Patent Publication Number: US-11028393-B2

Title: siRNA compositions that specifically down regulate expression of a variant of the PNPLA3 gene and methods of use thereof for treating a chronic liver disease

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a continuation of U.S. patent application Ser. No. 16/026,609, filed Jul. 3, 2017, which is a continuation of U.S. patent application Ser. No. 15/614,147, filed Jun. 5, 2017, now issued as U.S. Pat. No. 10,036,024, which claims priority to, and the benefit of, U.S. Provisional Patent Application Ser. No. 62/345,048, filed Jun. 3, 2016, the contents of each of which are incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     The invention provides siRNA compositions that specifically regulate expression of a variant of the PNPLA3 gene and methods of use thereof for treating a chronic liver disease or alcoholic liver disease (ALD). 
     BACKGROUND 
     Nonalcoholic Fatty Liver Disease (NAFLD) is a spectrum of chronic liver disorders, beginning as hepatic fat accumulation without significant alcohol consumption. A subset of patients with NAFLD have nonalcoholic steatohepatitis (NASH) which, over time and without treatment, may progress to cirrhosis and even hepatocellular carcinoma (HCC). It is estimated that 4-22% of HCC cases in the US are due to NASH, and about 2% of the U.S. population has NASH-derived cirrhosis, which is expected to become the leading cause of liver transplantation by 2020. Moreover, NAFLD/NASH is the central hallmark of obesity and type II diabetes which together affect over 50% of the US population, leading to a heavy economic burden. Unfortunately, therapeutic options for NASH are still very limited thus far, with only slight benefits observed from vitamin E or obeticholic acid treatment. Developing safe and effective treatments for NASH remain a significant unmet medical need. 
     SUMMARY 
     The invention recognizes that the patatin-like phospholipase domain containing 3 (PNPLA3) gene is strongly associated with chronic human liver disease (e.g., fatty liver disease, steatohepatitis, cirrhosis, alcoholic liver disease and hepatocellular carcinoma). Particularly, the 148 I&gt;M (rs738409 C&gt;G) mutation has been identified as the causal allele for these phenotypes. Overexpression of the 148M isoform is demonstrated herein to be a major cause of these pathogenic processes in both human hepatocytes and animal models. The invention provides small interfering RNA (siRNA) that can specifically recognize the mutant allele (148M) while having minimal effect on the wild-type allele (148I). In that manner, a novel therapeutic strategy for treating human chronic liver disease due to the overexpression of PNPLA3 148M isoform is provided. 
     In certain aspects, the invention provides compositions that include a small interfering RNA (siRNA) molecule that specifically binds mRNA corresponding to the rs738409 C&gt;G variant of the patatin-like phospholipase domain-containing (PNPLA3) gene, thereby downregulating expression of mutant allele. The rs738409 C&gt;G variant is also referred throughout as the 148M mutant allele or isoform, as opposed to the 148 wild-type allele or isoform. The siRNA molecule may be single stranded or double stranded. In certain embodiments, the siRNA molecule consists of the nucleotide sequence of at least one of SEQ ID NOs 1, 2 or 278-552. In certain embodiments, the siRNA molecule does not bind mRNA associated with the wild-type isoform of the PNPLA3 gene. In other embodiments, the siRNA molecule includes one or more non-naturally occurring nucleotides. 
     To facilitate effective delivery, the siRNA may be coupled to a pharmaceutically acceptable carrier system. In certain embodiments the pharmaceutically acceptable carrier system includes a nanoparticle to which the siRNA molecule is coupled. An exemplary nanoparticle includes low molecular weight polyethyleneimine (LPEI) or its derivatives (e.g., disulfide crosslinked polyethyleneimine (CLPEI)) and a lipid. In certain embodiments, the lipid is a bile acid, such as cholic acid, deoxycholic acid, and lithocholic acid. 
     Other aspects of the invention provide methods for treating a subject with a chronic liver disease that involve administrating a therapeutically effective amount of any of the above compositions to a subject having a chronic liver disease. Exemplary chronic liver diseases include fatty liver disease, steatohepatitis, cirrhosis, alcoholic liver disease (ALD), or hepatocellular carcinoma. 
     Other aspects of the invention provide an allele-specific DNA-based antisense oligo to downregulate expression of the 148M allele. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the sequence of wild type patatin-like phospholipase domain containing 3 (PNPLA3) gene as well as the sequence of the rs738409 C&gt;G (I148M) variant of the PNPLA3 gene. 
         FIG. 2A  shows a schematic of lipid-grafted LPEI preparation. 
         FIG. 2B  shows 1H-NMR analysis of a lipid-grafted LPEI preparation. 
         FIG. 3A  shows the specificity and potency of 148MSi in targeting PNPLA3148M as compared to PNPLA3 148M with transient transfection of siRNA and PNPLA3-Luc vectors into HEK293 cells for 48 h.  FIGS. 3B and 3C  show downregulation of endogenous transcription of PNPLA3 in HepG2 (148M homozygote) and HEK293 (148I homozygote) cells transfected with 20 pmole/ml 148MSi, control (Ctr-Si) or generic PNPLA3 siRNA set (Gen-Si, Santa Cruz) for 48 h.  FIGS. 3D-E  show reduction of intracellular triglycerides (TG) accumulation (ORO staining) by 148MSi targeting. After transfection, Huh-7 (148M homozygote) cells were co-incubated with free fatty acids [palmitic acid (0.3 mM) and oleic acid (0.9 mM)] and glucose (Glu) at 1 mM or 10 mM concentration for 48 h. *p&lt;0.05; **p&lt;0.01. 
         FIGS. 4A-C  shows transfection efficiency of LPEI- or LCA-LPEI polyplexes in NIH-3T3 cells. Cells were seeded at a density of 20,000 cells per well in a 24-well plate. After 2 days, cells in each well were treated with polyplexes consisting of pEGFP-C1 plasmid (0.2 μg) and polymers in 10/1 to 20/1 weight ratios±dermatan sulfate (DS, 0.2 μg) and incubated for 48 h. GFP expression was visualized with fluorescence microscope (top panel) and quantified by measuring the fluorescence of the supernatant of cell lysate. 
         FIG. 5  shows that genetic variation in PNPLA3 confers susceptibility to chronic liver disease. 
         FIG. 6  shows differences between the 148I phenotype and the 148M phenotype. 
         FIG. 7  shows vectors for compositions of the invention and their effectiveness in downregulating the 148M phenotype. 
         FIGS. 8A-D  show in vivo effectiveness of compositions of the invention in reducing hepatic PNPLA3 148M  expression and total reduction of total hepatic TG levels. 
     
    
    
     DETAILED DESCRIPTION 
     The invention provides siRNA compositions that specifically downregulate expression of a variant of the PNPLA3 gene and methods of use thereof for treating chronic liver disease. In certain aspects, the invention provides compositions including a small interfering RNA (siRNA) molecule that specifically binds mRNA transcribed from a rs738409 C&gt;G variant of a patatin-like phospholipase domain-containing (PNPLA3) gene. 
     The patatin-like phospholipase domain-containing (PNPLA3) gene refers to NCBI Gene ID: 80339.  FIG. 1  shows the sequence of wild type patatin-like phospholipase domain-containing (PNPLA3) gene as well as the sequence of the rs738409 C&gt;G (I148M) variant of the PNPLA3 gene. Genome-wide association studies (GWAS) in recent years have identified the rs738409 C&gt;G (I148M) variant of the patatin-like phospholipase domain-containing 3 (PNPLA3) gene as the strongest genetic risk allele for NAFLD/NASH, influencing degree of steatosis, grade of inflammation, stage of fibrosis and risk of HCC among all examined populations ( FIG. 5 ). Other studies have also demonstrated the strong association between 148M allele and ALD (Buch et al., (Nature Genetics, 47(12):1443-1447, 2015), the content of which is incorporated by reference herein in its entirety). Notably, NAFLD patients and diabetic patients carrying the 148M mutant allele have over 12- and 19-fold higher risk for the development of HCC, respectively, as compared to those who are 148I carriers, making this mutant allele the single largest genetic risk factor for HCC in the context of NASH. See Liu et al. (Journal of Hepatology, 61:75-81, 2014), the content of which is incorporated by reference herein in its entirety. The 148M allele frequency varies from ˜12% among African decedents, 24-40% among Caucasian and East Asian populations to ˜50% among Hispanic populations, accounting for a large variability in genetic susceptibility to NAFLD/NASH. Thus far, a number of mechanistic studies have validated the role of the PNPLA3 148M allele in the development of NAFLD. Accordingly, the present invention recognizes that targeting PNPLA3 may provide an ideal therapeutic option for the treatment of NAFLD/NASH. To date, no drug has been developed to target PNPLA3. 
     While the detailed molecular mechanism underlying the causal role of PNPLA3148M in NAFLD/NASH/ALD still remains incompletely understood, both in vitro cell line studies and in vivo studies using animal models have consistently demonstrated that induction of NAFLD phenotypes requires an “dominant-negative” effect of PNPLA3 (i.e., overexpression of PNPLA3 148M rather than PNPLA3 148I or gene deletion). More specifically, it has been validated that PNPLA3 148M leads to a loss-of-function of its triglycerides hydrolysis activity. However, knockout of the PNPLA3 gene in mice does not lead to NAFLD phenotypes. Overexpression of PNPLA3 148M rather than PNPLA3 148I have been found to induce NAFLD. Moreover, inducing the expression of a PNPLA3 catalytic activity-negative mutant, S47A in a knock-in mouse model parallels the effect of PNPAL3 148M, further highlighting the essential role of the high transcription level of PNPLA3 148M isoform in the development of NAFLD. Given the loss-of-function nature of 148M isoform, conventional therapeutic strategies (e.g., to develop agonist or antagonist chemicals targeting the PNPLA3 protein) are unlikely to block the pathogenic effect of PNPLA3 148M. Instead, specifically reducing the transcription of PNPLA3148M shows great potential. 
     The invention recognizes that due to its first-pass extract effect, the liver is the organ with the most successful siRNA delivery. Accordingly, the invention provides RNAi-based therapeutics targeting PNPLA3, especially with a capability of allele-specific downregulation of 148M transcription. In certain embodiments, it may be found that the siRNA molecules of the invention are highly specific and potent in downregulating expression of the PNPLA3 148M allele without effecting expression of the PNLPLA3 148I wild-type allele. In certain embodiments, novel nanoparticles capable of siRNA delivery may be used. 
     The siRNA molecules within the compositions of the invention specifically bind mRNA transcribed from the PNPLA3 148M allele. Here, the term specific or specifically, used in combination with e.g., binding, hybridization, or downregulating refers to binding of a target sequence or downregulation of a target gene&#39;s expression with minimal or no binding or downregulation of other nucleic acids or their expression. In particular, mRNA transcribed from the PNPLA3 148I allele (wild-type) is not bound and expression of wild-type PNPLA3 is not downregulated by siRNA of the invention that specifically downregulates expression of the PNPLA3 148M allele. Specific binding as used herein may refer to siRNA that hybridize to a target mRNA sequence under high stringency conditions. Nucleic acid hybridization may be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to base composition, length of complementary strands, and number of nucleotide base mismatches between hybridizing nucleic acids, as is readily appreciated by those skilled in the art. Stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon sequence length, washing temperature, and salt concentration. In general, longer sequences require higher temperatures for proper annealing, while shorter sequences need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below its melting temperature. The higher the degree of desired homology between the sequence and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995), the contents of which are incorporated by reference herein in their entirety. 
     Stringent conditions or high stringency conditions typically: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt&#39;s solution, sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. 
     Moderately stringent conditions may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989 (the contents of which are incorporated by reference herein in their entirety), and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt&#39;s solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37° C. to 50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as sequence length and the like. 
     Interfering RNA (which may be interchangeably referred to as RNAi or an interfering RNA sequence) refers to double-stranded RNA that is capable of silencing, reducing, or inhibiting expression of a target gene by any mechanism of action now known or yet to be disclosed. For example, RNAi may act by mediating the degradation of mRNAs which are complementary to the sequence of the RNAi when the RNAi is in the same cell as the target gene. As used herein, RNAi may refer to double-stranded RNA formed by two complementary RNA strands or by a single, self-complementary strand. RNAi may be substantially or completely complementary to the target mRNA or may comprise one or more mismatches upon alignment to the target mRNA. The sequence of the interfering RNA may correspond to the full length target mRNA, or any subsequence thereof. 
     The concept of RNAi includes small-interfering RNA, which, herein, may interchangeably be referred to as siRNA. siRNA is described for example in U.S. Pat. Nos. 9,328,347; 9,328,348; 9,289,514; 9,289,505; and 9,273,312, the content of each of which is incorporated by reference herein in its entirety. A siRNA may be any interfering RNA with a duplex length of about 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 18-23 nucleotides in length. Each complementary sequence of the double-stranded siRNA may be 15-60, 15-50, 15-40, 15-30, 15-25, or 18-23 nucleotides in length, but other noncomplementary sequences may be present. For example, siRNA duplexes may comprise 3′ overhangs of 1 to 4 or more nucleotides and/or 5′ phosphate termini comprising 1 to 4 or more nucleotides. A siRNA may be synthesized in any of a number of conformations. One skilled in the art would recognize the type of siRNA conformation to be used for a particular purpose. Examples of siRNA conformations include, but need not be limited to, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single-stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having complementary sense and antisense regions; or a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions. In the case of the circular polynucleotide, the polynucleotide may be processed either in vivo or in vitro to generate an active double-stranded siRNA molecule. 
     SiRNA can be chemically synthesized, may be encoded by a plasmid and transcribed, or may be vectored by a virus engineered to express the siRNA. A siRNA may be a single stranded molecule with complementary sequences that self-hybridize into duplexes with hairpin loops. siRNA can also be generated by cleavage of parent dsRNA through the use of an appropriate enzyme such as  E. coli  RNase III or Dicer (Yang et al, Proc. Natl. Acad. Sci. USA 99, 9942-9947 (2002); Calegari et al, Proc. Natl. Acad. Sci. USA 99, 14236-14240 (2002); Byrom et al, Ambion TechNotes 10, 4-6 (2003); Kawasaki et al, Nucleic Acids Res 31, 981-987 (2003); Knight et al, Science 293, 2269-2271 (2001); and Robertson et al, J Biol Chem 243, 82-91 (1968)). A parent dsRNA may be any double stranded RNA duplex from which a siRNA may be produced, such as a full or partial mRNA transcript. 
     A mismatch motif may be any portion of a siRNA sequence that is not 100% complementary to its target sequence. A siRNA may have zero, one, two, or three or more mismatch regions. The mismatch regions may be contiguous or may be separated by any number of complementary nucleotides. The mismatch motifs or regions may comprise a single nucleotide or may comprise two or more consecutive nucleotides. 
     A preferred example of a double stranded siRNA design of the invention is listed in Table 1: 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
             
            
               
                 1 
                 5′-CCUGCUUCAUGCCUUUCUACAGUGG-3′ 
               
               
                   
               
               
                 2 
                 3′-AAGGACGAAGUACGGAAAGAUGUCACC-5′ 
               
               
                   
               
            
           
         
       
     
     Exemplary DNA targets for siRNA molecules or antisense oligonucleotides of the invention may include the sequences listed in Table 2 below. 
     
       
         
           
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 SEQ ID NO 
                 DNA target Sequence 
               
               
                   
               
             
            
               
                   3 
                 GGCCTTGGTATGTTCCTGCTTCATG 
               
               
                   
               
               
                   4 
                 GCCTTGGTATGTTCCTGCTTCATG 
               
               
                   
               
               
                   5 
                 CCTTGGTATGTTCCTGCTTCATG 
               
               
                   
               
               
                   6 
                 CTTGGTATGTTCCTGCTTCATG 
               
               
                   
               
               
                   7 
                 TTGGTATGTTCCTGCTTCATG 
               
               
                   
               
               
                   8 
                 TGGTATGTTCCTGCTTCATG 
               
               
                   
               
               
                   9 
                 GGTATGTTCCTGCTTCATG 
               
               
                   
               
               
                  10 
                 GTATGTTCCTGCTTCATG 
               
               
                   
               
               
                  11 
                 TATGTTCCTGCTTCATG 
               
               
                   
               
               
                  12 
                 ATGTTCCTGCTTCATG 
               
               
                   
               
               
                  13 
                 TGTTCCTGCTTCATG 
               
               
                   
               
               
                  14 
                 GCCTTGGTATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  15 
                 CCTTGGTATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  16 
                 CTTGGTATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  17 
                 TTGGTATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  18 
                 TGGTATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  19 
                 GGTATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  20 
                 GTATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  21 
                 TATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  22 
                 ATGTTCCTGCTTCATGC 
               
               
                   
               
               
                  23 
                 TGTTCCTGCTTCATGC 
               
               
                   
               
               
                  24 
                 GTTCCTGCTTCATGC 
               
               
                   
               
               
                  25 
                 CCTTGGTATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  26 
                 CTTGGTATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  27 
                 TTGGTATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  28 
                 TGGTATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  29 
                 GGTATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  30 
                 GTATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  31 
                 TATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  32 
                 ATGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  33 
                 TGTTCCTGCTTCATGCC 
               
               
                   
               
               
                  34  
                 GTTCCTGCTTCATGCC 
               
               
                   
               
               
                  35 
                 TTCCTGCTTCATGCC 
               
               
                   
               
               
                  36 
                 CTTGGTATGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  37 
                 TTGGTATGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  38 
                 TGGTATGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  39 
                 GGTATGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  40 
                 GTATGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  41 
                 TATGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  42 
                 ATGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  43 
                 TGTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  44 
                 GTTCCTGCTTCATGCCT 
               
               
                   
               
               
                  45 
                 TTCCTGCTTCATGCCT 
               
               
                   
               
               
                  46 
                 TCCTGCTTCATGCCT 
               
               
                   
               
               
                  47 
                 TTGGTATGTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  48 
                 TGGTATGTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  49 
                 GGTATGTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  50 
                 GTATGTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  51 
                 TATGTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  52 
                 ATGTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  53 
                 TGTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  54 
                 GTTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  55 
                 TTCCTGCTTCATGCCTT 
               
               
                   
               
               
                  56 
                 TCCTGCTTCATGCCTT 
               
               
                   
               
               
                  57 
                 CCTGCTTCATGCCTT 
               
               
                   
               
               
                  58 
                 TGGTATGTTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  59 
                 GGTATGTTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  60 
                 GTATGTTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  61 
                 TATGTTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  62 
                 ATGTTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  63 
                 TGTTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  64 
                 GTTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  65 
                 TTCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  66 
                 TCCTGCTTCATGCCTTT 
               
               
                   
               
               
                  67 
                 CCTGCTTCATGCCTTT 
               
               
                   
               
               
                  68 
                 CTGCTTCATGCCTTT 
               
               
                   
               
               
                  69 
                 GGTATGTTCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  70 
                 GTATGTTCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  71 
                 TATGTTCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  72 
                 ATGTTCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  73 
                 TGTTCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  74 
                 GTTCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  75 
                 TTCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  76 
                 TCCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  77 
                 CCTGCTTCATGCCTTTC 
               
               
                   
               
               
                  78 
                 CTGCTTCATGCCTTTC 
               
               
                   
               
               
                  79 
                 TGCTTCATGCCTTTC 
               
               
                   
               
               
                  80 
                 GTATGTTCCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  81 
                 TATGTTCCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  82 
                 ATGTTCCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  83 
                 TGTTCCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  84 
                 GTTCCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  85 
                 TTCCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  86 
                 TCCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  87 
                 CCTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  88 
                 CTGCTTCATGCCTTTCT 
               
               
                   
               
               
                  89 
                 TGCTTCATGCCTTTCT 
               
               
                   
               
               
                  90 
                 GCTTCATGCCTTTCT 
               
               
                   
               
               
                  91 
                 TATGTTCCTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  92 
                 ATGTTCCTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  93 
                 TGTTCCTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  94 
                 GTTCCTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  95 
                 TTCCTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  96 
                 TCCTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  97 
                 CCTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  98 
                 CTGCTTCATGCCTTTCTA 
               
               
                   
               
               
                  99 
                 TGCTTCATGCCTTTCTA 
               
               
                   
               
               
                 100 
                 GCTTCATGCCTTTCTA 
               
               
                   
               
               
                 101 
                 CTTCATGCCTTTCTA 
               
               
                   
               
               
                 102 
                 ATGTTCCTGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 103 
                 TGTTCCTGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 104 
                 GTTCCTGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 105 
                 TTCCTGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 106 
                 TCCTGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 107 
                 CCTGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 108 
                 CTGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 109 
                 TGCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 110 
                 GCTTCATGCCTTTCTAC 
               
               
                   
               
               
                 111 
                 CTTCATGCCTTTCTAC 
               
               
                   
               
               
                 112 
                 TTCATGCCTTTCTAC 
               
               
                   
               
               
                 113 
                 TGTTCCTGCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 114 
                 GTTCCTGCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 115 
                 TTCCTGCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 116 
                 TCCTGCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 117 
                 CCTGCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 118 
                 CTGCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 119 
                 TGCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 120 
                 GCTTCATGCCTTTCTACA 
               
               
                   
               
               
                 121 
                 CTTCATGCCTTTCTACA 
               
               
                   
               
               
                 122 
                 TTCATGCCTTTCTACA 
               
               
                   
               
               
                 123 
                 TCATGCCTTTCTACA 
               
               
                   
               
               
                 124 
                 GTTCCTGCTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 125 
                 TTCCTGCTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 126 
                 TCCTGCTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 127 
                 CCTGCTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 128 
                 CTGCTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 129 
                 TGCTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 130 
                 GCTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 131 
                 CTTCATGCCTTTCTACAG 
               
               
                   
               
               
                 132 
                 TTCATGCCTTTCTACAG 
               
               
                   
               
               
                 133 
                 TCATGCCTTTCTACAG 
               
               
                   
               
               
                 134 
                 CATGCCTTTCTACAG 
               
               
                   
               
               
                 135 
                 TTCCTGCTTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 136 
                 TCCTGCTTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 137 
                 CCTGCTTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 138 
                 CTGCTTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 139 
                 TGCTTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 140 
                 GCTTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 141 
                 CTTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 142 
                 TTCATGCCTTTCTACAGT 
               
               
                   
               
               
                 143 
                 TCATGCCTTTCTACAGT 
               
               
                   
               
               
                 144 
                 CATGCCTTTCTACAGT 
               
               
                   
               
               
                 145 
                 ATGCCTTTCTACAGT 
               
               
                   
               
               
                 146 
                 TCCTGCTTCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 147 
                 CCTGCTTCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 148 
                 CTGCTTCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 149 
                 TGCTTCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 150 
                 GCTTCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 151 
                 CTTCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 152 
                 TTCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 153 
                 TCATGCCTTTCTACAGTG 
               
               
                   
               
               
                 154 
                 CATGCCTTTCTACAGTG 
               
               
                   
               
               
                 155 
                 ATGCCTTTCTACAGTG 
               
               
                   
               
               
                 156 
                 TGCCTTTCTACAGTG 
               
               
                   
               
               
                 157 
                 CCTGCTTCATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 158 
                 CTGCTTCATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 159 
                 TGCTTCATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 160 
                 GCTTCATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 161 
                 CTTCATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 162 
                 TTCATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 163 
                 TCATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 164 
                 CATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 165 
                 ATGCCTTTCTACAGTGG 
               
               
                   
               
               
                 166 
                 TGCCTTTCTACAGTGG 
               
               
                   
               
               
                 167 
                 GCCTTTCTACAGTGG 
               
               
                   
               
               
                 168 
                 CTGCTTCATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 169 
                 TGCTTCATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 170 
                 GCTTCATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 171 
                 CTTCATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 172 
                 TTCATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 173 
                 TCATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 174 
                 CATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 175 
                 ATGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 176 
                 TGCCTTTCTACAGTGGC 
               
               
                   
               
               
                 177 
                 GCCTTTCTACAGTGGC 
               
               
                   
               
               
                 178 
                 CCTTTCTACAGTGGC 
               
               
                   
               
               
                 179 
                 TGCTTCATGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 180 
                 GCTTCATGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 181 
                 CTTCATGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 182 
                 TTCATGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 183 
                 TCATGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 184 
                 CATGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 185 
                 ATGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 186 
                 TGCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 187 
                 GCCTTTCTACAGTGGCC 
               
               
                   
               
               
                 188 
                 CCTTTCTACAGTGGCC 
               
               
                   
               
               
                 189 
                 CTTTCTACAGTGGCC 
               
               
                   
               
               
                 190 
                 GCTTCATGCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 191 
                 CTTCATGCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 192 
                 TTCATGCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 193 
                 TCATGCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 194 
                 CATGCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 195 
                 ATGCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 196 
                 TGCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 197 
                 GCCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 198 
                 CCTTTCTACAGTGGCCT 
               
               
                   
               
               
                 199 
                 CTTTCTACAGTGGCCT 
               
               
                   
               
               
                 200 
                 TTTCTACAGTGGCCT 
               
               
                   
               
               
                 201 
                 CTTCATGCCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 202 
                 TTCATGCCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 203 
                 TCATGCCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 204 
                 CATGCCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 205 
                 ATGCCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 206 
                 TGCCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 207 
                 GCCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 208 
                 CCTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 209 
                 CTTTCTACAGTGGCCTT 
               
               
                   
               
               
                 210 
                 TTTCTACAGTGGCCTT 
               
               
                   
               
               
                 211 
                 TTCTACAGTGGCCTT 
               
               
                   
               
               
                 212 
                 TTCATGCCTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 213 
                 TCATGCCTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 214 
                 CATGCCTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 215 
                 ATGCCTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 216 
                 TGCCTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 217 
                 GCCTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 218 
                 CCTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 219 
                 CTTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 220 
                 TTTCTACAGTGGCCTTA 
               
               
                   
               
               
                 221 
                 TTCTACAGTGGCCTTA 
               
               
                   
               
               
                 222 
                 TCTACAGTGGCCTTA 
               
               
                   
               
               
                 223 
                 TCATGCCTTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 224 
                 CATGCCTTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 225 
                 ATGCCTTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 226 
                 TGCCTTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 227 
                 GCCTTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 228 
                 CCTTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 229 
                 CTTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 230 
                 TTTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 231 
                 TTCTACAGTGGCCTTAT 
               
               
                   
               
               
                 232 
                 TCTACAGTGGCCTTAT 
               
               
                   
               
               
                 233 
                 CTACAGTGGCCTTAT 
               
               
                   
               
               
                 234 
                 CATGCCTTTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 235 
                 ATGCCTTTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 236 
                 TGCCTTTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 237 
                 GCCTTTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 238 
                 CCTTTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 239 
                 CTTTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 240 
                 TTTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 241 
                 TTCTACAGTGGCCTTATC 
               
               
                   
               
               
                 242 
                 TCTACAGTGGCCTTATC 
               
               
                   
               
               
                 243 
                 CTACAGTGGCCTTATC 
               
               
                   
               
               
                 244 
                 TACAGTGGCCTTATC 
               
               
                   
               
               
                 245 
                 ATGCCTTTCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 246 
                 TGCCTTTCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 247 
                 GCCTTTCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 248 
                 CCTTTCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 249 
                 CTTTCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 250 
                 TTTCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 251 
                 TTCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 252 
                 TCTACAGTGGCCTTATCC 
               
               
                   
               
               
                 253 
                 CTACAGTGGCCTTATCC 
               
               
                   
               
               
                 254 
                 TACAGTGGCCTTATCC 
               
               
                   
               
               
                 255 
                 ACAGTGGCCTTATCC 
               
               
                   
               
               
                 256 
                 TGCCTTTCTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 257 
                 GCCTTTCTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 258 
                 CCTTTCTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 259 
                 CTTTCTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 260 
                 TTTCTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 261 
                 TTCTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 262 
                 TCTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 263 
                 CTACAGTGGCCTTATCCC 
               
               
                   
               
               
                 264 
                 TACAGTGGCCTTATCCC 
               
               
                   
               
               
                 265 
                 TCAGTGGCCTTATCCC 
               
               
                   
               
               
                 266 
                 CAGTGGCCTTATCCC 
               
               
                   
               
               
                 267 
                 GCCTTTCTACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 268 
                 CCTTTCTACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 269 
                 CTTTCTACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 270 
                 TTTCTACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 271 
                 TTCTACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 272 
                 TCTACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 273 
                 CTACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 274 
                 TACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 275 
                 ACAGTGGCCTTATCCCT 
               
               
                   
               
               
                 276 
                 CAGTGGCCTTATCCCT 
               
               
                   
               
               
                 277 
                 AGTGGCCTTATCCCT 
               
               
                   
               
            
           
         
       
     
     Examples of siRNA molecules targeting mRNAs transcribed from the DNA sequences listed in Table 2 are provided in Table 3 below. 
     
       
         
           
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 SEQ ID NO 
                 siRNA molecule sequence 
               
               
                   
               
             
            
               
                 278 
                 GGCCUUGGUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 279 
                 GCCUUGGUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 280 
                 CCUUGGUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 281 
                 CUUGGUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 282 
                 UUGGUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 283 
                 UGGUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 284 
                 GGUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 285 
                 GUAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 286 
                 UAUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 287 
                 AUGUUCCUGCUUCAUG 
               
               
                   
               
               
                 288 
                 UGUUCCUGCUUCAUG 
               
               
                   
               
               
                 289 
                 GCCUUGGUAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 290 
                 CCUUGGUAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 291 
                 CUUGGUAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 292 
                 UUGGUAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 293 
                 UGGUAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 294 
                 GGUAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 295 
                 GUAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 296 
                 UAUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 297 
                 AUGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 298 
                 UGUUCCUGCUUCAUGC 
               
               
                   
               
               
                 299 
                 GUUCCUGCUUCAUGC 
               
               
                   
               
               
                 300 
                 CCUUGGUAUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 301 
                 CUUGGUAUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 302 
                 UUGGUAUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 303 
                 UGGUAUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 304 
                 GGUAUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 305 
                 GUAUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 306 
                 UAUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 307 
                 AUGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 308 
                 UGUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 309 
                 GUUCCUGCUUCAUGCC 
               
               
                   
               
               
                 310 
                 UUCCUGCUUCAUGCC 
               
               
                   
               
               
                 311 
                 CUUGGUAUGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 312 
                 UUGGUAUGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 313 
                 UGGUAUGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 314 
                 GGUAUGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 315 
                 GUAUGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 316 
                 UAUGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 317 
                 AUGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 318 
                 UGUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 319 
                 GUUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 320 
                 UUCCUGCUUCAUGCCU 
               
               
                   
               
               
                 321 
                 UCCUGCUUCAUGCCU 
               
               
                   
               
               
                 322 
                 UUGGUAUGUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 323 
                 UGGUAUGUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 324 
                 GGUAUGUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 325 
                 GUAUGUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 326 
                 UAUGUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 327 
                 AUGUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 328 
                 UGUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 329 
                 GUUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 330 
                 UUCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 331 
                 UCCUGCUUCAUGCCUU 
               
               
                   
               
               
                 332 
                 CCUGCUUCAUGCCUU 
               
               
                   
               
               
                 333 
                 UGGUAUGUUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 334 
                 GGUAUGUUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 335 
                 GUAUGUUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 336 
                 UAUGUUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 337 
                 AUGUUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 338 
                 UGUUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 339 
                 GUUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 340 
                 UUCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 341 
                 UCCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 342 
                 CCUGCUUCAUGCCUUU 
               
               
                   
               
               
                 343 
                 CUGCUUCAUGCCUUU 
               
               
                   
               
               
                 344 
                 GGUAUGUUCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 345 
                 GUAUGUUCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 346 
                 UAUGUUCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 347 
                 AUGUUCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 348 
                 UGUUCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 349 
                 GUUCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 350 
                 UUCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 351 
                 UCCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 352 
                 CCUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 353 
                 CUGCUUCAUGCCUUUC 
               
               
                   
               
               
                 354 
                 UGCUUCAUGCCUUUC 
               
               
                   
               
               
                 355 
                 GUAUGUUCCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 356 
                 UAUGUUCCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 357 
                 AUGUUCCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 358 
                 UGUUCCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 359 
                 GUUCCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 360 
                 UUCCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 361 
                 UCCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 362 
                 CCUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 363 
                 CUGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 364 
                 UGCUUCAUGCCUUUCU 
               
               
                   
               
               
                 365 
                 GCUUCAUGCCUUUCU 
               
               
                   
               
               
                 366 
                 UAUGUUCCUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 367 
                 AUGUUCCUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 368 
                 UGUUCCUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 369 
                 GUUCCUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 370 
                 UUCCUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 371 
                 UCCUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 372 
                 CCUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 373 
                 CUGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 374 
                 UGCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 375 
                 GCUUCAUGCCUUUCUA 
               
               
                   
               
               
                 376 
                 CUUCAUGCCUUUCUA 
               
               
                   
               
               
                 377 
                 AUGUUCCUGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 378 
                 UGUUCCUGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 379 
                 GUUCCUGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 380 
                 UUCCUGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 381 
                 UCCUGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 382 
                 CCUGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 383 
                 CUGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 384 
                 UGCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 385 
                 GCUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 386 
                 CUUCAUGCCUUUCUAC 
               
               
                   
               
               
                 387 
                 UUCAUGCCUUUCUAC 
               
               
                   
               
               
                 388 
                 UGUUCCUGCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 389 
                 GUUCCUGCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 390 
                 UUCCUGCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 391 
                 UCCUGCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 392 
                 CCUGCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 393 
                 CUGCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 394 
                 UGCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 395 
                 GCUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 396 
                 CUUCAUGCCUUUCUACA 
               
               
                   
               
               
                 397 
                 UUCAUGCCUUUCUACA 
               
               
                   
               
               
                 398 
                 UCAUGCCUUUCUACA 
               
               
                   
               
               
                 399 
                 GUUCCUGCUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 400 
                 UUCCUGCUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 401 
                 UCCUGCUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 402 
                 CCUGCUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 403 
                 CUGCUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 404 
                 UGCUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 405 
                 GCUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 406 
                 CUUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 407 
                 UUCAUGCCUUUCUACAG 
               
               
                   
               
               
                 408 
                 UCAUGCCUUUCUACAG 
               
               
                   
               
               
                 409 
                 CAUGCCUUUCUACAG 
               
               
                   
               
               
                 410 
                 UUCCUGCUUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 411 
                 UCCUGCUUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 412 
                 CCUGCUUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 413 
                 CUGCUUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 414 
                 UGCUUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 415 
                 GCUUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 416 
                 CUUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 417 
                 UUCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 418 
                 UCAUGCCUUUCUACAGU 
               
               
                   
               
               
                 419 
                 CAUGCCUUUCUACAGU 
               
               
                   
               
               
                 420 
                 AUGCCUUUCUACAGU 
               
               
                   
               
               
                 421 
                 UCCUGCUUCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 422 
                 CCUGCUUCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 423 
                 CUGCUUCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 424 
                 UGCUUCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 425 
                 GCUUCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 426 
                 CUUCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 427 
                 UUCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 428 
                 UCAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 429 
                 CAUGCCUUUCUACAGUG 
               
               
                   
               
               
                 430 
                 AUGCCUUUCUACAGUG 
               
               
                   
               
               
                 431 
                 UGCCUUUCUACAGUG 
               
               
                   
               
               
                 432 
                 CCUGCUUCAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 433 
                 CUGCUUCAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 434 
                 UGCUUCAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 435 
                 GCUUCAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 436 
                 CUUCAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 437 
                 UUCAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 438 
                 UCAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 439 
                 CAUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 440 
                 AUGCCUUUCUACAGUGG 
               
               
                   
               
               
                 441 
                 UGCCUUUCUACAGUGG 
               
               
                   
               
               
                 442 
                 GCCUUUCUACAGUGG 
               
               
                   
               
               
                 443 
                 CUGCUUCAUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 444 
                 UGCUUCAUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 445 
                 GCUUCAUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 446 
                 CUUCAUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 447 
                 UUCAUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 448 
                 UCAUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 449 
                 CAUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 450 
                 AUGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 451 
                 UGCCUUUCUACAGUGGC 
               
               
                   
               
               
                 452 
                 GCCUUUCUACAGUGGC 
               
               
                   
               
               
                 453 
                 CCUUUCUACAGUGGC 
               
               
                   
               
               
                 454 
                 UGCUUCAUGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 455 
                 GCUUCAUGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 456 
                 CUUCAUGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 457 
                 UUCAUGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 458 
                 UCAUGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 459 
                 CAUGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 460 
                 AUGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 461 
                 UGCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 462 
                 GCCUUUCUACAGUGGCC 
               
               
                   
               
               
                 463 
                 CCUUUCUACAGUGGCC 
               
               
                   
               
               
                 464 
                 CUUUCUACAGUGGCC 
               
               
                   
               
               
                 465 
                 GCUUCAUGCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 466 
                 CUUCAUGCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 467 
                 UUCAUGCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 468 
                 UCAUGCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 469 
                 CAUGCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 470 
                 AUGCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 471 
                 UGCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 472 
                 GCCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 473 
                 CCUUUCUACAGUGGCCU 
               
               
                   
               
               
                 474 
                 CUUUCUACAGUGGCCU 
               
               
                   
               
               
                 475 
                 UUUCUACAGUGGCCU 
               
               
                   
               
               
                 476 
                 CUUCAUGCCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 477 
                 UUCAUGCCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 478 
                 UCAUGCCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 479 
                 CAUGCCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 480 
                 AUGCCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 481 
                 UGCCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 482 
                 GCCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 483 
                 CCUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 484 
                 CUUUCUACAGUGGCCUU 
               
               
                   
               
               
                 485 
                 UUUCUACAGUGGCCUU 
               
               
                   
               
               
                 486 
                 UUCUACAGUGGCCUU 
               
               
                   
               
               
                 487 
                 UUCAUGCCUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 488 
                 UCAUGCCUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 489 
                 CAUGCCUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 490 
                 AUGCCUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 491 
                 UGCCUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 492 
                 GCCUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 493 
                 CCUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 494 
                 CUUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 495 
                 UUUCUACAGUGGCCUUA 
               
               
                   
               
               
                 496 
                 UUCUACAGUGGCCUUA 
               
               
                   
               
               
                 497 
                 UCUACAGUGGCCUUA 
               
               
                   
               
               
                 498 
                 UCAUGCCUUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 499 
                 CAUGCCUUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 500 
                 AUGCCUUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 501 
                 UGCCUUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 502 
                 GCCUUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 503 
                 CCUUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 504 
                 CUUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 505 
                 UUUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 506 
                 UUCUACAGUGGCCUUAU 
               
               
                   
               
               
                 507 
                 UCUACAGUGGCCUUAU 
               
               
                   
               
               
                 508 
                 CUACAGUGGCCUUAU 
               
               
                   
               
               
                 509 
                 CAUGCCUUUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 510 
                 AUGCCUUUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 511 
                 UGCCUUUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 512 
                 GCCUUUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 513 
                 CCUUUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 514 
                 CUUUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 515 
                 UUUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 516 
                 UUCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 517 
                 UCUACAGUGGCCUUAUC 
               
               
                   
               
               
                 518 
                 CUACAGUGGCCUUAUC 
               
               
                   
               
               
                 519 
                 UACAGUGGCCUUAUC 
               
               
                   
               
               
                 520 
                 AUGCCUUUCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 521 
                 UGCCUUUCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 522 
                 GCCUUUCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 523 
                 CCUUUCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 524 
                 CUUUCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 525 
                 UUUCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 526 
                 UUCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 527 
                 UCUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 528 
                 CUACAGUGGCCUUAUCC 
               
               
                   
               
               
                 529 
                 UACAGUGGCCUUAUCC 
               
               
                   
               
               
                 530 
                 ACAGUGGCCUUAUCC 
               
               
                   
               
               
                 531 
                 UGCCUUUCUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 532 
                 GCCUUUCUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 533 
                 CCUUUCUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 534 
                 CUUUCUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 535 
                 UUUCUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 536 
                 UUCUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 537 
                 UCUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 538 
                 CUACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 539 
                 UACAGUGGCCUUAUCCC 
               
               
                   
               
               
                 540 
                 UCAGUGGCCUUAUCCC 
               
               
                   
               
               
                 541 
                 CAGUGGCCUUAUCCC 
               
               
                   
               
               
                 542 
                 GCCUUUCUACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 543 
                 CCUUUCUACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 544 
                 CUUUCUACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 545 
                 UUUCUACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 546 
                 UUCUACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 547 
                 UCUACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 548 
                 CUACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 549 
                 UACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 550 
                 ACAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 551 
                 CAGUGGCCUUAUCCCU 
               
               
                   
               
               
                 552 
                 AGUGGCCUUAUCCCU 
               
               
                   
               
            
           
         
       
     
     A siRNA molecule may be capable of inhibiting the expression of a target gene, such as the PNPLA3 148M allele. Herein, the terms “silencing” or “reducing” may be used interchangeably with “inhibiting.” To examine the extent of inhibition of expression by a siRNA, a siRNA of interest may be added to a test sample and monitored for expression along with a negative control sample to which the siRNA was not added. Preferably, a negative control sample will be similar to the test sample. More preferably, the negative control sample will be identical to the test sample. Examples of negative control samples include untreated samples, samples to which a siRNA-free buffer was added, or samples to which a negative control or mock siRNA was added. Expression in the test sample can then be compared to expression in the negative control sample. Expression may be measured by the detection of any expression product known in the art or yet to be disclosed. Typical expression products that may be detected include RNA and protein. 
     Methods known in the art for the detection and quantification of RNA expression in a sample include northern blotting and in situ hybridization (Parker and Barnes, Methods in Molecular Biology 106, 247-283 (1999) incorporated by reference herein in its entirety); RNAse protection assays (Hod, Biotechniques 13, 852-854 (1992) incorporated by reference herein in its entirety); and PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR) (Weis et al., Trends in Genetics 8, 263-264 (1992) incorporated by reference herein in its entirety). Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS). (See Mardis E R, Annu Rev Genomics Hum Genet 9, 387-402 (2008))(the content of which is incorporated by reference herein in its entirety). 
     Proteins, for example, can be detected and quantified through epitopes recognized by polyclonal and/or monoclonal antibodies used in methods such as ELISA, immunoblot assays, flow cytometric assays, immunohistochemical assays, radioimmuno assays, Western blot assays, an immunofluorescent assays, chemiluminescent assays and other polypeptide detection strategies. Proteins may also be detected by mass spectrometry assays (potentially coupled to immunoaffinity assays) including matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquid chromatography/quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS). Additionally, protein expression may be detected by tagging of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), (Kiernan et al, Anal Biochem 301, 49-56 (2002); Poutanen et al, Mass Spectrom 15, 1685-1692 (2001) the content of each of which is incorporated by reference herein in its entirety) or any other method of detecting protein. 
     In general, negative control samples are assigned a value of 100%. Inhibition of expression of a target gene may be achieved when the expression of the test sample relative to the control sample is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, less than 1% or 0%. Expression of a test sample relative to a negative control sample may also be presented in terms of fold reduction, such as a 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold less expression than the negative control sample. 
     Two or more nucleic acid sequences or subsequences may be referred to as being substantially identical, meaning that they are exactly the same or have a specified percentage of nucleotides that are the same. Substantially identical nucleotides may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more than 95% identity over a specified region when compared and aligned for maximum correspondence. This definition, when the context indicates, also refers to the complement of a sequence. Preferably, the substantial identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length. 
     SiRNA molecules can be provided in several forms including, e.g., as one or more isolated siRNA duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcriptional cassette in a DNA plasmid. The siRNA sequences may have overhangs (as 3′ or 5′ overhangs as described in Elbashir et al, Genes Dev 15, 188 (2001) or Nykanen et al, Cell 107, 309 (2001), the content of each of which is incorporated by reference herein in its entirety) or may lack overhangs (i.e., have blunt ends). 
     One or more DNA plasmids encoding one or more siRNA templates may be used to provide siRNA. siRNA can be transcribed as sequences that automatically fold into duplexes with hairpin loops from DNA templates in plasmids having RNA polymerase Ill transcriptional units, for example, based on the naturally occurring transcription units for small nuclear RNA U6 or human RNase P RNA H1 (Brummelkamp et al, Science 296, 550 (2002); Donze et al, Nucleic Acids Res 30, e46 (2002); Paddison et al, Genes Dev 16, 948 (2002); Yu et al, Proc Natl Acad Sci USA 99, 6047 (2002); Lee et al, Nat Biotech, 20, 500 (2002); Miyagishi et al, Nat Biotech 20, 497 (2002); Paul et al, Nat Biotech, 20, 505 (2002); and Sui et al, Proc Natl Acad Sci USA, 99, 5515 (2002); the content of each of which is incorporated by reference herein in its entirety). Typically, a transcriptional unit or cassette will contain an RNA transcript promoter sequence, such as an H1-RNA or a U6 promoter, operably linked to a template for transcription of a desired siRNA sequence and a termination sequence, comprised of 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal) (Brummelkamp et al (2002) supra). The selected promoter can provide for constitutive or inducible transcription. Compositions and methods for DNA-directed transcription of RNA interference molecules are described in detail in U.S. Pat. No. 6,573,099, incorporated by reference herein in its entirety. The transcriptional unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. Pat. Nos. 5,962,428 and 5,910,488, the content of each of which is incorporated by reference herein in its entirety. The selected plasmid can provide for transient or stable delivery of a nucleic acid to a target cell. It will be apparent to those of skill in the art that plasmids originally designed to express desired gene sequences can be modified to contain a transcriptional unit cassette for transcription of siRNA. 
     Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene 25, 263-269 (1983); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y., (2001), the content of each of which is incorporated by reference herein in its entirety) as are PCR methods (see, U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications, Innis et al, eds, (1990), the content of each of which is incorporated by reference herein in its entirety). Expression libraries are also well known to those of skill in the art. Additional basic texts disclosing the general methods of use in this invention include Sambrook and Russell (2001) supra; Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994), the content of each of which is incorporated by reference herein in its entirety. 
     A siRNA molecule may be chemically synthesized. In one example of chemical synthesis, a single-stranded nucleic acid that includes the siRNA duplex sequence can be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al, J Am Chem Soc, 109, 7845 (1987); Scaringe et al, Nucl Acids Res, 18, 5433 (1990); Wincott et al, Nucl Acids Res, 23, 2677-2684 (1995); and Wincott et al, Methods Mol Bio 74, 59 (1997), the content of each of which is incorporated by reference herein in its entirety. Synthesis of the single-stranded nucleic acid makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5′-end and phosphoramidites at the 3′-end. As a non-limiting example, small scale syntheses can be conducted on an Applied Biosystems synthesizer (Thermo Fisher Scientific, Waltham, Mass.) using a 0.2 micromolar scale protocol with a 2.5 min coupling step for 2′-O-methylated nucleotides. Alternatively, syntheses at the 0.2 micromolar scale can be performed on a 96-well plate synthesizer from Thermo Fisher Scientific. However, larger or smaller scale synthesis are also encompassed by the invention, including any method of synthesis now known or yet to be disclosed. Suitable reagents for synthesis of siRNA single-stranded molecules, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art. 
     In certain embodiments, siRNA can be synthesized via a tandem synthesis technique, wherein both strands are synthesized as a single continuous fragment or strand separated by a linker that is subsequently cleaved to provide separate fragments or strands that hybridize to form a siRNA duplex. Linkers may be any linker, including a polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siRNA can be readily adapted to both multiwell/multiplate synthesis platforms as well as large scale synthesis platforms employing batch reactors, synthesis columns, and the like. In some embodiments, siRNA can be assembled from two distinct single-stranded molecules, wherein one strand includes the sense strand and the other includes the antisense strand of the siRNA. For example, each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection. Either the sense or the antisense strand may contain additional nucleotides that are not complementary to one another and do not form a double stranded siRNA. In certain instances, siRNA molecules can be synthesized as a single continuous fragment, where the self-complementary sense and antisense regions hybridize to form a siRNA duplex having hairpin secondary structure. 
     A siRNA molecule may comprise a duplex having two complementary strands that form a double-stranded region with least one modified nucleotide in the double-stranded region. The modified nucleotide may be on one strand or both. If the modified nucleotide is present on both strands, it may be in the same or different positions on each strand. A modified siRNA may be less immunostimulatory than a corresponding unmodified siRNA sequence, but retains the capability of silencing the expression of a target sequence. 
     Examples of modified nucleotides suitable for use in the present invention include, but are not limited to, ribonucleotides having a 2′-O-methyl (2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl, 2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group. Modified nucleotides having a conformation such as those described in the art, for example in Saenger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984), incorporated by reference herein in its entirety, are also suitable for use in siRNA molecules. Other modified nucleotides include, without limitation: locked nucleic acid (LNA) nucleotides, G-clamp nucleotides, or nucleotide base analogs. LNA nucleotides include but need not be limited to 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides), 2′-O-(2-methoxyethyl) (MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy-2′-chloro (2Cl) nucleotides, and 2′-azido nucleotides. A G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex (Lin et al, J Am Chem Soc, 120, 8531-8532 (1998) incorporated by reference herein in its entirety). Nucleotide base analogs include for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (Loakes, Nucl Acids Res, 29, 2437-2447 (2001) incorporated by reference herein in its entirety). 
     A siRNA molecule may comprise one or more chemical modifications such as terminal cap moieties, phosphate backbone modifications, and the like. Examples of classes of terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl modifications, 4′,5′-methylene nucleotides, 1-(.beta.-D-erythrofuranosyl) nucleotides, 4′-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, .alpha.-nucleotides, modified base nucleotides, threo pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties, 3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties, 3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties, 5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties, 5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3 aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate, 5′ phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate, 5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate, and bridging or non-bridging methylphosphonate or 5′-mercapto moieties (see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al, Tetrahedron 49, 1925 (1993); the content of each of which is incorporated by reference herein in its entirety). Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al, Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al, Antisense Research, ACS, 24-39 (1994); the content of each of which is incorporated by reference herein in its entirety). Such chemical modifications can occur at the 5′-end and/or 3′-end of the sense strand, antisense strand, or both strands of the siRNA. 
     The sense and/or antisense strand of a siRNA may comprise a 3′-terminal overhang having 1 to 4 or more 2′-deoxyribonucleotides and/or any combination of modified and unmodified nucleotides. Additional examples of modified nucleotides and types of chemical modifications that can be introduced into the modified siRNA molecules of the present invention are described, e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos. 20040192626 and 20050282188, the content of each of which is incorporated by reference herein in its entirety. 
     A siRNA molecule may comprise one or more non-nucleotides in one or both strands of the siRNA. A non-nucleotide may be any subunit, functional group, or other molecular entity capable of being incorporated into a nucleic acid chain in the place of one or more nucleotide units that is not or does not comprise a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine, such as a sugar or phosphate. 
     Chemical modification of siRNA may comprise attaching a conjugate to a siRNA molecule. The conjugate can be attached at the 5′- and/or the 3′-end of the sense and/or the antisense strand of the siRNA via a covalent attachment such as a nucleic acid or non-nucleic acid linker. The conjugate can be attached to the siRNA through a carbamate group or other linking group (see, e.g., U.S. Patent Publication Nos. 20050074771, 20050043219, and 20050158727, the content of each of which is incorporated by reference herein in its entirety). A conjugate may be added to siRNA for any of a number of purposes. For example, the conjugate may be a molecular entity that facilitates the delivery of siRNA into a cell or may be a molecule that comprises a drug or label. Examples of conjugate molecules suitable for attachment to siRNA of the present invention include, without limitation, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S. Patent Publication Nos. 20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423; the content of each of which is incorporated by reference herein in its entirety). Other examples include the lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid, small molecule, oligosaccharide, carbohydrate cluster, intercalator, minor groove binder, cleaving agent, and cross-linking agent conjugate molecules described in U.S. Patent Publication Nos. 20050119470 and 20050107325, the content of each of which is incorporated by reference herein in its entirety. Other examples include the 2′-O-alkyl amine, 2′-O-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic amino acid conjugate molecules described in U.S. Patent Publication No. 20050153337, incorporated by reference herein in its entirety. Additional examples of conjugate molecules include a hydrophobic group, a membrane active compound, a cell penetrating compound, a cell targeting signal, an interaction modifier, or a steric stabilizer as described in U.S. Patent Publication No. 20040167090, incorporated by reference herein in its entirety. Further examples include the conjugate molecules described in U.S. Patent Publication No. 20050239739, incorporated by reference herein in its entirety. 
     The type of conjugate used and the extent of conjugation to the siRNA can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of the siRNA while retaining activity. As such, one skilled in the art can screen siRNA molecules having various conjugates attached thereto to identify siRNA conjugates having improved properties using any of a variety of well-known in vitro cell culture or in vivo animal models including the negative-controlled expression studies described above. 
     A siRNA may be incorporated into carrier systems containing siRNA molecules described herein. The carrier system may be a lipid-based carrier system such as a stabilized nucleic acid-lipid particle (e.g., SNALP or SPLP), cationic lipid or liposome nucleic acid complexes (i.e., lipoplexes), a liposome, a micelle, a virosome, or a mixture thereof. In other embodiments, the carrier system may be a polymer-based carrier system such as a cationic polymer-nucleic acid complex (i.e., polyplex). In additional embodiments, the carrier system can be a cyclodextrin-based carrier system such as a cyclodextrin polymer-nucleic acid complex (see US Patent Application Publication 20070218122, incorporated by reference herein in its entirety). In further embodiments, the carrier system may be a protein-based carrier system such as a cationic peptide-nucleic acid complex. A siRNA molecule may also be delivered as naked siRNA. 
     In certain embodiments, the carrier system can be a nanoparticle that includes low molecular weight polyethyleneimine (LPEI) or its derivatives (e.g., disulfide crosslinked polyethyleneimine (CLPEI)) and a lipid. The lipid may be a bile acid, such as cholic acid, deoxycholic acid, and lithocholic acid. Such carrier systems are described further in the Examples below. Other exemplary carrier systems are described for example in Wittrup et al. (Nature Reviews/Genetics, 16:543-552, 2015), the content of which is incorporated by reference herein in its entirety. 
     The compositions of the invention are particularly useful for treating a subject (e.g., a mammalian subject, e.g., human, child or adult) with a chronic liver disease or alcoholic liver disease (ALD). Chronic liver disease refers to diseases of the liver that last over a period of six months. It includes of a wide range of liver pathologies which include inflammation (chronic hepatitis), liver cirrhosis, and hepatocellular carcinoma. Alcoholic liver disease (ALD) typically occurs after years of heavy drinking. Over time, scarring and cirrhosis can occur. Cirrhosis is the final phase of alcoholic liver disease. There may be no symptoms, or symptoms may come on slowly, depending on how well the liver is working. Symptoms tend to be worse after a period of heavy drinking. Early symptoms include: fatigue and loss of energy; poor appetite and weight loss; nausea or belly pain; small, or red spider-like blood vessels on the skin As liver function worsens, symptoms may include: fluid buildup of the legs (edema) and in the abdomen (ascites); yellow color in the skin, mucous membranes, or eyes (jaundice); redness on the palms of the hands; easy bruising and abnormal bleeding; confusion or problems thinking; or pale or clay-colored stools. In men, symptoms may also include impotence, shrinking of the testicles, and breast swelling. 
     The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. 
     The phrases “systemic administration,” “administered systematically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient&#39;s system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. 
     When the compounds of the present invention are administered as pharmaceuticals, to humans and mammals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient, (e.g., a siRNA or antisense oligonucleotide of the invention) and/or derivative thereof, in combination with a pharmaceutically acceptable carrier. 
     The effective dosage of each agent can readily be determined by the skilled person, having regard to typical factors each as the age, weight, sex and clinical history of the patient. In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, doses of the compounds of this invention for a patient, when used for the indicated effects; will range from about 0.1 mg to about 250 mg per kilogram of body weight per day, more preferably from about 1 mg to about 60 mg per kg per day. 
     If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. 
     The pharmaceutical compositions of the invention include a “therapeutically effective amount” or a “prophylactically effective amount” of one or more of the compounds of the present invention, or functional derivatives thereof. An “effective amount” is the amount as defined herein in the definition section and refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., a diminishment or prevention of effects associated with neuropathic and/or inflammatory pain. A therapeutically effective amount of a compound of the present invention or functional derivatives thereof may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the therapeutic compound to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. 
     A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to, or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount. A prophylactically or therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the beneficial effects. 
     Dosage regimens may be adjusted to provide the optimum desired response (e.g. a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigency of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the patient. 
     The term “dosage unit” as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the compound, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals. 
     In some embodiments, therapeutically effective amount can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in other subjects. Generally, the therapeutically effective amount is sufficient to reduce or inhibit neuropathic and/or inflammatory pain in a subject. In some embodiments, the therapeutically effective amount is sufficient to eliminate neuropathic and/or inflammatory pain in a subject. Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol). A physician may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. The dose administered to a patient is sufficient to effect a beneficial therapeutic response in the patient over time, or, e.g., to reduce symptoms, or other appropriate activity, depending on the application. The dose is determined by the efficacy of the particular formulation, and the activity, stability or serum half-life of the compounds of the invention or functional derivatives thereof, and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose is also determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, formulation, or the like in a particular subject. Therapeutic compositions comprising one or more compounds of the invention or functional derivatives thereof are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, such as models of neuropathic and/or inflammatory pain, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art. In particular, dosages can be initially determined by activity, stability or other suitable measures of treatment vs. non-treatment (e.g., comparison of treated vs. untreated cells or animal models), in a relevant assay. Formulations are administered at a rate determined by the LD50 of the relevant formulation, and/or observation of any side-effects of compounds of the invention or functional derivatives thereof at various concentrations, e.g., as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses. 
     Administering typically involves administering pharmaceutically acceptable dosage forms, which means dosage forms of compounds described herein, and includes, for example, tablets, dragees, powders, elixirs, syrups, liquid preparations, including suspensions, sprays, inhalants tablets, lozenges, emulsions, solutions, granules, capsules, and suppositories, as well as liquid preparations for injections, including liposome preparations. Techniques and formulations generally may be found in Remington&#39;s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition, which is hereby incorporated by reference in its entirety. Administering may be carried out orally, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, or intranasally. Compounds may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions. 
     A pharmaceutical composition containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the techniques described in U.S. Pat. Nos. 4,256,108, 4,166,452 and 4,265,874 (the content of each of which is incorporated by reference herein in its entirety), to form osmotic therapeutic tablets for control release. 
     Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil. 
     Formulations may also include complexes of the parent (unionized) compounds with derivatives of β-cyclodextrin, especially hydroxypropyl-β-cyclodextrin. 
     An alternative oral formulation can be achieved using a controlled-release formulation, where the compound is encapsulated in an enteric coating. 
     Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such a polyoxyethylene with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin. 
     Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. 
     Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified, for example sweetening, flavoring and coloring agents, may also be present. 
     The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soya bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents. 
     Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer&#39;s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. 
     Each active agent may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols. 
     For topical use, creams, ointments, jellies, solutions or suspensions are suitable. Topical application includes the use of mouth washes and gargles. 
     The term “pharmaceutical composition” means a composition comprising a compound as described herein and at least one component comprising pharmaceutically acceptable carriers, diluents, adjuvants, excipients, or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms. The term “pharmaceutically acceptable carrier” is used to mean any carrier, diluent, adjuvant, excipient, or vehicle, as described herein. Examples of suspending agents include ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monosterate and gelatin. Examples of suitable carriers, diluents, solvents, or vehicles include water, ethanol, polyols, suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Examples of excipients include lactose, milk sugar, sodium citrate, calcium carbonate, and dicalcium phosphate. Examples of disintegrating agents include starch, alginic acids, and certain complex silicates. Examples of lubricants include magnesium stearate, sodium lauryl sulphate, talc, as well as high molecular weight polyethylene glycols. 
     The term “pharmaceutically acceptable” means it is, within the scope of sound medical judgment, suitable for use in contact with the cells of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. 
     INCORPORATION BY REFERENCE 
     References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. 
     EQUIVALENTS 
     Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof. 
     EXAMPLES 
     The PNPLA3 gene rs738409 C&gt;G polymorphism is associated with several types of liver disease (Shen, Journal of Lipid Research, 56:167-175, 2015), the content of which is incorporated by reference herein in its entirety. The G allele is associated with a significantly increased risk of chronic liver disease versus the C allele. Both the GC and GG genotypes are associated with a significantly increased risk of chronic liver disease versus the CC genotype. 
     Accordingly, the I148M mutation (rs738409 C&gt;G) in the PNPLA3 gene is a strong genetic risk factor for a series of chronic liver diseases, including nonalcoholic/alcoholic fatty liver disease, steatohepatitis, cirrhosis and hepatocellular carcinoma. It has been demonstrated that overexpression of the 148M isoform and not 148I is an important step for the manifestation of these phenotypes. Meanwhile, 148M has been demonstrated as a loss-of-function mutation that reduces the enzymatic activity (triglycerides hydrolase and other unknown function) as compared to the 148I isoform. This indicates that 148M plays a pathogenic role in liver disease etiology in a dominant-negative manner. 
     Without being limited by any particular theory of mechanism of action, it is believed that reducing the expression of the 148M isoform will lead to reversing disease progress from simple steatosis, steatohepatitis, cirrhosis and even liver cancer. Accordingly, the invention provides siRNA specifically targeting the 148M isoform to reduce its mRNA and protein expression. It has been found that the compositions of the invention have minimal effect on the wild-type 148I isoform. The data herein show that cell lines based model has confirmed that reducing the 148M isoform leads to reduced fat accumulation in human HepG2 cell line. 
     Example 1: In Vitro Systems for Pharmacological Testing of siRNA Molecules 
     The present invention identifies a potent siRNA specifically downregulating PNPLA3 148M isoform, namely 148MSi (SEQ ID NO.: 1) and associated sequences SEQ ID NOs: 2 and 278-552. The therapeutic potential of 148MSi may be further characterized through additional testing. To this end: 1) stable cell lines can be established constantly expressing PNPLA3148M-Luc and PNPLA3148I-Luc, which facilitates a subsequent high throughput optimization of 148MSi and its modified analogs in targeting PNPLA3 148M; and 2) The therapeutic effectiveness of 148MSi can be examined in vitro, which can collect key pharmacological parameters to further foster the basis of 148MSi as a therapeutic agent. 
     To establish stable cell lines and perform a high throughput screening for more candidate siRNA targeting PNPLA3 148M, lentivirus particles may be generated packing the vectors with each of the PNPLA3 148M-Luc and PNPLA3 148I-Luc fusion genes. The virus particles may be transduced into HEK293 cells. Cells constantly expressing the fusion reporter proteins can be selected using puromycin. Following the establishment of these stable cells, a high-throughput screening can be performed to further identify optimal siRNA candidates. Briefly, a series of siRNAs specifically targeting the 148M allele can be designed by altering their length and/or position to target, as well as by artificially introducing mutations into the siRNA sequence. These siRNAs may be transiently transfected into the stable cells to test their specificity and potency by comparing to that of the 148MSi. 
     It may be that the stable cells are able to serve as an in vitro model for rapid screening of siRNA candidates, which may lead to the identification of other siRNAs possessing equivalent to or even better therapeutic properties than 148MSi. Vectors may be created in the lab and transient transfection may be performed as below. 
     To examine the therapeutic effectiveness of 148MSi in vitro, the high specificity and potency of 148MSi may be demonstrated, as well as the potential of 148MSi treatment in reducing triglycerides (TG) accumulation (a hallmark of hepatic steatosis) under glucose induction of endogenous PNPLA3 148M in Huh-7 cells (See Examples below). However, glucose treatment may alter many pathways which might confound the effect of PNPLA3. Artificially increasing the PNPLA3 148M expression using adenoviral vectors without integrating into the genome would be useful to examine the focused effect of PNPLA3 148M on the induction of hepatic steatosis. PNPLA3 148M and PNPLA3 148I can be cloned into adenoviral vectors. PNPLA3 148M and PNPLA3 148I adenovirus particles can be generated to transduce the Huh-7 cells, respectively. 148MSi, in a series of concentrations, may be transfected into the cells using Lipofectamine 2000 (Thermal Fisher). The transfected cells can then be treated with free fatty acids (palmitic acid+oleic acid) for 48 hours, and TG accumulation may be measured using Oil Red O staining, and can be further compared between the cells transduced with different PNPLA3 isoforms. 
     Overexpression of PNPLA3148M isoform may significantly induce TG accumulation as compared to the 148I isoform. Transfection of 148MSi can significantly reduce TG accumulation in a dose-dependent manner and specifically in cells transduced with PNPLA3 148M but not PNPLA3 148I. Although Huh-7 is a PNPLA3 148M homozygote, the expression level of PNPLA3 may be insufficient to induce TG accumulation. Previous studies have shown that overexpression of PNPLA3 148M in hepatocyte indeed increased TG accumulation, and knockdown of PNPLA3 using generic commercial siRNAs can reverse that effect. 148MSi of the invention can outperform generic siRNAs in PNPLA3 148M targeting as shown below and the effect can likely be recapitulated in Huh-7. In one embodiment, short hairpin RNAs (shRNA) for 148MSi can be created and further packaged into adenovirus to perform co-transfection with PNPLA3 adenovirus. 
     Example 2: siRNA Targeting and Therapeutic Response In Vivo 
     To validate the cell-based data, useful mouse models can be created for in vivo evaluation of 148MSi. It has been reported that a short-term overexpression of PNPLA3 148M variant using an adenoviral vector leads to ˜3 fold increase in hepatic triglycerides. Transgenic mice that overexpress the PNPLA3 148M variant specifically in the liver also causes hepatic steatosis and dysregulated hepatic lipid metabolism. Thus, it is believed that overexpression of PNPLA3 148M variant in mouse liver should provide a useful tool for preclinical testing of the efficacy, pharmacodynamics, and pharmacokinetics. 
     Since adenoviral and lentiviral vectors can efficiently deliver gene products to the liver, their respective features (adenoviral vector does not integrate to the host genome but lentiviral vector does) can be advantageous to the development of both acute and chronic mouse models expressing human PNPLA3 148M ( FIG. 7 ). DNA constructs may be created for both systems, and large preparations can be made for animal injections. In order to validate the two models, PNPLA3 148I or PNPLA3 I148M adenoviruses (2×10 9  pfu/mouse) or lentiviruses (5×10 8  pfu/mouse) can be injected. For the adenoviral PNPLA3 model, mice may be fasted for 4 hours and sacrificed for blood and liver tissue collection 3 days after the injections. For the lentiviral PNPLA3 model, mice can be sacrificed 1 month after the injections. Serum and hepatic triglycerides and free fatty acids can be analyzed using commercial assay kits (Dako). Hepatic lipid droplets may be analyzed by H&amp;E and Oil Red 0 staining of liver sections. In order to facilitate longitudinal monitoring of the PNPLA3 expression, the two lentiviral vectors that carry PNPLA3 148I-Luc or PNPLA3 148M-Luc fusion gene mentioned in Example 1 above can be used. 
     Both adenoviral and lentiviral models for PNPLA3148M should develop hepatic steatosis manifested by increased liver triglycerides and lipid droplets. As an alternative strategy, an adeno-associated viral (AAV) vector system may be considered. 
     For the early phase of in vivo test, the adenoviral model can be used to assess the efficacy of PNPLA3 148M siRNAs. Two days after the adenoviral injection, 148MSi can be delivered using the Invivofectamine 3.0 reagent (Thermo Fisher, 1.5 mg/kg) into mice via tail vein injection. Two days later, animals can be sacrificed for blood and liver tissue collection. PNPLA3 knockdown efficiency may be analyzed by qPCR. Serum and liver TG can be analyzed as described above. To examine the long-term efficacy, the following lentiviral models may be used: PNPLA3 148I, PNPLA3 I148M, PNPLA3 148I-Luc and PNPLA3 148M-Luc. Two regimens can be performed on these 4 models: 1) Injection of 148MSi+Invivofectamine 3.0 (1.5 mg/kg) two days after the lentiviral injection and then weekly injection at a dose of 0.5 mg/kg for 1 month; 2) Injection of 148MSi+Invivofectamine 3.0 4 weeks after the lentiviral injection and then weekly injection at a dose of 0.5 mg/kg for 1 month. The luciferase signal of each mouse can be monitored weekly using, for example the Berthold LB981 NightOwl system (Berthold Technologies GmbH &amp; Co. KG, Germany). For assessing the potential toxicity, we can inject the same doses of siRNAs into wildtype C57BL6/J mice using the same regimens as mentioned above. The mice body weight can be monitored every two days for 1 month. At the end, animals may be euthanized for gross toxicity examination. Liver enzymes (ALT and AST) can be measured and liver histology can be examined. 
     These animal models should provide useful tools for siRNA tests. According to cellular data, 148MSi should knock down the PNPLA3 148M expression and reverse hepatic steatosis. Although serious toxicity from siRNA/Invivofectamine particles is not expected, any side effect may be noted and minimized by adjusting the dose regimens. 
     Example 3: Nanoparticle Vectors for siRNA Delivery to the Liver 
     Delivery of siRNA requires a carrier system that can protect siRNA from enzymatic degradation during circulation, prevent side effects due to non-specific distribution in off-target tissues, and help the siRNA to enter target cells. A ternary gene complex called DPH complex has been reported that includes nucleic acid, polycation (LPEI or disulfide-crosslinked polyethyleneimine, CLPEI), and polysaccharide. That complex has achieved superior gene transfection efficiency to that of commercial gene carriers like Lipofectamine or polyethyleneimine. However, due to its electrostatic nature of the complex, DPH is unstable in circulation and shows suboptimal gene transfection in vivo. It is believed that DPH with greater stability and smaller size can be produced by grafting lipid components to the polycation component such as LPEI ( FIG. 2 ). That belief is supported by the fact that cholesterol grafting reduces the size of polymeric micelle gene carriers from &gt;500 nm to &lt;200 nm by stabilizing the core-shell structure and increases colloidal stability. The lipid modification also facilitates cellular uptake of polyplexes, thereby increasing the gene transfection efficiency. Making a lipid-modified LPEI should help form small (&lt;100 nm) gene carriers, which can stably circulate, effectively reach the liver, and transfect hepatocytes. 
     Bile acids such as cholic acid, deoxycholic acid, and lithocholic acid (LCA), or their derivatives can be used given their biocompatibility, commercial availability, and chemical reactivity. For preparation of lipid-grafted LPEI, bile acids may be grafted as a NHS-activated bile acid to the secondary amine of LPEI ( FIG. 2A ). A LCA-grafted linear PEI (LPEI), which can be readily translated to conjugates of LCA and LPEI derivatives such as CLPEI, has also been synthesized. It has been confirmed that a ternary complex containing LCA-grafted LPEI (LCA-LPEI) performed better than that with LPEI in reporter gene transfection. The lipid content may be optimized considering its effect on particle stabilization and water solubility. It may be necessary to include a small quantity of water-miscible organic solvent such as DMSO to enhance the solubilization of the components and complex formation. Once a nanoparticulate gene carrier is formed, it is possible to remove the solvent via centrifugal filtration or dialysis. It is possible that the increased stability may interfere with intracellular dissociation of complex. Should this be the case, a lipid may be grafted to the secondary amine via disulfide bond, which can facilitate intracellular dissociation of the lipid. An amine derivative of bile acid may first be reacted with dithiobis [succinimidyl propionate] and then reacted with LPEI in the presence of N,N-diisopropylethylamine. Alternatively, LCA-LPEI can be produced using carbonyldiimidazole (CDA) as a coupling agent. Structures of synthetic intermediates and purified products can be confirmed by 1H-NMR analysis, based on the additional proton shifts indicating the steroid framework in 0.6-1.8 ppm ( FIG. 2B ). 
     Ternary complexes of siRNA, LCA-LPEI, and polysaccharides (hyaluronic acid (HA) or dermatan sulfate (DS)) may be formed following methods as shown in Xu, P., Quick, G., Yeo, Y, (2009), Gene delivery through the use of a hyaluronate-associated intracellularly degradable cross-linked polyethyleneimine, Biomaterials, 30(29):5834-5843, incorporated herein by reference in its entirety. Briefly, an siRNA-polymer binary complex may be first formed and then incubated with HA or DS to make a ternary complex. The transfection efficiency of 148Si can be tested in HepG2 cells with the PNPLA3-Luc as a reporter system. The ternary complexes may be characterized with respect to size and surface charge using Zetasizer Nano-ZS90. To examine particle size and its change in serum, the complexes can be incubated in 50% serum solution and sampled at different time points. To estimate chemical stability during circulation, the complexes may be incubated in the presence of serum, nucleases, and heparin (representing anionic glycosaminoglycans). The integrity of ternary complex can be tested with agarose gel electrophoresis as done in previous studies. See, Xu, P., et al., 2009, Gene delivery through the use of a hyaluronate-associated intracellularly degradable crosslinked polyethyleneimine, Biomaterials 30, 5834-5843 and Feng, M., et al., 2014, Stabilization of a hyaluronate-associated gene delivery system using calcium ions, Biomaterials Science, the content of each of which is incorporated by reference herein in its entirety. A complex that does not leach out siRNA upon the challenges may be considered stable. Cellular uptake of the siRNA/LCA-LPEI/HA (or DS) ternary complex can be evaluated with confocal microscopy and flow cytometry using fluorescently labeled siRNA and HepG2 cells. Uptake mechanism may be determined using inhibitors of different endocytosis pathways in each condition and changes in cellular uptake of complexes may be quantified with flow cytometry, and intracellular destination can be identified by co-localizing organelle markers with complexes under a confocal microscope. Cytotoxicity of the complexes may be assessed by incubating HepG2 cells with gene complexes equivalent to 1 to 100 μg/mL CLPEI with a MTT assay and comparing with cells treated with PBS, 148MSi, or LgCLPEI. 
     Derivatives of cholesterol and LCA have been synthesized. A LCA-grafted linear PEI (LPEI), which can be readily translated to LCA-CLPEI conjugates, has also been synthesized and it has been confirmed that a ternary complex containing LCA-grafted LPEI performed better than that with LPEI in reporter gene transfection. The lipid content may be optimized considering its effect on particle stabilization and water solubility. It may be necessary to include a small quantity of water-miscible organic solvent such as DMSO to enhance the solubilization of the components and complex formation. Once a nanoparticulate gene carrier is formed, it is possible to remove the solvent via centrifugal filtration or dialysis. It is possible that the increased stability may interfere with intracellular dissociation of complex. Should this be the case, a lipid may be grafted to the secondary amine via disulfide bond, which can facilitate intracellular dissociation of the lipid. An amine derivative of bile acid may first be reacted with dithiobis [succinimidyl propionate] and then reacted with CLPEI in the presence of N,N-diisopropylethylamine. 
     Example 4: Treating a Chronic Liver Disease with Compositions of the Invention 
     By using various genetic, genomics and systems-based approach, a number of polymorphisms, genes, lipids and miRNAs have been identified that are significantly associated with NAFLD and NASH susceptibility or drug response in treatment of NASH. Specifically for this application, it has been discovered that PNPLA3 148M has a high baseline expression level in human liver due to the linkage disequilibrium of rs738409 G (encoding the 148M isoform) and an intronic expression quantitative trait loci (eQTL) for PNPLA3, which confirms that a high transcription level of PNPLA3 148M is related to an increased risk for NAFLD/NASH. 
     LCA-LPEI has been successfully synthesized as well as a ternary complex of pEGFP, LCA-LPEI, and DS at varying polymer/DNA weight ratios (10/1 to 20/1 w/w). LCA-LPEI has showed greater gene transfection efficiency than LPEI. DS increased transfection for both LPEI and LCA-LPEI polyplexes at all levels of polymer/DNA ratios ( FIGS. 4A-C ). 
     Transient co-transfection of 148MSi and PNPLA3-Luciferase (PNPLA3-Luc) vectors into HEK293 cells has demonstrated that the 148M but not the 148I isoform was significantly down-regulated by 148MSi ( FIGS. 3A-C  and  FIG. 7 ). This regulation is dose-dependent with a IC50 (50% of mRNA to be cleared) of 8 pM ( FIG. 3A ). Further analyses demonstrated that 148MSi significantly downregulates endogenous level of PNPLA3 148M mRNA ( FIG. 3B ) but not the PNPLA3 148I wildtype ( FIG. 3C ). More importantly, 148MSi transient transfection significantly reduces the TG accumulation in HepG2 cells under induction by glucose ( FIGS. 3D-E ), as the baseline PNPLA3 expression in HepG2 is insufficient for inducing steatosis. 148MSi as an allele-specific siRNA has outperformed the gene knockdown effect of a commercial PNPLA3 siRNA set (Santa Cruz Biotechnology, Dallas, Tex., a mixture of multiple siRNA with unknown sequences) ( FIGS. 3B-C ). 
     Example 5: Treating a Chronic Liver Disease In Vivo with Compositions of the Invention 
     Compositions of the invention (e.g., mut-specific siRNA) were tested in vivo. The human PNPLA3 148M  was transduced into mice (n=9) liver via tail-vein injection of adenovirus particles. After 1 week of virus injection, siRNA or PBS (control) that was packed with INVIVOFECTAMINE 3.0 (an animal origin-free lipid nanoparticle designed for high efficiency in vivo delivery of siRNA and miRNA to mouse liver cells following tail vein injection; Thermo Fisher Scientific, Waltham, Mass.) were injected into the mice. Mice were sacrificed after 2 weeks, the human PNPLA3 gene expression as well as total triglycerides (TG) levels in the liver tissue were measured.  FIG. 8A  shows the significant (p&lt;0.05) reduction of hepatic PNPLA3 148M  expression after siRNA treatment.  FIGS. 8B through 8D  demonstrated the significant (p&lt;0.05) reduction of total hepatic TG levels after siRNA treatment.