Patent Publication Number: US-2022228141-A1

Title: Oligonucleotides for dgat2 modulation

Description:
RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/117,005, filed Nov. 23, 2020, the entire disclosure of which is hereby incorporated herein by reference. 
    
    
     STATEMENT OF FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under Grant No. DK103047 awarded by the National Institutes of Health. The Government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 29, 2022, is named 723374 UM9-261 ST25.txt and is 12,672 bytes in size. 
     FIELD OF THE DISCLOSURE 
     This disclosure relates to DGAT2 targeting sequences and methods for treating and preventing non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and lipodystrophy syndromes. 
     BACKGROUND 
     One of the many severe complications of type 2 diabetes is triglyceride accumulation in the liver (denoted non-alcoholic fatty liver disease, NAFLD), which can then lead to severe inflammation and fibrosis of the liver (denoted non-alcoholic steatohepatitis, NASH). At least some of the features of NASH appear to be driven by fat accumulation since human mutations in genes that cause NAFLD also lead to the development of NASH. The severity of NASH is underscored by the fact that it is rapidly becoming the leading cause of liver transplant. The development of NAFLD is associated with human obesity and well as lipodystrophies, and the severity of both NAFLD and NASH is often most severe in type 2 diabetes, which now affects 10% of the U.S. population. 
     The mechanisms in the liver that lead to NAFLD are multiple, and can include fatty acids entering liver from adipose tissues, from lipoproteins in the circulation, or from the synthesis of fatty acids in the liver from carbohydrate and other substrates (denoted de novo lipogenesis). Fatty acids in the liver are esterified into triglyceride (which accumulates and leads to NAFLD) through a synthesis pathway that joins fatty acids with glycerol. The last step in this triglyceride synthetic pathway in liver is catalyzed largely by the enzyme diacylglycerol 0-acyltransferase 2 (DGAT2), which joins the third fatty acid onto diacylglycerol to make triglyceride. Inhibition of DGAT2 may represent a useful therapeutic approach for the treatment of liver diseases, such as NAFLD and NASH. 
     Summary 
     In one aspect, the disclosure provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a Diacylglycerol O-Acyltransferase 2 (DGAT2) nucleic acid sequence of any one of SEQ ID NOs: 1-5. 
     In one aspect, the disclosure provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a Diacylglycerol O-Acyltransferase 2 (DGAT2) nucleic acid sequence of SEQ ID NO: 1. 
     In one aspect, the disclosure provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a Diacylglycerol O-Acyltransferase 2 (DGAT2) nucleic acid sequence of SEQ ID NO: 2. 
     In one aspect, the disclosure provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a Diacylglycerol O-Acyltransferase 2 (DGAT2) nucleic acid sequence of SEQ ID NO: 3. 
     In one aspect, the disclosure provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a Diacylglycerol O-Acyltransferase 2 (DGAT2) nucleic acid sequence of SEQ ID NO: 4. 
     In one aspect, the disclosure provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a Diacylglycerol O-Acyltransferase 2 (DGAT2) nucleic acid sequence of SEQ ID NO: 5. 
     In certain embodiments, the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 6-10. 
     In certain embodiments, the dsRNA comprises complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of the DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the dsRNA comprises no more than 3 mismatches with the DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the dsRNA comprises full complementarity to the DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. 
     In certain embodiments, the antisense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the sense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the antisense strand is 20 nucleotides in length. In certain embodiments, the antisense strand is 21 nucleotides in length. In certain embodiments, the antisense strand is 22 nucleotides in length. In certain embodiments, the sense strand is 15 nucleotides in length. In certain embodiments, the sense strand is 16 nucleotides in length. In certain embodiments, the sense strand is 18 nucleotides in length. In certain embodiments, the sense strand is 20 nucleotides in length. In certain embodiments, the sense strand is 21 nucleotides in length. 
     In certain embodiments, the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 15 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 16 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 18 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 20 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 21 base pairs. 
     In certain embodiments, the dsRNA comprises a blunt-end. In certain embodiments, the dsRNA comprises at least one single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises 2-nucleotide single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises 5-nucleotide single stranded nucleotide overhang. 
     In certain embodiments, the dsRNA comprises naturally occurring nucleotides. 
     In certain embodiments, the dsRNA comprises at least one modified nucleotide. 
     In certain embodiments, the modified nucleotide comprises a 2′-O-methyl modified nucleotide, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof. 
     In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage. 
     In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the dsRNA comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the dsRNA comprises 4-13 phosphorothioate internucleotide linkages. In certain embodiments, the dsRNA comprises 8 or 13 phosphorothioate internucleotide linkages. In certain embodiments, the antisense strand comprises 4 or 9 phosphorothioate internucleotide linkages. In certain embodiments, the sense strand comprises 4 phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage of Formula I: 
     
       
         
         
             
             
         
       
     
     wherein: 
     B is a base pairing moiety; 
     W is selected from the group consisting of O, OCH 2 , OCH, CH 2 , and CH; 
     X is selected from the group consisting of halo, hydroxy, and C 1-6  alkoxy; 
     Y is selected from the group consisting of O − , OH, OR, NW, NH 2 , S − , and SH; 
     Z is selected from the group consisting of O and CH 2 ; 
     R is a protecting group; and 
        is an optional double bond. 
     In certain embodiments, the dsRNA comprises at least 80% chemically modified nucleotides. In certain embodiments, the dsRNA is fully chemically modified. In certain embodiments, the dsRNA comprises at least 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises at least 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises about 70% to 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least 65% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2′-O-methyl nucleotide modifications. 
     In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments, the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of sense strand. In certain embodiments, the nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of the sense strand. 
     In certain embodiments, the antisense strand comprises a 5′ phosphate, a 5′-alkyl phosphonate, a 5′ alkylene phosphonate, or a 5′ alkenyl phosphonate. In certain embodiments, the antisense strand comprises a 5′ vinyl phosphonate. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate intemucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate intemucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 80% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 80% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate intemucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, 16 and 20 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 7 and 9-11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 7, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 50% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 65% 2′-O-methyl modifications; (7) the nucleotides at positions 3, 7, 9, 11, and 13 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 85% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 14, and 20 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate intemucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 75% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, 16, and 20 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 75% 2′-O-methyl modifications; (7) the nucleotides at positions 7, 9, 10, and llfrom the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the nucleotide at position 20 from the 5′ end of the antisense strand is not a 2′-methoxy-ribonucleotide. 
     In certain embodiments, a functional moiety is linked to the 5′ end and/or 3′ end of the antisense strand. In certain embodiments, a functional moiety is linked to the 5′ end and/or 3′ end of the sense strand. In certain embodiments, a functional moiety is linked to the 3′ end of the sense strand. 
     In certain embodiments, the functional moiety comprises an N-acetylgalactosamine (GalNAc) moiety. 
     In certain embodiments, the functional moiety comprises a hydrophobic moiety. 
     In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof. 
     In certain embodiments, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA). 
     In certain embodiments, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). 
     In certain embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. 
     In certain embodiments, the linker is a cleavable linker. In certain embodiments, the cleavable linker comprises a phosphodiester linkage, a disulfide linkage, an acid-labile linkage, or a photocleavable linkage. 
     In certain embodiments, the cleavable linker comprises a dTdT dinucleotide with phosphodiester intemucleotide linkages. 
     In certain embodiments, the acid-labile linkage comprises a β-thiopropionate linkage or a carboxydimethylmaleic anhydride (CDM) linkage. 
     In certain embodiments, the divalent or trivalent linker is selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     wherein n is 1, 2, 3, 4, or 5. 
     In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. 
     In certain embodiments, when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative. 
     In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     wherein X is O, S or BH 3 . 
     In certain embodiments, the nucleotides at positions 1 and 2 from the 3′ end of sense strand, and the nucleotides at positions 1 and 2 from the 5′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate linkages. 
     In certain embodiments, the dsRNA inhibits the expression of said DGAT2 gene by at least about 50%. 
     In certain embodiments, the dsRNA inhibits the expression of one or more of SREBP1c, FASN, SCD1, and ACC 1  genes by at least about 50%. 
     In another aspect, the disclosure provides a pharmaceutical composition for inhibiting the expression of Diacylglycerol O-Acyltransferase 2 (DGAT2) gene in an organism comprising the dsRNA recited above and a pharmaceutically acceptable carrier. 
     In certain embodiments, the dsRNA inhibits the expression of said DGAT2 gene by at least about 50%. In certain embodiments, the dsRNA inhibits the expression of said DGAT2 gene by at least about 80%. 
     In another aspect, the disclosure provides a method for inhibiting expression of DGAT2 gene in a cell, the method comprising: (a) introducing into the cell a double-stranded ribonucleic acid (dsRNA) recited above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the DGAT2 gene, thereby inhibiting expression of the DGAT2 gene in the cell. 
     In another aspect, the disclosure provides a method of treating or managing a disease associated with DGAT2 comprising administering to a patient in need of such treatment a therapeutically effective amount of said dsRNA recited above. 
     In certain embodiments, the disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), lipodystrophy, partial lipodystrophy, metabolic syndrome, cardiovascular disease, or a combination thereof. 
     In certain embodiments, the dsRNA is administered to one or both of the liver and white adipose tissue of the patient. 
     In certain embodiments, the dsRNA is administered by intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof. 
     In certain embodiments, administering the dsRNA causes a decrease in DGAT2 gene mRNA in one or more of the liver and white adipose tissue. In certain embodiments, administering the dsRNA causes a decrease in DGAT2 gene mRNA in one or more of hepatocytes and adipocytes. 
     In certain embodiments, the dsRNA inhibits the expression of said DGAT2 gene by at least about 50%. In certain embodiments, the dsRNA inhibits the expression of said DGAT2 gene by at least about 80%. 
     In certain embodiments, DGAT2 gene expression is inhibited by at least about 50% for four weeks post administration. In certain embodiments, DGAT2 gene expression is inhibited by at least about 50% for eight weeks post administration. In certain embodiments, DGAT2 gene expression is inhibited by at least about 50% for twelve weeks post administration. 
     In certain embodiments, the dsRNA is administered at a dose of about 1 mg/kg, about 3 mg/kg, or about 10 mg/kg. 
     In one aspect, the disclosure provides a vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes a dsRNA molecule substantially complementary to a DGAT2 nucleic acid sequence of SEQ ID NO:1-10. 
     In certain embodiments, the dsRNA molecule inhibits the expression of said DGAT2 gene by at least 30%. In certain embodiments, the dsRNA molecule inhibits the expression of said DGAT2 gene by at least about 50%. In certain embodiments, the dsRNA molecule inhibits the expression of said DGAT2 gene by at least about 80%. 
     In certain embodiments, the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of SEQ ID NO: 1-10. 
     In one aspect, the disclosure provides a cell comprising the vector recited above. 
     In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising the vector recited above and an AAV capsid. 
     In one aspect, the disclosure provides a method of treating or managing a disease associated with DGAT2 comprising administering to a patient in need of such treatment a therapeutically effective amount of a double-stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein: the antisense strand comprises a sequence substantially complementary to a Diacylglycerol O-Acyltransferase 2 (DGAT2) nucleic acid sequence; and the dsRNA molecule inhibits DGAT2 gene expression by at least about 50% for four weeks post administration. 
     In certain embodiments, the dsRNA molecule inhibits DGAT2 gene expression by at least about 50% for eight weeks post administration. In certain embodiments, the dsRNA molecule inhibits DGAT2 gene expression by at least about 50% for twelve weeks post administration. 
     In certain embodiments, the dsRNA molecule inhibits DGAT2 gene expression by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or about 90%. 
     In certain embodiments, the dsRNA is administered at a dose of about 0.1 mg/kg to about 100 mg/kg. 
     In certain embodiments, the dsRNA is administered at a dose of about 0.1 mg/kg, about 0.3 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, or about 30 mg/kg. 
     In certain embodiments, the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of SEQ ID NO: 1-10. 
     In certain embodiments, the antisense strand comprises complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of the DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the antisense strand comprises no more than 3 mismatches with the DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the antisense strand comprises full complementarity to the DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. 
     In certain embodiments, the antisense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the sense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the antisense strand is 20 nucleotides in length. In certain embodiments, the antisense strand is 21 nucleotides in length. In certain embodiments, the antisense strand is 22 nucleotides in length. In certain embodiments, the sense strand is 15 nucleotides in length. In certain embodiments, the sense strand is 16 nucleotides in length. In certain embodiments, the sense strand is 18 nucleotides in length. In certain embodiments, the sense strand is 20 nucleotides in length. 
     In certain embodiments, the dsRNA molecule comprises a double-stranded region of 15 base pairs to 20 base pairs. In certain embodiments, the dsRNA molecule comprises a double-stranded region of 15 base pairs. In certain embodiments, the dsRNA molecule comprises a double-stranded region of 16 base pairs. In certain embodiments, the dsRNA molecule comprises a double-stranded region of 18 base pairs. In certain embodiments, the dsRNA molecule comprises a double-stranded region of 20 base pairs. 
     In certain embodiments, the dsRNA comprises a blunt-end. 
     In certain embodiments, the dsRNA comprises at least one single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises 2-nucleotide single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises 5-nucleotide single stranded nucleotide overhang. 
     In certain embodiments, the dsRNA comprises at least one modified nucleotide. 
     In certain embodiments, the modified nucleotide comprises a 2′-O-methyl modified nucleotide, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof. 
     In certain embodiments, the dsRNA comprises at least one modified internucleotide linkage. 
     In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the dsRNA comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the dsRNA comprises 4-13 phosphorothioate internucleotide linkages. In certain embodiments, the dsRNA comprises 8 or 13 phosphorothioate internucleotide linkages. In certain embodiments, the antisense strand comprises 4 or 9 phosphorothioate internucleotide linkages. In certain embodiments, the sense strand comprises 4 phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises at least 80% chemically modified nucleotides. In certain embodiments, the dsRNA is fully chemically modified. In certain embodiments, the dsRNA comprises at least 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises at least 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises about 70% to 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least 65% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2′-O-methyl nucleotide modifications. 
     In certain embodiments, the antisense strand comprises a 5′ phosphate, a 5′-alkyl phosphonate, a 5′ alkylene phosphonate, or a 5′ alkenyl phosphonate. In certain embodiments, the antisense strand comprises a 5′ vinyl phosphonate. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 80% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 80% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, 16 and 20 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 7 and 9-11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 7, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In certain embodiments, the nucleotide at position 20 from the 5′ end of the antisense strand is not a 2′-methoxy-ribonucleotide. 
     In certain embodiments, a functional moiety is linked to the 5′ end and/or 3′ end of the antisense strand. In certain embodiments, a functional moiety is linked to the 5′ end and/or 3′ end of the sense strand. In certain embodiments, a functional moiety is linked to the 3′ end of the sense strand. 
     In certain embodiments, the functional moiety comprises an N-acetylgalactosamine (GalNAc) moiety. 
     In certain embodiments, the functional moiety comprises a hydrophobic moiety. 
     In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof. 
     In certain embodiments, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA). 
     In certain embodiments, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. 
         FIG. 1A - FIG. 1E  depict in vitro screens for cholesterol conjugated siRNA sequences targeting Diacylglycerol 0-acyltransferase 2 (DGAT2) gene. A cartoon depicting the cholesterol conjugated siRNAs that were used for the in vitro screenings is shown, ( FIG. 1A ). A schematic representation showing the target locations of the screening siRNAs on the mouse Dgat2 transcript for silencing, ( FIG. 1B ). Seven screening siRNAs at a concentration of 1.5 μM were tested in the FL83b mouse hepatocyte cell line, which identified DGAT2 1093, 1473 and DGAT2 1476 as efficacious targeting regions, ( FIG. 1C ). Results of a screen of the three identified target locations in HepG2 cells shows that targeting the 1473 and 1476 regions in the human DGAT2 transcript with siRNA silencing potently reduces human DGAT2 mRNA expression. Percent knockdowns, normalized against HPRT expression, are indicated, ( FIG. 1D ). Dose response curves in HepG2 cells done using 8 serially diluted doses of the two lead chemically modified siRNAs (1473 and 1476) are shown, the IC 50  values indicate strong potency ( FIG. 1E ). 
         FIG. 2A - FIG. 2B  depict in vivo Dgat2 silencing in mice with cholesterol conjugated, chemically modified siRNAs without a 5′ antisense strand vinylphosphonate (VP) modification. In this experiment, 8-week-old wild-type C 57 BL6/J mice on chow diet were subcutaneously injected with 10mg/kg cholesterol-conjugated non-targeting control siRNA (NTC), cholesterol-conjugated 1473 compound targeting Dgat2 (Chol-1473), or cholesterol-conjugated 1476 compound targeting Dgat2 (Chol-1476). Mice were sacrificed at 10 days after injection. Liver samples were processed for Dgat2 mRNA expression measurements and DGAT2 protein knockdown levels ( FIG. 2A  &amp;  FIG. 2B ). 
         FIG. 3A - FIG. 3C  depict in vivo DGAT2 silencing in mice with N-acetylgalactosamine (GalNAc) conjugated 1473 tool compound (P5 configuration with the VP modification). A cartoon of the GalNAc conjugated siRNAs that were used in the experiment is shown ( FIG. 3A ). A graph showing dose response and KD longevity of GalNAc-1473 is shown. Here, mice were injected either with the indicated doses, (10, 3, 1 mg/kg), or non-targeting control compound (10mg/kg) subcutaneously once and sacrificed at the indicated time points (4w, 8w, 12w) after injections. The DGAT2 silencing in liver was still abundant 12 weeks after single subcutaneous injection of GalNac-1473 ( FIG. 3B ). Graph showing de novo lipogenesis related gene expression changes upon DGAT2 knockdown in Wildtype mice ( FIG. 3C ). 
         FIG. 4A - FIG. 4C  depict the protocol for generating human hepatocyte engrafted NSG mice mouse model ( FIG. 4A ). The graphs ( FIG. 4B  &amp; C) show mouse and human DGAT2 transcript levels, respectively, 1 week after GalNac-1473 injections in to mice, which demonstrate significant silencing for the siRNA that has homology to both transcripts. 
         FIG. 5A -FIG. SF depict reduction in liver weights and lipid droplet/fat accumulation in an in vivo efficacy study in genetically obese mice with a Dgat2 knockdown and on a high fat diet. Here, 10-week-old genetically obese mice were subcutaneously injected with a 10mg/kg dose of GalNAc-NTC or GalNAc-1473 and switched to a high fat, high cholesterol Gubra Amylin NASH (GAN) diet for 3 weeks. Animals were bled weekly, and sacrificed at 3 weeks, and afterwards the injection and livers and plasma were used for analysis. 
         FIG. 5A  depicts graphs showing DGAT2 transcript levels and protein levels in the liver after GalNAc-1473 injection.  FIG. 5B  depicts graphs showing bodyweight comparisons (at start vs after 3 weeks on GAN diet).  FIG. 5C  depicts a significant decrease in the body to liver weight ratio upon DGAT2 silencing.  FIG. 5D  depicts an assessment of liver cell death by measuring Serum Activity of Alanine Aminotransferase (ALT).  FIG. 5E  depicts a histological analysis of mouse liver tissues by hematoxylin and eosin (H&amp;E) staining showing decreased liver fat accumulation in mice treated with GalNAc-siRNA1473.  FIG. 5F  depicts graphs showing liver triglyceride concentrations, diglyceride (DAG) concentrations, and the fatty acyl chain content in triglycerides, all as measured by mass-spectrometry, and showing a significant decrease in total liver triglycerides after DGAT2 silencing. 
         FIG. 6A - FIG. 6C  depict changes in de novo lipogenesis in genetically obese, NASH mice injected with GalNAc-NTC or GalNAc-1473. The graph shows changes in de novo lipogenesis related gene expression changes upon DGAT2 knockdown ( FIG. 6A ). Images and quantification of post translational and protein expression level analysis of transcription factors involved in de novo lipogenesis gene expression are shown ( FIG. 6B  &amp;  FIG. 6C  respectively). 
         FIG. 7A - FIG. 7C  depict phospholipid levels in ob/ob mice with NASH injected with GalNAc-NTC or GalNAc-1473. Phosphatidylcholine ( FIG. 7A ), phosphatidylethanolamine ( FIG. 7B ), and phosphatidylinositol ( FIG. 7C ) levels are shown. 
         FIG. 8  depicts DGAT2 mRNA levels in eight-week-old male C 57 BL6 mice injected with GalNAc-NTC or GalNAc-1473. The mice were injected with 10mg/kg of siRNA and DGAT2 silencing was examined in kidney, spleen, inguinal fat, epididymal fat and liver via qPCR. 
     
    
    
     DETAILED DESCRIPTION 
     Novel DGAT2 target sequences are provided. Also provided are novel RNA molecules, such as siRNAs compounds containing the same, which target the DGAT2 mRNA. 
     Unless otherwise specified, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to manufacturer&#39;s specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients. 
     Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting. 
     So that the disclosure may be more readily understood, certain terms are first defined. 
     The term “nucleoside” refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. Additional exemplary nucleosides include inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and N2,N2-dimethylguanosine (also referred to as “rare” nucleosides). The term “nucleotide” refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester or phosphorothioate linkage between 5′ and 3′ carbon atoms. 
     The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA. 
     As used herein, the term “small interfering RNA” (“”“siRNA”) (also referred to in the art as “short interfering RNAs”) refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs), which is capable of directing or mediating RNA interference. In certain embodiments, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, or between about 16-25 nucleotides (or nucleotide analogs), or between about 18-23 nucleotides (or nucleotide analogs), or between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA. 
     The term “nucleotide analog” or “altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of positions of the nucleotide that may be derivatized include: the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine; and the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified nucleotides (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) ; and other heterocyclically modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310. 
     Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotide. For example, the 2′ OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH 2 , NHR, NR 2 , or COOR, wherein R is substituted or unsubstituted C 1 -C 6  alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438. 
     The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions, which allow the nucleotide to perform its intended function, such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro. 
     The term “oligonucleotide” refers to a short polymer of nucleotides and/or nucleotide analogs. 
     The term “RNA analog” refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA, but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages, which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. For example, the nucleotides of the analog may comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and/or phosphorothioate linkages. Some RNA analogues include sugar- and/or backbone-modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate RNA interference. 
     As used herein, the term “”RNA interference“” (“RNAi”) refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA, which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes. 
     An RNAi agent, e.g., an RNA silencing agent, having a strand, which is ““sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi”)” means that the strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process. 
     As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to RNA molecules, which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. 
     As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms (e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules, which result in the inhibition or “”silencing“” of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi. 
     The term “discriminatory RNA silencing” refers to the ability of an RNA molecule to substantially inhibit the expression of a “first” or “target” polynucleotide sequence while not substantially inhibiting the expression of a “second” or “non-target” “polynucleotide sequence,” e.g., when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is the DNA sequence encoding the regulatory region (e.g. promoter or enhancer elements) of a target gene. In other embodiments, the target polynucleotide sequence is a target mRNA encoded by a target gene. 
     The term “in vitro” has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term “in vivo” also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism. 
     As used herein, the term “transgene” refers to any nucleic acid molecule, which is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from the cell. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. The term “transgene” also means a nucleic acid molecule that includes one or more selected nucleic acid sequences, e.g., DNAs, that encode one or more engineered RNA precursors, to be expressed in a transgenic organism, e.g., animal, which is partly or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but which is designed to be inserted into the animal&#39;s genome at a location which differs from that of the natural gene. A transgene includes one or more promoters and any other DNA, such as introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include an enhancer sequence. 
     A gene “involved” in a disease or disorder includes a gene, the normal or aberrant expression or function of which effects or causes the disease or disorder or at least one symptom of said disease or disorder. 
     As used herein, the term “target gene” is a gene whose expression is to be substantially inhibited or “silenced.” This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA of the target gene or translational repression of the target gene. The term “non-target gene” is a gene whose expression is not to be substantially silenced. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g. mRNA encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene may be a homologue (e.g. an orthologue or paralogue) of the target gene. 
     As described herein, the term “DGAT2” refers to the gene encoding for the enzyme, Diacylglycerol O-Acyltransferase 2. DGAT2 catalyzes the reaction that covalently joins diacylglycerol to long chain fatty acyl-CoAs in the final step of triglyceride synthesis. The DGAT2 gene is located on chromosome 11q13.5, is made up of 9 exons and is mainly expressed in the liver and white adipose tissue. The DGAT2 protein is 388 amino acids in length and has a molecular mass of approximately 43,831 Da. 
     The phrase “examining the function of a gene in a cell or organism” refers to examining or studying the expression, activity, function or phenotype arising therefrom. 
     As used herein, the term “RNA silencing agent” refers to an RNA, which is capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of a mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include small (&lt;50 b.p.), noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as precursors thereof. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression. 
     As used herein, the term “rare nucleotide” refers to a naturally occurring nucleotide that occurs infrequently, including naturally occurring deoxyribonucleotides or ribonucleotides that occur infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and 2,2N,N-dimethylguanosine. 
     The term “engineered,” as in an engineered RNA precursor, or an engineered nucleic acid molecule, indicates that the precursor or molecule is not found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by a human. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by mechanisms within a cell. Thus, an RNA precursor produced within a cell from a transgene that includes an engineered nucleic acid molecule is an engineered RNA precursor. 
     As used herein, the term “microRNA” (“miRNA”), also known in the art as “small temporal RNAs” (“stRNAs”), refers to a small (10-50 nucleotide) RNA, which are genetically encoded (e.g., by viral, mammalian, or plant genomes) and are capable of directing or mediating RNA silencing. An “miRNA disorder” shall refer to a disease or disorder characterized by an aberrant expression or activity of a miRNA. 
     As used herein, the term “dual functional oligonucleotide” refers to a RNA silencing agent having the formula T-L-μ, wherein T is an mRNA targeting moiety, L is a linking moiety, and μ is a miRNA recruiting moiety. As used herein, the terms “mRNA targeting moiety,” “targeting moiety,” “mRNA targeting portion” or “targeting portion” refer to a domain, portion or region of the dual functional oligonucleotide having sufficient size and sufficient complementarity to a portion or region of an mRNA chosen or targeted for silencing (i.e., the moiety has a sequence sufficient to capture the target mRNA). 
     As used herein, the terms “linking moiety” or “linking portion” refer to a domain, portion or region of the RNA-silencing agent which covalently joins or links the mRNA. 
     As used herein, the terms “miRNA recruiting moiety” or “miRNA targeting moiety” or “miRNA recruiting portion” refer to a domain, portion or region of the dual functional oligonucleotide having sufficient size and sufficient complementarity to a portion or region of a miRNA chosen or targeted for recruitment to the target mRNA. 
     As used herein, the term “antisense strand” of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or complementarity sufficient to trigger translational repression of the desired target mRNA. 
     The term “sense strand” or “second strand” of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is complementary to the antisense strand or first strand. Antisense and sense strands can also be referred to as first or second strands, the first or second strand having complementarity to the target sequence and the respective second or first strand having complementarity to said first or second strand. miRNA duplex intermediates or siRNA-like duplexes include a miRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a miRNA strand having sufficient complementarity to form a duplex with the miRNA strand. 
     As used herein, the term “guide strand” refers to a strand of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex or siRNA sequence, that enters into the RISC complex and directs cleavage of the target mRNA. 
     As used herein, the term “asymmetry,” as in the asymmetry of the duplex region of an RNA silencing agent (e.g., the stem of an shRNA), refers to an inequality of bond strength or base pairing strength between the termini of the RNA silencing agent (e.g., between terminal nucleotides on a first strand or stem portion and terminal nucleotides on an opposing second strand or stem portion), such that the 5′ end of one strand of the duplex is more frequently in a transient unpaired, e.g., single-stranded, state than the 5′ end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into a RISC complex. The strand whose 5′ end is less tightly paired to the complementary strand will preferentially be incorporated into RISC and mediate RNAi. 
     As used herein, the term “bond strength” or “base pair strength” refers to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex), due primarily to H-bonding, van der Waals interactions, and the like, between said nucleotides (or nucleotide analogs). 
     As used herein, the “5′ end,” as in the 5′ end of an antisense strand, refers to the 5′ terminal nucleotides, e.g., between one and about 5 nucleotides at the 5′ terminus of the antisense strand. As used herein, the “3′ end,” as in the 3′ end of a sense strand, refers to the region, e.g., a region of between one and about 5 nucleotides, that is complementary to the nucleotides of the 5′ end of the complementary antisense strand. 
     As used herein the term “destabilizing nucleotide” refers to a first nucleotide or nucleotide analog capable of forming a base pair with second nucleotide or nucleotide analog, such that the base pair is of lower bond strength than a conventional base pair (i.e., Watson-Crick base pair). In certain embodiments, the destabilizing nucleotide is capable of forming a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a wobble base pair with the second nucleotide. In yet other embodiments, the destabilizing nucleotide is capable of forming an ambiguous base pair with the second nucleotide. 
     As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of a RNA silencing agent and a target mRNA sequence), due primarily to H-bonding, van der Waals interactions, and the like between said nucleotides (or nucleotide analogs). As used herein, the term “bond strength” or “base pair strength” refers to the strength of the base pair. 
     As used herein, the term “mismatched base pair” refers to a base pair consisting of non-complementary or non-Watson-Crick base pairs, for example, not normal complementary G:C, A:T or A:U base pairs. As used herein the term “ambiguous base pair” (also known as a non-discriminatory base pair) refers to a base pair formed by a universal nucleotide. 
     As used herein, term “universal nucleotide” (also known as a “neutral nucleotide”) include those nucleotides (e.g. certain destabilizing nucleotides) having a base (a “universal base” or “neutral base”) that does not significantly discriminate between bases on a complementary polynucleotide when forming a base pair. Universal nucleotides are predominantly hydrophobic molecules that can pack efficiently into antiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portion of universal nucleotides typically comprise a nitrogen-containing aromatic heterocyclic moiety. 
     As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has a sequence (e.g. in the antisense strand, mRNA targeting moiety or miRNA recruiting moiety), which is sufficient to bind the desired target RNA, respectively, and to trigger the RNA silencing of the target mRNA. 
     As used herein, the term “translational repression” refers to a selective inhibition of mRNA translation. Natural translational repression proceeds via miRNAs cleaved from short hairpin RNA (shRNA) precursors. Both RNAi and translational repression are mediated by RNA-induced silencing complex (RISC). Both RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes. 
     Various methodologies of the instant disclosure include a step that involves comparing a value, level, feature, characteristic, property, etc. to a “suitable control,” referred to interchangeably herein as an “appropriate control.” A “suitable control” or “appropriate control” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent of the disclosure into a cell or organism. In another embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a “suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and example are illustrative only and not intended to be limiting. 
     Various aspects of the disclosure are described in further detail in the following subsections. 
     I. Novel Target Sequences 
     In certain exemplary embodiments, RNA silencing agents of the disclosure are capable of targeting a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10, as recited in Table 1. In certain exemplary embodiments, RNA silencing agents of the disclosure comprise double stranded RNA (dsRNA) molecules comprising a sense strand and an antisense strand, wherein the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of SEQ ID NO: 1-10, as recited in Table 1. Exemplary antisense and sense strands are recited in Tables 2-4. 
     The genomic sequence for each target sequence can be found in, for example, the publicly available database maintained by the NCBI. 
     II. siRNA Design 
     In some embodiments, siRNAs are designed as follows. First, a portion of the target gene (e.g., the DGAT2 gene), e.g., one or more of the target sequences set forth in Table 1 is selected. Cleavage of mRNA at these sites should eliminate translation of corresponding protein. Antisense strands were designed based on the target sequence and sense strands were designed to be complementary to the antisense strand. Hybridization of the antisense and sense strands forms the siRNA duplex. The antisense strand includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. In other embodiments, the antisense strand includes 20, 21, 22 or 23 nucleotides. The sense strand includes about 14 to 25 nucleotides, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In other embodiments, the sense strand is 15 nucleotides. In other embodiments, the sense strand is 18 nucleotides. In other embodiments, the sense strand is 20 nucleotides. The skilled artisan will appreciate, however, that siRNAs having a length of less than 19 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Accordingly, siRNAs of such length are also within the scope of the instant disclosure, provided that they retain the ability to mediate RNAi. Longer RNAi agents have been demonstrated to elicit an interferon or PKR response in certain mammalian cells, which may be undesirable. In certain embodiments, the RNAi agents of this disclosure do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types incapable of generating a PKR response or in situations where the PKR response has been down-regulated or dampened by alternative means. 
     The sense strand sequence can be designed such that the target sequence is essentially in the middle of the strand. Moving the target sequence to an off-center position can, in some instances, reduce efficiency of cleavage by the siRNA. Such compositions, i.e., less efficient compositions, may be desirable for use if off-silencing of the wild-type mRNA is detected. 
     The antisense strand can be the same length as the sense strand and includes complementary nucleotides. In one embodiment, the strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands align or anneal such that 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-nucleotide overhangs are generated, i.e., the 3′ end of the sense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides further than the 5′ end of the antisense strand and/or the 3′ end of the antisense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides further than the 5′ end of the sense strand. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material. 
     To facilitate entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5′ end of the sense strand and 3′ end of the antisense strand can be altered, e.g., lessened or reduced, as described in detail in U.S. Pat. Nos. 7,459,547, 7,772,203 and 7,732,593, entitled “Methods and Compositions for Controlling Efficacy of RNA Silencing” (filed Jun. 2, 2003) and U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705, entitled “Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2003), the contents of which are incorporated in their entirety by this reference. In one embodiment of these aspects of the disclosure, the base-pair strength is less due to fewer G:C base pairs between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand than between the 3′ end of the first or antisense strand and the 5′ end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one mismatched base pair between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pair strength is less due to at least one wobble base pair, e.g., G:U, between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one base pair comprising a rare nucleotide, e.g., inosine (I). In certain exemplary embodiments, the base pair is selected from the group consisting of an I:A, I:U and I:C. In yet another embodiment, the base pair strength is less due to at least one base pair comprising a modified nucleotide. In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A. 
     The design of siRNAs suitable for targeting the DGAT2 target sequences set forth in Table 1 is described in detail below. siRNAs can be designed according to the above exemplary teachings for any other target sequences found in the DGAT2 gene. Moreover, the technology is applicable to targeting any other target sequences, e.g., non-disease-causing target sequences. 
     To validate the effectiveness by which siRNAs destroy mRNAs (e.g., DGAT2 mRNA), the siRNA can be incubated with cDNA (e.g., DGAT2 cDNA) in a Drosophila-based in vitro mRNA expression system. Radiolabeled with  32 P, newly synthesized mRNAs (e.g., DGAT2mRNA) are detected autoradiographically on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA. Alternatively, control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. Sites of siRNA-mRNA complementation are selected which result in optimal mRNA specificity and maximal mRNA cleavage. 
     III. RNAi Agents 
     The present disclosure includes RNAi molecules, such as siRNA molecules designed, as described above. The siRNA molecules of the disclosure can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from e.g., shRNA, or by using recombinant human DICER enzyme, to cleave in vitro transcribed dsRNA templates into pools of 20-, 21- or 23-bp duplex RNA mediating RNAi. The siRNA molecules can be designed using any method known in the art. 
     In one aspect, instead of the RNAi agent being an interfering ribonucleic acid, the RNAi agent can encode an interfering ribonucleic acid, as described above. In other words, the RNAi agent can be a transcriptional template of the interfering ribonucleic acid. Thus, RNAi agents of the present disclosure can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3′ UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, Supra; Miyagishi et al., 2002; Paddison et al., 2002, supra; Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002, supra. More information about shRNA design and use can be found on the internet at the following addresses: katandin. cshl. org: 9331/RNAi/docs/BseRI-BamHI_Strategy .pdf and katandin. cshl. org:9331/RNAi/docs/Web_version_of_PCR_strategy 1. pdf). 
     Expression constructs of the present disclosure include any construct suitable for use in the appropriate expression system and include, but are not limited to, retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs can include one or more inducible promoters, RNA Pol III promoter systems, such as U6 snRNA promoters or H 1  RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct. (Tuschl, T., 2002, Supra). 
     Synthetic siRNAs can be delivered into cells by methods known in the art, including cationic liposome transfection and electroporation. To obtain longer term suppression of the target genes (e.g., DGAT2 genes) and to facilitate delivery under certain circumstances, one or more siRNA can be expressed within cells from recombinant DNA constructs. Such methods for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer-term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., H 1  or U6/snRNA promoter systems (Tuschl, T., 2002, supra) capable of expressing functional double-stranded siRNAs; (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5′-3′ and 3′-5′ orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by H 1  or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002), supra; Sui et al., 2002, supra). Constructs containing siRNA sequence under the control of T7 promoter also make functional siRNAs when co-transfected into the cells with a vector expressing T7 RNA polymerase (Jacque et al., 2002, supra). A single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the gene encoding DGAT2, targeting the same gene or multiple genes, and can be driven, for example, by separate Pollil promoter sites. 
     Animal cells express a range of noncoding RNAs of approximately 22 nucleotides termed micro RNA (miRNAs), which can regulate gene expression at the post transcriptional or translational level during animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop, probably by Dicer, an RNase III-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with sequence complementary to the target mRNA, a vector construct that expresses the engineered precursor can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., 2002, supra). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression (McManus et al., 2002, supra). MicroRNAs targeting polymorphisms may also be useful for blocking translation of mutant proteins in the absence of siRNA-mediated gene-silencing. Such applications may be useful in situations, for example, where a designed siRNA caused off-target silencing of wild type protein. 
     Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al., 2002, supra). Infection of HeLa cells by these recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of the recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. Id. In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into post-implantation mouse embryos (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be accomplished by “high-pressure” delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis et al., 2002.) Nanoparticles and liposomes can also be used to deliver siRNA into animals. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs into cells, e.g., neural cells (e.g., brain cells) (US Patent Applications 2014/0296486, 2010/0186103, 2008/0269149, 2006/0078542 and 2005/0220766). 
     The nucleic acid compositions of the disclosure include both unmodified siRNAs and modified siRNAs, such as crosslinked siRNA derivatives or derivatives having non-nucleotide moieties linked, for example, to their 3′ or 5′ ends. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative, as compared to the corresponding siRNA, and are useful for tracing the siRNA derivative in the cell, or improving the stability of the siRNA derivative compared to the corresponding siRNA. 
     Engineered RNA precursors, introduced into cells or whole organisms as described herein, will lead to the production of a desired siRNA molecule. Such an siRNA molecule will then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage and destruction. In this fashion, the mRNA will be targeted by the siRNA generated from the engineered RNA precursor and will be depleted from the cell or organism leading to a decrease in the concentration of the protein encoded by that mRNA in the cell or organism. The RNA precursors are typically nucleic acid molecules that individually encode either one strand of a dsRNA or encode the entire nucleotide sequence of an RNA hairpin loop structure. 
     The nucleic acid compositions of the disclosure can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability and/or half-life. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles). 
     The nucleic acid molecules of the present disclosure can also be labeled using any method known in the art. For instance, the nucleic acid compositions can be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine. The labeling can be carried out using a kit, e.g., the SILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can be radiolabeled, e.g., using  3 H,  32 P or another appropriate isotope. 
     Moreover, because RNAi is believed to progress via at least one single-stranded RNA intermediate, the skilled artisan will appreciate that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also be designed (e.g., for chemical synthesis), generated (e.g., enzymatically generated), or expressed (e.g., from a vector or plasmid) as described herein and utilized according to the claimed methodologies. Moreover, in invertebrates, RNAi can be triggered effectively by long dsRNAs (e.g., dsRNAs about 100-1000 nucleotides in length, such as about 200-500, for example, about 250, 300, 350, 400 or 450 nucleotides in length) acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27.) 
     IV. Anti-DGAT2 RNA Silencing Agents 
     In certain embodiment, the present disclosure provides novel anti-DGAT2 RNA silencing agents (e.g., siRNA and antisense oligonucleotides), methods of making said RNA silencing agents, and methods (e.g., research and/or therapeutic methods) for using said improved RNA silencing agents (or portions thereof) for RNA silencing of DGAT2 protein. The RNA silencing agents comprise an antisense strand (or portions thereof), wherein the antisense strand has sufficient complementary to a target DGAT2 mRNA to mediate an RNA-mediated silencing mechanism (e.g. RNAi). 
     In certain embodiments, siRNA compounds are provided having one or any combination of the following properties: (1) fully chemically-stabilized (i.e., no unmodified 2′-OH residues); (2) asymmetry; (3) 11-21 base pair duplexes; (4) at least 50% 2′-methoxy modifications, such as 70%-100% 2′-methoxy modifications, although an alternating pattern of chemically-modified nucleotides (e.g., 2′-fluoro and 2′-methoxy modifications), are also contemplated; and (5) single-stranded, fully phosphorothioated tails of 2-8 bases. In certain embodiments, the number of phosphorothioate modifications is varied from 4 to 16 total. In certain embodiments, the number of phosphorothioate modifications is varied from 8 to 13 total. 
     In certain embodiments, the siRNA compounds described herein can be conjugated to a variety of targeting agents, including, but not limited to, cholesterol, docosahexaenoic acid (DHA), phenyltropanes, cortisol, vitamin A, vitamin D, N-acetylgalactosamine (GalNac), and gangliosides. 
     Certain compounds of the disclosure having the structural properties described above, herein may be referred to as “hsiRNA-ASP” (hydrophobically-modified, small interfering RNA, featuring an advanced stabilization pattern). In addition, this hsiRNA-ASP pattern showed a dramatically improved distribution through several tissues, including, but not limited to, the liver, placenta, kidney, and spleen, making them accessible for therapeutic intervention. 
     The compounds of the disclosure can be described in the following aspects and embodiments. 
     In a first aspect, provided herein is a double stranded RNA (dsRNA) comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: 
     (1) the antisense strand comprises a sequence substantially complementary to aDGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; 
     (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; 
     (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; 
     (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; 
     (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: 
     (1) the antisense strand comprises a sequence substantially complementary to aDGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; 
     (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; 
     (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; 
     (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: 
     (1) the antisense strand comprises a sequence substantially complementary to aDGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; 
     (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; 
     (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; 
     (5) a portion of the antisense strand is complementary to a portion of the sense strand; 
     (6) the sense strand comprises 100% 2′-O-methyl modifications; and 
     (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: 
     (1) the antisense strand comprises a sequence substantially complementary to aDGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; 
     (2) the antisense strand comprises at least 80% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; 
     (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; 
     (5) a portion of the antisense strand is complementary to a portion of the sense strand; 
     (6) the sense strand comprises at least 80% 2′-O-methyl modifications; and 
     (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: 
     (1) the antisense strand comprises a sequence substantially complementary to aDGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; 
     (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; 
     (3) the nucleotides at positions 2, 6, 14, 16 and 20 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; 
     (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; 
     (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 7 and 9-11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: 
     (1) the antisense strand comprises a sequence substantially complementary to aDGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; 
     (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; 
     (3) the nucleotides at positions 2, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; 
     (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; 
     (5) a portion of the antisense strand is complementary to a portion of the sense strand; 
     (6) the sense strand comprises at least 70% 2′-O-methyl modifications; 
     (7) the nucleotides at positions 7, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and 
     (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 50% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 4, 5, 6, 8, 10, 12, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 65% 2′-O-methyl modifications; (7) the nucleotides at positions 3, 7, 9, 11, and 13 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 85% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 14, and 20 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises 100% 2′-O-methyl modifications; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In another aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a DGAT2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; (2) the antisense strand comprises at least 75% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, 16, and 20 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 75% 2′-O-methyl modifications; (7) the nucleotides at positions 7, 9, 10, and llfrom the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 
     In any of the above aspects of the disclosure, the antisense strand may comprise a length of 20 or 21 nucleotides. 
     In any of the above aspects of the disclosure, the sense strand may comprise a length of 16, 18, 19, 20, or 21 nucleotides. 
     In certain embodiments of the dsRNA, the nucleotide at position 20 from the 5′ end of the antisense strand is not a 2′-methoxy-ribonucleotide. 
     a) Design of Anti-DGAT2 siRNA Molecules 
     An siRNA molecule of the application is a duplex made of a sense strand and complementary antisense strand, the antisense strand having sufficient complementary to a DGAT2 mRNA to mediate RNAi. In certain embodiments, the siRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). In other embodiments, the siRNA molecule has a length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region. In certain embodiments, the strands are aligned such that there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases at the end of the strands, which do not align (i.e., for which no complementary bases occur in the opposing strand), such that an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues occurs at one or both ends of the duplex when strands are annealed. 
     Usually, siRNAs can be designed by using any method known in the art, for instance, by using the following protocol: 
     1. The siRNA should be specific for a target sequence, e.g., a target sequence set forth in the Examples. The first strand should be complementary to the target sequence, and the other strand is substantially complementary to the first strand. (See Examples for exemplary sense and antisense strands.) Exemplary target sequences are selected from any region of the target gene that leads to potent gene silencing. Regions of the target gene include, but are not limited to, the 5′ untranslated region (5′-UTR) of a target gene, the 3′ untranslated region (3′-UTR) of a target gene, an exon of a target gene, or an intron of a target gene. Cleavage of mRNA at these sites should eliminate translation of corresponding DGAT2 protein. Target sequences from other regions of the DGAT2 gene are also suitable for targeting. A sense strand is designed based on the target sequence. 
     2. The sense strand of the siRNA is designed based on the sequence of the selected target site. In certain embodiments, the sense strand includes about 15 to 25 nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In certain embodiments, the sense strand includes 15, 16, 17, 18, 19, or 20 nucleotides. In certain embodiments, the sense strand is 15 nucleotides in length. In certain embodiments, the sense strand is 18 nucleotides in length. In certain embodiments, the sense strand is 20 nucleotides in length. The skilled artisan will appreciate, however, that siRNAs having a length of less than 15 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Accordingly, siRNAs of such length are also within the scope of the instant disclosure, provided that they retain the ability to mediate RNAi. Longer RNA silencing agents have been demonstrated to elicit an interferon or Protein Kinase R (PKR) response in certain mammalian cells which may be undesirable. In certain embodiments, the RNA silencing agents of the disclosure do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNA silencing agents may be useful, for example, in cell types incapable of generating a PKR response or in situations where the PKR response has been down-regulated or dampened by alternative means. 
     The siRNA molecules of the disclosure have sufficient complementarity with the target sequence such that the siRNA can mediate RNAi. In general, siRNA containing nucleotide sequences sufficiently complementary to a target sequence portion of the target gene to effect RISC-mediated cleavage of the target gene are contemplated. Accordingly, in a certain embodiment, the antisense strand of the siRNA is designed to have a sequence sufficiently complementary to a portion of the target. For example, the antisense strand may have 100% complementarity to the target site. However, 100% complementarity is not required. Greater than 80% identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% complementarity, between the antisense strand and the target RNA sequence is contemplated. The present application has the advantage of being able to tolerate certain sequence variations to enhance efficiency and specificity of RNAi. In one embodiment, the antisense strand has 4, 3, 2, 1, or 0 mismatched nucleotide(s) with a target region, such as a target region that differs by at least one base pair between a wild-type and mutant allele, e.g., a target region comprising the gain-of-function mutation, and the other strand is identical or substantially identical to the first strand. Moreover, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective for mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition. 
     Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=number of identical positions/total number of positions×100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced. 
     The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. 
     In another embodiment, the alignment is optimized by introducing appropriate gaps and the percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. 
     3. The antisense or guide strand of the siRNA is routinely the same length as the sense strand and includes complementary nucleotides. In one embodiment, the guide and sense strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands of the siRNA can be paired in such a way as to have a 3′ overhang of 1 to 7 (e.g., 2, 3, 4, 5, 6 or 7), or 1 to 4, e.g., 2, 3 or 4 nucleotides. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material. Thus, in another embodiment, the nucleic acid molecules may have a 3′ overhang of 2 nucleotides, such as TT. The overhanging nucleotides may be either RNA or DNA. As noted above, it is desirable to choose a target region wherein the mutant:wild type mismatch is a purine:purine mismatch. 
     4. Using any method known in the art, compare the potential targets to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. One such method for such sequence homology searches is known as BLAST, which is available at National Center for Biotechnology Information website. 
     5. Select one or more sequences that meet your criteria for evaluation. 
     Further general information about the design and use of siRNA may be found in “The siRNA User Guide,” available at The Max-Plank-Institut fur Biophysikalische Chemie website. 
     Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing). Additional hybridization conditions include hybridization at 70° C. in 1xSSC or 50° C. in 1xSSC, 50% formamide followed by washing at 70° C. in 0.3xSSC or hybridization at 70° C. in 4xSSC or 50° C. in 4xSSC, 50% formamide followed by washing at 67° C. in 1xSSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (T m ) of the hybrid, where T m  is determined according to the following equations. For hybrids less than 18 base pairs in length, T m (° C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C)=81.5+16.6(log 10[Na+])+0.41(% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na + ] for 1× SSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley &amp; Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference. 
     Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. 
     6. To validate the effectiveness by which siRNAs destroy target mRNAs (e.g., wild-type or mutant DGAT2 mRNA), the siRNA may be incubated with target cDNA (e.g., DGAT2 cDNA) in a Drosophila-based in vitro mRNA expression system. Radiolabeled with  32 P, newly synthesized target mRNAs (e.g., DGAT2 mRNA) are detected autoradiographically on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA and use of non-target cDNA. Alternatively, control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. 
     Anti-DGAT2 siRNAs may be designed to target any of the target sequences described supra. Said siRNAs comprise an antisense strand, which is sufficiently complementary with the target sequence to mediate silencing of the target sequence. In certain embodiments, the RNA silencing agent is a siRNA. 
     In certain embodiments, the siRNA comprises a sense and an antisense strand comprising a sequence set forth in Tables 2-4. 
     Sites of siRNA-mRNA complementation are selected, which result in optimal mRNA specificity and maximal mRNA cleavage. 
     b) siRNA-Like Molecules 
     siRNA-like molecules of the disclosure have a sequence (i.e., have a strand having a sequence) that is “sufficiently complementary” to a target sequence of an DGAT2 mRNA to direct gene silencing either by RNAi or translational repression. siRNA-like molecules are designed in the same way as siRNA molecules, but the degree of sequence identity between the sense strand and target RNA approximates that observed between a miRNA and its target. In general, as the degree of sequence identity between a miRNA sequence and the corresponding target gene sequence is decreased, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi is increased. Therefore, in an alternative embodiment, where post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementarity sites) dispersed within the target mRNA (e.g. within the 3′-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes &amp; Dev., 2003). Since the mechanism of translational repression is cooperative, multiple complementarity sites (e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments. 
     The capacity of a siRNA-like duplex to mediate RNAi or translational repression may be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent at the site of complementarity. In one embodiment, where gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central portion of the complementarity site so that duplex formed by the miRNA guide strand and the target mRNA contains a central “bulge” (Doench J G et al., Genes &amp; Dev., 2003). In another embodiment 2, 3, 4, 5, or 6 contiguous or non-contiguous non-identical nucleotides are introduced. The non-identical nucleotide may be selected such that it forms a wobble base pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further embodiment, the “bulge” is centered at nucleotide positions 12 and 13 from the 5′ end of the miRNA molecule. 
     c) Short Hairpin RNA (shRNA) Molecules 
     In certain featured embodiments, the instant disclosure provides shRNAs capable of mediating RNA silencing of an DGAT2 target sequence with enhanced selectivity. In contrast to siRNAs, shRNAs mimic the natural precursors of micro RNAs (miRNAs) and enter at the top of the gene silencing pathway. For this reason, shRNAs are believed to mediate gene silencing more efficiently by being fed through the entire natural gene silencing pathway. 
     miRNAs are noncoding RNAs of approximately 22 nucleotides, which can regulate gene expression at the post transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNase III-type enzyme, or a homolog thereof. Naturally-occurring miRNA precursors (pre-miRNA) have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, that connects the two portions of the stem. In typical pre-miRNAs, the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide “loop” in one portion of the stem, and/or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other. Short hairpin RNAs, or engineered RNA precursors, of the present application are artificial constructs based on these naturally occurring pre-miRNAs, which are engineered to deliver desired RNA silencing agents (e.g., siRNAs of the disclosure). By substituting the stem sequences of the pre-miRNA with sequence complementary to the target mRNA, a shRNA is formed. The shRNA is processed by the entire gene silencing pathway of the cell, thereby efficiently mediating RNAi. 
     The requisite elements of a shRNA molecule include a first portion and a second portion having sufficient complementarity to anneal or hybridize to form a duplex or double-stranded stem portion. The two portions need not be fully or perfectly complementary. The first and second “stem” portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. 
     This latter portion is referred to as a “loop” portion in the shRNA molecule. The shRNA molecules are processed to generate siRNAs. shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide “loop” in a portion of the stem, for example a one-, two- or three-nucleotide loop. The stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription. 
     In shRNAs (or engineered precursor RNAs) of the instant disclosure, one portion of the duplex stem is a nucleic acid sequence that is complementary (or anti-sense) to the DGAT2 target sequence. In certain embodiments, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi). Thus, engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The antisense portion can be on the 5′ or 3′ end of the stem. The stem portions of a shRNA are about 15 to about 50 nucleotides in length. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In certain embodiments, the length of the stem portions should be 21 nucleotides or greater. When used in mammalian cells, the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway. In non-mammalian cells, the stem can be longer than 30 nucleotides. In fact, the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). In fact, a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). 
     The two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions can be, but need not be, fully or perfectly complementary. In addition, the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop in the shRNAs or engineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. 
     The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. In certain embodiments, a loop consists of or comprises a “tetraloop” sequence. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU. 
     In certain embodiments, shRNAs of the present application include the sequences of a desired siRNA molecule described supra. In other embodiments, the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from within the target RNA (e.g., DGAT2 mRNA), for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation. In general, the sequence can be selected from any portion of the target RNA (e.g., mRNA) including the 5′ UTR (untranslated region), coding sequence, or 3′ UTR. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA. This sequence can replace a stem portion of a wild-type pre-miRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized. For example, one can synthesize DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-miRNA. 
     Engineered RNA precursors include, in the duplex stem, the 21-22 or so nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo. Thus, the stem portion of the engineered RNA precursor includes at least 18 or 19 nucleotide pairs corresponding to the sequence of an exonic portion of the gene whose expression is to be reduced or inhibited. The two 3′ nucleotides flanking this region of the stem are chosen so as to maximize the production of the siRNA from the engineered RNA precursor and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro. 
     In certain embodiments, shRNAs of the disclosure include miRNA sequences, optionally end-modified miRNA sequences to enhance entry into RISC. The miRNA sequence can be similar or identical to that of any naturally occurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). Over one thousand natural miRNAs have been identified to date and together they are thought to comprise about 1% of all predicted genes in the genome. Many natural miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai E C et al., Genome Bio., 2003). An online registry provides a searchable database of all published miRNA sequences (The miRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs from humans and certain model organisms including  Drosophila melanogaster, Caenorhabditis elegans,  zebrafish,  Arabidopsis thalania,  Mus musculus, and  Rattus norvegicus  as described in International PCT Publication No. WO 03/029459. 
     Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as a double-stranded duplex, but only one strand is taken up by the RISC complex to direct gene silencing. Certain miRNAs, e.g., plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs and, hence, direct cleavage of the target mRNAs. Other miRNAs have less than perfect complementarity to their target mRNAs and, hence, direct translational repression of the target mRNAs. The degree of complementarity between a miRNA and its target mRNA is believed to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is predictive of a cleavage mechanism (Yekta et al., Science, 2004), whereas less than perfect complementarity is predictive of a translational repression mechanism. In certain embodiments, the miRNA sequence is that of a naturally-occurring miRNA sequence, the aberrant expression or activity of which is correlated with a miRNA disorder. 
     d) Dual Functional Oligonucleotide Tethers 
     In other embodiments, the RNA silencing agents of the present disclosure include dual functional oligonucleotide tethers useful for the intercellular recruitment of a miRNA. Animal cells express a range of miRNAs, noncoding RNAs of approximately 22 nucleotides, which can regulate gene expression at the post transcriptional or translational level. By binding a miRNA bound to RISC and recruiting it to a target mRNA, a dual functional oligonucleotide tether can repress the expression of genes involved e.g., in the arteriosclerotic process. The use of oligonucleotide tethers offers several advantages over existing techniques to repress the expression of a particular gene. First, the methods described herein allow an endogenous molecule (often present in abundance), a miRNA, to mediate RNA silencing. Accordingly, the methods described herein obviate the need to introduce foreign molecules (e.g., siRNAs) to mediate RNA silencing. Second, the RNA-silencing agents and the linking moiety (e.g., oligonucleotides such as the 2′-O-methyl oligonucleotide), can be made stable and resistant to nuclease activity. As a result, the tethers of the present disclosure can be designed for direct delivery, obviating the need for indirect delivery (e.g. viral) of a precursor molecule or plasmid designed to make the desired agent within the cell. Third, tethers and their respective moieties, can be designed to conform to specific mRNA sites and specific miRNAs. The designs can be cell and gene product specific. Fourth, the methods disclosed herein leave the mRNA intact, allowing one skilled in the art to block protein synthesis in short pulses using the cell&#39;s own machinery. As a result, these methods of RNA silencing are highly regulatable. 
     The dual functional oligonucleotide tethers (“tethers”) of the disclosure are designed such that they recruit miRNAs (e.g., endogenous cellular miRNAs) to a target mRNA so as to induce the modulation of a gene of interest. In certain embodiments, the tethers have the formula T-L-μ, wherein T is an mRNA targeting moiety, L is a linking moiety, and μ is a miRNA recruiting moiety. Any one or more moiety may be double stranded. In certain embodiments, each moiety is single stranded. 
     Moieties within the tethers can be arranged or linked (in the 5′ to 3′ direction) as depicted in the formula T-L-μ (i.e., the 3′ end of the targeting moiety linked to the 5′ end of the linking moiety and the 3′ end of the linking moiety linked to the 5′ end of the miRNA recruiting moiety). Alternatively, the moieties can be arranged or linked in the tether as follows: μ-T-L (i.e., the 3′ end of the miRNA recruiting moiety linked to the 5′ end of the linking moiety and the 3′ end of the linking moiety linked to the 5′ end of the targeting moiety). 
     The mRNA targeting moiety, T, as described above, is capable of capturing a specific target mRNA. According to the disclosure, expression of the target mRNA is undesirable, and, thus, translational repression of the mRNA is desired. The mRNA targeting moiety should be of sufficient size to effectively bind the target mRNA. The length of the targeting moiety will vary greatly, depending, in part, on the length of the target mRNA and the degree of complementarity between the target mRNA and the targeting moiety. In various embodiments, the targeting moiety is less than about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. In a certain embodiment, the targeting moiety is about 15 to about 25 nucleotides in length. 
     The miRNA recruiting moiety, μ, as described above, is capable of associating with a miRNA. According to the present application, the miRNA may be any miRNA capable of repressing the target mRNA. Mammals are reported to have over 250 endogenous miRNAs (Lagos-Quintana et al. (2002) Current Biol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; and Lim et al. (2003) Science 299:1540). In various embodiments, the miRNA may be any art-recognized miRNA. 
     The linking moiety, L, is any agent capable of linking the targeting moieties such that the activity of the targeting moieties is maintained. Linking moieties can be oligonucleotide moieties comprising a sufficient number of nucleotides, such that the targeting agents can sufficiently interact with their respective targets. Linking moieties have little or no sequence homology with cellular mRNA or miRNA sequences. Exemplary linking moieties include one or more 2′-O-methylnucleotides, e.g., 2′-β-methyladenosine, 2′-O-methylthymidine, 2′-O-methylguanosine or 2′-O-methyluridine. 
     e) Gene Silencing Oligonucleotides 
     In certain exemplary embodiments, gene expression (i.e., DGAT2 gene expression) can be modulated using oligonucleotide-based compounds comprising two or more single stranded antisense oligonucleotides that are linked through their 5′-ends that allow the presence of two or more accessible 3′-ends to effectively inhibit or decrease DGAT2 gene expression. Such linked oligonucleotides are also known as Gene Silencing Oligonucleotides (GSOs). (See, e.g., U.S. Pat. No. 8,431,544 assigned to Idera Pharmaceuticals, Inc., incorporated herein by reference in its entirety for all purposes.) 
     The linkage at the 5′ ends of the GSOs is independent of the other oligonucleotide linkages and may be directly via 5′, 3′ or 2′hydroxyl groups, or indirectly, via a non-nucleotide linker or a nucleoside, utilizing either the 2′ or 3′ hydroxyl positions of the nucleoside. Linkages may also utilize a functionalized sugar or nucleobase of a 5′ terminal nucleotide. 
     GSOs can comprise two identical or different sequences conjugated at their 5′-5′ ends via a phosphodiester, phosphorothioate or non-nucleoside linker. Such compounds may comprise 15 to 27 nucleotides that are complementary to specific portions of mRNA targets of interest for antisense down regulation of a gene product. GSOs that comprise identical sequences can bind to a specific mRNA via Watson-Crick hydrogen bonding interactions and inhibit protein expression. GSOs that comprise different sequences are able to bind to two or more different regions of one or more mRNA target and inhibit protein expression. Such compounds are comprised of heteronucleotide sequences complementary to target mRNA and form stable duplex structures through Watson-Crick hydrogen bonding. Under certain conditions, GSOs containing two free 3′-ends (5′-5′-attached antisense) can be more potent inhibitors of gene expression than those containing a single free 3′-end or no free 3′-end. 
     In some embodiments, the non-nucleotide linker is glycerol or a glycerol homolog of the formula HO—(CH 2 ) o —CH(OH)—(CH 2 ) p —OH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4 or from 1 to about 3. In some other embodiments, the non-nucleotide linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO—(CH 2 )m—C(O)NH—CH 2 —CH(OH)—CH 2 —NHC(O)—(CH 2 ) m —OH, wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 to about 6 or from 2 to about 4. 
     Some non-nucleotide linkers permit attachment of more than two GSO components. For example, the non-nucleotide linker glycerol has three hydroxyl groups to which GSO components may be covalently attached. Some oligonucleotide-based compounds of the disclosure, therefore, comprise two or more oligonucleotides linked to a nucleotide or a non-nucleotide linker. Such oligonucleotides according to the disclosure are referred to as being “branched.” 
     In certain embodiments, GSOs are at least 14 nucleotides in length. In certain exemplary embodiments, GSOs are 15 to 40 nucleotides long or 20 to 30 nucleotides in length. Thus, the component oligonucleotides of GSOs can independently be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length. 
     These oligonucleotides can be prepared by the art recognized methods, such as phosphoramidate or H-phosphonate chemistry, which can be carried out manually or by an automated synthesizer. These oligonucleotides may also be modified in a number of ways without compromising their ability to hybridize to mRNA. Such modifications may include at least one internucleotide linkage of the oligonucleotide being an alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidate, carboxymethyl ester, or a combination of these and other internucleotide linkages between the 5′ end of one nucleotide and the 3′ end of another nucleotide, in which the 5′ nucleotide phosphodiester linkage has been replaced with any number of chemical groups. 
     V. Modified Anti-DGAT2 RNA Silencing Agents 
     In certain aspects of the disclosure, an RNA silencing agent (or any portion thereof) of the present application, as described supra, may be modified, such that the activity of the agent is further improved. For example, the RNA silencing agents described in Section II supra, may be modified with any of the modifications described infra. The modifications can, in part, serve to further enhance target discrimination, to enhance stability of the agent (e.g., to prevent degradation), to promote cellular uptake, to enhance the target efficiency, to improve efficacy in binding (e.g., to the targets), to improve patient tolerance to the agent, and/or to reduce toxicity. 
     1) Modifications to Enhance Target Discrimination 
     In certain embodiments, the RNA silencing agents of the present application may be substituted with a destabilizing nucleotide to enhance single nucleotide target discrimination (see U.S. application Ser. No. 11/698,689, filed Jan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan. 25, 2006, both of which are incorporated herein by reference). Such a modification may be sufficient to abolish the specificity of the RNA silencing agent for a non-target mRNA (e.g. wild-type mRNA), without appreciably affecting the specificity of the RNA silencing agent for a target mRNA (e.g. gain-of-function mutant mRNA). 
     In certain embodiments, the RNA silencing agents of the present application are modified by the introduction of at least one universal nucleotide in the antisense strand thereof. Universal nucleotides comprise base portions that are capable of base pairing indiscriminately with any of the four conventional nucleotide bases (e.g. A, G, C, U). A universal nucleotide is contemplated because it has relatively minor effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base portion or an inosine analog base portion selected from the group consisting of deoxyinosine (e.g. 2′-deoxyinosine), 7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In certain embodiments, the universal nucleotide is an inosine residue or a naturally occurring analog thereof. 
     In certain embodiments, the RNA silencing agents of the disclosure are modified by the introduction of at least one destabilizing nucleotide within 5 nucleotides from a specificity-determining nucleotide (i.e., the nucleotide which recognizes the disease-related polymorphism). For example, the destabilizing nucleotide may be introduced at a position that is within 5, 4, 3, 2, or 1 nucleotide(s) from a specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position which is 3 nucleotides from the specificity-determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destablilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g. siRNAs and shRNAs), the destabilizing nucleotide may be introduced in the strand or strand portion that does not contain the specificity-determining nucleotide. In certain embodiments, the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide. 
     2) Modifications to Enhance Efficacy and Specificity 
     In certain embodiments, the RNA silencing agents of the disclosure may be altered to facilitate enhanced efficacy and specificity in mediating RNAi according to asymmetry design rules (see U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterations facilitate entry of the antisense strand of the siRNA (e.g., a siRNA designed using the methods of the present application or an siRNA produced from a shRNA) into RISC in favor of the sense strand, such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of an RNA silencing agent is enhanced by lessening the base pair strength between the antisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) of the RNA silencing agent relative to the bond strength or base pair strength between the antisense strand 3′ end (AS 3′) and the sense strand 5′ end (S′5) of said RNA silencing agent. 
     In one embodiment, the asymmetry of an RNA silencing agent of the present application may be enhanced such that there are fewer G:C base pairs between the 5′ end of the first or antisense strand and the 3′ end of the sense strand portion than between the 3′ end of the first or antisense strand and the 5′ end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one mismatched base pair between the 5′ end of the first or antisense strand and the 3′ end of the sense strand portion. In certain embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one wobble base pair, e.g., G:U, between the 5′ end of the first or antisense strand and the 3′ end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one base pair comprising a rare nucleotide, e.g., inosine (I). In certain embodiments, the base pair is selected from the group consisting of an I:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one base pair comprising a modified nucleotide. In certain embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A. 
     3) RNA Silencing Agents with Enhanced Stability 
     The RNA silencing agents of the present application can be modified to improve stability in serum or in growth medium for cell cultures. In order to enhance the stability, the 3′-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2′-deoxythymidine is tolerated and does not affect the efficiency of RNA interference. 
     In a one aspect, the present application features RNA silencing agents that include first and second strands wherein the second strand and/or first strand is modified by the substitution of internal nucleotides with modified nucleotides, such that in vivo stability is enhanced as compared to a corresponding unmodified RNA silencing agent. As defined herein, an “internal” nucleotide is one occurring at any position other than the 5′ end or 3′ end of nucleic acid molecule, polynucleotide or oligonucleotide. An internal nucleotide can be within a single-stranded molecule or within a strand of a duplex or double-stranded molecule. In one embodiment, the sense strand and/or antisense strand is modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet another embodiment, the sense strand and/or antisense strand is modified by the substitution of all of the internal nucleotides. 
     In one aspect, the present application features RNA silencing agents that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents may be fully chemically modified, i.e., 100% of the nucleotides are chemically modified. In another aspect, the present application features RNA silencing agents comprising 2′-OH ribose groups that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents comprise 2′-OH ribose groups that are about 80%, 85%, 90%, 95%, or 100% chemically modified. 
     In certain embodiments, the RNA silencing agents may contain at least one modified nucleotide analogue. The nucleotide analogues may be located at positions where the target-specific silencing activity, e.g., the RNAi mediating activity or translational repression activity is not substantially affected, e.g., in a region at the 5′-end and/or the 3′-end of the siRNA molecule. Moreover, the ends may be stabilized by incorporating modified nucleotide analogues. 
     Exemplary nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphodiester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphorothioate group. In exemplary sugar-modified ribonucleotides, the 2′ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2  or ON, wherein R is C 1 -C 6  alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. 
     In certain embodiments, the modifications are 2′-fluoro, 2′-amino and/or 2′-thio modifications. Modifications include 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine, 2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine, 2′-amino-adenosine, 2′-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and/or 5-amino-allyl-uridine. In a certain embodiment, the 2′-fluoro ribonucleotides are every uridine and cytidine. Additional exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-Ome nucleotides can also be used within modified RNA-silencing agent moieties of the instant disclosure. Additional modified residues include, deoxy-abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin. In a certain embodiment, the 2′ moiety is a methyl group such that the linking moiety is a 2′-O-methyl oligonucleotide. 
     In a certain embodiment, the RNA silencing agent of the present application comprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modified nucleotides that resist nuclease activities (are highly stable) and possess single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have 2′-0,4′-C-ethylene-bridged nucleic acids, with possible modifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increase the specificity of oligonucleotides by constraining the sugar moiety into the 3′-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10° C. per base. 
     In another exemplary embodiment, the RNA silencing agent of the present application comprises Peptide Nucleic Acids (PNAs). PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone, which is highly resistant to nuclease digestion and imparts improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500). 
     Also contemplated are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined. 
     In other embodiments, cross-linking can be employed to alter the pharmacokinetics of the RNA silencing agent, for example, to increase half-life in the body. Thus, the present application includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked. The present application also includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3′ terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like). Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA. 
     Other exemplary modifications include: (a) 2′ modification, e.g., provision of a 2′ OMe moiety on a U in a sense or antisense strand, or provision of a 2′ OMe moiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicated by the context); (b) modification of the backbone, e.g., with the replacement of an 0 with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; e.g., with the replacement of a 0 with an S; (c) replacement of the U with a C 5  amino linker; (d) replacement of an A with a G (sequence changes can be located on the sense strand and not the antisense strand in certain embodiments); and (d) modification at the 2′, 6′, 7′, or 8′ position. Exemplary embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. Yet other exemplary modifications include the use of a methylated P in a 3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification, e.g., provision of a 2′ 0 Me moiety and modification of the backbone, e.g., with the replacement of a 0 with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3′ overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl; modification with an abasic pyrrolidone in a 3′ overhang, e.g., at the 3′ terminus; modification with naproxen, ibuprofen, or other moieties which inhibit degradation at the 3′ terminus. 
     Heavily modified RNA silencing agents 
     In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified. 
     In certain embodiments, the RNA silencing agent is 2′-O-methyl rich, i.e., comprises greater than 50% 2′-O-methyl content. In certain embodiments, the RNA silencing agent comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2′-0-methyl nucleotide content. In certain embodiments, the RNA silencing agent comprises at least about 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent comprises between about 70% and about 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and sense strand. In certain embodiments, the antisense strand comprises at least about 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises between about 70% and about 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between about 70% and about 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between 100% 2′-O-methyl nucleotide modifications. 
     2′-O-methyl rich RNA silencing agents and specific chemical modification patterns are further described in U.S. Patent Publication No. 2020/0087663A1 and U.S.S.N. 16/999,759 (filed August 21, 2020), each of which is incorporated herein by reference. 
     Internucleotide linkage modifications 
     In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all of the internucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the RNA silencing agent comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 8-13 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand, each comprising a 5′ end and a 3′ end. In certain embodiments, the nucleotides at positions 1 and 2 from the 5′ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 3′ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-8 from the 3′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, or 1-8 from the 3′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-7 from the 3′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. 
     In one aspect, the disclosure provides a modified oligonucleotide, said oligonucleotide having a 5′ end, a 3′ end, that is complementary to a target, wherein the oligonucleotide comprises a sense and antisense strand, and at least one modified intersubunit linkage of Formula (I): 
     
       
         
         
             
             
         
       
     
     wherein: 
     B is a base pairing moiety; 
     W is selected from the group consisting of O, OCH 2 , OCH, CH 2 , and CH; 
     X is selected from the group consisting of halo, hydroxy, and C 1 -6 alkoxy; 
     Y is selected from the group consisting of O − , OH, OR, NW, NH 2 , and SH; 
     Z is selected from the group consisting of O and CH 2 ; 
     R is a protecting group; and 
        is an optional double bond. 
     In an embodiment of Formula (I), when W is CH,   is a double bond. 
     In an embodiment of Formula (I), when W selected from the group consisting of O, OCH 2 , OCH, CH 2 ,   is a single bond. 
     In an embodiment of Formula (I), when Y is O − , either Z or W is not O. 
     In an embodiment of Formula (I), Z is CH 2  and W is CH 2 . In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (II): 
     
       
         
         
             
             
         
       
     
     In an embodiment of Formula (I), Z is CH 2  and W is O. In another embodiment, wherein the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (III): 
     
       
         
         
             
             
         
       
     
     In an embodiment of Formula (I), Z is O and W is CH 2 . In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (IV): 
     
       
         
         
             
             
         
       
     
     In an embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula V: 
     
       
         
         
             
             
         
       
     
     In an embodiment of Formula (I), Z is O and W is OCH 2 . In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VI: 
     
       
         
         
             
             
         
       
     
     In an embodiment of Formula (I), Z is CH 2  and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VII: 
     
       
         
         
             
             
         
       
     
     In an embodiment of Formula (I), the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil. 
     In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5′ end, a 3′ end, that is complementary to a target, wherein the siRNA comprises a sense and antisense strand, and at least one modified intersubunit linkage of any one or more of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII). 
     In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5′ end, a 3′ end, that is complementary to a target and comprises a sense and antisense strand, wherein the siRNA comprises at least one modified intersubunit linkage is of Formula VIII: 
     
       
         
         
             
             
         
       
     
     wherein: 
     D is selected from the group consisting of O , OCH 2 , OCH, CH 2 , and CH; 
     C is selected from the group consisting of O − , OH, OW, NH − , NH 2 , and SH; 
     A is selected from the group consisting of O and CH 2 ; R 1  is a protecting group; 
        is an optional double bond; and 
     the intersubunit is bridging two optionally modified nucleosides. 
     In an embodiment, when C is O − , either A or D is not O. 
     In an embodiment, D is CH 2 . In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (IX): 
     
       
         
         
             
             
         
       
     
     In an embodiment, D is O. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (X): 
     
       
         
         
             
             
         
       
     
     In an embodiment, D is CH 2 . In another embodiment, the modified intersubunit linkage of Formula (VIII) is a modified intersubunit linkage of Formula (XI): 
     
       
         
         
             
             
         
       
     
     In an embodiment, D is CH. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (XII): 
     
       
         
         
             
             
         
       
     
     In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIV): 
     
       
         
         
             
             
         
       
     
     In an embodiment, D is OCH 2 . In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIII): 
     
       
         
         
             
             
         
       
     
     In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XXa): 
     
       
         
         
             
             
         
       
     
     In an embodiment of the modified siRNA linkage, each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine, and uridine. 
     In certain exemplary embodiments of Formula (I), W is O. In another embodiment, W is CH 2 . In yet another embodiment, W is CH. 
     In certain exemplary embodiments of Formula (I), X is OH. In another embodiment, X is OCH 3 . In yet another embodiment, X is halo. 
     In a certain embodiment of Formula (I), the modified siRNA does not comprise a 2′-fluoro substituent. 
     In an embodiment of Formula (I), Y is O − . In another embodiment, Y is OH. In yet another embodiment, Y is OR. In still another embodiment, Y is NW. In an embodiment, Y is NH 2 . In another embodiment, Y is S. In yet another embodiment, Y is SH. 
     In an embodiment of Formula (I), Z is O. In another embodiment, Z is CH 2 . 
     In an embodiment, the modified intersubunit linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the modified intersubunit linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the modified intersubunit linkage is inserted on position 10-11 of the antisense strand. In still another embodiment, the modified intersubunit linkage is inserted on position 19-20 of the antisense strand. In an embodiment, the modified intersubunit linkage is inserted on positions 5-6 and 18-19 of the antisense strand. 
     In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), C is 0 - . In another embodiment, C is OH. In yet another embodiment, C is OR&#39;. In still another embodiment, C is NH - . In an embodiment, C is NH 2 . In another embodiment, C is S. In yet another embodiment, C is SH. 
     In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), A is 0. In another embodiment, A is CH 2 . In yet another embodiment, C is OR&#39;. In still another embodiment, C is NH − . In an embodiment, C is NH 2 . In another embodiment, C is S. In yet another embodiment, C is SH. 
     In a certain embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is adenosine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is guanosine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is cytidine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is uridine. 
     In an embodiment of the modified siRNA linkage, wherein the linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the linkage is inserted on position 10-11 of the antisense strand. In still another embodiment, the linkage is inserted on position 19-20 of the antisense strand. In an embodiment, the linkage is inserted on positions 5-6 and 18-19 of the antisense strand. 
     In certain embodiments of Formula (I), the base pairing moiety B is adenine. In certain embodiments of Formula (I), the base pairing moiety B is guanine. In certain embodiments of Formula (I), the base pairing moiety B is cytosine. In certain embodiments of Formula (I), the base pairing moiety B is uracil. 
     In an embodiment of Formula (I), W is 0. In an embodiment of Formula (I), W is CH 2 . In an embodiment of Formula (I), W is CH. 
     In an embodiment of Formula (I), X is OH. In an embodiment of Formula (I), X is OCH 3 . In an embodiment of Formula (I), Xis halo. 
     In an exemplary embodiment of Formula (I), the modified oligonucleotide does not comprise a 2′-fluoro substituent. 
     In an embodiment of Formula (I), Y is 0 - . In an embodiment of Formula (I), Y is OH. In an embodiment of Formula (I), Y is OR. In an embodiment of Formula (I), Y is NW . In an embodiment of Formula (I), Y is NH 2 . In an embodiment of Formula (I), Y is S. In an embodiment of Formula (I), Y is SH. 
     In an embodiment of Formula (I), Z is 0. In an embodiment of Formula (I), Z is CH 2 . 
     In an embodiment of the Formula (I), the linkage is inserted on position 1-2 of the antisense strand. In another embodiment of Formula (I), the linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment of Formula (I), the linkage is inserted on position 10-11 of the antisense strand. In still another embodiment of Formula (I), the linkage is inserted on position 19-20 of the antisense strand. In an embodiment of Formula (I), the linkage is inserted on positions 5-6 and 18-19 of the antisense strand. 
     Modified intersubunit linkages are further described in WO 2020/198509 and U.S. Ser. No. 63/000,328 (filed Mar. 26, 2020), each of which is incorporated herein by reference. 
     4) Conjugated Functional Moieties 
     In other embodiments, RNA silencing agents may be modified with one or more functional moieties. A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the disclosure includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 5′ and/or 3′ terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles). 
     In a certain embodiment, the functional moiety is a hydrophobic moiety. In a certain embodiment, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In a certain embodiment, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA). In a certain embodiment, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). In a certain embodiment, the vitamin selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In a certain embodiment, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate. 
     In a certain embodiment, an RNA silencing agent of disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand that includes a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of an siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3′ end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. 
     In certain embodiments, the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting. A tethered ligand can include one or more modified bases or sugars that can function as intercalators. These can be located in an internal region, such as in a bulge of RNA silencing agent/target duplex. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings. The universal bases described herein can be included on a ligand. In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III). In some embodiments, a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. A tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo-5-acridine, has an increased affinity for the HIV Rev-response element (RRE). In some embodiments, the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent. A tethered ligand can be a poly-arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent. 
     Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier. In certain embodiments, the coupling is through a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated. In certain embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. 
     Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide. 
     Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or derivatives thereof, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines and substituted acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC 4 , texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides, guanidium aminoglycodies, artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (and thio analogs thereof), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20  fatty acids) and ethers thereof, e.g., C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20  alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu 3+  complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof. 
     In certain embodiments, the GalNAc is represented by the formula below: 
     
       
         
         
             
             
         
       
     
     Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-kB. 
     The ligand can be a substance, e.g., a drug, which can increase the uptake of the RNA silencing agent into the cell, for example, by disrupting the cell&#39;s cytoskeleton, e.g., by disrupting the cell&#39;s microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of the RNA silencing agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF □), interleukin-1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can bind a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. In a certain embodiment, the lipid based ligand binds HSA. A lipid-based ligand can bind HSA with a sufficient affinity such that the conjugate will be distributed to a non-kidney tissue. However, it is contemplated that the affinity not be so strong that the HSA-ligand binding cannot be reversed. In another embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand. 
     In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These can be useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL). 
     In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent can be an alpha-helical agent, which may have a lipophilic and a lipophobic phase. 
     The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to oligonucleotide agents can affect pharmacokinetic distribution of the RNA silencing agent, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. The peptide moiety can be an L-peptide or D-peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptidomimetic tethered to an RNA silencing agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized. 
     In certain embodiments, the functional moiety is linked to the 5′ end and/or 3′ end of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5′ end and/or 3′ end of an antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5′ end and/or 3′ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 3′ end of a sense strand of the RNA silencing agent of the disclosure. 
     In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3′ end of a sense strand by a linker. In certain embodiments, the linker is a cleavable linker. In certain embodiments, the cleavable linker comprises a phosphodiester linkage, a disulfide linkage, an acid-labile linkage, or a photocleavable linkage. 
     In certain embodiments, the cleavable linker comprises a dTdT dinucleotide with phosphodiester intemucleotide linkages. 
     In certain embodiments, the acid-labile linkage comprises a β-thiopropionate linkage or a carboxydimethylmaleic anhydride (CDM) linkage. 
     In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from: 
     
       
         
         
             
             
         
       
     
     wherein n is 1, 2, 3, 4, or 5. 
     In certain embodiments, the linker further comprises a phosphodiester or phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: 
     
       
         
         
             
             
         
       
     
     wherein X is O, S or BH 3 . 
     The various functional moieties of the disclosure and means to conjugate them to RNA silencing agents are described in further detail in WO2017/030973A1 and WO2018/031933A2, incorporated herein by reference. 
     Methods of Introducing Nucleic Acids, Vectors and Host Cells 
     RNA silencing agents of the disclosure may be directly introduced into the cell (e.g., a neural cell) (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced. 
     The RNA silencing agents of the disclosure can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or other-wise increase inhibition of the target gene. 
     Physical methods of introducing nucleic acids include injection of a solution containing the RNA, bombardment by particles covered by the RNA, soaking the cell or organism in a solution of the RNA, or electroporation of cell membranes in the presence of the RNA. A viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, and the like. Thus, the RNA may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, inhibit annealing of single strands, stabilize the single strands, or other-wise increase inhibition of the target gene. 
     RNA may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the RNA. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the RNA may be introduced. 
     The cell having the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands. 
     Depending on the particular target gene and the dose of double stranded RNA material delivered, this process may provide partial or complete loss of function for the target gene. A reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of gene expression refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene. Specificity refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, Enzyme Linked ImmunoSorbent Assay (ELISA), Western blotting, RadioImmunoAssay (RIA), other immunoassays, and Fluorescence Activated Cell Sorting (FACS). 
     For RNA-mediated inhibition in a cell line or whole organism, gene expression is conveniently assayed by use of a reporter or drug resistance gene whose protein product is easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present disclosure. Lower doses of injected material and longer times after administration of RNAi agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). Quantization of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell; mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region. 
     The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of material may yield more effective inhibition; lower doses may also be useful for specific applications. 
     In an exemplary aspect, the efficacy of an RNAi agent of the disclosure (e.g., an siRNA targeting an DGAT2 target sequence) is tested for its ability to specifically degrade target mRNA (e.g., DGAT2 mRNA and/or the production of DGAT2 protein) in cells, such as cells in the liver or white adipose tissue. In certain embodiments, cells in the liver or white adipose tissue include, but are not limited to, hepatocytes, Kupffer cells, hepatic stellate cells, liver endothelial cells, and adipocytes. Also suitable for cell-based validation assays are other readily transfectable cells, for example, HeLa cells or COS cells. Cells are transfected with human cDNAs (e.g., human DGAT2 cDNA). Standard siRNA, modified siRNA or vectors able to produce siRNA from U-looped mRNA are co-transfected. Selective reduction in target mRNA (e.g., DGAT2 mRNA) and/or target protein (e.g., DGAT2 protein) is measured. 
     Reduction of target mRNA or protein can be compared to levels of target mRNA or protein in the absence of an RNAi agent or in the presence of an RNAi agent that does not target DGAT2 mRNA. Exogenously-introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison purposes. 
     Recombinant Adeno-Associated Viruses and Vectors 
     In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs into cells, e.g., liver cells (e.g., hepatocytes and Kupffer cells). AAV is able to infect many different cell types, although the infection efficiency varies based upon serotype, which is determined by the sequence of the capsid protein. Several native AAV serotypes have been identified, with serotypes 1-9 being the most commonly used for recombinant AAV. AAV-2 is the most well-studied and published serotype. The AAV-DJ system includes serotypes AAV-DJ and AAV-DJ/8. These serotypes were created through DNA shuffling of multiple AAV serotypes to produce AAV with hybrid capsids that have improved transduction efficiencies in vitro (AAV-DJ) and in vivo (AAV-DJ/8) in a variety of cells and tissues. 
     rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. An rAAV can be suspended in a physiologically compatible carrier (i.e., in a composition), and may be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, a non-human primate (e.g., Macaque) or the like. In certain embodiments, a host animal is a non-human host animal. 
     Delivery of one or more rAAVs to a mammalian subject may be performed, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In certain embodiments, one or more rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. 
     The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In certain embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs each having one or more different transgenes. 
     An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of one or more rAAVs is generally in the range of from about 1 ml to about 100 ml of solution containing from about 10 9  to 10 16  genome copies. In some cases, a dosage between about 10 11  to 10 12  rAAV genome copies is appropriate. In certain embodiments, 10 12  rAAV genome copies is effective to target heart, liver, and pancreas tissues. In some cases, stable transgenic animals are produced by multiple doses of an rAAV. 
     In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., about 10 13  genome copies/mL or more). Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright et al. (2005) Molecular Therapy 12:171-178, the contents of which are incorporated herein by reference.) 
     “Recombinant AAV (rAAV) vectors” comprise, at a minimum, a transgene and its regulatory sequences, and 5′ and 3′ AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., siRNA) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue. 
     The AAV sequences of the vector typically comprise the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are usually about 145 basepairs in length. In certain embodiments, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present disclosure is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including mammalian AAV types described further herein. 
     VI. Methods of Treatment 
     In one aspect, the present disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) developing diseases associated with the dysregulation of lipid metabolism by inhibiting DGAT2 which catalyzes triglyceride synthesis. Diseases associated with lipid metabolism dysregulation include; Nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), dyslipidemia, lipodystrophy syndrome and metabolic syndrome (MetS) the latter of which is associated with an increased risk of developing atherosclerotic cardiovascular disease (CVD), stroke and type 2 diabetes. In general, treatment will result in a reduction in serum levels of at least one other hepatic enzyme besides DGAT2, (such as, e.g., Stearoyl-CoA desaturase-1 (SCD1) or Fatty acid synthase (FASN)), and/or a decrease in hepatic lipid accumulation. 
     “Treatment,” or “treating,” as used herein, is defined as the application or administration of a therapeutic agent (e.g., a RNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease. 
     In one aspect, the disclosure provides a method for preventing in a subject, a disease or disorder as described above, by administering to the subject a therapeutic agent (e.g., an RNAi agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression. 
     Another aspect of the disclosure pertains to methods treating subjects therapeutically, i.e., alter onset of symptoms of the disease or disorder. In an exemplary embodiment, the modulatory method of the disclosure involves contacting a hepatocyte expressing DGAT2 with a therapeutic agent (e.g., a RNAi agent or vector or transgene encoding same) that is specific for a target sequence within the gene (e.g., DGAT2 target sequences of Table 1), such that sequence specific interference with the gene is achieved. These methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). 
     VII. Pharmaceutical Compositions and Methods of Administration 
     The disclosure pertains to uses of the above-described agents for prophylactic and/or therapeutic treatments as described infra. Accordingly, the modulators (e.g., RNAi agents) of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule or modulatory compound and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. 
     A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, and subcutaneous. 
     The nucleic acid molecules of the disclosure can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et al., (2002), Supra. Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). The pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. 
     The nucleic acid molecules of the disclosure can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3′ UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al. (2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002), supra. 
     The expression constructs may be any construct suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or H1 RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, Tuschl (2002), Supra. 
     For example, compositions can include one or more species of a compound of the disclosure and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present disclosure may be administered by intravenous or subcutaneous injection. or. 
     It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following example, which is included for purposes of illustration only and is not intended to be limiting. 
     EXAMPLES 
     Example 1. In Vitro Identification of DGAT2 Targeting Sequences 
     The DGAT2 gene was used as a target for mRNA knockdown. A panel of cholesterol conjugated siRNA duplexes, with homology to both human and mouse DGAT2, targeting several different sequences of DGAT2 mRNA was developed and screened in human HepG2 and mouse FL83B hepatic cell lines, in vitro and compared to untreated control cells ( FIG. 1A ). The siRNA duplexes were designed to target the open reading frame (ORF) and 3′ untranslated region (3′UTR), (Table 1 and  FIG. 1B ). The siRNA duplexes were each tested at a concentration of 1.5 μM and the mRNA was evaluated with the QuantiGene gene expression assay (ThermoFisher, Waltham, Mass.) at the 72 hours timepoint.  FIG. 1C . depicts the results of the screen against mouse DGAT2 mRNA evaluating seven DGAT2 siRNA duplexes in FL83B mouse cells. Three sites were identified that yielded potent and efficacious silencing of DGAT2 mRNA relative to % untreated control. Three of the identified siRNA duplexes, oligo ID&#39;s 1093, 1473, and 1476, were further screened against human DGAT2 mRNA in the HepG2 cell line identifying two out of the three siRNA duplexes, ID&#39;s 1473, and 1476, as the most potent,  FIG. 1D . Dose-response curves were generated for the identified lead siRNA duplexes, 1473, and 1476, in the HepG2 cell line confirming their potency,  FIG. 1E . 
     Table 1 recites 120-nucleotide DGAT2 target regions, and the sense and anti-sense strands of the siRNA duplexes screened in  FIGS. 1 &amp; 2 . The antisense strands contain a 5′ uracil to enhance loading into RISC. 
     Example 2. In Vivo DGAT2 Silencing Validation with Cholesterol Conjugated siRNAs 
     To test whether the lead siRNA duplexes identified by the in vitro screens silence DGAT2 in vivo, 8-week-old wild-type C 57 BL6/J mice on chow diet were subcutaneously injected with 10mg/kg Cholesterol conjugated non- targeting control (Chol-NTC) or Cholesterol conjugated 1473 compound targeting DGAT2 (Chol-1473). Mice were sacrificed at 10 days after injection. Analysis of the liver samples for mRNA expression measurements and protein knockdown levels shows the modified siRNA duplexes potently and efficaciously silence mouse DGAT2 mRNA expression in vivo,  FIG. 2A  &amp;  FIG. 2B . 
     Example 3. In Vivo DGAT2 Silencing Validation with N-acetylgalactosamine (GalNAc) Conjugated 1473 Tool Compound 
     To test whether GalNAc conjugated compound 1473 could potently silence DGAT2 in vivo, 8-week-old, C 57 BL6/J wild type mice on chow diet were injected either with different doses of GalNAc-1473, (10, 3, 1 mg/kg), or non-targeting control compound (10mg/kg) subcutaneously once and sacrificed at 4, 8 or 12 weeks after injections ( FIG. 3A ). Liver samples were processed for mRNA expression measurements. The DGAT2 silencing in liver was still abundant 12 weeks after single subcutaneous injection of GalNac-1473  FIG. 3B , and the mRNA analysis showed that DGAT2 silencing led to expression changes in genes involved in de novo lipogenesis,  FIG. 3C . 
     Example 4. GalNAc-1473 Silencing of DGAT2 in Humanized NSG Mice Bearing Engrafted with Human Hepatocytes 
     To test the potency of GalNAc-1473 in simultaneously silencing both mouse and human DGAT2 transcripts, a human hepatocyte engrafted NSG mice mouse model was generated ( FIG. 4A ). The mice, which were on a chow diet for 5 weeks after the engraftment, were then injected with 10mg/kg GalNAc-1473, or non-targeting control compound subcutaneously once and sacrificed a week after injections and liver samples processed for mRNA expression measurements. RNA analysis for DGAT2 expression showed significant silencing in both homology ( FIG. 4B  &amp;  FIG. 4C ). 
     Example 5. In Vivo Efficacy Study of DGAT2 Silencing by GalNAc-1473 in a NAFLD/NASH Mouse Model 
     To test the efficacy of GalNAc-1473 in silencing DGAT2 in a NAFLD/NASH animal model, 10 week genetically obese (ob/ob) mice were injected subQ with 10 mg/kg dose of GalNac conjugated non-targeting control (GalNac-NTC) or GalNac-1473. The mice were then switched to a high fat, high cholesterol GAN diet on injection day to induce a metabolic NASH phenotype. Blood samples were taken after 1 week of injection and mice were sacrificed three weeks after injection. mRNA and protein analysis of liver tissue showed significant knockdown of DGAT2 in the GalNac-1473 injected mice,  FIG. 5A . Liver weight and liver to body weight measurements showed significant reduction in liver weights with DGAT2 silencing,  FIG. 5B  &amp;  FIG. 5C . Measurements of Alanine aminotransferase (ALT), a serum marker of liver damage showed no significant difference between the treated and control mice,  FIG. 5D . 
     For histological analyses, murine liver tissue specimens were fixed for 24 h in 4% formalin at room temperature, dehydrated by graded ethanol and embedded in paraffin. Tissue sections (thickness 5 μm) were deparaffinized with xylene and stained with haematoxylin and eosin. The H&amp;E staining, showed decreased liver fat accumulation in NASH mice treated with GalNAc-1473 compared to GalNac-NTC,  FIG. 5E . Analysis of hepatic triglyceride (TG) concentrations showed a significant decrease in total liver triglycerides after DGAT2 silencing, as shown in  FIG. 5F . Diglyceride (DAG) concentrations and the fatty acyl chain content in triglycerides, all as measured by mass-spectrometry, were also reduced, as shown in  FIG. 5F . 
     The levels of phospholipids in the ob/ob mice with NASH were also assessed. As shown in  FIG. 7A - FIG. 7C , phosphatidylcholine ( FIG. 7A ), phosphatidylethanolamine ( FIG. 7B ), and phosphatidylinositol ( FIG. 7C ) levels were all elevated in mice injected with GalNAc-1473 compared to GalNac-NTC, consistent with DGAT2 knockdown. 
     DGAT2 mRNA levels were also measured in eight-week-old male C 57 BL6 mice injected with GalNAc-NTC or GalNAc-1473. The mice were injected with 10 mg/kg of siRNA and DGAT2 silencing was examined in kidney, spleen, inguinal fat, epididymal fat and liver via qPCR. As shown in  FIG. 8 , DGAT2 was preferentially silenced in the liver. 
     Example 6. Effect of DGAT2 Silencing on the Transcription of Genes iInvolved in De Novo Lipogenesis in Mice with NASH 
     Gene expression analysis of liver tissue, from the NAFLD/NASH mice on the high fat, high cholesterol GAN diet treated with GalNac-NTC or GalNac-1473, showed significant changes in the expression of genes involved in hepatic de novo lipogenesis with DGAT2 knockdown,  FIG. 6A . There were also changes in post translational modifications and protein expression levels of transcription factors that have a role in de novo lipogenesis gene expression,  FIG. 6B  &amp;  FIG. 6C . 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 DGAT2 gene target regions and antisense and sense strands 
               
               
                 targeting said DGAT2 gene target regions. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Sense 
                 Sense 
               
               
                   
                 Target 
                 Target Region 
                 Antisense 
                 Sequence 
                 Sequence 
               
               
                 Sequence ID 
                 Region 
                 (20 nucleotide) 
                 Sequence 
                 (Asymmetric) 
                 (Blunt) 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 TGGAGGAACC 
                 ATGGGTGTCT 
                 UAUAACCCAC 
                 GGGUGUCUGU 
                 AUGGGUGU 
               
               
                 h1372 
                 AGCTGCAAAT 
                 GTGGGTTATT 
                 AGACACCCAU 
                 GGGUUAUA 
                 CUGUGGGU 
               
               
                   
                 CACTTTTTTGC 
                 (SEQ ID NO: 6) 
                 (SEQ ID NO: 11) 
                 (SEQ ID NO: 16) 
                 UAUA (SEQ 
               
               
                   
                 TCTGTAAATTT 
                   
                   
                   
                 ID NO: 21) 
               
               
                   
                 GGAAGTGTCA 
                   
                   
                   
                   
               
               
                   
                 TGGGTGTCTGT 
                   
                   
                   
                   
               
               
                   
                 GGGTTATTTA 
                   
                   
                   
                   
               
               
                   
                 AAAGAAATTA 
                   
                   
                   
                   
               
               
                   
                 TAACAATTTTG 
                   
                   
                   
                   
               
               
                   
                 CTAAACCATT 
                   
                   
                   
                   
               
               
                   
                 ACAATGTTAG 
                   
                   
                   
                   
               
               
                   
                 GTCTTT (SEQ 
                   
                   
                   
                   
               
               
                   
                 ID NO: 1) 
                   
                   
                   
                   
               
               
                   
               
               
                 DGAT2_m1473_ 
                 TGGAGGAA 
                 TGTGGGTTATT 
                 UUUCUUUU 
                 UGGGUUAU 
                 UGUGGGUU 
               
               
                 h1381 
                 CCAGCTGC 
                 TAAAAGAA 
                 AAAUAACC 
                 UUAAAAGA 
                 AUUUAAAA 
               
               
                   
                 AAATCACTT 
                 (SEQ ID NO: 7) 
                 CACA (SEQ 
                 AA (SEQ ID 
                 GAAA (SEQ 
               
               
                   
                 TTTTGCTCT 
                   
                 ID NO: 12) 
                 NO: 17) 
                 ID NO: 22) 
               
               
                   
                 GTAAATTTG 
                   
                   
                   
                   
               
               
                   
                 GAAGTGTC 
                   
                   
                   
                   
               
               
                   
                 ATGGGTGT 
                   
                   
                   
                   
               
               
                   
                 CTGTGGGTT 
                   
                   
                   
                   
               
               
                   
                 ATTTAAAA 
                   
                   
                   
                   
               
               
                   
                 GAAATTAT 
                   
                   
                   
                   
               
               
                   
                 AACAATTTT 
                   
                   
                   
                   
               
               
                   
                 GCTAAACC 
                   
                   
                   
                   
               
               
                   
                 ATTACAAT 
                   
                   
                   
                   
               
               
                   
                 GTTAGGTCT 
                   
                   
                   
                   
               
               
                   
                 TT (SEQ ID 
                   
                   
                   
                   
               
               
                   
                 NO: 2) 
                   
                   
                   
                   
               
               
                   
               
               
                 DGAT2_m1476_ 
                 TGGAGGAA 
                 GGGTTATTTAA 
                 UAAUUUCU 
                 GUUAUUUA 
                 GGGUUAUU 
               
               
                 h1384 
                 CCAGCTGC 
                 AAGAAATTA 
                 UUUAAAUA 
                 AAAGAAAU 
                 UAAAAGAA 
               
               
                   
                 AAATCACTT 
                 (SEQ ID NO: 8) 
                 ACCC (SEQ 
                 UA (SEQ ID 
                 AUUA (SEQ 
               
               
                   
                 TTTTGCTCT 
                   
                 ID NO: 13) 
                 NO: 18) 
                 ID NO: 23) 
               
               
                   
                 GTAAATTTG 
                   
                   
                   
                   
               
               
                   
                 GAAGTGTC 
                   
                   
                   
                   
               
               
                   
                 ATGGGTGT 
                   
                   
                   
                   
               
               
                   
                 CTGTGGGTT 
                   
                   
                   
                   
               
               
                   
                 ATTTAAAA 
                   
                   
                   
                   
               
               
                   
                 GAAATTAT 
                   
                   
                   
                   
               
               
                   
                 AACAATTTT 
                   
                   
                   
                   
               
               
                   
                 GCTAAACC 
                   
                   
                   
                   
               
               
                   
                 ATTACAAT 
                   
                   
                   
                   
               
               
                   
                 GTTAGGTCT 
                   
                   
                   
                   
               
               
                   
                 TT (SEQ ID 
                   
                   
                   
                   
               
               
                   
                 NO: 3) 
                   
                   
                   
                   
               
               
                   
               
               
                 DGAT2_m1093_ 
                 AGGAGGGC 
                 TGGGTCCAGA 
                 UGGAACUU 
                 GGUCCAGA 
                 UGGGUCCA 
               
               
                 h1022 
                 TCCTGGGG 
                 AGAAGTTCCA 
                 CUUCUGGA 
                 AGAAGUUC 
                 GAAGAAGU 
               
               
                   
                 CCGATGGG 
                 (SEQ ID NO: 9) 
                 CCCA (SEQ 
                 CA (SEQ ID 
                 UCCA (SEQ 
               
               
                   
                 TCCAGAAG 
                   
                 ID NO: 14) 
                 NO: 19) 
                 ID NO: 24) 
               
               
                   
                 AAGTTCCA 
                   
                   
                   
                   
               
               
                   
                 GAAATACA 
                   
                   
                   
                   
               
               
                   
                 TTGGTTTCG 
                   
                   
                   
                   
               
               
                   
                 CCCCATGC 
                   
                   
                   
                   
               
               
                   
                 ATCTTCCAT 
                   
                   
                   
                   
               
               
                   
                 GGTCG (SEQ 
                   
                   
                   
                   
               
               
                   
                 ID NO: 4) 
                   
                   
                   
                   
               
               
                   
               
               
                 DGAT_m1094_ 
                 AGGAGGGC 
                 GGGTCCAGAA 
                 UUGGAACU 
                 GUCCAGAA 
                 GGGUCCAG 
               
               
                 h1023 
                 TCCTGGGG 
                 GAAGTTCCAG 
                 UCUUCUGG 
                 GAAGUUCC 
                 AAGAAGUU 
               
               
                   
                 CCGATGGG 
                 (SEQ ID NO: 10) 
                 ACCC (SEQ 
                 AA (SEQ ID 
                 CCAA (SEQ 
               
               
                   
                 TCCAGAAG 
                   
                 ID NO: 15) 
                 NO: 20) 
                 ID NO: 25) 
               
               
                   
                 AAGTTCCA 
                   
                   
                   
                   
               
               
                   
                 GAAATACA 
                   
                   
                   
                   
               
               
                   
                 TTGGTTTCG 
                   
                   
                   
                   
               
               
                   
                 CCCCATGC 
                   
                   
                   
                   
               
               
                   
                 ATCTTCCAT 
                   
                   
                   
                   
               
               
                   
                 GGTCG (SEQ 
                   
                   
                   
                   
               
               
                   
                 ID NO: 5) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Modified DGAT2 mRNA targeting antisense (as) strand sequences 
               
               
                 with a 5′ phosphate and complementary cholesterol conjugated 
               
               
                 sense (s) strand sequences. 
               
            
           
           
               
               
            
               
                 OLIGO ID 
                 Modified Sequence 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 P(mU)#(fA)#(mU)(mA)(mA)(fC)(mC)(mC)(mA)(mC)(mA)(mG)(mA) 
               
               
                 h1372-as 
                 #(fC)#(mA)#(fC)#(mC)#(mC)#(mA)#(fU) (SEQ ID NO: 26) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 P(mU)#(fU)#(mU)(mC)(mU)(fU)(mU)(mU)(mA)(mA)(mA)(mU)(mA) 
               
               
                 h1381-as 
                 #(fA)#(mC)#(fC)#(mC)#(mA)#(mC)#(fA) (SEQ ID NO: 27) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 P(mU)#(fA)#(mA)(mU)(mU)(fU)(mC)(mU)(mU)(mU)(mU)(mA)(mA) 
               
               
                 h1384-as 
                 #(fA)#(mU)#(fA)#(mA)#(mC)#(mC)#(fC) (SEQ ID NO: 28) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 P(mU)#(fG)#(mG)(mA)(mA)(fC)(mU)(mU)(mC)(mU)(mU)(mC)(mU) 
               
               
                 h1022-as 
                 #(fG)#(mG)#(fA)#(mC)#(mC)#(mC)#(fA) (SEQ ID NO: 29) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 P(mU)#(fU)#(mG)(mG)(mA)(fA)(mC)(mU)(mU)(mC)(mU)(mU)(mC) 
               
               
                 h1023-as 
                 #(fU)#(mG)#(fG)#(mA)#(mC)#(mC)#(fC) (SEQ ID NO: 30) 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 (mG)#(mG)#(mG)(mU)(mG)(mU)(mC)(fU)(fG)(fU)(mG)(fG)(mG)(mU) 
               
               
                 h1372-s (A) 
                 (mU)(mA)#(mU)#(mA)-TegChol (SEQ ID NO: 31) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 (mU)#(mG)#(mG)(mG)(mU)(mU)(mA)(fU)(fU)(fU)(mA)(fA)(mA)(mA) 
               
               
                 h1381-s (A) 
                 (mG)(mA)#(mA)#(mA)-TegChol (SEQ ID NO: 32) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 (mG)#(mU)#(mU)(mA)(mU)(mU)(mU)(fA)(fA)(fA)(mA)(fG)(mA)(mA) 
               
               
                 h1384-s (A) 
                 (mA)(mU)#(mU)#(mA)-TegChol (SEQ ID NO: 33) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 (mG)#(mG)#(mU)(mC)(mC)(mA)(mG)(fA)(fA)(fG)(mA)(fA)(mG)(mU) 
               
               
                 h1022-s (A) 
                 (mU)(mC)#(mC)#(mA)-TegChol (SEQ ID NO: 34) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 (mG)#(mU)#(mC)(mC)(mA)(mG)(mA)(fA)(fG)(fA)(mA)(fG)(mU) 
               
               
                 h1023-s (A) 
                 (mU)(mC)(mC)#(mA)#(mA)-TegChol (SEQ ID NO: 35) 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 (mA)#(mU)#(mG)(mG)(mG)(mU)(mG)(mU)(mC)(fU)(fG)(fU)(mG)(fG) 
               
               
                 h1372-s (B) 
                 (mG)(mU)(mU)(mA)#(mU)#(mA)-TegChol (SEQ ID NO: 36) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 (mU)#(mG)#(mU)(mG)(mG)(mG)(mU)(mU)(mA)(fU)(fU)(fU)(mA)(fA) 
               
               
                 h1381-s (B) 
                 (mA)(mA)(mG)(mA)#(mA)#(mA)-TegChol (SEQ ID NO: 37) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 (mG)#(mG)#(mG)(mU)(mU)(mA)(mU)(mU)(mU)(fA)(fA)(fA)(mA)(fG) 
               
               
                 h1384-s (B) 
                 (mA)(mA)(mA)(mU)#(mU)#(mA)-TegChol (SEQ ID NO: 38) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 (mU)#(mG)#(mG)(mG)(mU)(mC)(mC)(mA)(mG)(fA)(fA)(fG)(mA)(fA) 
               
               
                 h1022-s (B) 
                 (mG)(mU)(mU)(mC)#(mC)#(mA)-TegChol (SEQ ID NO: 39) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 (mG)#(mG)#(mG)(mU)(mC)(mC)(mA)(mG)(mA)(fA)(fG)(fA)(mA)(fG) 
               
               
                 h1023-s (B) 
                 (mU)(mU)(mC)(mC)#(mA)#(mA)-TegChol (SEQ ID NO: 40) 
               
               
                   
               
               
                 “m” represents a 2′-O-methyl nucleotide modification; 
               
               
                 “f” represents a 2′-Fluoro nucleotide modification; 
               
               
                 “#” represents a phosphorothioate internucleotide linkage; 
               
               
                 “P” represents a 5′ phosphate; 
               
               
                 “TegChol” represents cholesterol with a TEG linker. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Modified DGAT2 mRNA targeting antisense (as) strand sequences with 
               
               
                 a 5′-(E)-vinylphosphonate (VP) and complementary cholesterol conjugated 
               
               
                 sense (s) strand sequences. 
               
            
           
           
               
               
            
               
                 OLIGO ID 
                 Modified Sequence 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 VP (mU)#(fA)#(mU)(mA)(mA)(fC)(mC)(mC)(mA)(mC)(mA)(mG)(mA) 
               
               
                 h1372-as 
                 #(fC)#(mA)#(fC)#(mC)#(mC)#(mA)#(fU) (SEQ ID NO: 41) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 VP(mU)#(fU)#(mU)(mC)(mU)(fU)(mU)(mU)(mA)(mA)(mA)(mU)(mA) 
               
               
                 h1381-as 
                 #(fA)#(mC)#(fC)#(mC)#(mA)#(mC)#(fA) (SEQ ID NO: 42) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 VP(mU)#(fA)#(mA)(mU)(mU)(fU)(mC)(mU)(mU)(mU)(mU)(mA)(mA) 
               
               
                 h1384-as 
                 #(fA)#(mU)#(fA)#(mA)#(mC)#(mC)#(fC) (SEQ ID NO: 43) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 VP(mU)#(fG)#(mG)(mA)(mA)(fC)(mU)(mU)(mC)(mU)(mU)(mC)(mU) 
               
               
                 h1022-as 
                 #(fG)#(mG)#(fA)#(mC)#(mC)#(mC)#(fA) (SEQ ID NO: 44) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 VP(mU)#(fU)#(mG)(mG)(mA)(fA)(mC)(mU)(mU)(mC)(mU)(mU)(mC) 
               
               
                 h1023-as 
                 #(fU)#(mG)#(fG)#(mA)#(mC)#(mC)#(fC) (SEQ ID NO: 45) 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 (mG)#(mG)#(mG)(mU)(mG)(mU)(mC)(fU)(fG)(fU)(mG)(fG)(mG)(mU) 
               
               
                 h1372-s (A) 
                 (mU)(mA)#(mU)#(mA)-TegChol (SEQ ID NO: 31) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 (mU)#(mG)#(mG)(mG)(mU)(mU)(mA)(fU)(fU)(fU)(mA)(fA)(mA)(mA) 
               
               
                 h1381-s (A) 
                 (mG)(mA)#(mA)#(mA)-TegChol (SEQ ID NO: 32) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 (mG)#(mU)#(mU)(mA)(mU)(mU)(mU)(fA)(fA)(fA)(mA)(fG)(mA)(mA) 
               
               
                 h1384-s (A) 
                 (mA)(mU)#(mU)#(mA)-TegChol (SEQ ID NO: 33) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 (mG)#(mG)#(mU)(mC)(mC)(mA)(mG)(fA)(fA)(fG)(mA)(fA)(mG)(mU) 
               
               
                 h1022-s (A) 
                 (mU)(mC)#(mC)#(mA)-TegChol (SEQ ID NO: 34) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 (mG)#(mU)#(mC)(mC)(mA)(mG)(mA)(fA)(fG)(fA)(mA)(fG)(mU) 
               
               
                 h1023-s (A) 
                 (mU)(mC)(mC)#(mA)#(mA)-TegChol (SEQ ID NO: 35) 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 (mA)#(mU)#(mG)(mG)(mG)(mU)(mG)(mU)(mC)(fU)(fG)(fU)(mG)(fG) 
               
               
                 h1372-s (B) 
                 (mG)(mU)(mU)(mA)#(mU)#(mA)-TegChol (SEQ ID NO: 36) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 (mU)#(mG)#(mU)(mG)(mG)(mG)(mU)(mU)(mA)(fU)(fU)(fU)(mA)(fA) 
               
               
                 h1381-s (B) 
                 (mA)(mA)(mG)(mA)#(mA)#(mA)-TegChol (SEQ ID NO: 37) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 (mG)#(mG)#(mG)(mU)(mU)(mA)(mU)(mU)(mU)(fA)(fA)(fA)(mA)(fG) 
               
               
                 h1384-s (B) 
                 (mA)(mA)(mA)(mU)#(mU)#(mA)-TegChol (SEQ ID NO: 38) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 (mU)#(mG)#(mG)(mG)(mU)(mC)(mC)(mA)(mG)(fA)(fA)(fG)(mA)(fA) 
               
               
                 h1022-s (B) 
                 (mG)(mU)(mU)(mC)#(mC)#(mA)-TegChol (SEQ ID NO: 39) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 (mG)#(mG)#(mG)(mU)(mC)(mC)(mA)(mG)(mA)(fA)(fG)(fA)(mA)(fG) 
               
               
                 h1023-s (B) 
                 (mU)(mU)(mC)(mC)#(mA)#(mA)-TegChol (SEQ ID NO: 40) 
               
               
                   
               
               
                 “m” represents a 2′-O-methyl nucleotide modification; 
               
               
                 “f” represents a 2′-Fluoro nucleotide modification; 
               
               
                 “#” represents a phosphorothioate internucleotide linkage; 
               
               
                 “VP” represents a 5′-(E)-vinylphosphonate; 
               
               
                 “TegChol” represents cholesterol with a TEG linker. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Modified DGAT2 mRNA targeting antisense (as) strand 
               
               
                 sequences with a 5′-(E)-vinylphosphonate (VP) and 
               
               
                 complementary GalNAc conjugated sense (s) strand sequences. 
               
            
           
           
               
               
            
               
                 OLIGO ID 
                 Modified Sequence 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 VP(mU)#(fA)#(mU)(mA)(mA)(fC)(mC)(mC)(mA)(mC)(mA)(mG)(mA) 
               
               
                 h1372-as 
                 #(fC)#(mA)#(fC)#(mC)#(mC)#(mA)#(fU) (SEQ ID NO: 41) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 VP(mU)#(fU)#(mU)(mC)(mU)(fU)(mU)(mU)(mA)(mA)(mA)(mU)(mA) 
               
               
                 h1381-as 
                 #(fA)#(mC)#(fC)#(mC)#(mA)#(mC)#(fA) (SEQ ID NO: 42) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 VP(mU)#(fA)#(mA)(mU)(mU)(fU)(mC)(mU)(mU)(mU)(mU)(mA)(mA) 
               
               
                 h1384-as 
                 #(fA)#(mU)#(fA)#(mA)#(mC)#(mC)#(fC) (SEQ ID NO: 43) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 VP(mU)#(fG)#(mG)(mA)(mA)(fC)(mU)(mU)(mC)(mU)(mU)(mC)(mU) 
               
               
                 h1022-as 
                 #(fG)#(mG)#(fA)#(mC)#(mC)#(mC)#(fA) (SEQ ID NO: 44) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 VP(mU)#(fU)#(mG)(mG)(mA)(fA)(mC)(mU)(mU)(mC)(mU)(mU)(mC) 
               
               
                 h1023-as 
                 #(fU)#(mG)#(fG)#(mA)#(mC)#(mC)#(fC) (SEQ ID NO: 45) 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 (mG)#(mG)#(mG)(mU)(mG)(mU)(mC)(fU)(fG)(fU)(mG)(fG)(mG)(mU) 
               
               
                 h1372-s (A) 
                 (mU)(mA)#(mU)#(mA)-GalNAc (SEQ ID NO: 46) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 (mU)#(mG)#(mG)(mG)(mU)(mU)(mA)(fU)(fU)(fU)(mA)(fA)(mA)(mA) 
               
               
                 h1381-s (A) 
                 (mG)(mA)#(mA)#(mA)-GalNAc (SEQ ID NO: 47) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 (mG)#(mU)#(mU)(mA)(mU)(mU)(mU)(fA)(fA)(fA)(mA)(fG)(mA)(mA) 
               
               
                 h1384-s (A) 
                 (mA)(mU)#(mU)#(mA)-GalNAc (SEQ ID NO: 48) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 (mG)#(mG)#(mU)(mC)(mC)(mA)(mG)(fA)(fA)(fG)(mA)(fA)(mG)(mU) 
               
               
                 h1022-s (A) 
                 (mU)(mC)#(mC)#(mA)-GalNAc (SEQ ID NO: 49) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 (mG)#(mU)#(mC)(mC)(mA)(mG)(mA)(fA)(fG)(fA)(mA)(fG)(mU) 
               
               
                 h1023-s (A) 
                 (mU)(mC)(mC)#(mA)#(mA)-GalNAc (SEQ ID NO: 50) 
               
               
                   
               
               
                 DGAT2_m1464_ 
                 (mA)#(mU)#(mG)(mG)(mG)(mU)(mG)(mU)(mC)(fU)(fG)(fU)(mG)(fG) 
               
               
                 h1372-s (B) 
                 (mG)(mU)(mU)(mA)#(mU)#(mA)-GalNAc (SEQ ID NO: 51) 
               
               
                   
               
               
                 DGAT2_m1473_ 
                 (mU)#(mG)#(mU)(mG)(mG)(mG)(mU)(mU)(mA)(fU)(fU)(fU)(mA)(fA) 
               
               
                 h1381-s (B) 
                 (mA)(mA)(mG)(mA)#(mA)#(mA)-GalNAc (SEQ ID NO: 52) 
               
               
                   
               
               
                 DGAT2_m1476_ 
                 (mG)#(mG)#(mG)(mU)(mU)(mA)(mU)(mU)(mU)(fA)(fA)(fA)(mA)(fG) 
               
               
                 h1384-s (B) 
                 (mA)(mA)(mA)(mU)#(mU)#(mA)-GalNAc (SEQ ID NO: 53) 
               
               
                   
               
               
                 DGAT2_m1093_ 
                 (mU)#(mG)#(mG)(mG)(mU)(mC)(mC)(mA)(mG)(fA)(fA)(fG)(mA)(fA) 
               
               
                 h1022-s (B) 
                 (mG)(mU)(mU)(mC)#(mC)#(mA)-GalNAc (SEQ ID NO: 54) 
               
               
                   
               
               
                 DGAT2_m1094_ 
                 (mG)#(mG)#(mG)(mU)(mC)(mC)(mA)(mG)(mA)(fA)(fG)(fA)(mA)(fG) 
               
               
                 h1023-s (B) 
                 (mU)(mU)(mC)(mC)#(mA)#(mA)-GalNAc (SEQ ID NO: 55) 
               
               
                   
               
               
                 “m” represents a 2′-O-methyl nucleotide modification; 
               
               
                 “f” represents a 2′-Fluoro nucleotide modification; 
               
               
                 “#” represents a phosphorothioate internucleotide linkage; 
               
               
                 “VP” represents a 5′-(E)-vinylphosphonate; 
               
               
                 “GalNAc” represents a GalNAc conjugate. 
               
            
           
         
       
     
     INCORPORATION BY REFERENCE 
     The contents of all cited references (including literature references, patents, patent applications, and websites) that maybe cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited therein. The disclosure will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, which are well known in the art. 
     The present disclosure also incorporates by reference in their entirety techniques well known in the field of molecular biology and drug delivery. These techniques include, but are not limited to, techniques described in the following publications: 
     Atwell et al. J. Mol. Biol. 1997, 270: 26-35; 
     Ausubel et al. (eds.), C URRENT  P ROTOCOLS  I N  M OLECULAR  B IOLOGY , John Wiley &amp; Sons, NY (1993); 
     Ausubel, F. M. et al. eds., S HORT  P ROTOCOLS  I N  M OLECULAR  B IOLOGY  (4th Ed. 1999) John Wiley &amp; Sons, NY. (ISBN 0-471-32938-X); 
     C ONTROLLED  D RUG  B IOAVAILABILITY , D RUG  P RODUCT  D ESIGN  A ND  P ERFORMANCE , Smolen and Ball (eds.), Wiley, New York (1984); 
     Giege, R. and Ducruix, A. Barrett, C RYSTALLIZATION  O F  N UCLEIC  A CIDS  A ND  P ROTEINS , a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, New York, (1999); 
     Goodson, in M EDICAL  A PPLICATIONS  O F  C ONTROLLED  R ELEASE , vol. 2, pp. 115-138 (1984); 
     Hammerling, et al., in: M ONOCLONAL  A NTIBODIES  A ND  T-C ELL  H YBRIDOMAS  563-681 (Elsevier, N.Y., 1981; 
     Harlow et al., A NTIBODIES : A L ABORATORY  M ANUAL , (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); 
     Kabat et al., S EQUENCES  O F  P ROTEINS  O F  I MMUNOLOGICAL  I NTEREST  (National Institutes of Health, Bethesda, Md. (1987) and (1991); 
     Kabat, E. A., et al. (1991) S EQUENCES  O F  P ROTEINS  O F  I MMUNOLOGICAL  I NTEREST , Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; 
     Kontermann and Dubel eds., A NTIBODY  E NGINEERING  (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5). 
     Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); 
     Lu and Weiner eds., C LONING  A ND  E XPRESSION  V ECTORS  F OR  G ENE  F UNCTION  A NALYSIS  (2001) BioTechniques Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X). 
     M EDICAL  A PPLICATIONS  O F  C ONTROLLED  R ELEASE , Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); 
     Old, R.W. &amp; S. B. Primrose, P RINCIPLES  O F  G ENE  M ANIPULATION : A N  I NTRODUCTION  T O  G ENETIC  E NGINEERING  (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4). 
     Sambrook, J. et al. eds., M OLECULAR  C LONING : A L ABORATORY  M ANUAL  (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6). 
     S USTAINED  A ND  C ONTROLLED  R ELEASE  D RUG  D ELIVERY  S YSTEMS , J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978 
     Winnacker, E. L. F ROM  G ENES  T O  C LONES : I NTRODUCTION  T O  G ENE  T ECHNOLOGY  (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4). 
     Equivalents 
     The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.