UCUGAAGAAAGGCAUGAA GCAUU 2788 D-1784 UGCUUCAUGCC UUUCUACAGA 2789 UCUGUAGAAAGGCAUGAA GCAUU 2790 D-1785 UCAUGCCUUUC UACAGUGGCA 2791 AGCCAAUGUAGAAAGGCA UGAUU 2792 D-1786 CUGCUUCAUGC CUUUCTACAA 2793 AUGUAGAAAGGCAUGAAG CAGUU 2794 D-1787 CUGCUUCAUGC CUUUCTACAA 2795 AUGUAGAAAGGCAUGAAG CAGUU 2796 D-1788 UGCUUCAUGCC 2797 UCUGUAGAAAGGCAUGAA 2798

- 95 047656

UUUCTACAGA GCAUU D-1789 UGCUUCAUGCC UUUCUACAGA 2799 UCUGUAGAAAGGCAUGAA GCAUU 2800 D-1790 UCAUGCCUUUC UACAGUGGCA 2801 AGCCAAUGUAGAAAGGCA UGAUU 2802 D-1791 CUGCUUCAUGC CUUUCUACAA 2803 AU[MEO- I]UAGAAAGGCAUGAAGCA GUU 2804 D-1792 CUGCUUCAUGC CUUUCUACAA 2805 AUGUAG[MEO- I]AAGGCAUGAAGCAGUU 2806 D-1793 CUGCUUCAUGC CUUACUACAA 2807 AUGUAGUAAGGCAUGAAG CAGUU 2808 D-1794 UGCUUCAUGCC UUUCUACAGA 2809 ACUGUAG[MEO- I]AAGGCAUGAA GCAUU 2810 D-1795 UGCUUCAUGCC UUCCUACAGA 2811 ACUGUAG[MEO- I]AAGGCAUGAA GCAUU 2812 D-1796 UGCUUCAUGCC UUUCUACAGA 2813 ACU[MEO- I]UAGAAAGGCAUGAAGCA UU 2814 D-1797 CUGCUUCAUGC CUUUCUACAA 2815 AUGU[MEO- I]GAAAGGCAUGAAGCAGU U 2816 D-1798 CUGCUUCAUGC CUUUCUACAA 2817 AUGU[MEO- I]GAAAGGCAUGAAGCAGU U 2818 D-1799 CUGCUUCAUGC CUUUCUACAA 2819 AUGU[MEO- I]GAAAGGCAUGAAGCAGU U 2820 D-1800 CUGCUUCAUGC CUUUCUACAA 2821 AUGUAG[MEO- I]AAGGCAUGAAGCAGUU 2822 D-1801 UCAUGCCUUUC UACAGUGGCA 2823 AGCCAAUGUAGAAAGGCA UGAUU 2824 D-1802 UCAUGCCUUUC UACAGUGGCA 2825 AGCCAAUGUAGAAAGGCA UGAUU 2826

- 96 047656

D-1803 CUGCUUCAUGG GAUUGUACAA 2827 AUGUACAAUCCCAUGAAG CAGUU 2828 D-1804 CUGCUUCAUGC CUUUCTACAA 2829 AUGU[MEO- I] GAAAGGC AUG AAGC AGU U 2830 D-1805 UGCUUCAUGCC UUUCUACAGA 2831 ACUGUA[MEO- I]AAAGGCAUGAAGCAUU 2832 D-1806 CUGCUUCAUGC CUTUCUACAA 2833 AUGUAGAAAGGCAUGAAG CAGUU 2834 D-1807 CUGCUUCAUGC CUTUCUACAA 2835 AUGUAGAAAGGCAUGAAG CAGUU 2836 D-1808 CUGCUUCAUGC CUUTCUACAA 2837 AUGUAG[MEO- I]AAGGCAUGAAGCAGUU 2838 D-1809 UCAUGCCUUUC UACAGUGGCA 2839 AGCCACUGUAGAAAGGCA UGAUU 2840 D-1810 UAUGUUCCUGC UUCAUGCCUA 2841 AAGGCAUGAAGCAGGAAC AUAUU 2842 D-1811 UGCUUCAUGCC UUUCUACAGA 2843 ACUGUAGAAAGGCAUGAA GCAUU 2844 D-1812 UGUUCCUGCUU CAUGCCUUUA 2845 AAGGCAUGAAGCAGGAAC AUU 2846 D-1813 CUGCUUCAUGC CUUUCUACAA 2847 AUGUAGAAAGGCAUGAAG CAGUU 2848 D-1814 UAUGUUCCUGC UUCAUGCCUA 2849 AAGGCAUGAAGCAGGAAC AUAUU 2850 D-1815 UGUUCCUGCUU CAUGCCUA 2851 AAGGCAUGAAGCAGGAAC AUU 2852 D-1816 UAUGUUCCUGC UUCAUGCCUUA 2853 AAGGCAUGAAGCAGGAAC AUAUU 2854 D-1817 UAUGUUCCUGC UUCAUGCCUA 2855 AAGGCAUGAAGCAGGAAC AUACC 2856 D-1818 UAUGUUCCUGC UUCAUGCCUUA 2857 AAGGCAUGAAGCAGGAAC AUACC 2858 D-1819 UAUGUUCCUGC 2859 AAGGCAUGAAGCAGGAAC 2860

- 97 047656

UUCAUGCCUA AUAUU D-1820 UAUGUUCCUGC UUCAUGCGUA 2861 AACGCAUGAAGCAGGAAC AUAUU 2862 D-1821 UAUGUUCCUGC UUCAUGGCUA 2863 AAGCCAUGAAGCAGGAAC AUAUU 2864 D-1822 UAUGUUCCUGC UUCAAGCCUA 2865 AAGGCUUGAAGCAGGAAC AUAUU 2866 D-1823 UAUGUUCCUGC UUCUUGCCUA 2867 AAGGCAAGAAGCAGGAAC AUAUU 2868 D-1824 UAUGUUCCUGC UUGAUGCCUA 2869 AAGGCAUCAAGCAGGAAC AUAUU 2870 D-1825 UAUGUUCCUGC UACAUGCCUA 2871 AAGGCAUGUAGCAGGAAC AUAUU 2872 D-1826 UAUGUUCCUGC AUCAUGCCUA 2873 AAGGCAUGAUGCAGGAAC AUAUU 2874 D-1827 UAUGUUCCUGG UUCAUGCCUA 2875 AAGGCAUGAACCAGGAAC AUAUU 2876 D-1828 UAUGUUCCUCC UUCAUGCCUA 2877 AAGGCAUGAAGGAGGAAC AUAUU 2878 D-1829 UAUGUUCCAGC UUCAUGCCUA 2879 AAGGCAUGAAGCUGGAAC AUAUU 2880 D-1830 UAUGUUCGUGC UUCAUGCCUA 2881 AAGGCAUGAAGCACGAAC AUAUU 2882 D-1831 UAUGUUGCUGC UUCAUGCCUA 2883 AAGGCAUGAAGCAGCAAC AUAUU 2884 D-1832 UAUGUACCUGC UUCAUGCCUA 2885 AAGGCAUGAAGCAGGUAC AUAUU 2886 D-1833 UAUGAUCCUGC UUCAUGCCUA 2887 AAGGCAUGAAGCAGGAUC AUAUU 2888 D-1834 UAUCUUCCUGC UUCAUGCCUA 2889 AAGGCAUGAAGCAGGAAG AUAUU 2890 D-1835 UAAGUUCCUGC UUCAUGCCUA 2891 AAGGCAUGAAGCAGGAAC UUAUU 2892 D-1836 UUUGUUCCUGC 2893 AAGGCAUGAAGCAGGAAC 2894

- 98 047656

UUCAUGCCUA AAAUU D-1837 AAUGUUCCUGC UUCAUGCCUA 2895 AAGGCAUGAAGCAGGAAC AUUUU 2896 D-1838 UCAUGCCUUUC UACAGUGGCA 2897 AGCCACUGUAGAAAGGCA UGAUU 2898 D-1839 UCAUGCCUUUC UACAGUGGGA 2899 ACCCACUGUAGAAAGGCA UGAUU 2900 D-1840 UCAUGCCUUUC UACAGUGCCA 2901 AGGCACUGUAGAAAGGCA UGAUU 2902 D-1841 UCAUGCCUUUC UACAGUCGCA 2903 AGCGACUGUAGAAAGGCA UGAUU 2904 D-1842 UCAUGCCUUUC UACAGAGGCA 2905 AGCCUCUGUAGAAAGGCA UGAUU 2906 D-1843 UCAUGCCUUUC UACACUGGCA 2907 AGCCAGUGUAGAAAGGCA UGAUU 2908 D-1844 UCAUGCCUUUC UACUGUGGCA 2909 AGCCACAGUAGAAAGGCA UGAUU 2910 D-1845 UCAUGCCUUUC UAGAGUGGCA 2911 AGCCACUCUAGAAAGGCA UGAUU 2912 D-1846 UCAUGCCUUUC UUCAGUGGCA 2913 AGCCACUGAAGAAAGGCA UGAUU 2914 D-1847 UCAUGCCUUUC AACAGUGGCA 2915 AGCCACUGUUGAAAGGCA UGAUU 2916 D-1848 UCAUGCCUUUG UACAGUGGCA 2917 AGCCACUGUACAAAGGCA UGAUU 2918 D-1849 UCAUGCCUAUC UACAGUGGCA 2919 AGCCACUGUAGAUAGGCA UGAUU 2920 D-1850 UCAUGCGUUUC UACAGUGGCA 2921 AGCCACUGUAGAAACGCA UGAUU 2922 D-1851 UCAUGGCUUUC UACAGUGGCA 2923 AGCCACUGUAGAAAGCCA UGAUU 2924 D-1852 UCAAGCCUUUC UACAGUGGCA 2925 AGCCACUGUAGAAAGGCU UGAUU 2926 D-1853 UCUUGCCUUUC 2927 AGCCACUGUAGAAAGGCA 2928

- 99 047656

UACAGUGGCA AGAUU D-1854 UGAUGCCUUUC UACAGUGGCA 2929 AGCCACUGUAGAAAGGCA UCAUU 2930 D-1855 ACAUGCCUUUC UACAGUGGCA 2931 AGCCACUGUAGAAAGGCA UGUUU 2932 D-1856 UCAUGCCUUAC UACAGUGGCA 2933 AGCCACUGUAGUAAGGCA UGAUU 2934 D-1857 AUGCCUUUCUA CAGUGGCUUA 2935 AGCCACUGUAGAAAGGCA UUU 2936 D-1858 AUGCCUUUCUA CAGUGGCA 2937 AGCCACUGUAGAAAGGCA UUU 2938 D-1859 UCAUGCCUUUC UACAGUGGCA 2939 AGCCACUGUAGAAAGGCA UGAUU 2940 D-1860 AAUGCCUUUCU ACAGUGGCU 2941 AGCCACUGUAGAAAGGCA UUU 2942 D-1861 UCAUGCCUUUC UACAGUGGCUA 2943 AGCCACUGUAGAAAGGCA UGAUU 2944 D-1862 UCAUGCCUUUC UACAGUGGCA 2945 AGCCACUGUAGAAAGGCA UGAAG 2946 D-1863 UCAUGCCUUUC UACAGUGGCUA 2947 AGCCACUGUAGAAAGGCA UGAAG 2948 D-1864 UCAUGCCUUUC UACAGUGGCUA 2949 AGCCACUGUAGAAAGGCA UGAUU 2950 D-1865 UCAUGCCUUUC UACAGUGGCA 2951 AGCCACUGUAGAAAGGCA UGAAG 2952 D-1866 UCAUGCCUUUC UACAGUGGCUA 2953 AGCCACUGUAGAAAGGCA UGAAG 2954 D-1867 CUGCUUCAUGC CUUUCUACAA 2955 AUGUA[MEO- I]AAAGGCAUGAAGCAGUU 2956 D-1868 GCGGCUUCGAC GGCUUCUAUU 2957 UAGAAGCCGUCGAAGCCG CUU 2958 D-1869 GCGGCUUCGAC GGGUUCUAUU 2959 UAGAACCCGUCGAAGCCG CUU 2960 D-1870 GCGGCUUCGAC 2961 UAGAAGGCGUCGAAGCCG 2962

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GCCUUCUAUU cuu D-1871 GCGGCUUCGAC CGCUUCUAUU 2963 UAGAAGCGGUCGAAGCCG CUU 2964 D-1872 GCGGCUUCCUG GGCUUCUAUU 2965 AUAGAAGCCCAGGAAGCC GCUU 2966 D-1873 AGCUUCAUGGG AUUGUACAUUU 2967 AUGUACAAUCCCAUGAAG CUU 2968 D-1874 UCAUGCCUUUG AUCACUGGCA 2969 AGCCAGUGAUCAAAGGCA UGAUU 2970 D-1875 GCGGCUUCCUG GGCUUCUAUU 2971 UAGAAGCCCAGGAAGCCG CUU 2972 D-1876 UGCUUCAUGCC UUUCUACAGA 2973 ACUGUAGAAAGGCAUGAA GCAUU 2974 D-1877 UCAUGCCUUUC UACAGUGGCA 2975 AGCCACUGUAGAAAGGCA UGAUU 2976 D-1878 CUGCUUCAUGC CUUUCUACAA 2977 AUGUAGAAAGGCAUGAAG CAGUU 2978 D-1879 CUGCUUCAUGC CUUUCUACAA 2979 AUGUAGAAAGGCAUGAAG CAGUU 2980 D-188O CUGCUUCAUGC CUUUCUACAA 2981 AUGUAGAAAGGCAUGAAG CAGUU 2982 D-1881 UCAUGCCUUUC UACAGUGGCA 2983 UGCCACUGUAGAAAGGCA UGAUU 2984 D-1882 CUGCUUCAUGC CUUUCUACAA 2985 AUGUAGAAAGGCAUGAAG CAGUU 2986 D-1883 UGCUUCAUGCC UUUCUACAGA 2987 UCUGUAGAAAGGCAUGAA GCAUU 2988 D-1884 CUGCUUCAUGC CUUUCUACAT 2989 AUGUAGAAAGGCAUGAAG CAGUU 2990

To enhance the activity and in vivo stability of the PNPLA3 siRNA sequences, chemical modifications were introduced into the PNPLA3 siRNA molecules. Specifically, 2'-O-methyl and 2'-fluoro modifications of the ribose sugar were introduced into specific positions of the PNPLA3 siRNA. Phosphorothioate internucleotide linkages were also introduced into the termini of the antisense and/or sense sequences. Table 2 below shows the modifications in the sense and antisense sequences for each of the modified PNPLA3 siRNAs. The nucleotide sequences in Table 2 and elsewhere in the application are listed according to the following notations: A, U, G, and C = the corresponding ribonucleotide; 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 = the corresponding 2'-O-methylribonucleotide; Af, Uf, Gf, and Cf = the corresponding 2'-deoxy-2'-fluoro(2'-fluoro)ribonucleotide; Ab = base-deleted nucleotide; MeO-I = 2'-methoxyinosine; GNA = glycolic nucleic acid; sGNA = glycolic nucleic acid with 3'-phosphorothioate; LNA = locked nucleic acid. The insertion of s in the sequence indicates that two adjacent nucleotides are linked by a phosphorothioate group (e.g., a phosphorothioate internucleotide linkage). Unless otherwise noted, all other nucleotides are linked by 3'-5'-phosphodiester groups. Each of the siRNA-based compounds in Table 2 contains a 19-bp duplex region containing either a two-nucleotide sticky end at the 3' end of both strands or a blunt end at one or both ends. GalNAc3K2AhxC6 is:

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Table 2

siRNA sequences targeting PNPLA3 with modifications

Duplex ks № Semantic sequence (5'-3') SEQ ID NO: (meaning of the word I) Antisense sequence (5'-3') SEQ ID NO: (antis MYSLO vaya) D- 2000 GfsgsGfcAfaUfaAfAfGfuAfcC fuGfcUfsusUf 167 asGfscAfgGfuAfcuuUfaUfuGfc CfcsUfsu 168 D- 2001 CfsgsGfcCfaAfuGfUfCfcAfcC faGfcUfsusUf 169 asGfscUfgGfuGfgacAfuUfgGfc CfgsUfsu 170 D- 2002 GfsgsUfcCfaGfcCfUfGfaAfcU fuCfuUfsusUf 171 asAfsgAfaGfuUfcagGfcUfgGfa CfcsUfsu 172 D- 2003 GfscsUfuCfaUfcCfCfCfuUfcU faCfaGfsusUf 173 csUfsgUfaGfaAfgggGfaUfgAfa GfcsUfsu 174 D- 2004 GfscsGfgCfuUfcCfUfGfgGfcU fuCfuAfsusUf 175 usAfsgAfaGfcCfcagGfaAfgCfc GfcsUfsu 176 D- 2005 GfscsCfuCfuGfaGfCfUfgAfgU fuGfgUfsusUf 177 asCfscAfaCfuCfagcUfcAfgAfg GfcsUfsu 178 D- 2006 GfsusGfaCfaAfcGfUfAfcCfcU fuCfaUfsusUf 179 asUfsgAfaGfgGfuacGfuUfgUfc AfcsUfsu 180 D- 2007 CfscsCfgCfcUfcCfAfGfgUfcCf cAfaAfsusUf 181 usUfsuGfgGfaCfcugGfaGfgCfg GfgsUfsu 182 D- 2008 CfsusUfcAfuCfcCfCfUfuCfuA fcAfgUfsusUf 183 asCfsuGfuAfgAfaggGfgAfuGfa AfgsUfsu 184 D- 2009 GfsgsUfaUfgUfuCfCfUfgCfuU fcAfuGfsusUf 185 csAfsuGfaAfgCfaggAfaCfaUfa CfcsUfsu 186 D- 2010 GfsusAfuGfuUfcCfUfGfcUfuC faUfgCfsusUf 187 gsCfsaUfgAfaGfcagGfaAfcAfu AfcsUfsu 188 D- 2011 UfsasUfgUfuCfcUfGfCfuUfcA fuGfcCfsusUf 189 gsGfscAfuGfaAfgcaGfgAfaCfa UfasUfsu 190 D- 2012 AfsusGfuUfcCfuGfCfUfuCfaU fgCfcCfsusUf 191 gsGfsgCfaUfgAfagcAfgGfaAfc AfusUfsu 192 D- 2013 UfsgsUfuCfcUfgCfUfUfcAfuG fcCfcUfsusUf 193 asGfsgGfcAfuGfaagCfaGfgAfa CfasUfsu 194 D- 2014 GfsusUfcCfuGfcUfUfCfaUfgC fcCfuUfsusUf 195 asAfsgGfgCfaUfgaaGfcAfgGfa AfcsUfsu 196 D- 2015 UfsusCfcUfgCfuUfCfAfuGfcC fcUfuCfsusUf 197 gsAfsaGfgGfcAfugaAfgCfaGfg AfasUfsu 198 D- 2016 UfscsCfuGfcUfuCfAfUfgCfcC fuUfcUfsusUf 199 asGfsaAfgGfgCfaugAfaGfcAfg GfasUfsu 200

- 102 047656

D- 2017 CfscsUfgCfuUfcAfUfGfcCfcU fuCfuAfsusUf 201 usAfsgAfaGfgGfcauGfaAfgCfa GfgsUfsu 202 D- 2018 CfsusGfcUfuCfaUfGfCfcCfuU fcUfaCfsusUf 203 g sUf s aGfaAfgGfgcaU fg AfaGfc AfgsUfsu 204 D- 2019 UfsgsCfuUfcAfuGfCfCfcUfuC fuAfcAfsusUf 205 usGfsuAfgAfaGfggcAfuGfaAfg CfasUfsu 206 D- 2020 GfscsUfuCfaUfgCfCfCfuUfcU faCfaGfsusUf 207 csUfsgUfaGfaAfgggCfaUfgAfa GfcsUfsu 208 D- 2021 CfsusUfcAfuGfcCfCfUfuCfuA fcAfgUfsusUf 209 asCfsuGfuAfgAfaggGfcAfuGfa AfgsUfsu 210 D- 2022 UfsusCfaUfgCfcCfUfUfcUfaC faGfuGfsusUf 211 csAfscUfgUfaGfaagGfgCfaUfg AfasUfsu 212 D- 2023 UfscsAfuGfcCfcUfUfCfuAfcA fgUfgGfsusUf 213 csCfsaCfuGfuAfgaaGfgGfcAfu GfasUfsu 214 D- 2024 CfsasUfgCfcCfuUfCfUfaCfaGf uGfgCfsusUf 215 gsCfscAfcUfgUfagaAfgGfgCfa UfgsUfsu 216 D- 2025 AfsusGfcCfcUfuCfUfAfcAfgU fgGfcCfsusUf 217 gsGfscCfaCfuGfuagAfaGfgGfc AfusUfsu 218 D- 2026 UfsgsCfcCfuUfcUfAfCfaGfuG fgCfcUfsusUf 219 asGfsgCfcAfcUfguaGfaAfgGfg CfasUfsu 220 D- 2027 GfscsCfcUfuCfuAfCfAfgUfgG fcCfuUfsusUf 221 asAfsgGfcCfaCfuguAfgAfaGfg GfcsUfsu 222 D- 2028 GfsgsUfaUfgUfuCfCfUfgCfuU IcAfuCfsusUf 223 gsAfsuGfaAfgCfaggAfaCfaUfa CfcsUfsu 224 D- 2029 GfsusAfuGfuUfcCfUfGfcUfuC faUfcCfsusUf 225 gsGfsaUfgAfaGfcagGfaAfcAfu AfcsUfsu 226 D- 2030 UfsasUfgUfuCfcUfGfCfuUfcA fuCfcCfsusUf 227 gsGfsgAfuGfaAfgcaGfgAfaCfa UfasUfsu 228 D- 2031 AfsusGfuUfcCfuGfCfUfuCfaU fcCfcCfsusUf 229 gsGfsgGfaUfgAfagcAfgGfaAfc AfusUfsu 230 D- 2032 UfsgsUfuCfcUfgCfUfUfcAfuC fcCfcUfsusUf 231 asGfsgGfgAfuGfaagCfaGfgAfa CfasUfsu 232 D- 2033 GfsusUfcCfuGfcUfUfCfaUfcC fcCfuUfsusUf 233 asAfsgGfgGfaUfgaaGfcAfgGfa AfcsUfsu 234

- 103 047656

D- 2034 UfsusCfcUfgCfuUfCfAfuCfcC fcUfuCfsusUf 235 gsAfsaGfgGfgAfugaAfgCfaGfg AfasUfsu 236 D- 2035 UfscsCfuGfcUfuCfAfUfcCfcC fuUfcUfsusUf 237 asGfsaAfgGfgGfaugAfaGfcAfg GfasUfsu 238 D- 2036 CfscsUfgCfuUfcAfUfCfcCfcU fuCfuAfsusUf 239 usAfsgAfaGfgGfgauGfaAfgCfa GfgsUfsu 240 D- 2037 CfsusGfcUfuCfaUfCfCfcCfuU fcUfaCfsusUf 241 gsUfsaGfaAfgGfggaUfgAfaGfc AfgsUfsu 242 D- 2038 UfsgsCfuUfcAfuCfCfCfcUfuC fuAfcAfsusUf 243 usGfsuAfgAfaGfgggAfuGfaAfg CfasUfsu 244 D- 2039 UfsusCfaUfcCfcCfUfUfcUfaCf aGfuGfsusUf 245 csAfscUfgUfaGfaagGfgGfaUfg AfasUfsu 246 D- 2040 UfscsAfuCfcCfcUfUfCfuAfcA fgUfgGfsusUf 247 csCfsaCfuGfuAfgaaGfgGfgAfu GfasUfsu 248 D- 2041 CfsasUfcCfcCfuUfCfUfaCfaGf uGfgCfsusUf 249 gsCfscAfcUfgUfagaAfgGfgGfa UfgsUfsu 250 D- 2042 UfscsCfcCfuUfcUfAfCfaGfuG fgCfcUfsusUf 251 asGfsgCfcAfcUfguaGfaAfgGfg GfasUfsu 252 D- 2043 GfsasUfcAfgGfaCfCfCfgAfgC fcGfaUfsusUf 253 asUfscGfgCfuCfgggUfcCfuGfa UfcsUfsu 254 D- 2044 UfsgsGfgCfuUfcUfAfCfcAfcG fuCfgUfsusUf 255 asCfsgAfcGfuGfguaGfaAfgCfc CfasUfsu 256 D- 2045 GfsasGfcGfaGfcAfCfGfcCfcCf gCfaUfsusUf 257 asUfsgCfgGfgGfcguGfcUfcGfc UfcsUfsu 258 D- 2046 UfsgsCfaCfuGfcGfUfCfgGfcG fuCfcUfsusUf 259 asGfsgAfcGfcCfgacGfcAfgUfg CfasUfsu 260 D- 2047 UfsgsGfaGfcAfgAfCfUfcUfgC faGfgUfsusUf 261 asCfscUfgCfaGfaguCfuGfcUfc CfasUfsu 262 D- 2048 UfsgsCfaGfgUfcCfUfCfuCfaG faUfcUfsusUf 263 asGfsaUfcUfgAfgagGfaCfcUfg CfasUfsu 264 D- 2049 CfscsCfgGfcCfaAfUfGfuCfcA fcCfaUfsusUf 265 asUfsgGfuGfgAfcauUfgGfcCfg GfgsUfsu 266 D- 2050 UfsusCfuAfcAfgUfGfGfcCfuU faUfcUfsusUf 267 asGfsaUfaAfgGfccaCfuGfuAfg AfasUfsu 268

- 104 047656

D- 2051 UfscsUfaCfaGfuGfGfCfcUfuA fuCfcUfsusUf 269 asGfsgAfuAfaGfgccAfcUfgUfa GfasUfsu 270 D- 2052 CfsusUfcCfuUfcAfGfAfgGfcG fuGfcUfsusUf 271 asGfscAfcGfcCfucuGfaAfgGfa AfgsUfsu 272 D- 2053 UfsusCfcUfuCfaGfAfGfgCfgU fgCfgAfsusUf 273 usCfsgCfaCfgCfcucUfgAfaGfg AfasUfsu 274 D- 2054 GfscsGfuGfcGfaUfAfUfgUfgG faUfgUfsusUf 275 asCfsaUfcCfaCfauaUfcGfcAfc GfcsUfsu 276 D- 2055 CfsgsUfgCfgAfuAfUfGfuGfg AfuGfgAfsusUf 277 usCfscAfuCfcAfcauAfuCfgCfa CfgsUfsu 278 D- 2056 UfsgsGfaUfgGfaGfGfAfgUfg AfgUfgAfsusUf 279 usCfsaCfuCfaCfuccUfcCfaUfcC fasUfsu 280 D- 2057 AfscsGfuAfcCfcUfUfCfaUfuG faUfgUfsusUf 281 asCfsaUfcAfaUfgaaGfgGfuAfc GfusUfsu 282 D- 2058 UfsgsGfaCfaUfcAfCfCfaAfgC fuCfaUfsusUf 283 asUfsgAfgCfuUfgguGfaUfgUfc CfasUfsu 284 D- 2059 CfsasCfcUfgCfgUfCfUfcAfgC faUfcUfsusUf 285 asGfsaUfgCfuGfagaCfgCfaGfg UfgsUfsu 286 D- 2060 AfscsCfuGfcGfuCfUfCfaGfcA fuCfcUfsusUf 287 asGfsgAfuGfcUfgagAfcGfcAfg GfusUfsu 288 D- 2061 CfscsAfgAfgAfcUfGfGfuGfaC faUfgUfsusUf 289 asCfsaUfgUfcAfccaGfuCfuCfu GfgsUfsu 290 D- 2062 AfsusGfgCfuUfcCfAfGfaUfaU IgCfcUfsusUf 291 asGfsgCfaUfaUfcugGfaAfgCfc AfusUfsu 292 D- 2063 CfscsGfcCfuCfcAfGfGfuCfcCf aAfaUfsusUf 293 asUfsuUfgGfgAfccuGfgAfgGfc GfgsUfsu 294 D- 2064 UfsasCfcUfgCfuGfGfUfgCfuG faGfgUfsusUf 295 asCfscUfcAfgCfaccAfgCfaGfg UfasUfsu 296 D- 2065 AfscsCfuGfcUfgGfUfGfcUfgA fgGfgUfsusUf 297 asCfscCfuCfaGfcacCfaGfcAfg GfusUfsu 298 D- 2066 CfsusCfuCfcAfcCfUfUfuCfcCf aGfuUfsusUf 299 asAfscUfgGfgAfaagGfuGfgAfg AfgsUfsu 300 D- 2067 UfsusUfuUfcAfcCfUfAfaCfuA faAfaUfsusUf 301 asUfsuUfuAfgUfuagGfuGfaAfa AfasUfsu 302

- 105 047656

D- 2068 CfgGfcCfaAfuGfUfCfcAfcCfa GfcUfsusUf 303 asGfscUfgGfuGfgacAfuUfgGfc CfgsUfsu 304 D- 2069 GfgUfcCfaGfcCfUfGfaAfcUfu CfuUfsusUf 305 asAfsgAfaGfuUfcagGfcUfgGfa CfcsUfsu 306 D- 2070 GfcGfgCfuUfcCfUfGfgGfcUfu CfuAfsusUf 307 usAfsgAfaGfcCfcagGfaAfgCfc GfcsUfsu 308 D- 2071 GfuGfaCfaAfcGfUfAfcCfcUfu CfaUfsusUf 309 asUfsgAfaGfgGfuacGfuUfgUfc AfcsUfsu 310 D- 2072 GfgUfaUfgUfuCfCfUfgCfuUfc AfuGfsusUf 311 csAfsuGfaAfgCfaggAfaCfaUfa CfcsUfsu 312 D- 2073 GfuAfuGfuUfcCfUfGfcUfuCfa UfgCfsusUf 313 gsCfsaUfgAfaGfcagGfaAfcAfu AfcsUfsu 314 D- 2074 UfgUfuCfcUfgCfUfUfcAfuGfc CfcUfsusUf 315 asGfsgGfcAfuGfaagCfaGfgAfa CfasUfsu 316 D- 2075 GfuUfcCfuGfcUfUfCfaUfgCfc CfuUfsusUf 317 asAfsgGfgCfaUfgaaGfcAfgGfa AfcsUfsu 318 D- 2076 CfcUfgCfuUfcAfUfGfcCfcUfu CfuAfsusUf 319 usAfsgAfaGfgGfcauGfaAfgCfa GfgsUfsu 320 D- 2077 GfcUfuCfaUfgCfCfCfuUfcUfa CfaGfsusUf 321 csUfsgUfaGfaAfgggCfaUfgAfa GfcsUfsu 322 D- 2078 CfuUfcAfuGfcCfCfUfuCfuAfc AfgUfsusUf 323 asCfsuGfuAfgAfaggGfcAfuGfa AfgsUfsu 324 D- 2079 UfuCfaUfgCfcCfUfUfcUfaCfa GfuGfsusUf 325 csAfscUfgUfaGfaagGfgCfaUfg AfasUfsu 326 D- 2080 AfuGfgCfuUfcCfAfGfaUfaUfg CfcUfsusUf 327 asGfsgCfaUfaUfcugGfaAfgCfc AfusUfsu 328 D- 2081 AfuGfcCfcUfuCfUfAfcAfgUfg GfcCfsusUf 329 gsGfscCfaCfuGfuagAfaGfgGfc AfusUfsu 330 D- 2082 GfcUfuCfaUfgCfCfCfuUfcUfa CfaUfsusUf 331 asUfsgUfaGfaAfgggCfaUfgAfa GfcsUfsu 332 D- 2083 {Phosphate} GfsgsAfaAfgAfcUf GfUfuCfc AfaAfaAfsu sUf 1295 {Φocφaτ}usUfsuUfuGfgAfaca GfuCfuUfuCfcsUfsu 1296 D- 2084 {GalNAc3K2AhxC6}ggsuaug UfuCfCfUfGfcuucaugsusu 1297 {Phosphate} csAfsuGfaAfGfcagg AfaCfauaccsusu 1298

- 106 047656

D- 2085 {GalNAc3K2AhxC6}guauguU fcCfUfGfCfuucaugcsusu 1299 [ Phosphate [gsCfsaUfgAfAfgcag GfaAfcauacsusu 1300 D- 2086 {GalNAc3K2AhxC6}uguuccU fgCfUfUfCfaugcccususu 1301 {Phosphate} as GfsgGfcAfUfgaagC faGfgaacasusu 1302 D- 2087 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacagsusu 1303 {Phosphate} csUfsgUfaGfAfaggg CfaUfgaagcsusu 1304 D- 2088 {GalN Ac3 K2AhxC6} cuucauGf cCfCfUfUfcuacagususu 1305 {Phosphate} asCfsuGfuAfGfaagg GfcAfugaagsusu 1306 D- 2089 {GalNAc3K2AhxC6}gcggcuU fcCfUfGfGfgcuucuasusu 1307 {Φocφaτ}usAfsgAfaGfCfccagG faAfgccgcsusu 1308 D- 2090 {GalN Ac3 K2AhxC6} guuccuG fcUfUfCfAfugcccuususu 1309 {Phosphate} as AfsgGfgCfAfugaa GfcAfggaacsusu 1310 D- 2091 {GalNAc3K2AhxC6}AfuGfgC fuUfcCfAfGfaUfaUfgCfcUfsus Uf 1311 {Phosphate} as GfsgCfaUfaUfcugG faAfgCfcAfusUfsu 1312 D- 2092 {GalN Ac3 K2AhxC6} ccugcuUf cAfUfGfCfccuucuasusu 1313 {Φocφaτ}usAfsgAfaGfGfgcau GfaAfgcaggsusu 1314 D- 2093 {GalNAc3K2AhxC6}uucaugCf cCfUfUfCfuacaguususu 1315 {Phosphate} asAf scUfgUfAfgaag GfgCfaugaasusu 1316 D- 2094 {GalNAc3K2AhxC6}uucaugCf cCfUfUfCfuacagugsusu 1317 {Phosphate} cs AfscUfgUfAfgaag GfgCfaugaasusu 1318 D- 2095 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacaususu 1319 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1320 D- 2096 {GalNAc3K2AhxC6}gguccaGf cCfUfGfAfacuucuususu 1321 {Phosphate} asAfsgAfaGfUfucag GfcUfggaccsusu 1322 D- 2097 {GalNAc3K2AhxC6}GfcGfgC fuUfcCfUfGfGfgcUfuCfuAfsu sUf 1323 {Φocφaτ}usAfsgAfaGfCfccagG faAfgCfcGfcsUfsu 1324 D- 2098 {GalNAc3K2AhxC6}GfcGfgC fuUfcCfuGfGfgcUfuCfuAfsus Uf 1325 {Φocφaτ}usAfsgAfaGfCfccAfg GfaAfgCfcGfcsUfsu 1326 D- 2099 {GalNAc3K2AhxC6}GfuUfcC fuGfcUfUfCfAfugCfcCfuUfsus Uf 1327 {Phosphate} as AfsgGfgCfAfugaa GfcAfgGfaAfcsUfsu 1328

- 107 047656

D- 2100 {GalNAc3K2AhxC6}GfuUfcC fuGfcUfuCfAfugCfcCfuUfsus Uf 1329 {Phosphate} asAfsgGfgCfAfugAfa GfcAfgGfaAfcsUfsu 1330 D- 2101 {GalNAc3K2AhxC6}CfcUfgC fuUfcAfUfGfCfccUfuCfuAfsus Uf 1331 {Φocφaτ}usAfsgAfaGfGfgcau GfaAfgCfaGfgsUfsu 1332 D- 2102 {GalNAc3K2AhxC6}CfcUfgC fuUfcAfuGfCfccUfuCfuAfsus Uf 1333 {Phosphate} us AfsgAfaGfGfgcAfu GfaAfgCfaGfgsUfsu 1334 D- 2103 {GalNAc3K2AhxC6}augcccUf uCfUfAfCfaguggccsusu 1335 i Phosphate JgsGfscCfaCfUf'guag A faGfggcaususu 1336 D- 2104 {GalN Ac3 K2AhxC6} auggcuU fcCfAfGfAfuaugccususu 1337 {Phosphate} as GfsgCfaUfAfucug GfaAfgccaususu 1338 D- 2105 {GalNAc3K2AhxC6}gugacaAf cGfUfAfCfccuucaususu 1339 {Phosphate} asUfsgAfaGfGfguac GfuUfgucacsusu 1340 D- 2106 {GalNAc3K2AhxC6}guauguU fcCfUfGfCfuucaugcsusu 1341 {Φocφaτ}gsCfsaUfgAfAfgcAfg GfaAfcauacsusu 1342 D- 2107 {GalNAc3K2AhxC6}guauguU fcCfuGfCfuucaugcsusu 1343 {Φocφaτ}gsCfsaUfgAfAfgcag GfaAfcauacsusu 1344 D- 2108 {GalNAc3K2AhxC6}guauguU fcCfuGfCfuucaugcsusu 1345 {Φocφaτ}gsCfsaUfgAfAfgcAfg GfaAfcauacsusu 1346 D- 2109 {GalNAc3K2AhxC6}guauguU fcCfUfGfCfuucaugcscsu 1347 {Phosphate} asGfsgCfaUfgAfAfgc agGfaAfcauacsusu 1348 D- 2110 {GalNAc3K2AhxC6}ugguauG fuUfCfCfUfgcuucausgsu 1349 {Φocφaτ}gsCfsaUfgAfaGfCfag gaAfcAfuaccasusu 1350 D- 2111 {GalNAc3K2AhxC6}guauguU fcCfUfGfCfuucausgsu 1351 {Φocφaτ}gsCfsaUfgAfAfgcag GfaAfcauacsusu 1352 D- 2112 {GalNAc3K2AhxC6}guauguU fcCfUfGfCfuucaugcs {invAb} 1353 {Φocφaτ}gsCfsaUfgAfAfgcag GfaAfcauacsusu 1354 D- 2113 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfCfUfucUfaCfaUfsus Uf 1355 {Phosphate} asUfsgUfaGfAfagg CfaUfgAfaGfcsUfsu 1356 D- 2114 {GalNAc3K2AhxC6}GfcUfuC faUfgCfcCfUfucUfaCfaUfsusU 1357 {Phosphate} asUfsgUfaGfAfagGfg CfaUfgAfaGfcsUfsu 1358

- 108 047656

f D- 2115 {GalN Ac3 K2AhxC6} gcuucaUf gCfCfCfUfucuacaususu 1359 {Phosphate} asusguaGfAfagggCfa Ufgaagcsusu 1360 D- 2116 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucUfaCfaUfsusUf 1361 {Phosphate} asusguaGfAfagggCfa Ufgaagcsusu 1362 D- 2117 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfCfUfucuacaususu 1363 {Phosphate} asusguaGfAfagggCfa Ufgaagcsusu 1364 D- 2118 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacaususu 1365 {Phosphate} asusguaGfAfagggCfa UfgAfaGfcsUfsu 1366 D- 2119 {GalN Ac3 K2AhxC6} gcuucaUf gCfCfCfUfucuacaususu 1367 {Phosphate} asUfsgUfaGfAfagg CfaUfgAfaGfcsUfsu 1368 D- 2120 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfCfUfucUfaCfaUfsus Uf 1369 {Phosphate} asusguaGfAfagggCfa Ufgaagcsusu 1370 D- 2121 {GalN Ac3 K2 AhxC 6} GfcUfuC faUfgCfCfCfUfucuacaususu 1371 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1372 D- 2122 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucUfaCfaUfsusUf 1373 {Phosphate} asusguaGfAfaggCfa UfgAfaGfcsUfsu 1374 D- 2123 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucUfaCfaUfsusUf 1375 {Phosphate} asUfsgUfaGfAfagg CfaUfgaagcsusu 1376 D- 2124 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfCfUfucuacaususu 1377 {Phosphate} asusguaGfAfagggCfa UfgAfaGfcsUfsu 1378 D- 2125 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfCfUfucuacaususu 1379 {Phosphate} asUfsgUfaGfAfagg CfaUfgAfaGfcsUfsu 1380 D- 2126 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucUfaCfaUfsusUf 1381 {Phosphate} asUfsgUfaGfAfagg CfaUfgAfaGfcsUfsu 1382 D- 2127 (GalNAc3K2AhxC6 ] GfcUfuC faUfgCfCfCfUfucUfaCfaUfsus Uf 1383 {Phosphate} asUfsgUfaGfAfagg CfaUfgaagcsusu 1384 D- 2128 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfCfUfucUfaCfaUfsus Uf 1385 {Phosphate} asusguaGfAfaggCfa UfgAfaGfcsUfsu 1386 D- 2129 {GalN Ac3 K2AhxC6} gcuucaUf g[dC]CfCfuucuacaususu 1387 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1388

- 109 047656

D- 2130 {GalNAc3K2AhxC6}gcuucaUf gCfCf[dC]Ufucuacaususu 1389 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1390 D- 2131 {GalNAc3K2AhxC6}gcuucaUf gCf[dC]CfUfucuacaususu 1391 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1392 D- 2132 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacaususu 1393 {Phosphate} asUfsgUfaGfAfagGfg CfaUfgaagcsusu 1394 D- 2133 {GalNAc3K2AhxC6}gcuucaUf gCfcCfUfucuacaususu 1395 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1396 D- 2134 {GalN Ac3 K2AhxC6} gcuucaUf gCfcCfUfucuacaususu 1397 {Φocφaτ}asUfsgUfaGfAfagGfg CfaUfgaagcsusu 1398 D- 2135 {GalNAc3K2AhxC6}GfscsUfu CfaUfgCfcCfUfucUfaCfaUfsus Uf 1399 {Phosphate} asUfsgUfaGfAfagg CfaUfgAfaGfcsUfsu 1400 D- 2136 {GalNAc3K2AhxC6}GfscsUfu CfaUfgCfCfCfUfucUfaCfaUfs usUf 1401 {Phosphate} asUfsgUfaGfAfagGfg CfaUfgAfaGfcsUfsu 1402 D- 2137 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacagsusu 1403 {Phosphate} as AfscUfgUfaGfAfag ggCfaUfgaagcsusu 1404 D- 2138 {GalNAc3K2AhxC6}cugcuuCf aUfGfCfCfcuucuacsasu 1405 {Phosphate} asUfsgUfaGfaAfGfgg caUfgAfagcagsusu 1406 D- 2139 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacsasu 1407 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1408 D- 2140 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacaus {invAb} 1409 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1410 D- 2141 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacauuus {invAb} 1411 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1412 D- 2142 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacaususu 1413 {Phosphate} AfsusGfuAfgAfaggg CfaUfgaagcsusu 1414 D- 2143 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfuCfuacaususu 1415 {Phosphate} AfsusGfuAfgAfaggg CfaUfgaagcsusu 1416 D- 2144 {GalNAc3K2AhxC6}gcuucaug CfCfCfUfucuacaususu 1417 {Phosphate} asUfsgUfaGfAfagg CfaUfgaagcsusu 1418 D- 2145 {GalNAc3K2AhxC6}gcuucaUf gCfCfCfUfucuacaususu 1419 {Phosphate} asUfsgUfaGfAfaggg Cfaugaagcsusu 1420

- 110 047656

D- 2146 {GalNAc3K2AhxC6}gcuucaUf gCfCfCIUfucuacaususu 1421 {Phosphate} asusgUfaGfAfagggCf aUfgaagcsusu 1422 D- 2147 {GalNAc3K2AhxC6}gcuucaUf gCfCfCIUfucuacaususu 1423 {Phosphate} asUfsgUfagAfagggCf aUfgaagcsusu 1424 D- 2148 {GalNAc3K2AhxC6}gcuucaUf gCfCfCIUfucuacaususu 1425 {Phosphate} asusgUfagAfagggCfa Ufgaagcsusu 1426 D- 2149 {GalNAc3K2AhxC6}gcuucaUf gCfCfCIUfucuacaususu 1427 {Phosphate} asUfsguagAfagggCfa Ufgaagcsusu 1428 D- 2150 {GalN Ac3 K2AhxC6} gcuucaUf gCfCfCIUfucuacaususu 1429 {Φocφaτ}asusguagAfagggCfaU fgaagcsusu 1430 D- 2151 {GalNAc3K2AhxC6}guauguU fcCfUfGfCfuucaugcuus{invAb } 1431 {Φocφaτ}gsCfsaUfgAfAfgcag GfaAfcauacsusu 1432 D- 2152 {GalN Ac3 K2 AhxC6} gguaugU fuCfCfUfGfcuucauususu 1433 {Phosphate} aAfsuGfaAfGfcaggAf aCfauaccsusu 1434 D- 2153 {GalNAc3K2AhxC6}guauguU fcCfUfGfCfuucaugususu 1435 {Phosphate} asCfsaUfgAfAfgcagG faAfcauacsusu 1436 D- 2154 {GalNAc3K2AhxC6}cggccaAf uGfUfCfCfaccagcususu 1437 {Phosphate} asGfscUfgGIUfggac AfuUfggccgsusu 1438 D- 2155 {GalNAc3K2AhxC6}uggagcA fgAfCfUfCfugcaggususu 1439 { Phosphate} asCfscUfgCfAfgaguC fuGfcuccasusu 1440 D- 2156 {GalNAc3K2AhxC6}acguacCf cUIUfCfAfuugaugususu 1441 {Phosphate} aCfsaUfcAfAfugaaGf gGfuacgususu 1442 D- 2157 {GalNAc3K2AhxC6}ccagagAf cUfGfGfUfgacaugususu 1443 {Phosphate} asCfsaUfgUfCfaccaG fuCfucuggsusu 1444 D- 2158 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfUfUfucuacausus u 1445 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcsusu 1446 D- 2159 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfUfUfucuacausus u 1447 {Phosphate} as AfsaUfgUfaGfAfaa ggCfaUfgaagcsusu 1448 D- 2160 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfcUfUfUfCfuacausu SU 1449 {Phosphate} as AfsaUfgUfAfgaaaG fgCfaUfgaagcsusu 1450

- 111 047656

D- 2161 {GalNAc3K2AhxC6}ugcuucA fuGfCfCfUfuucuacasusu 1451 [ Phosphate JusGfsuAfgAfAfaggc AfuGfaagcasusu 1452 D- 2162 {GalNAc3K2AhxC6}uauguuC fcUfGfCfUfucaugcususu 1453 {Phosphate} as GfscAfuGfAfagcaG fgAfacauasusu 1454 D- 2163 {GalNAc3K2AhxC6}uuccugCf uUfCfAfUfgccuuuususu 1455 {Phosphate} as Afsa AfgGfCfauga A fgCfaggaasusu 1456 D- 2164 {GalNAc3K2AhxC6}ucaugcCf uUfUfCfUfacagugususu 1457 {Phosphate} asCfsaCfuGfUfagaaA fgGfcaugasusu 1458 D- 2165 {GalNAc3K2AhxC6}caugccUf uUfCfUfAfcaguggususu 1459 [ Phosphate jasCfscAfcUfGfuagaA faGfgcaugsusu 1460 D- 2166 {GalN Ac3 K2AhxC6} augccuUf uCfUfAfCfaguggcususu 1461 {Phosphate} asGfscCfaCfUfguagA faAfggcaususu 1462 D- 2167 {GalNAc3K2AhxC6}gguaugU fuCfCfUfGfcuucauasusu 1463 {Φocφaτ}usAfsuGfaAfGfcagg AfaCfauaccsusu 1464 D- 2168 {GalN Ac3 K2AhxC6} guauguU fcCfUfGfCfuucaugasusu 1465 {Φocφaτ}usCfsaUfgAfAfgcag GfaAfcauacsusu 1466 D- 2169 {GalNAc3K2AhxC6}uauguuC fcUfGfCfUfucaugcasusu 1467 [ Phosphate [usGfscAfuGfAfagca GfgAfacauasusu 1468 D- 2170 {GalNAc3K2AhxC6}uuccugCf uUfCfAfUfgccuuuasusu 1469 [Phosphate [us Afsa AfgGfCfauga AfgCfaggaasusu 1470 D- 2171 {GalN Ac3 K2AhxC6} cugcuuCf aUfGfCfCfuuucuaasusu 1471 {Φocφaτ}usUfsaGfaAfAfggca UfgAfagcagsusu 1472 D- 2172 {GalNAc3K2AhxC6}gcuucaUf gCfCfUfUfucuacaasusu 1473 {Φocφaτ}usUfsgUfaGfAfaagg CfaUfgaagcsusu 1474 D- 2173 {GalNAc3K2AhxC6}uucaugCf cUfUfUfCfuacaguasusu 1475 [Phosphate} us AfscIJfgUfAfgaaa GfgCfaugaasusu 1476 D- 2174 (GalN Ac3 K2AhxC6} ucaugcCf uUfUfCfUfacagugasusu 1477 {Phosphate} usCfsaCfuGfUfagaaA fgGfcaugasusu 1478 D- 2175 {GalNAc3K2AhxC6}caugccUf uUfCfUfAfcaguggasusu 1479 [ Phosphate [usCfscAfcUfGfuaga A faGfgcaugsusu 1480 D- 2176 {GalNAc3K2AhxC6}augccuUf uCfUfAfCfaguggcasusu 1481 {Φocφaτ}usGfscCfaCfUfguagA faAfggcaususu 1482 D- 2177 {GalNAc3K2AhxC6}acguacCf cUfUfCfAfuugaugasusu 1483 {Φocφaτ}usCfsaUfcAfAfugaaG fgGfuacgususu 1484

- 112 047656

D- 2178 {GalNAc3K2AhxC6}ccagagAf cUfGfGfUfgacaugasusu 1485 [ Phosphate [usCfsaUfgUfCfaccaG fuCfucuggsusu 1486 D- 2179 {GalN Ac3 K2AhxC6} auggcuU fcCfAfGfAfuaugccasusu 1487 {Φocφaτ}usGfsgCfaUfAfucug GfaAfgccaususu 1488 D- 2180 {GalNAc3K2AhxC6}guuccuG fcUfUfCfAfugccuuususu 1489 {Phosphate} as AfsaGfgCfAfugaaG fcAfggaacsusu 1490 D- 2181 {GalN Ac3 K2AhxC6} ccugcuUf cAfUfGfCfcuuucuasusu 1491 {Φocφaτ}usAfsgAfaAfGfgcau GfaAfgcaggsusu 1492 D- 2182 {GalNAc3K2AhxC6}gcuucaUf gCfCfUfUfucuacaususu 1493 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcsusu 1494 D- 2183 {GalNAc3K2AhxC6}cuucauGf cCfUfUfUfcuacagususu 1495 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1496 D- 2184 {GalNAc3K2AhxC6}uucaugCf cUfUfUfCfuacaguususu 1497 {Phosphate} as AfscUfgUfAfgaaaG fgCfaugaasusu 1498 D- 2185 {GalNAc3K2AhxC6}gcuucaUf cCfCfUfUfucuacaususu 1499 {Phosphate} asUfsgUfaGfAfaagg GfaUfgaagcsusu 1500 D- 2186 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfCfUfucuacausus u 1501 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1502 D- 2187 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfCfUfucuacausus u 1503 {Phosphate} asAfsaUfgUfaGfAfag ggCfaUfgaagcsusu 1504 D- 2188 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfcCfUfUfCfuacausu SU 1505 {Phosphate} as AfsaUfgUfAfgaag GfgCfaUfgaagcsusu 1506 D- 2189 {GalNAc3K2AhxC6} [invAb]g cggcuUfcCfUfGfGfgcuucuasus u 1507 {Φocφaτ}usAfsgAfaGfCfccagG faAfgccgcsusu 1508 D- 2190 {GalNAc3K2AhxC6} [invAb]C fuGfcGfgCfuUfcCfuGfGfgcUf uCfsusAf 1509 {Φocφaτ}usAfsgAfaGfCfccAfg GfaAfgCfcGfcAfgsUfsu 1510 D- 2191 {GalNAc3K2AhxC6}cugcggCf uUfCfCfUfgggcuucus {invAb} 1511 {Φocφaτ}usAfsgAfaGfcCfCfag gaAfgCfcgcagsusu 1512 D- {GalNAc3K2AhxC6} [invAb]c 1513 {Φocφaτ}usAfsgAfaGfcCfCfag 1514

- 113 047656

2192 ugcggCfuUfCfCfUfgggcuucsus a gaAfgCfcgcagsusu D- 2193 {GalNAc3K2AhxC6} [invAb]g cggcuUfcCfUfGfGfgcUfuCfuA fsusUf 1515 {Phosphate; us AfsgAfaGfCfccagG faAfgccgcsusu 1516 D- 2194 {GalNAc3K2AhxC6}cugcggCf uUfCfCfUfggGfcUfuCfus {inv Ab} 1517 {Φocφaτ}usAfsgAfaGfcCfCfag gaAfgCfcgcagsusu 1518 D- 2195 {GalNAc3K2AhxC6} [invAbJc ugcggCfuUfCfCfUfggGfcUfuC fsusAf 1519 {Phosphate} us AfsgAfaGfcCfCfag gaAfgCfcgcagsusu 1520 D- 2196 {GalNAc3K2AhxC6}[invAb]G fcGfgCfuUfcCfuGfGfgcUfuCf uAfsusUf 1521 {Phosphate; us AfsgAfaGfCfccAfg GfaAfgCfcGfcsUfsu 1522 D- 2197 {GalNAc3K2AhxC6}CfuGfcG fgCfuUfcCfUfggGfcUfuCfus {i nvAb} 1523 {Φocφaτ}usAfsgAfaGfcCfCfag GfaAfgCfcGfcAfgsUfsu 1524 D- 2198 {GalNAc3K2AhxC6} [invAb]C fuGfcGfgCfuUfcCfUfggGfcUf uCfsusAf 1525 {Φocφaτ}usAfsgAfaGfcCfCfag GfaAfgCfcGfcAfgsUfsu 1526 D- 2199 {GalNAc3K2AhxC6} [invAb]G fcGfgCfuUfcCfUfGfGfgcuucu asusu 1527 {Φocφaτ}usAfsgAfaGfCfccagG faAfgCfcGfcsUfsu 1528 D- 2200 {GalNAc3K2AhxC6} [invAb]C fuGfcGfgCfuUfCfCfUfgggcuu csusa 1529 {Φocφaτ}usAfsgAfaGfcCfCfag gaAfgCfcGfcAfgsUfsu 1530 D- 2201 {GalNAc3K2AhxC6} [invAb]c ugcggcuUfcCfUfGfGfgcUfuCf usAf 1531 {Φocφaτ}usAfsgAfaGfCfccagG faAfgccgcagsusu 1532 D- 2202 {GalNAc3K2AhxC6} [invAb]C fuGfcGfgCfuUfcCfUfGfGfgcu ucsusa 1533 {Φocφaτ}usAfsgAfaGfCfccagG faAfgCfcGfcAfgsUfsu 1534 D- 2203 {GalNAc3K2AhxC6} [invAb]a uggcuUfcCfAfGfAfuaugccusus 1535 {Phosphate} as GfsgCfaUfAfucug GfaAfgccaususu 1536

- 114 047656

u D- 2204 {GalNAc3K2AhxC6} [invAb] A fcAfuGfgCfuUfcCfaGfAfuaUf gCfscsUf 1537 {Phosphate} as GfsgCfaUfAfucUfg GfaAfgCfcAfuGfusUfsu 1538 D- 2205 {GalNAc3K2AhxC6}acauggCf uUfCfCfAfgauaugccs {invAb} 1539 {Phosphate} as GfsgCfaUfaUfCfug gaAfgCfcaugususu 1540 D- 2206 {GalN Ac3 K2AhxC6} [invAb] a cauggCfuUfCfCfAfgauaugcscs u 1541 {Phosphate} asGfsgCfaUfaUfCfug gaAfgCfcaugususu 1542 D- 2207 {GalNAc3K2AhxC6} [invAb]a uggcuUfcCfAfGfAfuaUfgCfcU fsusUf 1543 {Phosphate} as GfsgCfaUfAfucug GfaAfgccaususu 1544 D- 2208 {GalNAc3K2AhxC6}acauggCf uUfCfCfAfgaUfaUfgCfc s {inv Ab} 1545 {Phosphate} asGfsgCfaUfaUfCfug gaAfgCfcaugususu 1546 D- 2209 {GalN Ac3 K2AhxC6} [invAb] a cauggCfuUfCfCfAfgaUfaUfgC fscsUf 1547 {Phosphate} asGfsgCfaUfaUfCfug gaAfgCfcaugususu 1548 D- 2210 {GalNAc3K2AhxC6} [invAb] A fuGfgCfuUfcCfaGfAfuaUfgCf cUfsusUf 1549 {Phosphate} as GfsgCfaUfAfucUfg GfaAfgCfcAfusUfsu 1550 D- 2211 {GalNAc3K2AhxC6} AfcAfuG fgCfuUfcCfAfgaUfaUfgCfcs {i nvAb} 1551 {Phosphate} asGfsgCfaUfaUfCfug GfaAfgCfcAfuGfusUfsu 1552 D- 2212 {GalNAc3K2AhxC6} [invAb] A fcAfuGfgCfuUfcCfAfgaUfaUf gCfscsUf 1553 {Phosphate} asGfsgCfaUfaUfCfug GfaAfgCfcAfuGfusUfsu 1554 D- 2213 {GalNAc3K2AhxC6] [invAb] A fuGfgCfuUfcCfAfGfAfuaugcc ususu 1555 {Phosphate} asGfsgCfaUfAfucug GfaAfgCfcAfusUfsu 1556 D- 2214 {GalNAc3K2AhxC6} AfcAfuG fgCfuUfCfCfAfgauaugccs{inv Ab} 1557 {Phosphate} asGfsgCfaUfaUfCfug gaAfgCfcAfuGfusUfsu 1558 D- {GalNAc3K2AhxC6} [invAb] A 1559 {Phosphate} asGfsgCfaUfaUfCfug 1560

- 115 047656

2215 fcAfuGfgCfuUfCfCfAfgauaug cscsu gaAfgCfcAfuGfusUfsu D- 2216 {GalNAc3K2AhxC6} [invAb]a cauggcuUfcCfAfGfAfuaugcscs u 1561 {Phosphate} as GfsgCfaUfAfucug GfaAfgccaugususu 1562 D- 2217 {GalNAc3K2AhxC6} [invAb]a cauggcuUfcCfAfGfAfuaUfgCf csUf 1563 {Phosphate} asGfsgCfaUfAfucug GfaAfgccaugususu 1564 D- 2218 (GalNAc3K2AhxC6] [invAb] A fcAfuGfgCfuUfcCfAfGfAfuau gcscsu 1565 {Phosphate} asGfsgCfaUfAfucug GfaAfgCfcAfuGfusUfsu 1566 D- 2219 {GalNAc3K2AhxC6}[invAb]a cguacCfcUfUfCfAfuugaugusus u 1567 {Phosphate} asCfsaUfcAfAfugaaG fgGfuacgususu 1568 D- 2220 {GalNAc3K2AhxC6} [invAb]C faAfcGfuAfcCfcUfuCfAfuuGf aUfsgsUf 1569 {Phosphate} asCfsaUfcAfAfugAfa GfgGfuAfcGfuUfgsUfsu 1570 D- 2221 {GalNAc3K2AhxC6} [invAb]c aacguAfcCfCfUfUfcauugausgs u 1571 {Phosphate} asCfsaUfcAfaUfGfaa ggGfuAfcguugsusu 1572 D- 2222 {GalNAc3K2AhxC6} [invAb]a cguacCfcUfUfCfAfuuGfaUfgU fsusUf 1573 {Phosphate} asCfsaUfcAfAfugaaG fgGfuacgususu 1574 D- 2223 {GalN Ac3 K2 AhxC 6} caacgu Af cCfCfUfUfcaUfuGfaUfgs {inv Ab} 1575 {Phosphate} asCfsaUfcAfaUfGfaa ggGfuAfcguugsusu 1576 D- 2224 {GalNAc3K2AhxC6} [invAb] A fcGfuAfcCfcUfuCfAfuuGfaUf gUfsusUf 1577 {Phosphate} asCfsaUfcAfAfugAfa GfgGfuAfcGfusUfsu 1578 D- 2225 {GalNAc3K2AhxC6}CfaAfcG fu AfcCfcUfUfcaUfuGfaUfgs {i nvAb} 1579 {Phosphate} asCfsaUfcAfaUfGfaa GfgGfuAfcGfuUfgsUfsu 1580 D- 2226 {GalNAc3K2AhxC6} [invAb]C faAfcGfuAfcCfcUfUfcaUfuGf 1581 {Phosphate} asCfsaUfcAfaUfGfaa GfgGfuAfcGfuUfgsUfsu 1582

- 116 047656

aUfsgsUf D- 2227 {GalNAc3K2AhxC6} [invAb] A fcGfuAfcCfcUfUfCfAfuugaug ususu 1583 {Phosphate} asCfsaUfcAfAfugaaG fgGfuAfcGfusUfsu 1584 D- 2228 {GalNAc3K2AhxC6}CfaAfcG fuAfcCfCfUfUfcauugaugs {inv Ab} 1585 {Phosphate} asCfsaUfcAfaUfGfaa ggGfuAfcGfuUfgsUfsu 1586 D- 2229 {GalN Ac3 K2 AhxC6} [inv Ab] C faAfcGfuAfcCfCfUfUfcauugau sgsu 1587 {Phosphate} asCfsaUfcAfaUfGfaa ggGfuAfcGfuUfgsUfsu 1588 D- 2230 {GalNAc3K2AhxC6} [invAb]c aacguacCfcUfUfCfAfuugausgs u 1589 {Phosphate} asCfsaUfcAfAfugaaG fgGfuacguugsusu 1590 D- 2231 {GalNAc3K2AhxC6} [invAb]c aacguacCfcU fU fCfAfuu GfaU f gsUf 1591 {Phosphate} asCfsaUfcAfAfugaaG fgGfuacguugsusu 1592 D- 2232 {GalNAc3K2AhxC6} [invAb]C faAfcGfuAfcCfcUfUfCfAfuug ansgsu 1593 {Phosphate} asCfsaUfcAfAfugaaG fgGfuAfcGfuUfgsUfsu 1594 D- 2233 {GalN Ac3 K2AhxC6} cugcuuca UfgCfCfUfUfucuacsasu 1595 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1596 D- 2234 {GalN AsZ K2AhxC6} cugcuuca UfgCfCfUfUfucUfaCfsasUf 1597 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1598 D- 2235 {GalN Ac3 K2AhxC6} [invAb] G fcUfuCfaUfgCfcUfUfucUfaCfa UfsusUf 1599 {Phosphate} asUfsgUfaGfAfaaGfg CfaUfgAfaGfcsUfsu 1600 D- 2236 {GalN Ac3 K2 AhxC6} [invAb] G fcUfuCfaUfgCfCfUfUfucuacau SUSU 1601 { Phosphate} asUfsgUfaGfAfaaggC faUfgAfaGfcsUfsu 1602 D- 2237 {GalNAc3K2AhxC6}CfuGfcU fuCfaUfgCfcUfUfucUfaCfsasU f 1603 {Phosphate} asUfsgUfaGfAfaaGfg CfaUfgAfaGfcAfgsUfsu 1604 D- 2238 {GalNAc3K2AhxC6}CfuGfcU fuCfaUfgCfCfUfUfucuacsasu 1605 {Phosphate} asUfsgUfaGfAfaaggC faUfgAfaGfcAfgsUfsu 1606

- 117 047656

D- 2239 {GalNAc3K2AhxC6} [invAb]C fuGfcUfuCfaUfgCfCfUfUfucu acsasu 1607 {Phosphate} asUfsgUfaGfAfaaggC faUfgAfaGfcAfgsUfsu 1608 D- 2240 {GalNAc3K2AhxC6}CfuGfcU fuCfaUfgCfCfUfUfucuacas {in vAb} 1609 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1610 D- 2241 {GalNAc3K2AhxC6} [invAb]C fuGfcUfuCfaUfgCfCfUfUfucu acsasu 1611 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1612 D- 2242 {GalNAc3K2AhxC6}[invAb]c ugcuucaUfgCf[dC]UfUfucuacs asu 1613 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1614 D- 2243 {GalN Ac3 K2AhxC6} cugcuuca UfgCfCfUfUfucuacsasu 1615 {Phosphate} asUfsgUfaGfAfaaGfg CfaUfgaagcagsusu 1616 D- 2244 {GalNAc3K2AhxC6} [invAb]c ugcuucaUfgCfCfUfUfucuacsas u 1617 {Phosphate} asUfsgUfaGfAfaaGfg CfaUfgaagcagsusu 1618 D- 2245 {GalN Ac3 K2AhxC6} cugcuuca UfgCfcUfUfucuacsasu 1619 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1620 D- 2246 {GalN Ac3 K2AhxC6} cugcuuca UfgCfcUfUfucuacas {invAb} 1621 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1622 D- 2247 {GalNAc3K2AhxC6} [invAb]c ugcuucaUfgCfcUfUfucuacsasu 1623 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1624 D- 2248 {GalN Ac3 K2AhxC6} cugcuuca UfgCfCfUfUfucuacsasu 1625 {Phosphate} asUfsgUfagAfaaggCf aUfgaagcagsusu 1626 D- 2249 {GalN Ac3 K2AhxC6} cugcuuca UfgCfCfUfUfucuacas {invAb} 1627 {Phosphate} asUfsgUfagAfaaggCf aUfgaagcagsusu 1628 D- 2250 {GalNAc3K2AhxC6} [invAb]c ugcuucaUfgCfCfUfUfucuacsas u 1629 {Phosphate} asUfsgUfagAfaaggCf aUfgaagcagsusu 1630 D- 2251 {GalN Ac3 K2AhxC6} cugcuuca UfgCfCfUfUfucuacas {invAb} 1631 {Phosphate} asUfsguagAfaaggCfa Ufgaagcagsusu 1632 D- 2252 {GalNAc3K2AhxC6} [invAb]c ugcuucaUfgCfCfUfUfucuacsas 1633 {Phosphate} asUfsguagAfaaggCfa Ufgaagcagsusu 1634

- 118 047656

u D- 2253 {GalNAc3K2AhxC6} [invAb]G fcUfuCfaUfgCfcUfUfucUfaCfa UfsusUf 1635 {Phosphate} asUfsgUfagAfaaGfgC faUfgAfaGfcsUfsu 1636 D- 2254 {GalNAc3K2AhxC6} [invAb]G fcUfuCfaUfgCfCfUfUfucuacau SUSU 1637 {Phosphate} asUfsgUfagAfaaggCf aU fg AfaGfc sUf su 1638 D- 2255 {GalN Ac3 K2 AhxC6} [inv Ab] G fcUfuCfaUfgCfCfUfUfucuacau SUSU 1639 { Phosphate} asUfsgUfagAfaaggCf aUfgaagcsusu 1640 D- 2256 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfUfUfucuacausus u 1641 {Phosphate} asUfsgUfagAfaaGfgC faUfgaagcsusu 1642 D- 2257 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfcUfUfucuacaususu 1643 {Phosphate} asUfsgUfagAfaaGfgC faUfgaagcsusu 1644 D- 2258 {GalNAc3K2AhxC6}cugcuuca UfgCfCfCfUfucuacsasu 1645 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcagsusu 1646 D- 2259 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfCfUfucUfaCfaU fsusUf 1647 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1648 D- 2260 {GalNAc3K2AhxC6} [invAb]G fcUfuCfaUfgCfcCfUfucUfaCfa UfsusUf 1649 {Phosphate} asUfsgUfaGfAfagGfg CfaUfgAfaGfcsUfsu 1650 D- 2261 {GalNAc3K2AhxC6} [invAb]G fcUfuCfaUfgCfCfCfUfucuacau SUSU 1651 {Phosphate} asUfsgUfaGfAfagg CfaUfgAfaGfcsUfsu 1652 D- 2262 {GalN Ac3 K2 AhxC6} cugcuuca UfgCfCfCfUfucuacas {inv Ab} 1653 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcagsusu 1654 D- 2263 {GalNAc3K2AhxC6} [invAb]c ugcuucaUfgCfCfCfUfucuacsas u 1655 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcagsusu 1656 D- 2264 {GalNAc3K2AhxC6} [invAb]G fcUfuCfaUfgCfCfCfUfucuacau SUSU 1657 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1658

- 119 047656

D- 2265 {GalN Ac3 K2AhxC6} auguucCf uGfCfUfUfcaugccususu 1659 {Phosphate} as GfsgCfaUfGfaagcA fgGfaacaususu 1660 D- 2266 {GalNAc3K2AhxC6}uguuccU fgCfUfUfCfaugccuususu 1661 {Phosphate} as AfsgGfcAfUfgaagC faGfgaacasusu 1662 D- 2267 {GalNAc3K2AhxC6}ugccuuU fcUfAfCfAfguggccususu 1663 {Phosphate} as GfsgCfcAfCfiiguaG faAfaggcasusu 1664 D- 2268 {Biotin- C6 JcguacuUfcGfUfCfCfuugua ugsusu 1665 {Phosphate} csAfsuAfcAfAfggac GfaAfguacgsusu 1666 D- 2269 {GalNAc3K2AhxC6} [invAblg cuucaU fgCf [dC] CfUfucuacau s usu 1667 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1668 D- 2270 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfCfUfucuacausus u 1669 {Phosphate} asUfsgUfaGfAfagGfg CfaUfgaagcsusu 1670 D- 2271 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfcCfUfucuacaususu 1671 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1672 D- 2272 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfCfUfucuacausus u 1673 {Phosphate} asUfsgUfagAfagggCf aUfgaagcsusu 1674 D- 2273 {GalNAc3K2AhxC6} [invAb]g cuucaUfgCfCfCfUfucuacausus u 1675 {Phosphate} asUfsguagAfagggCfa Ufgaagcsusu 1676 D- 2274 {GalNAc3K2AhxC6} [invAb]G fcUfuCfaUfgCfcCfUfucuacaus usu 1677 {Phosphate} asUfsgUfaGfAfaggg CfaUfgaagcsusu 1678 D- 2275 {sGalNAc3K2AhxC6 } [invAb] ccugcuUfcAfUfGfCfcuUfuCfu AfsusUf 1679 {Φocφaτ}usAfsgAfaAfGfgcau GfaAfgcaggsusu 1680 D- 2276 {sGalNAc3K2AhxC6 } [invAb] CfcUfgCfuUfcAfuGfCfcuUfuC fuAfsusUf 1681 {Φocφaτ}usAfsgAfaAfGfgcAfu GfaAfgCfaGfgsUfsu 1682 D- 2277 {sGalNAc3K2AhxC6 }UfuCfc UfgCfuUfcAfUfgcCfuUfuCfus 1683 {Φocφaτ}usAfsgAfaAfgGfCfau GfaAfgCfaGfgAfasUfsu 1684

- 120 047656

{invAb} D- 2278 {sGalNAc3K2AhxC6} [invAb] CfcUfgCfuUfcAfUfGfCfcuuuc uasusu 1685 {Phosphate} us AfsgAfaAfGfgcau GfaAfgCfaGfgsUfsu 1686 D- 2279 {sGalNAc3K2AhxC6 JUfuCfc UfgCfuUfCfAfUfgccuuucus {in vAb] 1687 {Φocφaτ}usAfsgAfaAfgGfCfau gaAfgCfaGfgAfasUfsu 1688 D- 2280 {sGalNAc3K2AhxC6} [invAb] augccuUfuCfUfAfCfaguggcusu SU 1689 {Phosphate} asGfscCfaCfUfguagA faAfggcaususu 1690 D- 2281 {sGalNAc3K2AhxC6} [invAb] augccuUfuCfUfAfCfagUfgGfc UfsusUf 1691 {Phosphate} asGfscCfaCfUfguagA faAfggcaususu 1692 D- 2282 {sGalNAc3K2AhxC6} [invAb] AfuGfcCfuUfuCfu AfCfagU fg GfcUfsusUf 1693 {Phosphate} asGfscCfaCfUfguAfg AfaAfgGfc AfusUfsu 1694 D- 2283 {sGalNAc3K2AhxC6 JUfcAfu GfcCfuUfuCfUfacAfgUfgGfcs {invAb} 1695 {Phosphate} as GfscCfaCfuGfUfag AfaAfgGfcAfuGfasUfsu 1696 D- 2284 {sGalNAc3 K2 AhxC6} [invAb] AfuGfcCfuUfuCfUfAfCfagugg cususu 1697 {Phosphate} asGfscCfaCfUfguagA faAfgGfcAfusUfsu 1698 D- 2285 {sGalNAc3K2AhxC6} [invAb] UfcAfuGfcCfuUfUfCfUfacagu ggscsu 1699 {Phosphate} asGfscCfaCfuGfUfag aaAfgGfcAfuGfasUfsu 1700 D- 2286 {sGalNAc3K2AhxC6} [invAb] ugcuucAfuGfCfCfUfuucuacasg SU 1701 {Phosphate} asCfsuGfuAfgAfAfag gcAfuGfaagcasusu 1702 D- 2287 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfUfUfUfcuAfcAfg UfsusUf 1703 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1704 D- 2288 {sGalNAc3K2AhxC6 JUfgCfu UfcAfuGfcCfUfuuCfuAfcAfgs {invAb} 1705 {Phosphate} asCfsuGfuAfgAfAfag GfcAfuGfaAfgCfasUfsu 1706

- 121 047656

D- 2289 {sGalNAc3 K2 AhxC6} [invAb] CfuUfcAfuGfcCfUfUfUfcuaca gususu 1707 {Phosphate} asCfsuGfuAfGfaaagG fcAfuGfaAfgsUfsu 1708 D- 2290 {sGalNAc3K2AhxC6 JUfgCfu UfcAfuGfCfCfUfuucuacags{in vAb] 1709 {Phosphate} asCfsuGfuAfgAfAfag gcAfuGfaAfgCfasUfsu 1710 D- 2291 {sGalNAc3K2AhxC6} [invAb] UfgCfuUfcAfuGfCfCfUfuucua casgsu 1711 {Phosphate} asCfsuGfuAfgAfAfag gcAfuGfaAfgCfasUfsu 1712 D- 2292 {sGalNAc3K2AhxC6} [invAb] ugcuucauGfcCfUfUfUfcuacasg SU 1713 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagcasusu 1714 D- 2293 {sGalNAc3K2AhxC6} [invAb] ugcuucauGfcCfUfUfUfcuAfcA fgsUf 1715 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagcasusu 1716 D- 2294 {sGalNAc3K2AhxC6} [invAb] UfgCfuUfcAfuGfcCfUfUfUfcu acasgsu 1717 {Phosphate} asCfsuGfuAfGfaaagG fcAfuGfaAfgCfasUfsu 1718 D- 2295 {sGalNAc3K2AhxC6} [invAb] guuccuGfcUfUfCfAfugccuuus usu 1719 {Phosphate} as AfsaGfgCfAfugaaG fcAfggaacsusu 1720 D- 2296 {sGalNAc3K2AhxC6} [invAb] ccugcuUfcAfUfGfCfcuuucuasu SU 1721 {Φocφaτ}usAfsgAfaAfGfgcau GfaAfgcaggsusu 1722 D- 2297 {sGalN Ac3K2AhxC6} [invAb] cuucauGfcCfUfUfUfcuacagusu SU 1723 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1724 D- 2298 {sGalNAc3K2AhxC6} [invAb] UfuCfcUfgCfuUfcAfuGfCfcu UfuCfsusAf 1725 {Φocφaτ}usAfsgAfaAfGfgcAfu GfaAfgCfaGfgAfasUfsu 1726 D- 2299 {sGalNAc3K2AhxC6]uuccug CfuUfCfAfUfgccuuucus {invA b} 1727 {Φocφaτ}usAfsgAfaAfgGfCfau gaAfgCfaggaasusu 1728 D- {sGalNAc3K2AhxC6} [invAb] 1729 {Φocφaτ}usAfsgAfaAfgGfCfau 1730

- 122 047656

2300 uuccugCfuUfCfAfUfgccuuucsu sa gaAfgCfaggaasusu D- 2301 {sGalNAc3K2AhxC6}uuccug CfuUfCfAfUfgcCfuUfuCfus {i nvAb} 1731 {Phosphate} us AfsgAfaAfgGfCfau gaAfgCfaggaasusu 1732 D- 2302 {sGalNAc3K2AhxC6} [invAb] uuccugCfuUfCfAfUfgcCfuUfu CfsusAf 1733 {Phosphate} us AfsgAfaAfgGfCfau gaAfgCfaggaasusu 1734 D- 2303 (sGalNAc3K2AhxC6} [invAb] UfuCfcUfgCfuUfcAfUfgcCfu UfuCfsusAf 1735 { Phosphate] us AfsgAfaAfgGfCfau GfaAfgCfaGfgAfasUfsu 1736 D- 2304 {sGa!NAc3K2AhxC6] [invAb] UfuCfcUfgCfuUfCfAfUfgccuu ucsusa 1737 {Φocφaτ}usAfsgAfaAfgGfCfau gaAfgCfaGfgAfasUfsu 1738 D- 2305 {sGalNAc3K2AhxC6} [invAb] uuccugcuUfcAfUfGfCfcuuucsu sa 1739 {Φocφaτ}usAfsgAfaAfGfgcau GfaAfgcaggaasusu 1740 D- 2306 {sGalNAc3K2AhxC6} [invAb] uuccugcuUfcAfUfGfCfcuUfuC fusAf 1741 {Φocφaτ}usAfsgAfaAfGfgcau GfaAfgcaggaasusu 1742 D- 2307 {sGalNAc3K2AhxC6} [invAb] UfuCfcUfgCfuUfcAfUfGfCfcu uucsusa 1743 {Φocφaτ}usAfsgAfaAfGfgcau GfaAfgCfaGfgAfasUfsu 1744 D- 2308 {sGalNAc3K2AhxC6} [invAb] UfcAfuGfcCfuUfuCfuAfCfag UfgGfscsUf 1745 {Phosphate] asGfscCfaCfUfguAfg AfaAfgGfcAfuGfasUfsu 1746 D- 2309 {sGalNAc3K2AhxC6}ucaugcC fuUfUfCfUfacaguggcs {invAb} 1747 { Phosphate] asGfscCfaCfuGfUfag aaAfgGfcaugasusu 1748 D- 2310 {sGalNAc3K2AhxC6} [invAb] ucaugcCfuUfUfCfUfacaguggsc SU 1749 { Phosphate] asGfscCfaCfuGfUfag aaAfgGfcaugasusu 1750 D- 2311 {sGalNAc3K2AhxC6]ucaugcC fuUfUfCfUfacAfgUfgGfcs {inv Ab] 1751 {Phosphate] asGfscCfaCfuGfUfag aaAfgGfcaugasusu 1752

- 123 047656

D- 2312 {sGalNAc3 K2 AhxC6} [inv Ab] ucaugcCfuUfUfCfUfacAfgUfg GfscsUf 1753 {Phosphate} asGfscCfaCfuGfUfag aaAfgGfcaugasusu 1754 D- 2313 {sGalNAc3K2AhxC6} [inv Ab] UfcAfuGfcCfuUfuCfUfacAfg UfgGfscsUf 1755 {Phosphate} asGfscCfaCfuGfUfag AfaAfgGfcAfuGfasUfsu 1756 D- 2314 {sGalNAc3K2AhxC6 JUfcAfu GfcCfuUfUfCfUfacaguggcs {in vAb] 1757 {Phosphate} asGfscCfaCfuGfUfag aaAfgGfcAfuGfasUfsu 1758 D- 2315 {sGalNAc3K2AhxC6} [inv Ab] ucaugccuUfuCfUfAfCfaguggsc SU 1759 {Phosphate} asGfscCfaCfUfguagA faAfggcaugasusu 1760 D- 2316 {sGalNAc3K2AhxC6} [inv Ab] ucaugccuUfuCfUfAfCfagUfgG fcsUf 1761 {Phosphate} asGfscCfaCfUfguagA faAfggcaugasusu 1762 D- 2317 {sGalNAc3K2AhxC6} [inv Ab] UfcAfuGfcCfuUfuCfUfAfCfag uggscsu 1763 {Phosphate} asGfscCfaCfUfguagA faAfgGfcAfuGfasUfsu 1764 D- 2318 {sGalNAc3K2AhxC6} [inv Ab] UfgCfuUfcAfuGfcCfuUfUfcu AfcAfsgsUf 1765 {Phosphate} asCfsuGfuAfGfaaAfg GfcAfuGfaAfgCfasUfsu 1766 D- 2319 {sGalNAc3K2AhxC6]ugcuuc AfuGfCfCfUfuucuacags{invA b} 1767 {Phosphate} asCfsuGfuAfgAfAfag gcAfuGfaagcasusu 1768 D- 2320 {sGalNAc3K2AhxC6]ugcuuc AfuGfCfCfUfuuCfuAfc Afgs {i nvAb] 1769 {Phosphate} asCfsuGfuAfgAfAfag gcAfuGfaagcasusu 1770 D- 2321 {sGalNAc3K2AhxC6} [inv Ab] ugcuucAfuGfCfCfUfuuCfuAfc AfsgsUf 1771 {Phosphate} asCfsuGfuAfgAfAfag gcAfuGfaagcasusu 1772 D- 2322 {sGalNAc3K2AhxC6} [inv Ab] CfuUfcAfuGfcCfuUfUfcuAfc AfgUfsusUf 1773 {Phosphate} asCfsuGfuAfGfaaAfg GfcAfuGfaAfgsUfsu 1774 D- {sGalNAc3K2AhxC6} [inv Ab] 1775 {Phosphate} asCfsuGfuAfgAfAfag 1776

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2323 UfgCfuUfcAfuGfcCfUfuuCfu AfcAfsgsUf GfcAfuGfaAfgCfasUfsu D- 2324 {sGalNAc3K2AhxC6} [invAb] gcuucaUfgCfCfUfUfucuacausu SU 1777 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcsusu 1778 D- 2325 {sGalNAc3K2AhxC6} [invAb] caugccUfuUfCfUfAfcaguggusu SU 1779 {Phosphate} asCfscAfcUfGfiiagaA faGfgcaugsusu 1780 D- 2326 (sGalNAc3K2AhxC6} [invAb] cugcuuCfaUfGfCfCfuuucuaasu SU 1781 {Phosphate} usUfsaGfaAfAfggca UfgAfagcagsusu 1782 D- 2327 {sGa!NAc3K2AhxC6] [invAb] gcuucaUfgCfCfUfUfucuacaasu SU 1783 {Φocφaτ}usUfsgUfaGfAfaagg CfaUfgaagcsusu 1784 D- 2328 {sGalNAc3K2AhxC6} [invAb] uucaugCfcUfUfUfCfuacaguasu SU 1785 {Φocφaτ}usAfscUfgUfAfgaaa GfgCfaugaasusu 1786 D- 2329 {sGalNAc3K2AhxC6} [invAb] guuccuGfcUfUfCfAfugCfcUfu UfsusUf 1787 {Phosphate} as AfsaGfgCfAfugaaG fcAfggaacsusu 1788 D- 2330 {sGalNAc3K2AhxC6]auguuc CfuGfCfUfUfcaUfgCfcUfus {in vAb] 1789 {Phosphate} as AfsaGfgCfaUfGfaa gcAfgGfaacaususu 1790 D- 2331 {sGalNAc3K2AhxC6} [invAb] auguucCfuGfCf U fU fcaU fgCfc UfsusUf 1791 {Phosphate} as AfsaGfgCfaUfGfaa gcAfgGfaacaususu 1792 D- 2332 {sGalNAc3K2AhxC6} [invAb] GfuUfcCfuGfcUfuCfAfugCfc UfuUfsusUf 1793 {Phosphate} as AfsaGfgCfAfugAfa GfcAfgGfaAfcsUfsu 1794 D- 2333 {sGalNAc3K2AhxC6} AfuGfu UfcCfuGfcUfUfcaUfgCfcUfus {invAb} 1795 {Phosphate} as AfsaGfgCfaUfGfaa GfcAfgGfaAfcAfusUfsu 1796 D- 2334 {sGalNAc3K2AhxC6} [invAb] AfuGfuUfcCfuGfcUfUfcaUfg 1797 {Phosphate} as AfsaGfgCfaUfGfaa GfcAfgGfaAfcAfusUfsu 1798

- 125 047656

CfcUfsusUf D- 2335 {sGalNAc3K2AhxC6} [invAb] GfuUfcCfuGfcUfUfCfAfugccu uususu 1799 {Phosphate} as AfsaGfgCfAfugaaG fcAfgGfaAfcsUfsu 1800 D- 2336 {sGalNAc3K2AhxC6} AfuGfu UfcCfuGfCfUfUfcaugccuus {in vAb] 1801 {Phosphate} as AfsaGfgCfaUfGfaa gcAfgGfaAfcAfusUfsu 1802 D- 2337 {sGalNAc3K2AhxC6} [invAb] AfuGfuUfcCfuGfCfUfUfcaugc cususu 1803 {Phosphate} as AfsaGfgCfaUfGfaa gcAfgGfaAfcAfusUfsu 1804 D- 2338 {sGalNAc3K2AhxC6} [invAb] auguuccuGfcUfUfCfAfugccusu SU 1805 {Phosphate} as AfsaGfgCfAfugaaG fcAfggaacaususu 1806 D- 2339 {sGalNAc3K2AhxC6} [invAb] auguuccuGfcUfUfCfAfugCfcU fusUf 1807 {Phosphate} as AfsaGfgCfAfugaaG fcAfggaacaususu 1808 D- 2340 {sGalNAc3K2AhxC6} [invAb] GfcUfuCfaUfgCfcUfuUfCfuaC faGfsusAf 1809 {Φocφaτ}usAfscUfgUfAfgaAfa GfgCfaUfgAfaGfcsUfsu 1810 D- 2341 {sGalNAc3 K2 AhxC6} [invAb] gcuucaUfgCfCfUfUfucuacagsu sa 1811 [Phosphate] us AfscUfgUfaGfAfaa ggCfaUfgaagcsusu 1812 D- 2342 {sGalNAc3K2AhxC6} [invAb] uucaugCfcUfUfUfCfuaCfaGfu AfsusUf 1813 {Φocφaτ}usAfscUfgUfAfgaaa GfgCfaugaasusu 1814 D- 2343 {sGalNAc3K2AhxC6} [invAb] gcuucaUfgCfCfUfUfucUfaCfa GfsusAf 1815 {Φocφaτ}usAfscUfgUfaGfAfaa ggCfaUfgaagcsusu 1816 D- 2344 {sGalNAc3K2AhxC6}GfcUfu CfaUfgCfCfUfUfucuacagus {in vAb] 1817 {Φocφaτ}usAfscUfgUfaGfAfaa ggCfaUfgAfaGfcsUfsu 1818 D- 2345 {sGalNAc3K2AhxC6} [invAb] GfcGfgCfuUfcCfUfGfGfgcuuc uasusu 1819 {Φocφaτ}usAfsgAfaGfCfccagG faAfgCfcGfcsUfsu 1820

- 126 047656

D- 2346 {sGalNAc3 K2 AhxC6} [inv Ab] auggcuUfcCfAfGfAfuaugccusu SU 1821 {Phosphate} as GfsgCfaUfAfucug GfaAfgccaususu 1822 D- 2347 {sGalNAc3K2AhxC6} [inv Ab] acauggCfuUfCfCfAfgaUfaUfg CfscsUf 1823 {Phosphate} asGfsgCfaUfaUfCfug gaAfgCfcaugususu 1824 D- 2348 {sGalNAc3K2AhxC6} [inv Ab] AfcAfuGfgCfuUfcCfAfGfAfua ugcscsu 1825 {Phosphate} as GfsgCfaUfAfucug GfaAfgCfcAfuGfusUfsu 1826 D- 2349 {sGalNAc3K2AhxC6} [inv Ab] caacguAfcCfCfUfUfcauugausg SU 1827 {Phosphate} asCfsaUfcAfaUfGfaa ggGfuAfcguugsusu 1828 D- 2350 {sGalNAc3K2AhxC6} [inv Ab] caacguacCfcUfUfCfAfuuGfaU fgsUf 1829 {Phosphate} asCfsaUfcAfAfugaaG fgGfuacguugsusu 1830 D- 2351 {sGalNAc3K2AhxC6} [inv Ab] acguacCfcUfUfCfAfuugaugusu SU 1831 {Phosphate} asCfsaUfcAfAfugaaG fgGfuacgususu 1832 D- 2352 {sGalNAc3K2AhxC6]cugcuuc aUfgCfCfUfUfucuacas {inv Ab} 1833 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1834 D- 2353 {sGalNAc3K2AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucuacsa SU 1835 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1836 D- 2354 {sGalNAc3K2AhxC6} [inv Ab] gcuucaUfgCfCfUfUfucUfaCfa UfsusUf 1837 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcsusu 1838 D- 2355 {sGalNAc3K2AhxC6]cugcuuc aUfgCfCfUfUfucUfaCfas {inv Ab] 1839 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1840 D- 2356 {sGalNAc3K2AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucUfaC fasUf 1841 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1842 D- 2357 {sGalNAc3K2AhxC6 Jcugcuuc aUfgCfCfUfUfucuacas {inv Ab} 1843 { Phosphate} asUfsguagAfaaggCfa Ufgaagcagsusu 1844

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D- 2358 {sGalNAc3 K2 AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucuacas {inv Ab} 1845 {Phosphate} asUfsgUfaGfAfaaggC faUfgaagcagsusu 1846 D- 2359 {sGalNAc3K2AhxC6} [inv Ab] gcuucaUfgCfCfUfUfucuacausu SU 1847 {Phosphate} asUfsgUfaGfAfaaGfg CfaUfgaagcsusu 1848 D- 2360 {sGalNAc3K2AhxC6} [inv Ab] CfuUfcAfuGfcCfUfUfUfcuaca gususu 1849 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1850 D- 2361 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{invAb} 1851 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagcasusu 1852 D- 2362 {sGalNAc3K2AhxC6}[invAb] cugcuucaUfgCfCfUfUfucuacsa SU 1853 {Phosphate} asUfsgUfaGfAfaaGfg CfaUfgaagcagsusu 1854 D- 2363 {sGalNAc3K2AhxC6}cugcuuc aUfgCfcUfUfucuacas {inv Ab} 1855 {Phosphate} asUfsgUfaGfAfaaggc aUfgaagcagsusu 1856 D- 2364 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab} 1857 {Phosphate} asUfsgUfaGfAfaaGfg CfaUfgaagcagsusu 1858 D- 2365 {sGalNAc3K2AhxC6} [inv Ab] cuucauCfcCfUfUfUfcuacagusu SU 1859 {Phosphate} asCfsuGfuAfGfaaagG fgAfugaagsusu 1860 D- 2366 {sGalNAc3K2AhxC6} [inv Ab] gcuucaUfcCfCfUfUfucuacausu SU 1861 {Phosphate} asUfsgUfaGfAfaagg GfaUfgaagcsusu 1862 D- 2367 {sGalNAc3K2AhxC6}ugcuuca uCfcCfUfUfUfcuacags {inv Ab} 1863 {Phosphate} asCfsuGfuAfGfaaagG fgAfugaagcasusu 1864 D- 2368 {sGalNAc3K2AhxC6}cugcuuc aUfcCfCfUfUfucuacas {inv Ab ] 1865 {Phosphate} asUfsgUfaGfAfaagg CfaUfgaagcagsusu 1866 D- 2369 {sGalNAc3K2AhxC6} [inv Ab] acauugCfuCfUfUfUfcaccugasu SU 1867 {Φocφaτ}usCfsaGfgUfGfaaagA fgCfaaugususu 1868 D- 2370 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab} 1869 asUfsgUfaGfAfaaggCfaUfgaagc agsusu 1870 D- {sGalNAc3K2AhxC6]cugcuuc 1871 asUfsgUfaGfAfaaggCfaUfgaagc 1872

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2371 aUfgCfCfUfUfucuacas {invAb} asgsusu D- 2372 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 1873 asUfsgsUfaGfAfaaggCfaUfgaag cagsusu 1874 D- 2373 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 1875 asUfsgUfaGfAfaaggCfaUfgaagc agusu 1876 D- 2374 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 1877 asUfsgsUfaGfAfaaggCfaUfgaag cagusu 1878 D- 2375 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 1879 asUfgUfaGfAfaaggCfaUfgaagca gsusu 1880 D- 2376 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 1881 asUfgUfaGfAfaaggCfaUfgaagca sgsusu 1882 D- 2377 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacsas {invAb } 1883 asUfsgUfaGfAfaaggCfaUfgaagc agsusu 1884 D- 2378 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuascsas {invA b} 1885 asUfsgUfaGfAfaaggCfaUfgaagc agsusu 1886 D- 2379 {sGalNAc3K2AhxC6}csugcuu caUfgCfCfUfUfucuacas{invAb } 1887 asUfsgUfaGfAfaaggCfaUfgaagc agsusu 1888 D- 2380 {sGalNAc3K2AhxC6}csusgcu ucaUfgCfCfUfUfucuacas {invA b} 1889 asUfsgUfaGfAfaaggCfaUfgaagc agsusu 1890 D- 2381 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCf[dT]UfUfcuacagu SUSU 1891 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1892 D- 2382 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfUfUfUfcuacagusu SU 1893 {Phosphate} asCfsuGfuAfGfaaAfg GfcAfugaagsusu 1894 D- 2383 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfuUfUfcuacagusus u 1895 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1896 D- 2384 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfUfUfUfcuacagusu 1897 {Phosphate} asCfsuGfuaGfaaagGf cAfugaagsusu 1898

- 129 047656

SU D- 2385 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfUfUfUfcuacagusu SU 1899 {Phosphate} asCfsuguaGfaaagGfc Afugaagsusu 1900 D- 2386 {sGalNAc3K2AhxC6} [invAb] cuucauGfc[dC]UfUfUfcuacagu SUSU 1901 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1902 D- 2387 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfUf[dT]Ufcuacagu SUSU 1903 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1904 D- 2388 {sGalNAc3K2AhxC6} [invAb] gcuucaUfgGfGfAfUfucuacausu SU 1905 {Phosphate} asUfsgUfaGfAfauccC faUfgaagcsusu 1906 D- 2389 {sGalNAc3K2AhxC6} [invAb] cuucauGfcGfAfAfUfcuacagusu SU 1907 {Phosphate} asCfsuGfuAfGfauuc GfcAfugaagsusu 1908 D- 2390 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 1909 {Φocφaτ}usUfsgUfaGfAfaagg CfaUfgaagcagsusu 1910 D- 2391 {sGalNAc3K2AhxC6} [invAb] cugcuucaUfgCfCfUfUfucuacsa sa 1911 {Φocφaτ}usUfsgUfaGfAfaagg CfaUfgaagcagsusu 1912 D- 2392 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invDA } 1913 {Φocφaτ}usUfsgUfaGfAfaagg CfaUfgaagcagsusu 1914 D- 2393 {sGalNAc3K2AhxC6}cugcuuc aUfgGfGfAfU fucuacas {invAb } 1915 {Phosphate} asUfsgUfaGfAfauccC faUfgaagcagsusu 1916 D- 2394 {sGalNAc3K2AhxC6} [invAb] cugcuucaUfgGfGfAfUfucuacsa SU 1917 {Phosphate} asUfsgUfaGfAfauccC faUfgaagcagsusu 1918 D- 2395 {sGa!NAc3K2AhxC6] [invAb] CfuUfcAfuGfcCfUfUfUfcuAfc AfgUfsusUf 1919 {Phosphate} asCfsuGfuAfGfaaagG fcAfugaagsusu 1920 D- {sGalNAc3K2AhxC6} [invAb] 1921 {Phosphate} asCfsuGfuAfGfaaagG 1922

- 130 047656

2396 cuucauGfcCfUfUfUfcuAfcAfg UfsusUf fcAfuGfaAfgsUfsu D- 2397 {sGalNAc3K2AhxC6} [invAb] gcuucaUfgCfCfUfUfucuacausu SU 1923 asUfsgUfaGfAfaaggCfaUfgaagc SUSU 1924 D- 2398 {sGalNAc3K2AhxC6} [invAb] ugcuucauGfcCfUfUfUfcuacags {invAb} 1925 asCfsuGfuAfGfaaagGfcAfugaag casusu 1926 D- 2399 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfUfUfUfcuacagusu su 1927 {Phosphate} asCfsuGfuAfGfaaagG fcAfuGfaAfgsUfsu 1928 D- 2400 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcu Afc Afgs {inv Ab} 1929 asCfsuGfuAfGfaaagGfcAfugaag casusu 1930 D- 2401 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb} 1931 asCfsuGfuAfGfaaAfgGfcAfuga agcasusu 1932 D- 2402 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb} 1933 asCfsuguaGfaaagGfcAfugaagca susu 1934 D- 2403 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfUfUfUfcuacagusu su 1935 asCfsuGfuAfGfaaagGfcAfugaag SUSU 1936 D- 2404 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb} 1937 asCfsuGfuAfGfaaagGfcAfugaag casusu 1938 D- 2405 {GalN Ac3 K2AhxC6} augccuuu CfuAfCfAfGfuggcusus{ invAb } 1939 {Phosphate} asGfscCfaCfUfguAfg AfaAfgGfcAfuGfasUfsu 1940 D- 2406 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invAb } 1941 asGfscCfaCfUfguagAfaAfggcau gasusu 1942 D- 2407 {sGalNAc3K2AhxC6} [invAb] augccuUfuCfUfAfCfaguggcusu su 1943 asGfscCfaCfUfguagAfaAfggcau SUSU 1944 D- 2408 {sGalNAc3K2AhxC6} [invAb] augccuUfuCfUfAfCfagUfgGfc 1945 asGfscCfaCfUfguagAfaAfggcau SUSU 1946

- 131 047656

UfsusUf D- 2409 {sGalNAc3K2AhxC6} [invAb] ucaugccuUfuCfUfAfCfaguggsc SU 1947 asGfscCfaCfUfguagAfaAfggcau gasusu 1948 D- 2410 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfagUfgGfcs {inv Ab} 1949 asGfscCfaCfUfguagAfaAfggcau gasusu 1950 D- 2411 {sGalNAc3K2AhxC6} [invAb] augccuUfuCfUfAfCfaguggcusu SU 1951 asGfscCfaCfUfguAfgAfaAfggc aususu 1952 D- 2412 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invAb } 1953 asGfscCfaCfUfguAfgAfaAfggc augasusu 1954 D- 2413 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invAb } 1955 asGfsccacUfguagAfaAfggcauga susu 1956 D- 2414 {sGalNAc3K2AhxC6} [invAb] ucaugccuUfuCfUfAfCfaguggcs {invAb} 1957 asGfscCfaCfUfguagAfaAfggcau gasusu 1958 D- 2415 {sGalNAc3 K2 AhxC6} [invAb] augccuUfuCfUfAfCfaguggcasu SU 1959 usGfscCfaCfUfguagAfaAfggcau SUSU 1960 D- 2416 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invAb } 1961 usGfscCfaCfUfguagAfaAfggcau gasusu 1962 D- 2417 {sGalNAc3K2AhxC6} [invAb] ucaugccuUfuCfUfAfCfaguggsc sa 1963 usGfscCfaCfUfguagAfaAfggcau gasusu 1964 D- 2418 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invDA } 1965 usGfscCfaCfUfguagAfaAfggcau gasusu 1966 D- 2419 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 1967 asUfsguagAfaaggCfaUfgaagcag SUSU 1968 D- {sGalNAc3K2AhxC6}cugcuuc 1969 usUfsguagAfaaggCfaUfgaagcag 1970

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2420 aUfgCfCfUfUfucuacas {invAb} SUSU D- 2421 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucu acas {invD A } 1971 usUfsguagAfaaggCfaUfgaagcag SUSU 1972 D- 2422 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invAb } 1973 usGfsccacUfguagAfaAfggcauga SUSU 1974 D- 2423 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invDA } 1975 usGfsccacUfguagAfaAfggcauga SUSU 1976 D- 2424 {sGalNAc3K2AhxC6} [invAb] cuucauGfcCfuUfUfcuacagusus u 1977 {Phosphate} asCfsuGfuAfGfaaAfg GfcAfugaagsusu 1978 D- 2425 {sGalNAc3K2AhxC6]cugcuuc aUfgCfCfUfUfucuacas {invAb} 1979 asUfsgUfa[Ab]AfaaggCfaUfgaa gcagsusu 1980 D- 2426 {sGalNAc3K2AhxC6]cugcuuc aUfgCfCfUfUfucuacas {invAb} 1981 asUfsgua[Ab]AfaaggCfaUfgaag cagsusu 1982 D- 2427 {sGa!NAc3K2AhxC6]ugcuuca uGfcCfUfUfUfcuacags{ invAb] 1983 asCf suGfu [Ab] GfaaagGfc Afuga agcasusu 1984 D- 2428 {sGalNAc3K2AhxC6]ugcuuca uGfcCfUfUfUfcuacags{ invAb] 1985 asCf sugu [Ab] GfaaagGfc Afugaa gcasusu 1986 D- 2429 {sGalNAc3K2AhxC6]ucaugcc uUfuCfUfAfCfaguggcs {invAb } 1987 asGfscCfa[Ab]UfguagAfaAfggc augasusu 1988 D- 2430 {sGalNAc3K2AhxC6]ucaugcc uUfuCfUfAfCfaguggcs {invAb } 1989 asGfscca[Ab]UfguagAfaAfggca ugasusu 1990 D- 2431 {sGalNAc3K2AhxC6]cugcuuc aUfgCfCfUfUfucuacas {invAb} 1991 asUfs[GNA- G]uagAfaaggCfaUfgaagcagsusu 1992 D- 2432 {sGalNAc3K2AhxC6]cugcuuc aUfgCfCfUfUfucuacas {invAb ] 1993 asUfsg[GNA- U]agAfaaggCfaUfgaagcagsusu 1994 D- 2433 {sGalNAc3K2AhxC6]cugcuuc aUfgCfCfUfUfucuacas {invAb} 1995 asUfsgu[GNA- A]gAfaaggCfaUfgaagcagsusu 1996 D- {sGalNAc3K2AhxC6]cugcuuc 1997 asUfsgua[GNA- 1998

- 133 047656

2434 aUfgCfCfUfUfucuacas {inv Ab} G]AfaaggCfaUfgaagcagsusu D- 2435 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab} 1999 asUfsguag[GNA- A]aaggCfaUfgaagcagsusu 2000 D- 2436 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab} 2001 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2002 D- 2437 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab} 2003 asUfsgUfagAfaaggCfaUfgaagca gsusu 2004 D- 2438 {sGalNAc3K2AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucuacsa SU 2005 asUfsguagAfaaggCfaUfgaagcag SUSU 2006 D- 2439 {sGalNAc3K2AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucuacas {inv Ab} 2007 asUfsguagAfaaggCfaUfgaagcag SUSU 2008 D- 2440 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab ] 2009 asUfsguagAfaaGfgCfaUfgaagca gsusu 2010 D- 2441 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucUfaCfas {inv Ab} 2011 asUfsguagAfaaggCfaUfgaagcag SUSU 2012 D- 2442 {sGalNAc3K2AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucUfaC fasUf 2013 asUfsguagAfaaggCfaUfgaagcag SUSU 2014 D- 2443 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucUfaCfas {inv Ab} 2015 asUfsguagAfaaGfgCfaUfgaagca gsusu 2016 D- 2444 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab} 2017 asUfsgUfaGfAfaaGfgCfaUfgaa gcagsusu 2018 D- 2445 {sGalNAc3K2AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucuacsa SU 2019 asUfsgUfaGfAfaaGfgCfaUfgaa gcagsusu 2020 D- 2446 {sGalNAc3K2AhxC6} [inv Ab] cugcuucaUfgCfCfUfUfucuacas {inv Ab} 2021 asUfsgUfaGfAfaaggCfaUfgaagc agsusu 2022 D- 2447 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {inv Ab ] 2023 as [Ab] guagAfaaggCfaUfgaagca gsusu 2024

- 134 047656

D- 2448 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2025 asUfs[Ab]uagAfaaggCfaUfgaag cagsusu 2026 D- 2449 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2027 asUfsg[Ab]agAfaaggCfaUfgaag cagsusu 2028 D- 2450 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2029 asUfsgu[Ab]gAfaaggCfaUfgaag cagsusu 2030 D- 2451 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2031 asUfsguag[Ab]aaggCfaUfgaagc agsusu 2032 D- 2452 {sGalNAc3 K2 AhxC6} cugcuuc aUfgCfCfUfUfucuacas {invAb} 2033 asUfsguagAf[Ab]aggCfaUfgaag cagsusu 2034 D- 2453 {sGalNAc3K2AhxC6}caacgua cCfcUfUfCfAfuugaugs{ invAb } 2035 asCfsaucaAfugaaGfgGfuacguug SUSU 2036 D- 2454 {sGalNAc3K2AhxC6}caacgua cCfcUfUfCfAfuugaugs {invAb } 2037 asCfsaUfcAfAfugaaGfgGfuacgu ugsusu 2038 D- 2455 {sGalNAc3K2AhxC6}acauggc uUfcCfAfGfAfuaugccs{ invAb } 2039 asGfsgcauAfucugGfaAfgccaugu SUSU 2040 D- 2456 {sGalNAc3K2AhxC6}acauggc uUfcCfAfGfAfuaugccs{ invAb } 2041 asGfsgCfaUfAfucugGfaAfgccau gususu 2042 D- 2457 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2043 asUfsguaGfAfaaggCfaUfgaagca gsusu 2044 D- 2458 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucUfacas {invA b} 2045 asUfsguagAfaaggCfaUfgaagcag SUSU 2046 D- 2459 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuaCfas{invA b} 2047 asUfsguagAfaaggCfaUfgaagcag SUSU 2048 D- 2460 {sGalNAc3K2AhxC6}cugcggc uUfcCfUfGfGfgcuucus {invAb } 2049 usAfsgAfaGfCfccagGfaAfgccgc agsusu 2050 D- (sGalNAc3K2AhxC6} cugcggc 2051 usAfsgaagCfccagGfaAfgccgcag 2052

- 135 047656

2461 uUfcCfUfGfGfgcuucus {invAb } SUSU D- 2462 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb} 2053 asCfsuGfuaGfaaagGfcAfugaagc asusu 2054 D- 2463 {sGalNAc3K2AhxC6} [invAb] ugcuucauGfcCfUfUfUfcuacasg SU 2055 asCfsuGfuaGfaaagGfcAfugaagc asusu 2056 D- 2464 {sGalNAc3K2AhxC6} [invAb] ugcuucauGfcCfUfUfUfcuacags {invAb} 2057 asCfsuGfuaGfaaagGfcAfugaagc asusu 2058 D- 2465 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb] 2059 asCfsuGfuaGfaaAfgGfcAfugaag casusu 2060 D- 2466 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuAfcAfgs {inv Ab} 2061 asCfsuGfuaGfaaagGfcAfugaagc asusu 2062 D- 2467 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuAfcAfgs {inv Ab} 2063 asCfsuGfuAfGfaaagGfcAfuGfa AfgCfasUfsu 2064 D- 2468 {sGalNAc3K2AhxC6}cugcuu CfaUfGfCfcuuucuacsasu 2065 asUfsguaGfaaAfggcaUfgAfagca gsusu 2066 D- 2469 {sGalNAc3K2AhxC6}cugcuu CfaUfGfCfcuuucuacsasu 2067 asUfsguaGfaaaggcaUfgAfagcag SUSU 2068 D- 2470 {sGalNAc3K2AhxC6}cugcuu CfaUfGfCfcuuucuacsasu 2069 asUfsguaGfaAfAfggcaUfgAfag cagsusu 2070 D- 2471 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb] 2071 usCfsuguaGfaaagGfcAfugaagca SUSU 2072 D- 2472 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{invDA } 2073 usCfsuguaGfaaagGfcAfugaagca SUSU 2074 D- 2473 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucu acas {invDT } 2075 asUfsguagAfaaggCfaUfgaagcag SUSU 2076 D- 2474 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb ] 2077 asUfsguagAf[sGNA- A]aggCfaUfgaagcagsusu 2078

- 136 047656

D- 2475 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2079 asUfsguagAfs[GNA- A]aggCfaUfgaagcagsusu 2080 D- 2476 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2081 asUfsguagAfs[sGNA- A]aggCfaUfgaagcagsusu 2082 D- 2477 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2083 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2084 D- 2478 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2085 asUfsguagAf[GNA- A]AfggCfaUfgaagcagsusu 2086 D- 2479 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2087 asUfsguaga[GNA- A]aggCfaUfgaagcagsusu 2088 D- 2480 csusgcuucaUfgCfCfUfuucuaca s{ invAb} 2089 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2090 D- 2481 csusgcuucaUfgCfCfUfuucuaca s{ invAb} 2091 asUfsguaga[GNA- A]aggCfaUfgaagcagsusu 2092 D- 2482 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2093 asUfsguaga[GNA- A]AfggCfaUfgaagcagsusu 2094 D- 2483 csusgcuucaUfgCfCfUfuucuaca s{ invAb} 2095 asUfsguagAf[GNA- A]AfggCfaUfgaagcagsusu 2096 D- 2484 csusgcuucaUfgCfCfUfuucuaca s{ invAb} 2097 asUfsguaga[GNA- A]AfggCfaUfgaagcagsusu 2098 D- 2485 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2099 asUfsgUfaGfAf[GNA- A]aggCfaUfgaagcagsusu 2100 D- 2486 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2101 asUfsgUfagAf[GNA- AJaGfgCfaUfgaagcagsusu 2102 D- 2487 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2103 asUfsgUfagAffGNA- A]aggCfaUfgaagcagsusu 2104 D- 2488 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2105 asUfsgUfaGfAf[GNA- A]aGfgCfaUfgaagcagsusu 2106 D- 2489 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2107 asUfsguagAf[GNA- A]aGfgCfaUfgaagcagsusu 2108 D- 2490 csusgcuucaUfgCfCfUfUfucUfa Cfas{ invAb} 2109 asUfsguagAf[GNA- A]aGfgCfaUfgaagcagsusu 2110 D- 2491 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2111 asUfsguagAf[GNA- A]a[dG]gCfaUfgaagcagsusu 2112

- 137 047656

D- 2492 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2113 asUfsguagAf[GNA- A] [dA]ggCfaUfgaagcagsusu 2114 D- 2493 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2115 asUfsguagAf[GNA- A]ag[dG]CfaUfgaagcagsusu 2116 D- 2494 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2117 asUfsguaGfAf[GNA- A]aggCfaUfgaagcagsusu 2118 D- 2495 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2119 asUfsguaGfa[GNA- A]aggCfaUfgaagcagsusu 2120 D- 2496 csusgcuucaUfgCfCfUfUfucuac as {invAb} 2121 asUfsguaGfa[GNA- A]AfggCfaUfgaagcagsusu 2122 D- 2497 csusgcuuCfaUfGfCfcuuucuaca s{ invAb} 2123 asUfsguaGfa[GNA- A]aggcaUfgAfagcagsusu 2124 D- 2498 csusgcuuCfaUfGfCfcuuucuaca s{ invAb} 2125 asUfsguaGfa[GNA- A]AfggcaUfgAfagcagsusu 2126 D- 2499 csusgcuuCfaUfgCfcuuucuacas{ invAb} 2127 asUfsguaga[GNA- A]aggCfaUfgAfagcagsusu 2128 D- 2500 csusgcuuCfaUfgCfcuuucuacas{ invAb} 2129 asUfsguagaaaggCfaUfgAfagcag SUSU 2130 D- 2501 usgscuucauGfcCfUfUfUfcuaca gs{invDA} 2131 us[sGNA- C]uguaGfaaagGfcAfugaagcasus u 2132 D- 2502 usgscuucauGfcCfUfUfUfcuaca gs{invDA} 2133 usCfs[GNA-U] guaGfaaagGfc Afugaagcasusu 2134 D- 2503 usgscuucauGfcCfUfUfUfcuaca gs{invDA} 2135 usCfsuglGNA- U]aGfaaagGfc Afugaagcasusu 2136 D- 2504 usgscuucauGfcCfUfUfUfcuaca gs{invDA} 2137 usCfsugu[GNA- A]GfaaagGfcAfugaagcasusu 2138 D- 2505 usgscuucauGfcCfUfUfUfcuaca gs{invDA} 2139 usCfsuguaGf[GNA- A] aagGfcAfugaagcasusu 2140 D- 2506 usgscuucauGfcCfUfUfUfcuaca gs{invDA} 2141 us [Ab] uguaGfaaagGfc Afugaagc asusu 2142 D- 2507 usgscuucauGfcCfUfUfUfcuaca gs{invDA} 2143 usCfs[Ab]guaGfaaagGfcAfugaa gcasusu 2144 D- usgscuucauGfcCfUfUfUfcuaca 2145 usCfsu[Ab]uaGfaaagGfcAfugaa 2146

- 138 047656

2508 gs{invDA] gcasusu D- 2509 usgscuucauGfcCfUfUfUfcuaca gs{invDA] 2147 usCfsug[Ab]aGfaaagGfcAfugaa gcasusu 2148 D- 2510 usgscuucauGfcCfUfUfUfcuaca gs{invDA] 2149 usCfsugu[Ab]GfaaagGfcAfugaa gcasusu 2150 D- 2511 usgscuucauGfcCfUfUfUfcuaca gs{invDA] 2151 usCfsugua[Ab]aaagGfcAfugaag casusu 2152 D- 2512 usgscuucauGfcCfUfUfUfcuaca gs{invDA] 2153 usCfsuguaGf[Ab]aagGfcAfugaa gcasusu 2154 D- 2513 uscsaugccuUfuCfUfAfCfagugg cs{invDT} 2155 asGfsccacUfguagAfaAfggcauga SUSU 2156 D- 2514 uscsaugccuUfuCfUfAfCfagugg cs{invAb} 2157 usGfsccacUfguagAfaAfggcauga SUSU 2158 D- 2515 uscsaugccuUfuCfUfAfCfagugg cs{invDA] 2159 usGfsccacUfguagAfaAfggcauga susu 2160 D- 2516 uscscugcuuCfaUfGfCfCfuuucu as{invDT} 2161 asUfsagaaAfggcaUfgAfagcagga SUSU 2162 D- 2517 uscscugcuuCfaUfGfCfCfuuucu as{invAb} 2163 usUfsagaaAfggcaUfgAfagcagga SUSU 2164 D- 2518 uscscugcuuCfaUfGfCfCfuuucu as{invDA] 2165 usUfsagaaAfggcaUfgAfagcagga susu 2166 D- 2519 usasuguuccUfgCfUfUfCfaugcc us{invDT} 2167 asAfsggcaUfgaagCfaGfgaacaua susu 2168 D- 2520 usasuguuccUfgCfUfUfCfaugcc us{invAb} 2169 usAfsggcaUfgaagCfaGfgaacaua susu 2170 D- 2521 usasuguuccUfgCfUfUfCfaugcc us{invDA) 2171 usAfsggcaUfgaagCfaGfgaacaua susu 2172 D- 2522 {sGalNAc3K2AhxC6} uccugcu uCfaUfGfCfCfuuucuas{ inv Ab} 2173 asUfsagaaAfggcaUfgAfagcagga susu 2174 D- 2523 {sGalNAc3K2AhxC6}uauguuc cUfgCfUfUfCfaugccus{ inv Ab} 2175 asAfsggcaUfgaagCfaGfgaacaua susu 2176 D- 2524 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invDT } 2177 asGfsccacUfguagAfaAfggcauga susu 2178

- 139 047656

D- 2525 {sGalNAc3K2AhxC6}uccugcu uCfaUfGfCfCfuuucuas{ invDT } 2179 asUfsagaaAfggcaUfgAfagcagga SUSU 2180 D- 2526 {sGalNAc3K2AhxC6}uccugcu uCfaUfGfCfCfuuucuas{ invDA } 2181 usUfsagaaAfggcaUfgAfagcagga SUSU 2182 D- 2527 {sGalNAc3K2AhxC6}uauguuc cUfgCfUfUfCfaugccus{ invDA } 2183 usAfsggcaUfgaagCfaGfgaacaua SUSU 2184 D- 2528 usgscuucauGfcCfUfUfUfcuaca gs{ invDA} 2185 usCfsugua[GNA- G] aaagGfcAfugaagcasusu 2186 D- 2529 cugcuucaUfgCfCfUfUfsucuaca s{ invAb} 2187 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2188 D- 2530 cugcuucaUfgCfCfUfsUfucuaca s{ invAb} 2189 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2190 D- 2531 cugcuucaUfgCfCfUfsUfsucuac as {invAb} 2191 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2192 D- 2532 cugcuucaUfgCfCfUfsUfucuaca s{ invAb} 2193 asUfsguagAfs[GNA- A]aggCfaUfgaagcagsusu 2194 D- 2533 cugcuucaUfgCfCfUfsUfsucuac as {invAb} 2195 asUfsguagAfs[GNA- A]aggCfaUfgaagcagsusu 2196 D- 2534 cugcuucaUfgCfCfUfUfsucuaca s{ invAb} 2197 asUfsguagAf[sGNA- A]aggCfaUfgaagcagsusu 2198 D- 2535 cugcuucaUfgCfCfUfsUfsucuac as {invAb} 2199 asUfsguagAf[sGNA- A]aggCfaUfgaagcagsusu 2200 D- 2536 cugcuucaUfgCfCfUfUfsucuaca s{ invAb} 2201 asUfsguagAfs[sGNA- A]aggCfaUfgaagcagsusu 2202 D- 2537 cugcuucaUfgCfCfUfsUfucuaca s{ invAb} 2203 asUfsguagAfs[sGNA- A]aggCfaUfgaagcagsusu 2204 D- 2538 cugcuucaUfgCfCfUf[LNA- T]ucuacas{ invAb} 2205 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2206 D- 2539 cugcuucaUfgCfCf[LNA- T] Ufucuacas {invAb} 2207 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2208 D- cugcuucaUfgCfCfUfUf[LNA- 2209 asUfsguagAf[GNA- 2210

- 140 047656

2540 T]cuacas{invAb} A] aggCfaUfgaagcagsusu D- 2541 cugcuucaUfgCfCfUfUfuc[LNA -T]acas{invAb} 2211 asUfsgu[GNA- A]gAfaaggCfaUfgaagcagsusu 2212 D- 2542 cugcuucaUfgCfCfUfUfucu[LN A-A]cas{invAb} 2213 asUfsg[GNA- U]agAfaaggCfaUfgaagcagsusu 2214 D- 2543 cugcuucaUfgCfCfUfUfucuac[s LNA-A]{invAb} 2215 as[sGNA- U]guagAfaaggCfaUfgaagcagsus u 2216 D- 2544 cugcuucaUfgCfCfUfUf[LNA- T]cuacas{invAb} 2217 asUfsguag[Ab]aaggCfaUfgaagc agsusu 2218 D- 2545 cugcuucaUfgCfCfUfUfuc[LNA -T]acas{invAb} 2219 asUfsgu[Ab]gAfaaggCfaUfgaag cagsusu 2220 D- 2546 cugcuucaUfgCfCfUfUfucu[LN A-A]cas{invAb} 2221 asUfsg[Ab]agAfaaggCfaUfgaag cagsusu 2222 D- 2547 cugcuucaUfgCfCfUfUfucUfaC fas {inv Ab} 2223 asUfsgUfagAf[GNA- A] aggCfaUfgaagcagsusu 2224 D- 2548 cugcuucaUfgCfCfUfUfucUfaC fas {inv Ab} 2225 asUfsguagAf[GNA- A] aggCfaUfgaagcagsusu 2226 D- 2549 cugcuucaUfgCfCfUfs[LNAT] ucuacas {inv Ab} 2227 asUfsguagAf[GNA- A] aggCfaUfgaagcagsusu 2228 D- 2550 cugcuucaUfgCfCfUfs[sLNAT] ucuacas {inv Ab} 2229 asUfsguagAf[GNA- A] aggCfaUfgaagcagsusu 2230 D- 2551 cugcuucaUfgCfCfUffLNATJ ucuacas {inv Ab} 2231 asUfsguagAf[sGNA- AJaggCfaUfgaagcagsusu 2232 D- 2552 cugcuucaUfgCfCfUf[LNAT] ucuacas {inv Ab} 2233 asUfsguagAfs[GNA- A] aggCfaUfgaagcagsusu 2234 D- 2553 usgscuucauGfcCfUfUfUfcu[L NA-A]cags{invDA} 2235 usCfsug[Ab]aGfaaagGfc Afugaa gcasusu 2236 D- 2554 usgscuucauGfcCfUfUfUfc[LN A-T] acags {invD A} 2237 usCfsugu[Ab]GfaaagGfc Afugaa gcasusu 2238 D- 2555 usgscuucauGfcCfUfUf[LNAT] cuacag s {invD A} 2239 usCfsuguaGf[Ab]aagGfcAfugaa gcasusu 2240 D- 2556 usgscuucauGfcCfUfUfUfcuac} LNA-A] gs {invD A} 2241 usCfs[GNA-U] guaGfaaagGfc Afugaagcasusu 2242

- 141 047656

D- 2557 usgscuucauGfcCfUfUfUfcu[L NA-A]cags{invDA} 2243 usCfsug[GNA- U]aGfaaagGfcAfugaagcasusu 2244 D- 2558 usgscuucauGfcCfUfUfUfc[LN A-T]acags {invDA} 2245 usCfsugu[GNA- A]GfaaagGfcAfugaagcasusu 2246 D- 2559 usgscuucauGfcCfUfUfUfcuaca [sLNA-G]{ invDA} 2247 us [Ab] uguaGfaaagGfc Afugaagc asusu 2248 D- 2560 usgscuucauGfcCfUfUfUfcuac[ LNA-A]gs{ invDA} 2249 usCfs[Ab]guaGfaaagGfcAfugaa gcasusu 2250 D- 2561 ucaugccuUfuCfUfAfCfa[LNA- G]uggcs{ invAb} 2251 asGfscca[Ab]UfguagAfaAfggca ugasusu 2252 D- 2562 ucaugccuUfuCfUfAfCfag[LNA -T]ggcs{ invAb} 2253 asGfscca[Ab]UfguagAfaAfggca ugasusu 2254 D- 2563 ucaugccuUfuCfUfAfCfasgsugg cs{ invAb} 2255 asGfscca[Ab]UfguagAfaAfggca ugasusu 2256 D- 2564 {GalNAc3K2AhxC6}GfgUfaU fgUfuCfCfUfGfcuUfcAfuUfsu sUf 2349 {Phosphate} asAfsuGfaAfGfcagg AfaCfaUfaCfcsUfsu 2350 D- 2565 {GalNAc3K2AhxC6}GfgUfaU fgUfuCfCfUfGfcuUfcAfuAfsu sUf 2351 {Φocφaτ}usAfsuGfaAfGfcagg AfaCfaUfaCfcsUfsu 2352 D- 2566 {GalNAc3K2AhxC6}GfuAfuG fuUfcCfUfGfCfuuCfaUfgUfsus Uf 2353 {Phosphate} asCfsaUfgAfAfgcagG faAfcAfuAfcsUfsu 2354 D- 2567 {GalNAc3K2AhxC6}UfaUfgU fuCfcUfGfCfUfucAfuGfcAfsus Uf 2355 {Phosphate} usGfscAfuGfAfagca GfgAfaCfaUfasUfsu 2356 D- 2568 {GalNAc3K2AhxC6} AfuGfuU fcCfuGfCfUfUfcaUfgCfcUfsus Uf 2357 { Phosphate} asGfsgCfaUfGfaagcA fgGfaAfcAfusUfsu 2358 D- 2569 {GalNAc3K2AhxC6}UfgUfuC fcUfgCfUfUfCfauGfcCfuUfsus Uf 2359 {Phosphate} as AfsgGfcAfUfgaagC faGfgAfaCfasUfsu 2360 D- 2570 {GalNAc3K2AhxC6}GfuUfcC fuGfcUfUfCfAfugCfcUfuUfsu 2361 {Phosphate} as AfsaGfgCfAfugaaG fcAfgGfaAfcsUfsu 2362

- 142 047656

sUf D- 2571 {GalNAc3K2AhxC6}UfuCfcU fgCfuUfCfAfUfgcCfuUfuUfsu sUf 2363 {Phosphate} as AfsaAfgGfCfaugaA fgCfaGfgAfasUfsu 2364 D- 2572 {GalNAc3K2AhxC6}UfuCfcU fgCfuUfCfAfUfgcCfuUfuAfsu sUf 2365 {Φocφaτ}usAfsaAfgGfCfauga AfgCfaGfgAfasUfsu 2366 D- 2573 {GalNAc3K2AhxC6}UfcCfuG fcUfuCfAfUfGfccUfuUfcUfsus Uf 2367 {Phosphate} asGfsaAfaGfGfcaugA faGfcAfgGfasUfsu 2368 D- 2574 {GalNAc3K2AhxC6}CfcUfgC fuUfcAfUfGfCfcuUfuCfuAfsu sUf 2369 {Φocφaτ}usAfsgAfaAfGfgcau GfaAfgCfaGfgsUfsu 2370 D- 2575 {GalNAc3K2AhxC6}CfuGfcU fuCfaUfGfCfCfuuUfcUfaUfsus Uf 2371 {Phosphate} asUfsaGfaAfAfggcaU fgAfaGfcAfgsUfsu 2372 D- 2576 {GalNAc3K2AhxC6}CfuGfcU fuCfaUfGfCfCfuuUfcUfaAfsus Uf 2373 {Φocφaτ}usUfsaGfaAfAfggca UfgAfaGfcAfgsUfsu 2374 D- 2577 {GalNAc3K2AhxC6}UfgCfuU fcAfuGfCfCfUfuuCfuAfcAfsus Uf 2375 {Φocφaτ}usGfsuAfgAfAfaggc AfuGfaAfgCfasUfsu 2376 D- 2578 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfUfUfucUfaCfaUfsus Uf 2377 { Phosphate} asUfsgUfaGfAfaaggC faUfgAfaGfcsUfsu 2378 D- 2579 {GalNAc3K2AhxC6}GfcUfuC faUfgCfCfUfUfucUfaCfaAfsus Uf 2379 {Φocφaτ}usUfsgUfaGfAfaagg CfaUfgAfaGfcsUfsu 2380 D- 2580 {GalNAc3K2AhxC6}CfuUfcA fuGfcCfUfUfUfcuAfcAfgUfsu sUf 2381 {Phosphate} asCfsuGfuAfGfaaagG fcAfuGfaAfgsUfsu 2382 D- 2581 {GalNAc3K2AhxC6}UfuCfaU fgCfcUfUfUfCfuaCfaGfuUfsus Uf 2383 {Phosphate} as AfscUfgUfAfgaaaG fgCfaUfgAfasUfsu 2384

- 143 047656

D- 2582 {GalNAc3K2AhxC6}UfuCfaU fgCfcUfUfUfCfuaCfaGfuAfsus Uf 2385 [Phosphate} us AfscUfgUfAfgaaa GfgCfaUfgAfasUfsu 2386 D- 2583 {GalNAc3K2AhxC6}UfcAfuG fcCfuUfUfCfUfacAfgUfgUfsus Uf 2387 {Phosphate} asCfsaCfuGfUfagaaA fgGfcAfuGfasUfsu 2388 D- 2584 {GalNAc3K2AhxC6}UfcAfuG fcCfuUfUfCfUfacAfgUfgAfsus Uf 2389 [ Phosphate [usCfsaCfuGfUfagaa A fgGfcAfuGfasUfsu 2390 D- 2585 {GalNAc3K2AhxC6}CfaUfgC fcUfuUfCfUfAfcaGfuGfgUfsu sUf 2391 {Phosphate} asCfscAfcUfGfuagaA faGfgCfaUfgsUfsu 2392 D- 2586 {GalNAc3K2AhxC6}CfaUfgC fcUfuUfCfUfAfcaGfuGfgAfsu sUf 2393 {Phosphate JusCfscAfcUfGfuaga A faGfgCfaUfgsUfsu 2394 D- 2587 {GalNAc3K2AhxC6} AfuGfcC fuUfuCfUfAfCfagUfgGfcUfsu sUf 2395 {Phosphate} asGfscCfaCfUfguagA faAfgGfcAfusUfsu 2396 D- 2588 {GalNAc3K2AhxC6} AfuGfcC fuUfuCfUfAfCfagUfgGfcAfsu sUf 2397 {Φocφaτ}usGfscCfaCfUfguagA faAfgGfcAfusUfsu 2398 D- 2589 {GalNAc3K2AhxC6}UfgCfcU fuUfcUfAfCfAfguGfgCfcUfsu sUf 2399 {Phosphate} asGfsgCfcAfCfuguaG faAfaGfgCfasUfsu 2400 D- 2590 {GalNAc3K2AhxC6}GfcCfuU fuCfuAfCfAfGfugGfcCfuUfsu sUf 2401 {Phosphate} as AfsgGfcCfAfcugu AfgAfaAfgGfcsUfsu 2402 D- 2591 {GalNAc3K2AhxC6}GfgUfaU fgUfuCfCfUfGfcuUfcAfuCfsus Uf 2403 {Φocφaτ}gsAfsuGfaAfGfcagg AfaCfaUfaCfcsUfsu 2404 D- 2592 {GalNAc3K2AhxC6}GfuAfuG fuUfcCfUfGfCfuuCfaUfcCfsus Uf 2405 {Φocφaτ}gsGfsaUfgAfAfgcag GfaAfcAfuAfcsUfsu 2406 D- {GalNAc3K2AhxC6}UfaUfgU 2407 {Φocφaτ}gsGfsgAfuGfAfagca 2408

- 144 047656

2593 fuCfcUfGfCfUfucAfuCfcCfsus Uf GfgAfaCfaUfasUfsu D- 2594 {GalNAc3K2AhxC6} AfuGfuU fcCfuGfCfUfUfcaUfcCfcCfsus Uf 2409 {<I>oc()aT}gsGfsgGfaUfGfaagc AfgGfaAfcAfusUfsu 2410 D- 2595 {GalNAc3K2AhxC6}UfgUfuC fcUfgCfUfUfCfauCfcCfcUfsus Uf 2411 {Phosphate} asGfsgGfgAfUfgaag CfaGfgAfaCfasUfsu 2412 D- 2596 (GalNAc3K2AhxC6] GfuUfcC fuGfcUfUfCfAfucCfcCfuUfsus Uf 2413 {Phosphate} asAfsgGfgGfAfugaa GfcAfgGfaAfcsUfsu 2414 D- 2597 {GalNAc3K2AhxC6}UfuCfcU fgCfuUfCfAfUfccCfcUfuCfsus Uf 2415 {Φocφaτ}gsAfsaGfgGfGfauga AfgCfaGfgAfasUfsu 2416 D- 2598 {GalNAc3K2AhxC6}UfcCfuG fcUfuCfAfUfCfccCfuUfcUfsus Uf 2417 {Phosphate} asGfsaAfgGfGfgaug AfaGfcAfgGfasUfsu 2418 D- 2599 {GalNAc3K2AhxC6}CfcUfgC fuUfcAfUfCfCfccUfuCfuAfsus Uf 2419 {Φocφaτ}usAfsgAfaGfGfggau GfaAfgCfaGfgsUfsu 2420 D- 2600 {GalNAc3K2AhxC6}CfuGfcU fuCfaUfCfCfCfcuUfcUfaCfsus Uf 2421 {Φocφaτ}gsUfsaGfaAfGfggga UfgAfaGfcAfgsUfsu 2422 D- 2601 {GalNAc3K2AhxC6}UfgCfuU fcAfuCfCfCfCfuuCfuAfcAfsus Uf 2423 {Φocφaτ}usGfsuAfgAfAfgggg AfuGfaAfgCfasUfsu 2424 D- 2602 {GalNAc3K2AhxC6}GfcUfuC faUfcCfCfCfUfucUfaCfaGfsus Uf 2425 {Phosphate} csUfsgUfaGfAfagg GfaUfgAfaGfcsUfsu 2426 D- 2603 {GalNAc3K2AhxC6}CfuUfcA fuCfcCfCfUfUfcuAfcAfgUfsus Uf 2427 {Phosphate} asCfsuGfuAfGfaagg GfgAfuGfaAfgsUfsu 2428 D- 2604 {GalNAc3K2AhxC6}UfuCfaU fcCfcCfUfUfCfuaCfaGfuGfsus 2429 {Phosphate} cs AfscUfgUfAfgaag GfgGfaUfgAfasUfsu 2430

- 145 047656

Uf D- 2605 {GalNAc3K2AhxC6}UfcAfuC fcCfcUfUfCfUfacAfgUfgGfsus Uf 2431 {Phosphate} csCfsaCfuGfUfagaaG fgGfgAfuGfasUfsu 2432 D- 2606 {GalNAc3K2AhxC6}CfaUfcC fcCfuUfCfUfAfcaGfuGfgCfsus Uf 2433 {Phosphate {gsCfscAfcUfGfuaga A fgGfgGfaUfgsUfsu 2434 D- 2607 {GalNAc3K2AhxC6} AfuCfcC fcUfuCfUfAfCfagUfgGfcCfsus Uf 2435 {Φocφaτ}gsGfscCfaCfUfguagA faGfgGfgAfusUfsu 2436 D- 2608 {GalNAc3K2AhxC6}UfcCfcC fuUfcUfAfCfAfguGfgCfcUfsu sUf 2437 {Phosphate} as GfsgCfcAfCfiiguaG faAfgGfgGfasUfsu 2438 D- 2609 {GalNAc3K2AhxC6}CfcCfcU fuCfuAfCfAfGfugGfcCfuUfsu sUf 2439 {Phosphate} as AfsgGfcCfAfcugu AfgAfaGfgGfgsUfsu 2440 D- 2610 cugcuucaUfgCfCfUfUfucua[L NA-mC]as{invAb} 2805 asUfs[GNA- G]uagAfaaggCfaUfgaagcagsusu 2806 D- 2611 cugcuucaUfgCfCfUfUfucua[L N A-mC ] a s {inv Ab} 2807 asUfs[Ab]uagAfaaggCfaUfgaag cagsusu 2808 D- 2612 usgscuucauGfcCfUfUfUfcua[L N A-mC ] ag s {invD A} 2809 usCfsu[Ab]uaGfaaagGfcAfugaa gcasusu 2810 D- 2613 usgscuucauGfcCfUfUfUfcua[L N A-mC ] ag s {invD A} 2811 usCfsu[GNA- G]uaGfaaagGfcAfugaagcasusu 2812 D- 2614 ucaugccuUfuCfUfAfCfasguggc s{invAb} 2813 asGfscca[Ab]UfguagAfaAfggca ugasusu 2814 D- 2615 ucaugccuUfuCfUfAfCfagsuggc s{invAb} 2815 asGfscca[Ab]UfguagAfaAfggca ugasusu 2816 D- 2616 ucaugccuUfuCfUfAfCfas [LNA -G]uggcs{invAb} 2817 asGfscca[Ab]UfguagAfaAfggca ugasusu 2818 D- 2617 ucaugccuUfuCfUfAfCfas[sLN A-G]uggcs{ inv Ab} 2819 asGfscca[Ab]UfguagAfaAfggca ugasusu 2820 D- 2618 cugcuucaUfgCfCfUfUfucuacas {inv Ab} 2821 as[sMeO- I] guagAfaaggCfaU fgaagcagsusu 2822

- 146 047656

D- 2619 cugcuucaUfgCfCfUfUfucuacas {inv Ab} 2823 asUfsg[MeO- I]agAfaaggCfaUfgaagcagsusu 2824 D- 2620 cugcuucaUfgCfCfUfUfucuacas {inv Ab} 2825 asUfsgu[MeO- I]gAfaaggCfaUfgaagcagsusu 2826 D- 2621 cugcuucaUfgCfCfUfUfucuacas {inv Ab} 2827 asUfsguafMeO- I]AfaaggCfaUfgaagcagsusu 2828 D- 2622 cugcuucaUfgCfCfUfUfucuacas {inv Ab} 2829 asUfsguagAf[MeO- I]aggCfaUfgaagcagsusu 2830 D- 2623 cugcuucaUfgCfCfUfUfucuaccs {inv Ab} 2831 as[sMcO- I] guagAfaaggCfaU fgaagcagsusu 2832 D- 2624 cugcuucaUfgCfCfUfUfucuccas {inv Ab} 2833 asUfsg[MeO- I]agAfaaggCfaUfgaagcagsusu 2834 D- 2625 cugcuucaUfgCfCfUfUfccuacas {inv Ab} 2835 asUfsguag[MeO- I]aaggCfaUfgaagcagsusu 2836 D- 2626 cugcuucaUfgCfCfUfCfucuacas {inv Ab} 2837 asUfsguagAf[McO- I]aggCfaUfgaagcagsusu 2838 D- 2627 cugcuucaUfgCfCfUfUfucuacus {inv Ab} 2839 asAfsguagAfaaggCfaUfgaagcag SUSU 2840 D- 2628 cugcuucaUfgCfCfUfUfucuucas {inv Ab} 2841 asUfsgaagAfaaggCfaUfgaagcag SUSU 2842 D- 2629 cugcuucaUfgCfCfUfUfucaacas {inv Ab} 2843 asUfsguugAfaaggCfaUfgaagcag SUSU 2844 D- 2630 cugcuucaUfgCfCfUfAfucuacas {inv Ab} 2845 asUfsguagAfuaggCfaUfgaagcag SUSU 2846 D- 2631 ugcuucauGfcCfUfUfUfcuacacs {inv Ab} 2847 as[sMeO- I]uguaGfaaagGfcAfugaagcasusu 2848 D- 2632 ugcuucauGfcCfUfUfUfcuacags {inv Ab} 2849 asCfs[MeO- I]guaGfaaagGfcAfugaagcasusu 2850 D- 2633 ugcuucauGfcCfUfUfUfcuacags {inv Ab} 2851 asCfsug[MeO- I]aGfaaagGfcAfugaagcasusu 2852 D- 2634 ugcuucauGfcCfUfUfUfcuacags {inv Ab} 2853 asCfsugu[MeO- I]GfaaagGfcAfugaagcasusu 2854 D- 2635 ugcuucauGfcCfUfUfUfcuacags {inv Ab} 2855 asCfsugua[MeO- I]aaagGfcAfugaagcasusu 2856

- 147 047656

D- 2636 ugcuucauGfcCfUfUfUfcuaccgs {invAb} 2857 asCfs[MeO- I]guaGfaaagGfcAfugaagcasusu 2858 D- 2637 ugcuucauGfcCfUfUfUfcuccags {invAb} 2859 asCfsug[MeO- I]aGfaaagGfcAfugaagcasusu 2860 D- 2638 ugcuucauGfcCfUfUfUfccacags {invAb} 2861 asCfsugufMeO- I]GfaaagGfcAfugaagcasusu 2862 D- 2639 cugcuucaUfgCfCfUfUfucuaaas {invAb} 2863 asUfsuuagAfaaggCfaUfgaagcag SUSU 2864 D- 2640 cugcuucaUfgCfCfUfUfuauacas {invAb} 2865 asUfsguauAfaaggCfaUfgaagcag SUSU 2866 D- 2641 cugcuucaUfgCfCfUfsUfucuaca s{ invAb} 2867 asUfsguagAf[sGNA- A]aggCfaUfgaagcagsusu 2868 D- 2642 cugcuucaUfgCfCfUf[LNAT] ucuacas {invAb} 2869 asUfsguagAfs[sGNA- A]aggCfaUfgaagcagsusu 2870 D- 2643 usgscuucauGfcCfUfUfUfcuaca [sLNA-G]{invDA} 2871 us[sGNA- C]uguaGfaaagGfcAfugaagcasus u 2872 D- 2644 usgscuucauGfcCfUfUffLNAT] cuacag s {invD A} 2873 usCfsuguaGffGNA- A] aagGfcAfugaagcasusu 2874 D- 2645 ucaugccuUfuCfUfAfCf[LNA- A]guggcs{ invAb} 2875 asGfscca[Ab]UfguagAfaAfggca ugasusu 2876 D- 2646 cugcuucaUfgCfCfUfUfu[LNAmC]uacas{ invAb} 2877 asUfsgua[Ab]AfaaggCfaUfgaag cagsusu 2878 D- 2647 ucaugccuUfuCfUfAfCfal sLNA -G]uggcs{invAb} 2879 asGfscca[AbJUfguagAfaAfggca ugasusu 2880 D- 2648 cugcuucaUfgCfCfUfUfucuacas {invAb} 2881 asUfs[MeO- I]uagAfaaggCfaUfgaagcagsusu 2882 D- 2649 cugcuucaUfgCfCfUfUfucuagas {invAb} 2883 asUfscuagAfaaggCfaUfgaagcag SUSU 2884 D- 2650 cugcuucaUfgCfCfUfUfacuacas {invAb} 2885 asUfsguagUfaaggCfaUfgaagcag SUSU 2886 D- 2651 ugcuucauGfcCfUfUfUfcuacags {invAb} 2887 asCfsu[MeO- I]uaGfaaagGfcAfugaagcasusu 2888 D- ugcuucauGfcCfUfUfUfcuacags 2889 asCfsuguaGf[MeO- 2890

- 148 047656

2652 {invAb} I]aagGfcAfugaagcasusu D- 2653 ugcuucauGfcCfUfUfCfcuacags {invAb} 2891 asCfsuguaGf[MeO- I]aagGfcAfugaagcasusu 2892 D- 2654 cugcuucaUfgCfCfUfUfucuauas {invAb} 2893 asUfsauagAfaaggCfaUfgaagcag SUSU 2894 D- 2655 cugcuucaUfgCfCfUfUfuuuacas {invAb} 2895 asUfsguaaAfaaggCfaUfgaagcag SUSU 2896 D- 2656 cugcuucaUfgCfCfUfUfucuacas {invAb} 2897 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2898 D- 2657 cugcuucaUfgCfCfUfUfsucuaca s{ invAb} 2899 asUfsguagAfs[GNA- A]aggCfaUfgaagcagsusu 2900 D- 2658 cugcuucaUfgCfCfUfUf[LNA- T]cuacas{ invAb} 2901 asUfsguag[GNA- A]aaggCfaUfgaagcagsusu 2902 D- 2659 cugcuucaUfgCfCfUf[LNAT] ucuacas {invAb} 2903 asUfsguagAf[Ab]aggCfaUfgaag cagsusu 2904 D- 2660 cugcuucaUfgCfCfUfUfucuac[s LNA-A]{ invAb} 2905 as [ s Ab] g uag AfaaggCfaU fgaagc agsusu 2906 D- 2661 cugcuucaUfgCfCfUfUfu[LNAmC]uacas{invAb} 2907 asUfsgua[GNA- G] AfaaggCfaUfgaagcagsusu 2908 D- 2662 usgscuucauGfcCfUfUfUf[LNA -mC]uacags{invDA} 2909 usCfsugua[Ab]aaagGfcAfugaag casusu 2910 D- 2663 cugcuucaUfgCfCfUfUfucuacas {invAb} 2911 asUfsguag[MeO- I]aaggCfaUfgaagcagsusu 2912 D- 2664 cugcuucaUfgCfCfUfsUfsucuac as{invAb} 2913 asUfsguagAfs[sGNA- A]aggCfaUfgaagcagsusu 2914 D- 2665 cugcuucaUfgCfCfUf[sLNAT] ucuacas {invAb} 2915 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2916 D- 2666 {sGalNAc3K2AhxC6}cusgcuu caUfgCfCfUfUfucUfaCfas {inv Ab} 2917 asUfsguagAf[GNA- A]aGfgCfaUfgaagcagsusu 2918 D- 2667 {sGalNAc3K2AhxC6}cugcuu CfaUfGfCfCfuuucuacas {invAb } 2919 asUfsgUfaGfa[GNA- A]aggcaUfgAfagcagsusu 2920 D- {sGalNAc3K2AhxC6}cugcuu 2921 asUfsgUfaGfa[GNA- 2922

- 149 047656

2668 CfaUfGfCfCfuuucuacas {invAb } A]AfggcaUfgAfagcagsusu D- 2669 {sGalNAc3K2AhxC6}cugcuu CfaUfgCfcuuucuacas {invAb} 2923 asUfsguaga[GNA- A]aggCfaUfgAfagcagsusu 2924 D- 2670 {sGalNAc3K2AhxC6}cugcuuc aUfgCfcU fuucuacas {invAb} 2925 asUfsguagAf[GNA- A]AfggCfaUfgaagcagsusu 2926 D- 2671 {sGalNAc3K2AhxC6}cugcuuc aUfgCfcU fuucuacas {invAb} 2927 asUfsguaga[GNA- A]AfggCfaUfgaagcagsusu 2928 D- 2672 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUf[LNA- T] ucuacas {invAb} 2929 asUfsguagAf[GNA- A]aggCfaUfgaagcagsusu 2930 D- 2673 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucua[LNAmC] as {invAb} 2931 asUfs[GNA- G]uagAfaaggCfaUfgaagcagsusu 2932 D- 2674 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfa[LNAG]uggcs{ invAb} 2933 asGfscca[Ab]UfguagAfaAfggca ugasusu 2934 D- 2675 {sGalNAc3K2AhxC6}cugcuuc aUfgGfGfAfU fucuacas {invAb } 2935 asUfsguagAfauccCfaUfgaagcag SUSU 2936 D- 2676 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2937 asUfsguaGfa[GNA- A]AfggCfaUfgaagcagsusu 2938 D- 2677 {sGalNAc3K2AhxC6}guauguu cCfuGfCfUfUfcaugccs {invAb} 2939 asGfsgcauGfaagcAfgGfaacauac SUSU 2940 D- 2678 {sGalNAc3K2AhxC6}uguccug cUfuCfAfUfGfccuuucs{ invAb } 2941 asGfsaaagGfcaugAfaGfcaggaca SUSU 2942 D- 2679 {sGalNAc3K2AhxC6}gcuucau _g CfcUfUfUfCfuacagus{ invAb} 2943 asAfscuguAfgaaaGfgCfaugaagc SUSU 2944 D- 2680 {sGalNAc3K2AhxC6}cuucaug cCfuUfUfCfUfacagugs{ invAb} 2945 asCfsacugUfagaaAfgGfcaugaag SUSU 2946 D- 2681 {sGalNAc3 K2 AhxC6} uucaugc cUfuUfCfUfAfcaguggs {invAb ) 2947 asCfscacuGfuagaAfaGfgcaugaa SUSU 2948

- 150 047656

D- 2682 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucaacas {invAb} 2949 asUfsguugAfaaggCfaUfgaagcag SUSU 2950 D- 2683 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuccas {invAb} 2951 asUfsg[MeO- I]agAfaaggCfaUfgaagcagsusu 2952 D- 2684 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuccags{ invAb} 2953 asCfsug[MeO- I]aGfaaagGfcAfugaagcasusu 2954 D- 2685 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2955 asUfsgu[MeO- I]gAfaaggCfaUfgaagcagsusu 2956 D- 2686 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb} 2957 asCfsugu[McO- I]GfaaagGfcAfugaagcasusu 2958 D- 2687 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2959 asUfsgua[MeO- I]AfaaggCfaUfgaagcagsusu 2960 D- 2688 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb} 2961 asCfsugua[MeO- I]aaagGfcAfugaagcasusu 2962 D- 2689 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfccuacas {invAb} 2963 asUfsguag[McO- I]aaggCfaUfgaagcagsusu 2964 D- 2690 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfCfucuacas {invAb} 2965 asUfsguagAf[MeO- I]aggCfaUfgaagcagsusu 2966 D- 2691 {sGalNAc3K2AhxC6}usgscuu cauGfcCfUfUfUfcu [LNAA]cags{invDA} 2967 usCfsug[Ab]aGfaaagGfcAfugaa gcasusu 2968 D- 2692 {sGalNAc3K2AhxC6}usgscuu cauGfcCfUfUfUfcuac[LNAA]gs{invDA} 2969 usCfs[GNA-U] guaGfaaagGfc Afugaagcasusu 2970 D- 2693 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfasgsuggcs{invA b} 2971 asGfscca[Ab]UfguagAfaAfggca ugasusu 2972 D- 2694 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfuc[LNA- T]acas{ invAb} 2973 asUfsgu[GNA- A]gAfaaggCfaUfgaagcagsusu 2974 D- 2695 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfuc[LNA- T]acas{ invAb} 2975 asUfsgu[Ab]gAfaaggCfaUfgaag cagsusu 2976 D- {sGalNAc3K2AhxC6}usgscuu 2977 usCfsugu[GNA- 2978

- 151 047656

2696 cauGfcCfUfUfUfc[LNA- T]acags{invDA} A] GfaaagGfcAfugaagcasusu D- 2697 {sGalNAc3K2AhxC6}usgscuu cauGfcCfUfUfUfcua[LNAmC]ags{invDA} 2979 usCfsu[GNA- G]uaGfaaagGfcAfugaagcasusu 2980 D- 2698 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfas[sLNAG]uggcs{ invAb} 2981 asGfscca[Ab]UfguagAfaAfggca ugasusu 2982 D- 2699 (sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2983 asUfs[MeO- I]uagAfaaggCfaUfgaagcagsusu 2984 D- 2700 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacas {invAb} 2985 asUfsguag[MeO- I]aaggCfaUfgaagcagsusu 2986 D- 2701 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfacuacas {invAb} 2987 asUfsguagUfaaggCfaUfgaagcag SUSU 2988 D- 2702 (sGalNAc3K2AhxC6} ugcuuca uGfcCfUfUfUfcuacags{ invAb} 2989 asCfsuguaGf[MeO- I]aagGfc Afugaagcasusu 2990 D- 2703 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfCfcuacags {invAb} 2991 asCfsuguaGf[MeO- I]aagGfcAfugaagcasusu 2992 D- 2704 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUfcuacags{ invAb} 2993 asCfsu[MeO- I]uaGfaaagGfcAfugaagcasusu 2994 D- 2705 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucusacas {invAb } 2995 asUfsgu[MeO- I]gAfaaggCfaUfgaagcagsusu 2996 D- 2706 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucsuacas {invAb } 2997 asUfsgu[MeO- I]gAfaaggCfaUfgaagcagsusu 2998 D- 2707 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucsusacas {invA b} 2999 asUfsgu[MeO- I]gAfaaggCfaUfgaagcagsusu 3000 D- 2708 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfuscuacas {invAb } 3001 asUfsguag[MeO- I]aaggCfaUfgaagcagsusu 3002 D- 2709 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfa[sLNA- 3003 asGfscca[Ab]UfguagAfaAfggca ugasusu 3004

- 152 047656

G]uggcs{ invAb} D- 2710 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCf[LNAA]guggcs{ invAb} 3005 asGfscca[Ab]UfguagAfaAfggca ugasusu 3006 D- 2711 {sGalNAc3K2AhxC6}cugcuuc aUfgGfGfAfU fuguacas {invAb } 3007 asUfsguacAfauccCfaUfgaagcag SUSU 3008 D- 2712 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfuc[LNA- T]acas{ invAb} 3009 asUfsgu[MeO- I]gAfaaggCfaUfgaagcagsusu 3010 D- 2713 {sGalNAc3K2AhxC6}ugcuuca uGfcCfUfUfUf[LNAmC]uacags {invAb} 3011 asCfsugua[MeO- I]aaagGfcAfugaagcasusu 3012 D- 2714 {sGalNAc3K2AhxC6}cugcuu CfaUfGfCfCfu[LNA- T] ucuacas {invAb} 3013 asUfsgUfaGfa[GNA- A]aggcaUfgAfagcagsusu 3014 D- 2715 {sGalNAc3K2AhxC6}cugcuu CfaUfGfCfCfu[LNA- T] ucuacas {invAb} 3015 asUfsgUfaGfa[GNA- A] Afggc aU fg Afagcag su su 3016 D- 2716 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUf[LNAT]cuacas{ invAb} 3017 asUfsguag[MeO- I]aaggCfaUfgaagcagsusu 3018 D- 2717 {sGalNAc3K2AhxC6}ucaugcC fuUfuCfuacaguggcs {invAb} 3019 asGfsccacuguagAfaAfgGfcauga susu 3020 D- 2718 {sGalNAc3K2AhxC6}uauguu CfcUfgCfuucaugccus {invAb} 3021 asAfsggcaugaagCfaGfgAfacaua SUSU 3022 D- 2719 {sGalNAc3K2AhxC6}ugcuuc AfuGfcCfuuucuacags {invAb) 3023 asCfsuguagaaagGfcAfuGfaagca susu 3024 D- 2720 {sGalNAc3K2AhxC6}uguucc UfgCfUfUfCfaugccuuus {invA b} 3025 asAfsggcaUfgaagCfaGfgaacasus u 3026 D- 2721 {sGalNAc3K2AhxC6}cugcuu CfaUfgCfcuuucuacas {invAb} 3027 asUfsguagaaaggCfaUfgAfagcag susu 3028 D- {sGalNAc3K2AhxC6}uauguuc 3029 asAfsggcaUfgaagCfaGfgaacaua 3030

- 153 047656

2722 cUfgCfuUfCfaugccus {inv Ab} susu D- 2723 {sGalNAc3K2AhxC6}uguucc UfgCfUfUfCfaugccus {inv Ab} 3031 asAfsggcaUfgaagCfaGfgaacasus u 3032 D- 2724 {sGalNAc3K2AhxC6}uauguu CfcUfgCfuucaugccuus {inv Ab} 3033 asAfsggcaugaagCfaGfgAfacaua susu 3034 D- 2725 {sGalNAc3K2AhxC6}uauguu CfcUfgCfuucaugccus {inv Ab} 3035 asAfsggcaugaagCfaGfgAfacaua scsc 3036 D- 2726 {sGalNAc3K2AhxC6}uauguu CfcUfgCfuucaugccuus {inv Ab} 3037 asAfsggcaugaagCfaGfgAfacaua scsc 3038 D- 2727 uauguuccUfgCfUfUfCfaugccus {inv Ab} 3039 asAfsggcaUfgaagCfaGfgaacaua susu 3040 D- 2728 uauguuccUfgCfUfUfCfaugcgus {inv Ab} 3041 asAfscgcaUfgaagCfaGfgaacauas usu 3042 D- 2729 uauguuccUfgCfUfUfCfauggcus {inv Ab} 3043 asAfsgccaUfgaagCfaGfgaacauas usu 3044 D- 2730 uauguuccUfgCfUfUfCfaagccus {inv Ab} 3045 asAfsggcuUfgaagCfaGfgaacaua susu 3046 D- 2731 uauguuccUfgCfUfUfCfuugccus {inv Ab} 3047 asAfsggcaAfgaagCfaGfgaacaua susu 3048 D- 2732 uauguuccUfgCfUfUfGfaugccus {inv Ab} 3049 asAfsggcaUfcaagCfaGfgaacauas usu 3050 D- 2733 uauguuccUfgCfUfAfCfaugccus {inv Ab} 3051 asAfsggcaUfguagCfaGfgaacaua susu 3052 D- 2734 uauguuccUfgCfAfUfCfaugccus {inv Ab} 3053 asAfsggcaUfgaugCfaGfgaacaua susu 3054 D- 2735 uauguuccUfgGfUfUfCfaugccus {inv Ab] 3055 asAfsggcaUfgaacCfaGfgaacauas usu 3056 D- 2736 uauguuccUfcCfUfUfCfaugccus {inv Ab} 3057 asAfsggcaUfgaagGfaGfgaacaua susu 3058 D- 2737 uauguuccAfgCfUfUfCfaugccus {inv Ab} 3059 asAfsggcaUfgaagCfuGfgaacaua susu 3060 D- 2738 uauguucgUfgCfUfUfCfaugccus {inv Ab} 3061 asAfsggcaUfgaagCfaCfgaacauas usu 3062 D- uauguugcUfgCfUfUfCfaugccus 3063 asAfsggcaUfgaagCfaGfcaacauas 3064

- 154 047656

2739 {invAb} usu D- 2740 uauguaccUfgCfUfUfCfaugccus {invAb} 3065 asAfsggcaUfgaagCfaGfguacaua SUSU 3066 D- 2741 uaugauccUfgCfUfUfCfaugccus {invAb} 3067 asAfsggcaUfgaagCfaGfgaucaua SUSU 3068 D- 2742 uaucuuccUfgCfUfUfCfaugccus {invAb} 3069 asAfsggcaUfgaagCfaGfgaagaua SUSU 3070 D- 2743 uaaguuccUfgCfUfUfCfaugccus {invAb} 3071 asAfsggcaUfgaagCfaGfgaacuua SUSU 3072 D- 2744 uuuguuccUfgCfUfUfCfaugccus {invAb} 3073 asAfsggcaUfgaagCfaGfgaacaaas usu 3074 D- 2745 aauguuccUfgCfUfUfCfaugccus {invAb} 3075 asAfsggcaUfgaagCfaGfgaacauu SUSU 3076 D- 2746 ucaugccuUfuCfUfAfCfaguggcs {invAb} 3077 asGfsccacUfguagAfaAfggcauga SUSU 3078 D- 2747 ucaugccuUfuClUfAfCfagugggs {invAb} 3079 asCfsccacUlguagAfaAlggcauga SUSU 3080 D- 2748 ucaugccuUfuCfUfAfCfagugccs {invAb} 3081 asGfsgcacUfguagAfaAfggcauga SUSU 3082 D- 2749 ucaugccuUfuCfUfAfCfagucgcs {invAb} 3083 asGfscgacUfguagAfaAfggcauga SUSU 3084 D- 2750 ucaugccuUfuClUfAfCfagaggcs {invAb} 3085 as Gfsee ucU 1g uag AfaAfggc auga SUSU 3086 D- 2751 ucaugccuUfuCfUfAfCfacuggcs {invAb} 3087 asGfsccagUfguagAfaAfggcauga SUSU 3088 D- 2752 ucaugccuUfuCfUfAfCfuguggcs {invAb} 3089 asGfsccacAfguagAfaAfggcauga SUSU 3090 D- 2753 ucaugccuUfuClUfAfGfaguggcs {invAb} 3091 asGfsccacUfcuagAfaAfggcauga SUSU 3092 D- 2754 ucaugccuUfuCfUfUfCfaguggcs {invAb} 3093 asGfsccacUfgaagAfaAfggcauga SUSU 3094 D- 2755 ucaugccuUfuCfAfAfCfaguggcs {invAb} 3095 asGfsccacUfguugAfaAfggcauga SUSU 3096 D- ucaugccuUfuGfUfAfCfaguggcs 3097 asGfsccacUfguacAfaAfggcauga 3098

- 155 047656

2756 {invAb} SUSU D- 2757 ucaugccuAfuCfUfAfCfaguggcs {invAb} 3099 asGfsccacUfguagAfuAfggcauga SUSU 3100 D- 2758 ucaugcguUfuCfUfAfCfaguggcs {invAb} 3101 asGfsccacUfguagAfaAfcgcauga SUSU 3102 D- 2759 ucauggcuUfuCfUfAfCfaguggcs {invAb} 3103 asGfsccacUfguagAfaAfgccauga SUSU 3104 D- 2760 ucaagccuUfuCfUfAfCfaguggcs {invAb} 3105 asGfsccacUfguagAfaAfggcuuga SUSU 3106 D- 2761 ucuugccuUfuCfUfAfCfaguggcs {invAb} 3107 asGfsccacUfguagAfaAfggcaaga SUSU 3108 D- 2762 ugaugccuUfuCfUfAfCfaguggcs {invAb} 3109 asGfsccacUfguagAfaAfggcauca SUSU 3110 D- 2763 acaugccuUfuCfUfAfCfaguggcs {invAb} 3111 asGfsccacUfguagAfaAfggcaugu SUSU 3112 D- 2764 ucaugccuUfaCfUfAfCfaguggcs {invAb} 3113 asGfsccacUfguagUfaAfggcauga SUSU 3114 D- 2765 {sGalNAc3K2AhxC6}augccu UfuCfUf AfCfaguggcusus {inv Ab} 3115 asGfsccacUfguagAfaAfggcausu SU 3116 D- 2766 {sGalNAc3K2AhxC6}augccu UfuCfUfAfCfaguggcs {invAb} 3117 asGfsccacUfguagAfaAfggcausu SU 3118 D- T1(I {sGalNAc3K2AhxC6}ucaugcc uUfuCfu AfCfaguggcs {invAb} 3119 asGfsccacUfguagAfaAfggcuuga SUSU 3120 D- 2768 {sGalNAc3K2AhxC6} [invAb] augccuUfUfCfUfacaguggscsu 3121 asGfsccacUfguagaaAfgGfcausu SU 3122 D- 2769 {sGalNAc3K2AhxC6]ucaugcC fuUfuCfuacaguggcus{ invAb} 3123 asGfsccacuguagAfaAfgGfcauga SUSU 3124 D- 2770 {sGalNAc3K2AhxC6}ucaugcC fuUfuCfuacaguggcs {invAb} 3125 asGfsccacuguagAfaAfgGfcauga sasc 3126 D- 2771 {sGalNAc3K2AhxC6}ucaugcC fuUfuCfuacaguggcus{ invAb} 3127 asGfsccacuguagAfaAfgGfcauga sasc 3128 D- 2772 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcus {invA 3129 asGfsccacUfguagAfaAfggcauga SUSU 3130

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b} D- 2773 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcs {invAb } 3131 asGfsccacUfguagAfaAfggcauga sasc 3132 D- 2774 {sGalNAc3K2AhxC6}ucaugcc uUfuCfUfAfCfaguggcus {invA b} 3133 asGfsccacUfguagAfaAfggcauga sasc 3134 D- 2775 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfu[LNAmC]uacas{invAb} 3135 asUfsgua[MeO- I]AfaaggCfaUfgaagcagsusu 3136 D- 2776 {GalNAc3K2AhxC6}gcggcuU IcGfAfCfGfgcuucuasusu 3137 [Phosphate] us AfsgAfaGfCfcguc GfaAfgccgcsusu 3138 D- 2777 {GalNAc3K2AhxC6}gcggcuU fcGfAfCfGfgguucuasusu 3139 [Phosphate] us AfsgAfaCfCfcgucG faAfgccgcsusu 3140 D- 2778 (GalN Ac3 K2AhxC6} gcggcuU fcGfAfCfGfccuucuasusu 3141 {Phosphate} us AfsgAfaGfGfcguc GfaAfgccgcsusu 3142 D- 2779 {GalNAc3K2AhxC6}gcggcuU IcGfAfCfCfgcuucuasusu 3143 {Φocφaτ}usAfsgAfaGfCfgguc GfaAfgccgcsusu 3144 D- 2780 {GalNAc3K2AhxC6}gcggcuU fcCfUfGfGfgcuucuasusu 3145 [sfnvAb]usAfgAfaGfCfccagGfa Afgccgcsusu 3146 D- 2781 {sGalNAc3K2AhxC6} [invAb] gcuucaUfgGfGfAfUfuguacaus usu 3147 {Phosphate} asUfsgUfaCfAfauccC faUfgaagcsusu 3148 D- 2782 {sGalNAc3K2AhxC6}ucaugcc uUfuGfA fUI'C facuggcs {invAb } 3149 asGfsccagUfgaucAfaAfggcauga SUSU 3150 D- 2783 {sGalNAc3K2AhxC6}cugcuuc aUfgCfCfUfUfucuacasc 3151 csUfsguagAfaaggCfaUfgaagcag SUSU 3152 D- 2784 {sGalNAc3K2AhxC6} [invAb] cuucauGfcGfAfAfUfcaAfcAfg UfsusUf 3153 {Phosphate} asCfsuGfuUfGfauuc GfcAfugaagsusu 3154 D- 2785 {sGalNAc3 K2 AhxC6} ggccuu AfuCfCfCfuccuuccususa 3155 usAfsaggAfaGfGfagggAfuAfag gccsasc 3156 D- {sGalNAc3K2AhxC6}gugucu 3157 usUfsuggAfaUfGfgaacUfcAfga 3158 2786 GfaGfUfUfccauuccasasa cacscsa

Example 2. Efficiency of selected PNPLA3 siRNA molecules in RNA FISH analysis.

A panel of fully chemically modified siRNAs, including siRNA spanning rs738409 and/or rs738408 of the PNPLA3 SNP, was generated and tested for mRNA knockdown activity and selectivity in vitro. Each siRNA duplex consisted of two strands: a sense or 'passenger' strand and an antisense or 'guide' strand. The strands were 21 or 23 nucleotides long with 19 complementary base pairs. In some cases, 3' overhangs consisting of two base pairs were present. siRNAs were generated with the natural 2'-OH group in the ribose of each nucleotide replaced by either 2'-OMe or 2'-P group. Phosphodiester internucleotide linkages in one or both strands were optionally replaced with phosphorothioates to reduce degradation of the molecule by exonucleases.

The potency of each siRNA molecule in reducing PNPLA3 expression was assessed using an in vitro siRNA transfection assay in a 384-well format followed by RNA-targeted fluorescence in situ hybridization (RNA FISH) to determine IC ₅₀ values and maximal activity. The assay was performed on the human hepatocellular carcinoma cell line Hep3B (ATCC HB-8064) and on Chinese hamster ovary (CHO) cells expressing human PNPLA3 I148I. The human hepatocellular carcinoma cell line HepB3 was maintained in EMEM (ATCC 30-2003) supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic at 37°C and 5% CO ₂ . CHO cells expressing human PNPLA3 I148I were maintained in medium containing 50% CD-CHO (Life Technologies), 50% Ex-Cell CHO

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Medium (Sigma), 8 mM L-glutamine, 1x HT, 1% antibiotic/antimycotic and 10 µg/ml puromycin, at

37°C and 5% CO2.

For Hep3B cell assays, transfection complexes consisting of siRNA molecules and Lipofectamine RNAiMAX transfection reagent (Life Technologies) in EMEM (ATCC 30-2003) were added to 384-well plates (PerkinElmer) at 10 μg/well according to the manufacturer's recommendations. For human CHO cell assays, transfection complexes consisting of siRNA molecules and Lipofectamine RNAiMAX transfection reagent in F12K medium (Mediatech) were added to 384-well plates at 10 μg/well according to the manufacturer's recommendations. Cells were diluted to 67,000 cells/mL in antibiotic/no antibiotic medium and 30 μL were added to each well for a final density of 2,000 cells/well in 40 μL of medium. After 20 min incubation at room temperature, the plates were transferred to an incubator with 37°C and 5% CO2 conditions. Transfection mixtures with Hep3B cells were incubated for 72 h, and transfection mixtures with CHO cells with human PNPLA3 I148I were incubated for 48 h.

Upon harvest, cells were fixed in 8% formaldehyde fixative (Thermo Scientific) for 15 min at room temperature. Plates were then dehydrated with sequential washes of 50, 70, and 100% ethanol. Plates were then sealed and stored at -20°C.

RNA FISH analysis was performed using the Affymetrix QuantiGene® View RNA HC Screening Assay (QVP0011), the Affymetrix View HC Signal Amplification Kit 3-plex (QVP0213) from Affymetrix, and gene-specific probes from Affymetrix: 0.33 ml View RNA type 6 (650 tag) human VA6-20279-01 PNPLA3 and 0.44 ml View RNA type 1 (488 tag) human VA1-10148-01 PPIB.

Plate contents were first rehydrated with sequential washes with 100, 70, and 50% ethanol. Cells were then washed with PBS and then permeabilized and protease digested according to kit instructions. Targeted working probe sets were prepared according to the manufacturer's protocol, added to wells, and incubated for 3 h at 40°C. The manufacturer's protocol was used for sequential hybridizations with working probe sets, working preamplifiers, working amplifiers, and working LPs. Finally, nuclei were counterstained (Hoechst 33342 and Cell Mask Blue; Molecular Probes). Plates were incubated for 30 min at room temperature, washed with PBS, added with 80 μl PBS, and then sealed for visualization.

All plates were visualized using the pera Phenix High Content Screening System (PerkinElmer) using the UV channel for Hoechst 33342 and Cell Mask Blue, the 488 nm channel for type 1 probes, and the 647 nm channel for type 6 probes.

RNA FISH data were analyzed using Columbus software and images were generated using Genedata Screener. The assay results for CHO cells transfected with PNPLA3 I148I are shown in Table 3. The assay results for CHO cells transfected with PNPLA3 I148M are shown in Table 4. PNPLA3 knockdown is shown as percent knockdown compared to control. Negative values indicate decreased PNPLA3 levels.

Table 3

RNA FISH analysis of CHO cells transfected with PNPLA3 I148I

Duplex No. IC50 (µM) PNPLA3 knockdown (%) . D-2001 .0589 -33.9 D-2002 .0158 -67.2 D-2003 .0427 -43.4 D-2004 .00835 -63.5 D-2006 .0177 -77.8

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D-2008 .125 -10.7 D-2009 .00769 -45.5 D-2010 .00558 -80 D-2011 .035 -3 D-2012 >0.5 -3.7 D-2013 .036 -6.4 D-2014 .0122 -58.2 D-2015 >0.5 8 D-2016 >0.5 8 D-2017 .0153 -73.2 D-2018 .00386 -31.5 D-2019 .125 D-2020 >0.5 D-2021 >0.5 8 D-2022 >0.167 -6.8 D-2023 .0257 -36.9 D-2024 >0.5 4 D-2025 >0.5 -2.5 D-2026 .022 -35.1 D-2027 .00172 -16.5 D-2028 >0.5 10 D-2029 .0106 -56.2 D-2032 .00205 -52.9 D-2033 .0107 -61.7 D-2034 >0.5 6 D-2035 >0.5 -3.8 D-2036 .00665 -55.9 D-2037 >0.5 4 D-2038 >0.5 10 D-2039 .0116 -23.9 D-2040 >0.5 -25.4 D-2041 >0.5 9 D-2042 .00959 -25.5 D-2043 >0.5 9

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D-2044 .00552 -29 D-2045 >0.5 9 D-2046 >0.5 9 D-2047 >0.5 -5.9 D-2048 .00618 -56.3 D-2049 >0.5 12 D-2050 >0.5 10 D-2051 >0.5 -17.2 D-2052 >0.5 -8.3 D-2053 >0.5 11 D-2054 >0.5 -14.9 D-2055 >0.5 -10.6 D-2056 >0.5 10 D-2057 .00485 -59.2 D-2058 .014 -53 D-2059 >0.5 -4.9 D-2060 >0.5 18 D-2061 .00795 -44.8 D-2062 .000668 -74.6 D-2063 >0.5 -21.8 D-2064 >0.5 9 D-2065 >0.5 -10.5 D-2066 .0412 -42.2 D-2067 >0.5 10 Table 4 RNA FISH analysis CHO cells transfected with PNPLA3 I148M Duplex No. IC50 (µM) PNPLA3 knockdown (%) D-2000 .0316 -29.2 D-2001 .0131 -81.8 D-2002 .00216 -90.5 D-2003 .022 -50.4 D-2004 .00429 -88 D-2005 >0.5 15 D-2006 .00301 -89.2 D-2007 >0.5 6

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D-2009 .00274 -86.9 D-2010 .00203 -93.3 D-2011 .000694 -11.9 D-2012 >0.5 -18 D-2013 .011 -66.4 D-2014 .0057 -84.3 D-2015 >0.5 -13.5 D-2016 >0.5 -12.4 D-2017 .00448 -89.4 D-2018 .0104 -36.2 D-2019 .0302 -7.7 D-2020 .01 -78.9 D-2021 .00435 -83.5 D-2022 .00628 -88.6 D-2023 .0143 -44.3 D-2024 >0.5 11 D-2025 .00355 -58.2 D-2026 .000867 -39.4 D-2027 >0.5 32 D-2028 .00205 -89.9 D-2029 .0019 -94 D-2030 >0.5 -9.4 D-2031 >0.5 4

RNA FISH was also performed on a liver cell line containing the double mutation PNPLA3rs738408-rs738409, as well as on a wild-type control cell line Hep3B. Hep3B and HepG2 cells (purchased from ATCC) were cultured in minimal essential medium (MEM from Corning for Hep3B cells and EMEM from ATCC for HepG2 cells) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% penicillin-streptomycin (PS, Corning). siRNA transfection was performed as follows: 1 μl of test siRNAs and 4 μl of plain MEM or EMEM, depending on the cell line, were added to PDL (PerkinElmer) coated CellCarrier-384 Ultra assay plates from BioMek FX (Beckman Coulter). 5 μl of Lipofectamine RNAiMAX (Thermo Fisher Scientific) pre-diluted in plain MEM or EMEM (i.e., 0.035 μl RNAiMAX in 5 μl MEM for Hep3B cells and 0.06 μl RNAiMAX in 5 μl EMEM for HepG2 cells) were then dispensed into the assay plates using the Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific). After 20 min of siRNA/RNAiMAX mixture incubation at room temperature (RT), 30 μl of Hep3B cells or HepG2 cells (2000 cells per well) in MEM or EMEM supplemented with 10% FBS and 1% PS were added to the transfection complex using the Multidrop Combi Reagent Dispenser. The assay plates were incubated for 20 min at RT before being placed in the incubator. Cells were then incubated for 72 h at 37°C and 5% CO ₂ . ViewRNA ISH analysis of cells was performed according to the manufacturer's protocol (Thermo Fisher Scientific) using a self-assembled automated liquid-based FISH assay platform. Briefly, cells were fixed in 4% formaldehyde (Thermo Fisher Scientific) for 15 min at RT, permeabilized with detergent for 3 min at RT, and then treated with protease solution for 10 min at RT. Target-specific probe pairs (Thermo Fisher Scientific) were incubated for 3 h, while preamplifiers, amplifiers, and labeled probes (Thermo Fisher Scientific) were incubated for 1 h each. All hybridization steps were performed at 40°C in a Cytomat 2 C-LIN automated incubator (Thermo Fisher Scientific). Following hybridization reactions, cells were stained with Hoechst and CellMask Blue (Thermo Fisher Scientific) for 30 min and then visualized with Opera Phenix (PerkinElmer). Images were analyzed using the Columbus Image Data Storage and Analysis System (PerkinElmer) to obtain the mean number of spots per cell. Spot counts were normalized using high-count (containing phosphate-buffered saline from Corning) and low-count (no target probe pairs) control wells. Normalized values against total siRNA concentrations were plotted, and the data were fitted with a four-parameter sigmoid model in Genedata Screener (Genedata) to obtain IC ₅₀ and maximum activity values. The assay results for HepG2 cells are shown in Table 5, and the assay results for Hep3B cells are shown in Table 6.

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PNPLA3 knockdown is shown as percent knockdown compared to control. Negative values indicate decreased PNPLA3 levels. In cases where a duplex was assayed more than once, the mean IC50 value is shown with standard deviation.

Table 5

RNA FISH analysis of HepG2 hepatocytes

Duplex No. IC50 (µM) PNPLA3 knockdown (%) D-2001 .0132 -70.5 D-2003 .0458 -46.9 D-2004 .0049 -74.2 D-2006 .00283 -69.6 D-2009 .00448 -62.2 D-2010 .00206 -52 D-2013 .00319 -71.7 D-2014 .00164 -67.4 D-2017 .00222 -60.9 D-2018 .00717 -52.7 D-2020 .00664 -68.3 D-2021 .00559 -61.5 D-2022 .004 -30.5 D-2023 >0.5 -18 D-2025 .0041 -61.9 D-2026 .00937 -34.6 D-2043 >0.1 -5,394 D-2045 >0.1 -37,195 D-2046 >0.1 0.94 D-2047 >0.1 -11,446 D-2048 0,000355 -56,824 D-2049 Not defined -30,881 D-2050 >0.1 -11,814 D-2051 0,000903 -73,749 D-2052 0,000235 -38,467 D-2053 0,000142 -19,405 D-2054 >0.1 -29,069 D-2055 >0.1 -31,986 D-2056 Not defined -38,596 D-2057 0,000197 -87,199 D-2058 0,00126 -40,177 D-2059 >0.1 -11,162 D-2060 >0.1 -8,147 D-2069 0,0329 -62,804 D-2072 0,00377 -47,552 D-2078 >0.1 -25,077 D-2073 0,00602 -54,092 D-2084 0,000549 -45,854 D-2084 0,0018 -60,106 D-2085 0,000122 -38,539

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D-2085 0,0076 -68.83 D-2086 0,00182 -25,524 D-2087 0,00103 -53,242 D-2088 0,00111 -26,614 D-2089 0,000776 -55,436 D-2089 0.000457 +/- 0.000174 -62.57 +/- 1.46 D-2090 Not defined -56,094 D-2092 0,000979 -52,168 D-2099 0,000727 -43,233 D-2100 0,000152 -40,651 D-2104 0,000712 -72,734 D-2105 0,000129 -75,994 D-2128 0,00154 -60,927 D-2138 0,0142 -63,864 D-2149 0,000518 -74,288 D-2152 0,00524 -64,758 D-2153 0,00149 -72,861 D-2155 >0.1 -32,937 D-2156 0,0183 -75,145 D-2157 > 0.0333 -37,881 D-2158 0,00368 -79,231 D-2159 0,00167 -85,848 D-2160 0,00442 -37,827 D-2172 0,0000615 -74,462 D-2182 0,000211 -79,605 D-2183 0,000159 -80,865 D-2187 0,00109 -67,777 D-2188 0,000728 -71,503 D-2189 0,000224 -58,772 D-2190 0,000803 -72,508 D-2192 0,00125 -33,021 D-2193 0,000152 -74,239 D-2194 0,00385 -50,848 D-2196 0,000362 -70,158

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D-2197 0,000180 -54.44 D-2198 0,000269 -45,556 D-2199 0,000198 -81,082 D-2201 0,000356 -82.2 D-2202 0,000123 -62,466 D-2203 0,00476 -68,112 D-2204 0,000300 -63,383 D-2205 0,000442 -70,712 D-2206 0,000969 -74,194 D-2208 0,000202 -64,046 D-2209 0,000752 -63,083 D-2210 0,0000991 -60,426 D-2211 0,0000948 -71,098 D-2213 0,00119 -65.57 D-2214 0,000293 -72,959 D-2215 0,000307 -64,561 D-2217 0,00198 -87,273 D-2218 0,000186 -64,522 D-2219 0,000577 -74,879 D-2220 0,000189 -74,278 D-2221 0,000173 -54,639 D-2223 0,00131 -69,834 D-2224 0,000527 -84,911 D-2225 0,000561 -68,969 D-2226 0,00109 -75,536 D-2227 0,000227 -85,915 D-2228 0,00202 -50,227 D-2229 Not defined -59,847 D-2230 0,000156 -53,194 D-2231 0,000351 -65,054 D-2232 0,0000619 -63,991 D-2233 0,0000982 -74,304 D-2234 0,000290 -81,992 D-2235 0,000127 -83,593

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D-2236 0,000243 -80,826 D-2237 0,0000711 -62,814 D-2238 0,0000690 -76,476 D-2239 0,000123 -74,747 D-2240 0,000120 -79,301 D-2241 0,000201 -88,769 D-2242 0,000179 -84,503 D-2243 0,000264 -88,712 D-2244 0,0000605 -82,556 D-2245 0,000125 -83.93 D-2246 0,000122 -84.41 D-2247 0,000100 -81,777 D-2248 0,000141 -79.18 D-2249 0,0000710 -85,093 D-2250 0,000494 -81,724 D-2251 0,000196 -87,601 D-2252 0,0000796 -62.87 D-2253 0,000501 -78,699 D-2254 0,0000979 -67,617 D-2255 0,000359 -81.18 D-2256 0,000610 -98,206 D-2257 0,000151 -76,718 D-2258 0,000325 -87,548 D-2259 0,000124 -82,545 D-2260 0,000109 -85.47 D-2261 0,000231 -75,386 D-2262 0,000162 -72,407 D-2263 0,000271 -74,968 D-2264 0,000132 -76,418 D-2265 >0.1 -17,144 D-2266 Not defined -58,475 D-2267 >0.1 -3,991 D-2280 0,0000390 -85,368 D-2286 0,00304 -89,536

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D-2287 0,00142 -85,435 D-2287 0,000261 -66,501 D-2288 0,000839 -81,988 D-2289 0,0012 -83,437 D-2289 0,00168 -69,471 D-2290 0,00157 -73,089 D-2291 0,0112 -64,108 D-2292 0,00411 -66,681 D-2292 0,00138 -56,253 D-2293 0,00112 -61,118 D-2294 0,000408 -70,684 D-2295 0,000195 -65,493 D-2297 0,00165 -61,821 D-2297 0,000684 -69,444 D-2318 0,000986 -69.28 D-2319 0,00136 -67,654 D-2320 0,00129 -83,723 D-2323 0,00194 -74,525 D-2324 0,00209 -63,745 D-2325 0,000301 -81,302 D-2327 0,000306 -77,959 D-2327 0,000610 -56,293 D-2328 0,000131 -72,631 D-2345 0,000131 -72,677 D-2346 0,0000274 -71,459 D-2347 0,000640 -78,414 D-2348 0,000101 -64,627 D-2349 0,000487 -52,131 D-2350 0,000102 -68,668 D-2351 Not defined -70,229 D-2352 0,000332 -81,083 D-2352 0.0027713 +/- 0.00426 -68.138+/- 9.28 D-2353 0,000410 -83,001 D-2353 0,00215 -64,343

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D-2354 0.003405 +/- 0.00185 -72.56 +/- 7.42 D-2354 0,000362 -72,776 D-2355 0,00215 -69,321 D-2358 0,0011 -61,081 D-2359 0,000654 -76,258 D-2360 Not defined -59,027 D-2361 0,00414 -57.88 D-2362 0,000772 -87,512 D-2364 0,00104 -53,895 D-2370 0,0024 -83,423 D-2371 0,00418 -72,262 D-2372 0,00492 -67,648 D-2373 0,000906 -64,099 D-2374 0,00282 -64,049 D-2375 0,00125 -59,813 D-2376 0,00171 -63,025 D-2377 0,00168 -61,597 D-2378 0,00505 -72,446 D-2379 0,00511 -80,576 D-2380 0,0497 -82,835 D-2381 0,000944 -71,788 D-2382 0,000830 -65,087 D-2383 0,00118 -71,393 D-2386 0,0000367 -44,073 D-2387 0,00224 -74.24 D-2390 0,00188 -69,313 D-2391 0,000493 -72,314 D-2393 0.008875 +/- 0.0112 -48.855 +/- 15.6 D-2401 0,000125 -69,217 D-2409 0,00479 -68,703 D-2402 >0.5 21.01 +/- 6.08 D-2410 0,00533 -64,812 D-2411 0,00283 -64,889 D-2412 0,00211 -67,781

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D-2413 0.006575 +/- 0.0000212 -64.021 +/- 15.8 D-2417 0,00558 -50,293 D-2418 0,00566 -59,533 D-2419 >0.5 -15.364 +/- 7.56 D-2423 0.000498 +/- 0.000414 -65.15 +/- 17.5 D-2426 >0.1 -15,207 D-2430 0,0000511 -63,602 D-2444 0.000262 +/- 0.000192 -67.206+/- 9.13 D-2454 0.001795 +/- 0.000106 -51.619+/- 0.163 D-2456 0,000274 -61,291 D-2472 0,000314 -70,624 D-2473 >0.1 -19.624 +/- 7.27 D-2474 0,000339 -37,846 D-2475 0.002249+/- 0.00231 -41.661 +/- 8.64 D-2476 0,000594 -26.48 D-2477 0,000547 -61,592 D-2478 0,000168 -65,904 D-2479 0,0005565 -65.14 D-2480 0,000907 -53,271 D-2481 0,0004005 -59.5 D-2482 0,000493 -61,712 D-2483 0.0004015 +/- 0.000218 -66,803 +/-.197 D-2484 0,0003375 -67.4 D-2485 0,0002865 -44,529 D-2486 0,0012575 -62,847 D-2487 0,000454 -54,396 D-2488 0,0002415 -66,315 D-2489 >0.5 -2.973 +/- 1.98 D-2490 0,0011555 -62,566 D-2491 0,0006005 -60,304 D-2492 0,0007775 -47,748 D-2493 0,001465 -48.8 D-2494 0,0008985 -50,707

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D-2495 0,001317 -55.5 D-2496 0,0001374 -68,949 D-2497 0.00013615+/- 0.0000691 -72.763 +/- 4.02 D-2498 0.000332 +/- 0.000259 -82.507+/- 6.15 D-2499 0,0004155 -75.4 D-2500 0.0000742 +/- 0.0000407 -74.968 +/- 5.41 D-2501 >0.5 -1.648+/- 2.2 D-2502 0.0002705 +/- 0.000252 -68.406 +/- 5.36 D-2503 0.000313 +/- 0.000198 -67,858 +/- 8,2 D-2504 0.0006445 +/- 0.000385 -59.733 +/- 7.96 D-2505 0,0004985 -53.3 D-2506 >0.1 -16.588 +/- 14.9 D-2507 0,002136 -64,978 D-2508 >0.1 -20.61 +/-11.1 D-2509 0,001242 -65,112 D-2510 0,0015895 -56,416 D-2511 0,00166 -19,901 D-2512 0,000593 -0.374 D-2513 0.0001497 +/- 0.000123 -69.663 +/- 11.6 D-2514 0.000204 +/- 0.0000594 -69.924 +/- 13.7 D-2515 Ο.ΟΟΟ331 +/- 0.000129 -71.29+/-8.74 D-2516 0.000348 +/- 0.000249 -65.905 +/- 14.8 D-2517 0.000452 +/- 0.000322 -68.212+/- 11.2 D-2518 0,0006965 -73,226 D-2519 >0.5 -1.486+/- 4.67 D-2520 0,000098 -75,127 D-2521 0.000717 +/- 0.000378 -61.216+/- 12.8 D-2522 0.000661 +/- 0.000748 -56,771 +/- 16.9 D-2523 0,0003685 -73,312 D-2524 0.0002745 +/- 0.0000686 -65.341 +/- 12.7 D-2525 0.003855 +/- 0.0033 -44.288 +/- 19.0 D-2526 0.0006115+/- 0.000204 -57.77 +/- 13.2 D-2527 0,000333 -77,405 D-2528 0.0002665 +/- 0.000105 -62.407 +/- 8.65

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D-2529 0.0076967 +/- 0.00555 -44.194+/- 6.73 D-2530 0,0132 -42.657 +/- 0.222 D-2531 0,00312 -32,789 +/- 12.0 D-2532 0.005795 +/- 0.00268 -44.051 +/- 2.82 D-2533 0.005515+/- 0.00356 -43,145 D-2534 0,0129 -28,734+/- 16,4 D-2535 0.003195 +/- 0.000233 -29.79 D-2536 0.005865 +/- 0.00456 -25,918 D-2537 0,0133 -25.755 +/- 3.32 D-2538 >0.1 -40.955 +/- 5.3 D-2539 0.004785+/- 0.00183 -34.334+/- 11.0 D-2540 0.0044 +/- 0.00075 -39,221 D-2541 0.002735+/- 0.00141 -62.39 +/- 3.03 D-2542 0.01323 +/- 0.0108 -30,917+/- 1,57 D-2543 >0.1 -8.54 +/- 8.37 D-2544 0,017 -23,757 +/- 2.2 D-2545 0.01642 +/- 0.0164 -52.394 +/- 7.21 D-2546 >0.1 -25.351 +/- 7.84 D-2547 0,00412 -25.042 +/- 5.28 D-2548 0.00411 +/- 0.00184 -40.406 +/- 1.26 D-2549 0.02285 +/- 0.00318 -53,713+/- 14,1 D-2550 0.015935 +/- 0.0115 -41,907 D-2551 0,00795 -26.39 D-2552 0.01165+/- 0.00841 -41,306 D-2553 0,00361 -70,457 D-2554 0,00656 -42,866 D-2555 >0.1 -11,425 D-2556 0,00336 -63,996 D-2557 >0.1 -2,734 D-2558 0,00251 -66,775 D-2559 >0.1 -7,034 D-2560 0,0234 -44,657 D-2561 0,00154 -74,225 D-2562 0,00454 -66,578

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D-2563 0,00182 -77,025 D-2564 0,009 -64,021 D-2565 0,00609 -70,965 D-2566 0,00255 -52.65 D-2567 0,00584 -56,603 D-2568 0,0157 -63,002 D-2569 0,00327 -64,898 D-2570 0,00144 -55,424 D-2571 >0.1 -18,661 D-2572 0,00557 -25,668 D-2573 0,0115 -55,329 D-2574 0,00289 -69.84 D-2575 0,00378 -69,491 D-2576 0,00527 -64,841 D-2577 0,00511 -46,449 D-2578 0,0026 -67,821 D-2579 0,00402 -67,057 D-2580 0,00119 -64,422 D-2581 0,00915 -73,008 D-2582 0,000823 -77,053 D-2583 0,00851 -66,555 D-2584 0,00513 -54,442 D-2585 0,0154 -67,707 D-2586 0,00701 -69,624 D-2587 0,00732 -66,627 D-2588 0,00226 -70,854

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D-2589 0,00837 -31,221 D-2590 0,0249 -41,857 D-2591 0,009 -64,021 D-2592 0,00609 -70,965 D-2593 0,00255 -52.65 D-2594 0,00584 -56,603 D-2595 0,0157 -63,002 D-2596 0,00327 -64,898 D-2597 0,00144 -55,424 D-2598 >0.1 -18,661 D-2599 0,00557 -25,668 D-2600 0,0115 -55,329 D-2601 0,00289 -69.84 D-2602 0,00378 -69,491 D-2603 0,00527 -64,841 D-2604 0,00511 -46,449 D-2605 0,0026 -67,821 D-2606 0,00402 -67,057 D-2607 0,00119 -64,422 D-2608 0,00915 -73,008 D-2609 0,000823 -77,053 D-2610 0,00141 -37.663 +/- 39.4 D-2611 >0.1 -2.829 +/- 0.006 D-2612 >0.1 -15,894 D-2613 0,00809 -58,833 D-2614 0,00204 -73,042 D-2615 0,00306 -70,087 D-2616 7,95E-04 -71,014

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D-2617 0,00218 -75,769 D-2618 0,0429 -31.19+/- 2.61 D-2619 0.02495 +/- 0.0192 -59.645 +/- 13.4 D-2620 0.0055 +/- 0.00329 -73.308 +/- 5.33 D-2621 0.004705 +/- 0.00176 -63.053 +/- 7.66 D-2622 0.004525 +/- 0.00231 -66.895 +/- 7.78 D-2623 0,00435 -31.366+/- 14.5 D-2624 0.0056 +/- 0.0016 -65.16+/- 10.9 D-2625 0.00764+/- 0.00115 -73.065 +/- 9.97 D-2626 0.021645 +/- 0.0267 -71.117+/-3.63 D-2627 0.00651 +/- 0.00489 -63.009 +/- 8.83 D-2628 0.01334 +/- 0.00588 -48,771 +/- 2.35 D-2629 0.005504 +/- 0.00434 -74.111 +/- 4.61 D-2630 0.007965 +/- 0.00684 -70.17+/- 5.32 D-2631 >0.1 -8,645 D-2632 >0.1 -37,841 D-2633 0,0111 -53,354 D-2634 0,0187 -63,686 D-2635 0,00797 -58,605 D-2636 >0.1 -34,727 D-2637 0,00541 -61,225 D-2638 0,00397 -62,221 D-2639 0.0295 +/- 0.0187 -51.328+/- 12.8 D-2640 0,0212 -37,799+/- 16.1 D-2641 0,00164 -21,913+/- 14,1 D-2642 >0.1 -11.733 +/- 23.5 D-2643 >0.1 -23,009 D-2644 0,00165 -46,514 D-2645 0,00242 -75,956 D-2646 >0.1 -15,539+/- 1.03 D-2647 0,00243 -73,363 D-2648 0.00117 +/- 0.0000849 -74.63 +/- 1.55 D-2649 0.007455 +/- 0.00756 -46.662 +/- 2.86 D-2650 0.003845 +/- 0.00122 -67,914+/- 1,87

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D-2651 0,000449 -70,274 D-2652 0,0014 -66.6 D-2653 0,00299 -59,695 D-2654 0,00431 -26.338 +/- 4.54 D-2655 0.00254 +/- 0.0012 -52.632 +/- 0.938 D-2656 0.003275+/- 0.00112 -37.351 +/- 0.79 D-2657 0.007435 +/- 0.00357 -37.313+/-3.03 D-2658 >0.1 -22.486 +/- 10.6 D-2659 >0.1 -10.905 +/- 3.7 D-2660 >0.1 -4.489 +/- 8.45 D-2661 0.00875 +/- 0.00728 -56,877+/- 9,41 D-2662 >0.1 -8,133 D-2663 0.00268 +/- 0.000778 -73,024+/- 1,16 D-2664 0,008 -30,908+/- 1,19 D-2665 0.004675 +/- 0.000516 -39.914 +/- 3.72 D-2666 0.004085 +/- 0.000106 -46.423 +/- 3.66 D-2667 0.017107 +/- 0.0145 -72.967 +/- 2.96 D-2668 0.00589 +/- 0.000919 -73,779 +/- 2.9 D-2669 0.0035033 +/- 0.00167 -67.153 +/- 0.761 D-2670 0,0129 -59.149+/- 0.371 D-2671 0.0157 +/- 0.00156 -57.5 +/- 3.88 D-2672 >0.1 -3.262 +/- 6.36 D-2673 0.00461 +/- 0.00173 -50.295+/- 14.1 D-2674 0,00279 -54.886 +/- 32.2 D-2675 >0.1 -13.5 +/- 21.4 D-2676 0.015075 +/- 0.0156 -50.255 +/- 8.86 D-2677 >0.1 -22,123 D-2678 >0.1 -43,995 D-2679 0,00268 -83,804 D-2680 >0.1 -47,702 D-2681 >0.1 -38,246 D-2682 0.00305 +/- 0.000255 -70.849+/- 11.3 D-2683 0,00363 -59.037 +/- 15.6 D-2684 0,00763 -74.35

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D-2685 0.00585 +/- 0.00379 -72.725 +/- 18.8 D-2686 >0.1 -6,429 D-2687 0.0031933 +/- 0.000225 -70.122+/-5.47 D-2688 0.0023063 +/- 0.00152 -65.267 +/- 8.73 D-2689 0.0025767 +/- 0.000721 -66.016+/- 11.4 D-2690 0.0032367 +/- 0.00272 -63.04+/- 13.4 D-2691 >0.1 -63,208 D-2692 0,00376 -62,833 D-2693 0.002915+/- 0.00131 -71.229+/- 10.9 D-2694 0.003245 +/- 0.000573 -69,264 D-2695 0.01652 +/- 0.0179 -51.139+/- 17.8 D-2696 0,0024 12.88 D-2697 0,013 -63,845 D-2698 0.003285 +/- 0.000304 -79.24 D-2699 0.002205+/- 0.00117 -67.306 +/- 0.216 D-2700 0.0022297 +/- 0.00129 -67.949 +/- 7.66 D-2701 0.0026037 +/- 0.00196 -54.669 +/- 7.49 D-2702 0,00448 -64,496 D-2703 0,00254 -65,869 D-2704 0,00257 -71,285 D-2705 0.002505 +/- 0.000346 -64.722 +/- 4.62 D-2706 0.003865 +/- 0.00264 -63.247 +/- 10.0 D-2707 0.003425 +/- 0.000318 -65.82+/- 2.07 D-2708 0.0055112+/- 0.00582 -66.395 +/- 10.4 D-2709 0,00243 -76,778 D-2710 0,000912 -78,289 D-2711 >0.1 4.37 +/- 9.89 D-2712 0.0046104 +/- 0.00608 -65.876 +/- 9.59 D-2713 0.0010115+/- 0.00102 -67,741 +/- 2.46 D-2714 0.0063585 +/- 0.00869 -66.904 +/- 0.044 D-2715 0.0061022 +/- 0.00862 -74,054 D-2716 0.00338 +/- 0.00308 -70.054 +/- 9.47 D-2717 0.0033 +/- 0.00155 -72.932 +/- 7.6 D-2718 0,0155 -58,784

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D-2719 0,00527 -57,281 D-2720 0,0215 -41.76 D-2721 0.01829+/- 0.0132 -64.649 +/- 3.84 D-2722 0,0164 -53,261 D-2723 0,00733 -47.56 D-2724 0,00564 -37.19 D-2725 0,00776 -42.77 D-2726 0,00675 -42,811 D-2727 0,00667 -57,414 D-2728 0,0438 -37,169 D-2729 0,00454 -25,538 D-2730 0,0241 -47,501 D-2731 0,0088 -44,866 D-2732 0,0326 -44.68 D-2733 0,0217 -25,146 D-2734 >0.1 -14,582 D-2735 0,0202 -28,294 D-2736 0,014 -38,251 D-2737 >0.1 -17,833 D-2738 0,00514 -62,424 D-2739 0,0317 -41,409 D-2740 0,0111 -59,253 D-2741 0,0111 -59,687 D-2742 0,0229 -82,351 D-2743 0,00933 -59,271 D-2744 0,0215 -54.24 D-2745 0,00619 -57,406 D-2746 0,00654 -69,738 D-2747 0,0307 -63,431 D-2748 0,0152 -59,477 D-2749 0,0329 -45,543 D-2750 0,0149 -47,589 D-2751 0,0261 -54,747 D-2752 0,0106 -65,912

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D-2753 0,0113 -66,051 D-2754 0,0201 -65,582 D-2755 >0.1 -25,144 D-2756 0,0179 -65,965 D-2757 0,0251 -40,795 D-2758 0,0312 -40.55 D-2759 0,00936 -63,545 D-2760 0,00859 -74.99 D-2761 0,0115 -71,716 D-2762 0,00777 -68,169 D-2763 0,0106 -72,426 D-2764 0,0327 -58,009 D-2766 0.003215 +/- 0.000516 -77,976 D-2767 0.00376 +/- 0.000552 -74,346 D-2768 0.00396 +/- 0.0000566 -76,319 D-2769 0.005655 +/- 0.00165 -72.844 +/- 3.26 D-2770 0.0041 +/- 0.000778 -79.301 +/- 1.36 D-2771 0.00891 +/- 0.00137 -70,223 D-2772 0.004855 +/- 0.00000707 -72,651 D-2773 0.004115+/- 0.000544 -77,716 +/- 5,2 D-2774 0.00467 +/- 0.000354 -76.397 +/- 3.67

Table 6

RNA FISH analysis of Hep3B hepatocytes

Duplex No. IC50 (mkM) PNPLA3 knockdown (%) D-2001 .00842 -37 D-2003 .0158 -32.1 D-2004 .00266 -32.4 D-2006 .00948 -54.1 D-2009 .00228 -29.5 D-2010 .00219 -37.2 D-2013 .00524 -31.5 D-2014 .00148 -37.6 D-2017 .00333 -37.6 D-2018 .00315 -21.3

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D-2020 >0.5 6 D-2021 >0.5 -1.6 D-2022 .00272 -30.9 D-2023 >0.5 24 D-2025 .0101 -30.3 D-2026 .00551 -23 D-2413 -45,832 D-2419 >0.5 -4,545 D-2426 >0.1 -15,207 D-2454 0,00187 -51,735 D-2473 >0.1 -21,333 D-2522 0,00504 -43,036 D-2523 0,0122 -52,084 D-2564 0,01 -63,199 D-2565 0,00938 -58,392 D-2566 0,00343 -61,484 D-2567 0,0175 -53,489 D-2568 >0.1 -18,367 D-2569 0,0195 -62,568 D-2570 0,0127 -77,141 D-2571 >0.1 -15,922 D-2572 >0.1 -12,434 D-2573 >0.1 -14,649 D-2574 0,0215 -52,515 D-2575 0,0203 -53,175 D-2576 0,018 -48,137 D-2577 >0.1 -16,105 D-2578 >0.1 -21,309 D-2579 >0.1 -17,510 D-2580 >0.1 -24,616 D-2581 >0.1 -13,987 D-2582 0,0574 -30,543 D-2583 >0.1 -23,990 D-2584 >0.1 -6,715

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D-2585 >0.1 -17,518 D-2586 >0.1 -24,518 D-2587 0,0391 -58,478 D-2588 0,0218 -56,609 D-2589 >0.1 -17,418 D-2590 >0.1 -21,161 D-2591 0,0167 -59,366 D-2592 0,0104 -61,548 D-2593 Not defined -61,879 D-2594 0,0211 -43,856 D-2595 0,0272 -63,020 D-2596 >0.1 -10,278 D-2597 0,0546 -31,743 D-2598 Not defined -47,517 D-2599 0,00489 -70,825 D-2600 >0.1 -8,522 D-2601 0,0364 -31,836 D-2602 0,00577 -65,062 D-2603 0,01 -58,287 D-2604 0,00353 -40,649 D-2605 0,0113 -50,691 D-2606 >0.1 -5,097 D-2607 0,0261 -49,898 D-2608 >0.1 -23,747 D-2609 >0.1 -23,804 D-2620 >0.5 -21,969 D-2637 >0.1 -12.63 D-2638 >0.1 -16.25 D-2639 >0.1 -22,905 D-2640 >0.1 -14,572 D-2666 >0.5 -14,898 D-2667 >0.5 -14,537 D-2668 >0.5 -18,779 D-2669 >0.5 -8,624

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D-2670 >0.5 -16,641 D-2671 >0.5 -8,665 D-2672 >0.5 4.92 D-2673 >0.5 -8,024 D-2675 >0.5 -17,999 D-2676 >0.5 -22,989 D-2677 >0.5 -11,245 D-2678 >0.5 -10,209 D-2679 -46,578 D-2680 >0.5 -12,289 D-2681 >0.5 -9,564 D-2682 0.184 -57,848 D-2683 >0.5 -19,024 D-2684 >0.5 -5,026 D-2685 0,0395 -34,673 D-2686 >0.5 -12,298 D-2687 >0.5 -7,153 D-2688 >0.5 -12,895 D-2689 >0.5 -20,429 D-2690 >0.5 -8,608 D-2691 >0.5 -7,608 D-2692 >0.5 -7,763 D-2693 >0.5 -12,294 D-2694 >0.5 -10,718 D-2695 -25,618 D-2696 >0.5 -7,666 D-2697 >0.5 -11,529 D-2698 >0.5 -11,875 D-2699 >0.5 -4,132 D-2700 -34,399 D-2701 >0.5 -4,434 D-2702 >0.5 -9,919 D-2703 >0.5 -9,667 D-2704 >0.5 -1,989

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D-2705 >0.5 -8.5 D-2706 >0.167 D-2707 >0.167 D-2708 0.103 -32,624 D-2709 >0.5 -2,042 D-2710 >0.5 -5,079 D-2712 >0.5 -1,399 D-2717 >0.5 -1,399 D-2718 0,00521 -38,552 D-2719 0,013 D-2720 >0.5 -2.52 D-2721 >0.5 -3,882 D-2722 >0.5 -6,527 D-2723 >0.5 -16,917 D-2724 0,0227 -46,386 D-2725 0,0187 D-2726 0,0105 D-2727 0,0124 -38,592 D-2728 0,00741 -30,796 D-2729 0,0152 -36.3 D-2730 >0.5 -17,996 D-2731 0,00433 -45,007 D-2732 >0.5 -4,662 D-2733 >0.5 -8,838 D-2734 >0.5 -11,614 D-2735 >0.5 -15,778 D-2736 >0.5 -17,337 D-2737 >0.5 -11,139 D-2717 >0.5 -6,604 D-2738 0,00878 -30,636 D-2739 >0.5 -12,614 D-2740 0,013 -57,307 D-2741 0,00938 -48,272 D-2742 0,0271

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D-2743 0,00623 -41,449 D-2744 0,015 -37,951 D-2745 0,00611 -53,267 D-2746 0,0108 -63,434 D-2747 >0.5 -13,578 D-2748 0,0382 -38.04 D-2749 >0.5 -8,412 D-2750 >0.5 -2.25 D-2751 >0.5 -12,014 D-2752 >0.5 10.68 D-2753 >0.5 -3,443 D-2754 0,0704 -40,897 D-2755 >0.5 -9,405 D-2756 >0.5 -18,327 D-2757 >0.5 -10,531 D-2758 >0.5 -13,929 D-2759 0,0174 -39,948 D-2760 0,048 -66,415 D-2761 >0.5 -14,128 D-2762 0,0132 -61,074 D-2763 0,0333 -67,071 D-2764 >0.5 -10,771 D-2413 -45,832 D-2419 >0.5 -4,545

Example 3. Droplet digital PCR analysis of siRNA for PNPLA3-rs738409 and PNPLA3-rs738409-rs738408.

Following the manufacturer's protocol, thawed primary human hepatocytes (Xenotech/Sekisui donor lot HC3-38) in OptiThaw medium (Cat# K8000 Xenotech) were centrifuged and aspirated, resuspended in OptiPlate Hepatocyte Medium (Cat# K8200 Xenotech) and added to 96-well collagen-coated plates (Cat# Greiner 655950). After a 2-4 hour incubation period, the medium was removed and replaced with OptiCulture Hepatocyte Medium (Cat# K8300 Xenotech). GalNAc-conjugated siRNA was freely taken up by the cells (without transfection reagent) 2-4 hours after addition of OptiCulture medium. Cells were incubated for 24-72 h at 37°C and 5% CO2. Cells were then lysed with Qiagen RLT buffer (79216) + 1% 2-mercaptoethanol (Sigma, M-3148) and lysates were stored at -20°C. RNA purification was performed using a Qiagen QIACube HT instrument (9001793) and a Qiagen RNeasy 96 QIACube HT Kit (74171) according to the manufacturer's instructions. Samples were analyzed using the QIAxpert system (9002340). cDNA was synthesized from RNA samples using the Applied Biosystems cDNA High Throughput Reverse Transcription Kit (4368813), with reactions performed according to the manufacturer's instructions and the concentration of RNA added depending on the sample. Reverse transcription was performed in a BioRad tetrad thermal cycler (model no. PTC-0240G) under the following conditions: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min, followed by an optional indefinite hold at 4°C.

Droplet digital PCR (ddPCR) was performed using the BioRad AutoDG QX200 Droplet Digital PCR System according to the manufacturer's instructions. Reaction mixtures were prepared in a clear 96-well PCR plate from Eppendorf (951020303) using BioRad ddPCR Supermix for probes (1863010) and fluorescently labeled qPCR assay mixes for PNPLA3 (IDT Hs.PT.58.21464637, primer to probe ratio 3.6:1 and TBP (IDT Hs.PT.53a.20105486, primer to probe ratio 3.6:1) and RNase-free water (Ambion, AM9937). The final primer/probe concentration was 900 nM/250 nM, respectively, and the starting cDNA concentration varied among wells. Droplets were generated using a BioRad Auto DG droplet generator (1864101) configured for manufacturer's recommended consumables (BioRad DG32 cartridges 1864108, BioRad tips 1864121, Eppendorf blue 96-well PCR plate 951020362, BioRad probe droplet generation oil 1864110 and droplet assembly

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BioRad). Droplets were amplified on a BioRad C1000 touch thermal cycler (1851197) using the following conditions: enzyme activation at 95°C for 10 min, denaturation at 94°C for 30 sec followed by annealing/elongation at 60°C for 1 min, 40 cycles at a heating/cooling rate of 2°C/sec, enzyme inactivation at 98°C for 10 min followed (optionally) by an indefinite hold at 4°C. Samples were then read on a BioRad QX200 Droplet Reader measuring the FAM/HEX signal, which correlates with the concentration of PNPLA3 or TBP. Data analysis was performed using the BioRad QuantaSoft software package. Samples were gated by channel (fluorescent label) to determine the concentration per sample. Each sample was then expressed as a ratio of the gene of interest (PNPLA3) concentration to the housekeeping gene (TBP) concentration to control for differences in sample loading. The data were then imported into Genedata Screener, where each siRNA tested was normalized to the median values of the neutral control wells (containing buffer only). IC ₅₀ values are presented in Table 7.

Table 7

ddPCR analysis performed on primary hepatocytes

Duplex No. IC50 (µM) % PNPLA3 knockdown D-2068 .0339 -49,628 D-2069 .00408 -52,997 D-2070 .00433 -42,193 D-2072 .00884 -53.16 D-2073 >2.0 -7,435 D-2078 .0044 -43,123 D-2084 0,00499 -38,791 D-2085 0,00539 -64,312 D-2086 >2.0 -14,938 D-2087 >2.0 -25,465 D-2088 0.207 -34,944 D-2089 0,0107 -38,791 D-2090 0,0218 -38,977 D-2091 0,0508 -41,209 D-2092 0,00192 -44 D-2093 0,00634 -30,233 D-2094 >2.0 4.93 D-2095 0,00181 -59,814 D-2096 0,0181 -52,807 D-2099 0,00549 -39,296 D-2100 0,0142 -55,281 D-2158 0,0681 -48,649 D-2159 0,0325 -36,036 D-2160 > 0.667 -13,514 D-2161 >2.0 -24,229 D-2162 0,0726 -28,634 D-2163 > 0.667 -16.3 D-2164 >2.0 -15,418 D-2165 0,00644 -26,872 D-2166 0,00192 -30,045 D-2167 > 0.667 -6,726 D-2168 >2.0 -15,418

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D-2169 >2.0 -13,004 D-2170 >2.0 -9,417 D-2171 0,00505 -44,395 D-2172 0,003 -55,336 D-2173 0,00598 -46,188 D-2174 >2.0 -9,009 D-2175 0,017 -27,928 D-2176 0,00452 -35,426 D-2177 >2.0 4,5 D-2178 >2.0 1,8 D-2179 >2.0 -6,306 D-2180 0,00546 -40,969 D-2181 0,00152 -43,119 D-2182 0,00317 -54,128 D-2183 0,00232 -53,211 D-2184 0,0109 -50,459 D-2185 >2.0 -7,339 D-2186 0,0021 -48,624 D-2187 >2.0 -11,009 D-2188 >2.0 1.32 D-2191 0,0984 -66,923 D-2192 0,124 -64,231 D-2193 0,138 -60,606 D-2194 0,0478 -54,182 D-2195 0,0801 -47,515 D-2199 0,0517 -62,973 D-2201 0,0165 -72,404 D-2202 0,00946 -49,459 D-2203 0,0241 -58,545 D-2204 0,0382 -45,576 D-2205 0,0222 -50,946 D-2206 0,0459 -46,081 D-2209 0,0867 -46,622 D-2212 0.358 -60

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D-2216 0,0826 -58,942 D-2218 0,0242 -63,462 D-2220 0,0113 -69,333 D-2221 0,138 -55,541 D-2224 0,0198 -66,486 D-2225 0.245 -68,077 D-2228 0,155 -44,606 D-2229 0,0651 -38,909 D-2231 0,0512 -56,892 D-2232 0,0678 -67,981 D-2233 0,00802 -57,182 D-2234 0,00473 -55,947 D-2235 0,00816 -62,115 D-2236 0,00245 -51,542 D-2237 0,00495 -60 D-2238 0,00561 -63,017 D-2239 0,00453 -55,537 D-2240 0,00584 -56,116 D-2241 0,00755 -54.76 D-2242 0,0137 -56,332 D-2243 0,00329 -57,118 D-2244 0,0127 -56,909 D-2245 0,00697 -58,364 D-2246 0,00713 -56,828 D-2247 0,00875 -57,797 D-2248 0,0098 -58.59 D-2249 0,00603 -57,759 D-2250 0,0105 -62,155 D-2251 0,00521 -59,914 D-2252 0,00988 -58,678 D-2253 0,00481 -57,118 D-2254 0,00721 -56,332 D-2255 0,00788 -52,838 D-2256 0,00831 -55,455

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D-2257 0,00503 -54,545 D-2258 0,00626 -54,545 D-2259 0,00401 -55,947 D-2260 0,00379 -52,423 D-2261 0,00151 -54.31 D-2262 0,00292 -53,448 D-2263 0,00607 -59,483 D-2264 0,00703 -59,504 D-2265 >4.0 -20,524 D-2266 0,0129 -32,727 D-2267 0,107 -27,273 D-2275 0,00359 -45,701 D-2276 0,00416 -43,891 D-2277 0,00218 -49.14 D-2278 0,00743 -42,986 D-2279 0,0116 -53,846 D-2280 0,00347 -36,652 D-2281 0,0134 -37,557 D-2282 0,00864 -34,842 D-2283 0,00738 -49,558 D-2284 0,0202 -38,053 D-2285 0,00543 -45,487 D-2286 0,00934 -47,611 D-2287 0,00652 -55,575 D-2288 0,0259 -61,593 D-2289 0,00549 -53,805 D-2290 0,00476 -51,062 D-2291 0,0105 -42,584 D-2292 0,0059 -45,455 D-2293 0,0117 -45,646 D-2294 0,0109 -52,823 D-2295 0.01246 +/- 0.015 -59.847 +/- 15.2 D-2296 0,0279 -41,346 D-2297 0,00529 -55,926

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D-2298 0,00838 -35,577 D-2299 0,00832 -40,865 D-2300 0,00371 -40,096 D-2301 0,00563 -38,365 D-2302 0,00639 -40,385 D-2303 0,00669 -41.25 D-2304 0,00212 -38,462 D-2305 0,00573 -37,736 D-2306 0,0645 -33,962 D-2307 0,00232 -33,019 D-2308 0,00181 -31,132 D-2309 0,0447 -45,283 D-2310 0,00655 -42,453 D-2311 0,00613 -41,509 D-2312 0,00941 -50 D-2313 0,0218 -41,784 D-2314 0,0142 -42,723 D-2315 0,0182 -31,455 D-2316 >4.0 -23,005 D-2317 0,0228 -37,089 D-2318 0,00809 -45,352 D-2319 0,0165 -44,601 D-2320 0,0184 -38,373 D-2321 0,00766 -41,627 D-2322 0,00815 -46,507 D-2323 0,0168 -48,325 D-2324 0,00663 -62,254 D-2325 0,00716 -39,367 D-2326 0,044 -52,036 D-2327 0,00282 -65,701 D-2328 0,00411 -53,991 D-2329 0,012 -48,357 D-2330 0,019 -42,593 D-2331 0,00448 -47,418 D-2332 0,00944 -37,327 D-2333 0,00514 -37,327 D-2334 0.154 -38,249 D-2335 0,0089 -40,092 D-2336 0,0169 -48,148 D-2337 0,00274 -46,296 D-2338 0,0225 -42,723 D-2339 0.00222 +/- 0.00136 -43.218 +/- 4.81 D-2340 0,0136 -56,561 D-2341 0,0222 -50,226 D-2342 0,0273 -55,385 D-2343 0,0164 -41,784 D-2344 0,0314 -60,282 D-2345 0,0103 -65.1 D-2346 0,0427 -63.9 D-2347 0,00446 -49.4 D-2348 0,113 -53.4 D-2349 0,0176 -56.3 D-2350 0,00467 -49.4

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Example 4. Screening the efficacy of selected PNPLA3 siRNA molecules in a humanized mouse model.

Adeno-associated virus (AAV; serotype AAV8 or AAV7; endotoxin-free, Amgen) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) to levels not exceeding 4e11-1e12 viral particles per animal was injected intravenously into the tail vein of male C57BL/6NCrl mice (Charles River Laboratories Inc.) to stimulate expression of either human PNPLA3 ^WT (PNPLA3-WT), PNPLA3 ^rs738409 (PNPLA3-I148M), or PNPLA3 ^{rs738409-rs738408} (PNPLA3-I148M DM) in the liver. Mice were on average 10-12 weeks old and n=4-6 animals per group. Each screening step included at least two control groups treated with vehicle: empty AAV vector and AAV-PNPLA3 ^WT or PNPLA3 ^rs738409 and PNPLA3 rs738409-rs738408 treated ^with vehicle. All siRNAs were tested for functionality against AAV-PNPLA3 ^{WT ,} PNPLA3 ^rs738409 and/or ^{PNPLA3 rs738409 -rs738408 .} Two weeks after AAV injection, mice were treated with a single dose of D-2324 siRNA (0.5 mM) via subcutaneous injection at a dose of 0.5, 1.0, 3.0, or 5.0 mg/kg animal diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). On days 8, 15, 22, 28, or 42 after siRNA injection, liver tissues were collected from animals, snap-frozen in liquid nitrogen, processed for purified RNA extraction using the QIACube HT instrument (Qiagen, 9001793) and the RNeasy 96 QIACube HT kit (Qiagen, 74171) according to the manufacturer's instructions. Samples were analyzed using the QIAxpert system (Qiagen, 9002340). RNA was treated with RNase-free RQ1 DNase (Promega, M6101) and prepared for Real-Time qPCR using the RNA-to-C _T ™ 1-Step kit TaqMan™ (Applied Biosystems, 4392653). Real-Time qPCR was performed on a QuantStudio Real-Time PCR instrument. Results were calculated from human PNPLA3 expression normalized to mouse Gapdh (Invitrogen TaqMan™ kits, hs00228747_m1 and 4352932E, respectively) and are presented as relative knockdown of human PNPLA3 mRNA expression compared to vehicle-treated controls. For comparison, endogenous expression of the mouse Pnpla3 gene was determined (Invitrogen, Mm00504420_m1).

For liver triglyceride analysis, approximately 0.05–0.1 mg of frozen liver collected from an animal was homogenized in 1 mL of isopropanol. After 1 h of incubation on ice, samples were centrifuged at 10,000 rpm in a microcentrifuge and the supernatants were transferred to a clean 96-well deep-well plate. Triglyceride content was determined using Infinity Triglyceride Reagent (Thermo Fisher Scientific, TR22421) and Triglyceride Standard (Pointe Scientific, T7531-STD) according to the manufacturer's instructions. Results are presented as milligrams of triglyceride per milligram of tissue.

Fig. 1A-1D. Examples of five siRNA molecules that were screened for both dose-dependent mRNA knockdown and functional stability in vivo. Mice expressing human PNPLA3 ^{rs738409-rs738408} were treated with siRNA two weeks after intravenous injections of AAV. N=6 mice per group; data are presented as mean and standard error of the mean. (A) siRNA was administered at 0.5, 1.0, 3.0, or 5.0 mg/kg body weight subcutaneously into the abdomen of the mouse. Four weeks after siRNA treatment, mice were sacrificed and liver tissues were collected and processed for gene expression analysis. Data represent the average relative knockdown of human PNPLA3 ^rs 7 ³⁸ 4 ^09-rs 7 ³⁸ 4 ⁰⁸ in each group of mice compared to the vehicle-treated control group. (B) Liver tissues collected from the same four-week treatment group were also processed for triglyceride content to assess the functional efficacy of the agent. Data represent the average triglyceride content expressed as milligrams per gram of treated tissue. (C) siRNA was administered at 1.0 and 3.0 mg/kg body weight subcutaneously in the abdomen of parallel cohort animals. Tissue was collected from mice six weeks after siRNA administration to compare the stability of the siRNA molecules in vivo. Livers were collected and processed for gene expression analysis. Data represent the average relative knockdown of human PNPLA3 ^rs 7 ³⁸ 4 ^09,-rs 7 ³⁸ 4 ⁰⁸ in each group of mice compared to vehicle-treated controls. (D) Liver tissues isolated from the same six-week-treated group were also processed for triglyceride content to assess functional efficacy over time. Data represent average triglyceride content expressed as milligrams per gram of treated tissue.

Relative knockdown data are shown in Tables 8–12 and show relative knockdown at days 8, 15, 22, 28, and 42, respectively, and across doses. PNPLA3 knockdown is expressed as a percentage with negative values indicating decreased PNPLA3 levels.

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Day 8, PNPLA3 knockdown assay

Table 8

Duplex number AAV based vector Particles AAV/animal Administered dose (mg/kg) Knockdown PNPLA3 (%) D-2092 PNPLA3-I148M 1.00E+12 3 -91.51 D-2092 PNPLA3-I148M 1.00E+12 5 -92.64 D-2095 PNPLA3-I148M 1.00E+12 3 -79.21 D-2095 PNPLA3-I148M 1.00E+12 5 -86.38 D-2068 PNPLA3-I148M 4.00E+11 5 -34.15 D-2069 PNPLA3-I148M 4.00E+11 5 -82.74 D-2070 PNPLA3-I148M 4.00E+11 5 -79.64 D-2071 PNPLA3-I148M 4.00E+11 5 -48.80 D-2071 PNPLA3-WT 4.00E+11 5 -59.43 D-2075 PNPLA3-I148M 4.00E+11 5 -79.10 D-2075 PNPLA3-WT 4.00E+11 5 -60,60 D-2079 PNPLA3-I148M 4.00E+11 5 -50.08 D-2072 PNPLA3-I148M 4.00E+11 5 -44.40 D-2072 PNPLA3-WT 4.00E+11 5 -43.75 D-2077 PNPLA3-I148M 4.00E+11 5 -28.46 D-2076 PNPLA3-I148M 4.00E+11 5 -83.25 D-2078 PNPLA3-I148M 4.00E+11 5 -49.10 D-2078 PNPLA3-WT 4.00E+11 5 -3.45 D-2073 PNPLA3-I148M 4.00E+11 5 -39.42 D-2074 PNPLA3-I148M 4.00E+11 5 -46.68 D-2074 PNPLA3-WT 4.00E+11 5 -5.11

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D-2084 PNPLA3-I148M 4,00E+ll 5 -87.49 D-2084 PNPLA3-I148M 8.00E+ll 5 -88.75 D-2084 PNPLA3-I148M 8.00E+ll 1 -16.49 D-2084 PNPLA3-WT 8.00E+ll 1 -15.42 D-2085 PNPLA3-I148M 4,00E+ll 5 -77.14 D-2085 PNPLA3-T148M 8.00E+l 1 5 -84.42 D-2085 PNPLA3-I148M 8.00E+ll 1 -25.19 D-2085 PNPLA3-WT 8.00E+ll 1 -21.14 D-2086 PNPLA3-I148M 4,00E+ll 5 -64.54 D-2086 PNPLA3-I148M 4,00E+ll 5 -52.95 D-2087 PNPLA3-I148M 4,00E+ll 5 -20.15 D-2088 PNPLA3-I148M 4,00E+ll 5 -47.18 D-2088 PNPLA3-I148M 8.00E+ll 5 -66.96 D-2089 PNPLA3-I148M 8.00E+ll 5 -85.47 D-2089 PNPLA3-WT 8.00E+ll 1 -21.01 D-2089 PNPLA3-I148M 8.00E+ll 1 -34.21 D-2089 PNPLA3-I148M l,00E+12 5 -90.55 D-2090 PNPLA3-I148M 8.00E+ll 5 -89.58 D-2090 PNPLA3-I148M 8.00E+ll 1 -50.13 D-2090 PNPLA3-WT 8.00E+ll 1 8.76 D-2091 PNPLA3-WT 8.00E+ll 5 -35.70 D-2092 PNPLA3-I148M 8.00E+ll 5 -92.34 D-2092 PNPLA3-I148M 8.00E+ll 1 -51.35 D-2092 PNPLA3-WT 8.00E+ll 1 -42.88 D-2093 PNPLA3-I148M 8.00E+ll 5 -83.87 D-2094 PNPLA3-I148M 8.00E+ll 5 -70.12 D-2095 PNPLA3-T148M 8.00E+11 1 -29.95 D-2095 PNPLA3-WT 8.00E+ll 1 67.40 D-2095 PNPLA3-I148M 8.00E+ll 5 -85.42 D-2096 PNPLA3-WT 8.00E+ll 5 -90.44 D-2081 PNPLA3-I148M 8.00E+ll 5 6.25 D-2081 PNPLA3-I148M 8.00E+ll 5 -11.81 D-2097 PNPLA3-I148M 8.00E+ll 5 -87.61 D-2098 PNPLA3-I148M 8.00E+ll 5 -79.84

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D-2099 PNPLA3-I148M 8.00E+ll 5 -84.36 D-2100 PNPLA3-I148M 8.00E+ll 5 -79.67 D-2101 PNPLA3-I148M 8.00E+ll 5 -89.64 D-2102 PNPLA3-I148M 8.00E+ll 5 -61.49 D-2103 PNPLA3-I148M 8.00E+ll 5 -19.65 D-2104 PNPLA3-T148M 8,00E+l1 5 -79.70 D-2104 PNPLA3-I148M Ι,ΟΟΕ+12 5 -82.87 D-2105 PNPLA3-I148M 8.00E+ll 5 -84.49 D-2105 PNPLA3-I148M Ι,ΟΟΕ+12 5 -87.71 D-2152 PNPLA3-I148M Ι,ΟΟΕ+12 5 -39.35 D-2153 PNPLA3-I148M Ι,ΟΟΕ+12 5 -79.04 D-2154 PNPLA3-I148M Ι,ΟΟΕ+12 5 -66.72 D-2155 PNPLA3-I148M Ι,ΟΟΕ+12 5 -44.68 D-2156 PNPLA3-I148M Ι,ΟΟΕ+12 5 -84.72 D-2157 PNPLA3-I148M Ι,ΟΟΕ+12 5 -17.25 D-2280 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -99.70 D-2280 PNPLA3 WT Ι,ΟΟΕ+12 5 -51.13 D-2295 PNPLA3-WT Ι,ΟΟΕ+12 5 -35.90 D-2295 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -94.68 D-2296 PNPLA3-WT Ι,ΟΟΕ+12 5 -23.24 D-2296 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -92.78 D-2297 PNPLA3-WT Ι,ΟΟΕ+12 5 43.71 D-2297 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -94.59 D-2324 PNPLA3-WT Ι,ΟΟΕ+12 5 6.53 D-2324 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -97.39 D-2326 PNPLA3-WT Ι,ΟΟΕ+12 5 -8.25 D-2326 PNPLA3-T148M DM Ι,ΟΟΕ+12 5 -77.82 D-2328 PNPLA3-WT Ι,ΟΟΕ+12 5 -2.12 D-2328 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -92.49

Table 9

Day 15, PNPLA3 knockdown assay

Duplex number AAV based vector Particles AAV/animal Administered dose (mg/kg) Knockdown PNPLA3 (%) D-2092 PNPLA3-H48M Ι,ΟΟΕ+12 3 -87.58 D-2092 PNPLA3-T148M Ι,ΟΟΕ+12 5 -93.96

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D-2095 PNPLA3-I148M l,00E+12 3 -81.73 D-2095 PNPLA3-I148M l,00E+12 5 -72.99 D-2131 PNPLA3-I148M l,00E+12 5 -66.02 D-2186 PNPLA3-I148M l,00E+12 5 -87.67 D-2089 PNPLA3-I148M l,00E+12 5 -95.01 D-2104 PNPLA3-I148M 1.00E+12 5 -76.01 D-2105 PNPLA3-I148M l,00E+12 5 -72.67 D-2123 PNPLA3-I148M l,00E+12 5 -56.93 D-2128 PNPLA3-I148M l,00E+12 5 -37.26 D-2138 PNPLA3-I148M l,00E+12 5 -62.16 D-2149 PNPLA3-I148M 1.00E+12 5 -72.34 D-2156 PNPLA3-I148M l,00E+12 5 -75.81 D-2259 PNPLA3-I148M l,00E+12 5 -79.15 D-2260 PNPLA3-I148M l,00E+12 5 -69.97 D-2261 PNPLA3-I148M l,00E+12 5 -50.40 D-2262 PNPLA3-I148M l,00E+12 5 -84.36 D-2263 PNPLA3-I148M l,00E+12 5 -77.08 D-2264 PNPLA3-I148M l,00E+12 5 -40.86 D-2269 PNPLA3-I148M l,00E+12 5 -37.55 D-2270 PNPLA3-I148M l,00E+12 5 -77.42 D-2271 PNPLA3-I148M Ι,ΟΟΕ+12 5 -26.87 D-2272 PNPLA3-I148M l,00E+12 5 -56.05 D-2280 PNPLA3-I148M DM l,00E+12 3 -86.30 D-2287 PNPLA3-I148M DM l,00E+12 3 -94.73 D-2289 PNPLA3-I148M DM l,00E+12 3 -93.48 D-2292 PNPLA3-I148M DM l,00E+12 3 -82.48 D-2297 PNPLA3-T148M DM 1.00E+12 3 -72.61 D-2322 PNPLA3-I148M DM l,00E+12 3 -32.92 D-2324 PNPLA3-I148M DM l,00E+12 3 -87.56 D-2327 PNPLA3-I148M DM l,00E+12 3 -86.70 D-2345 PNPLA3-WT l,00E+12 5 -83.48 D-2346 PNPLA3-WT Ι,ΟΟΕ+12 5 -75.93 D-2347 PNPLA3-WT l,00E+12 5 -22.83 D-2348 PNPLA3-WT l,00E+12 5 -20.09

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D-2349 PNPLA3-WT Ι,ΟΟΕ+12 5 -67.70 D-2350 PNPLA3-WT Ι,ΟΟΕ+12 5 -57.51 D-2351 PNPLA3-WT Ι,ΟΟΕ+12 5 -56.11 D-2352 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -92.21 D-2353 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -89.55 D-2354 PNPLA3-T148M DM Ι,ΟΟΕ+12 3 -90.21 D-2358 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -95.10 D-2359 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -91.17 D-2360 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -63.98 D-2361 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -92.47 D-2362 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -95.10 D-2364 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -95.31 D-2370 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -95.99 D-2370 PNPLA3 WT Ι,ΟΟΕ+12 3 11.88 D-2371 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -91.14 D-2372 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -93.71 D-2373 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -73.80 D-2374 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -75.98 D-2375 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -96.68 D-2376 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -96.78 D-2377 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -96.88 D-2378 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -97.60 D-2379 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -94.73 D-2380 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -94.73 D-2381 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -76.24 D-2382 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -80.33 D-2383 PNPLA3-T148M DM Ι,ΟΟΕ+12 3 -71.98 D-2384 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -87.24 D-2385 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -78.77 D-2386 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -70.58 D-2387 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -67.09 D-2390 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -92.97 D-2391 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -94.10 D-2392 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -92.14

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D-2395 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -85.63 D-2396 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -94.63 D-2397 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -92.90 D-2398 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -95.48 D-2399 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -93.40 D-2400 PNPLA3-T148M DM 1.00E+12 3 -97.55 D-2401 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -96.98 D-2402 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -97.25 D-2403 PNPLA3 WT Ι,ΟΟΕ+12 3 36.90 D-2404 PNPLA3-I148M DM l,00E+12 3 -95.08 D-2405 PNPLA3-I148M DM l,00E+12 3 -96.93 D-2406 PNPLA3 WT Ι,ΟΟΕ+12 3 28,80 D-2395 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -17.25 D-2396 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -93.70 D-2413 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -94.80 D-2415 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -90.95 D-2418 PNPLA3-I148M DM l,00E+12 3 -88.45 D-2419 PNPLA3 WT Ι,ΟΟΕ+12 3 43.72 D-2453 PNPLA3 WT Ι,ΟΟΕ+12 3 -81.33 D-2454 PNPLA3 WT l,00E+12 3 -83.38 D-2455 PNPLA3 WT Ι,ΟΟΕ+12 3 -68.85 D-2456 PNPLA3 WT Ι,ΟΟΕ+12 3 -89.03 D-2460 PNPLA3 WT Ι,ΟΟΕ+12 3 -68.65 D-2461 PNPLA3 WT Ι,ΟΟΕ+12 3 -47.85

Table 10

Day 22, PNPLA3 knockdown assay

Number of duplex AAV based vector Particles AAV/animal Administered dose (mg/kg) Knockdown PNPLA3 (%) D-2092 PNPLA3-H48M Ι,ΟΟΕ+12 3 -74.61 D-2092 PNPLA3-I148M Ι,ΟΟΕ+12 5 -85.78 D-2095 PNPLA3-I148M Ι,ΟΟΕ+12 3 -27.78 D-2095 PNPLA3-I148M Ι,ΟΟΕ+12 5 -33.60 D-2324 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -56.68 D-2324 PNPLA3-H48M DM Ι,ΟΟΕ+12 5 -88.02 D-2089 PNPLA3-I148M Ι,ΟΟΕ+12 5 -80.86 D-2104 PNPLA3-H48M Ι,ΟΟΕ+12 5 -65.61 D-2105 PNPLA3-I148M Ι,ΟΟΕ+12 5 -39.67 D-2280 PNPLA3-H48M DM Ι,ΟΟΕ+12 5 -84.12 D-2297 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -58.01 D-2297 PNPLA3-H48M DM Ι,ΟΟΕ+12 5 -76.43 D-2352 PNPLA3-T148M DM Ι,ΟΟΕ+12 5 -92.74 D-2353 PNPLA3-H48M DM Ι,ΟΟΕ+12 5 -84.54 D-2354 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -89.06 D-2355 PNPLA3-I148M DM Ι,ΟΟΕ+12 5 -95.53 D-2356 PNPLA3-H48M DM Ι,ΟΟΕ+12 5 -94.99 D-2357 PNPLA3-H48M DM Ι,ΟΟΕ+12 5 -97.37

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Day 28, PNPLA3 knockdown assay

Table 11

Duplex number AAV based vector Particles AAV/animal Administered dose (mg/kg) Knockdown PNPLA3 (%) D-2092 PNPLA3-H48M Ι,ΟΟΕ+12 3 -72.57 D-2092 PNPLA3-I148M Ι,ΟΟΕ+12 5 -82.74 D-2095 PNPLA3-H48M Ι,ΟΟΕ+12 3 -32.93 D-2095 PNPLA3-H48M Ι,ΟΟΕ+12 5 -55.31 D-2089 PNPLA3-H48M Ι,ΟΟΕ+12 5 -63.49 D-2104 PNPLA3-I148M Ι,ΟΟΕ+12 5 -44.39 D-2105 PNPLA3-I148M Ι,ΟΟΕ+12 5 -39.80 D-2370 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -89.28 D-2400 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -88.23 D-2401 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -91.95 D-2402 PNPLA3-I148M DM Ι,ΟΟΕ+12 0,5 -50.65 D-2402 PNPLA3-H48M DM Ι,ΟΟΕ+12 1 -69.37 D-2402 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -96.43 D-2402 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -85.44 D-2402 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -90.52 D-2404 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -95.67 D-2419 PNPLA3-H48M DM Ι,ΟΟΕ+12 0,5 -68.83 D-2419 PNPLA3-H48M DM Ι,ΟΟΕ+12 1 -76.88 D-2419 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -97.53 D-2419 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -93.95

- 195 047656

D-2419 PNPLA3-I148M DM l,00E+12 3 -89.66 D-2419 PNPLA3-I148M DM l,00E+12 3 -93.25 D-2420 PNPLA3-I148M DM l,00E+12 3 -95.93 D-2421 PNPLA3-I148M DM l,00E+12 0,5 -50.01 D-2421 PNPLA3-1148M DM l,00E+12 1 -66.30 D-2421 PNPLA3-I148M DM 1.00E+12 3 -94.99 D-2421 PNPLA3-I148M DM l,00E+12 3 -80.05 D-2421 PNPLA3 WT l,00E+12 3 -36.65 D-2421 PNPLA3-I148M DM l,00E+12 3 -91.08 D-2425 PNPLA3-I148M DM l,00E+12 3 -16.07 D-2426 PNPLA3-I148M DM 1.00E+12 3 -37.54 D-2427 PNPLA3-I148M DM l,00E+12 3 -25.19 D-2428 PNPLA3-I148M DM l,00E+12 3 -16.71 D-2437 PNPLA3-I148M DM l,00E+12 3 -93.78 D-2438 PNPLA3-I148M DM l,00E+12 3 -90.63 D-2439 PNPLA3-I148M DM 1.00E+12 3 -88.10 D-2440 PNPLA3-I148M DM l,00E+12 3 -95.25 D-2441 PNPLA3-I148M DM l,00E+12 3 -90.13 D-2442 PNPLA3-I148M DM l,00E+12 3 -57.24 D-2443 PNPLA3-I148M DM l,00E+12 3 -95.43 D-2444 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -90.58 D-2445 PNPLA3-I148M DM l,00E+12 3 -84.55 D-2446 PNPLA3-I148M DM l,00E+12 3 -81.50 D-2427 PNPLA3-I148M DM l,00E+12 3 -90.09 D-2462 PNPLA3-I148M DM l,00E+12 3 -89.20 D-2463 PNPLA3-1148M DM l,00E+12 3 -41.25 D-2464 PNPLA3-I148M DM l,00E+12 3 -60.53 D-2465 PNPLA3-I148M DM l,00E+12 3 -91.35 D-2466 PNPLA3-I148M DM l,00E+12 3 -93.68 D-2467 PNPLA3-I148M DM l,00E+12 3 -85.15 D-2472 PNPLA3-I148M DM l,00E+12 0,5 -46.58 D-2472 PNPLA3-I148M DM Ι,ΟΟΕ+12 1 -74.47 D-2472 PNPLA3-I148M DM l,00E+12 3 -88.79 D-2472 PNPLA3 WT l,00E+12 3 -23.16

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D-2472 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -90.54 D-2473 PNPLA3-H48M DM l,00E+12 0,5 -57.95 D-2473 PNPLA3-I148M DM l,00E+12 1 -71.96 D-2473 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -91.70 D-2473 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -85.94 D-2473 PNPLA3 WT 1.00E+12 3 -18.70 D-2675 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -25.9 D-2677 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -3,807 D-2678 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -31.76 D-2679 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -88.48 D-2680 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -56.44 D-2681 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -35.71 D-2682 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -71.43 D-2683 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -68.62 D-2685 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -29.93 D-2687 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -75.05 D-2689 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -81.12 D-2690 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -79.08 D-2694 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -4,531 D-2695 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 29.23 D-2668 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -65.16 D-2669 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -78.36 D-2670 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -56.05 D-2671 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -56.87

Table 12

Day 42, PNPLA3 knockdown assay

Duplex number AAV based vector Particles AAV/animal Administered dose (mg/kg) Knockdown PNPLA3 (%) D-2402 PNPLA3-H48M DM Ι,ΟΟΕ+12 1 -57.74 D-2402 PNPLA3-H48M DM Ι,ΟΟΕ+12 3 -83.75 D-2419 PNPLA3-H48M DM Ι,ΟΟΕ+12 1 -71.07 D-2419 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -70.8 D-2421 PNPLA3-I148M DM Ι,ΟΟΕ+12 1 -62.21 D-2421 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -80.12 D-2472 PNPLA3-T148M DM Ι,ΟΟΕ+12 1 -60.54 D-2472 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -74.77 D-2473 PNPLA3-I148M DM Ι,ΟΟΕ+12 1 -54.55 D-2473 PNPLA3-I148M DM Ι,ΟΟΕ+12 3 -81.13

Example 5. Prevention of occurrence and rescue of NAFLD by application of siRNA molecules in a humanized mouse model with PNPLA3 ^{rs738409-rs738408} .

The NAFLD/NASH American Lifestyle-Induced Obesity Syndrome or ALIOS model was developed by feeding mice a diet high in trans fat (45% of total fat) and sugar (Tetri 2008). For these studies, eight- to ten-week-old male C57BL/6NCrl mice (Charles River Laboratories Inc.) were injected with either the empty AAV vector or the AAV8-PNPLA3 ^{rs738409-rs738408} vector as described previously. At the time of AAV injection, mice were either maintained on normal chow or were fed the ALIOS diet (Envigo, TD.06303) in combination with drinking water supplemented with a mixture of 55% fructose and 45% glucose (Sigma, F0127 and G7021, respectively) until tissue collection. Previous experiments have shown that overexpression of PNPLA3 ^{rs738409-rs738408} in this context both accelerates and worsens NAFLD phenotypes (data not shown).

Two weeks after AAV injection and initiation of the indicated diet, mice were treated with a single subcutaneous injection of siRNA D-2324 (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) at 5.0 mg/kg animal or vehicle control. Dosing was repeated every two weeks until tissue collection.

- 197 047656 At the time of tissue collection, body weights were recorded, serum was collected by cardiac puncture under isoflurane anesthesia, and liver weights were recorded. The median lobe was fixed with 10% neutral buffered formalin, processed, and embedded in paraffin. The remainder of the liver was snap frozen for content and gene expression analysis as previously described.

Snap-frozen liver tissue was processed for RNA and gene expression analysis as previously described. Results are shown for both raw Ct value and relative mRNA expression of the indicated gene normalized to mouse Gapdh. (Invitrogen TaqMan™ Assays: human PNPLA3, hs00228747_m1; mouse Pnpla3, Mm00504420_m1; mouse Gapdh, 4352932E).

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

Serum analysis included analysis of TIMP1, a biomarker associated with NASH, and NASH-associated fibrosis (Youssani 2011). TIMP1 ELISA (R&D Systems, M™100) was performed according to the manufacturer's instructions.

Fig. 2A-2G. To assess the ability of the siRNA molecule D-2324, specific for PNPLA3 ^rs738409 ' rs738408, to prevent the development of phenotypes associated with NAFLD and overexpression

PNPLA3 ^{rs738409-rs738408} , mice were injected with the AAV8 empty vector (EV) or the AAV8-PNPLA3 ^{rs738409-rs738408} vector or vehicle and maintained on standard chow or switched to the ALIOS diet. Two weeks after AAV injections, mice were treated with siRNA or vehicle every other week for six weeks for a total of three injection cycles. Tissue was collected from mice at the eight-week time point. Results are presented as group mean and standard error, with N=8 per group. Asterisks denote statistical significance for the AAV8-PNPLA3 ^{rs738409-rs738408} cohort, which was calculated by one-way analysis of variance with Dunnett’s multiple comparison test. (A) Ratio of liver weight (gram) to body weight (gram) at tissue collection. 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 liver by qPCR. (Left) Raw Ct value and (Right) relative fold expression of mRNA normalized to mouse Gapdh; comparison between ALIOS diet-fed groups, namely PNPLA3 ^{rs738409-rs738408} +vehicle and PNPLA3 ^{rs738409-rs738408} +siRNA. (C) qPCR analysis of mouse Pnpla3 mRNA expression in liver indicates that the expression level of endogenous Pnpla3 is not significantly altered by AAV-mediated overexpression or siRNA-induced silencing. (Left) Raw Ct value and (Right) relative fold expression of mRNA normalized to mouse Gapdh comparing the standard diet without AAV group with the ALIOS diet, namely PNPLA3 ^{rs738409-rs738408} +vehicle and PNPLA3 ^{rs738409-rs738408} +siRNA groups. (D) Liver triglyceride content is expressed as milligrams of triglyceride per gram of liver tissue. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, *=0.0393, AAV-PNPLA3 ^{rs738409-rs738408} +siRNA, **=0.0063. (E) Serum TIMP1 levels are expressed as picograms per milliliter of serum. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, ****<0.0001. (F) Histologic evidence of steatosis by hematoxylin and eosin staining, scored as: within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAVPNPLA3 ^{rs738409-rs738408} +siRNA, **=0.0012. (G) Histologic evidence of inflammation by hematoxylin and eosin staining, scored as: within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle group, ****<0.0001, AAV-PNPLA3 ^{rs738409rs738408} +siRNA, ****<0.0001.

Fig. 3A-3G. To assess the ability of the PNPLA3 ^{rs738409-rs738408} -specific siRNA molecule to prevent further progression of PNPLA3 ^{rs738409-rs738408} -mediated disease, mice were injected with an AAV8 empty vector (EV) or an AAV8-PNPLA3 ^{rs738409-rs738408} vector or vehicle after disease onset and maintained on a standard chow or switched to the ALIOS diet. Eight weeks after the AAV injections and diet changes, mice were treated with siRNA or vehicle biweekly for an additional eight weeks for a total of four injection cycles. Mice were harvested for tissue at the sixteen-week time point. Although no changes in steatosis were observed when siRNA treatment was initiated after disease induction, several other disease-associated endpoints were significantly reduced. Results are presented as means and standard errors, with N=8 per group. Asterisks indicate statistical significance for the AAV8-PNPLA3 ^{rs738409-rs738408} cohort, which was calculated by one-way ANOVA with Dunnett's multiple comparison test. (A) Ratio of liver weight (grams) to body weight (grams) at tissue collection. 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 liver by qPCR. (Left) Raw Ct value and (right) relative fold expression of mRNA normalized to mouse Gapdh; comparison between ALIOS diet-fed groups, namely PNPLA3 ^{rs738409-rs738408} +vehicle and PNPLA3 ^{rs738409-rs738408} +siRNA. (C) qPCR analysis of mouse Pnpla3 mRNA expression in liver indicates that the expression level of endogenous Pnpla3 is not significantly altered by AAV-mediated overexpression or siRNA-induced silencing. (Left) Raw Ct value and (Right) relative fold expression of mRNA normalized to mouse Gapdh comparing the standard diet without AAV group with the ALIOS diet, namely PNPLA3 ^{rs738409-rs738408} +vehicle and PNPLA3 ^{rs738409-rs738408} +siRNA groups. (D) Liver triglyceride content is presented as milligrams of triglyceride per gram of liver tissue. Adjusted P-values: AAV-EV+vehicle, not significant, AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, *=0.0403. (E) Serum TIMP1 levels are expressed as picograms per milliliter of serum. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, **=0.0027, AAV-PNPLA3 ^{rs738409rs73840} 8+siRNA, **=0.002. (F) Histologic evidence of steatosis by hematoxylin and eosin staining, scored as: within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, not significant. (G) Histological evidence of inflammation by hematoxylin and eosin staining, scored as: within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, **=0.0068.

Fig. 4A-4D. To assess the ability of the PNPLA3 ^{rs738409-rs738408} -specific siRNA molecule to rescue disease-associated phenotypes caused by PNPLA3 ^{rs738409-rs738408} overexpression, livers and serum from ALIOS diet-fed mice treated with AAV8-PNPLA3 ^{rs738409-rs738408} plus vehicle for eight weeks were compared with livers and serum from AAV8-PNPLA3 ^{rs738409-rs738408} -fed ALIOS diet-fed mice treated with siRNA for sixteen weeks. Although no changes in steatosis were observed at this time point with siRNA treatment, liver triglyceride levels, serum TIMP1, and inflammation were statistically lower at the sixteen-week time point compared to the eight-week vehicle-treated control groups. Results are presented as mean values and standard errors, with N=8 per group. Asterisks denote statistical significance for the eight-week AAV8-PNPLA3 ^{rs738409-rs738408} with vehicle cohort, which was calculated by one-way ANOVA with Dunnett’s multiple comparisons test. (A) Liver triglyceride content is presented as milligrams of triglyceride per gram of liver tissue. Adjusted P-values: 16 weeks AAV-PNPLA3 ^{rs738409-rs738408} + vehicle, not significant; 16 weeks AAV-PNPLA3 ^{rs738409-rs738408} +siRNA, **= 0.0011. (B) Serum Timp1 levels are expressed as picograms per milliliter of serum. Adjusted P-values: 16 weeks AAV-PNPLA3 ^{rs738409-rs738408} +vehicle, not significant; 16 weeks AAV-PNPLA3 ^{rs738409-rs738408} +siRNA, *= 0.0134. (C) Histologic evidence of steatosis by hematoxylin and eosin staining, scored as: within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P-values: 16 weeks AAV-PNPLA3 ^{rs738409-rs738408} +vehicle, not significant; 16 weeks AAV-PNPLA3 ^{rs738409-rs738408} +siRNA, not significant. (D) Histologic evidence of inflammation by hematoxylin and eosin staining, scored as: within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P-values: 16 weeks AAV-PNPLA3 ^{rs738409-rs738408} +vehicle, not significant; 16 weeks AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, *=0.0112.

Example 6. Prevention of liver fibrosis by using siRNA molecules in a humanized mouse model with PNPLA3 ^{rs738409-rs738408} .

The AMLN diet, developed by Amylin Pharmaceuticals (Clapper 2013), is a modified version of the ALIOS diet. The diet contains ten-fold increased cholesterol (2%) and additional sucrose. Mice fed the AMLN diet developed mild to moderate fibrosis after 20 to 30 weeks (Clapper, Mells and Kristiansen). For these studies, eight- to ten-week-old male C57BL/6NCrl mice (Charles River Laboratories Inc.) were injected with either an empty AAV vector or an AAV-PNPLA3 ^{rs738409-rs738408} vector, as described previously, to accelerate disease onset. During AAV injection, mice were maintained on standard chow or were fed the Envigo diet

TD.170748 in combination with drinking water supplemented with a mixture of 55% fructose and 45% glucose (Sigma,

F0127 and G7021, respectively), until killed.

Two weeks after AAV injection and initiation of the indicated diet, mice were treated with a single subcutaneous injection of siRNA D-2324 (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136) at 5.0 mg/kg or vehicle control. Dosing was repeated every two weeks until tissue collection. Body weights were recorded at tissue collection, serum was collected by cardiac puncture under isoflurane anesthesia, and liver weights were recorded. The median lobe was fixed with 10% neutral buffered formalin, processed, and embedded in paraffin. The remainder of the liver was snap frozen for gene expression analysis.

Snap-frozen liver tissue was processed for RNA and gene expression analysis as described previously. Results are shown as both raw Ct value and relative mRNA expression of the indicated gene normalized to mouse Gapdh. (Invitrogen TaqMan™ Assays: 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).

Formalin-fixed tissues were processed for hematoxylin and eosin and Masson's trichrome staining (Dako, CS70030-2, CS70130-2, AR17311-2, respectively) according to the manufacturer's instructions. Smooth muscle actin staining was performed without antigen retrieval and using a DAKO automated stainer. Sections were processed with peroxidase-1 and Sniper (Biocare, PX968 and BS966, respectively) and stained with monoclonal alpha-smooth muscle actin antibodies (Sigma, F3777), followed by rabbit anti-FITC antibody (Invitrogen, 711900), Envision-Rabbit HRP polymer (Dako, K4003), DAB+ (Dako, K3468) and hematoxylin. The degree of steatosis, inflammation, oval cell/bile duct hyperplasia, and aSMA-positive cell count were assessed by a board-certified pathologist.

Serum levels of mouse TIMP1 (R&D Systems, M™100) and mouse cytokeratin 18-M30 (Cusabio, CSB-E14265m) were assayed 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).

Fig. 5A-5L. To evaluate the ability of the PNPLA3 ^{rs738409-rs738408} -specific siRNA molecule to prevent the development of early fibrosis, mice were injected with the AAV8 empty vector (EV), AAV8PNPLA3 ^{rs738409-rs738408} vector, or vehicle and maintained on standard chow or AMLN diet. Two weeks after AAV injections, mice were treated with D-2324 siRNA or vehicle every other week for six weeks, for a total of six injection cycles. Tissue was collected from mice at the ten-week time point. Results are presented as mean values and standard error, standard chow without AAV + vehicle and AMLN chow with AAV8-PNPLA3 ^{rs738409-rs738408} vector + vehicle, N=8 per group; AMLN diet with AAV8-PNPLA3 ^{rs738409-rs738408} vector +vehicle and with AAV8-PNPLA3 ^{rs738409-rs738408} vector +siRNA, N=12 per group. Asterisks indicate statistical significance for the AAV8PNPLA3 ^{rs738409-rs738408} plus vehicle treated cohort, which was calculated by one-way ANOVA with Dunnett's multiple comparisons test. (A) Ratio of liver weight (grams) to body weight (grams) at the time of tissue collection. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV- PNPLA3 ^{rs738409rs738408} +siRNA, ****<0.0001.

(B) Confirmation of human PNPLA3 mRNA expression and silencing in liver by qPCR. (Left) Raw Ct value and (Right) relative fold expression of mRNA normalized to mouse Gapdh; comparison between PNPLA3 ^{rs738409-rs738408} +vehicle and PNPLA3 ^{rs738409rs73840} 8 +siRNA groups. (C) qPCR analysis of mouse Pnpla3 mRNA expression in liver indicates that the expression level of endogenous Pnpla3 is not significantly altered by AAV-mediated overexpression or siRNA-induced silencing. (Left) Raw Ct value and (Right) relative fold expression of mRNA normalized to mouse Gapdh; Comparison of the standard diet group without AAV with the groups fed the AMLN diet, namely PNPLA3 ^{rs738409-rs738408} +vehicle and PNPLA3 ^{rs738409-rs738408} +siRNA. (D) Serum Timp1 levels are presented as picograms per milliliter of serum. Adjusted P-values: no AAV +vehicle group, ****<0.0001, AAV-EV +vehicle, ****<0.0001, AAV- PNPLA3 ^{rs738409rs738408} +siRNA, ****<0.0001. (E) Serum CK18m30 levels are presented as picograms per milliliter of serum. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, ****<0.0001. (F) Histological evidence of inflammation by hematoxylin and eosin 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) Histologic evidence of oval cell/bile duct hyperplasia by hematoxylin and eosin staining graded as: within normal (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) Immunohistochemistry staining with anti-smooth muscle actin antibody graded as: within normal (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 ^{rs738409rs738408} +siRNA, ***=0.0002. (I) Masson's trichrome staining for fibrosis scored as: within normal limits (0), minimal (1), mild (2), moderate (3), and severe (4). Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, not significant, AAV-PNPLA3 ^{rs738409-rs738408} +siRNA, not significant. (J) Mouse Col1a1 mRNA expression in liver assessed by qPCR. Relative fold expression of mRNA normalized to mouse Gapdh. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, ****<0.0001. (K) Mouse Col3a1 mRNA expression in liver assessed by qPCR. Relative fold expression of mRNA normalized to mouse Gapdh. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ****<0.0001, AAV- PNPLA3 ^{rs738409-rs738408} +siRNA, ****<0.0001. (L) Mouse Col4a1 mRNA expression in liver assessed by qPCR. Relative fold expression of mRNA normalized to mouse Gapdh. Adjusted P-values: No AAV+vehicle group, ****<0.0001, AAV-EV+vehicle, ***<0.0005, AAV-PNPLA3 ^{rs738409-rs738408} +siRNA, **<0.0041.

Example 7. Screening of PNPLA3 siRNA molecules using a mouse bioluminescence imaging model.

Male BALB/c mice (Charles River Laboratories Inc.), generally 10–12 weeks old, were challenged with adeno-associated virus (AAV; serotype AAVDJ8; endotoxin-free, Amgen in-house) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). AAV was administered at 5e11 to 7.5e11 viral particles per animal and was administered intravenously by tail vein injection. AAV-based constructs were constructed with the murine cytomegalovirus CMV promoter and a firefly luciferase reporter gene containing at the 3' end a series of nucleotides containing regions of either the human PNPLA3 ^WT (reference allele) or the human PNPLA3 ^{rs738409-rs738408} (minor allele) target sequences and other human PNPLA3 target sequences of interest not spanning the SNPs, as detailed in Figs. 6 and 7.

Two weeks after AAV injection, mice were injected with RediJect D-luciferin (PerkinElmer, 770504) according to the manufacturer's instructions. The bioluminescent signal in mice was determined using the IVIS In Vivo Spectral Imaging System (PerkinElmer) and analyzed using Living Image software (PerkinElmer). Mice were then randomized into groups with n=5 based on total flux signal [photons/second] in the liver region. Following randomization to treatment groups, mice were injected with a single dose of siRNA (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190-136). siRNA was injected subcutaneously at the indicated milligrams per kilogram of animal weight. A vehicle-treated control group was included for each screening cycle and each AAV type.

At 1, 2, 3, and 4 weeks after siRNA injection, mice were re-imaged and total flux intensity [f/s] measurements were collected using the same defined region of interest established for baseline. For each animal, the relative percent knockdown was determined by calculating the percent change in total flux intensity at 1, 2, 3, or 4 weeks relative to the total flux intensity for that animal at baseline, normalized to the mean change in total flux intensity relative to baseline in the vehicle-treated control group for the same time point. For example, an animal treated with siRNA has a relative knockdown calculated as follows: (total flux intensity for the animal at week 2)/(total flux intensity for the animal at baseline), normalized to the mean of (total flux intensity for vehicle-treated animals at week 2/total flux intensity for vehicle-treated animals at baseline). 8 shows illustrative images of animals injected with AAV expressing either target sequences of the human PNPLA3 minor allele or target sequences of the reference allele, and the change in overall flux intensity over time both before and after siRNA treatment.

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PNPLA3 knockdown assay

Table 13

Number DUPLEX AAV based vector AAV particles/animal Introduced dose (mg/kg) Nokda un PNPL AZ, day 8 (%) Nokda Un PNPL AZ, day 15 (%) Nokda Un PNPL AZ, day 22 (%) Nokda Un PNPL AZ, day 29 (%) Nokda Un PNPL AZ, day 43 (%) D- PNPLA3- 5E+11 5 -98.03 -94.77 -94.15 -65.40 2324 D- 2370 I148M DM PNPLA3- I148M DM 5E+11 5 -96.48 -96.52 -95.40 -69.31 D2419 PNPLA3- I148M DM 5E+11 5 -97.66 -97.09 -96.84 -90.52 D- 2324 PNPLA3- I148M DM 5E+11 3 -96.39 -91.43 -87.71 -61.75 D2370 PNPLA3- I148M DM 5E+11 3 -97.42 -95.98 -95.51 -85.66 D2419 PNPLA3- I148M DM 5E+11 3 -97.37 -95.32 -95.52 -81.81 D2421 PNPLA3- I148M DM 5E+11 3 -96.13 -93.99 -95.26 -85.49 D2404 D2402 PNPLA3-I148M DM PNPLA3-I148M DM 5E+11 5E+11 3 3 -97.77 -97.69 -96.82 -96.69 -96.83 -96.81 -55.46 D- 2472 PNPLA3- I148M DM 5E+11 3 -95.89 -93.61 -92.05 -68.74 D- 2443 PNPLA3- I148M DM 5E+11 3 -97.33 -96.51 -95.82 -84.56 D- 2466 PNPLA3- T148M DM 5E+11 3 -97.96 -97.13 -96.75 -79.62 D- 2473 PNPLA3- I148M DM 5E+11 3 -97.18 -95.95 -95.79 -68.96 D- 2324 PNPLA3- I148M DM 5E+11 1 -90.86 -82.53 -80.56 -74.32 D2370 PNPLA3- I148M DM 5E+11 1 -93.49 -90.34 -89.17 -56.45 D2419 PNPLA3- I148M DM 5E+11 1 -89.12 -84.16 -87.52 -47.52 D2421 PNPLA3- I148M DM 5E+11 1 -83.03 -70.41 -74.45 -26.47 D2404 PNPLA3- I148M DM 5E+11 1 -94.34 -91.18 -90.01 -59.48 D- 2402 PNPLA3- I148M DM 5E+11 1 -87.41 -79.59 -84.95 -32.94 D- 2472 PNPLA3- I148M DM 5E+11 1 -87.28 -79.86 -76.77 -33.42 D- 2443 PNPLA3- I148M DM 5E+11 1 -48.96 -46.63 -69.39 -34.70 D2466 PNPLA3- I148M DM 5E+11 1 -90.36 -83.03 -85.21 -44.20 D- 2473 PNPLA3- I148M DM 5E+11 1 -80.89 -76.26 -84.45 -56.63 D2454 PNPLA3 WT 5E+11 3 -97.89 -85.94 -92.41 -44.16 D- PNPLA3 5E+11 3 -98.67 -90.86 -94.56 -72.71

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2453 WT D- 2456 PNPLA3 WT 5E+11 3 -97.75 -84.03 -87.90 -40.07 D- 2455 PNPLA3 WT 5E+11 3 -89.75 -61.34 -80.50 -38.33 D- 2454 PNPLA3 WT 5E+11 1 -94.43 -73.24 -89.57 -60.76 D- 2453 PNPLA3 WT 5E+11 1 -94.15 -72.63 -80.99 5,10 D- 2456 PNPLA3 WT 5E+11 1 -79.90 -50.52 -69.95 -20.01 D- 2455 PNPLA3 WT 5E+11 1 -71.82 -1.32 -55.65 17.39 D- 2419 PNPLA3- I148M DM 7.5E+11 3 -98,00 -97.61 -96.67 -91.38 D- 2666 PNPLA3- I148M DM 7.5E+11 3 -94.32 -94.07 -90.69 -80.56 D- 2667 PNPLA3- I148M DM 7.5E+11 3 -95.49 -94.77 -93.58 -85.70 D- 2676 PNPLA3- I148M DM 7.5E+11 3 -94.63 -91.64 -89.02 -72.10 D- 2699 PNPLA3- T148M DM 7.5E+11 3 -90.85 -85.48 -57.10 D- 2700 PNPLA3- I148M DM 7.5E+11 3 -97.65 -96.86 -93.76 -87.13 D- 2701 PNPLA3- I148M DM 7.5E+11 3 -97.37 -96.59 -95,00 -88.29 D- 2705 PNPLA3- I148M DM 7.5E+11 3 -95.74 -91.67 -86.98 -75.38 D- 2706 PNPLA3- I148M DM 7.5E+11 3 -95.51 -91.01 -85.19 -68.36 D- 2707 PNPLA3- I148M DM 7.5E+11 3 -94.33 -89.29 -85.91 -66.08 D- 2708 PNPLA3- I148M DM 7.5E+11 3 -97.72 -96.34 -95.70 -89.66 D- 2684 PNPLA3- I148M DM 7.5E+11 3 -94.66 -89.59 -85.28 -55.31 D- 2686 PNPLA3- I148M DM 7.5E+11 3 -96.29 -94.61 -89.34 -51.49 D- 2688 PNPLA3- I148M DM 7.5E+11 3 -95.97 -94.43 -93.01 -82.63 D- 2691 PNPLA3- I148M DM 7.5E+11 3 -94.47 -86.71 -81.93 -64.98 D- 2692 PNPLA3- I148M DM 7.5E+11 3 -96.52 -94.01 -89.97 -80.83 D- 2696 PNPLA3- I148M DM 7.5E+11 3 -96.94 -94.79 -92.89 -83.76 D- PNPLA3- 7.5E+11 3 -91.39 -83.82 -75.89 -51.50

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2697 I148M DM D- 2419 PNPLA3- I148M DM 7.5E+11 3 -95.52 -93.54 -91.46 -74.42 D- 2523 PNPLA3- I148M DM 7.5E+11 3 -94.27 -91.79 -66.89 -50.87 D- 2413 PNPLA3- I148M DM 7.5E+11 3 -94.04 -95.17 -76.04 -53.22 D- 2523 PNPLA3- I148M DM 7.5E+11 1 -65.61 -86.75 78.32 163.55 D- 2413 PNPLA3- I148M DM 7.5E+11 1 -83.37 -85.51 -26.85 -9.13 D- 2667 PNPLA3- I148M DM 7.5E+11 1 -58.87 -59.41 -4.15 35,34 D2419 PNPLA3- I148M DM 7.5E+11 1 -91.58 -90.56 -73.21 -54.90 D- 2688 PNPLA3- I148M DM 7.5E+11 1 -73.57 -84.01 -37.46 -1.49 D- 2708 PNPLA3- I148M DM 7.5E+11 1 -84.06 -86.91 -65.11 -62.60 D2669 PNPLA3- I148M DM 7.5E+11 1 -0.36 15.49 220.82 268,45 D2711 PNPLA3- T148M DM 7.5E+11 3 -0.01 49,59 238,44 212.77 D- 2713 PNPLA3- I148M DM 7.5E+11 3 -91.72 -90.51 -63.93 -30.60 D2716 PNPLA3- I148M DM 7.5E+11 3 -86.43 -87.62 -53.78 -44.36 D2674 PNPLA3- I148M DM 7.5E+11 3 -80.59 -81.95 -36.01 0.59 D- 2698 PNPLA3- I148M DM 7.5E+11 3 -85.22 -71.73 483.18 807,64 D- 2693 PNPLA3- I148M DM 7.5E+11 3 -75.98 -59.22 39,49 110.57 D2456 PNPLA3 WT 7.5E+11 3 -85.45 -78.57 -73.40 -58.06 D- 2523 PNPLA3 WT 7.5E+11 3 -27.40 -30.01 -40.22 -49.59 D- 2413 PNPLA3 WT 7.5E+11 3 -36.68 -27.93 -10.77 12.54 D2419 PNPLA3 WT 7.5E+11 3 115.08 100.96 101.39 64.16 D2679 PNPLA3 WT 7.5E+11 3 -34.16 -6.82 17.45 17.46 D- 2688 PNPLA3 WT 7.5E+11 3 135.68 67,66 73.67 48.09 D2708 PNPLA3 WT 7.5E+11 3 69.43 40.96 50.89 52,15 D- PNPLA3 7.5E+11 3 189.68 50,72 148.32 147.67

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2669 WT D2711 PNPLA3 WT 7.5E+11 3 109.67 114.26 75.39 93.47 D2713 PNPLA3 WT 7.5E+11 3 115.32 48.86 18.67 1.36 D- 2716 PNPLA3 WT 7.5E+11 3 205.93 114.38 114.09 83,88 D2674 PNPLA3 WT 7.5E+11 3 81,00 67.18 109.44 65.24 D2698 PNPLA3 WT 7.5E+11 3 105.88 76.39 62.37 37.65 D- 2693 PNPLA3 WT 7.5E+11 3 148.52 125.48 116.64 61.43 D- 2690 PNPLA3 WT 7.5E+11 3 260.84 226.65 132.56 143.47 D- 2419 PNPLA3- I148M DM 7.5E+11 1 -93.56 -89.52 -84.43 -63.34 D- 2717 PNPLA3- I148M DM 7.5E+11 1 -80.51 -67.95 -58.97 -22.43 D2718 PNPLA3- I148M DM 7.5E+11 1 -71.69 -55.51 -36.08 38.24 D2721 PNPLA3- T148M DM 7.5E+11 1 -63.71 -62.50 -48.52 -12.97 D- 2719 PNPLA3- I148M DM 7.5E+11 1 -78.95 -70.97 -65.38 -13.45 D2713 PNPLA3- I148M DM 7.5E+11 1 -70.43 -64.51 -43.23 -0.48 D2712 PNPLA3- I148M DM 7.5E+11 1 -79.63 -68.64 -51.82 -8.94 D2720 PNPLA3- I148M DM 7.5E+11 1 -65.95 -86.70 -40.92 2.24 D2722 PNPLA3- I148M DM 7.5E+11 1 -80.32 -75.04 -63.28 -34.56 D- 2723 PNPLA3- I148M DM 7.5E+11 1 -72.69 -55.59 -39.98 35,01 D- 2724 PNPLA3- I148M DM 7.5E+11 1 -87.97 -78.29 -73.04 -42.89 D- 2725 PNPLA3- I148M DM 7.5E+11 1 -79.65 -70.69 -32.50 -35.65 D2726 PNPLA3- I148M DM 7.5E+11 1 -40.03 -38.86 -14.74 55.71 D2716 PNPLA3- I148M DM 7.5E+11 1 34.75 92.81 181,00 440,62 D- 2454 PNPLA3 WT 7.5E+11 3 -98.16 -95.32 -90.10 -88.50 D- 2523 PNPLA3 WT 7.5E+11 3 -44.09 -18.41 133.97 45,50 D- PNPLA3 7.5E+11 3 -64.12 -40.89 38,07 -3.05

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2413 WT D2717 PNPLA3 WT 7.5E+11 3 -47.67 -19.71 109.49 42.75 D2718 PNPLA3 WT 7.5E+11 3 -77.04 -60.05 -3.84 -40.18 D2721 PNPLA3 WT 7.5E+11 3 -47.41 -49.33 10.37 -41.12 D2719 PNPLA3 WT 7.5E+11 3 -29.15 -27.68 72,52 -20.99 D2720 PNPLA3 WT 7.5E+11 3 -67.46 -46.37 66.09 -20.66 D2726 PNPLA3 WT 7.5E+11 3 -83.36 -81.38 -37.21 -61.84 D- 2723 PNPLA3 WT 7.5E+11 3 -76.09 -57.32 -7.17 -45.85 D- 2419 PNPLA3- I148M DM 7.5E+11 3 -96.66 -97.62 -93.93 -88.77 D- 2765 PNPLA3- I148M DM 7.5E+11 3 -96.64 -97.81 -95.29 -87.53 D2766 PNPLA3- I148M DM 7.5E+11 3 -97.30 -98.15 -95.74 -90.07 D- 2724 PNPLA3- T148M DM 7.5E+11 3 -97.41 -97.40 -94.77 -74.92 D- 2768 PNPLA3- I148M DM 7.5E+11 3 -93.09 -95.62 -90.36 -74.62 D2769 PNPLA3- I148M DM 7.5E+11 3 -96.94 -97.93 -95.17 -89.60 D2770 PNPLA3- I148M DM 7.5E+11 3 -89.23 -94.14 -90.41 -79.60 D- 2771 PNPLA3- I148M DM 7.5E+11 3 -97.22 -97.80 -95.95 -90.02 D2772 PNPLA3- I148M DM 7.5E+11 3 -97.47 -97.84 -95.09 -92.03 D- 2773 PNPLA3- I148M DM 7.5E+11 3 -97.51 -97.95 -96.18 -93.12 D- 2774 PNPLA3- I148M DM 7.5E+11 3 -96.78 -97.86 -95.36 -91.98 D- 2413 PNPLA3- I148M DM 7.5E+11 3 -97.94 -98.01 -96.24 -91.62 D2419 PNPLA3- I148M DM 7.5E+11 3 -95.82 -97.64 -93.55 -89.59 D- 2717 PNPLA3- I148M DM 7.5E+11 3 -96.64 -97.39 -95.19 -90.49 D- 2453 PNPLA3 WT 7.5E+11 1,5 -94.74 -92.67 -88.92 -82.19 D- 2724 PNPLA3 WT 7.5E+11 3 20.64 74,00 49,42 77.34 D- PNPLA3 7.5E+11 3 -27.08 53.78 58.28 17.26

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2522 WT D- 2765 PNPLA3 WT 7.5E+11 3 -65.40 -34.32 -35.49 -46.32 D2766 PNPLA3 WT 7.5E+11 3 -11.64 30.74 34,37 27.78 D2767 PNPLA3 WT 7.5E+11 3 14.66 60.91 75,79 39.35 D2768 PNPLA3 WT 7.5E+11 3 50,61 196.44 345,66 149.29 D2769 PNPLA3 WT 7.5E+11 3 30,51 68.93 90.25 35.98 D- 2770 PNPLA3 WT 7.5E+11 3 25,01 69.81 95,00 99.26 D- 2771 PNPLA3 WT 7.5E+11 3 60.93 158.57 281,70 147.49 D- 2772 PNPLA3 WT 7.5E+11 3 -3.32 58,08 89.41 85.72 D- 2773 PNPLA3 WT 7.5E+11 3 -36.28 -20.77 21.91 -22.36 D2774 PNPLA3 WT 7.5E+11 3 2.83 51.07 71.49 65,75 D2413 PNPLA3 WT 7.5E+11 3 -5.20 44.59 50.74 28.08 D- 2419 PNPLA3 WT 7.5E+11 3 101,03 115,15 32.98 -16.47 D2717 PNPLA3 WT 7.5E+11 1,5 21,80 107.39 173.59 87.94

Example 8. Confirmation of the efficacy of PNPLA3 ^{rs738409-rs738408} -selective siRNA molecule in a chimeric humanized mouse liver model.

To evaluate the efficacy of siRNA targeting the human PNPLA3 minor allele in hepatocytes in vivo, livers from chimeric humanized 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). The male mice were approximately four months old at the start of the study; at least three months after transplantation. Mice were determined to have a human hepatocyte replacement rate of 90-95% by PhoenixBio and, based on previous genotyping, were heterozygous for PNPLA3 ^rs738409 (lot BD195). Upon arrival, mice were fed standard ProLab RMH 3000 chow as recommended by PhoenixBio. After a one-week acclimation period, the diet was changed to a high-fat, high-fructose NASH-inducing diet (Research Diets, D19021301). After one week on the NASH-inducing diet, mice were randomized based on body weight measurements. Mice were treated with a single subcutaneous injection of siRNA (0.5 mM) diluted in phosphate-buffered saline (Thermo Fisher Scientific, 14190136) at a dose of 3.0 or 10.0 mg/kg body weight, or mice received vehicle only. Two or four weeks after siRNA injection, liver tissues were collected from animals, snap-frozen in liquid nitrogen, processed for purified RNA extraction using the QIAcube Automated DNA/RNA Isolation and Purification System (Qiagen) and the RNeasy Mini QIAcube Kit (Qiagen, 74116) according to the manufacturer's instructions. Samples were analyzed using a NanoDrop™ 8000 Spectrophotometer (Thermo Scientific, ND-8000-GL). RNA was processed

DNA was purified with RNase-free RQ1 DNase (Promega, M6101) and prepared for droplet digital PCR (ddPCR) according to the manufacturer's instructions. AccuScript High Fidelity Single Strand cDNA Synthesis Kit (Thermo Fisher, 200820) was used to perform the reverse transcription reaction and PCR reactions were performed using ddPCR Supermix for Probes (BioRad, 1863010). ddPCR was performed using the AutoDG Droplet Digital PCR System (BioRad, QX200). The following TaqMan™ assays were purchased from Invitrogen: human PNPLA3 (Hs00228747_m1), human ASGR1 (Hs1005019 m1), mouse Asgr1 (Mm01245581 m1), and human PNPLA3 rs738409 minor/reference allele discrimination assay (C 7241 10). The following assays were purchased from Integrated DNA Technologies Inc.: human TBP (Hs. PT 53a.20105486; primer to probe ratio is 3.6:1), human HPRT1 (Hs.PT.39a.22214821; primer to probe ratio is 3.6:1), and mouse Hprt (Mm.PT.39a.22214828; primer to probe ratio is 3.6:1). Results for human PNPLA3, HPRT, and ASGR1 and mouse Hprt and Asgr1 are presented as copies per 20 microliter reaction, normalized to human TBP. Data for human PNPLA3, human HPRT, and mouse Hprt are also presented as the relative percentage of mRNA expression knockdown compared to vehicle-treated controls.

For liver triglyceride analysis, approximately 0.05-0.1 mg of frozen liver collected from mice was homogenized in 1 ml of isopropanol. After one hour of incubation on ice, the samples were centrifuged at 10,000 rpm in a microcentrifuge and the supernatants were transferred to clean

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96-well deep well plate. Triglyceride content was determined using colorimetric Infinity Triglyceride Reagent (Thermo Fisher Scientific, TR22421) and Triglyceride Standard (Pointe

Scientific, T7531-STD) in accordance with the manufacturer's instructions, and a microplate reader

SpectraMax Plus with SoftMax Pro6 software (Molecular Devices). Results are presented as milligrams of triglycerides per gram of liver tissue.

Figure 9 shows an example of an siRNA molecule, D-2419, demonstrating both dose-dependent and allele-selective knockdown of mRNA and functional efficacy in vivo. After one week on a NASH-inducing diet, mice heterozygous for human PNPLA3 ^{rs738409-rs738408} were treated with siRNA or vehicle. (A) The siRNA molecule, D-2419, was administered at 3.0 and 10.0 mg/kg body weight subcutaneously into the abdomen of the mouse. Two and four weeks after siRNA treatment, mice were sacrificed and liver tissues were collected and processed for analysis. Using ddPCR and the TaqMan® two-dye allele-specific reagent to distinguish between minor and reference alleles of PNPLA3, data demonstrate both dose-dependent and allele-selective knockdown of PNPLA3 ^{rs738409-rs738408} and no measurable change for PNPLA3 ^WT . 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=re significant. (B) Data represent mean relative percentage mRNA knockdown and standard error of the mean for the human PNPLA3 ^{rs738409-rs738408} allele compared to PNPLA3 ^WT determined in the vehicle-treated control group. Values are expressed relative to the mean of vehicle-treated control two weeks prior to assay, with all values normalized to human TBP. (C) Liver tissues isolated from the two-week treatment group were processed for triglyceride content to assess functional efficacy. Data represent milligrams of triglyceride/g liver. N=5 mice per group; data are presented as mean and standard error of the mean. One-way ANOVA, **=0.01, NS=re significant. (D) To control for efficient GalNAc-mediated siRNA delivery, D-2787, siRNA cross-reacting with human and mouse HPRT and Hprt, respectively, was delivered at 10 mg/kg and liver tissues were harvested after two weeks. Data represent HPRT mRNA and Hprt mRNA copies in D-2787-treated mice (N=4) compared to vehicle-treated mice (N=5). Data are presented as mean and SEM. One-way ANOVA, *=0.01. (E) Data represent mean relative percentage mRNA knockdown and SEM for human HPRT and mouse Hprt mRNA, respectively, adjusted for vehicle-treated controls; all data are normalized to human TBP. (F) To confirm GalNAc receptor expression on hepatocytes in PXB® mice, mouse Asgr1 mRNA and human ASGR1 mRNA levels were assessed in the absence and presence of D-2419 at both two weeks and four weeks after siRNA injection. N=5 mice per group; data are presented as mean and SEM.

Claims (43)

1. An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises the antisense sequence of SEQ ID NO: 2982 or 2618 and wherein the RNAi construct suppresses the expression of patatin-like phospholipase domain-containing protein 3 (PNPLA3). 2. The RNAi construct of claim 1, wherein the antisense strand comprises a region that is complementary to the PNPLA3 mRNA sequence. 3. An RNAi construct according to any of the preceding claims, wherein the sense strand comprises the sense sequence of SEQ ID NO: 2981 or 2617. 4. An RNAi construct according to any one of the preceding claims, wherein the construct preferentially suppresses the minor allele of PNPLA3-rs738409. 5. The RNAi construct of claim 4, wherein the construct suppresses the minor allele of PNPLA3-rs738409 by at least 10% more than the major allele. 6. An RNAi construct according to any one of the preceding claims, wherein the construct preferentially suppresses the minor allele of PNPLA3-rs738408. 7. The RNAi construct of claim 6, wherein the construct suppresses the two minor alleles of PNPLA3-rs738409-rs738408 by at least 10% more than the major allele. 8. An RNAi construct according to any one of the preceding claims, wherein the sense strand comprises a sequence that is sufficiently complementary to a sequence of the antisense strand to form a duplex region of 15 to 30 base pairs in length. 9. The RNAi construct according to claim 8, wherein the length of the duplex region is from 17 to 24 base pairs. 10. The RNAi construct according to claim 8, wherein the length of the duplex region is from 19 to 21 base pairs. 11. The RNAi construct according to claim 10, wherein the length of the duplex region is 19 base pairs. - 208 047656 12. The RNAi construct according to claim 10, wherein the length of the duplex region is 20 base pairs. 13. The RNAi construct according to claim 10, wherein the length of the duplex region is 21 base pairs. 14. An RNAi construct according to any one of claims 8 to 13, wherein the length of each of the sense strand and the antisense strand is from 15 to 30 nucleotides. 15. The RNAi construct of claim 14, wherein the length of each of the sense strand and the antisense strand is from 19 to 27 nucleotides. 16. The RNAi construct of claim 14, wherein the length of each of the sense strand and the antisense strand is from 21 to 25 nucleotides. 17. The RNAi construct of claim 14, wherein the length of each of the sense strand and the antisense strand is from 21 to 23 nucleotides. 18. An RNAi construct according to any one of claims 1 to 17, wherein the RNAi construct comprises at least one blunt end. 19. An RNAi construct according to any one of claims 1 to 17, wherein the RNAi construct comprises at least one nucleotide sticky end of 1 to 4 unpaired nucleotides. 20. The RNAi construct of claim 19, wherein the nucleotide sticky end comprises two unpaired nucleotides. 21. The RNAi construct of claim 19 or 20, wherein the RNAi construct comprises a nucleotide sticky end 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. 22. The RNAi construct of any one of claims 19 to 21, wherein the nucleotide sticky end comprises the dinucleotide 5'-UU-3' or the dinucleotide 5'-dTdT-3'. 23. The RNAi construct according to any one of claims 1 to 22, wherein the RNAi construct comprises at least one modified nucleotide. 24. The RNAi construct of claim 23, wherein the modified nucleotide is a 2'-modified nucleotide. 25. The RNAi construct of claim 23, wherein the modified nucleotide is a 2'-fluoro-modified nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-O-methoxyethyl-modified nucleotide, a 2'-O-allyl-modified nucleotide, a bicyclic nucleic acid (BNA), a glycolic nucleic acid (GNA), an inverted base, or combinations thereof. 26. The RNAi construct of claim 25, wherein the modified nucleotide is a 2'-O-methyl-modified nucleotide, a 2'-O-methoxyethyl-modified nucleotide, a 2'-fluoro-modified nucleotide, or combinations thereof. 27. The RNAi construct of claim 23, wherein all nucleotides in the sense and antisense strands are modified nucleotides. 28. The RNAi construct of claim 27, wherein the modified nucleotides are 2'-O-methyl-modified nucleotides, 2'-fluoro-modified nucleotides, or combinations thereof. 29. The RNAi construct according to any one of claims 1 to 28, wherein the RNAi construct comprises at least one phosphorothioate internucleotide linkage. 30. The RNAi construct of claim 29, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3' end of the antisense strand. 31. The RNAi construct of claim 29, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3' and 5' end of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5' end of the sense strand. 32. The RNAi construct of any one of claims 1 to 31, wherein the antisense strand and the sense strand consist of SEQ ID NO: 2982 and 2981, respectively. 33. The RNAi construct of any one of claims 1 to 31, wherein the antisense strand and the sense strand consist of SEQ ID NO: 2618 and 2617, respectively. 34. The RNAi construct of any one of claims 1 to 33, wherein the RNAi construct provides a reduction in the expression level of PNPLA3 in liver cells after incubation with the RNAi construct compared to the expression level of PNPLA3 in liver cells that were incubated with a control RNAi construct. 35. The RNAi construct of claim 34, wherein the liver cells are Hep3B or HepG2 cells. 36. The RNAi construct of any one of claims 1 to 35, wherein the RNAi construct suppresses PNPLA3 expression by at least 10% at 5 nM in Hep3B cells in vitro. 37. The RNAi construct of any one of claims 1 to 35, wherein the RNAi construct suppresses PNPLA3 expression by at least 10% at 5 nM in HepG2 cells in vitro. 38. The RNAi construct of any one of claims 1 to 35, wherein the RNAi construct suppresses PNPLA3 expression in Hep3B cells with an _IC50 value of less than 1 nM. 39. An RNAi construct according to any one of claims 1 to 35, wherein the RNAi construct suppresses the expression of - 209 047656 PNPLA3 in HepG2 cells with an IC ₅₀ value of less than 1 nM. 40. A pharmaceutical composition comprising an RNAi construct according to any one of claims 1 to 39 and a pharmaceutically acceptable carrier, excipient or diluent. 41. Use of an RNAi construct according to any one of claims 1 to 39 for producing a medicinal product for the treatment of liver disease. 42. The use according to claim 41, wherein the level of PNPLA3 expression in hepatocytes is reduced after administration of the RNAi construct compared to the level of PNPLA3 expression in a patient not receiving the RNAi construct. 43. The use according to claim 41, wherein the liver disease is non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).