Patent Publication Number: US-2020283766-A1

Title: Compositions and Methods for Inhibiting Expression of Transthyretin

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 16/276,541, filed Feb. 14, 2019, (pending), which is a continuation of U.S. patent application Ser. No. 15/380,571, filed Dec. 15, 2016, now U.S. Pat. No. 10,240,152, issued Mar. 26, 2019, which is a continuation of U.S. patent application Ser. No. 14/965,825, filed Dec. 10, 2015, (abandoned), which is a continuation of U.S. patent application Ser. No. 14/220,829, filed Mar. 20, 2014, now U.S. Pat. No. 9,234,196, issued Jan. 12, 2016, which is a continuation of U.S. patent application Ser. No. 13/410,262, filed Mar. 1, 2012, now U.S. Pat. No. 8,741,866, issued Jun. 3, 2014, which is a continuation of U.S. patent application Ser. No. 12/582,669, filed Oct. 20, 2009, now U.S. Pat. No. 8,168,775, issued May 1, 2012, which claims the benefit of U.S. Provisional Application No. 61/106,956, filed Oct. 20, 2008; U.S. Provisional Application No. 61/115,738, filed Nov. 18, 2008; U.S. Provisional Application No. 61/156,670, filed Mar. 2, 2009; U.S. Provisional Application No. 61/185,545, filed Jun. 9, 2009; U.S. Provisional Application No. 61/242,783, filed Sep. 15, 2009; and U.S. Provisional Application No. 61/244,794, filed Sep. 22, 2009, all of which are incorporated herein by reference, in their entirety, for all purposes. 
    
    
     FIELD OF THE INVENTION 
     The invention relates to a double-stranded ribonucleic acid (dsRNA) targeting a transthyretin (TTR) gene, and methods of using the dsRNA to inhibit expression of TTR. 
     REFERENCE TO SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 16, 2020, is named AYL078C6_sequencelisting.txt, which contains 1410 sequences and is 323,584 bytes in size. 
     BACKGROUND OF THE INVENTION 
     Transthyretin (TTR) is a secreted thyroid hormone-binding protein. TTR binds and transports retinol binding protein (RBP)/Vitamin A, and serum thyroxine (T4) in plasma and cerebrospinal fluid. 
     Both normal-sequence TTR and variant-sequence TTR cause amyloidosis. Normal-sequence TTR causes cardiac amyloidosis in people who are elderly and is termed senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA)). SSA often is accompanied by microscopic deposits in many other organs. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of clinically significant TTR amyloidosis (also called ATTR (amyloidosis-transthyretin type)). More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis. 
     The liver is the major site of TTR expression. Other significant sites of expression include the choroid plexus, retina and pancreas. 
     TTR amyloidosis manifests in various forms. When the peripheral nervous system is affected more prominently, the disease is termed familial amyloidotic polyneuropathy (FAP). When the heart is primarily involved but the nervous system is not, the disease is called familial amyloidotic cardiomyopathy (FAC). A third major type of TTR amyloidosis is called leptomeningeal/CNS (Central Nervous System) amyloidosis. 
     Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in  C. elegans.  dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.),  Drosophila  (see, e.g., Yang, D., et al.,  Curr. Biol.  (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). 
     U.S. 20070207974 discloses functional and hyperfunctional siRNAs. U.S. 20090082300 discloses antisense molecules directed against TTR. U.S. Pat. No. 7,250,496 discloses microRNAs directed against TTR. 
     SUMMARY OF THE INVENTION 
     In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to a part of a mRNA encoding transthyretin (TTR), wherein said region of complementarity is less than 30 nucleotides in length and the antisense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:170, SEQ ID NO:450, SEQ ID NO:730, or SEQ ID NO:1010. In a related embodiment, the sense strand comprises 15 or more contiguous nucleotides of SEQ ID NO:169, 
     SEQ ID NO:449, SEQ ID NO:729, or SEQ ID NO:1009. In yet another related embodiment, the sense strand consists of SEQ ID NO:449 and the antisense strand consists of SEQ ID NO:450. In yet another related embodiment, the sense strand consists of SEQ ID NO:729 and the antisense strand consists of SEQ ID NO:730. In still another related embodiment, the sense strand consists of SEQ ID NO:1009 and the antisense strand consists of SEQ ID NO:1010. In yet another related embodiment, the dsRNA comprises a sense strand selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16, and an antisense strand selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16. 
     In certain embodiments, the region of complementarity between the antisense strand of the dsRNA and the mRNA encoding transthyretin is 19 nucleotides in length. In another embodiment, the region of complementary consists of SEQ ID NO:169. In other embodiments, each strand of the dsRNA is 19, 20, 21, 22, 23, or 24 nucleotides in length. In still another embodiment, each strand is 21 nucleotides in length. 
     In certain embodiments, the dsRNA for inhibiting expression of transthyretin does not cleave a TTR mRNA between the adenine nucleotide at position 637 of SEQ ID NO:1331 and the guanine nucleotide at position 638 of SEQ ID NO:1331. In other embodiments, the dsRNA cleaves a TTR mRNA between the guanine nucleotide at position 636 of SEQ ID NO:1331 and the adenine nucleotide at position 637 of SEQ ID NO:1331. In certain embodiments, the dsRNA anneals to a TTR mRNA between the guanine nucleotide at position 628 of SEQ ID NO:1331 and the uracil nucleotide at position 646 of SEQ ID NO: 1331. 
     In still other related embodiments, the invention provides dsRNA as described above for inhibiting expression of transthyretin wherein the dsRNA comprises one or more modified nucleotides. In related embodiments, at least one modified nucleotide (or nucleotides) is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In another related embodiment, the modified nucleotide is chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. In certain embodiments, the dsRNA comprises at least one 2′-O-methyl modified nucleotide. 
     In other embodiments, a dsRNA as described above for inhibiting expression of transthyretin is conjugated to a ligand, or formulated in a lipid formulation. In certain embodiments, the lipid formulation may be a LNP formulation, a LNP01 formulation, a XTC-SNALP formulation, or a SNALP formulation. In related embodiments, the XTC-SNALP formulation is as follows: using 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (XTC) with XTC/DPPC/Cholesterol/PEG-cDMA in a ratio of 57.1/7.1/34.4/1.4 and a lipid:siRNA ratio of about 7. In still other related embodiments, the sense strand of the dsRNA consists of SEQ ID NO:1009 and the antisense strand consists of SEQ ID NO:1010, and the dsRNA is formulated in a XTC-SNALP formulation as follows: using 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC) with a XTC/DPPC/Cholesterol/PEG-cDMA in a ratio of 57.1/7.1/34.4/1.4 and a lipid:siRNA ratio of about 7. Alternatively, a dsRNA such as those described above can be formulated in a LNP09 formulation as follows: using XTC/DSPC/Chol/PEG 2000 -C14 in a ratio of 50/10/38.5/1.5 mol % and a lipid:siRNA ratio of about 11:1. In another variation, the dsRNA is formulated in a LNP11 formulation as follows: using MC3/DSPC/Chol/PEG 2000 -C14 in a ratio of 50/10/38.5/1.5 mol % and a lipid:siRNA ratio of about 11:1. In still another embodiment, the dsRNA is formulated in a LNP09 formulation or a LNP11 formulation and reduces TTR mRNA levels by about 85 to 90% at a dose of 0.3 mg/kg, relative to a PBS control group. In yet another embodiment, the dsRNA is formulated in a LNP09 formulation or a LNP11 formulation and reduces TTR mRNA levels by about 50% at a dose of 0.1 mg/kg, relative to a PBS control group. In yet another embodiment, the dsRNA is formulated in a LNP09 formulation or a LNP11 formulation and reduces TTR protein levels in a dose-dependent manner relative to a PBS control group as measured by a western blot. In yet another embodiment, the dsRNA is formulated in a SNALP formulation as follows: using DlinDMA with a DLinDMA/DPPC/Cholesterol/PEG2000-cDMA in a ratio of 57.1/7.1/34.4/1.4 and a lipid:siRNA ratio of about 7. 
     In certain embodiments, the invention provides a dsRNA such as those described above for inhibiting expression of transthyretin, wherein administration of the dsRNA to a cell results in about 95% inhibition of TTR mRNA expression as measured by a real time PCR assay, wherein the cell is a HepG2 cell or a Hep3B cell, and wherein the concentration of the dsRNA is 10 nM. In related embodiments, administration of the dsRNA to a cell results in about 74% inhibition of TTR mRNA expression as measured by a branched DNA assay, wherein the cell is a HepG2 cell or a Hep3B cell, and wherein the concentration of the dsRNA is 10 nM. In other related embodiments, the dsRNA has an IC50 of less than 10 pM in a HepG2 cell, wherein the concentration of the dsRNA is 10 nM. In still other related embodiments, the dsRNA has an ED50 of about 1 mg/kg. In still other related embodiments, administration of the dsRNA reduces TTR mRNA by about 80% in cynomolgus monkey liver, wherein the concentration of the dsRNA is 3 mg/kg. In still other related embodiments, administration of the dsRNA does not result in immunostimulatory activity in human peripheral blood mononuclear cells (PBMCs) as measured by IFN-αlpha and TNF-αlpha ELISA assays. In still other related embodiments, administration of the dsRNA reduces liver TTR mRNA levels by about 97% or serum TTR protein levels by about 90%, wherein the concentration of the dsRNA is 6 mg/kg. In still other related embodiments, administration of the dsRNA reduces liver TTR mRNA levels and/or serum TTR protein levels up to 22 days, wherein the concentration of the dsRNA is 6 mg/kg or 3 mg/kg. In still other related embodiments, the dsRNA suppresses serum TTR protein levels up to day 14 post-treatment when administered to a subject in need thereof at 1 mg/kg or 3 mg/kg. In still other related embodiments, the dsRNA reduces TTR expression by 98.9% in a Hep3B cell at a concentration of 0.1 nM as measured by real-time PCR. In still other related embodiments, the dsRNA reduces TTR expression by 99.4% in a Hep3B cell at a concentration of 10 nM as measured by real-time PCR. 
     In other embodiments, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein said dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to a part of a mRNA encoding transthyretin (TTR), wherein said region of complementarity is less than 30 nucleotides in length and wherein the dsRNA comprises a sense strand selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16, and an antisense strand selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16. 
     In another embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein said dsRNA comprises an antisense strand comprising a region complementary to 15-30 nucleotides of nucleotides 618-648 of SEQ ID NO: 1331 and wherein said antisense strand base pairs with the guanine at position 628 of SEQ ID NO:1331. 
     In certain embodiments, the invention provides a cell containing any of the dsRNAs described in the Summary, above. In certain other embodiments, the invention provides a vector comprising a nucleotide sequence that encodes at least one strand of any of the dsRNAs described in the Summary, above. In certain embodiments, the vector is in a cell. 
     In other embodiments, the invention provides a pharmaceutical composition for inhibiting expression of a TTR gene comprising any of the dsRNAs described in the Summary, above, and a pharmaceutically acceptable carrier. In related embodiments, the invention provides a pharmaceutical composition for inhibiting expression of a TTR gene comprising a dsRNA and a SNALP formulation, wherein the dsRNA comprises an antisense strand which is less than 30 nucleotides in length and comprises 15 or more contiguous nucleotides of SEQ ID NO:170, SEQ ID NO:450, SEQ ID NO:730, or SEQ ID NO:1010, and wherein the SNALP formulation comprises DlinDMA, DPPC, Cholesterol and PEG2000-cDMA in a ratio of 57.1/7.1/34.4/1.4 respectively. 
     In yet another embodiment, the invention provides a method of inhibiting TTR expression in a cell, the method comprising: (a) contacting the cell with any of dsRNAs described in the Summary, above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TTR gene, thereby inhibiting expression of the TTR gene in the cell. 
     In yet another embodiment, the invention provides a method of treating a disorder mediated by TTR expression comprising administering to a human in need of such treatment a therapeutically effective amount of any of the dsRNAs describe in the Summary, above. In related embodiments, the dsRNA is administered to the human at about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg. In yet another related embodiment, the dsRNA is administered to the human at about 1.0 mg/kg. In yet another related embodiment, the human being treated has transthyretin amyloidosis, and/or a liver disorder. In a related embodiment, the human is further provided a liver transplant. In yet another embodiment, administration of the dsRNA reduces TTR mRNA by about 80% in human liver, wherein the concentration of the dsRNA is 3 mg/kg. In yet another related embodiment, administration of the dsRNA does not result in immunostimulatory activity in the human as measured by IFN-αlpha and TNF-αlpha ELISA assays. In yet another related embodiment, administration of the dsRNA reduces liver TTR mRNA levels by about 97% or serum TTR protein levels by about 90%, wherein the concentration of the dsRNA is 6 mg/kg. In yet another related embodiment, administration of the dsRNA reduces liver TTR mRNA levels and/or serum TTR protein levels up to 22 days, wherein the concentration of the dsRNA is 6 mg/kg or 3 mg/kg. In yet another related embodiment, the dsRNA is formulated in a LNP09 formulation as follows: using XTC/DSPC/Chol/PEG 2000 -C14 in a ratio of 50/10/38.5/1.5 mol % and a lipid:siRNA ratio of about 11:1. In yet another related embodiment, the dsRNA is formulated in a LNP11 formulation as follows: using MC3/DSPC/Chol/PEG 2000 -C14 in a ratio of 50/10/38.5/1.5 mol % and a lipid:siRNA ratio of about 11:1. In yet another related embodiment, the dsRNA is formulated in a LNP09 formulation or a LNP11 formulation and reduces TTR mRNA levels by about 85 to 90% at a dose of 0.3 mg/kg, relative to a PBC control group. In yet another related embodiment, the dsRNA is formulated in a LNP09 formulation or a LNP11 formulation and reduces TTR mRNA levels by about 50% at a dose of 0.1 mg/kg, relative to a PBC control group. In still another related embodiment, the dsRNA is formulated in a LNP09 formulation or a LNP11 formulation and reduces TTR protein levels in a dose-dependent manner relative to a PBC control group as measured by a western blot. In still another related embodiment, administration of the dsRNA suppresses serum TTR protein levels up to day 14 post-treatment when administered to human at 1 mg/kg or 3 mg/kg. In still another related embodiment, the dsRNA is formulated in a SNALP formulation as follows: using DlinDMA with a DLinDMA/DPPC/Cholesterol/PEG2000-cDMA in a ratio of 57.1/7.1/34.4/1.4 and a lipid:siRNA ratio of about 7. 
     In another embodiment, the invention provides the use of a dsRNA for treating a disorder mediated by TTR expression comprising administering to a human in need of such treatment a therapeutically effective amount of any of the dsRNAs described in the Summary, above. In related embodiments, the dsRNA is administered to the human at about 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg/kg. In a particular related embodiment, the dsRNA is administered to the human at about 1.0 mg/kg. In another related embodiment, the human has transthyretin amyloidosis, and/or a liver disorder. In yet another embodiment of the use provided by the invention, the treated human is further provided a liver transplant. 
     In yet another embodiment, the invention provides the use of a dsRNA in a method for inhibiting TTR expression in a cell, wherein the method comprises (a) contacting the cell with a dsRNA described in the Summary, above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a TTR gene, thereby inhibiting expression of the TTR gene in the cell. 
     The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a graph of TNFalpha and IFNalpha levels in cultured human PBMCs following transfection with TTR siRNAs. 
         FIGS. 2A and 2B  are dose response curves for AD-18324 and AD-18328, respectively, in HepG2 cells. 
         FIG. 3  is a dose response curve for AD-18246 in HepG2 cells. 
         FIG. 4A  and  FIG. 4B  show inhibition of liver mRNA and plasma protein levels, respectively, in transgenic H129-mTTR-KO/iNOS-KO/hTTR mice by an intravenous bolus administration of TTR-dsRNA (AD-18324, AD-18328 and AD-18246) formulated in LNP01. 
         FIG. 5  is a graph summarizing the measurements of TTR mRNA levels in livers of non-human primates following 15-minute intravenous infusion of TTR-dsRNA (AD-18324 and AD-18328) formulated in SNALP. 
         FIG. 6A  and  FIG. 6B  show inhibition of human V30M TTR liver mRNA and serum protein levels, respectively, in transgenic mice by an intravenous bolus administration of SNALP-18328. Group means were determined, normalized to the PBS control group, and then plotted. Error bars represent standard deviations. The percentage reduction of the group mean, relative to PBS, is indicated for the SNALP-1955 and SNALP-18328 groups. (***p&lt;0.001, One-way ANOVA, with Dunn&#39;s post-hoc test). 
         FIG. 7A  and  FIG. 7B  show the durability of reduction of human V30M TTR liver mRNA and serum protein levels, respectively, in transgenic mice over 22 days following a single intravenous bolus administration of SNALP-18328. Group means were determined. TTR/GAPDH mRNA levels were normalized to day 0 levels and plotted. The percent reduction of normalized TTR mRNA levels relative to SNALP-1955 for each time point were calculated and are indicated for the SNALP-18328 groups. (***p&lt;0.001, One-way ANOVA, with Dunn&#39;s post-hoc test). 
         FIG. 8  shows the timecourse of TTR serum protein levels in non-human primates over 14 days following a single 15-minute intravenous infusion of SNALP-18328. 
         FIG. 9  shows reduction of TTR-immunoreactivity in various tissues of human V30M TTR/HSF-1 knock-out mice following intravenous bolus administration of SNALP-18328. E, esophagus; S, stomach; I1, intestine/duodenum; I4, intestine/colon; N, nerve; D, dorsal root ganglia. 
         FIG. 10  shows the measurements of TTR mRNA levels in livers of non-human primates following 15-minute intravenous infusion of XTC-SNALP-18328. 
         FIGS. 11A and 11B  show the measurements of TTR mRNA and serum protein levels, respectively, in livers of non-human primates following 15-minute intravenous infusion of LNP09-18328 or LNP11-18328.  FIG. 11C  shows the timecourse of TTR serum protein levels over 28 days following a 15-minute intravenous infusion of 0.3 mg/kg LNP09-18328, as compared to the PBS control group. 
         FIG. 12  shows the sequence of human TTR mRNA (Ref. Seq. NM_000371.3, SEQ ID NO:1331). 
         FIGS. 13A and 13B  are the sequences of human and rat TTR mRNA, respectively.  FIG. 13A  is the sequence of human TTR mRNA (Ref. Seq. NM_000371.2, SEQ ID NO:1329).  FIG. 13B  is the sequence of rat TTR mRNA (Ref. Seq. NM_012681.1, SEQ ID NO:1330). 
         FIG. 14  shows the nucleotide alignment of NM_000371.3 (SEQ ID NO: 1331), NM_000371.2 (SEQ ID NO: 1329), and AD-18328 (SEQ ID NO: 1410). 
         FIG. 15  illustrates symptoms and mutations in TTR associated with familial amyloidotic neuropathy, familial amyloidotic cardiomyopathy and CNS amyloidosis. 
         FIG. 16  shows reduction of TTR mRNA levels in the liver with SNALP-18534 with different infusion durations. Groups of animals (n=4/group) were administered 1 mg/kg SNALP-18534 via a 15-minute, or 1, 2, or 3 hour infusion. Forty-eight hours later, rats were euthanized and livers harvested. TTR and GAPDH mRNA levels were measured from liver lysates using the Quantigene bDNA assay. The ratio of TTR to GAPDH mRNA levels was calculated for each animal. Group means were determined and normalized to a PBS control group, and then plotted. Error bars represent standard deviations. (***p&lt;0.001, One-way ANOVA with Bonferroni post-hoc test, relative to PBS). 
         FIG. 17  shows the measurements of TTR mRNA levels in livers of rats following 15-minute intravenous infusion of LNP07-18534 or LNP08-18534. 
         FIG. 18  shows in vivo inhibition of endogenous TTR mRNA levels in livers of Sprague-Dawley Rats following a 15-min IV infusion of LNP09-18534 or LNP11-18534. Groups of animals (n=4/group) were intravenously administered 0.01, 0.03, 0.1, or 0.3 mg/kg LNP09-18534, LNP-11-18534; or PBS via a 15-minute infusion. Forty-eight hours later, animals were euthanized and livers harvested. TTR and GAPDH mRNA levels were measured from liver biopsy lysates using the Quantigene bDNA assay. The ratio of TTR to GAPDH mRNA levels was calculated for each animal. Group means were determined, normalized to the PBS control group, and then plotted. Error bars represent standard deviations. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention provides dsRNAs and methods of using the dsRNAs for inhibiting the expression of a TTR gene in a cell or a mammal where the dsRNA targets a TTR gene. The invention also provides compositions and methods for treating pathological conditions and diseases, such as a TTR amyloidosis, in a mammal caused by the expression of a TTR gene. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). 
     The dsRNAs of the compositions featured herein include an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an mRNA transcript of a TTR gene. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in pathologies associated with TTR expression in mammals. Very low dosages of TTR dsRNAs in particular can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a TTR gene. Using cell-based assays, the present inventors have demonstrated that dsRNAs targeting TTR can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of a TTR gene. Thus, methods and compositions including these dsRNAs are useful for treating pathological processes that can be mediated by down regulating TTR, such as in the treatment of a liver disorder or a TTR amyloidosis, e.g., FAP. 
     The methods and compositions containing a TTR dsRNA are useful for treating pathological processes mediated by TTR expression, such as a TTR amyloidosis. In an embodiment, a method of treating a disorder mediated by TTR expression includes administering to a human in need of such treatment a therapeutically effective amount of a dsRNA targeted to TTR. In an embodiment, a dsRNA is administered to the human at about 0.01, 0.1, 0.5, 1.0, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mg/kg. 
     The following detailed description discloses how to make and use the compositions containing dsRNAs to inhibit the expression of a TTR gene, as well as compositions and methods for treating diseases and disorders caused by the expression of this gene. The pharmaceutical compositions featured in the invention include a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of a TTR gene, together with a pharmaceutically acceptable carrier. The compositions featured in the invention also include a dsRNA having an antisense strand having a region of complementarity which is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of a TTR gene. 
     The sense strand of a dsRNA can include 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides of SEQ ID NO:169, SEQ ID NO:449, SEQ ID NO:729, or SEQ ID NO:1009. The antisense strand of a dsRNA can include 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides of SEQ ID NO:170, SEQ ID NO:450, SEQ ID NO:730, or SEQ ID NO:1010. In an embodiment, the sense strand of a dsRNA can consist of SEQ ID NO:449 or fragments thereof and the antisense strand can consist of SEQ ID NO:450 or fragments thereof. In an embodiment, the sense strand of a dsRNA can consist of SEQ ID NO:729 or fragments thereof and the antisense strand can consist of SEQ ID NO:730 or fragments thereof. In an embodiment, the sense strand of a dsRNA can consist of SEQ ID NO:1009 or fragments thereof and the antisense strand can consist of SEQ ID NO:1010 or fragments thereof. 
     In an embodiment, a dsRNA can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In an embodiment, a modified nucleotide can include a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and/or a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. In an embodiment, a modified nucleotide can include a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and/or a non-natural base comprising nucleotide. 
     In an embodiment, the region of complementary of a dsRNA is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more nucleotides in length. In an embodiment, the region of complementary includes 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides of SEQ ID NO:169. 
     In an embodiment, each strand of a dsRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. In an embodiment, the dsRNA includes a sense strand, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotide fragment thereof, selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16, and an antisense strand, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotide fragment thereof, selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16. 
     In an embodiment, administration of a dsRNA to a cell results in about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more inhibition of TTR mRNA expression as measured by a real time PCR assay. In an embodiment, administration of a dsRNA to a cell results in about 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or more inhibition of TTR mRNA expression as measured by a real time PCR assay. In an embodiment, administration of a dsRNA to a cell results in about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more inhibition of TTR mRNA expression as measured by a branched DNA assay. In an embodiment, administration of a dsRNA to a cell results in about 40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95% or more inhibition of TTR mRNA expression as measured by a branched DNA assay. 
     In an embodiment, a dsRNA has an IC50 of less than 0.01 pM, 0.1 pM, 1 pM, 5 pM, 10 pM, 100 pM, or 1000 pM. In an embodiment, a dsRNA has an ED50 of about 0.01, 0.1, 1, 5, or 10 mg/kg. 
     In an embodiment, administration of a dsRNA can reduce TTR mRNA by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more in cynomolgus monkeys. In an embodiment, administration of a dsRNA reduces liver TTR mRNA levels by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more or serum TTR protein levels by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more. In an embodiment, administration of a dsRNA reduces liver TTR mRNA levels and/or serum TTR protein levels up to 1, 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 days. 
     In an embodiment, a dsRNA is formulated in a LNP formulation and reduces TTR mRNA levels by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more at a dose of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/kg, relative to a PBC control group. In an embodiment, a dsRNA is formulated in a LNP formulation and reduces TTR protein levels about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more relative to a PBC control group as measured by a western blot. In an embodiment, a dsRNA suppresses serum TTR protein levels up to day 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 post-treatment when administered to a subject in need thereof at 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 mg/kg. 
     Accordingly, in some aspects, pharmaceutical compositions containing a TTR dsRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a TTR gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of a TTR gene are featured in the invention. 
     I. Definitions 
     For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail. 
     “G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., deoxyribothymine. However, it will be understood that the term “ribonucleotide” or “nucleotide” or “deoxyribonucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention. 
     As used herein, “transthyretin” (“TTR”) refers to a gene in a cell. TTR is also known as ATTR, HsT2651, PALB, prealbumin, TBPA, and transthyretin (prealbumin, amyloidosis type I). The sequence of a human TTR mRNA transcript can be found at NM_000371. The sequence of mouse TTR mRNA can be found at NM_013697.2, and the sequence of rat TTR mRNA can be found at NM_012681.1. 
     As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TTR gene, including mRNA that is a product of RNA processing of a primary transcription product. 
     As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature. 
     As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides. 
     This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein. 
     “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing. 
     The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use. 
     As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding TTR) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a TTR mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding TTR. 
     The term “double-stranded RNA” or “dsRNA,” as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims. 
     The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to a dsRNA as described above. 
     As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. 
     The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus. 
     The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand. 
     As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as a dsRNA or a plasmid from which a dsRNA is transcribed. SNALP are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15, 2008. These applications are hereby incorporated by reference. “Introducing into a cell,” when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein or known in the art. 
     The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to a TTR gene, herein refer to the at least partial suppression of the expression of a TTR gene, as manifested by a reduction of the amount of mRNA which may be isolated from a first cell or group of cells in which a TTR gene is transcribed and which has or have been treated such that the expression of a TTR gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of 
     
       
         
           
             
               
                 
                   
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     Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to TTR gene expression, e.g., the amount of protein encoded by a TTR gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, TTR gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a TTR gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference. 
     For example, in certain instances, expression of a TTR gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a TTR gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a TTR gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide featured in the invention. 
     As used herein in the context of TTR expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by TTR expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by TTR expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition, such as the slowing the progression of a TTR amyloidosis, such as FAP. Symptoms of TTR amyloidosis include sensory neuropathy (e.g. paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and corneal lattice dystrophy. 
     As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by TTR expression or an overt symptom of pathological processes mediated by TTR expression. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by TTR expression, the patient&#39;s history and age, the stage of pathological processes mediated by TTR expression, and the administration of other anti-pathological processes mediated by TTR expression agents. 
     As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter. For example, a therapeutically effective amount of a dsRNA targeting TTR can reduce TTR serum levels by at least 25%. In another example, a therapeutically effective amount of a dsRNA targeting TTR can improve liver function or renal function by at least 25%. 
     The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. 
     As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed. 
     II. Double-Stranded Ribonucleic Acid (dsRNA) 
     As described in more detail herein, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a TTR gene in a cell or mammal, e.g., in a human having a TTR amyloidosis, where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of a TTR gene, and where the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and where said dsRNA, upon contact with a cell expressing said TTR gene, inhibits the expression of said TTR gene by at least 30% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of a TTR gene can be reduced by at least 30% when measured by an assay as described in the Examples below. For example, expression of a TTR gene in cell culture, such as in Hep3B cells, can be assayed by measuring TTR mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay. The dsRNA of the invention can further include one or more single-stranded nucleotide overhangs. 
     The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a TTR gene, the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ. 
     Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is 25-30 nucleotides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ. 
     The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand. 
     A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. 
     In one embodiment, a TTR gene is a human TTR gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Tables 3A, 3B, 4, 6A, 6B, or 7, and the antisense strand is one of the antisense sequences of Tables 3A, 3B, 4, 6A, 6B, or 7. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 3A, 3B, 4, 6A, 6B, or 7 can readily be determined using the target sequence and the flanking TTR sequence. 
     The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 3A, 3B, 4, 6A, 6B, and 7, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Tables 3A, 3B, 4, 6A, 6B, or 7 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 3, 4, 6 or 7, and differing in their ability to inhibit the expression of a TTR gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired TTR target sequence can readily be made using the corresponding TTR antisense sequence and a complementary sense sequence. 
     In addition, the dsRNAs provided in Tables 3A, 3B, 4, 6A, 6B, or 7 identify a site in a TTR that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 3A, 3B, 4, 6A, 6B, or 7 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a TTR gene. 
     The dsRNA featured in the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA featured in the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a TTR gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a TTR gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a TTR gene is important, especially if the particular region of complementarity in a TTR gene is known to have polymorphic sequence variation within the population. 
     Modifications 
     In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Eds.), John Wiley &amp; Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. 
     Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 1 -amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 1 -5′ linkages, 2 1 -5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. 
     Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference 
     Modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2  component parts. 
     Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference. 
     In other suitable dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500. 
     Other embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH 2 —NH—CH 2 —, —CH 2 —N(CH 3 )—O—CH 2 [known as a methylene (methylimino) or MMI backbone], —CH 2 —O—N(CH 3 )—CH 2 —, —CH 2 —N(CH 3 )—N(CH 3 )—CH 2 — and —N(CH 3 )—CH 2 —CH 2 [wherein the native phosphodiester backbone is represented as —O—P—O—CH 2 —] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. 
     Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C 1  to C 10  alkyl or C 2  to C 10  alkenyl and alkynyl. Particularly preferred are O[(CH 2 ) n O] m CH 3 , O(CH 2 ) n OCH 3 , O(CH 2 ) n NH 2 , O(CH 2 ) n CH 3 , O(CH 2 ) n ONH 2 , and O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C 1  to C 10  lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , SO 2 CH 3 , ONO 2 , NO 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH 2 CH 2 OCH 3 , also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Hely. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH 2 ) 2 ON(CH 3 ) 2  group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH 2 —O—CH 2 —N(CH 2 ) 2 , also described in examples herein below. 
     Other preferred modifications include 2′-methoxy (2′-OCH 3 ), 2′-aminopropoxy (2′-OCH 2 CH 2 CH 2 NH 2 ) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety. 
     dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley &amp; Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2-O-methoxyethyl sugar modifications. 
     Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference. 
     Conjugates 
     Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &amp; Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937). 
     Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference. 
     It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within a dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. 
     Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. The cleavage site on the target mRNA of a dsRNA can be determined using methods generally known to one of ordinary skill in the art, e.g., the 5′-RACE method described in Soutschek et al.,  Nature;  2004, Vol. 432, pp. 173-178 (which is herein incorporated by reference for all purposes). In an embodiment, using the 5′-RACE method described by Soutschek et al., ALN-18328 was determined to cleave a TTR mRNA between the guanine nucleotide at position 636 of SEQ ID NO:1331 (NM_000371.3) and the adenine nucleotide at position 637 of SEQ ID NO:1331. In an embodiment, it was determined that ALN-18328 does not cleave a TTR mRNA between the adenine nucleotide at position 637 of SEQ ID NO:1331 and the guanine nucleotide at position 638 of SEQ ID NO:1331. 
     In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides &amp; Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate. 
     Vector Encoded dsRNAs 
     In another aspect, TTR dsRNA molecules are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,  TIG.  (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al.,  Proc. Natl. Acad. Sci. USA  (1995) 92:1292). 
     The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure. 
     The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al.,  Curr. Topics Micro. Immunol.  (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992),  Cell  68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al.,  Science  (1985) 230:1395-1398; Danos and Mulligan,  Proc. Natl. Acad. Sci. USA  (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19 ; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. Nos. 4,868,116; 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection. 
     Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. 
     For example, lentiviral vectors featured in the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors featured in the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference. 
     Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference. 
     Viral vectors can be derived from AV and AAV. In one embodiment, the dsRNA featured in the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. 
     A suitable AV vector for expressing the dsRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002),  Nat. Biotech.  20: 1006-1010. 
     Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference. 
     The promoter driving dsRNA expression in either a DNA plasmid or viral vector featured in the invention may be a eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)). 
     In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene. 
     Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell. 
     dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single TTR gene or multiple TTR genes over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance. 
     TTR specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, 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 gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene 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. 
     III. Pharmaceutical Compositions Containing dsRNA 
     In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a TTR gene, such as pathological processes mediated by TTR expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion. 
     The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of TTR genes. 
     In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.0059 mg/kg, 0.01 mg/kg, 0.0295 mg/kg, 0.05 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.628 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. 
     In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.3 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg , 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg. 
     In one embodiment, the dosage is between 0.005 mg/kg and 1.628 mg/kg. For example, the dsRNA can be administered at a dose of 0.0059 mg/kg, 0.0295 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, or 1.628 mg/kg. 
     In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. For example, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg. 
     The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. 
     The effect of a single dose on TTR levels is long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals, or at not more than 5, 6, 7, 8, 9, or 10 week intervals. 
     The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein. 
     Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by TTR expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human TTR. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human TTR. 
     The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. 
     The dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein. 
     Administration 
     The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration. 
     The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver). 
     The present invention includes pharmaceutical compositions that can be delivered by injection directly into the brain. The injection can be by stereotactic injection into a particular region of the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus), or the dsRNA can be delivered into multiple regions of the central nervous system (e.g., into multiple regions of the brain, and/or into the spinal cord). The dsRNA can also be delivered into diffuse regions of the brain (e.g., diffuse delivery to the cortex of the brain). 
     In one embodiment, a dsRNA targeting TTR can be delivered by way of a cannula or other delivery device having one end implanted in a tissue, e.g., the brain, e.g., the substantia nigra, cortex, hippocampus, striatum, corpus callosum or globus pallidus of the brain. The cannula can be connected to a reservoir of the dsRNA composition. The flow or delivery can be mediated by a pump, e.g., an osmotic pump or minipump, such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, a pump and reservoir are implanted in an area distant from the tissue, e.g., in the abdomen, and delivery is effected by a conduit leading from the pump or reservoir to the site of release. Infusion of the dsRNA composition into the brain can be over several hours or for several days, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery to the brain are described, for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014. 
     Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C 1-10  alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference. 
     Liposomal Formulations 
     There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. 
     Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo. 
     In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores. 
     Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes. 
     Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act. 
     Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin. 
     Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis 
     Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985). 
     Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274). 
     One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol. 
     Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265). 
     Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466). 
     Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G M1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters,  1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765). 
     Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside G M1 , galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G M1  or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al). 
     Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C 1215G , that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces. 
     A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene. 
     Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin. 
     Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). 
     If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class. 
     If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps. 
     If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. 
     If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides. 
     The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). 
     Nucleic Acid Lipid Particles 
     In one embodiment, a TTR dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964. 
     In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. 
     The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle. 
     In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference. 
     In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio. 
     The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle. 
     The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci 2 ), a PEG-dimyristyloxypropyl (Ci 4 ), a PEG-dipalmityloxypropyl (Ci 6 ), or a PEG-distearyloxypropyl (C[ 8 ). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle. 
     In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle. 
     LNP01 
     In one embodiment, the lipidoid ND98·4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4. 
     
       
         
         
             
             
         
       
     
     LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference. 
     Additional exemplary lipid-siRNA formulations are as follows: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                   
                 cationic lipid/non-cationic 
                   
               
               
                   
                   
                 lipid/cholesterol/PEG-lipid conjugate 
               
               
                   
                 Cationic Lipid 
                 Lipid:siRNA ratio 
                 Process 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 SNALP 
                 1,2-Dilinolenyloxy-N,N- 
                 DLinDMA/DPPC/Cholesterol/PEG-cDMA 
                   
               
               
                   
                 dimethylaminopropane (DLinDMA) 
                 (57.1/7.1/34.4/1.4) 
               
               
                   
                   
                 lipid:siRNA~7:1 
               
               
                 SNALP- 
                 2,2-Dilinoleyl-4-dimethylaminoethyl- 
                 XTC/DPPC/Cholesterol/PEG-cDMA 
               
               
                 XTC 
                 [1,3]-dioxolane (XTC) 
                 57.1/7.1/34.4/1.4 
               
               
                   
                   
                 lipid:siRNA~7:1 
               
               
                 LNP05 
                 2,2-Dilinoleyl-4-dimethylaminoethyl- 
                 XTC/DSPC/Cholesterol/PEG-DMG 
                 Extrusion 
               
               
                   
                 [1,3]-dioxolane (XTC) 
                 57.5/7.5/31.5/3.5 
               
               
                   
                   
                 lipid:siRNA~6:1 
               
               
                 LNP06 
                 2,2-Dilinoleyl-4-dimethylaminoethyl- 
                 XTC/DSPC/Cholesterol/PEG-DMG 
                 Extrusion 
               
               
                   
                 [1,3]-dioxolane (XTC) 
                 57.5/7.5/31.5/3.5 
               
               
                   
                   
                 lipid:siRNA~11:1 
               
               
                 LNP07 
                 2,2-Dilinoleyl-4-dimethylaminoethyl- 
                 XTC/DSPC/Cholesterol/PEG-DMG 
                 In-line 
               
               
                   
                 [1,3]-dioxolane (XTC) 
                 60/7.5/31/1.5, 
                 mixing 
               
               
                   
                   
                 lipid:siRNA~6:1 
               
               
                 LNP08 
                 2,2-Dilinoleyl-4-dimethylaminoethyl- 
                 XTC/DSPC/Cholesterol/PEG-DMG 
                 In-line 
               
               
                   
                 [1,3]-dioxolane (XTC) 
                 60/7.5/31/1.5, 
                 mixing 
               
               
                   
                   
                 lipid:siRNA~11:1 
               
               
                 LNP09 
                 2,2-Dilinoleyl-4-dimethylaminoethyl- 
                 XTC/DSPC/Cholesterol/PEG-DMG 
                 In-line 
               
               
                   
                 [1,3]-dioxolane (XTC) 
                 50/10/38.5/1.5 
                 mixing 
               
               
                   
                   
                 Lipid:siRNA 10:1 
               
               
                 LNP10 
                 (3aR,5s,6aS)-N,N-dimethyl-2,2- 
                 ALN100/DSPC/Cholesterol/PEG-DMG 
                 In-line 
               
               
                   
                 di((9Z,12Z)-octadeca-9,12- 
                 50/10/38.5/1.5 
                 mixing 
               
               
                   
                 dienyl)tetrahydro-3aH- 
                 Lipid:siRNA 10:1 
               
               
                   
                 cyclopenta[d][1,3] 
               
               
                   
                 dioxol-5-amine (ALN100) 
               
               
                 LNP11 
                 (6Z,9Z,28Z,31Z)-heptatriaconta- 
                 MC-3/DSPC/Cholesterol/PEG-DMG 
                 In-line 
               
               
                   
                 6,9,28,31-tetraen-19-yl 4- 
                 50/10/38.5/1.5 
                 mixing 
               
               
                   
                 (dimethylamino)butanoate (MC3) 
                 Lipid:siRNA 10:1 
               
               
                 LNP12 
                 1,1′-(2-(4-(2-((2-(bis(2- 
                 Tech G1/DSPC/Cholesterol/PEG-DMG 
                 In-line 
               
               
                   
                 hydroxydodecyl)amino)ethyl)(2- 
                 50/10/38.5/1.5 
                 mixing 
               
               
                   
                 hydroxydodecyl)amino)ethyl)piperazin- 
                 Lipid:siRNA 10:1 
               
               
                   
                 1-yl)ethylazanediyl)didodecan-2-ol 
               
               
                   
                 (Tech G1) 
               
               
                   
               
            
           
         
       
     
     LNP09 formulations and XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, which is hereby incorporated by reference. LNP11 formulations and MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, which is hereby incorporated by reference. 
     Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically &gt;85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm. 
     Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e. g. , p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference. 
     Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. 
     Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma. 
     The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. 
     The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. 
     Emulsions 
     The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington&#39;s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion. 
     Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). 
     Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285). 
     Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate. 
     A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). 
     Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase. 
     Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin. 
     The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions. 
     In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington&#39;s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271). 
     The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously. 
     Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil. 
     Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids. 
     Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above. 
     Penetration Enhancers 
     In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. 
     Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Each of the above mentioned classes of penetration enhancers are described below in greater detail. 
     Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252). 
     Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654). 
     Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman &amp; Gilman&#39;s The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington&#39;s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583). 
     Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51). 
     Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626). 
     Carriers 
     Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA &amp; Nucl. Acid Drug Dev., 1996, 6, 177-183. 
     Excipients 
     In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc). 
     Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. 
     Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used. 
     Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like. 
     Other Components 
     The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation. 
     Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. 
     In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more dsRNA compounds and (b) one or more anti-cytokine biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target IL1β (e.g., anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab). 
     Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred. 
     The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. 
     In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by TTR expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein. 
     Methods for Treating Diseases Caused by Expression of a TTR Gene 
     The invention relates in particular to the use of a dsRNA targeting TTR and compositions containing at least one such dsRNA for the treatment of a TTR-mediated disorder or disease. For example, a dsRNA targeting a TTR gene can be useful for the treatment of a TTR amyloidosis, such as familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/CNS amyloidosis, amyloidosis VII form (also known as leptomeningeal or meningocerebrovascular amyloidosis), hyperthyroxinemia, and cardiac amyloidosis (also called senile systemic amyloidosis (SSA) and senile cardiac amyloidosis (SCA)). 
       FIG. 15  illustrates symptoms and mutations in TTR associated with familial amyloidotic neuropathy, familial amyloidotic cardiomyopathy and CNS amyloidosis. The invention includes compositions and methods for treatment of these diseases and symptoms, and directed to these mutant versions of TTR. 
     A dsRNA targeting a TTR gene is also used for treatment of symptoms and disorders, such as TTR amyloidosis. Symptoms associated with such amyloidosis include, e.g., seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, gastrointestinal dysfunction (e.g., gastric ulcers, diarrhea, constipation, malabsorption), weight loss, hepatomegaly, lymphadenopathy, goiter, vitreous opacities, renal insufficiency (including proteinuria and kidney failure), nephropathy, cranial nerve dysfunction, corneal lattice dystrophy, and congestive heart failure with generalized weakness and difficulties breathing from fluid retention. 
     Owing to the inhibitory effects on TTR expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life. 
     The invention further relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating a TTR amyloidosis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. In one example, a dsRNA targeting TTR can be administered in combination with a liver transplant. In other examples, a dsRNA targeting TTR can be administered in combination with a pharmaceutical or therapeutic method for treating a symptom of a TTR disease, such as diuretics, ACE (angiotensin converting enzyme) inhibitors, angiotensin receptor blockers (ARBs), or dialysis, e.g., for management of renal function. 
     The dsRNA and an additional therapeutic agent can be administered in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein. 
     The invention features a method of administering a dsRNA targeting TTR to a patient having a disease or disorder mediated by TTR expression, such as a TTR amyloidosis, e.g., FAP. Administration of the dsRNA can stabilize and improve peripheral neurological function, for example, in a patient with FAP. Patients can be administered a therapeutic amount of dsRNA, such as 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dsRNA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, 25 minute, 60 minute, 120 minute or 180 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the dsRNA can reduce TTR levels in the blood or urine of the patient by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more. 
     Before administration of a full dose of the dsRNA, patients can be administered a smaller dose, such as a dose that is 5% of the full dose, and monitored for adverse effects, such as an allergic reaction or a change in liver function. For example, in patients monitored for changes in liver function, a low incidence of LFT (Liver Function Test) change (e.g., a 10-20% incidence of LFT) is acceptable (e.g., a reversible, 3-fold increase in ALT (alanine aminotransferase) and/or AST (aspartate aminotransferase) levels). 
     Many TTR-associated diseases and disorders are hereditary. Therefore, a patient in need of a TTR dsRNA can be identified by taking a family history. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a TTR dsRNA. A DNA test may also be performed on the patient to identify a mutation in the TTR gene, before a TTR dsRNA is administered to the patient. 
     The patient may have a biopsy performed before receiving a TTR dsRNA. The biopsy can be, for example, on a tissue, such as the gastric mucosa, peripheral nerve, skin, abdominal fat, liver, or kidney, and the biopsy may reveal amyloid plaques, which are indicative of a TTR-mediated disorder. Upon the identification of amyloid plaques, the patient is administered a TTR dsRNA. 
     Methods for Inhibiting Expression of a TTR Gene 
     In yet another aspect, the invention provides a method for inhibiting the expression of a TTR gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target TTR gene is silenced. 
     When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. 
     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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the dsRNAs and methods featured in the invention, 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 examples are illustrative only and not intended to be limiting. 
     EXAMPLES 
     Example 1. dsRNA Synthesis 
     Source of Reagents 
     Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology. 
     siRNA Synthesis 
     Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S.L. et al. (Edrs.), John Wiley &amp; Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany). 
     Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use. 
     For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows: 
     Diethyl-2-azabutane-1,4-dicarboxylate AA 
     
       
         
         
             
             
         
       
     
     A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%). 
     3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB 
     
       
         
         
             
             
         
       
     
     Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB. 
     3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC 
     
       
         
         
             
             
         
       
     
     3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt. 
     3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD 
     
       
         
         
             
             
         
       
     
     The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%). 
     1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE 
     
       
         
         
             
             
         
       
     
     Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH 2 PO 4 .H 2 O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%). 
     [6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF 
     
       
         
         
             
             
         
       
     
     Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl) (89%). 
     (6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG 
     
       
         
         
             
             
         
       
     
     Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%). 
     Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH 
     
       
         
         
             
             
         
       
     
     Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt%, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step. 
     Cholesterol Derivatised CPG AI 
     
       
         
         
             
             
         
       
     
     Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g). 
     The synthesis of siRNAs bearing a 5 1 -12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer. 
     Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Abbreviations of nucleotide monomers used in nucleic 
               
               
                 acid sequence representation. It will be understood 
               
               
                 that these monomers, when present in an oligonucleotide, 
               
               
                 are mutually linked by 5′-3′-phosphodiester bonds. 
               
            
           
           
               
               
               
            
               
                   
                 Abbreviation 
                 Nucleotide(s) 
               
               
                   
                   
               
               
                   
                 A 
                 adenosine-3′-phosphate 
               
               
                   
                 C 
                 cytidine-3′-phosphate 
               
               
                   
                 G 
                 guanosine-3′-phosphate 
               
               
                   
                 T 
                 5-methyluridine-3′-phosphate 
               
               
                   
                 U 
                 uridine-3′-phosphate 
               
               
                   
                 N 
                 any nucleotide (G, A, C, or T) 
               
               
                   
                 a 
                 2′-O-methyladenosine-3′-phosphate 
               
               
                   
                 c 
                 2′-O-methylcytidine-3′-phosphate 
               
               
                   
                 g 
                 2′-O-methylguanosine-3′-phosphate 
               
               
                   
                 u 
                 2′-O-methyluridine-3′-phosphate 
               
               
                   
                 dT 
                 2′-deoxythymidine-3′-phosphate 
               
               
                   
                 sT; sdT 
                 2′-deoxy-thymidine-5′phosphate-phosphorothioate 
               
               
                   
                   
               
            
           
         
       
     
     Example 2A 
     TTR siRNA Design 
     Transcripts 
     siRNA design was carried out to identify siRNAs targeting the gene transthyretin from human (symbol TTR) and rat (symbol Ttr). The design used the TTR transcripts NM_000371.2 (SEQ ID NO:1329) (human) and NM_012681.1 (SEQ ID NO:1330) (rat) from the NCBI Refseq collection. The siRNA duplexes were designed with 100% identity to their respective TTR genes. 
     siRNA Design and Specificity Prediction 
     The predicted specificity of all possible 19mers was determined for each sequence. The TTR siRNAs were used in a comprehensive search against the human and rat transcriptomes (defined as the set of NM_ and XM_ records within the NCBI Refseq set) using the FASTA algorithm. The Python script ‘offtargetFasta.py’ was then used to parse the alignments and generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8; mismatches in the putative cleavage sites 10 and 11 are given a penalty of 1.2, and mismatches in positions 12-19 a penalty of 1. Mismatches in position 1 are not considered. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNA strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific, and between 2.2 and 2.8 as moderately specific. In picking which oligos to synthesize, off-target scores of the antisense strand were sorted from high to low, and the 144 best (lowest off-target score) oligo pairs from human, and the best 26 pairs from rat were selected. 
     siRNA Sequence Selection 
     A total of 140 sense and 140 antisense human TTR derived siRNA oligos were synthesized and formed into duplexes. A total of 26 sense and 26 antisense rat TTR derived siRNA oligos were synthesized and formed into duplexes. Duplexes included The oligos are presented in Tables 2-4 (human TTR) and Tables 5-7 (rat TTR). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Identification numbers for human TTR dsRNAs 
               
               
                 See Table 4 for sequences and modifications of oligos. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Sense 
                 Antisense 
               
               
                   
                 Duplex # 
                 Oligo # 
                 Oligo # 
               
               
                   
                   
               
               
                   
                 AD-18243 
                 A-32153 
                 A-32154 
               
               
                   
                 AD-18244 
                 A-32155 
                 A-32156 
               
               
                   
                 AD-18245 
                 A-32157 
                 A-32158 
               
               
                   
                 AD-18246 
                 A-32159 
                 A-32160 
               
               
                   
                 AD-18247 
                 A-32163 
                 A-32164 
               
               
                   
                 AD-18248 
                 A-32165 
                 A-32166 
               
               
                   
                 AD-18249 
                 A-32167 
                 A-32168 
               
               
                   
                 AD-18250 
                 A-32169 
                 A-32170 
               
               
                   
                 AD-18251 
                 A-32171 
                 A-32172 
               
               
                   
                 AD-18252 
                 A-32175 
                 A-32176 
               
               
                   
                 AD-18253 
                 A-32177 
                 A-32178 
               
               
                   
                 AD-18254 
                 A-32179 
                 A-32180 
               
               
                   
                 AD-18255 
                 A-32181 
                 A-32182 
               
               
                   
                 AD-18256 
                 A-32183 
                 A-32184 
               
               
                   
                 AD-18257 
                 A-32187 
                 A-32188 
               
               
                   
                 AD-18258 
                 A-32189 
                 A-32190 
               
               
                   
                 AD-18259 
                 A-32191 
                 A-32192 
               
               
                   
                 AD-18260 
                 A-32193 
                 A-32194 
               
               
                   
                 AD-18261 
                 A-32195 
                 A-32196 
               
               
                   
                 AD-18262 
                 A-32199 
                 A-32200 
               
               
                   
                 AD-18263 
                 A-32201 
                 A-32202 
               
               
                   
                 AD-18264 
                 A-32203 
                 A-32204 
               
               
                   
                 AD-18265 
                 A-32205 
                 A-32206 
               
               
                   
                 AD-18266 
                 A-32207 
                 A-32208 
               
               
                   
                 AD-18267 
                 A-32211 
                 A-32212 
               
               
                   
                 AD-18268 
                 A-32213 
                 A-32214 
               
               
                   
                 AD-18269 
                 A-32215 
                 A-32216 
               
               
                   
                 AD-18270 
                 A-32217 
                 A-32218 
               
               
                   
                 AD-18271 
                 A-32219 
                 A-32220 
               
               
                   
                 AD-18272 
                 A-32221 
                 A-32222 
               
               
                   
                 AD-18273 
                 A-32223 
                 A-32224 
               
               
                   
                 AD-18274 
                 A-32225 
                 A-32226 
               
               
                   
                 AD-18275 
                 A-32227 
                 A-32228 
               
               
                   
                 AD-18276 
                 A-32229 
                 A-32230 
               
               
                   
                 AD-18277 
                 A-32231 
                 A-32232 
               
               
                   
                 AD-18278 
                 A-32233 
                 A-32234 
               
               
                   
                 AD-18279 
                 A-32235 
                 A-32236 
               
               
                   
                 AD-18280 
                 A-32237 
                 A-32238 
               
               
                   
                 AD-18281 
                 A-32239 
                 A-32240 
               
               
                   
                 AD-18282 
                 A-32241 
                 A-32242 
               
               
                   
                 AD-18283 
                 A-32243 
                 A-32244 
               
               
                   
                 AD-18284 
                 A-32247 
                 A-32248 
               
               
                   
                 AD-18285 
                 A-32249 
                 A-32250 
               
               
                   
                 AD-18286 
                 A-32251 
                 A-32252 
               
               
                   
                 AD-18287 
                 A-32253 
                 A-32254 
               
               
                   
                 AD-18288 
                 A-32255 
                 A-32256 
               
               
                   
                 AD-18289 
                 A-32259 
                 A-32260 
               
               
                   
                 AD-18290 
                 A-32261 
                 A-32262 
               
               
                   
                 AD-18291 
                 A-32263 
                 A-32264 
               
               
                   
                 AD-18292 
                 A-32265 
                 A-32266 
               
               
                   
                 AD-18293 
                 A-32267 
                 A-32268 
               
               
                   
                 AD-18294 
                 A-32269 
                 A-32270 
               
               
                   
                 AD-18295 
                 A-32271 
                 A-32272 
               
               
                   
                 AD-18296 
                 A-32273 
                 A-32274 
               
               
                   
                 AD-18297 
                 A-32275 
                 A-32276 
               
               
                   
                 AD-18298 
                 A-32277 
                 A-32278 
               
               
                   
                 AD-18299 
                 A-32279 
                 A-32280 
               
               
                   
                 AD-18300 
                 A-32281 
                 A-32282 
               
               
                   
                 AD-18301 
                 A-32283 
                 A-32284 
               
               
                   
                 AD-18302 
                 A-32285 
                 A-32286 
               
               
                   
                 AD-18303 
                 A-32287 
                 A-32288 
               
               
                   
                 AD-18304 
                 A-32289 
                 A-32290 
               
               
                   
                 AD-18305 
                 A-32291 
                 A-32292 
               
               
                   
                 AD-18306 
                 A-32295 
                 A-32296 
               
               
                   
                 AD-18307 
                 A-32297 
                 A-32298 
               
               
                   
                 AD-18308 
                 A-32299 
                 A-32300 
               
               
                   
                 AD-18309 
                 A-32301 
                 A-32302 
               
               
                   
                 AD-18310 
                 A-32303 
                 A-32304 
               
               
                   
                 AD-18311 
                 A-32307 
                 A-32308 
               
               
                   
                 AD-18312 
                 A-32309 
                 A-32310 
               
               
                   
                 AD-18313 
                 A-32311 
                 A-32312 
               
               
                   
                 AD-18314 
                 A-32313 
                 A-32314 
               
               
                   
                 AD-18315 
                 A-32315 
                 A-32316 
               
               
                   
                 AD-18316 
                 A-32319 
                 A-32320 
               
               
                   
                 AD-18317 
                 A-32321 
                 A-32322 
               
               
                   
                 AD-18318 
                 A-32323 
                 A-32324 
               
               
                   
                 AD-18319 
                 A-32325 
                 A-32326 
               
               
                   
                 AD-18320 
                 A-32327 
                 A-32328 
               
               
                   
                 AD-18321 
                 A-32331 
                 A-32332 
               
               
                   
                 AD-18322 
                 A-32333 
                 A-32334 
               
               
                   
                 AD-18323 
                 A-32335 
                 A-32336 
               
               
                   
                 AD-18324 
                 A-32337 
                 A-32338 
               
               
                   
                 AD-18325 
                 A-32339 
                 A-32340 
               
               
                   
                 AD-18326 
                 A-32341 
                 A-32342 
               
               
                   
                 AD-18327 
                 A-32343 
                 A-32344 
               
               
                   
                 AD-18328 
                 A-32345 
                 A-32346 
               
               
                   
                 AD-18329 
                 A-32347 
                 A-32348 
               
               
                   
                 AD-18330 
                 A-32349 
                 A-32350 
               
               
                   
                 AD-18331 
                 A-32351 
                 A-32352 
               
               
                   
                 AD-18332 
                 A-32353 
                 A-32354 
               
               
                   
                 AD-18333 
                 A-32355 
                 A-32356 
               
               
                   
                 AD-18334 
                 A-32357 
                 A-32358 
               
               
                   
                 AD-18335 
                 A-32359 
                 A-32360 
               
               
                   
                 AD-18336 
                 A-32363 
                 A-32364 
               
               
                   
                 AD-18337 
                 A-32367 
                 A-32368 
               
               
                   
                 AD-18338 
                 A-32369 
                 A-32370 
               
               
                   
                 AD-18339 
                 A-32371 
                 A-32372 
               
               
                   
                 AD-18340 
                 A-32373 
                 A-32374 
               
               
                   
                 AD-18341 
                 A-32375 
                 A-32376 
               
               
                   
                 AD-18342 
                 A-32379 
                 A-32380 
               
               
                   
                 AD-18343 
                 A-32381 
                 A-32382 
               
               
                   
                 AD-18344 
                 A-32383 
                 A-32384 
               
               
                   
                 AD-18345 
                 A-32385 
                 A-32386 
               
               
                   
                 AD-18346 
                 A-32387 
                 A-32388 
               
               
                   
                 AD-18347 
                 A-32391 
                 A-32392 
               
               
                   
                 AD-18348 
                 A-32393 
                 A-32394 
               
               
                   
                 AD-18349 
                 A-32395 
                 A-32396 
               
               
                   
                 AD-18350 
                 A-32397 
                 A-32398 
               
               
                   
                 AD-18351 
                 A-32399 
                 A-32400 
               
               
                   
                 AD-18352 
                 A-32401 
                 A-32402 
               
               
                   
                 AD-18353 
                 A-32403 
                 A-32404 
               
               
                   
                 AD-18354 
                 A-32405 
                 A-32406 
               
               
                   
                 AD-18355 
                 A-32407 
                 A-32408 
               
               
                   
                 AD-18356 
                 A-32409 
                 A-32410 
               
               
                   
                 AD-18357 
                 A-32411 
                 A-32412 
               
               
                   
                 AD-18358 
                 A-32415 
                 A-32416 
               
               
                   
                 AD-18359 
                 A-32417 
                 A-32418 
               
               
                   
                 AD-18360 
                 A-32419 
                 A-32420 
               
               
                   
                 AD-18361 
                 A-32421 
                 A-32422 
               
               
                   
                 AD-18362 
                 A-32423 
                 A-32424 
               
               
                   
                 AD-18363 
                 A-32427 
                 A-32428 
               
               
                   
                 AD-18364 
                 A-32429 
                 A-32430 
               
               
                   
                 AD-18446 
                 A-32161 
                 A-32162 
               
               
                   
                 AD-18447 
                 A-32173 
                 A-32174 
               
               
                   
                 AD-18448 
                 A-32185 
                 A-32186 
               
               
                   
                 AD-18449 
                 A-32197 
                 A-32198 
               
               
                   
                 AD-18450 
                 A-32209 
                 A-32210 
               
               
                   
                 AD-18451 
                 A-32245 
                 A-32246 
               
               
                   
                 AD-18452 
                 A-32257 
                 A-32258 
               
               
                   
                 AD-18453 
                 A-32293 
                 A-32294 
               
               
                   
                 AD-18454 
                 A-32305 
                 A-32306 
               
               
                   
                 AD-18455 
                 A-32317 
                 A-32318 
               
               
                   
                 AD-18456 
                 A-32329 
                 A-32330 
               
               
                   
                 AD-18457 
                 A-32361 
                 A-32362 
               
               
                   
                 AD-18458 
                 A-32365 
                 A-32366 
               
               
                   
                 AD-18459 
                 A-32377 
                 A-32378 
               
               
                   
                 AD-18460 
                 A-32389 
                 A-32390 
               
               
                   
                 AD-18461 
                 A-32401 
                 A-32402 
               
               
                   
                 AD-18462 
                 A-32413 
                 A-32414 
               
               
                   
                 AD-18463 
                 A-32425 
                 A-32426 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3A  
               
             
            
               
                   
               
               
                 Sense and antisense strand sequences of human TTR dsRNAs 
               
               
                 Strand: s = sense; as = antisense; 
               
               
                 Position: position of 5′ base on transcript 
               
               
                 (NM_000371.2, SEQ ID NO: 1329) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
                 Sequence with 3′ 
                 SEQ 
               
               
                   
                   
                 Sequence 
                 ID 
                 dinucleotide overhang 
                 ID 
               
               
                 Strand 
                 Position 
                 (5′ to 3′) 
                 NO: 
                 (5′ to 3′) 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 S 
                 100 
                 CCGGUGAAUCCAAGUGUCC 
                 1 
                 CCGGUGAAUCCAAGUGUCCNN 
                 281 
               
               
                   
               
               
                 as 
                 118 
                 GGACACUUGGAUUCACCGG 
                 2 
                 GGACACUUGGAUUCACCGGNN 
                 282 
               
               
                   
               
               
                 S 
                 11 
                 ACUCAUUCUUGGCAGGAUG 
                 3 
                 ACUCAUUCUUGGCAGGAUGNN 
                 283 
               
               
                   
               
               
                 as 
                 29 
                 CAUCCUGCCAAGAAUGAGU 
                 4 
                 CAUCCUGCCAAGAAUGAGUNN 
                 284 
               
               
                   
               
               
                 S 
                 111 
                 AAGUGUCCUCUGAUGGUCA 
                 5 
                 AAGUGUCCUCUGAUGGUCANN 
                 285 
               
               
                   
               
               
                 as 
                 129 
                 UGACCAUCAGAGGACACUU 
                 6 
                 UGACCAUCAGAGGACACUUNN 
                 286 
               
               
                   
               
               
                 S 
                 13 
                 UCAUUCUUGGCAGGAUGGC 
                 7 
                 UCAUUCUUGGCAGGAUGGCNN 
                 287 
               
               
                   
               
               
                 as 
                 31 
                 GCCAUCCUGCCAAGAAUGA 
                 8 
                 GCCAUCCUGCCAAGAAUGANN 
                 288 
               
               
                   
               
               
                 s 
                 130 
                 AAGUUCUAGAUGCUGUCCG 
                 9 
                 AAGUUCUAGAUGCUGUCCGNN 
                 289 
               
               
                   
               
               
                 as 
                 148 
                 CGGACAGCAUCUAGAACUU 
                 10 
                 CGGACAGCAUCUAGAACUUNN 
                 290 
               
               
                   
               
               
                 s 
                 132 
                 GUUCUAGAUGCUGUCCGAG 
                 11 
                 GUUCUAGAUGCUGUCCGAGNN 
                 291 
               
               
                   
               
               
                 as 
                 150 
                 CUCGGACAGCAUCUAGAAC 
                 12 
                 CUCGGACAGCAUCUAGAACNN 
                 292 
               
               
                   
               
               
                 s 
                 135 
                 CUAGAUGCUGUCCGAGGCA 
                 13 
                 CUAGAUGCUGUCCGAGGCANN 
                 293 
               
               
                   
               
               
                 as 
                 153 
                 UGCCUCGGACAGCAUCUAG 
                 14 
                 UGCCUCGGACAGCAUCUAGNN 
                 294 
               
               
                   
               
               
                 s 
                 138 
                 GAUGCUGUCCGAGGCAGUC 
                 15 
                 GAUGCUGUCCGAGGCAGUCNN 
                 295 
               
               
                   
               
               
                 as 
                 156 
                 GACUGCCUCGGACAGCAUC 
                 16 
                 GACUGCCUCGGACAGCAUCNN 
                 296 
               
               
                   
               
               
                 s 
                 14 
                 CAUUCUUGGCAGGAUGGCU 
                 17 
                 CAUUCUUGGCAGGAUGGCUNN 
                 297 
               
               
                   
               
               
                 as 
                 32 
                 AGCCAUCCUGCCAAGAAUG 
                 18 
                 AGCCAUCCUGCCAAGAAUGNN 
                 298 
               
               
                   
               
               
                 s 
                 140 
                 UGCUGUCCGAGGCAGUCCU 
                 19 
                 UGCUGUCCGAGGCAGUCCUNN 
                 299 
               
               
                   
               
               
                 as 
                 158 
                 AGGACUGCCUCGGACAGCA 
                 20 
                 AGGACUGCCUCGGACAGCANN 
                 300 
               
               
                   
               
               
                 s 
                 146 
                 CCGAGGCAGUCCUGCCAUC 
                 21 
                 CCGAGGCAGUCCUGCCAUCNN 
                 301 
               
               
                   
               
               
                 as 
                 164 
                 GAUGGCAGGACUGCCUCGG 
                 22 
                 GAUGGCAGGACUGCCUCGGNN 
                 302 
               
               
                   
               
               
                 s 
                 152 
                 CAGUCCUGCCAUCAAUGUG 
                 23 
                 CAGUCCUGCCAUCAAUGUGNN 
                 303 
               
               
                   
               
               
                 as 
                 170 
                 CACAUUGAUGGCAGGACUG 
                 24 
                 CACAUUGAUGGCAGGACUGNN 
                 304 
               
               
                   
               
               
                 s 
                 164 
                 CAAUGUGGCCGUGCAUGUG 
                 25 
                 CAAUGUGGCCGUGCAUGUGNN 
                 305 
               
               
                   
               
               
                 as 
                 182 
                 CACAUGCACGGCCACAUUG 
                 26 
                 CACAUGCACGGCCACAUUGNN 
                 306 
               
               
                   
               
               
                 s 
                 178 
                 AUGUGUUCAGAAAGGCUGC 
                 27 
                 AUGUGUUCAGAAAGGCUGCNN 
                 307 
               
               
                   
               
               
                 as 
                 196 
                 GCAGCCUUUCUGAACACAU 
                 28 
                 GCAGCCUUUCUGAACACAUNN 
                 308 
               
               
                   
               
               
                 s 
                 2 
                 CAGAAGUCCACUCAUUCUU 
                 29 
                 CAGAAGUCCACUCAUUCUUNN 
                 309 
               
               
                   
               
               
                 as 
                 20 
                 AAGAAUGAGUGGACUUCUG 
                 30 
                 AAGAAUGAGUGGACUUCUGNN 
                 310 
               
               
                   
               
               
                 s 
                 21 
                 GGCAGGAUGGCUUCUCAUC 
                 31 
                 GGCAGGAUGGCUUCUCAUCNN 
                 311 
               
               
                   
               
               
                 as 
                 39 
                 GAUGAGAAGCCAUCCUGCC 
                 32 
                 GAUGAGAAGCCAUCCUGCCNN 
                 312 
               
               
                   
               
               
                 s 
                 210 
                 GAGCCAUUUGCCUCUGGGA 
                 33 
                 GAGCCAUUUGCCUCUGGGANN 
                 313 
               
               
                   
               
               
                 as 
                 228 
                 UCCCAGAGGCAAAUGGCUC 
                 34 
                 UCCCAGAGGCAAAUGGCUCNN 
                 314 
               
               
                   
               
               
                 s 
                 23 
                 CAGGAUGGCUUCUCAUCGU 
                 35 
                 CAGGAUGGCUUCUCAUCGUNN 
                 315 
               
               
                   
               
               
                 as 
                 41 
                 ACGAUGAGAAGCCAUCCUG 
                 36 
                 ACGAUGAGAAGCCAUCCUGNN 
                 316 
               
               
                   
               
               
                 s 
                 24 
                 AGGAUGGCUUCUCAUCGUC 
                 37 
                 AGGAUGGCUUCUCAUCGUCNN 
                 317 
               
               
                   
               
               
                 as 
                 42 
                 GACGAUGAGAAGCCAUCCU 
                 38 
                 GACGAUGAGAAGCCAUCCUNN 
                 318 
               
               
                   
               
               
                 s 
                 245 
                 AGAGCUGCAUGGGCUCACA 
                 39 
                 AGAGCUGCAUGGGCUCACANN 
                 319 
               
               
                   
               
               
                 as 
                 263 
                 UGUGAGCCCAUGGAGCUCU 
                 40 
                 UGUGAGCCCAUGGAGCUCUNN 
                 320 
               
               
                   
               
               
                 s 
                 248 
                 GCUGCAUGGGCUCACAACU 
                 41 
                 GCUGCAUGGGCUCACAACUNN 
                 321 
               
               
                   
               
               
                 as 
                 266 
                 AGUUGUGAGCCCAUGGAGC 
                 42 
                 AGUUGUGAGCCCAUGGAGCNN 
                 322 
               
               
                   
               
               
                 s 
                 25 
                 GGAUGGCUUCUCAUCGUCU 
                 43 
                 GGAUGGCUUCUCAUCGUCUNN 
                 323 
               
               
                   
               
               
                 as 
                 43 
                 AGACGAUGAGAAGCCAUCC 
                 44 
                 AGACGAUGAGAAGCCAUCCNN 
                 324 
               
               
                   
               
               
                 s 
                 251 
                 GCAUGGGCUCACAACUGAG 
                 45 
                 GCAUGGGCUCACAACUGAGNN 
                 325 
               
               
                   
               
               
                 as 
                 269 
                 CUCAGUUGUGAGCCCAUGC 
                 46 
                 CUCAGUUGUGAGCCCAUGCNN 
                 326 
               
               
                   
               
               
                 s 
                 253 
                 AUGGGCUCACAACUGAGGA 
                 47 
                 AUGGGCUCACAACUGAGGANN 
                 327 
               
               
                   
               
               
                 as 
                 271 
                 UCCUCAGUUGUGAGCCCAU 
                 48 
                 UCCUCAGUUGUGAGCCCAUNN 
                 328 
               
               
                   
               
               
                 s 
                 254 
                 UGGGCUCACAACUGAGGAG 
                 49 
                 UGGGCUCACAACUGAGGAGNN 
                 329 
               
               
                   
               
               
                 as 
                 272 
                 CUCCUCAGUUGUGAGCCCA 
                 50 
                 CUCCUCAGUUGUGAGCCCANN 
                 330 
               
               
                   
               
               
                 s 
                 270 
                 GAGGAAUUUGUAGAAGGGA 
                 51 
                 GAGGAAUUUGUAGAAGGGANN 
                 331 
               
               
                   
               
               
                 as 
                 288 
                 UCCCUUCUACAAAUUCCUC 
                 52 
                 UCCCUUCUACAAAUUCCUCNN 
                 332 
               
               
                   
               
               
                 s 
                 276 
                 UUUGUAGAAGGGAUAUACA 
                 53 
                 UUUGUAGAAGGGAUAUACANN 
                 333 
               
               
                   
               
               
                 as 
                 294 
                 UGUAUAUCCCUUCUACAAA 
                 54 
                 UGUAUAUCCCUUCUACAAANN 
                 334 
               
               
                   
               
               
                 s 
                 277 
                 UUGUAGAAGGGAUAUACAA 
                 55 
                 UUGUAGAAGGGAUAUACAANN 
                 335 
               
               
                   
               
               
                 as 
                 295 
                 UUGUAUAUCCCUUCUACAA 
                 56 
                 UUGUAUAUCCCUUCUACAANN 
                 336 
               
               
                   
               
               
                 s 
                 278 
                 UGUAGAAGGGAUAUACAAA 
                 57 
                 UGUAGAAGGGAUAUACAAANN 
                 337 
               
               
                   
               
               
                 as 
                 296 
                 UUUGUAUAUCCCUUCUACA 
                 58 
                 UUUGUAUAUCCCUUCUACANN 
                 338 
               
               
                   
               
               
                 s 
                 281 
                 AGAAGGGAUAUACAAAGUG 
                 59 
                 AGAAGGGAUAUACAAAGUGNN 
                 339 
               
               
                   
               
               
                 as 
                 299 
                 CACUUUGUAUAUCCCUUCU 
                 60 
                 CACUUUGUAUAUCCCUUCUNN 
                 340 
               
               
                   
               
               
                 s 
                 295 
                 AAGUGGAAAUAGACACCAA 
                 61 
                 AAGUGGAAAUAGACACCAANN 
                 341 
               
               
                   
               
               
                 as 
                 313 
                 UUGGUGUCUAUUUCCACUU 
                 62 
                 UUGGUGUCUAUUUCCACUUNN 
                 342 
               
               
                   
               
               
                 s 
                 299 
                 GGAAAUAGACACCAAAUCU 
                 63 
                 GGAAAUAGACACCAAAUCUNN 
                 343 
               
               
                   
               
               
                 as 
                 317 
                 AGAUUUGGUGUCUAUUUCC 
                 64 
                 AGAUUUGGUGUCUAUUUCCNN 
                 344 
               
               
                   
               
               
                 s 
                 300 
                 GAAAUAGACACCAAAUCUU 
                 65 
                 GAAAUAGACACCAAAUCUUNN 
                 345 
               
               
                   
               
               
                 as 
                 318 
                 AAGAUUUGGUGUCUAUUUC 
                 66 
                 AAGAUUUGGUGUCUAUUUCNN 
                 346 
               
               
                   
               
               
                 s 
                 303 
                 AUAGACACCAAAUCUUACU 
                 67 
                 AUAGACACCAAAUCUUACUNN 
                 347 
               
               
                   
               
               
                 as 
                 321 
                 AGUAAGAUUUGGUGUCUAU 
                 68 
                 AGUAAGAUUUGGUGUCUAUNN 
                 348 
               
               
                   
               
               
                 s 
                 304 
                 UAGACACCAAAUCUUACUG 
                 69 
                 UAGACACCAAAUCUUACUGNN 
                 349 
               
               
                   
               
               
                 as 
                 322 
                 CAGUAAGAUUUGGUGUCUA 
                 70 
                 CAGUAAGAUUUGGUGUCUANN 
                 350 
               
               
                   
               
               
                 s 
                 305 
                 AGACACCAAAUCUUACUGG 
                 71 
                 AGACACCAAAUCUUACUGGNN 
                 351 
               
               
                   
               
               
                 as 
                 323 
                 CCAGUAAGAUUUGGUGUCU 
                 72 
                 CCAGUAAGAUUUGGUGUCUNN 
                 352 
               
               
                   
               
               
                 s 
                 317 
                 UUACUGGAAGGCACUUGGC 
                 73 
                 UUACUGGAAGGCACUUGGCNN 
                 353 
               
               
                   
               
               
                 as 
                 335 
                 GCCAAGUGCCUUCCAGUAA 
                 74 
                 GCCAAGUGCCUUCCAGUAANN 
                 354 
               
               
                   
               
               
                 s 
                 32 
                 UUCUCAUCGUCUGCUCCUC 
                 75 
                 UUCUCAUCGUCUGCUCCUCNN 
                 355 
               
               
                   
               
               
                 as 
                 50 
                 GAGGAGCAGACGAUGAGAA 
                 76 
                 GAGGAGCAGACGAUGAGAANN 
                 356 
               
               
                   
               
               
                 s 
                 322 
                 GGAAGGCACUUGGCAUCUC 
                 77 
                 GGAAGGCACUUGGCAUCUCNN 
                 357 
               
               
                   
               
               
                 as 
                 340 
                 GAGAUGCCAAGUGCCUUCC 
                 78 
                 GAGAUGCCAAGUGCCUUCCNN 
                 358 
               
               
                   
               
               
                 s 
                 326 
                 GGCACUUGGCAUCUCCCCA 
                 79 
                 GGCACUUGGCAUCUCCCCANN 
                 359 
               
               
                   
               
               
                 as 
                 344 
                 UGGGGAGAUGCCAAGUGCC 
                 80 
                 UGGGGAGAUGCCAAGUGCCNN 
                 360 
               
               
                   
               
               
                 s 
                 333 
                 GGCAUCUCCCCAUUCCAUG 
                 81 
                 GGCAUCUCCCCAUUCCAUGNN 
                 361 
               
               
                   
               
               
                 as 
                 351 
                 AUGGAAUGGGGAGAUGCCTT 
                 82 
                 AUGGAAUGGGGAGAUGCCTTNN 
                 362 
               
               
                   
               
               
                 s 
                 334 
                 GCAUCUCCCCAUUCCAUGA 
                 83 
                 GCAUCUCCCCAUUCCAUGANN 
                 363 
               
               
                   
               
               
                 as 
                 352 
                 UCAUGGAAUGGGGAGAUGC 
                 84 
                 UCAUGGAAUGGGGAGAUGCNN 
                 364 
               
               
                   
               
               
                 s 
                 335 
                 CAUCUCCCCAUUCCAUGAG 
                 85 
                 CAUCUCCCCAUUCCAUGAGNN 
                 365 
               
               
                   
               
               
                 as 
                 353 
                 CUCAUGGAAUGGGGAGAUG 
                 86 
                 CUCAUGGAAUGGGGAGAUGNN 
                 366 
               
               
                   
               
               
                 s 
                 336 
                 AUCUCCCCAUUCCAUGAGC 
                 87 
                 AUCUCCCCAUUCCAUGAGCNN 
                 367 
               
               
                   
               
               
                 as 
                 354 
                 GCUCAUGGAAUGGGGAGAU 
                 88 
                 GCUCAUGGAAUGGGGAGAUNN 
                 368 
               
               
                   
               
               
                 s 
                 338 
                 CUCCCCAUUCCAUGAGCAU 
                 89 
                 CUCCCCAUUCCAUGAGCAUNN 
                 369 
               
               
                   
               
               
                 as 
                 356 
                 AUGCUCAUGGAAUGGGGAG 
                 90 
                 AUGCUCAUGGAAUGGGGAGNN 
                 370 
               
               
                   
               
               
                 s 
                 341 
                 CCCAUUCCAUGAGCAUGCA 
                 91 
                 CCCAUUCCAUGAGCAUGCANN 
                 371 
               
               
                   
               
               
                 as 
                 359 
                 UGCAUGCUCAUGGAAUGGG 
                 92 
                 UGCAUGCUCAUGGAAUGGGNN 
                 372 
               
               
                   
               
               
                 s 
                 347 
                 CCAUGAGCAUGCAGAGGUG 
                 93 
                 CCAUGAGCAUGCAGAGGUGNN 
                 373 
               
               
                   
               
               
                 as 
                 365 
                 CACCUCUGCAUGCUCAUGG 
                 94 
                 CACCUCUGCAUGCUCAUGGNN 
                 374 
               
               
                   
               
               
                 s 
                 352 
                 AGCAUGCAGAGGUGGUAUU 
                 95 
                 AGCAUGCAGAGGUGGUAUUNN 
                 375 
               
               
                   
               
               
                 as 
                 370 
                 AAUACCACCUCUGCAUGCU 
                 96 
                 AAUACCACCUCUGCAUGCUNN 
                 376 
               
               
                   
               
               
                 s 
                 354 
                 CAUGCAGAGGUGGUAUUCA 
                 97 
                 CAUGCAGAGGUGGUAUUCANN 
                 377 
               
               
                   
               
               
                 as 
                 372 
                 UGAAUACCACCUCUGCAUG 
                 98 
                 UGAAUACCACCUCUGCAUGNN 
                 378 
               
               
                   
               
               
                 s 
                 355 
                 AUGCAGAGGUGGUAUUCAC 
                 99 
                 AUGCAGAGGUGGUAUUCACNN 
                 379 
               
               
                   
               
               
                 as 
                 373 
                 GUGAAUACCACCUCUGCAU 
                 100 
                 GUGAAUACCACCUCUGCAUNN 
                 380 
               
               
                   
               
               
                 s 
                 362 
                 GGUGGUAUUCACAGCCAAC 
                 101 
                 GGUGGUAUUCACAGCCAACNN 
                 381 
               
               
                   
               
               
                 as 
                 380 
                 GUUGGCUGUGAAUACCACC 
                 102 
                 GUUGGCUGUGAAUACCACCNN 
                 382 
               
               
                   
               
               
                 s 
                 363 
                 GUGGUAUUCACAGCCAACG 
                 103 
                 GUGGUAUUCACAGCCAACGNN 
                 383 
               
               
                   
               
               
                 as 
                 381 
                 CGUUGGCUGUGAAUACCAC 
                 104 
                 CGUUGGCUGUGAAUACCACNN 
                 384 
               
               
                   
               
               
                 s 
                 364 
                 UGGUAUUCACAGCCAACGA 
                 105 
                 UGGUAUUCACAGCCAACGANN 
                 385 
               
               
                   
               
               
                 as 
                 382 
                 UCGUUGGCUGUGAAUACCA 
                 106 
                 UCGUUGGCUGUGAAUACCANN 
                 386 
               
               
                   
               
               
                 s 
                 365 
                 GGUAUUCACAGCCAACGAC 
                 107 
                 GGUADUCACAGCCAACGACNN 
                 387 
               
               
                   
               
               
                 as 
                 383 
                 GUCGUUGGCUGUGAAUACC 
                 108 
                 GUCGUUGGCUGUGAAUACCNN 
                 388 
               
               
                   
               
               
                 s 
                 366 
                 GUAUUCACAGCCAACGACU 
                 109 
                 GUAUUCACAGCCAACGACUNN 
                 389 
               
               
                   
               
               
                 as 
                 384 
                 AGUCGUUGGCUGUGAAUAC 
                 110 
                 AGUCGUUGGCUGUGAAUACNN 
                 390 
               
               
                   
               
               
                 s 
                 367 
                 UAUUCACAGCCAACGACUC 
                 111 
                 UAUUCACAGCCAACGACUCNN 
                 391 
               
               
                   
               
               
                 as 
                 385 
                 GAGUCGUUGGCUGUGAAUA 
                 112 
                 GAGUCGUUGGCUGUGAAUANN 
                 392 
               
               
                   
               
               
                 s 
                 370 
                 UCACAGCCAACGACUCCGG 
                 113 
                 UCACAGCCAACGACUCCGGNN 
                 393 
               
               
                   
               
               
                 as 
                 388 
                 CCGGAGUCGUUGGCUGUGA 
                 114 
                 CCGGAGUCGUUGGCUGUGANN 
                 394 
               
               
                   
               
               
                 s 
                 390 
                 CCCCGCCGCUACACCAUUG 
                 115 
                 CCCCGCCGCUACACCAUUGNN 
                 395 
               
               
                   
               
               
                 as 
                 408 
                 CAAUGGUGUAGCGGCGGGG 
                 116 
                 CAAUGGUGUAGCGGCGGGGNN 
                 396 
               
               
                   
               
               
                 s 
                 4 
                 GAAGUCCACUCAUUCUUGG 
                 117 
                 GAAGUCCACUCAUUCUUGGNN 
                 397 
               
               
                   
               
               
                 as 
                 22 
                 CCAAGAAUGAGUGGACUUC 
                 118 
                 CCAAGAAUGAGUGGACUUCNN 
                 398 
               
               
                   
               
               
                 s 
                 412 
                 CCCUGCUGAGCCCCUACUC 
                 119 
                 CCCUGCUGAGCCCCUACUCNN 
                 399 
               
               
                   
               
               
                 as 
                 430 
                 GAGUAGGGGCUCAGCAGGG 
                 120 
                 GAGUAGGGGCUCAGCAGGGNN 
                 400 
               
               
                   
               
               
                 s 
                 417 
                 CUGAGCCCCUACUCCUAUU 
                 121 
                 CUGAGCCCCUACUCCUAUUNN 
                 401 
               
               
                   
               
               
                 as 
                 435 
                 AAUAGGAGUAGGGGCUCAG 
                 122 
                 AAUAGGAGUAGGGGCUCAGNN 
                 402 
               
               
                   
               
               
                 s 
                 418 
                 UGAGCCCCUACUCCUAUUC 
                 123 
                 UGAGCCCCUACUCCUAUUCNN 
                 403 
               
               
                   
               
               
                 as 
                 436 
                 GAAUAGGAGUAGGGGCUCA 
                 124 
                 GAAUAGGAGUAGGGGCUCANN 
                 404 
               
               
                   
               
               
                 s 
                 422 
                 CCCCUACUCCUAUUCCACC 
                 125 
                 CCCCUACUCCUAUUCCACCNN 
                 405 
               
               
                   
               
               
                 as 
                 440 
                 GGUGGAAUAGGAGUAGGGG 
                 126 
                 GGUGGAAUAGGAGUAGGGGNN 
                 406 
               
               
                   
               
               
                 s 
                 425 
                 CUACUCCUAUUCCACCACG 
                 127 
                 CUACUCCUAUUCCACCACGNN 
                 407 
               
               
                   
               
               
                 as 
                 443 
                 CGUGGUGGAAUAGGAGUAG 
                 128 
                 CGUGGUGGAAUAGGAGUAGNN 
                 408 
               
               
                   
               
               
                 s 
                 426 
                 UACUCCUAUUCCACCACGG 
                 129 
                 UACUCCUAUUCCACCACGGNN 
                 409 
               
               
                   
               
               
                 as 
                 444 
                 CCGUGGUGGAAUAGGAGUA 
                 130 
                 CCGUGGUGGAAUAGGAGUANN 
                 410 
               
               
                   
               
               
                 s 
                 427 
                 ACUCCUAUUCCACCACGGC 
                 131 
                 ACUCCUAUUCCACCACGGCNN 
                 411 
               
               
                   
               
               
                 as 
                 445 
                 GCCGUGGUGGAAUAGGAGU 
                 132 
                 GCCGUGGUGGAAUAGGAGUNN 
                 412 
               
               
                   
               
               
                 s 
                 429 
                 UCCUAUUCCACCACGGCUG 
                 133 
                 UCCUAUUCCACCACGGCUGNN 
                 413 
               
               
                   
               
               
                 as 
                 447 
                 CAGCCGUGGUGGAAUAGGA 
                 134 
                 CAGCCGUGGUGGAAUAGGANN 
                 414 
               
               
                   
               
               
                 s 
                 432 
                 UAUUCCACCACGGCUGUCG 
                 135 
                 UAUUCCACCACGGCUGUCGNN 
                 415 
               
               
                   
               
               
                 as 
                 450 
                 CGACAGCCGUGGUGGAAUA 
                 136 
                 CGACAGCCGUGGUGGAAUANN 
                 416 
               
               
                   
               
               
                 s 
                 433 
                 AUUCCACCACGGCUGUCGU 
                 137 
                 AUUCCACCACGGCUGUCGUNN 
                 417 
               
               
                   
               
               
                 as 
                 451 
                 ACGACAGCCGUGGUGGAAU 
                 138 
                 ACGACAGCCGUGGUGGAAUNN 
                 418 
               
               
                   
               
               
                 s 
                 437 
                 CACCACGGCUGUCGUCACC 
                 139 
                 CACCACGGCUGUCGUCACCNN 
                 419 
               
               
                   
               
               
                 as 
                 455 
                 GGUGACGACAGCCGUGGUG 
                 140 
                 GGUGACGACAGCCGUGGUGNN 
                 420 
               
               
                   
               
               
                 s 
                 438 
                 ACCACGGCUGUCGUCACCA 
                 141 
                 ACCACGGCUGUCGUCACCANN 
                 421 
               
               
                   
               
               
                 as 
                 456 
                 UGGUGACGACAGCCGUGGU 
                 142 
                 UGGUGACGACAGCCGUGGUNN 
                 422 
               
               
                   
               
               
                 s 
                 439 
                 CCACGGCUGUCGUCACCAA 
                 143 
                 CCACGGCUGUCGUCACCAANN 
                 423 
               
               
                   
               
               
                 as 
                 457 
                 UUGGUGACGACAGCCGUGG 
                 144 
                 UUGGUGACGACAGCCGUGGNN 
                 424 
               
               
                   
               
               
                 s 
                 441 
                 ACGGCUGUCGUCACCAAUC 
                 145 
                 ACGGCUGUCGUCACCAAUCNN 
                 425 
               
               
                   
               
               
                 as 
                 459 
                 GAUUGGUGACGACAGCCGU 
                 146 
                 GAUUGGUGACGACAGCCGUNN 
                 426 
               
               
                   
               
               
                 s 
                 442 
                 CGGCUGUCGUCACCAAUCC 
                 147 
                 CGGCUGUCGUCACCAAUCCNN 
                 427 
               
               
                   
               
               
                 as 
                 460 
                 GGAUUGGUGACGACAGCCG 
                 148 
                 GGAUUGGUGACGACAGCCGNN 
                 428 
               
               
                   
               
               
                 s 
                 449 
                 CGUCACCAAUCCCAAGGAA 
                 149 
                 CGUCACCAAUCCCAAGGAANN 
                 429 
               
               
                   
               
               
                 as 
                 467 
                 UUCCUUGGGAUUGGUGACG 
                 150 
                 UUCCUUGGGAUUGGUGACGNN 
                 430 
               
               
                   
               
               
                 s 
                 455 
                 CAAUCCCAAGGAAUGAGGG 
                 151 
                 CAAUCCCAAGGAAUGAGGGNN 
                 431 
               
               
                   
               
               
                 as 
                 473 
                 CCCUCAUUCCUUGGGAUUG 
                 152 
                 CCCUCAUUCCUUGGGAUUGNN 
                 432 
               
               
                   
               
               
                 s 
                 491 
                 CCUGAAGGACGAGGGAUGG 
                 153 
                 CCUGAAGGACGAGGGAUGGNN 
                 433 
               
               
                   
               
               
                 as 
                 509 
                 CCAUCCCUCGUCCUUCAGG 
                 154 
                 CCAUCCCUCGUCCUUCAGGNN 
                 434 
               
               
                   
               
               
                 s 
                 497 
                 GGACGAGGGAUGGGAUUUC 
                 155 
                 GGACGAGGGAUGGGAUUUCNN 
                 435 
               
               
                   
               
               
                 as 
                 515 
                 GAAAUCCCAUCCCUCGUCC 
                 156 
                 GAAAUCCCAUCCCUCGUCCNN 
                 436 
               
               
                   
               
               
                 s 
                 5 
                 AAGUCCACUCAUUCUUGGC 
                 157 
                 AAGUCCACUCAUUCUUGGCNN 
                 437 
               
               
                   
               
               
                 as 
                 23 
                 GCCAAGAAUGAGUGGACUU 
                 158 
                 GCCAAGAAUGAGUGGACUUNN 
                 438 
               
               
                   
               
               
                 s 
                 508 
                 GGGAUUUCAUGUAACCAAG 
                 159 
                 GGGAUUUCAUGUAACCAAGNN 
                 439 
               
               
                   
               
               
                 as 
                 526 
                 CUUGGUUACAUGAAAUCCC 
                 160 
                 CUUGGUUACAUGAAAUCCCNN 
                 440 
               
               
                   
               
               
                 s 
                 509 
                 GGAUUUCAUGUAACCAAGA 
                 161 
                 GGAUUUCAUGUAACCAAGANN 
                 441 
               
               
                   
               
               
                 as 
                 527 
                 UCUUGGUUACAUGAAAUCC 
                 162 
                 UCUUGGUUACAUGAAAUCCNN 
                 442 
               
               
                   
               
               
                 s 
                 514 
                 UCAUGUAACCAAGAGUAUU 
                 163 
                 UCAUGUAACCAAGAGUAUUNN 
                 443 
               
               
                   
               
               
                 as 
                 532 
                 AAUACUCUUGGUUACAUGA 
                 164 
                 AAUACUCUUGGUUACAUGANN 
                 444 
               
               
                   
               
               
                 s 
                 516 
                 AUGUAACCAAGAGUAUUCC 
                 165 
                 AUGUAACCAAGAGUAUUCCNN 
                 445 
               
               
                   
               
               
                 as 
                 534 
                 GGAAUACUCUUGGUUACAU 
                 166 
                 GGAAUACUCUUGGUUACAUNN 
                 446 
               
               
                   
               
               
                 s 
                 517 
                 UGUAACCAAGAGUAUUCCA 
                 167 
                 UGUAACCAAGAGUAUUCCANN 
                 447 
               
               
                   
               
               
                 as 
                 535 
                 UGGAAUACUCUUGGUUACA 
                 168 
                 UGGAAUACUCUUGGUUACANN 
                 448 
               
               
                   
               
               
                 s 
                 518 
                 GUAACCAAGAGUAUUCCAU 
                 169 
                 GUAACCAAGAGUAUUCCAUNN 
                 449 
               
               
                   
               
               
                 as 
                 536 
                 AUGGAAUACUCUUGGUUAC 
                 170 
                 AUGGAAUACUCUUGGUUACNN 
                 450 
               
               
                   
               
               
                 s 
                 54 
                 UGCCUUGCUGGACUGGUAU 
                 171 
                 UGCCUUGCUGGACUGGUAUNN 
                 451 
               
               
                   
               
               
                 as 
                 72 
                 AUACCAGUCCAGCAAGGCA 
                 172 
                 AUACCAGUCCAGCAAGGCANN 
                 452 
               
               
                   
               
               
                 s 
                 543 
                 UAAAGCAGUGUUUUCACCU 
                 173 
                 UAAAGCAGUGUUUUCACCUNN 
                 453 
               
               
                   
               
               
                 as 
                 561 
                 AGGUGAAAACACUGCUUUA 
                 174 
                 AGGUGAAAACACUGCUUUANN 
                 454 
               
               
                   
               
               
                 s 
                 55 
                 GCCUUGCUGGACUGGUAUU 
                 175 
                 GCCUUGCUGGACUGGUAUUNN 
                 455 
               
               
                   
               
               
                 as 
                 73 
                 AAUACCAGUCCAGCAAGGC 
                 176 
                 AAUACCAGUCCAGCAAGGCNN 
                 456 
               
               
                   
               
               
                 s 
                 551 
                 UGUUUUCACCUCAUAUGCU 
                 177 
                 UGUUUUCACCUCAUAUGCUNN 
                 457 
               
               
                   
               
               
                 as 
                 569 
                 AGCAUAUGAGGUGAAAACA 
                 178 
                 AGCADAUGAGGUGAAAACANN 
                 458 
               
               
                   
               
               
                 s 
                 552 
                 GUUUUCACCUCAUAUGCUA 
                 179 
                 GUUUUCACCUCAUAUGCUANN 
                 459 
               
               
                   
               
               
                 as 
                 570 
                 UAGCAUAUGAGGUGAAAAC 
                 180 
                 UAGCAUAUGAGGUGAAAACNN 
                 460 
               
               
                   
               
               
                 s 
                 553 
                 UUUUCACCUCAUAUGCUAU 
                 181 
                 UUUUCACCUCAUAUGCUAUNN 
                 461 
               
               
                   
               
               
                 as 
                 571 
                 AUAGCAUAUGAGGUGAAAA 
                 182 
                 AUAGCAUAUGAGGUGAAAANN 
                 462 
               
               
                   
               
               
                 s 
                 555 
                 UUCACCUCAUAUGCUAUGU 
                 183 
                 UUCACCUCAUAUGCUAUGUNN 
                 463 
               
               
                   
               
               
                 as 
                 573 
                 ACAUAGCAUAUGAGGUGAA 
                 184 
                 ACAUAGCAUAUGAGGUGAANN 
                 464 
               
               
                   
               
               
                 s 
                 557 
                 CACCUCAUAUGCUAUGUUA 
                 185 
                 CACCUCAUAUGCUAUGUUANN 
                 465 
               
               
                   
               
               
                 as 
                 575 
                 UAACAUAGCAUAUGAGGUG 
                 186 
                 UAACAUAGCAUAUGAGGUGNN 
                 466 
               
               
                   
               
               
                 s 
                 56 
                 CCUUGCUGGACUGGUAUUU 
                 187 
                 CCUUGCUGGACUGGUAUUUNN 
                 467 
               
               
                   
               
               
                 as 
                 74 
                 AAAUACCAGUCCAGCAAGG 
                 188 
                 AAAUACCAGUCCAGCAAGGNN 
                 468 
               
               
                   
               
               
                 s 
                 563 
                 AUAUGCUAUGUUAGAAGUC 
                 189 
                 AUAUGCUAUGUUAGAAGUCNN 
                 469 
               
               
                   
               
               
                 as 
                 581 
                 GACUUCUAACAUAGCAUAU 
                 190 
                 GACUUCUAACAUAGCAUAUNN 
                 470 
               
               
                   
               
               
                 s 
                 564 
                 UAUGCUAUGUUAGAAGUCC 
                 191 
                 UAUGCUAUGUUAGAAGUCCNN 
                 471 
               
               
                   
               
               
                 as 
                 582 
                 GGACUUCUAACAUAGCAUA 
                 192 
                 GGACUUCUAACAUAGCAUANN 
                 472 
               
               
                   
               
               
                 s 
                 566 
                 UGCUAUGUUAGAAGUCCAG 
                 193 
                 UGCUAUGUUAGAAGUCCAGNN 
                 473 
               
               
                   
               
               
                 as 
                 584 
                 CUGGACUUCUAACAUAGCA 
                 194 
                 CUGGACUUCUAACAUAGCANN 
                 474 
               
               
                   
               
               
                 s 
                 57 
                 CUUGCUGGACUGGUAUUUG 
                 195 
                 CUUGCUGGACUGGUAUUUGNN 
                 475 
               
               
                   
               
               
                 as 
                 75 
                 CAAAUACCAGUCCAGCAAG 
                 196 
                 CAAAUACCAGUCCAGCAAGNN 
                 476 
               
               
                   
               
               
                 s 
                 578 
                 AGUCCAGGCAGAGACAAUA 
                 197 
                 AGUCCAGGCAGAGACAAUANN 
                 477 
               
               
                   
               
               
                 as 
                 596 
                 AUUGUCUCUGCCUGGACUTT 
                 198 
                 AUUGUCUCUGCCUGGACUTTNN 
                 478 
               
               
                   
               
               
                 s 
                 580 
                 UCCAGGCAGAGACAAUAAA 
                 199 
                 UCCAGGCAGAGACAAUAAANN 
                 479 
               
               
                   
               
               
                 as 
                 598 
                 UUUAUUGUCUCUGCCUGGA 
                 200 
                 UUUADUGUCUCUGCCUGGANN 
                 480 
               
               
                   
               
               
                 s 
                 607 
                 GUGAAAGGCACUUUUCAUU 
                 201 
                 GUGAAAGGCACUUUUCAUUNN 
                 481 
               
               
                   
               
               
                 as 
                 625 
                 AAUGAAAAGUGCCUUUCAC 
                 202 
                 AAUGAAAAGUGCCUUUCACNN 
                 482 
               
               
                   
               
               
                 s 
                 62 
                 UGGACUGGUAUUUGUGUCU 
                 203 
                 UGGACUGGUAUUUGUGUCUNN 
                 483 
               
               
                   
               
               
                 as 
                 80 
                 AGACACAAAUACCAGUCCA 
                 204 
                 AGACACAAAUACCAGUCCANN 
                 484 
               
               
                   
               
               
                 s 
                 77 
                 GUCUGAGGCUGGCCCUACG 
                 205 
                 GUCUGAGGCUGGCCCUACGNN 
                 485 
               
               
                   
               
               
                 as 
                 95 
                 CGUAGGGCCAGCCUCAGAC 
                 206 
                 CGUAGGGCCAGCCUCAGACNN 
                 486 
               
               
                   
               
               
                 s 
                 79 
                 CUGAGGCUGGCCCUACGGG 
                 207 
                 CUGAGGCUGGCCCUACGGGNN 
                 487 
               
               
                   
               
               
                 as 
                 97 
                 CCCGUAGGGCCAGCCUCAG 
                 208 
                 CCCGUAGGGCCAGCCUCAGNN 
                 488 
               
               
                   
               
               
                 s 
                 81 
                 GAGGCUGGCCCUACGGGCA 
                 209 
                 GAGGCUGGCCCUACGGGCANN 
                 489 
               
               
                   
               
               
                 as 
                 99 
                 UGCCCGUAGGGCCAGCCUC 
                 210 
                 UGCCCGUAGGGCCAGCCUCNN 
                 490 
               
               
                   
               
               
                 s 
                 82 
                 AGGCUGGCCCUACGGGCAC 
                 211 
                 AGGCUGGCCCUACGGGCACNN 
                 491 
               
               
                   
               
               
                 as 
                 100 
                 GUGCCCGUAGGGCCAGCCU 
                 212 
                 GUGCCCGUAGGGCCAGCCUNN 
                 492 
               
               
                   
               
               
                 s 
                 84 
                 GCUGGCCCUACGGGCACCG 
                 213 
                 GCUGGCCCUACGGGCACCGNN 
                 493 
               
               
                   
               
               
                 as 
                 102 
                 CGGUGCCCGUAGGGCCAGC 
                 214 
                 CGGUGCCCGUAGGGCCAGCNN 
                 494 
               
               
                   
               
               
                 s 
                 85 
                 CUGGCCCUACGGGCACCGG 
                 215 
                 CUGGCCCUACGGGCACCGGNN 
                 495 
               
               
                   
               
               
                 as 
                 103 
                 CCGGUGCCCGUAGGGCCAG 
                 216 
                 CCGGUGCCCGUAGGGCCAGNN 
                 496 
               
               
                   
               
               
                 s 
                 87 
                 GGCCCUACGGGCACCGGUG 
                 217 
                 GGCCCUACGGGCACCGGUGNN 
                 497 
               
               
                   
               
               
                 as 
                 105 
                 CACCGGUGCCCGUAGGGCC 
                 218 
                 CACCGGUGCCCGUAGGGCCNN 
                 498 
               
               
                   
               
               
                 s 
                 9 
                 CCACUCAUUCUUGGCAGGA 
                 219 
                 CCACUCAUUCUUGGCAGGANN 
                 499 
               
               
                   
               
               
                 as 
                 27 
                 UCCUGCCAAGAAUGAGUGG 
                 220 
                 UCCUGCCAAGAAUGAGUGGNN 
                 500 
               
               
                   
               
               
                 s 
                 90 
                 CCUACGGGCACCGGUGAAU 
                 221 
                 CCUACGGGCACCGGUGAAUNN 
                 501 
               
               
                   
               
               
                 as 
                 108 
                 AUUCACCGGUGCCCGUAGG 
                 222 
                 AUUCACCGGUGCCCGUAGGNN 
                 502 
               
               
                   
               
               
                 s 
                 91 
                 CUACGGGCACCGGUGAAUC 
                 223 
                 CUACGGGCACCGGUGAAUCNN 
                 503 
               
               
                   
               
               
                 as 
                 109 
                 GAUUCACCGGUGCCCGUAG 
                 224 
                 GAUUCACCGGUGCCCGUAGNN 
                 504 
               
               
                   
               
               
                 s 
                 92 
                 UACGGGCACCGGUGAAUCC 
                 225 
                 UACGGGCACCGGUGAAUCCNN 
                 505 
               
               
                   
               
               
                 as 
                 110 
                 GGAUUCACCGGUGCCCGUA 
                 226 
                 GGAUUCACCGGUGCCCGUANN 
                 506 
               
               
                   
               
               
                 s 
                 93 
                 ACGGGCACCGGUGAAUCCA 
                 227 
                 ACGGGCACCGGUGAAUCCANN 
                 507 
               
               
                   
               
               
                 as 
                 111 
                 UGGAUUCACCGGUGCCCGU 
                 228 
                 UGGAIJUCACCGGUGCCCGUNN 
                 508 
               
               
                   
               
               
                 s 
                 97 
                 GCACCGGUGAAUCCAAGUG 
                 229 
                 GCACCGGUGAAUCCAAGUGNN 
                 509 
               
               
                   
               
               
                 as 
                 115 
                 CACUUGGAUUCACCGGUGC 
                 230 
                 CACUUGGAUUCACCGGUGCNN 
                 510 
               
               
                   
               
               
                 s 
                 98 
                 CACCGGUGAAUCCAAGUGU 
                 231 
                 CACCGGUGAAUCCAAGUGUNN 
                 511 
               
               
                   
               
               
                 as 
                 116 
                 ACACUUGGAUUCACCGGUG 
                 232 
                 ACACUUGGAUUCACCGGUGNN 
                 512 
               
               
                   
               
               
                 s 
                 167 
                 UGUGGCCAUGCAUGUGUUC 
                 233 
                 UGUGGCCAUGCAUGUGUUCNN 
                 513 
               
               
                   
               
               
                 as 
                 185 
                 GAACACAUGCAUGGCCACA 
                 234 
                 GAACACAUGCAUGGCCACANN 
                 514 
               
               
                   
               
               
                 s 
                 168 
                 GUGGCCAUGCAUGUGUUCA 
                 235 
                 GUGGCCAUGCAUGUGUUCANN 
                 515 
               
               
                   
               
               
                 as 
                 186 
                 UGAACACAUGCAUGGCCAC 
                 236 
                 UGAACACAUGCAUGGCCACNN 
                 516 
               
               
                   
               
               
                 s 
                 171 
                 GCCAUGCAUGUGUUCAGAA 
                 237 
                 GCCAUGCAUGUGUUCAGAANN 
                 517 
               
               
                   
               
               
                 as 
                 189 
                 UUCUGAACACAUGCAUGGC 
                 238 
                 UUCUGAACACAUGCAUGGCNN 
                 518 
               
               
                   
               
               
                 s 
                 432 
                 UAUUCCACCACGGCUGUCA 
                 239 
                 UAUUCCACCACGGCUGUCANN 
                 519 
               
               
                   
               
               
                 as 
                 449 
                 UGACAGCCGUGGUGGAAUA 
                 240 
                 UGACAGCCGUGGUGGAAUANN 
                 520 
               
               
                   
               
               
                 s 
                 447 
                 GUCAUCACCAAUCCCAAGG 
                 241 
                 GUCAUCACCAAUCCCAAGGNN 
                 521 
               
               
                   
               
               
                 as 
                 465 
                 CCUUGGGAUUGGUGAUGAC 
                 242 
                 CCUUGGGAUUGGUGAUGACNN 
                 522 
               
               
                   
               
               
                 s 
                 115 
                 GUCCUCUGAUGGUCAAAGU 
                 243 
                 GUCCUCUGAUGGUCAAAGUNN 
                 523 
               
               
                   
               
               
                 as 
                 133 
                 ACUUUGACCAUCAGAGGAC 
                 244 
                 ACUUUGACCAUCAGAGGACNN 
                 524 
               
               
                   
               
               
                 s 
                 122 
                 GAUGGUCAAAGUUCUAGAU 
                 245 
                 GAUGGUCAAAGUUCUAGAUNN 
                 525 
               
               
                   
               
               
                 as 
                 140 
                 AUCUAGAACUUUGACCAUC 
                 246 
                 AUCUAGAACUUUGACCAUCNN 
                 526 
               
               
                   
               
               
                 s 
                 139 
                 AUGCUGUCCGAGGCAGUCC 
                 247 
                 AUGCUGUCCGAGGCAGUCCNN 
                 527 
               
               
                   
               
               
                 as 
                 157 
                 GGACUGCCUCGGACAGCAU 
                 248 
                 GGACUGCCUCGGACAGCAUNN 
                 528 
               
               
                   
               
               
                 s 
                 172 
                 CCGUGCAUGUGUUCAGAAA 
                 249 
                 CCGUGCAUGUGUUCAGAAANN 
                 529 
               
               
                   
               
               
                 as 
                 190 
                 UUUCUGAACACAUGCACGG 
                 250 
                 UUUCUGAACACAUGCACGGNN 
                 530 
               
               
                   
               
               
                 s 
                 238 
                 AGUCUGGAGAGCUGCAUGG 
                 251 
                 AGUCUGGAGAGCUGCAUGGNN 
                 531 
               
               
                   
               
               
                 as 
                 256 
                 CCAUGCAGCUCUCCAGACU 
                 252 
                 CCAUGCAGCUCUCCAGACUNN 
                 532 
               
               
                   
               
               
                 s 
                 252 
                 CAUGGGCUCACAACUGAGG 
                 253 
                 CAUGGGCUCACAACUGAGGNN 
                 533 
               
               
                   
               
               
                 as 
                 270 
                 CCUCAGUUGUGAGCCCAUG 
                 254 
                 CCUCAGUUGUGAGCCCAUGNN 
                 534 
               
               
                   
               
               
                 s 
                 33 
                 UCUCAUCGUCUGCUCCUCC 
                 255 
                 UCUCAUCGUCUGCUCCUCCNN 
                 535 
               
               
                   
               
               
                 as 
                 51 
                 GGAGGAGCAGACGAUGAGA 
                 256 
                 GGAGGAGCAGACGAUGAGANN 
                 536 
               
               
                   
               
               
                 s 
                 340 
                 CCCCAUUCCAUGAGCAUGC 
                 257 
                 CCCCAUUCCAUGAGCAUGCNN 
                 537 
               
               
                   
               
               
                 as 
                 358 
                 GCAUGCUCAUGGAAUGGGG 
                 258 
                 GCAUGCUCAUGGAAUGGGGNN 
                 538 
               
               
                   
               
               
                 s 
                 421 
                 GCCCCUACUCCUAUUCCAC 
                 259 
                 GCCCCUACUCCUAUUCCACNN 
                 539 
               
               
                   
               
               
                 as 
                 439 
                 GUGGAAUAGGAGUAGGGGC 
                 260 
                 GUGGAAUAGGAGUAGGGGCNN 
                 540 
               
               
                   
               
               
                 s 
                 431 
                 CUAUUCCACCACGGCUGUC 
                 261 
                 CUAUUCCACCACGGCUGUCNN 
                 541 
               
               
                   
               
               
                 as 
                 449 
                 GACAGCCGUGGUGGAAUAG 
                 262 
                 GACAGCCGUGGUGGAAUAGNN 
                 542 
               
               
                   
               
               
                 s 
                 440 
                 CACGGCUGUCGUCACCAAU 
                 263 
                 CACGGCUGUCGUCACCAAUNN 
                 543 
               
               
                   
               
               
                 as 
                 458 
                 AUUGGUGACGACAGCCGUG 
                 264 
                 AUUGGUGACGACAGCCGUGNN 
                 544 
               
               
                   
               
               
                 s 
                 496 
                 AGGACGAGGGAUGGGAUUU 
                 265 
                 AGGACGAGGGAUGGGAUUUNN 
                 545 
               
               
                   
               
               
                 as 
                 514 
                 AAAUCCCAUCCCUCGUCCU 
                 266 
                 AAAUCCCAUCCCUCGUCCUNN 
                 546 
               
               
                   
               
               
                 s 
                 556 
                 UCACCUCAUAUGCUAUGUU 
                 267 
                 UCACCUCAUAUGCUAUGUUNN 
                 547 
               
               
                   
               
               
                 as 
                 574 
                 AACAUAGCAUAUGAGGUGA 
                 268 
                 AACADAGCAUAUGAGGUGANN 
                 548 
               
               
                   
               
               
                 s 
                 559 
                 CCUCAUAUGCUAUGUUAGA 
                 269 
                 CCUCAUAUGCUAUGUUAGANN 
                 549 
               
               
                   
               
               
                 as 
                 577 
                 UCUAACAUAGCAUAUGAGG 
                 270 
                 UCUAACAUAGCAUAUGAGGNN 
                 550 
               
               
                   
               
               
                 s 
                 570 
                 AUGUUAGAAGUCCAGGCAG 
                 271 
                 AUGUUAGAAGUCCAGGCAGNN 
                 551 
               
               
                   
               
               
                 as 
                 588 
                 CUGCCUGGACUUCUAACAU 
                 272 
                 CUGCCUGGACUUCUAACAUNN 
                 552 
               
               
                   
               
               
                 s 
                 78 
                 UCUGAGGCUGGCCCUACGG 
                 273 
                 UCUGAGGCUGGCCCUACGGNN 
                 553 
               
               
                   
               
               
                 as 
                 96 
                 CCGUAGGGCCAGCCUCAGA 
                 274 
                 CCGUAGGGCCAGCCUCAGANN 
                 554 
               
               
                   
               
               
                 s 
                 87 
                 GGCCCUACGGGCACCGGUG 
                 275 
                 GGCCCUACGGGCACCGGUGNN 
                 555 
               
               
                   
               
               
                 as 
                 105 
                 CACCGGUGCCCGUAGGGCC 
                 276 
                 CACCGGUGCCCGUAGGGCCNN 
                 556 
               
               
                   
               
               
                 s 
                 95 
                 GGGCACCGGUGAAUCCAAG 
                 277 
                 GGGCACCGGUGAAUCCAAGNN 
                 557 
               
               
                   
               
               
                 as 
                 113 
                 CUUGGAUUCACCGGUGCCC 
                 278 
                 CUUGGAUUCACCGGUGCCCNN 
                 558 
               
               
                   
               
               
                 s 
                 167 
                 CCAUGCAUGUGUUCAGAAA 
                 279 
                 CCAUGCAUGUGUUCAGAAANN 
                 559 
               
               
                   
               
               
                 as 
                 185 
                 UUUCUGAACACAUGCAUGG 
                 280 
                 UUUCUGAACACAUGCAUGGNN 
                 560 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3B 
               
             
            
               
                   
               
               
                 Sense and antisense strand 
               
               
                 sequences of human TTR dsRNAs 
               
               
                 Strand: s = sense; as = antisense; 
               
               
                 Position: position of 5′ base on transcript 
               
               
                 (NM_000371.2, SEQ ID NO: 1329) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Sequence with 
                 SEQ 
               
               
                   
                   
                   
                 3′ deoxythimidine 
                 ID 
               
               
                   
                 Strand 
                 Position 
                 overhang (5′ to 3′) 
                 NO: 
               
               
                   
               
               
                   
                 s 
                 100 
                 CCGGUGAAUCCAAGUGUCCdTdT 
                 561 
               
               
                   
               
               
                   
                 as 
                 118 
                 GGACACUUGGAUUCACCGGdTdT 
                 562 
               
               
                   
               
               
                   
                 s 
                  11 
                 ACUCAUUCUUGGCAGGAUGdTdT 
                 563 
               
               
                   
               
               
                   
                 as 
                  29 
                 CAUCCUGCCAAGAAUGAGUdTdT 
                 564 
               
               
                   
               
               
                   
                 s 
                 111 
                 AAGUGUCCUCUGAUGGUCAdTdT 
                 565 
               
               
                   
               
               
                   
                 as 
                 129 
                 UGACCAUCAGAGGACACUUdTdT 
                 566 
               
               
                   
               
               
                   
                 s 
                  13 
                 UCAUUCUUGGCAGGAUGGCdTdT 
                 567 
               
               
                   
               
               
                   
                 as 
                  31 
                 GCCAUCCUGCCAAGAAUGAdTdT 
                 568 
               
               
                   
               
               
                   
                 s 
                 130 
                 AAGUUCUAGAUGCUGUCCGdTdT 
                 569 
               
               
                   
               
               
                   
                 as 
                 148 
                 CGGACAGCAUCUAGAACUUdTdT 
                 570 
               
               
                   
               
               
                   
                 s 
                 132 
                 GUUCUAGAUGCUGUCCGAGdTdT 
                 571 
               
               
                   
               
               
                   
                 as 
                 150 
                 CUCGGACAGCAUCUAGAACdTdT 
                 572 
               
               
                   
               
               
                   
                 s 
                 135 
                 CUAGAUGCUGUCCGAGGCAdTdT 
                 573 
               
               
                   
               
               
                   
                 as 
                 153 
                 UGCCUCGGACAGCAUCUAGdTdT 
                 574 
               
               
                   
               
               
                   
                 s 
                 138 
                 GAUGCUGUCCGAGGCAGUCdTdT 
                 575 
               
               
                   
               
               
                   
                 as 
                 156 
                 GACUGCCUCGGACAGCAUCdTdT 
                 576 
               
               
                   
               
               
                   
                 s 
                  14 
                 CAUUCUUGGCAGGAUGGCUdTdT 
                 577 
               
               
                   
               
               
                   
                 as 
                  32 
                 AGCCAUCCUGCCAAGAAUGdTdT 
                 578 
               
               
                   
               
               
                   
                 s 
                 140 
                 UGCUGUCCGAGGCAGUCCUdTdT 
                 579 
               
               
                   
               
               
                   
                 as 
                 158 
                 AGGACUGCCUCGGACAGCAdTdT 
                 580 
               
               
                   
               
               
                   
                 s 
                 146 
                 CCGAGGCAGUCCUGCCAUCdTdT 
                 581 
               
               
                   
               
               
                   
                 as 
                 164 
                 GAUGGCAGGACUGCCUCGGdTdT 
                 582 
               
               
                   
               
               
                   
                 s 
                 152 
                 CAGUCCUGCCAUCAAUGUGdTdT 
                 583 
               
               
                   
               
               
                   
                 as 
                 170 
                 CACAUUGAUGGCAGGACUGdTdT 
                 584 
               
               
                   
               
               
                   
                 s 
                 164 
                 CAAUGUGGCCGUGCAUGUGdTdT 
                 585 
               
               
                   
               
               
                   
                 as 
                 182 
                 CACAUGCACGGCCACAUUGdTdT 
                 586 
               
               
                   
               
               
                   
                 s 
                 178 
                 AUGUGUUCAGAAAGGCUGCdTdT 
                 587 
               
               
                   
               
               
                   
                 as 
                 196 
                 GCAGCCUUUCUGAACACAUdTdT 
                 588 
               
               
                   
               
               
                   
                 s 
                   2 
                 CAGAAGUCCACUCAUUCUUdTdT 
                 589 
               
               
                   
               
               
                   
                 as 
                  20 
                 AAGAAUGAGUGGACUUCUGdTdT 
                 590 
               
               
                   
               
               
                   
                 s 
                  21 
                 GGCAGGAUGGCUUCUCAUCdTdT 
                 591 
               
               
                   
               
               
                   
                 as 
                  39 
                 GAUGAGAAGCCAUCCUGCCdTdT 
                 592 
               
               
                   
               
               
                   
                 s 
                 210 
                 GAGCCAUUUGCCUCUGGGAdTdT 
                 593 
               
               
                   
               
               
                   
                 as 
                 228 
                 UCCCAGAGGCAAAUGGCUCdTdT 
                 594 
               
               
                   
               
               
                   
                 s 
                  23 
                 CAGGAUGGCUUCUCAUCGUdTdT 
                 595 
               
               
                   
               
               
                   
                 as 
                  41 
                 ACGAUGAGAAGCCAUCCUGdTdT 
                 596 
               
               
                   
               
               
                   
                 s 
                  24 
                 AGGAUGGCUUCUCAUCGUCdTdT 
                 597 
               
               
                   
               
               
                   
                 as 
                  42 
                 GACGAUGAGAAGCCAUCCUdTdT 
                 598 
               
               
                   
               
               
                   
                 s 
                 245 
                 AGAGCUGCAUGGGCUCACAdTdT 
                 599 
               
               
                   
               
               
                   
                 as 
                 263 
                 UGUGAGCCCAUGCAGCUCUdTdT 
                 600 
               
               
                   
               
               
                   
                 s 
                 248 
                 GCUGCAUGGGCUCACAACUdTdT 
                 601 
               
               
                   
               
               
                   
                 as 
                 266 
                 AGUUGUGAGCCCAUGCAGCdTdT 
                 602 
               
               
                   
               
               
                   
                 s 
                  25 
                 GGAUGGCUUCUCAUCGUCUdTdT 
                 603 
               
               
                   
               
               
                   
                 as 
                  43 
                 AGACGAUGAGAAGCCAUCCdTdT 
                 604 
               
               
                   
               
               
                   
                 s 
                 251 
                 GCAUGGGCUCACAACUGAGdTdT 
                 605 
               
               
                   
               
               
                   
                 as 
                 269 
                 CUCAGUUGUGAGCCCAUGCdTdT 
                 606 
               
               
                   
               
               
                   
                 s 
                 253 
                 AUGGGCUCACAACUGAGGAdTdT 
                 607 
               
               
                   
               
               
                   
                 as 
                 271 
                 UCCUCAGUUGUGAGCCCAUdTdT 
                 608 
               
               
                   
               
               
                   
                 s 
                 254 
                 UGGGCUCACAACUGAGGAGdTdT 
                 609 
               
               
                   
               
               
                   
                 as 
                 272 
                 CUCCUCAGUUGUGAGCCCAdTdT 
                 610 
               
               
                   
               
               
                   
                 s 
                 270 
                 GAGGAAUUUGUAGAAGGGAdTdT 
                 611 
               
               
                   
               
               
                   
                 as 
                 288 
                 UCCCUUCUACAAAUUCCUCdTdT 
                 612 
               
               
                   
               
               
                   
                 s 
                 276 
                 UUUGUAGAAGGGAUAUACAdTdT 
                 613 
               
               
                   
               
               
                   
                 as 
                 294 
                 UGUAUAUCCCUUCUACAAAdTdT 
                 614 
               
               
                   
               
               
                   
                 s 
                 277 
                 UUGUAGAAGGGAUAUACAAdTdT 
                 615 
               
               
                   
               
               
                   
                 as 
                 295 
                 UUGUAUAUCCCUUCUACAAdTdT 
                 616 
               
               
                   
               
               
                   
                 s 
                 278 
                 UGUAGAAGGGAUAUACAAAdTdT 
                 617 
               
               
                   
               
               
                   
                 as 
                 296 
                 UUUGUAUAUCCCUUCUACAdTdT 
                 618 
               
               
                   
               
               
                   
                 s 
                 281 
                 AGAAGGGAUAUACAAAGUGdTdT 
                 619 
               
               
                   
               
               
                   
                 as 
                 299 
                 CACUUUGUAUAUCCCUUCUdTdT 
                 620 
               
               
                   
               
               
                   
                 s 
                 295 
                 AAGUGGAAAUAGACACCAAdTdT 
                 621 
               
               
                   
               
               
                   
                 as 
                 313 
                 UUGGUGUCUAUUUCCACUUdTdT 
                 622 
               
               
                   
               
               
                   
                 s 
                 299 
                 GGAAAUAGACACCAAAUCUdTdT 
                 623 
               
               
                   
               
               
                   
                 as 
                 317 
                 AGAUUUGGUGUCUAUUUCCdTdT 
                 624 
               
               
                   
               
               
                   
                 s 
                 300 
                 GAAAUAGACACCAAAUCUUdTdT 
                 625 
               
               
                   
               
               
                   
                 as 
                 318 
                 AAGAUUUGGUGUCUAUUUCdTdT 
                 626 
               
               
                   
               
               
                   
                 s 
                 303 
                 AUAGACACCAAAUCUUACUdTdT 
                 627 
               
               
                   
               
               
                   
                 as 
                 321 
                 AGUAAGAUUUGGUGUCUAUdTdT 
                 628 
               
               
                   
               
               
                   
                 s 
                 304 
                 UAGACACCAAAUCUUACUGdTdT 
                 629 
               
               
                   
               
               
                   
                 as 
                 322 
                 CAGUAAGAUUUGGUGUCUAdTdT 
                 630 
               
               
                   
               
               
                   
                 s 
                 305 
                 AGACACCAAAUCUUACUGGdTdT 
                 631 
               
               
                   
               
               
                   
                 as 
                 323 
                 CCAGUAAGAUUUGGUGUCUdTdT 
                 632 
               
               
                   
               
               
                   
                 s 
                 317 
                 UUACUGGAAGGCACUUGGCdTdT 
                 633 
               
               
                   
               
               
                   
                 as 
                 335 
                 GCCAAGUGCCUUCCAGUAAdTdT 
                 634 
               
               
                   
               
               
                   
                 s 
                  32 
                 UUCUCAUCGUCUGCUCCUCdTdT 
                 635 
               
               
                   
               
               
                   
                 as 
                  50 
                 GAGGAGCAGACGAUGAGAAdTdT 
                 636 
               
               
                   
               
               
                   
                 s 
                 322 
                 GGAAGGCACUUGGCAUCUCdTdT 
                 637 
               
               
                   
               
               
                   
                 as 
                 340 
                 GAGAUGCCAAGUGCCUUCCdTdT 
                 638 
               
               
                   
               
               
                   
                 s 
                 326 
                 GGCACUUGGCAUCUCCCCAdTdT 
                 639 
               
               
                   
               
               
                   
                 as 
                 344 
                 UGGGGAGAUGCCAAGUGCCdTdT 
                 640 
               
               
                   
               
               
                   
                 s 
                 333 
                 GGCAUCUCCCCAUUCCAUGdTdT 
                 641 
               
               
                   
               
               
                   
                 as 
                 351 
                 AUGGAAUGGGGAGAUGCCTTdTdT 
                 642 
               
               
                   
               
               
                   
                 s 
                 334 
                 GCAUCUCCCCAUUCCAUGAdTdT 
                 643 
               
               
                   
               
               
                   
                 as 
                 352 
                 UCAUGGAAUGGGGAGAUGCdTdT 
                 644 
               
               
                   
               
               
                   
                 s 
                 335 
                 CAUCUCCCCAUUCCAUGAGdTdT 
                 645 
               
               
                   
               
               
                   
                 as 
                 353 
                 CUCAUGGAAUGGGGAGAUGdTdT 
                 646 
               
               
                   
               
               
                   
                 s 
                 336 
                 AUCUCCCCAUUCCAUGAGCdTdT 
                 647 
               
               
                   
               
               
                   
                 as 
                 354 
                 GCUCAUGGAAUGGGGAGAUdTdT 
                 648 
               
               
                   
               
               
                   
                 s 
                 338 
                 CUCCCCAUUCCAUGAGCAUdTdT 
                 649 
               
               
                   
               
               
                   
                 as 
                 356 
                 AUGCUCAUGGAAUGGGGAGdTdT 
                 650 
               
               
                   
               
               
                   
                 s 
                 341 
                 CCCAUUCCAUGAGCAUGCAdTdT 
                 651 
               
               
                   
               
               
                   
                 as 
                 359 
                 UGCAUGCUCAUGGAAUGGGdTdT 
                 652 
               
               
                   
               
               
                   
                 s 
                 347 
                 CCAUGAGCAUGCAGAGGUGdTdT 
                 653 
               
               
                   
               
               
                   
                 as 
                 365 
                 CACCUCUGCAUGCUCAUGGdTdT 
                 654 
               
               
                   
               
               
                   
                 s 
                 352 
                 AGCAUGCAGAGGUGGUAUUdTdT 
                 655 
               
               
                   
               
               
                   
                 as 
                 370 
                 AAUACCACCUCUGCAUGCUdTdT 
                 656 
               
               
                   
               
               
                   
                 s 
                 354 
                 CAUGGAGAGGUGGUAUUCAdTdT 
                 657 
               
               
                   
               
               
                   
                 as 
                 372 
                 UGAAUACCACCUCUGCAUGdTdT 
                 658 
               
               
                   
               
               
                   
                 s 
                 355 
                 AUGCAGAGGUGGUAUUCACdTdT 
                 659 
               
               
                   
               
               
                   
                 as 
                 373 
                 GUGAAUACCACCUCUGCAUdTdT 
                 660 
               
               
                   
               
               
                   
                 s 
                 362 
                 GGUGGUAUUCACAGCCAACdTdT 
                 661 
               
               
                   
               
               
                   
                 as 
                 380 
                 GUUGGCUGUGAAUACCACCdTdT 
                 662 
               
               
                   
               
               
                   
                 s 
                 363 
                 GUGGUAUUCACAGCCAACGdTdT 
                 663 
               
               
                   
               
               
                   
                 as 
                 381 
                 CGUUGGCUGUGAAUACCACdTdT 
                 664 
               
               
                   
               
               
                   
                 s 
                 364 
                 UGGUAUUCACAGCCAACGAdTdT 
                 665 
               
               
                   
               
               
                   
                 as 
                 382 
                 UCGUUGGCUGUGAAUACCAdTdT 
                 666 
               
               
                   
               
               
                   
                 s 
                 365 
                 GGUAUUCACAGCCAACGACdTdT 
                 667 
               
               
                   
               
               
                   
                 as 
                 383 
                 GUCGUUGGCUGUGAAUACCdTdT 
                 668 
               
               
                   
               
               
                   
                 s 
                 366 
                 GUAUUCACAGCCAACGACUdTdT 
                 669 
               
               
                   
               
               
                   
                 as 
                 384 
                 AGUCGUUGGCUGUGAAUACdTdT 
                 670 
               
               
                   
               
               
                   
                 s 
                 367 
                 UAUUCACAGCCAACGACUCdTdT 
                 671 
               
               
                   
               
               
                   
                 as 
                 385 
                 GAGUCGUUGGCUGUGAAUAdTdT 
                 672 
               
               
                   
               
               
                   
                 s 
                 370 
                 UCACAGCCAACGACUCCGGdTdT 
                 673 
               
               
                   
               
               
                   
                 as 
                 388 
                 CCGGAGUCGUUGGCUGUGAdTdT 
                 674 
               
               
                   
               
               
                   
                 s 
                 390 
                 CCCCGCCGCUACACCAUUGdTdT 
                 675 
               
               
                   
               
               
                   
                 as 
                 408 
                 CAAUGGUGUAGCGGCGGGGdTdT 
                 676 
               
               
                   
               
               
                   
                 s 
                   4 
                 GAAGUCCACUCAUUCUUGGdTdT 
                 677 
               
               
                   
               
               
                   
                 as 
                  22 
                 CCAAGAAUGAGUGGACUUCdTdT 
                 678 
               
               
                   
               
               
                   
                 s 
                 412 
                 CCCUGCUGAGCCCCUACUCdTdT 
                 679 
               
               
                   
               
               
                   
                 as 
                 430 
                 GAGUAGGGGCUCAGCAGGGdTdT 
                 680 
               
               
                   
               
               
                   
                 s 
                 417 
                 CUGAGCCCCUACUCCUAUUdTdT 
                 681 
               
               
                   
               
               
                   
                 as 
                 435 
                 AAUAGGAGUAGGGGCUCAGdTdT 
                 682 
               
               
                   
               
               
                   
                 s 
                 418 
                 UGAGCCCCUACUCCUAUUCdTdT 
                 683 
               
               
                   
               
               
                   
                 as 
                 436 
                 GAAUAGGAGUAGGGGCUCAdTdT 
                 684 
               
               
                   
               
               
                   
                 s 
                 422 
                 CCCCUACUCCUAUUCCACCdTdT 
                 685 
               
               
                   
               
               
                   
                 as 
                 440 
                 GGUGGAAUAGGAGUAGGGGdTdT 
                 686 
               
               
                   
               
               
                   
                 s 
                 425 
                 CUACUCCUAUUCCACCACGdTdT 
                 687 
               
               
                   
               
               
                   
                 as 
                 443 
                 CGUGGUGGAAUAGGAGUAGdTdT 
                 688 
               
               
                   
               
               
                   
                 s 
                 426 
                 UACUCCUAUUCCACCACGGdTdT 
                 689 
               
               
                   
               
               
                   
                 as 
                 444 
                 CCGUGGUGGAAUAGGAGUAdTdT 
                 690 
               
               
                   
               
               
                   
                 s 
                 427 
                 ACUCCUAUUCCACCACGGCdTdT 
                 691 
               
               
                   
               
               
                   
                 as 
                 445 
                 GCCGUGGUGGAAUAGGAGUdTdT 
                 692 
               
               
                   
               
               
                   
                 s 
                 429 
                 UCCUAUUCCACCACGGCUGdTdT 
                 693 
               
               
                   
               
               
                   
                 as 
                 447 
                 CAGCCGUGGUGGAAUAGGAdTdT 
                 694 
               
               
                   
               
               
                   
                 s 
                 432 
                 UAUUCCACCACGGCUGUCGdTdT 
                 695 
               
               
                   
               
               
                   
                 as 
                 450 
                 CGACAGCCGUGGUGGAAUAdTdT 
                 696 
               
               
                   
               
               
                   
                 s 
                 433 
                 AUUCCACCACGGCUGUCGUdTdT 
                 697 
               
               
                   
               
               
                   
                 as 
                 451 
                 ACGACAGCCGUGGUGGAAUdTdT 
                 698 
               
               
                   
               
               
                   
                 s 
                 437 
                 CACCACGGCUGUCGUCACCdTdT 
                 699 
               
               
                   
               
               
                   
                 as 
                 455 
                 GGUGACGACAGCCGUGGUGdTdT 
                 700 
               
               
                   
               
               
                   
                 s 
                 438 
                 ACCACGGCUGUCGUCACCAdTdT 
                 701 
               
               
                   
               
               
                   
                 as 
                 456 
                 UGGUGACGACAGCCGUGGUdTdT 
                 702 
               
               
                   
               
               
                   
                 s 
                 439 
                 CCACGGCUGUCGUCACCAAdTdT 
                 703 
               
               
                   
               
               
                   
                 as 
                 457 
                 UUGGUGACGACAGCCGUGGdTdT 
                 704 
               
               
                   
               
               
                   
                 s 
                 441 
                 ACGGCUGUCGUCACCAAUCdTdT 
                 705 
               
               
                   
               
               
                   
                 as 
                 459 
                 GAUUGGUGACGACAGCCGUdTdT 
                 706 
               
               
                   
               
               
                   
                 s 
                 442 
                 CGGCUGUCGUCACCAAUCCdTdT 
                 707 
               
               
                   
               
               
                   
                 as 
                 460 
                 GGAUUGGUGACGACAGCCGdTdT 
                 708 
               
               
                   
               
               
                   
                 s 
                 449 
                 CGUCACCAAUCCCAAGGAAdTdT 
                 709 
               
               
                   
               
               
                   
                 as 
                 467 
                 UUCCUUGGGAUUGGUGACGdTdT 
                 710 
               
               
                   
               
               
                   
                 s 
                 455 
                 CAAUCCCAAGGAAUGAGGGdTdT 
                 711 
               
               
                   
               
               
                   
                 as 
                 473 
                 CCCUCAUUCCUUGGGAUUGdTdT 
                 712 
               
               
                   
               
               
                   
                 s 
                 491 
                 CCUGAAGGACGAGGGAUGGdTdT 
                 713 
               
               
                   
               
               
                   
                 as 
                 509 
                 CCAUCCCUCGUCCUUCAGGdTdT 
                 714 
               
               
                   
               
               
                   
                 s 
                 497 
                 GGACGAGGGAUGGGAUUUCdTdT 
                 715 
               
               
                   
               
               
                   
                 as 
                 515 
                 GAAAUCCCAUCCCUCGUCCdTdT 
                 716 
               
               
                   
               
               
                   
                 s 
                   5 
                 AAGUCCACUCAUUCUUGGCdTdT 
                 717 
               
               
                   
               
               
                   
                 as 
                  23 
                 GCCAAGAAUGAGUGGACUUdTdT 
                 718 
               
               
                   
               
               
                   
                 s 
                 508 
                 GGGAUUUCAUGUAACCAAGdTdT 
                 719 
               
               
                   
               
               
                   
                 as 
                 526 
                 CUUGGUUACAUGAAAUCCCdTdT 
                 720 
               
               
                   
               
               
                   
                 s 
                 509 
                 GGAUUUCAUGUAACCAAGAdTdT 
                 721 
               
               
                   
               
               
                   
                 as 
                 527 
                 UCUUGGUUACAUGAAAUCCdTdT 
                 722 
               
               
                   
               
               
                   
                 s 
                 514 
                 UCAUGUAACCAAGAGUAUUdTdT 
                 723 
               
               
                   
               
               
                   
                 as 
                 532 
                 AAUACUCUUGGUUACAUGAdTdT 
                 724 
               
               
                   
               
               
                   
                 s 
                 516 
                 AUGUAACCAAGAGUAUUCCdTdT 
                 725 
               
               
                   
               
               
                   
                 as 
                 534 
                 GGAAUACUCUUGGUUACAUdTdT 
                 726 
               
               
                   
               
               
                   
                 s 
                 517 
                 UGUAACCAAGAGUAUUCCAdTdT 
                 727 
               
               
                   
               
               
                   
                 as 
                 535 
                 UGGAAUACUCUUGGUUACAdTdT 
                 728 
               
               
                   
               
               
                   
                 s 
                 518 
                 GUAACCAAGAGUAUUCCAUdTdT 
                 729 
               
               
                   
               
               
                   
                 as 
                 536 
                 AUGGAAUACUCUUGGUUACdTdT 
                 730 
               
               
                   
               
               
                   
                 s 
                  54 
                 UGCCUUGCUGGACUGGUAUdTdT 
                 731 
               
               
                   
               
               
                   
                 as 
                  72 
                 AUACCAGUCCAGCAAGGCAdTdT 
                 732 
               
               
                   
               
               
                   
                 s 
                 543 
                 UAAAGCAGUGUUUUCACCUdTdT 
                 733 
               
               
                   
               
               
                   
                 as 
                 561 
                 AGGUGAAAACACUGCUUUAdTdT 
                 734 
               
               
                   
               
               
                   
                 s 
                  55 
                 GCCUUGCUGGACUGGUAUUdTdT 
                 735 
               
               
                   
               
               
                   
                 as 
                  73 
                 AAUACCAGUCCAGCAAGGCdTdT 
                 736 
               
               
                   
               
               
                   
                 s 
                 551 
                 UGUUUUCACCUCAUAUGCUdTdT 
                 737 
               
               
                   
               
               
                   
                 as 
                 569 
                 AGCAUAUGAGGUGAAAACAdTdT 
                 738 
               
               
                   
               
               
                   
                 s 
                 552 
                 GUUUUCACCUCAUAUGCUAdTdT 
                 739 
               
               
                   
               
               
                   
                 as 
                 570 
                 UAGCAUAUGAGGUGAAAACdTdT 
                 740 
               
               
                   
               
               
                   
                 s 
                 553 
                 UUUUCACCUCAUAUGCUAUdTdT 
                 741 
               
               
                   
               
               
                   
                 as 
                 571 
                 AUAGCAUAUGAGGUGAAAAdTdT 
                 742 
               
               
                   
               
               
                   
                 s 
                 555 
                 UUCACCUCAUAUGCUAUGUdTdT 
                 743 
               
               
                   
               
               
                   
                 as 
                 573 
                 ACAUAGCAUAUGAGGUGAAdTdT 
                 744 
               
               
                   
               
               
                   
                 s 
                 557 
                 CACCUCAUAUGCUAUGUUAdTdT 
                 745 
               
               
                   
               
               
                   
                 as 
                 575 
                 UAACAUAGCAUAUGAGGUGdTdT 
                 746 
               
               
                   
               
               
                   
                 s 
                  56 
                 CCUUGCUGGACUGGUAUUUdTdT 
                 747 
               
               
                   
               
               
                   
                 as 
                  74 
                 AAAUACCAGUCCAGCAAGGdTdT 
                 748 
               
               
                   
               
               
                   
                 s 
                 563 
                 AUAUGCUAUGUUAGAAGUCdTdT 
                 749 
               
               
                   
               
               
                   
                 as 
                 581 
                 GACUUCUAACAUAGCAUAUdTdT 
                 750 
               
               
                   
               
               
                   
                 s 
                 564 
                 UAUGCUAUGUUAGAAGUCCdTdT 
                 751 
               
               
                   
               
               
                   
                 as 
                 582 
                 GGACUUCUAACAUAGCAUAdTdT 
                 752 
               
               
                   
               
               
                   
                 s 
                 566 
                 UGCUAUGUUAGAAGUCCAGdTdT 
                 753 
               
               
                   
               
               
                   
                 as 
                 584 
                 CUGGACUUCUAACAUAGCAdTdT 
                 754 
               
               
                   
               
               
                   
                 s 
                  57 
                 CUUGCUGGACUGGUAUUUGdTdT 
                 755 
               
               
                   
               
               
                   
                 as 
                  75 
                 CAAAUACCAGUCCAGCAAGdTdT 
                 756 
               
               
                   
               
               
                   
                 s 
                 578 
                 AGUCCAGGCAGAGACAAUAdTdT 
                 757 
               
               
                   
               
               
                   
                 as 
                 596 
                 AUUGUCUCUGCCUGGACUTTdTdT 
                 758 
               
               
                   
               
               
                   
                 s 
                 580 
                 UCCAGGCAGAGACAAUAAAdTdT 
                 759 
               
               
                   
               
               
                   
                 as 
                 598 
                 UUUAUUGUCUCUGCCUGGAdTdT 
                 760 
               
               
                   
               
               
                   
                 s 
                 607 
                 GUGAAAGGCACUUUUCAUUdTdT 
                 761 
               
               
                   
               
               
                   
                 as 
                 625 
                 AAUGAAAAGUGCCUUUCACdTdT 
                 762 
               
               
                   
               
               
                   
                 s 
                  62 
                 UGGACUGGUAUUUGUGUCUdTdT 
                 763 
               
               
                   
               
               
                   
                 as 
                  80 
                 AGACACAAAUACCAGUCCAdTdT 
                 764 
               
               
                   
               
               
                   
                 s 
                  77 
                 GUCUGAGGCUGGCCCUACGdTdT 
                 765 
               
               
                   
               
               
                   
                 as 
                  95 
                 CGUAGGGCCAGCCUCAGACdTdT 
                 766 
               
               
                   
               
               
                   
                 s 
                  79 
                 CUGAGGCUGGCCCUACGGGdTdT 
                 767 
               
               
                   
               
               
                   
                 as 
                  97 
                 CCCGUAGGGCCAGCCUCAGdTdT 
                 768 
               
               
                   
               
               
                   
                 s 
                  81 
                 GAGGCUGGCCCUACGGGCAdTdT 
                 769 
               
               
                   
               
               
                   
                 as 
                  99 
                 UGCCCGUAGGGCCAGCCUCdTdT 
                 770 
               
               
                   
               
               
                   
                 s 
                  82 
                 AGGCUGGCCCUACGGGCACdTdT 
                 771 
               
               
                   
               
               
                   
                 as 
                 100 
                 GUGCCCGUAGGGCCAGCCUdTdT 
                 772 
               
               
                   
               
               
                   
                 s 
                  84 
                 GCUGGCCCUACGGGCACCGdTdT 
                 773 
               
               
                   
               
               
                   
                 as 
                 102 
                 CGGUGCCCGUAGGGCCAGCdTdT 
                 774 
               
               
                   
               
               
                   
                 s 
                  85 
                 CUGGCCCUACGGGCACCGGdTdT 
                 775 
               
               
                   
               
               
                   
                 as 
                 103 
                 CCGGUGCCCGUAGGGCCAGdTdT 
                 776 
               
               
                   
               
               
                   
                 s 
                  87 
                 GGCCCUACGGGCACCGGUGdTdT 
                 777 
               
               
                   
               
               
                   
                 as 
                 105 
                 CACCGGUGCCCGUAGGGCCdTdT 
                 778 
               
               
                   
               
               
                   
                 s 
                   9 
                 CCACUCAUUCUUGGCAGGAdTdT 
                 779 
               
               
                   
               
               
                   
                 as 
                  27 
                 UCCUGCCAAGAAUGAGUGGdTdT 
                 780 
               
               
                   
               
               
                   
                 s 
                  90 
                 CCUACGGGCACCGGUGAAUdTdT 
                 781 
               
               
                   
               
               
                   
                 as 
                 108 
                 AUUCACCGGUGCCCGUAGGdTdT 
                 782 
               
               
                   
               
               
                   
                 s 
                  91 
                 CUACGGGCACCGGUGAAUCdTdT 
                 783 
               
               
                   
               
               
                   
                 as 
                 109 
                 GAUUCACCGGUGCCCGUAGdTdT 
                 784 
               
               
                   
               
               
                   
                 s 
                  92 
                 UACGGGCACCGGUGAAUCCdTdT 
                 785 
               
               
                   
               
               
                   
                 as 
                 110 
                 GGAUUCACCGGUGCCCGUAdTdT 
                 786 
               
               
                   
               
               
                   
                 s 
                  93 
                 ACGGGCACCGGUGAAUCCAdTdT 
                 787 
               
               
                   
               
               
                   
                 as 
                 111 
                 UGGAUUCACCGGUGCCCGUdTdT 
                 788 
               
               
                   
               
               
                   
                 s 
                  97 
                 GCACCGGUGAAUCCAAGUGdTdT 
                 789 
               
               
                   
               
               
                   
                 as 
                 115 
                 CACUUGGAUUCACCGGUGCdTdT 
                 790 
               
               
                   
               
               
                   
                 s 
                  98 
                 CACCGGUGAAUCCAAGUGUdTdT 
                 791 
               
               
                   
               
               
                   
                 as 
                 116 
                 ACACUUGGAUUCACCGGUGdTdT 
                 792 
               
               
                   
               
               
                   
                 s 
                 167 
                 UGUGGCCAUGCAUGUGUUCdTdT 
                 793 
               
               
                   
               
               
                   
                 as 
                 185 
                 GAACACAUGCAUGGCCACAdTdT 
                 794 
               
               
                   
               
               
                   
                 s 
                 168 
                 GUGGCCAUGCAUGUGUUCAdTdT 
                 795 
               
               
                   
               
               
                   
                 as 
                 186 
                 UGAACACAUGCAUGGCCACdTdT 
                 796 
               
               
                   
               
               
                   
                 s 
                 171 
                 GCCAUGCAUGUGUUCAGAAdTdT 
                 797 
               
               
                   
               
               
                   
                 as 
                 189 
                 UUCUGAACACAUGCAUGGCdTdT 
                 798 
               
               
                   
               
               
                   
                 s 
                 432 
                 UAUUCCACCACGGCUGUCAdTdT 
                 799 
               
               
                   
               
               
                   
                 as 
                 449 
                 UGACAGCCGUGGUGGAAUAdTdT 
                 800 
               
               
                   
               
               
                   
                 s 
                 447 
                 GUCAUCACCAAUCCCAAGGdTdT 
                 801 
               
               
                   
               
               
                   
                 as 
                 465 
                 CCUUGGGAUUGGUGAUGACdTdT 
                 802 
               
               
                   
               
               
                   
                 s 
                 115 
                 GUCCUCUGAUGGUCAAAGUdTdT 
                 803 
               
               
                   
               
               
                   
                 as 
                 133 
                 ACUUUGACCAUCAGAGGACdTdT 
                 804 
               
               
                   
               
               
                   
                 s 
                 122 
                 GAUGGUCAAAGUUCUAGAUdTdT 
                 805 
               
               
                   
               
               
                   
                 as 
                 140 
                 AUCUAGAACUUUGACCAUCdTdT 
                 806 
               
               
                   
               
               
                   
                 s 
                 139 
                 AUGCUGUCCGAGGCAGUCCdTdT 
                 807 
               
               
                   
               
               
                   
                 as 
                 157 
                 GGACUGCCUCGGACAGCAUdTdT 
                 808 
               
               
                   
               
               
                   
                 s 
                 172 
                 CCGUGCAUGUGUUCAGAAAdTdT 
                 809 
               
               
                   
               
               
                   
                 as 
                 190 
                 UUUCUGAACACAUGCACGGdTdT 
                 810 
               
               
                   
               
               
                   
                 s 
                 238 
                 AGUCUGGAGAGCUGCAUGGdTdT 
                 811 
               
               
                   
               
               
                   
                 as 
                 256 
                 CCAUGCAGCUCUCCAGACUdTdT 
                 812 
               
               
                   
               
               
                   
                 s 
                 252 
                 CAUGGGCUCACAACUGAGGdTdT 
                 813 
               
               
                   
               
               
                   
                 as 
                 270 
                 CCUCAGUUGUGAGCCCAUGdTdT 
                 814 
               
               
                   
               
               
                   
                 s 
                  33 
                 UCUCAUCGUCUGCUCCUCCdTdT 
                 815 
               
               
                   
               
               
                   
                 as 
                  51 
                 GGAGGAGCAGACGAUGAGAdTdT 
                 816 
               
               
                   
               
               
                   
                 s 
                 340 
                 CCCCAUUCCAUGAGCAUGCdTdT 
                 817 
               
               
                   
               
               
                   
                 as 
                 358 
                 GCAUGCUCAUGGAAUGGGGdTdT 
                 818 
               
               
                   
               
               
                   
                 s 
                 421 
                 GCCCCUACUCCUAUUCCACdTdT 
                 819 
               
               
                   
               
               
                   
                 as 
                 439 
                 GUGGAAUAGGAGUAGGGGCdTdT 
                 820 
               
               
                   
               
               
                   
                 s 
                 431 
                 CUAUUCCACCACGGCUGUCdTdT 
                 821 
               
               
                   
               
               
                   
                 as 
                 449 
                 GACAGCCGUGGUGGAAUAGdTdT 
                 822 
               
               
                   
               
               
                   
                 s 
                 440 
                 CACGGCUGUCGUCACCAAUdTdT 
                 823 
               
               
                   
               
               
                   
                 as 
                 458 
                 AUUGGUGACGACAGCCGUGdTdT 
                 824 
               
               
                   
               
               
                   
                 s 
                 496 
                 AGGACGAGGGAUGGGAUUUdTdT 
                 825 
               
               
                   
               
               
                   
                 as 
                 514 
                 AAAUCCCAUCCCUCGUCCUdTdT 
                 826 
               
               
                   
               
               
                   
                 s 
                 556 
                 UCACCUCAUAUGCUAUGUUdTdT 
                 827 
               
               
                   
               
               
                   
                 as 
                 574 
                 AACAUAGCAUAUGAGGUGAdTdT 
                 828 
               
               
                   
               
               
                   
                 s 
                 559 
                 CCUCAUAUGCUAUGUUAGAdTdT 
                 829 
               
               
                   
               
               
                   
                 as 
                 577 
                 UCUAACAUAGCAUAUGAGGdTdT 
                 830 
               
               
                   
               
               
                   
                 s 
                 570 
                 AUGUUAGAAGUCCAGGCAGdTdT 
                 831 
               
               
                   
               
               
                   
                 as 
                 588 
                 CUGCCUGGACUUCUAACAUdTdT 
                 832 
               
               
                   
               
               
                   
                 s 
                  78 
                 UCUGAGGCUGGCCCUACGGdTdT 
                 833 
               
               
                   
               
               
                   
                 as 
                  96 
                 CCGUAGGGCCAGCCUCAGAdTdT 
                 834 
               
               
                   
               
               
                   
                 s 
                  87 
                 GGCCCUACGGGCACCGGUGdTdT 
                 835 
               
               
                   
               
               
                   
                 as 
                 105 
                 CACCGGUGCCCGUAGGGCCdTdT 
                 836 
               
               
                   
               
               
                   
                 s 
                  95 
                 GGGCACCGGUGAAUCCAAGdTdT 
                 837 
               
               
                   
               
               
                   
                 as 
                 113 
                 CUUGGAUUCACCGGUGCCCdTdT 
                 838 
               
               
                   
               
               
                   
                 s 
                 167 
                 CCAUGCAUGUGUUCAGAAAdTdT 
                 839 
               
               
                   
               
               
                   
                 as 
                 185 
                 UUUCUGAACACAUGCAUGGdTdT 
                 840 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Chemically modified sense and antisense 
               
               
                 strand sequences of human TTR dsRNAs 
               
               
                 See Table 2 for duplex #. Strand: s = sense; 
               
               
                 as = antisense; Position: position of 5′ base on 
               
               
                 transcript (NM_000371.2, SEQ ID NO: 1329) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 SEQ 
               
               
                   
                   
                 Po- 
                 Sequence 
                 ID 
               
               
                 Strand 
                 Oligo # 
                 sition 
                 (5′ to 3′) 
                 NO: 
               
               
                   
               
               
                 s 
                 A-32153 
                 100 
                 ccGGuGAAuccAAGuGuccdTdT 
                  841 
               
               
                   
               
               
                 as 
                 A-32154 
                 118 
                 GGAcACUUGGAUUcACCGGdTdT 
                  842 
               
               
                   
               
               
                 s 
                 A-32155 
                  11 
                 AcucAuucuuGGcAGGAuGdTdT 
                  843 
               
               
                   
               
               
                 as 
                 A-32156 
                  29 
                 cAUCCUGCcAAGAAUGAGUdTdT 
                  844 
               
               
                   
               
               
                 s 
                 A-32157 
                 111 
                 AAGuGuccucuGAuGGucAdTdT 
                  845 
               
               
                   
               
               
                 as 
                 A-32158 
                 129 
                 UGACcAUcAGAGGAcACUUdTdT 
                  846 
               
               
                   
               
               
                 s 
                 A-32163 
                  13 
                 ucAuucuuGGcAGGAuGGcdTdT 
                  847 
               
               
                   
               
               
                 as 
                 A-32164 
                  31 
                 GCcAUCCUGCcAAGAAUGAdTdT 
                  848 
               
               
                   
               
               
                 s 
                 A-32165 
                 130 
                 AAGuucuAGAuGcuGuccGdTdT 
                  849 
               
               
                   
               
               
                 as 
                 A-32166 
                 148 
                 CGGAcAGcAUCuAGAACUUdTdT 
                  850 
               
               
                   
               
               
                 s 
                 A-32167 
                 132 
                 GuucuAGAuGcuGuccGAGdTdT 
                  851 
               
               
                   
               
               
                 as 
                 A-32168 
                 150 
                 CUCGGAcAGcAUCuAGAACdTdT 
                  852 
               
               
                   
               
               
                 s 
                 A-32169 
                 135 
                 cuAGAuGcuGuccGAGGcAdTdT 
                  853 
               
               
                   
               
               
                 as 
                 A-32170 
                 153 
                 UGCCUCGGAcAGcAUCuAGdTdT 
                  854 
               
               
                   
               
               
                 s 
                 A-32171 
                 138 
                 GAuGcuGuccGAGGcAGucdTdT 
                  855 
               
               
                   
               
               
                 as 
                 A-32172 
                 156 
                 GACUGCCUCGGAcAGcAUCdTdT 
                  856 
               
               
                   
               
               
                 s 
                 A-32175 
                  14 
                 cAuucuuGGcAGGAuGGcudTdT 
                  857 
               
               
                   
               
               
                 as 
                 A-32176 
                  32 
                 AGCcAUCCUGCcAAGAAUGdTdT 
                  858 
               
               
                   
               
               
                 s 
                 A-32177 
                 140 
                 uGcuGuccGAGGcAGuccudTdT 
                  859 
               
               
                   
               
               
                 as 
                 A-32178 
                 158 
                 AGGACUGCCUCGGAcAGcAdTdT 
                  860 
               
               
                   
               
               
                 s 
                 A-32179 
                 146 
                 ccGAGGcAGuccuGccAucdTdT 
                  861 
               
               
                   
               
               
                 as 
                 A-32180 
                 164 
                 GAUGGcAGGACUGCCUCGGdTdT 
                  862 
               
               
                   
               
               
                 s 
                 A-32181 
                 152 
                 cAGuccuGccAucAAuGuGdTdT 
                  863 
               
               
                   
               
               
                 as 
                 A-32182 
                 170 
                 cAcAUUGAUGGcAGGACUGdTdT 
                  864 
               
               
                   
               
               
                 s 
                 A-32183 
                 164 
                 cAAuGuGGccGuGcAuGuGdTdT 
                  865 
               
               
                   
               
               
                 as 
                 A-32184 
                 182 
                 cAcAUGcACGGCcAcAUUGdTdT 
                  866 
               
               
                   
               
               
                 s 
                 A-32187 
                 178 
                 AuGuGuucAGAAAGGcuGcdTdT 
                  867 
               
               
                   
               
               
                 as 
                 A-32188 
                 196 
                 GcAGCCUUUCUGAAcAcAUdTdT 
                  868 
               
               
                   
               
               
                 s 
                 A-32189 
                   2 
                 cAGAAGuccAcucAuucuudTdT 
                  869 
               
               
                   
               
               
                 as 
                 A-32190 
                  20 
                 AAGAAUGAGUGGACUUCUGdTdT 
                  870 
               
               
                   
               
               
                 s 
                 A-32191 
                  21 
                 GGcAGGAuGGcuucucAucdTdT 
                  871 
               
               
                   
               
               
                 as 
                 A-32192 
                  39 
                 GAUGAGAAGCcAUCCUGCCdTdT 
                  872 
               
               
                   
               
               
                 s 
                 A-32193 
                 210 
                 GAGccAuuuGccucuGGGAdTdT 
                  873 
               
               
                   
               
               
                 as 
                 A-32194 
                 228 
                 UCCcAGAGGcAAAUGGCUCdTdT 
                  874 
               
               
                   
               
               
                 s 
                 A-32195 
                  23 
                 cAGGAuGGcuucucAucGudTdT 
                  875 
               
               
                   
               
               
                 as 
                 A-32196 
                  41 
                 ACGAUGAGAAGCcAUCCUGdTdT 
                  876 
               
               
                   
               
               
                 s 
                 A-32199 
                  24 
                 AGGAuGGcuucucAucGucdTdT 
                  877 
               
               
                   
               
               
                 as 
                 A-32200 
                  42 
                 GACGAUGAGAAGCcAUCCUdTdT 
                  878 
               
               
                   
               
               
                 s 
                 A-32201 
                 245 
                 AGAGcuGcAuGGGcucAcAdTdT 
                  879 
               
               
                   
               
               
                 as 
                 A-32202 
                 263 
                 UGUGAGCCcAUGcAGCUCUdTdT 
                  880 
               
               
                   
               
               
                 s 
                 A-32203 
                 248 
                 GcuGcAuGGGcucAcAAcudTdT 
                  881 
               
               
                   
               
               
                 as 
                 A-32204 
                 266 
                 AGUUGUGAGCCcAUGcAGCdTdT 
                  882 
               
               
                   
               
               
                 s 
                 A-32205 
                  25 
                 GGAuGGcuucucAucGucudTdT 
                  883 
               
               
                   
               
               
                 as 
                 A-32206 
                  43 
                 AGACGAUGAGAAGCcAUCCdTdT 
                  884 
               
               
                   
               
               
                 s 
                 A-32207 
                 251 
                 GcAuGGGcucAcAAcuGAGdTdT 
                  885 
               
               
                   
               
               
                 as 
                 A-32208 
                 269 
                 CUcAGUUGUGAGCCcAUGCdTdT 
                  886 
               
               
                   
               
               
                 s 
                 A-32211 
                 253 
                 AuGGGcucAcAAcuGAGGAdTdT 
                  887 
               
               
                   
               
               
                 as 
                 A-32212 
                 271 
                 UCCUcAGUUGUGAGCCcAUdTdT 
                  888 
               
               
                   
               
               
                 s 
                 A-32213 
                 254 
                 uGGGcucAcAAcuGAGGAGdTdT 
                  889 
               
               
                   
               
               
                 as 
                 A-32214 
                 272 
                 CUCCUcAGUUGUGAGCCcAdTdT 
                  890 
               
               
                   
               
               
                 s 
                 A-32215 
                 270 
                 GAGGAAuuuGuAGAAGGGAdTdT 
                  891 
               
               
                   
               
               
                 as 
                 A-32216 
                 288 
                 UCCCUUCuAcAAAUUCCUCdTdT 
                  892 
               
               
                   
               
               
                 s 
                 A-32217 
                 276 
                 uuuGuAGAAGGGAuAuAcAdTdT 
                  893 
               
               
                   
               
               
                 as 
                 A-32218 
                 294 
                 UGuAuAUCCCUUCuAcAAAdTdT 
                  894 
               
               
                   
               
               
                 s 
                 A-32219 
                 277 
                 uuGuAGAAGGGAuAuAcAAdTdT 
                  895 
               
               
                   
               
               
                 as 
                 A-32220 
                 295 
                 UUGuAuAUCCCUUCuAcAAdTdT 
                  896 
               
               
                   
               
               
                 s 
                 A-32221 
                 278 
                 uGuAGAAGGGAuAuAcAAAdTdT 
                  897 
               
               
                   
               
               
                 as 
                 A-32222 
                 296 
                 UUUGuAuAUCCCUUCuAcAdTdT 
                  898 
               
               
                   
               
               
                 s 
                 A-32223 
                 281 
                 AGAAGGGAuAuAcAAAGuGdTdT 
                  899 
               
               
                   
               
               
                 as 
                 A-32224 
                 299 
                 cACUUUGuAuAUCCCUUCUdTdT 
                  900 
               
               
                   
               
               
                 s 
                 A-32225 
                 295 
                 AAGuGGAAAuAGAcAccAAdTdT 
                  901 
               
               
                   
               
               
                 as 
                 A-32226 
                 313 
                 UUGGUGUCuAUUUCcACUUdTdT 
                  902 
               
               
                   
               
               
                 s 
                 A-32227 
                 299 
                 GGAAAuAGAcAccAAAucudTdT 
                  903 
               
               
                   
               
               
                 as 
                 A-32228 
                 317 
                 AGAUUUGGUGUCuAUUUCCdTdT 
                  904 
               
               
                   
               
               
                 s 
                 A-32229 
                 300 
                 GAAAuAGAcAccAAAucuudTdT 
                  905 
               
               
                   
               
               
                 as 
                 A-32230 
                 318 
                 AAGAUUUGGUGUCuAUUUCdTdT 
                  906 
               
               
                   
               
               
                 s 
                 A-32231 
                 303 
                 AuAGAcAccAAAucuuAcudTdT 
                  907 
               
               
                   
               
               
                 as 
                 A-32232 
                 321 
                 AGuAAGAUUUGGUGUCuAUdTdT 
                  908 
               
               
                   
               
               
                 s 
                 A-32233 
                 304 
                 uAGAcAccAAAucuuAcuGdTdT 
                  909 
               
               
                   
               
               
                 as 
                 A-32234 
                 322 
                 cAGuAAGAUUUGGUGUCuAdTdT 
                  910 
               
               
                   
               
               
                 s 
                 A-32235 
                 305 
                 AGAcAccAAAucuuAcuGGdTdT 
                  911 
               
               
                   
               
               
                 as 
                 A-32236 
                 323 
                 CcAGuAAGAUUUGGUGUCUdTdT 
                  912 
               
               
                   
               
               
                 s 
                 A-32237 
                 317 
                 uuAcuGGAAGGcAcuuGGcdTdT 
                  913 
               
               
                   
               
               
                 as 
                 A-32238 
                 335 
                 GCcAAGUGCCUUCcAGuAAdTdT 
                  914 
               
               
                   
               
               
                 s 
                 A-32239 
                  32 
                 uucucAucGucuGcuccucdTdT 
                  915 
               
               
                   
               
               
                 as 
                 A-32240 
                  50 
                 GAGGAGcAGACGAUGAGAAdTdT 
                  916 
               
               
                   
               
               
                 s 
                 A-32241 
                 322 
                 GGAAGGcAcuuGGcAucucdTdT 
                  917 
               
               
                   
               
               
                 as 
                 A-32242 
                 340 
                 GAGAUGCcAAGUGCCUUCCdTdT 
                  918 
               
               
                   
               
               
                 s 
                 A-32243 
                 326 
                 GGcAcuuGGcAucuccccAdTdT 
                  919 
               
               
                   
               
               
                 as 
                 A-32244 
                 344 
                 UGGGGAGAUGCcAAGUGCCdTdT 
                  920 
               
               
                   
               
               
                 s 
                 A-32247 
                 333 
                 GGcAucuccccAuuccAuGdTdT 
                  921 
               
               
                   
               
               
                 as 
                 A-32248 
                 351 
                 cAUGGAAUGGGGAGAUGCCdTdT 
                  922 
               
               
                   
               
               
                 s 
                 A-32249 
                 334 
                 GcAucuccccAuuccAuGAdTdT 
                  923 
               
               
                   
               
               
                 as 
                 A-32250 
                 352 
                 UcAUGGAAUGGGGAGAUGCdTdT 
                  924 
               
               
                   
               
               
                 s 
                 A-32251 
                 335 
                 cAucuccccAuuccAuGAGdTdT 
                  925 
               
               
                   
               
               
                 as 
                 A-32252 
                 353 
                 CUcAUGGAAUGGGGAGAUGdTdT 
                  926 
               
               
                   
               
               
                 s 
                 A-32253 
                 336 
                 AucuccccAuuccAuGAGcdTdT 
                  927 
               
               
                   
               
               
                 as 
                 A-32254 
                 354 
                 GCUcAUGGAAUGGGGAGAUdTdT 
                  928 
               
               
                   
               
               
                 s 
                 A-32255 
                 338 
                 cuccccAuuccAuGAGcAudTdT 
                  929 
               
               
                   
               
               
                 as 
                 A-32256 
                 356 
                 AUGCUcAUGGAAUGGGGAGdTdT 
                  930 
               
               
                   
               
               
                 s 
                 A-32259 
                 341 
                 cccAuuccAuGAGcAuGcAdTdT 
                  931 
               
               
                   
               
               
                 as 
                 A-32260 
                 359 
                 UGcAUGCUcAUGGAAUGGGdTdT 
                  932 
               
               
                   
               
               
                 s 
                 A-32261 
                 347 
                 ccAuGAGcAuGcAGAGGuGdTdT 
                  933 
               
               
                   
               
               
                 as 
                 A-32262 
                 365 
                 cACCUCUGcAUGCUcAUGGdTdT 
                  934 
               
               
                   
               
               
                 s 
                 A-32263 
                 352 
                 AGcAuGcAGAGGuGGuAuudTdT 
                  935 
               
               
                   
               
               
                 as 
                 A-32264 
                 370 
                 AAuACcACCUCUGcAUGCUdTdT 
                  936 
               
               
                   
               
               
                 s 
                 A-32265 
                 354 
                 cAuGcAGAGGuGGuAuucAdTdT 
                  937 
               
               
                   
               
               
                 as 
                 A-32266 
                 372 
                 UGAAuACcACCUCUGcAUGdTdT 
                  938 
               
               
                   
               
               
                 s 
                 A-32267 
                 355 
                 AuGcAGAGGuGGuAuucAcdTdT 
                  939 
               
               
                   
               
               
                 as 
                 A-32268 
                 373 
                 GUGAAuACcACCUCUGcAUdTdT 
                  940 
               
               
                   
               
               
                 s 
                 A-32269 
                 362 
                 GGuGGuAuucAcAGccAAcdTdT 
                  941 
               
               
                   
               
               
                 as 
                 A-32270 
                 380 
                 GUUGGCUGUGAAuACcACCdTdT 
                  942 
               
               
                   
               
               
                 s 
                 A-32271 
                 363 
                 GuGGuAuucAcAGccAAcGdTdT 
                  943 
               
               
                   
               
               
                 as 
                 A-32272 
                 381 
                 CGUUGGCUGUGAAuACcACdTdT 
                  944 
               
               
                   
               
               
                 s 
                 A-32273 
                 364 
                 uGGuAuucAcAGccAAcGAdTdT 
                  945 
               
               
                   
               
               
                 as 
                 A-32274 
                 382 
                 UCGUUGGCUGUGAAuACcAdTdT 
                  946 
               
               
                   
               
               
                 s 
                 A-32275 
                 365 
                 GGuAuucAcAGccAAcGAcdTdT 
                  947 
               
               
                   
               
               
                 as 
                 A-32276 
                 383 
                 GUCGUUGGCUGUGAAuACCdTdT 
                  948 
               
               
                   
               
               
                 s 
                 A-32277 
                 366 
                 GuAuucAcAGccAAcGAcudTdT 
                  949 
               
               
                   
               
               
                 as 
                 A-32278 
                 384 
                 AGUCGUUGGCUGUGAAuACdTdT 
                  950 
               
               
                   
               
               
                 s 
                 A-32279 
                 367 
                 uAuucAcAGccAAcGAcucdTdT 
                  951 
               
               
                   
               
               
                 as 
                 A-32280 
                 385 
                 GAGUCGUUGGCUGUGAAuAdTdT 
                  952 
               
               
                   
               
               
                 s 
                 A-32281 
                 370 
                 ucAcAGccAAcGAcuccGGdTdT 
                  953 
               
               
                   
               
               
                 as 
                 A-32282 
                 388 
                 CCGGAGUCGUUGGCUGUGAdTdT 
                  954 
               
               
                   
               
               
                 s 
                 A-32283 
                 390 
                 ccccGccGcuAcAccAuuGdTdT 
                  955 
               
               
                   
               
               
                 as 
                 A-32284 
                 408 
                 cAAUGGUGuAGCGGCGGGGdTdT 
                  956 
               
               
                   
               
               
                 s 
                 A-32285 
                   4 
                 GAAGuccAcucAuucuuGGdTdT 
                  957 
               
               
                   
               
               
                 as 
                 A-32286 
                  22 
                 CcAAGAAUGAGUGGACUUCdTdT 
                  958 
               
               
                   
               
               
                 s 
                 A-32287 
                 412 
                 cccuGcuGAGccccuAcucdTdT 
                  959 
               
               
                   
               
               
                 as 
                 A-32288 
                 430 
                 GAGuAGGGGCUcAGcAGGGdTdT 
                  960 
               
               
                   
               
               
                 s 
                 A-32289 
                 417 
                 cuGAGccccuAcuccuAuudTdT 
                  961 
               
               
                   
               
               
                 as 
                 A-32290 
                 435 
                 AAuAGGAGuAGGGGCUcAGdTdT 
                  962 
               
               
                   
               
               
                 s 
                 A-32291 
                 418 
                 uGAGccccuAcuccuAuucdTdT 
                  963 
               
               
                   
               
               
                 as 
                 A-32292 
                 436 
                 GAAuAGGAGuAGGGGCUcAdTdT 
                  964 
               
               
                   
               
               
                 s 
                 A-32295 
                 422 
                 ccccuAcuccuAuuccAccdTdT 
                  965 
               
               
                   
               
               
                 as 
                 A-32296 
                 440 
                 GGUGGAAuAGGAGuAGGGGdTdT 
                  966 
               
               
                   
               
               
                 s 
                 A-32297 
                 425 
                 cuAcuccuAuuccAccAcGdTdT 
                  967 
               
               
                   
               
               
                 as 
                 A-32298 
                 443 
                 CGUGGUGGAAuAGGAGuAGdTdT 
                  968 
               
               
                   
               
               
                 s 
                 A-32299 
                 426 
                 uAcuccuAuuccAccAcGGdTdT 
                  969 
               
               
                   
               
               
                 as 
                 A-32300 
                 444 
                 CCGUGGUGGAAuAGGAGuAdTdT 
                  970 
               
               
                   
               
               
                 s 
                 A-32301 
                 427 
                 AcuccuAuuccAccAcGGcdTdT 
                  971 
               
               
                   
               
               
                 as 
                 A-32302 
                 445 
                 GCCGUGGUGGAAuAGGAGUdTdT 
                  972 
               
               
                   
               
               
                 s 
                 A-32303 
                 429 
                 uccuAuuccAccAcGGcuGdTdT 
                  973 
               
               
                   
               
               
                 as 
                 A-32304 
                 447 
                 cAGCCGUGGUGGAAuAGGAdTdT 
                  974 
               
               
                   
               
               
                 s 
                 A-32307 
                 432 
                 uAuuccAccAcGGcuGucGdTdT 
                  975 
               
               
                   
               
               
                 as 
                 A-32308 
                 450 
                 CGAcAGCCGUGGUGGAAuAdTdT 
                  976 
               
               
                   
               
               
                 s 
                 A-32309 
                 433 
                 AuuccAccAcGGcuGucGudTdT 
                  977 
               
               
                   
               
               
                 as 
                 A-32310 
                 451 
                 ACGAcAGCCGUGGUGGAAUdTdT 
                  978 
               
               
                   
               
               
                 s 
                 A-32311 
                 437 
                 cAccAcGGcuGucGucAccdTdT 
                  979 
               
               
                   
               
               
                 as 
                 A-32312 
                 455 
                 GGUGACGAcAGCCGUGGUGdTdT 
                  980 
               
               
                   
               
               
                 s 
                 A-32313 
                 438 
                 AccAcGGcuGucGucAccAdTdT 
                  981 
               
               
                   
               
               
                 as 
                 A-32314 
                 456 
                 UGGUGACGAcAGCCGUGGUdTdT 
                  982 
               
               
                   
               
               
                 s 
                 A-32315 
                 439 
                 ccAcGGcuGucGucAccAAdTdT 
                  983 
               
               
                   
               
               
                 as 
                 A-32316 
                 457 
                 UUGGUGACGAcAGCCGUGGdTdT 
                  984 
               
               
                   
               
               
                 s 
                 A-32319 
                 441 
                 AcGGcuGucGucAccAAucdTdT 
                  985 
               
               
                   
               
               
                 as 
                 A-32320 
                 459 
                 GAUUGGUGACGAcAGCCGUdTdT 
                  986 
               
               
                   
               
               
                 s 
                 A-32321 
                 442 
                 cGGcuGucGucAccAAuccdTdT 
                  987 
               
               
                   
               
               
                 as 
                 A-32322 
                 460 
                 GGAUUGGUGACGAcAGCCGdTdT 
                  988 
               
               
                   
               
               
                 s 
                 A-32323 
                 449 
                 cGucAccAAucccAAGGAAdTdT 
                  989 
               
               
                   
               
               
                 as 
                 A-32324 
                 467 
                 UUCCUUGGGAUUGGUGACGdTdT 
                  990 
               
               
                   
               
               
                 s 
                 A-32325 
                 455 
                 cAAucccAAGGAAuGAGGGdTdT 
                  991 
               
               
                   
               
               
                 as 
                 A-32326 
                 473 
                 CCCUcAUUCCUUGGGAUUGdTdT 
                  992 
               
               
                   
               
               
                 s 
                 A-32327 
                 491 
                 ccuGAAGGAcGAGGGAuGGdTdT 
                  993 
               
               
                   
               
               
                 as 
                 A-32328 
                 509 
                 CcAUCCCUCGUCCUUcAGGdTdT 
                  994 
               
               
                   
               
               
                 s 
                 A-32331 
                 497 
                 GGAcGAGGGAuGGGAuuucdTdT 
                  995 
               
               
                   
               
               
                 as 
                 A-32332 
                 515 
                 GAAAUCCcAUCCCUCGUCCdTdT 
                  996 
               
               
                   
               
               
                 s 
                 A-32333 
                   5 
                 AAGuccAcucAuucuuGGcdTdT 
                  997 
               
               
                   
               
               
                 as 
                 A-32334 
                  23 
                 GCcAAGAAUGAGUGGACUUdTdT 
                  998 
               
               
                   
               
               
                 s 
                 A-32335 
                 508 
                 GGGAuuucAuGuAAccAAGdTdT 
                  999 
               
               
                   
               
               
                 as 
                 A-32336 
                 526 
                 CUUGGUuAcAUGAAAUCCCdTdT 
                 1000 
               
               
                   
               
               
                 s 
                 A-32337 
                 509 
                 GGAuuucAuGuAAccAAGAdTdT 
                 1001 
               
               
                   
               
               
                 as 
                 A-32338 
                 527 
                 UCUUGGUuAcAUGAAAUCCdTdT 
                 1002 
               
               
                   
               
               
                 s 
                 A-32339 
                 514 
                 ucAuGuAAccAAGAGuAuudTdT 
                 1003 
               
               
                   
               
               
                 as 
                 A-32340 
                 532 
                 AAuACUCUUGGUuAcAUGAdTdT 
                 1004 
               
               
                   
               
               
                 s 
                 A-32341 
                 516 
                 AuGuAAccAAGAGuAuuccdTdT 
                 1005 
               
               
                   
               
               
                 as 
                 A-32342 
                 534 
                 GGAAuACUCUUGGUuAcAUdTdT 
                 1006 
               
               
                   
               
               
                 s 
                 A-32343 
                 517 
                 uGuAAccAAGAGuAuuccAdTdT 
                 1007 
               
               
                   
               
               
                 as 
                 A-32344 
                 535 
                 UGGAAuACUCUUGGUuAcAdTdT 
                 1008 
               
               
                   
               
               
                 s 
                 A-32345 
                 518 
                 GuAAccAAGAGuAuuccAudTdT 
                 1009 
               
               
                   
               
               
                 as 
                 A-32346 
                 536 
                 AUGGAAuACUCUUGGUuACdTdT 
                 1010 
               
               
                   
               
               
                 s 
                 A-32347 
                  54 
                 uGccuuGcuGGAcuGGuAudTdT 
                 1011 
               
               
                   
               
               
                 as 
                 A-32348 
                  72 
                 AuACcAGUCcAGcAAGGcAdTdT 
                 1012 
               
               
                   
               
               
                 s 
                 A-32349 
                 543 
                 uAAAGcAGuGuuuucAccudTdT 
                 1013 
               
               
                   
               
               
                 as 
                 A-32350 
                 561 
                 AGGUGAAAAcACUGCUUuAdTdT 
                 1014 
               
               
                   
               
               
                 s 
                 A-32351 
                  55 
                 GccuuGcuGGAcuGGuAuudTdT 
                 1015 
               
               
                   
               
               
                 as 
                 A-32352 
                  73 
                 AAuACcAGUCcAGcAAGGCdTdT 
                 1016 
               
               
                   
               
               
                 s 
                 A-32353 
                 551 
                 uGuuuucAccucAuAuGcudTdT 
                 1017 
               
               
                   
               
               
                 as 
                 A-32354 
                 569 
                 AGcAuAUGAGGUGAAAAcAdTdT 
                 1018 
               
               
                   
               
               
                 s 
                 A-32355 
                 552 
                 GuuuucAccucAuAuGcuAdTdT 
                 1019 
               
               
                   
               
               
                 as 
                 A-32356 
                 570 
                 uAGcAuAUGAGGUGAAAACdTdT 
                 1020 
               
               
                   
               
               
                 s 
                 A-32357 
                 553 
                 uuuucAccucAuAuGcuAudTdT 
                 1021 
               
               
                   
               
               
                 as 
                 A-32358 
                 571 
                 AuAGcAuAUGAGGUGAAAAdTdT 
                 1022 
               
               
                   
               
               
                 s 
                 A-32359 
                 555 
                 uucAccucAuAuGcuAuGudTdT 
                 1023 
               
               
                   
               
               
                 as 
                 A-32360 
                 573 
                 AcAuAGcAuAUGAGGUGAAdTdT 
                 1024 
               
               
                   
               
               
                 s 
                 A-32363 
                 557 
                 cAccucAuAuGcuAuGuuAdTdT 
                 1025 
               
               
                   
               
               
                 as 
                 A-32364 
                 575 
                 uAAcAuAGcAuAUGAGGUGdTdT 
                 1026 
               
               
                   
               
               
                 s 
                 A-32367 
                  56 
                 ccuuGcuGGAcuGGuAuuudTdT 
                 1027 
               
               
                   
               
               
                 as 
                 A-32368 
                  74 
                 AAAuACcAGUCcAGcAAGGdTdT 
                 1028 
               
               
                   
               
               
                 s 
                 A-32369 
                 563 
                 AuAuGcuAuGuuAGAAGucdTdT 
                 1029 
               
               
                   
               
               
                 as 
                 A-32370 
                 581 
                 GACUUCuAAcAuAGcAuAUdTdT 
                 1030 
               
               
                   
               
               
                 s 
                 A-32371 
                 564 
                 uAuGcuAuGuuAGAAGuccdTdT 
                 1031 
               
               
                   
               
               
                 as 
                 A-32372 
                 582 
                 GGACUUCuAAcAuAGcAuAdTdT 
                 1032 
               
               
                   
               
               
                 s 
                 A-32373 
                 566 
                 uGcuAuGuuAGAAGuccAGdTdT 
                 1033 
               
               
                   
               
               
                 as 
                 A-32374 
                 584 
                 CUGGACUUCuAAcAuAGcAdTdT 
                 1034 
               
               
                   
               
               
                 s 
                 A-32375 
                  57 
                 cuuGcuGGAcuGGuAuuuGdTdT 
                 1035 
               
               
                   
               
               
                 as 
                 A-32376 
                  75 
                 cAAAuACcAGUCcAGcAAGdTdT 
                 1036 
               
               
                   
               
               
                 s 
                 A-32379 
                 578 
                 AGuccAGGcAGAGAcAAuAdTdT 
                 1037 
               
               
                   
               
               
                 as 
                 A-32380 
                 596 
                 uAUUGUCUCUGCCUGGACUdTdT 
                 1038 
               
               
                   
               
               
                 s 
                 A-32381 
                 580 
                 uccAGGcAGAGAcAAuAAAdTdT 
                 1039 
               
               
                   
               
               
                 as 
                 A-32382 
                 598 
                 UUuAUUGUCUCUGCCUGGAdTdT 
                 1040 
               
               
                   
               
               
                 s 
                 A-32383 
                 607 
                 GuGAAAGGcAcuuuucAuudTdT 
                 1041 
               
               
                   
               
               
                 as 
                 A-32384 
                 625 
                 AAUGAAAAGUGCCUUUcACdTdT 
                 1042 
               
               
                   
               
               
                 s 
                 A-32385 
                  62 
                 uGGAcuGGuAuuuGuGucudTdT 
                 1043 
               
               
                   
               
               
                 as 
                 A-32386 
                  80 
                 AGAcAcAAAuACcAGUCcAdTdT 
                 1044 
               
               
                   
               
               
                 s 
                 A-32387 
                  77 
                 GucuGAGGcuGGcccuAcGdTdT 
                 1045 
               
               
                   
               
               
                 as 
                 A-32388 
                  95 
                 CGuAGGGCcAGCCUcAGACdTdT 
                 1046 
               
               
                   
               
               
                 s 
                 A-32391 
                  79 
                 cuGAGGcuGGcccuAcGGGdTdT 
                 1047 
               
               
                   
               
               
                 as 
                 A-32392 
                  97 
                 CCCGuAGGGCcAGCCUcAGdTdT 
                 1048 
               
               
                   
               
               
                 s 
                 A-32393 
                  81 
                 GAGGcuGGcccuAcGGGcAdTdT 
                 1049 
               
               
                   
               
               
                 as 
                 A-32394 
                  99 
                 UGCCCGuAGGGCcAGCCUCdTdT 
                 1050 
               
               
                   
               
               
                 s 
                 A-32395 
                  82 
                 AGGcuGGcccuAcGGGcAcdTdT 
                 1051 
               
               
                   
               
               
                 as 
                 A-32396 
                 100 
                 GUGCCCGuAGGGCcAGCCUdTdT 
                 1052 
               
               
                   
               
               
                 s 
                 A-32397 
                  84 
                 GcuGGcccuAcGGGcAccGdTdT 
                 1053 
               
               
                   
               
               
                 as 
                 A-32398 
                 102 
                 CGGUGCCCGuAGGGCcAGCdTdT 
                 1054 
               
               
                   
               
               
                 s 
                 A-32399 
                  85 
                 cuGGcccuAcGGGcAccGGdTdT 
                 1055 
               
               
                   
               
               
                 as 
                 A-32400 
                 103 
                 CCGGUGCCCGuAGGGCcAGdTdT 
                 1056 
               
               
                   
               
               
                 s 
                 A-32401 
                  87 
                 GGcccuAcGGGcAccGGuGdTdT 
                 1057 
               
               
                   
               
               
                 as 
                 A-32402 
                 105 
                 cACCGGUGCCCGuAGGGCCdTdT 
                 1058 
               
               
                   
               
               
                 s 
                 A-32403 
                   9 
                 ccAcucAuucuuGGcAGGAdTdT 
                 1059 
               
               
                   
               
               
                 as 
                 A-32404 
                  27 
                 UCCUGCcAAGAAUGAGUGGdTdT 
                 1060 
               
               
                   
               
               
                 s 
                 A-32405 
                  90 
                 ccuAcGGGcAccGGuGAAudTdT 
                 1061 
               
               
                   
               
               
                 as 
                 A-32406 
                 108 
                 AUUcACCGGUGCCCGuAGGdTdT 
                 1062 
               
               
                   
               
               
                 s 
                 A-32407 
                  91 
                 cuAcGGGcAccGGuGAAucdTdT 
                 1063 
               
               
                   
               
               
                 as 
                 A-32408 
                 109 
                 GAUUcACCGGUGCCCGuAGdTdT 
                 1064 
               
               
                   
               
               
                 s 
                 A-32409 
                  92 
                 uAcGGGcAccGGuGAAuccdTdT 
                 1065 
               
               
                   
               
               
                 as 
                 A-32410 
                 110 
                 GGAUUcACCGGUGCCCGuAdTdT 
                 1066 
               
               
                   
               
               
                 s 
                 A-32411 
                  93 
                 AcGGGcAccGGuGAAuccAdTdT 
                 1067 
               
               
                   
               
               
                 as 
                 A-32412 
                 111 
                 UGGAUUcACCGGUGCCCGUdTdT 
                 1068 
               
               
                   
               
               
                 s 
                 A-32415 
                  97 
                 GcAccGGuGAAuccAAGuGdTdT 
                 1069 
               
               
                   
               
               
                 as 
                 A-32416 
                 115 
                 cACUUGGAUUcACCGGUGCdTdT 
                 1070 
               
               
                   
               
               
                 s 
                 A-32417 
                  98 
                 cAccGGuGAAuccAAGuGudTdT 
                 1071 
               
               
                   
               
               
                 as 
                 A-32418 
                 116 
                 AcACUUGGAUUcACCGGUGdTdT 
                 1072 
               
               
                   
               
               
                 s 
                 A-32419 
                 167 
                 uGuGGccAuGcAuGuGuucdTdT 
                 1073 
               
               
                   
               
               
                 as 
                 A-32420 
                 185 
                 GAAcAcAUGcAUGGCcAcAdTdT 
                 1074 
               
               
                   
               
               
                 s 
                 A-32421 
                 168 
                 GuGGccAuGcAuGuGuucAdTdT 
                 1075 
               
               
                   
               
               
                 as 
                 A-32422 
                 186 
                 UGAAcAcAUGcAUGGCcACdTdT 
                 1076 
               
               
                   
               
               
                 s 
                 A-32423 
                 171 
                 GccAuGcAuGuGuucAGAAdTdT 
                 1077 
               
               
                   
               
               
                 as 
                 A-32424 
                 189 
                 UUCUGAAcAcAUGcAUGGCdTdT 
                 1078 
               
               
                   
               
               
                 s 
                 A-32427 
                 432 
                 uAuuccAccAcGGcuGucAdTdT 
                 1079 
               
               
                   
               
               
                 as 
                 A-32428 
                 449 
                 UGAcAGCCGUGGUGGAAuAdTdT 
                 1080 
               
               
                   
               
               
                 s 
                 A-32429 
                 447 
                 GucAucAccAAucccAAGGdTdT 
                 1081 
               
               
                   
               
               
                 as 
                 A-32430 
                 465 
                 CCUUGGGAUUGGUGAUGACdTdT 
                 1082 
               
               
                   
               
               
                 s 
                 A-32159 
                 115 
                 GuccucuGAuGGucAAAGudTdT 
                 1083 
               
               
                   
               
               
                 as 
                 A-32160 
                 133 
                 ACUUUGACcAUcAGAGGACdTdT 
                 1084 
               
               
                   
               
               
                 s 
                 A-32161 
                 122 
                 GAuGGucAAAGuucuAGAudTdT 
                 1085 
               
               
                   
               
               
                 as 
                 A-32162 
                 140 
                 AUCuAGAACUUUGACcAUCdTdT 
                 1086 
               
               
                   
               
               
                 s 
                 A-32173 
                 139 
                 AuGcuGuccGAGGcAGuccdTdT 
                 1087 
               
               
                   
               
               
                 as 
                 A-32174 
                 157 
                 GGACUGCCUCGGAcAGcAUdTdT 
                 1088 
               
               
                   
               
               
                 s 
                 A-32185 
                 172 
                 ccGuGcAuGuGuucAGAAAdTdT 
                 1089 
               
               
                   
               
               
                 as 
                 A-32186 
                 190 
                 UUUCUGAAcAcAUGcACGGdTdT 
                 1090 
               
               
                   
               
               
                 s 
                 A-32197 
                 238 
                 AGucuGGAGAGcuGcAuGGdTdT 
                 1091 
               
               
                   
               
               
                 as 
                 A-32198 
                 256 
                 CcAUGcAGCUCUCcAGACUdTdT 
                 1092 
               
               
                   
               
               
                 s 
                 A-32209 
                 252 
                 cAuGGGcucAcAAcuGAGGdTdT 
                 1093 
               
               
                   
               
               
                 as 
                 A-32210 
                 270 
                 CCUcAGUUGUGAGCCcAUGdTdT 
                 1094 
               
               
                   
               
               
                 s 
                 A-32245 
                  33 
                 ucucAucGucuGcuccuccdTdT 
                 1095 
               
               
                   
               
               
                 as 
                 A-32246 
                  51 
                 GGAGGAGcAGACGAUGAGAdTdT 
                 1096 
               
               
                   
               
               
                 s 
                 A-32257 
                 340 
                 ccccAuuccAuGAGcAuGcdTdT 
                 1097 
               
               
                   
               
               
                 as 
                 A-32258 
                 358 
                 GcAUGCUcAUGGAAUGGGGdTdT 
                 1098 
               
               
                   
               
               
                 s 
                 A-32293 
                 421 
                 GccccuAcuccuAuuccAcdTdT 
                 1099 
               
               
                   
               
               
                 as 
                 A-32294 
                 439 
                 GUGGAAuAGGAGuAGGGGCdTdT 
                 1100 
               
               
                   
               
               
                 s 
                 A-32305 
                 431 
                 cuAuuccAccAcGGcuGucdTdT 
                 1101 
               
               
                   
               
               
                 as 
                 A-32306 
                 449 
                 GAcAGCCGUGGUGGAAuAGdTdT 
                 1102 
               
               
                   
               
               
                 s 
                 A-32317 
                 440 
                 cAcGGcuGucGucAccAAudTdT 
                 1103 
               
               
                   
               
               
                 as 
                 A-32318 
                 458 
                 AUUGGUGACGAcAGCCGUGdTdT 
                 1104 
               
               
                   
               
               
                 s 
                 A-32329 
                 496 
                 AGGAcGAGGGAuGGGAuuudTdT 
                 1105 
               
               
                   
               
               
                 as 
                 A-32330 
                 514 
                 AAAUCCcAUCCCUCGUCCUdTdT 
                 1106 
               
               
                   
               
               
                 s 
                 A-32361 
                 556 
                 ucAccucAuAuGcuAuGuudTdT 
                 1107 
               
               
                   
               
               
                 as 
                 A-32362 
                 574 
                 AAcAuAGcAuAUGAGGUGAdTdT 
                 1108 
               
               
                   
               
               
                 s 
                 A-32365 
                 559 
                 ccucAuAuGcuAuGuuAGAdTdT 
                 1109 
               
               
                   
               
               
                 as 
                 A-32366 
                 577 
                 UCuAAcAuAGcAuAUGAGGdTdT 
                 1110 
               
               
                   
               
               
                 s 
                 A-32377 
                 570 
                 AuGuuAGAAGuccAGGcAGdTdT 
                 1111 
               
               
                   
               
               
                 as 
                 A-32378 
                 588 
                 CUGCCUGGACUUCuAAcAUdTdT 
                 1112 
               
               
                   
               
               
                 s 
                 A-32389 
                  78 
                 ucuGAGGcuGGcccuAcGGdTdT 
                 1113 
               
               
                   
               
               
                 as 
                 A-32390 
                  96 
                 CCGuAGGGCcAGCCUcAGAdTdT 
                 1114 
               
               
                   
               
               
                 s 
                 A-32401 
                  87 
                 GGcccuAcGGGcAccGGuGdTdT 
                 1115 
               
               
                   
               
               
                 as 
                 A-32402 
                 105 
                 cACCGGUGCCCGuAGGGCCdTdT 
                 1116 
               
               
                   
               
               
                 s 
                 A-32413 
                  95 
                 GGGcAccGGuGAAuccAAGdTdT 
                 1117 
               
               
                   
               
               
                 as 
                 A-32414 
                 113 
                 CUUGGAUUcACCGGUGCCCdTdT 
                 1118 
               
               
                   
               
               
                 s 
                 A-32425 
                 167 
                 ccAuGcAuGuGuucAGAAAdTdT 
                 1119 
               
               
                   
               
               
                 as 
                 A-32426 
                 185 
                 UUUCUGAAcAcAUGcAUGGdTdT 
                 1120 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Identification numbers for rat TTR dsRNAs 
               
               
                 See Table 7 for sequences. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Sense 
                 Antisense 
               
               
                   
                 Duplex # 
                 Oligo # 
                 Oligo # 
               
               
                   
                   
               
               
                   
                 AD-18529 
                 A-32745 
                 A-32746 
               
               
                   
                 AD-18530 
                 A-32747 
                 A-32748 
               
               
                   
                 AD-18531 
                 A-32749 
                 A-32750 
               
               
                   
                 AD-18532 
                 A-32751 
                 A-32752 
               
               
                   
                 AD-18533 
                 A-32753 
                 A-32754 
               
               
                   
                 AD-18534 
                 A-32755 
                 A-32756 
               
               
                   
                 AD-18535 
                 A-32757 
                 A-32758 
               
               
                   
                 AD-18536 
                 A-32759 
                 A-32760 
               
               
                   
                 AD-18537 
                 A-32761 
                 A-32762 
               
               
                   
                 AD-18538 
                 A-32763 
                 A-32764 
               
               
                   
                 AD-18539 
                 A-32159 
                 A-32160 
               
               
                   
                 AD-18540 
                 A-32765 
                 A-32766 
               
               
                   
                 AD-18541 
                 A-32767 
                 A-32768 
               
               
                   
                 AD-18542 
                 A-32769 
                 A-32770 
               
               
                   
                 AD-18543 
                 A-32771 
                 A-32772 
               
               
                   
                 AD-18544 
                 A-32773 
                 A-32774 
               
               
                   
                 AD-18545 
                 A-32775 
                 A-32776 
               
               
                   
                 AD-18546 
                 A-32777 
                 A-32778 
               
               
                   
                 AD-18547 
                 A-32779 
                 A-32780 
               
               
                   
                 AD-18548 
                 A-32781 
                 A-32782 
               
               
                   
                 AD-18549 
                 A-32783 
                 A-32784 
               
               
                   
                 AD-18550 
                 A-32785 
                 A-32786 
               
               
                   
                 AD-18551 
                 A-32787 
                 A-32788 
               
               
                   
                 AD-18552 
                 A-32791 
                 A-32792 
               
               
                   
                 AD-18553 
                 A-32793 
                 A-32794 
               
               
                   
                 AD-18554 
                 A-32795 
                 A-32796 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6A 
               
             
            
               
                   
               
               
                 Sense and antisense strand sequences 
               
               
                 for rat TTR dsRNAs 
               
               
                 Strand: s = sense; as = antisense; 
               
               
                 Position: position of 5′ base on transcript 
               
               
                 (NM_012681.1, SEQ ID NO: 1330) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 SEQ 
                 Sequence with 3′ 
                 SEQ 
               
               
                   
                   
                 Sequence 
                 ID 
                 dinucleotide overhang 
                 ID 
               
               
                 Strand 
                 Position 
                 (5′ to 3′) 
                 NO: 
                 (5′ to 3′) 
                 NO: 
               
               
                   
               
               
                 s 
                 115 
                 GUCCUCUGAUGGUCAAAGU 
                 1121 
                 GUCCUCUGAUGGUCAAAGUNN 
                 1173 
               
               
                   
               
               
                 as 
                 133 
                 ACUUUGACCAUCAGAGGAC 
                 1122 
                 ACUUUGACCAUCAGAGGACNN 
                 1174 
               
               
                   
               
               
                 s 
                 537 
                 UUCUUGCUCUAUAAACCGU 
                 1123 
                 UUCUUGCUCUAUAAACCGUNN 
                 1175 
               
               
                   
               
               
                 as 
                 555 
                 ACGGUUUAUAGAGCAAGAA 
                 1124 
                 ACGGUUUAUAGAGCAAGAANN 
                 1176 
               
               
                   
               
               
                 s 
                 543 
                 CUCUAUAAACCGUGUUAGC 
                 1125 
                 CUCUAUAAACCGUGUUAGCNN 
                 1177 
               
               
                   
               
               
                 as 
                 561 
                 GCUAACACGGUUUAUAGAG 
                 1126 
                 GCUAACACGGUUUAUAGAGNN 
                 1178 
               
               
                   
               
               
                 s 
                 392 
                 UCGCCACUACACCAUCGCA 
                 1127 
                 UCGCCACUACACCAUCGCANN 
                 1179 
               
               
                   
               
               
                 as 
                 410 
                 UGCGAUGGUGUAGUGGCGA 
                 1128 
                 UGCGAUGGUGUAGUGGCGANN 
                 1180 
               
               
                   
               
               
                 s 
                 538 
                 UCUUGCUCUAUAAACCGUG 
                 1129 
                 UCUUGCUCUAUAAACCGUGNN 
                 1181 
               
               
                   
               
               
                 as 
                 556 
                 CACGGUUUAUAGAGCAAGA 
                 1130 
                 CACGGUUUAUAGAGCAAGANN 
                 1182 
               
               
                   
               
               
                 s 
                 541 
                 UGCUCUAUAAACCGUGUUA 
                 1131 
                 UGCUCUAUAAACCGUGUUANN 
                 1183 
               
               
                   
               
               
                 as 
                 559 
                 UAACACGGUUUAUAGAGCA 
                 1132 
                 UAACACGGUUUAUAGAGCANN 
                 1184 
               
               
                   
               
               
                 s 
                 532 
                 CAGUGUUCUUGCUCUAUAA 
                 1133 
                 CAGUGUUCUUGCUCUAUAANN 
                 1185 
               
               
                   
               
               
                 as 
                 550 
                 UUAUAGAGCAAGAACACUG 
                 1134 
                 UUAUAGAGCAAGAACACUGNN 
                 1186 
               
               
                   
               
               
                 s 
                 542 
                 GCUCUAUAAACCGUGUUAG 
                 1135 
                 GCUCUAUAAACCGUGUUAGNN 
                 1187 
               
               
                   
               
               
                 as 
                 560 
                 CUAACACGGUUUAUAGAGC 
                 1136 
                 CUAACACGGUUUAUAGAGCNN 
                 1188 
               
               
                   
               
               
                 s 
                 134 
                 CCUGGAUGCUGUCCGAGGC 
                 1137 
                 CCUGGAUGCUGUCCGAGGCNN 
                 1189 
               
               
                   
               
               
                 as 
                 152 
                 GCCUCGGACAGCAUCCAGG 
                 1138 
                 GCCUCGGACAGCAUCCAGGNN 
                 1190 
               
               
                   
               
               
                 s 
                 119 
                 UCUGAUGGUCAAAGUCCUG 
                 1139 
                 UCUGAUGGUCAAAGUCCUGNN 
                 1191 
               
               
                   
               
               
                 as 
                 137 
                 CAGGACUUUGACCAUCAGA 
                 1140 
                 CAGGACUUUGACCAUCAGANN 
                 1192 
               
               
                   
               
               
                 s 
                 241 
                 CUGGAGAGCUGCACGGGCU 
                 1141 
                 CUGGAGAGCUGCACGGGCUNN 
                 1193 
               
               
                   
               
               
                 as 
                 259 
                 AGCCCGUGCAGCUCUCCAG 
                 1142 
                 AGCCCGUGCAGCUCUCCAGNN 
                 1194 
               
               
                   
               
               
                 s 
                 544 
                 UCUAUAAACCGUGUUAGCA 
                 1143 
                 UCUAUAAACCGUGUUAGCANN 
                 1195 
               
               
                   
               
               
                 as 
                 562 
                 UGCUAACACGGUUUAUAGA 
                 1144 
                 UGCUAACACGGUUUAUAGANN 
                 1196 
               
               
                   
               
               
                 s 
                 530 
                 AACAGUGUUCUUGCUCUAU 
                 1145 
                 AACAGUGUUCUUGCUCUAUNN 
                 1197 
               
               
                   
               
               
                 as 
                 548 
                 AUAGAGCAAGAACACUGUU 
                 1146 
                 AUAGAGCAAGAACACUGUUNN 
                 1198 
               
               
                   
               
               
                 s 
                 118 
                 CUCUGAUGGUCAAAGUCCU 
                 1147 
                 CUCUGAUGGUCAAAGUCCUNN 
                 1199 
               
               
                   
               
               
                 as 
                 136 
                 AGGACUUUGACCAUCAGAG 
                 1148 
                 AGGACUUUGACCAUCAGAGNN 
                 1200 
               
               
                   
               
               
                 s 
                 140 
                 UGCUGUCCGAGGCAGCCCU 
                 1149 
                 UGCUGUCCGAGGCAGCCCUNN 
                 1201 
               
               
                   
               
               
                 as 
                 158 
                 AGGGCUGCCUCGGACAGCA 
                 1150 
                 AGGGCUGCCUCGGACAGCANN 
                 1202 
               
               
                   
               
               
                 s 
                 239 
                 GUCUGGAGAGCUGCACGGG 
                 1151 
                 GUCUGGAGAGCUGCACGGGNN 
                 1203 
               
               
                   
               
               
                 as 
                 257 
                 CCCGUGCAGCUCUCCAGAC 
                 1152 
                 CCCGUGCAGCUCUCCAGACNN 
                 1204 
               
               
                   
               
               
                 s 
                 531 
                 ACAGUGUUCUUGCUCUAUA 
                 1153 
                 ACAGUGUUCUUGCUCUAUANN 
                 1205 
               
               
                   
               
               
                 as 
                 549 
                 UAUAGAGCAAGAACACUGU 
                 1154 
                 UAUAGAGCAAGAACACUGUNN 
                 1206 
               
               
                   
               
               
                 s 
                 117 
                 CCUCUGAUGGUCAAAGUCC 
                 1155 
                 CCUCUGAUGGUCAAAGUCCNN 
                 1207 
               
               
                   
               
               
                 as 
                 135 
                 GGACUUUGACCAUCAGAGG 
                 1156 
                 GGACUUUGACCAUCAGAGGNN 
                 1208 
               
               
                   
               
               
                 s 
                 131 
                 AGUCCUGGAUGCUGUCCGA 
                 1157 
                 AGUCCUGGAUGCUGUCCGANN 
                 1209 
               
               
                   
               
               
                 as 
                 149 
                 UCGGACAGCAUCCAGGACU 
                 1158 
                 UCGGACAGCAUCCAGGACUNN 
                 1210 
               
               
                   
               
               
                 s 
                 217 
                 UUGCCUCUGGGAAGACCGC 
                 1159 
                 UUGCCUCUGGGAAGACCGCNN 
                 1211 
               
               
                   
               
               
                 as 
                 235 
                 GCGGUCUUCCCAGAGGCAA 
                 1160 
                 GCGGUCUUCCCAGAGGCAANN 
                 1212 
               
               
                   
               
               
                 s 
                 242 
                 UGGAGAGCUGCACGGGCUC 
                 1161 
                 UGGAGAGCUGCACGGGCUCNN 
                 1213 
               
               
                   
               
               
                 as 
                 260 
                 GAGCCCGUGCAGCUCUCCA 
                 1162 
                 GAGCCCGUGCAGCUCUCCANN 
                 1214 
               
               
                   
               
               
                 s 
                 244 
                 GAGAGCUGCACGGGCUCAC 
                 1163 
                 GAGAGCUGCACGGGCUCACNN 
                 1215 
               
               
                   
               
               
                 as 
                 262 
                 GUGAGCCCGUGCAGCUCUC 
                 1164 
                 GUGAGCCCGUGCAGCUCUCNN 
                 1216 
               
               
                   
               
               
                 s 
                 246 
                 GAGCUGCACGGGCUCACCA 
                 1165 
                 GAGCUGCACGGGCUCACCANN 
                 1217 
               
               
                   
               
               
                 as 
                 264 
                 UGGUGAGCCCGUGCAGCUC 
                 1166 
                 UGGUGAGCCCGUGCAGCUCNN 
                 1218 
               
               
                   
               
               
                 s 
                 399 
                 UACACCAUCGCAGCCCUGC 
                 1167 
                 UACACCAUCGCAGCCCUGCNN 
                 1219 
               
               
                   
               
               
                 as 
                 417 
                 GCAGGGCUGCGAUGGUGUA 
                 1168 
                 GCAGGGCUGCGAUGGUGUANN 
                 1220 
               
               
                   
               
               
                 s 
                 132 
                 GUCCUGGAUGCUGUCCGAG 
                 1169 
                 GUCCUGGAUGCUGUCCGAGNN 
                 1221 
               
               
                   
               
               
                 as 
                 150 
                 CUCGGACAGCAUCCAGGAC 
                 1170 
                 CUCGGACAGCAUCCAGGACNN 
                 1222 
               
               
                   
               
               
                 s 
                 245 
                 AGAGCUGCACGGGCUCACC 
                 1171 
                 AGAGCUGCACGGGCUCACCNN 
                 1223 
               
               
                   
               
               
                 as 
                 263 
                 GGUGAGCCCGUGCAGCUCU 
                 1172 
                 GGUGAGCCCGUGCAGCUCUNN 
                 1224 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6B 
               
             
            
               
                   
               
               
                 Sense and antisense strand sequences 
               
               
                 for rat TTR dsRNAs 
               
               
                 Strand: s = sense; as = antisense; 
               
               
                 Position: position of 5′ base on transcript 
               
               
                 (NM_012681.1, SEQ ID NO: 1330) 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Sequence with 
                 SEQ 
               
               
                   
                   
                 3′ deoxythimidine 
                 ID 
               
               
                 Strand 
                 Position 
                 overhang (5′ to 3′) 
                 NO: 
               
               
                   
               
               
                 s 
                 115 
                 GUCCUCUGAUGGUCAAAGUdTdT 
                 1225 
               
               
                   
               
               
                 as 
                 133 
                 ACUUUGACCAUCAGAGGACdTdT 
                 1226 
               
               
                   
               
               
                 s 
                 537 
                 UUCUUGCUCUAUAAACCGUdTdT 
                 1227 
               
               
                   
               
               
                 as 
                 555 
                 ACGGUUUAUAGAGCAAGAAdTdT 
                 1228 
               
               
                   
               
               
                 s 
                 543 
                 CUCUAUAAACCGUGUUAGCdTdT 
                 1229 
               
               
                   
               
               
                 as 
                 561 
                 GCUAACACGGUUUAUAGAGdTdT 
                 1230 
               
               
                   
               
               
                 s 
                 392 
                 UCGCCACUACACCAUCGCAdTdT 
                 1231 
               
               
                   
               
               
                 as 
                 410 
                 UGCGAUGGUGUAGUGGCGAdTdT 
                 1232 
               
               
                   
               
               
                 s 
                 538 
                 UCUUGCUCUAUAAACCGUGdTdT 
                 1233 
               
               
                   
               
               
                 as 
                 556 
                 CACGGUUUAUAGAGCAAGAdTdT 
                 1234 
               
               
                   
               
               
                 s 
                 541 
                 UGCUCUAUAAACCGUGUUAdTdT 
                 1235 
               
               
                   
               
               
                 as 
                 559 
                 UAACACGGUUUAUAGAGCAdTdT 
                 1236 
               
               
                   
               
               
                 s 
                 532 
                 CAGUGUUCUUGCUCUAUAAdTdT 
                 1237 
               
               
                   
               
               
                 as 
                 550 
                 UUAUAGAGCAAGAACACUGdTdT 
                 1238 
               
               
                   
               
               
                 s 
                 542 
                 GCUCUAUAAACCGUGUUAGdTdT 
                 1239 
               
               
                   
               
               
                 as 
                 560 
                 CUAACACGGUUUAUAGAGCdTdT 
                 1240 
               
               
                   
               
               
                 s 
                 134 
                 CCUGGAUGCUGUCCGAGGCdTdT 
                 1241 
               
               
                   
               
               
                 as 
                 152 
                 GCCUCGGACAGCAUCCAGGdTdT 
                 1242 
               
               
                   
               
               
                 s 
                 119 
                 UCUGAUGGUCAAAGUCCUGdTdT 
                 1243 
               
               
                   
               
               
                 as 
                 137 
                 CAGGACUUUGACCAUCAGAdTdT 
                 1244 
               
               
                   
               
               
                 s 
                 241 
                 CUGGAGAGCUGCACGGGCUdTdT 
                 1245 
               
               
                   
               
               
                 as 
                 259 
                 AGCCCGUGCAGCUCUCCAGdTdT 
                 1246 
               
               
                   
               
               
                 s 
                 544 
                 UCUAUAAACCGUGUUAGCAdTdT 
                 1247 
               
               
                   
               
               
                 as 
                 562 
                 UGCUAACACGGUUUAUAGAdTdT 
                 1248 
               
               
                   
               
               
                 s 
                 530 
                 AACAGUGUUCUUGCUCUAUdTdT 
                 1249 
               
               
                   
               
               
                 as 
                 548 
                 AUAGAGCAAGAACACUGUUdTdT 
                 1250 
               
               
                   
               
               
                 s 
                 118 
                 CUCUGAUGGUCAAAGUCCUdTdT 
                 1251 
               
               
                   
               
               
                 as 
                 136 
                 AGGACUUUGACCAUCAGAGdTdT 
                 1252 
               
               
                   
               
               
                 s 
                 140 
                 UGCUGUCCGAGGCAGCCCUdTdT 
                 1253 
               
               
                   
               
               
                 as 
                 158 
                 AGGGCUGCCUCGGACAGCAdTdT 
                 1254 
               
               
                   
               
               
                 s 
                 239 
                 GUCUGGAGAGCUGCACGGGdTdT 
                 1255 
               
               
                   
               
               
                 as 
                 257 
                 CCCGUGCAGCUCUCCAGACdTdT 
                 1256 
               
               
                   
               
               
                 s 
                 531 
                 ACAGUGUUCUUGCUCUAUAdTdT 
                 1257 
               
               
                   
               
               
                 as 
                 549 
                 UAUAGAGCAAGAACACUGUdTdT 
                 1258 
               
               
                   
               
               
                 s 
                 117 
                 CCUCUGAUGGUCAAAGUCCdTdT 
                 1259 
               
               
                   
               
               
                 as 
                 135 
                 GGACUUUGACCAUCAGAGGdTdT 
                 1260 
               
               
                   
               
               
                 s 
                 131 
                 AGUCCUGGAUGCUGUCCGAdTdT 
                 1261 
               
               
                   
               
               
                 as 
                 149 
                 UCGGACAGCAUCCAGGACUdTdT 
                 1262 
               
               
                   
               
               
                 s 
                 217 
                 UUGCCUCUGGGAAGACCGCdTdT 
                 1263 
               
               
                   
               
               
                 as 
                 235 
                 GCGGUCUUCCCAGAGGCAAdTdT 
                 1264 
               
               
                   
               
               
                 s 
                 242 
                 UGGAGAGCUGCACGGGCUCdTdT 
                 1265 
               
               
                   
               
               
                 as 
                 260 
                 GAGCCCGUGCAGCUCUCCAdTdT 
                 1266 
               
               
                   
               
               
                 s 
                 244 
                 GAGAGCUGCACGGGCUCACdTdT 
                 1267 
               
               
                   
               
               
                 as 
                 262 
                 GUGAGCCCGUGCAGCUCUCdTdT 
                 1268 
               
               
                   
               
               
                 s 
                 246 
                 GAGCUGCACGGGCUCACCAdTdT 
                 1269 
               
               
                   
               
               
                 as 
                 264 
                 UGGUGAGCCCGUGCAGCUCdTdT 
                 1270 
               
               
                   
               
               
                 s 
                 399 
                 UACACCAUCGCAGCCCUGCdTdT 
                 1271 
               
               
                   
               
               
                 as 
                 417 
                 GCAGGGCUGCGAUGGUGUAdTdT 
                 1272 
               
               
                   
               
               
                 s 
                 132 
                 GUCCUGGAUGCUGUCCGAGdTdT 
                 1273 
               
               
                   
               
               
                 as 
                 150 
                 CUCGGACAGCAUCCAGGACdTdT 
                 1274 
               
               
                   
               
               
                 s 
                 245 
                 AGAGCUGCACGGGCUCACCdTdT 
                 1275 
               
               
                   
               
               
                 as 
                 263 
                 GGUGAGCCCGUGCAGCUCUdTdT 
                 1276 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Chemically modified sense and antisense 
               
               
                 strand sequences for rat TTR dsRNAs 
               
               
                 See Table 5 for duplex # (dsRNA name). 
               
               
                 Strand: s = sense; as = antisense; 
               
               
                 Position: position of 5′ base on transcript 
               
               
                 (NM_012681.1, SEQ ID NO: 1330) 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 SEQ 
               
               
                   
                 Oligo 
                 Po- 
                   
                 ID 
               
               
                 Strand 
                 # 
                 sition 
                 Sequence (5′ to 3′) 
                 NO: 
               
               
                   
               
               
                 s 
                 A-32159 
                 115 
                 GuccucuGAuGGucAAAGudTdT 
                 1277 
               
               
                   
               
               
                 as 
                 A-32160 
                 133 
                 ACUUUGACcAUcAGAGGACdTdT 
                 1278 
               
               
                   
               
               
                 s 
                 A-32745 
                 537 
                 uucuuGcucuAuAAAccGudTdT 
                 1279 
               
               
                   
               
               
                 as 
                 A-32746 
                 555 
                 ACGGUUuAuAGAGcAAGAAdTdT 
                 1280 
               
               
                   
               
               
                 s 
                 A-32747 
                 543 
                 cucuAuAAAccGuGuuAGcdTdT 
                 1281 
               
               
                   
               
               
                 as 
                 A-32748 
                 561 
                 GCuAAcACGGUUuAuAGAGdTdT 
                 1282 
               
               
                   
               
               
                 s 
                 A-32749 
                 392 
                 ucGccAcuAcAccAucGcAdTdT 
                 1283 
               
               
                   
               
               
                 as 
                 A-32750 
                 410 
                 UGCGAUGGUGuAGUGGCGAdTdT 
                 1284 
               
               
                   
               
               
                 s 
                 A-32751 
                 538 
                 ucuuGcucuAuAAAccGuGdTdT 
                 1285 
               
               
                   
               
               
                 as 
                 A-32752 
                 556 
                 cACGGUUuAuAGAGcAAGAdTdT 
                 1286 
               
               
                   
               
               
                 s 
                 A-32753 
                 541 
                 uGcucuAuAAAccGuGuuAdTdT 
                 1287 
               
               
                   
               
               
                 as 
                 A-32754 
                 559 
                 uAAcACGGUUuAuAGAGcAdTdT 
                 1288 
               
               
                   
               
               
                 s 
                 A-32755 
                 532 
                 cAGuGuucuuGcucuAuAAdTdT 
                 1289 
               
               
                   
               
               
                 as 
                 A-32756 
                 550 
                 UuAuAGAGcAAGAAcACUGdTdT 
                 1290 
               
               
                   
               
               
                 s 
                 A-32757 
                 542 
                 GcucuAuAAAccGuGuuAGdTdT 
                 1291 
               
               
                   
               
               
                 as 
                 A-32758 
                 560 
                 CuAAcACGGUUuAuAGAGCdTdT 
                 1292 
               
               
                   
               
               
                 s 
                 A-32759 
                 134 
                 ccuGGAuGcuGuccGAGGcdTdT 
                 1293 
               
               
                   
               
               
                 as 
                 A-32760 
                 152 
                 GCCUCGGAcAGcAUCcAGGdTdT 
                 1294 
               
               
                   
               
               
                 s 
                 A-32761 
                 119 
                 ucuGAuGGucAAAGuccuGdTdT 
                 1295 
               
               
                   
               
               
                 as 
                 A-32762 
                 137 
                 cAGGACUUUGACcAUcAGAdTdT 
                 1296 
               
               
                   
               
               
                 s 
                 A-32763 
                 241 
                 cuGGAGAGcuGcAcGGGcudTdT 
                 1297 
               
               
                   
               
               
                 as 
                 A-32764 
                 259 
                 AGCCCGUGcAGCUCUCcAGdTdT 
                 1298 
               
               
                   
               
               
                 s 
                 A-32765 
                 544 
                 ucuAuAAAccGuGuuAGcAdTdT 
                 1299 
               
               
                   
               
               
                 as 
                 A-32766 
                 562 
                 UGCuAAcACGGUUuAuAGAdTdT 
                 1300 
               
               
                   
               
               
                 s 
                 A-32767 
                 530 
                 AAcAGuGuucuuGcucuAudTdT 
                 1301 
               
               
                   
               
               
                 as 
                 A-32768 
                 548 
                 AuAGAGcAAGAAcACUGUUdTdT 
                 1302 
               
               
                   
               
               
                 s 
                 A-32769 
                 118 
                 cucuGAuGGucAAAGuccudTdT 
                 1303 
               
               
                   
               
               
                 as 
                 A-32770 
                 136 
                 AGGACUUUGACcAUcAGAGdTdT 
                 1304 
               
               
                   
               
               
                 s 
                 A-32771 
                 140 
                 uGcuGuccGAGGcAGcccudTdT 
                 1305 
               
               
                   
               
               
                 as 
                 A-32772 
                 158 
                 AGGGCUGCCUCGGAcAGcAdTdT 
                 1306 
               
               
                   
               
               
                 s 
                 A-32773 
                 239 
                 GucuGGAGAGcuGcAcGGGdTdT 
                 1307 
               
               
                   
               
               
                 as 
                 A-32774 
                 257 
                 CCCGUGcAGCUCUCcAGACdTdT 
                 1308 
               
               
                   
               
               
                 s 
                 A-32775 
                 531 
                 AcAGuGuucuuGcucuAuAdTdT 
                 1309 
               
               
                   
               
               
                 as 
                 A-32776 
                 549 
                 uAuAGAGcAAGAAcACUGUdTdT 
                 1310 
               
               
                   
               
               
                 s 
                 A-32777 
                 117 
                 ccucuGAuGGucAAAGuccdTdT 
                 1311 
               
               
                   
               
               
                 as 
                 A-32778 
                 135 
                 GGACUUUGACcAUcAGAGGdTdT 
                 1312 
               
               
                   
               
               
                 s 
                 A-32779 
                 131 
                 AGuccuGGAuGcuGuccGAdTdT 
                 1313 
               
               
                   
               
               
                 as 
                 A-32780 
                 149 
                 UCGGAcAGcAUCcAGGACUdTdT 
                 1314 
               
               
                   
               
               
                 s 
                 A-32781 
                 217 
                 uuGccucuGGGAAGAccGcdTdT 
                 1315 
               
               
                   
               
               
                 as 
                 A-32782 
                 235 
                 GCGGUCUUCCcAGAGGcAAdTdT 
                 1316 
               
               
                   
               
               
                 s 
                 A-32783 
                 242 
                 uGGAGAGcuGcAcGGGcucdTdT 
                 1317 
               
               
                   
               
               
                 as 
                 A-32784 
                 260 
                 GAGCCCGUGcAGCUCUCcAdTdT 
                 1318 
               
               
                   
               
               
                 s 
                 A-32785 
                 244 
                 GAGAGcuGcAcGGGcucAcdTdT 
                 1319 
               
               
                   
               
               
                 as 
                 A-32786 
                 262 
                 GUGAGCCCGUGcAGCUCUCdTdT 
                 1320 
               
               
                   
               
               
                 s 
                 A-32787 
                 246 
                 GAGcuGcAcGGGcucAccAdTdT 
                 1321 
               
               
                   
               
               
                 as 
                 A-32788 
                 264 
                 UGGUGAGCCCGUGcAGCUCdTdT 
                 1322 
               
               
                   
               
               
                 s 
                 A-32791 
                 399 
                 uAcAccAucGcAGcccuGcdTdT 
                 1323 
               
               
                   
               
               
                 as 
                 A-32792 
                 417 
                 GcAGGGCUGCGAUGGUGuAdTdT 
                 1324 
               
               
                   
               
               
                 s 
                 A-32793 
                 132 
                 GuccuGGAuGcuGuccGAGdTdT 
                 1325 
               
               
                   
               
               
                 as 
                 A-32794 
                 150 
                 CUCGGAcAGcAUCcAGGACdTdT 
                 1326 
               
               
                   
               
               
                 s 
                 A-32795 
                 245 
                 AGAGcuGcAcGGGcucAccdTdT 
                 1327 
               
               
                   
               
               
                 as 
                 A-32796 
                 263 
                 GGUGAGCCCGUGcAGCUCUdTdT 
                 1328 
               
               
                   
               
            
           
         
       
     
     Synthesis of TTR Sequences 
     TTR sequences were synthesized on MerMade 192 synthesizer at 1 μmol scale. For all the sequences in the list, ‘endolight’ chemistry was applied as detailed below.
         All pyrimidines (cytosine and uridine) in the sense strand were replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)   In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides   A two base dTdT extension at 3′ end of both sense and antisense sequences was introduced   The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software       

     The synthesis of TTR sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. The synthesis of the above sequences was performed at lum scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator. 
     The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and triethylamine.3HF in the second step. The crude sequences thus obtained were precipitated using acetone: ethanol mix and the pellet were re-suspended in 0.5M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS and the resulting mass data confirmed the identity of the sequences. A selected set of samples were also analyzed by IEX chromatography. 
     The next step in the process was purification. All sequences were purified on an AKTA explorer purification system using Source 15Q column. A single peak corresponding to the full length sequence was collected in the eluent and was subsequently analyzed for purity by ion exchange chromatography. 
     The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted TTR sequences were analyzed for concentration and purity. The single strands were then annealed to form TTR-dsRNA. 
     Example 2B 
     In Vitro Screening of TTR siRNAs for mRNA Suppression 
     Human TTR targeting dsRNAs (Table 2) were assayed for inhibition of endogenous TTR expression in HepG2 and Hep3B cells, using qPCR (real time PCR) and bDNA (branched DNA) assays to quantify TTR mRNA. Rodent TTR targeting dsRNA (Table 5) were synthesized and assayed for inhibition of endogenous TTR expression using bDNA assays in H.4.II.E cells. Results from single dose assays were used to select a subset of TTR dsRNA duplexes for dose response experiments to calculate IC50&#39;s. IC50 results were used to select TTR dsRNAs for further testing. 
     Cell Culture and Transfections: 
     The hepatocyte cell lines HepG2, Hep3B and H.4.II.E cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO 2  in Dulbecco&#39;s modified Eagle&#39;s medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. H.4.II.E cells were also grown in Earle&#39;s minimal essential medium. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat # 13778-150) and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotics containing 4×10 4  (HepG2), 2×10 4  (Hep3B) or 2×10 4  (H.4.II.E) cells were then added. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM final duplex concentration and dose response experiments were done with 10, 1, 0.5, 0.1,0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001 nM. 
     Total RNA Isolation Using MagMAX-96 Total RNA Isolation Kit (Applied Biosystems, Foster City Calif., Part #: AM1830): 
     Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 μl of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA it was eluted with 50 μl of water. 
     cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813): 
     A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold. 
     Real Time PCR: 
     2 μl of cDNA was added to a master mix of 1 μl 18S TaqMan Probe (Applied Biosystems Cat #4319413E), 1 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914 M1) and 10 μl TaqMan Universal PCR Master Mix (Applied Biosystems Cat #4324018) per well in a MicroAmp Optical 96 well plate (Applied Biosystems cat #4326659). Real time PCR was done in an ABI 7000 Prism or an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔ Ct(RQ) assay. All reactions were done in triplicate. 
     Real time data were analyzed using the ΔΔ Ct method and normalized to assays performed from cells transfected with 10 nM BlockIT fluorescent Oligo (Invitrogen Cat #2013) or 10 nM AD-1955 (a control duplex that targets the non-mammalian luciferase gene) to calculate fold change. 
     Branched DNA Assays—QuantiGene 1.0 (Panomics, Fremont, Calif. cat #: QG0004)—Used to Screen Rodent Specific Duplexes 
     H.4.II.E cells (ATCC) were transfected with 10 nM siRNA. After removing media, H.4.II.E were lysed in 100 μl of Diluted Lysis Mixture (a mixture of 1 volume of Lysis mixture, 2 volume of nuclease-free water and 10 ul of Proteinase-K per ml for the final concentration of 20 mg/ml) then incubated at 65° C. for 35 minutes. Then, 80 μl of Working Probe Set (a mixture of TTR or GAPDH probe) and 20 ul of cell-lysate were added into the Capture Plate. Capture Plates were incubated at 53° C.±1° C. overnight (approximately 16-20 hrs). Capture Plates were washed 3 times with 1× Wash Buffer (a mixture of nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 1000 rpm. 100 μl of Amplifier Working Reagent was added into the Capture Plate, which was then sealed and incubated for 1 hour at 46° C.±1° C. Wash and dry steps were repeated after 1 hour of incubation and 100 μl of Label Solution Reagent was added. The plate was then washed, dried and 100 μl Substrate (a mixture of Lithium Lauryl Sulfate and Substrate solution) was added. Capture Plates were placed in the incubator for 30 minutes at 46° C.±1° C. Capture Plates were then removed from the incubator and incubated at room temperature for 30 minutes. Finally, the Capture Plates were read using the Victor Luminometer (Perkin Elmer, Waltham, Mass.). 
     Branched DNA Assays—QuantiGene 2.0 (Panomics Cat #: QS0011): Used to Screen All Other Duplexes 
     After a 24 hour incubation at the dose or doses stated, media was removed and cells were lysed in 100 μl Lysis Mixture (1 volume lysis mixture, 2 volumes nuclease-free water and 10 μl of Proteinase-K/ml for a final concentration of 20 mg/ml) then incubated at 65° C. for 35 minutes. 20 μl Working Probe Set (TTR probe for gene target and GAPDH for endogenous control) and 80 μl of cell-lysate were then added to the Capture Plates. Capture Plates were incubated at 55° C.±1° C. (approx. 16-20 hrs). The next day, the Capture Plates were washed 3 times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 240 g. 100 μl of pre-Amplifier Working Reagent was added to the Capture Plates, which were sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C. Following a 1 hour incubation, the wash step was repeated, then 100 μl Amplifier Working Reagent was added. After 1 hour, the wash and dry steps were repeated, and 100 μl Label Probe was added. Capture plates were incubated 50° C.±1° C. for 1 hour. The plates were then washed with 1× Wash Buffer and dried, and then 100 μl Substrate was added to the Capture Plates. Capture Plates were read using the SpectraMax Luminometer (Molecular Devices, Sunnyvale, Calif.) following 5 to 15 minutes incubation. 
     bDNA Data Analysis: 
     bDNA data were analyzed by (i) subtracting the average background from each triplicate sample, (ii) averaging the resultant triplicate GAPDH (control probe) and TTR (experimental probe) values, and then (iii) taking the ratio: (experimental probe-background)/(control probe-background). 
     Results 
     A summary of the single dose and IC50 results for TTR-dsRNAs (TTR siRNAs) are presented below in Table 8. Single dose results are expressed as % TTR mRNA relative to control, assayed in HepG2 cells. IC50s were determined in HepG2 and/or Hep3B cells, as indicated. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Single dose and IC50 results of in vitro screens of TTR siRNAs 
               
            
           
           
               
               
               
            
               
                   
                 Single Dose at 10 nM 
                   
               
               
                   
                 % relative to control 
                 IC50 (nM) 
               
            
           
           
               
               
               
               
            
               
                   
                 HepG2 
                 HepG2 
                 Hep3B 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Duplex # 
                 qPCR 
                 bDNA 
                 qPCR 
                 bDNA 
                 qPCR 
                 bDNA 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 AD-18243 
                 50.35 
                 141.53 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18244 
                 64.26 
                 158.55 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18245 
                 56.89 
                 107.22 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18246 
                 10.53 
                 32.51* 
                 0.265 
                 0.086 
                 ND 
                 ND 
               
               
                 AD-18247 
                 125.56 
                 69.57 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18248 
                 127.78 
                 66.97 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18249 
                 48.77 
                 48.76 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18250 
                 96.94 
                 86.42 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18251 
                 170.41 
                 129.15 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18252 
                 73.52 
                 81.90 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18253 
                 25.25 
                 61.25 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18254 
                 95.13 
                 103.96 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18255 
                 119.46 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18256 
                 42.64 
                 95.67 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18257 
                 146.25 
                 141.75 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18258 
                 10.20 
                 13.41* 
                 0.007 
                 0.005 
                 0.004 
                 0.005 
               
               
                 AD-18259 
                 9.30 
                 20.91* 
                 0.102 
                 0.005 
                 ND 
                 ND 
               
               
                 AD-18260 
                 125.37 
                 81.36 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18261 
                 14.27 
                 19.40* 
                 0.210 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18262 
                 84.95 
                 104.05 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18263 
                 16.32 
                 23.25* 
                 0.110 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18264 
                 104.18 
                 83.69 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18265 
                 41.62 
                 64.87 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18266 
                 39.98 
                 110.53 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18267 
                 149.64 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18268 
                 152.93 
                 174.04 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18269 
                 37.27 
                 92.28 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18270 
                 99.44 
                 164.75 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18271 
                 18.89 
                 28.33* 
                 0.503 
                 0.004 
                 ND 
                 ND 
               
               
                 AD-18272 
                 128.32 
                 132.58 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18273 
                 115.78 
                 201.95 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18274 
                 8.97 
                 20.04* 
                 0.009 
                 0.176 
                 0.036 
                 0.012 
               
               
                 AD-18275 
                 4.09 
                 22.25* 
                 0.026 
                 0.118 
                 ND 
                 ND 
               
               
                 AD-18276 
                 19.73 
                 45.22* 
                 0.198 
                 0.677 
                 ND 
                 ND 
               
               
                 AD-18277 
                 10.55 
                 26.31* 
                 0.121 
                 0.426 
                 ND 
                 ND 
               
               
                 AD-18278 
                 108.86 
                 116.26 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18279 
                 66.59 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18280 
                 103.26 
                 170.52 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18281 
                 87.98 
                 123.88 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18282 
                 82.47 
                 140.32 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18283 
                 106.54 
                 182.78 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18284 
                 106.93 
                 151.78 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18285 
                 26.58 
                 60.05* 
                 ND 
                 0.089 
                 ND 
                 ND 
               
               
                 AD-18286 
                 109.95 
                 173.66 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18287 
                 54.23 
                 155.45 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18288 
                 73.52 
                 174.09 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18289 
                 103.36 
                 174.76 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18290 
                 17.06 
                 52.04* 
                 1.253 
                 0.181 
                 ND 
                 ND 
               
               
                 AD-18291 
                 7.71 
                 169.29* 
                 1.304 
                 0.019 
                 ND 
                 ND 
               
               
                 AD-18292 
                 7.51 
                 210.03* 
                 0.604 
                 0.005 
                 ND 
                 ND 
               
               
                 AD-18293 
                 3.61 
                 62.53* 
                 0.078 
                 0.003 
                 ND 
                 ND 
               
               
                 AD-18294 
                 111.53 
                 107.56 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18295 
                 115.88 
                 105.37 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18296 
                 57.03 
                 38.03 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18297 
                 87.69 
                 73.87 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18298 
                 10.39 
                 7.25* 
                 0.455 
                 0.008 
                 ND 
                 ND 
               
               
                 AD-18299 
                 18.79 
                 18.06* 
                 0.895 
                 0.014 
                 ND 
                 ND 
               
               
                 AD-18300 
                 108.70 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18301 
                 114.22 
                 70.50 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18302 
                 116.19 
                 122.40 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18303 
                 124.89 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18304 
                 132.99 
                 89.54 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18305 
                 153.10 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18306 
                 159.22 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18307 
                 116.83 
                 84.57 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18308 
                 156.72 
                 87.80 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18309 
                 113.22 
                 101.97 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18310 
                 132.33 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18311 
                 161.68 
                 92.92 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18312 
                 103.01 
                 71.17 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18313 
                 120.65 
                 53.26 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18314 
                 116.33 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18315 
                 115.13 
                 ND 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18316 
                 118.73 
                 122.34 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18317 
                 114.03 
                 121.10 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18318 
                 80.85 
                 122.57 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18319 
                 119.14 
                 148.87 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18320 
                 22.86 
                 55.43* 
                 ND 
                 0.023 
                 0.403 
                 ND 
               
               
                 AD-18321 
                 6.44 
                 31.56* 
                 0.001 
                 0.033 
                 ND 
                 ND 
               
               
                 AD-18322 
                 54.21 
                 100.46 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18323 
                 6.37 
                 28.71* 
                 0.005 
                 0.023 
                 ND 
                 ND 
               
               
                 AD-18324 
                 2.53 
                 15.98* 
                 0.002 
                 0.006 
                 0.005 
                 0.014 
               
               
                 AD-18325 
                 2.52 
                 11.96* 
                 0.001 
                 0.016 
                 ND 
                 ND 
               
               
                 AD-18326 
                 18.34 
                 43.16* 
                 0.025 
                 0.186 
                 ND 
                 ND 
               
               
                 AD-18327 
                 18.28 
                 13.90* 
                 0.044 
                 0.215 
                 ND 
                 ND 
               
               
                 AD-18328 
                 4.53 
                 26.04* 
                 0.003 
                 0.004 
                 0.006 
                 0.006 
               
               
                 AD-18329 
                 96.93 
                 131.54 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18330 
                 11.80 
                 45.18* 
                  0.0004 
                 0.010 
                 0.020 
                 ND 
               
               
                 AD-18331 
                 117.77 
                 163.07 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18332 
                 11.53 
                 35.09* 
                 0.001 
                 0.076 
                 0.065 
                 ND 
               
               
                 AD-18333 
                 12.24 
                 46.94* 
                 0.001 
                 0.115 
                 0.075 
                 ND 
               
               
                 AD-18334 
                 16.27 
                 55.28* 
                  0.0004 
                 0.181 
                 1.071 
                 ND 
               
               
                 AD-18335 
                 53.52 
                 112.80 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18336 
                 6.39 
                 33.00* 
                 0.001 
                 0.112 
                 0.081 
                 ND 
               
               
                 AD-18337 
                 51.77 
                 105.33 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18338 
                 48.21 
                 102.86 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18339 
                 6.48 
                 26.56* 
                 0.004 
                 0.002 
                 0.018 
                 0.029 
               
               
                 AD-18340 
                 4.53 
                 30.76* 
                 0.002 
                 0.002 
                 ND 
                 ND 
               
               
                 AD-18341 
                 31.27 
                 100.41 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18342 
                 7.60 
                 42.89* 
                 ND 
                 0.016 
                 0.076 
                 ND 
               
               
                 AD-18343 
                 3.42 
                 17.45* 
                 ND 
                 0.001 
                 ND 
                 ND 
               
               
                 AD-18344 
                 75.08 
                 134.31 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18345 
                 13.62 
                 42.75* 
                 0.002 
                 0.013 
                 ND 
                 ND 
               
               
                 AD-18346 
                 59.25 
                 121.10 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18347 
                 91.23 
                 139.54 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18348 
                 89.95 
                 159.29 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18349 
                 108.01 
                 144.96 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18350 
                 123.65 
                 125.87 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18351 
                 108.36 
                 104.02 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18352 
                 87.82 
                 128.72 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18353 
                 14.40 
                 65.77 
                 0.012 
                 0.027 
                 ND 
                 ND 
               
               
                 AD-18354 
                 99.27 
                 123.53 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18355 
                 135.04 
                 150.88 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18356 
                 100.76 
                 178.96 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18357 
                 125.30 
                 162.85 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18358 
                 103.15 
                 136.01 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18359 
                 34.74 
                 140.48 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18360 
                 103.86 
                 146.86 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18361 
                 105.74 
                 152.74 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18362 
                 106.96 
                 188.22 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18363 
                 124.22 
                 58.46 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18364 
                 113.75 
                 66.87 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18446 
                 29.73 
                 13.30 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18447 
                 109.74 
                 53.63 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18448 
                 22.96 
                 8.81 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18449 
                 112.59 
                 50.11 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18450 
                 89.41 
                 34.89 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18451 
                 74.35 
                 23.88 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18452 
                 125.25 
                 54.86 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18453 
                 126.98 
                 56.31 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18454 
                 113.88 
                 52.48 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18455 
                 163.00 
                 48.89 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18456 
                 15.70 
                 10.52 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18457 
                 12.86 
                 8.22 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18458 
                 13.00 
                 7.00 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18459 
                 14.41 
                 10.72 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18460 
                 121.16 
                 74.87 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18461 
                 100.53 
                 71.87 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18462 
                 47.75 
                 29.35 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                 AD-18463 
                 58.98 
                 44.79 
                 ND 
                 ND 
                 ND 
                 ND 
               
               
                   
               
               
                 ND: no data; 
               
               
                 *indicates result that represents average of two experiments. 
               
            
           
         
       
     
     The dose response data used to identify the IC50 for 5 TTR-dsRNAs (AD-18258, AD-18274, AD-18324, AD-18328, and AD-18339), are presented in detail below in Table 9. All 5 siRNAs were determined to have pM IC50s. The IC50 data for dsRNAs in Table 8 is a summary of the data presented in Table 9 below. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Dose response data for 5 TTR-dsRNAs 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 % inhibition relative to control AD-1955 
                   
               
               
                   
                 Detection 
                 Dose of duplex (nM) 
                 IC50 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Cell type 
                 method 
                 10 
                 1 
                 0.5 
                 0.1 
                 0.05 
                 0.01 
                 0.005 
                 0.001 
                 0.0005 
                 0.0001 
                 0.00005 
                 0.00001 
                 (nM) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Duplex AD-18258 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 HepG2 
                 qPCR 
                 14.4 
                 14.1 
                 16.2 
                 23.9 
                 27.26 
                 40.19 
                 68.46 
                 78.1 
                 74.48 
                 104.37 
                 98.28 
                 113.68 
                 0.007 
               
               
                 HepG2 
                 bDNA 
                 14.3 
                 14.5 
                 11.1 
                 12.8 
                 18.82 
                 19.77 
                 51.21 
                 56.03 
                 63.63 
                 58.35 
                 43.64 
                 51.05 
                 0.005 
               
               
                 Hep3B 
                 qPCR 
                 11.9 
                 8.62 
                 12.4 
                 16.4 
                 28.35 
                 30.49 
                 58.36 
                 54.57 
                 81.26 
                 89.43 
                 81.85 
                 101.87 
                 0.004 
               
               
                 Hep3B 
                 bDNA 
                 7.65 
                 7.5 
                 11.3 
                 12.6 
                 28.85 
                 27.89 
                 64.57 
                 73.48 
                 72.03 
                 91.44 
                 86.71 
                 89.31 
                 0.005 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Duplex AD-18274 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 HepG2 
                 qPCR 
                 6.68 
                 8.45 
                 11.7 
                 24.2 
                 42.08 
                 49.89 
                 56.95 
                 62.99 
                 64.47 
                 54.92 
                 67.39 
                 72.67 
                 0.009 
               
               
                 HepG2 
                 bDNA 
                 27.5 
                 69 
                 25.2 
                 34.2 
                 73.03 
                 103.4 
                 121.57 
                 97.31 
                 154.93 
                 156.7 
                 Nd 
                 152.25 
                 0.176 
               
               
                 Hep3B 
                 qPCR 
                 7.58 
                 17 
                 15.6 
                 43.9 
                 42.22 
                 60.55 
                 78.8 
                 77.81 
                 79.97 
                 85.84 
                 86.13 
                 83.99 
                 0.036 
               
               
                 Hep3B 
                 bDNA 
                 3.77 
                 4.92 
                 7.51 
                 15 
                 35.21 
                 51.66 
                 72.45 
                 70.12 
                 78.31 
                 77.52 
                 90.72 
                 83.01 
                 0.012 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Duplex AD-18324 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 HepG2 
                 qPCR 
                 2.07 
                 2.27 
                 2.74 
                 6.36 
                 8.18 
                 15.23 
                 28.82 
                 52.79 
                 90.86 
                 94.72 
                 116.07 
                 98.97 
                 0.002 
               
               
                 HepG2 
                 bDNA 
                 14.5 
                 7.88 
                 11.8 
                 15.9 
                 17.2 
                 46.44 
                 40.4 
                 91.86 
                 0 
                 95.57 
                 0 
                 52.15 
                 0.006 
               
               
                 Hep3B 
                 qPCR 
                 2.07 
                 3.48 
                 5.76 
                 16.2 
                 18.73 
                 44.54 
                 49.77 
                 68.88 
                 63.48 
                 76.61 
                 74.7 
                 77.83 
                 0.005 
               
               
                 Hep3B 
                 bDNA 
                 3.48 
                 3.8 
                 5.15 
                 15.2 
                 30.84 
                 55.36 
                 74.75 
                 99.39 
                 88.89 
                 110.83 
                 96.55 
                 110.26 
                 0.014 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Duplex AD-18328 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 HepG2 
                 qPCR 
                 5.85 
                 3.97 
                 3.32 
                 5.62 
                 8 
                 16.75 
                 55.01 
                 39.76 
                 122.41 
                 102.37 
                 114.02 
                 124.09 
                 0.003 
               
               
                 HepG2 
                 bDNA 
                 12.3 
                 10.7 
                 10.7 
                 11.9 
                 20.06 
                 25 
                 69.52 
                 57.29 
                 112.28 
                 98.14 
                 142.26 
                 148.92 
                 0.004 
               
               
                 Hep3B 
                 qPCR 
                 3.17 
                 5.52 
                 11.7 
                 13.8 
                 27.68 
                 39.58 
                 61.21 
                 61.87 
                 90.51 
                 87.56 
                 106.03 
                 108.72 
                 0.006 
               
               
                 Hep3B 
                 bDNA 
                 3.08 
                 3.66 
                 4.19 
                 7.25 
                 21.05 
                 22.1 
                 73.74 
                 63.19 
                 105.55 
                 96.27 
                 105.97 
                 96.46 
                 0.006 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Duplex AD-18339 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 HepG2 
                 qPCR 
                 6.27 
                 7.28 
                 Nd 
                 11 
                 15.25 
                 38.69 
                 38.78 
                 71.7 
                 84.09 
                 62.2 
                 75.61 
                 85.46 
                 0.004 
               
               
                 HepG2 
                 bDNA 
                 15.1 
                 8.14 
                 5.13 
                 6.89 
                 12.17 
                 32.14 
                 42.98 
                 64.01 
                 60.76 
                 79.95 
                 81.97 
                 95.43 
                 0.002 
               
               
                 Hep3B 
                 qPCR 
                 8.3 
                 9.47 
                 13.2 
                 34.5 
                 44.54 
                 77.38 
                 81.04 
                 81.41 
                 93.95 
                 81.04 
                 75.61 
                 78.28 
                 0.018 
               
               
                 Hep3B 
                 bDNA 
                 10.5 
                 9.43 
                 11.7 
                 27.1 
                 44.88 
                 72.32 
                 79.88 
                 79.6 
                 87.46 
                 96.53 
                 95.13 
                 89.88 
                 0.029 
               
               
                   
               
            
           
         
       
     
     A summary of the single dose results for rodent specific TTR-dsRNAs (TTR siRNAs) are presented below in Table 10. Single dose results are expressed as % TTR mRNA relative to control, assayed in rat H.4.II.E cells, after transfection of rodent specific TTR siRNAs at 10 nM. These results show that some rodent specific TTR siRNAs are effective in suppressing endogenous rat TTR mRNA in vitro. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Single dose results of in vitro screen of 
               
               
                 rodent specific TTR-dsRNAs (TTR siRNAs) 
               
            
           
           
               
               
               
            
               
                   
                   
                 % Relative to 
               
               
                   
                 Duplex # 
                 control at 10 nM 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 AD-18529 
                 19.83 
               
               
                   
                 AD-18530 
                 44.49 
               
               
                   
                 AD-18531 
                 6.01 
               
               
                   
                 AD-18532 
                 24.06 
               
               
                   
                 AD-18533 
                 37.78 
               
               
                   
                 AD-18534 
                 8.19 
               
               
                   
                 AD-18535 
                 10.18 
               
               
                   
                 AD-18536 
                 16.13 
               
               
                   
                 AD-18537 
                 15.88 
               
               
                   
                 AD-18538 
                 19.93 
               
               
                   
                 AD-18539 
                 49.24 
               
               
                   
                 AD-18540 
                 2.99 
               
               
                   
                 AD-18541 
                 1.32 
               
               
                   
                 AD-18542 
                 6.3 
               
               
                   
                 AD-18543 
                 16.46 
               
               
                   
                 AD-18544 
                 17.55 
               
               
                   
                 AD-18545 
                 3.53 
               
               
                   
                 AD-18546 
                 2.75 
               
               
                   
                 AD-18547 
                 7.01 
               
               
                   
                 AD-18548 
                 5.02 
               
               
                   
                 AD-18549 
                 1.61 
               
               
                   
                 AD-18550 
                 9.58 
               
               
                   
                 AD-18551 
                 7.74 
               
               
                   
                 AD-18552 
                 3.74 
               
               
                   
                 AD-18553 
                 50.39 
               
               
                   
                 AD-18554 
                 111.06 
               
               
                   
                   
               
            
           
         
       
     
     Example 3 
     In Vitro Assay of TTR siRNAs for Induction of TNF-α and IFN-α Secretion 
     To evaluate potential for immunostimulation, TTR siRNAs were assayed in vitro for induction of TNF-α and IFN-α secretion. 
     Human PBMC were isolated from freshly collected buffy coats obtained from healthy donors (Research Blood Components, Inc., Boston, Mass.) by a standard Ficoll-Hypaque density centrifugation. Freshly isolated cells (1×10 5 /well/100 μl) were seeded in 96-well plates and cultured in RPMI 1640 GlutaMax medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen). 
     siRNAs were transfected into PBMC using DOTAP transfection reagent (Roche Applied Science). The DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/DOTAP complexes were incubated as specified by the manufacturer&#39;s instructions and subsequently added to PBMC (50 μl/well) which were then cultured for 24 hours. Positive and negative control siRNAs were included in all assays. AD-5048 was used as a positive control siRNA. AD-5048 corresponds to a sequence that targets human Apolipoprotein B (Soutschek et al., 2004) and elicits secretion of both IFN-α and TNF-α in this assay. AD-1955, which does not elicit IFN-α and TNF-α secretion in this assay, was used as a negative control siRNA. All siRNAs were used at a final concentration of 133 nM. The ratio of RNA to transfection reagent was 16.5 pmoles per μg of DOTAP. 
     Cytokines were detected and quantified in culture supernatants with a commercially available ELISA kit for IFN-α (BMS216INST) and TNF-α (BMS223INST), both from Bender MedSystems (Vienna, Austria). TTR siRNA cytokine induction is expressed as percent IFN-α or TNF-α produced relative to the positive control siRNA AD-5048. 
     IFN-α and TNF-α stimulation results for a number of TTR siRNAs are presented in  FIG. 1  (mean of quadruplicate wells±SD) and below in Table 11 (percentage compared with AD-5048). None of the TTR siRNAs evaluated induced significant TNF-α or IFN-α secretion by cultured human PBMCs. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 IFN-α and TNF-α stimulation results for TTR siRNAs 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 IFN-α 
                 TNF-α 
               
               
                   
                 Duplex # 
                 (% of AD-5048) 
                 (% of AD-5048) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 AD-18246 
                 0 
                 4 
               
               
                   
                 AD-18258 
                 0 
                 0 
               
               
                   
                 AD-18259 
                 0 
                 0 
               
               
                   
                 AD-18261 
                 0 
                 0 
               
               
                   
                 AD-18263 
                 0 
                 0 
               
               
                   
                 AD-18271 
                 0 
                 0 
               
               
                   
                 AD-18274 
                 2 
                 1 
               
               
                   
                 AD-18275 
                 0 
                 0 
               
               
                   
                 AD-18276 
                 0 
                 0 
               
               
                   
                 AD-18277 
                 0 
                 0 
               
               
                   
                 AD-18285 
                 0 
                 0 
               
               
                   
                 AD-18290 
                 0 
                 0 
               
               
                   
                 AD-18291 
                 0 
                 0 
               
               
                   
                 AD-18292 
                 0 
                 0 
               
               
                   
                 AD-18293 
                 0 
                 0 
               
               
                   
                 AD-18298 
                 0 
                 0 
               
               
                   
                 AD-18299 
                 0 
                 0 
               
               
                   
                 AD-18320 
                 0 
                 0 
               
               
                   
                 AD-18321 
                 0 
                 0 
               
               
                   
                 AD-18323 
                 0 
                 0 
               
               
                   
                 AD-18324 
                 0 
                 0 
               
               
                   
                 AD-18325 
                 0 
                 0 
               
               
                   
                 AD-18326 
                 0 
                 0 
               
               
                   
                 AD-18327 
                 0 
                 0 
               
               
                   
                 AD-18328 
                 0 
                 0 
               
               
                   
                 AD-18330 
                 0 
                 0 
               
               
                   
                 AD-18332 
                 1 
                 0 
               
               
                   
                 AD-18333 
                 0 
                 1 
               
               
                   
                 AD-18334 
                 0 
                 1 
               
               
                   
                 AD-18336 
                 1 
                 0 
               
               
                   
                 AD-18339 
                 0 
                 0 
               
               
                   
                 AD-18340 
                 0 
                 0 
               
               
                   
                 AD-18342 
                 0 
                 0 
               
               
                   
                 AD-18343 
                 0 
                 0 
               
               
                   
                 AD-18345 
                 0 
                 0 
               
               
                   
                 AD-18353 
                 0 
                 0 
               
               
                   
                 AD-18448 
                 0 
                 0 
               
               
                   
                 AD-18456 
                 0 
                 0 
               
               
                   
                 AD-18457 
                 0 
                 0 
               
               
                   
                 AD-18458 
                 0 
                 0 
               
               
                   
                 AD-18459 
                 0 
                 0 
               
               
                   
                   
               
            
           
         
       
     
     The five lead TTR targeting dsRNAs (TTR siRNAs) were selected based on IC50s in the pM range in the human hepatocyte cell lines HepG2 and Hep3B, and the absence of immunostimulatory activity. Duplexes without any mismatches are more likely to achieve significant knockdown of the target transcript than duplexes with mismatches between the oligo and the mRNA. To better enable interpretation of cross-species toxicology data and to have the broadest applicability to human patients, duplexes that have 100% identity in orthologous genes from rat, cynomolgus monkey and human, and that do not target regions with known polymorphisms are generally preferred. The five lead compounds were selected based on IC50 in hepatocyte cell lines in the pM range, the absence of immunostimulatory activity, specificity to the human TTR transcripts, and absence of known polymorphisms (mutations) in the region of the mRNA targeted by the duplex. In the case of TTR, no 19 base oligos were found with complete identity in human, rat and cynomolgus monkey. A summary of these data are presented in Table 12, which also includes information on known TTR mutations in the region targeted by the duplex and cross-species reactivity. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Summary of data for five most potent TTR dsRNAs. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 IC50 (qPCR): 
                 IC50 (bDNA): 
                   
                 Mutations 
                 Cross-species 
               
               
                 Duplex # 
                 nM HepG2 
                 nM HepG2 
                 IFNa/TNFa 
                 not covered 
                 reactivity 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 AD-18258 
                 0.007 
                 0.005 
                 Negative 
                 None 
                 Cyno: 1 mismatch @ 
               
               
                   
                   
                   
                   
                 (non-coding 
                 position 14 A to G 
               
               
                   
                   
                   
                   
                 region) 
                 Rat: no homology at 
               
               
                   
                   
                   
                   
                   
                 any position 
               
               
                 AD-18274 
                 0.009 
                 0.176 
                 Negative 
                 Lys70Asn; 
                 Cyno: no mismatch 
               
               
                   
                   
                   
                   
                 Val71Ala; 
                 Rat: no homology at 
               
               
                   
                   
                   
                   
                 Ile73Val; 
                 any position 
               
               
                   
                   
                   
                   
                 Asp74His 
               
               
                 AD-18324 
                 0.002 
                 0.006 
                 Negative 
                 None 
                 Cyno: no mismatch 
               
               
                   
                   
                   
                   
                 (non-coding 
                 Rat: no homology at 
               
               
                   
                   
                   
                   
                 region) 
                 any position 
               
               
                 AD-18328 
                 0.003 
                 0.004 
                 Negative 
                 None 
                 Cyno: no mismatch 
               
               
                   
                   
                   
                   
                 (non-coding 
                 Rat: 7 mismatches 
               
               
                   
                   
                   
                   
                 region) 
               
               
                 AD-18339 
                 0.004 
                 0.002 
                 Negative 
                 None 
                 None 
               
               
                   
                   
                   
                   
                 (non-coding 
               
               
                   
                   
                   
                   
                 region) 
               
               
                   
               
            
           
         
       
     
     Example 4 
     In Vivo Reduction of Liver TTR mRNA and Plasma TTR Protein by LNP01-18324, LNP01-18328 and LNP01-18246 in Transgenic Mice 
     Two TTR siRNAs, AD-18324 and AD-18328, were chosen for in vivo evaluation. These duplexes exhibited potent dose-dependent silencing in vitro in hepatocyte cell lines (e.g. HepG2).  FIG. 2A  and  FIG. 2B  show the dose responses in HepG2 cells after transfection with AD-18324 ( FIG. 2A ) or AD-18328 ( FIG. 2B ) where the doses are expressed in nM on the x-axis and the responses are expressed as fraction TTR mRNA remaining relative to control, on the y-axis. In HepG2 cells, the IC50s of AD-18324 and AD-18328 were determined to be 2 pM and 3 pM, respectively. The TTR target sites for both lead dsRNA candidates are in the 3′ untranslated region of the TTR mRNA, in a region where there are no reported mutations in the literature. 
     The sequences of each strand of the two lead candidates are reproduced below from the Tables. Strand: s=sense; as=antisense; Position: position of 5′ base on transcript NM_000371.2. 
     
       
         
           
               
               
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                   
                   
                 SEQ 
               
               
                 Duplex 
                   
                 Oligo 
                 Po- 
                 Sequence 
                 ID 
               
               
                 # 
                 Strand 
                 # 
                 sition* 
                 5′ to 3′ 
                 NO: 
               
               
                   
               
             
            
               
                 AD-18324 
                 s 
                 A-32337 
                 509 
                 GGAuuucAuGuA 
                 1001 
               
               
                   
                   
                   
                   
                 AccAAGAdTdT 
                   
               
               
                   
               
               
                 AD-18324 
                 as 
                 A-32338 
                 527 
                 UCUUGGUuAcAU 
                 1002 
               
               
                   
                   
                   
                   
                 GAAAUCCdTdT 
                   
               
               
                   
               
               
                 AD-18328 
                 s 
                 A-32345 
                 518 
                 GuAAccAAGAGu 
                 1009 
               
               
                   
                   
                   
                   
                 AuuccAudTdT 
                   
               
               
                   
               
               
                 AD-18328 
                 as 
                 A-32346 
                 536 
                 AUGGAAuACUCU 
                 1010 
               
               
                   
                   
                   
                   
                 UGGUuACdTdT 
               
               
                   
               
            
           
         
       
     
     In addition, a rodent cross-reactive TTR dsRNA, AD-18246, was chosen for further evaluation in vivo. AD-18246 targets a sequence beginning at position 88 of the open reading frame, where there are three mutations reported in the literature. A dose response curve for AD-18246 in HepG2 cells is shown in  FIG. 3 . AD-18246 is substantially less potent than AD-18324 and AD-18328; the IC50 of AD-18246 was determined to be 265 pM. 
     AD-18324, AD-18328, and AD-18246 were administered to transgenic mice after formulation in LNP01. 3-5 month old H129-mTTR-KO/iNOS-KO/hTTR transgenic mice (mouse transthyretin knock-out/ inducible nitric oxide synthase knock-out/human transthyretin transgenic) were intravenously (IV) administered 200 μl of LNP01-formulated transthyretin-specific siRNA (AD-18324, AD-18328, or AD-18246), LNP01-formulated control siRNA targeting the non-mammalian luciferase gene (AD-1955) or PBS via the tail vein at concentrations of 1.0 mg/kg, 3.0 mg/kg, or 6.0 mg/kg for siRNAs AD-18324 and AD-18328, 3.0 mg/kg for siRNA AD-18246, and 6.0 mg/kg for siRNA AD-1955. LNP01 is a lipidoid formulation comprised of ND98, Cholesterol, and PEG-Ceramide C16. 
     After approximately forty-hours, mice were anesthetized with 200 μl of ketamine, and then exsanguinated by severing the right caudal artery. Whole blood was isolated and plasma was isolated and stored at −80° C. until assaying. Liver tissue was collected, flash-frozen and stored at −80° C. until processing. 
     Efficacy of treatment was evaluated by (i) measurement of TTR mRNA in liver at 48 hours post-dose, and (ii) measurement of TTR protein in plasma at prebleed and at 48 hours post-dose. TTR liver mRNA levels were assayed utilizing the Branched DNA assays-QuantiGene 2.0 (Panomics cat #: QS0011). Briefly, mouse liver samples were ground and tissue lysates were prepared. Liver lysis mixture (a mixture of 1 volume of lysis mixture, 2 volume of nuclease-free water and 10 ul of Proteinase-K/ml for a final concentration of 20 mg/ml) was incubated at 65° C. for 35 minutes. 20 μl of Working Probe Set (TTR probe for gene target and GAPDH for endogenous control) and 80 μl of tissue-lysate were then added into the Capture Plate. Capture Plates were incubated at 55° C.±1° C. (aprx. 16-20 hrs). The next day, the Capture Plate were washed 3 times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 240 g. 100 μl of pre-Amplifier Working Reagent was added into the Capture Plate, which was sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C. Following 1 hour incubation, the wash step was repeated, then 100 μl of Amplifier Working Reagent was added. After 1 hour, the wash and dry steps were repeated, and 100 μl of Label Probe was added. Capture plates were incubated 50° C. ±1° C. for 1 hour. The plate was then washed with 1× Wash Buffer, dried and 100 μl Substrate was added into the Capture Plate. Capture Plates were read using the SpectraMax Luminometer following a 5 to 15 minute incubation. bDNA data were analyzed by subtracting the average background from each triplicate sample, averaging the resultant triplicate GAPDH (control probe) and TTR (experimental probe) values, and then computing the ratio: (experimental probe-background)/(control probe-background). 
     TTR plasma levels were assayed utilizing the commercially available kit “AssayMax Human Prealbumin ELISA Kit” (AssayPro, St. Charles, Mo., Catalog # EP3010-1) according to manufacturer&#39;s guidelines. Briefly, mouse plasma was diluted 1:10,000 in 1× mix diluents and added to pre-coated plates along with kit standards, and incubated for 2 hours at room temperature followed by 5× washes with kit wash buffer. Fifty microliters of biotinylated prealbumin antibody was added to each well and incubated for 1 hr at room temperature, followed by 5× washes with wash buffer. Fifty microliters of streptavidin-peroxidase conjugate was added to each well and plates were incubated for 30 minutes at room temperature followed by washing as previously described. The reaction was developed by the addition of 50 μl/well of chromogen substrate and incubation for 10 minutes at room temperature with stopping of reaction by the addition of 50 μl/well of stop solution. Absorbance at 450 nm was read on a Versamax microplate reader (Molecular Devices, Sunnyvale, Calif.) and data were analyzed utilizing the Softmax 4.6 software package (Molecular Devices). 
     LNP01-18324 and LNP01-18328 were found to reduce liver TTR mRNA ( FIG. 4A ) and plasma TTR protein ( FIG. 4B ) levels in a dose-dependent manner with IV bolus administration. The mRNA ED50 of LNP01-18328 was determined to be ˜1 mg/kg whereas the ED50 of LNP01-18324 was determined to be ˜2 mg/kg. The effects of LNP01-18324 and LNP01-18328 were specific, because the control, LNP01-1955 at 6 mg/kg, did not significantly affect liver TTR mRNA levels, as compared with the PBS group. LNP01-18324 and LNP01-18328 reduced plasma TTR protein levels relative to the PBS group, with potencies that were similar to those on TTR mRNA levels. At 3 mg/kg, LNP01-18246 reduced liver TTR mRNA levels to a lessor extent than 3 mg/kg LNP01-18324 or LNP01-18328. 
     These results demonstrate that LNP01-18324 and LNP01-18328, administered by IV bolus, substantially reduce human TTR mRNA expressed by the transgenic mouse liver, which results in reduction of human TTR protein in the circulation. 
     Example 5 
     In Vivo Reduction of Wild-Type TTR mRNA in the Non-Human Primate Liver by SNALP-18324 and SNALP-18328 
     To evaluate the efficacy of TTR siRNAs AD-18324 and AD-18328 in non-human primates on liver TTR mRNA levels, the siRNAs were formulated in SNALP and administered by 15-minute IV infusion. Cynomolgus monkeys ( Macaca fascicularis ) (2 to 5 kg, 3 animals per group) were administered 15-minute IV infusions of SNALP-18324 (0.3, 1.0 or 3.0 mg/kg), SNALP-18328 (0.3, 1 or 3 mg/kg), or SNALP-1955 (3 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase). At forty-eight hours post-dosing, monkeys were anesthetized with sodium pentobarbital and exsanguinated. Liver tissue for TTR mRNA determination was collected, flash-frozen, and stored at −80° C. until processing. 
     TTR mRNA levels in the liver were assayed utilizing a custom designed Branched DNA assay, utilizing the QuantiGenel.0 technology. Briefly, monkey liver samples were ground and tissue lysates were prepared. Liver lysis mixture (1 volume lysis mixture, 2 volume nuclease-free water, and 10 μl of Proteinase-K/ml for a final concentration of 20 mg/ml) was incubated at 65° C. for 35 minutes. 20 μl Working Probe Set (TTR probe for gene target and GAPDH for endogenous control) and 80 μl tissue-lysate were then added into the Capture Plate. Capture Plates were incubated at 55° C.±1° C. (approx. 16-20 hrs). The next day, the Capture Plates were washed three times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 240 g. 100 μl of pre-Amplifier Working Reagent was added into the Capture Plate, which was sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C. Following a 1-hour incubation, the wash step was repeated, and then 100 μl Amplifier Working Reagent was added. After 1 hour, the wash and dry steps were repeated, and 100 μl Label Probe was added. Capture plates were incubated 50° C.±1° C. for 1 hour. The plates were then washed with 1× Wash Buffer and dried, and then 100 μl Substrate was added into the Capture Plate. Capture Plates were read using the SpectraMax Luminometer following a 5 to 15 minute incubation. bDNA data were analyzed by (i) subtracting the average background from each triplicate sample, (ii) averaging the resultant GAPDH (control probe) and TTR (experimental probe) values, and then (iii) taking the ratio: (experimental probe-background)/(control probe-background). 
     The results are shown in  FIG. 5 . SNALP-18324 and SNALP-18328 reduced TTR mRNA levels in the liver in a dose-dependent manner, compared to the negative control SNALP-1955. The mRNA ED5Os of SNALP-18328 and SNALP-18324 were determined to be ˜0.3 and ˜1 mg/kg, respectively. 
     These results demonstrate that SNALP-18324 and SNALP-18328 are effective in suppressing wild-type TTR mRNA in non-human primate liver when administered by IV infusion. 
     Example 6 
     In Vivo Reduction of Mutant (V30M) TTR mRNA and Protein by SNALP-18328 in the Transgenic Mouse 
     To evaluate the efficacy of TTR siRNA AD-18328 on mutant (V30M) TTR mRNA in the liver and mutant (V30M) TTR protein in the serum, AD-18328 was formulated in SNALP and administered by IV bolus to V30M hTTR transgenic mice. 8 to 12-week old V30M hTTR transgenic mice (5 animals/ group) were intravenously (IV) administered 200 !al SNALP-18328 (0.03, 0.3 or 3 mg/kg), SNALP-1955 (3 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase), or PBS. Mice used were the Mus musculus strain H129-hTTR KO from Institute of Molecular and Cellular Biology, Porto, Portugal. Briefly, hTTR H129 transgenic mice were crossed with a H129 endogenous TTR KO mice (null mice to generate the H129-hTTR transgenic mice, in a null mouse TTR background (Maeda, S., (2003), Use of genetically altered mice to study the role of serum amyloid P component in amyloid deposition. Amyloid Suppl. 1, 17-20.). 
     At 48 hrs post-injection, animals in all five treatment groups were given a lethal dose of ketamine/xylazine. Serum samples were collected and stored at −80° C. until analysis. Liver tissue was collected, flash-frozen and stored at −80° C. until processing. 
     For TTR mRNA quantitation, frozen liver tissue was ground into powder, and lysates were prepared. TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer&#39;s instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group means of the normalized values were then further normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression. 
     For TTR protein quantitation, serum was assayed using the AssayPro (St. Charles, Mo.) Assaymax PreAlbumin ELISA Kit according to the manufacturer&#39;s protocol. 
     The results are shown in  FIG. 6A  and  FIG. 6B  for liver mRNA and serum protein, respectively. SNALP-18328 treated V30M hTTR transgenic mice had a dose-dependent and significant decrease in liver TTR mRNA levels relative to the PBS control group, reaching a maximum reduction of 97% (p&lt;0.001) at 3 mg/kg SNALP-18328, and a 50% reduction (ED50) at ˜0.15 mg/kg SNALP-18328. Serum TTR protein was also suppressed in a dose-dependent manner, with a maximum reduction of serum TTR protein of 99% (p&lt;0.01) (relative to pre-dose levels) at 3 mg/kg SNALP-18328, consistent with the reduction in TTR mRNA levels. SNALP-1955 at 3 mg/kg did not have a statistically significant effect on either TTR mRNA or protein levels, compared to PBS. 
     These results demonstrate that SNALP-18328, when administered IV, is active in suppressing mutant V30M TTR mRNA in the transgenic mouse liver, which results in reduction of mutant V30M TTR protein in the circulation. 
     Example 7 
     Durability of TTR mRNA and Protein Suppression by SNALP-18328 in the Transgenic Mouse 
     To evaluate the durability of TTR mRNA and protein suppression by SNALP-18328, AD-18328 was formulated in SNALP and administered by IV bolus to V30M hTTR transgenic mice. At various timepoints post-dose, liver TTR mRNA levels and serum TTR protein levels were quantified. 8- to 12-week old V30M hTTR transgenic mice (4 animals/group) were intravenously (IV) administered 200 μl SNALP-18328 (1 mg/kg) or SNALP-1955 (1 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase). Mice used were Mus musculus strain H129-hTTR KO from Institute of Molecular and Cellular Biology, Porto, Portugal. Briefly, hTTR H129 transgenic mice were crossed with a H129 endogenous TTR KO mice (null mice to generate the H129-hTTR transgenic mice, in a null mouse TTR background (Maeda, S., (2003), Use of genetically altered mice to study the role of serum amyloid P component in amyloid deposition. Amyloid Suppl. 1, 17-20). Days 3, 8, 15, or 22 post-dose, animals in both treatment groups were given a lethal dose of ketamine/xylazine. Serum samples were collected and stored at −80° C. until analysis. Liver tissue was collected, flash-frozen and stored at −80° C. until processing. 
     For TTR mRNA quantitation, frozen liver tissue was ground into powder, and lysates were prepared. TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer&#39;s instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group means of the normalized values were then further normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression. 
     For TTR protein quantitation, serum was assayed using the AssayPro (St. Charles, Mo.) Assaymax PreAlbumin ELISA Kit according to the manufacturer&#39;s protocol. 
     The results are shown in  FIG. 7A  and  FIG. 7B  for liver mRNA and serum protein, respectively. A single IV bolus administration of SNALP-18328 in the hTTR V30M transgenic mouse resulted in durable inhibition of TTR mRNA levels in the liver and TTR protein levels in the serum. Compared to the control group (1 mg/ml SNALP-1955), a single IV administration of SNALP-18328 at 1 mg/kg significantly reduced relative TTR mRNA levels on Days 3, 8, 15 and 22 post-dose by 96% (p&lt;0.001), 90% (p&lt;0.001), 82% (p&lt;0.001) and 73% (p&lt;0.001), respectively, and did not return to baseline levels at termination of the study (Day 22 post-dose). Protein levels also decreased with a maximum reduction of serum TTR of 97% (p&lt;0.001) (relative to SNALP-1955) at Day 3 post-dose. At Days 8, 15, and 22 post-dose, TTR protein levels were suppressed by 72% (p&lt;0.05), 32% (p&lt;0.05), and 40% (p&lt;0.001), respectively, relative to SNALP-1955. 
     These results demonstrate that a single IV administration of SNALP-18328 produces durable suppression of target liver mRNA and serum protein levels in the V30M hTTR transgenic mouse, with significant reductions of both liver TTR mRNA and serum TTR protein at 22 days post-dose. 
     Example 8 
     Durability of Serum TTR Protein Suppression by SNALP-18328 in the Non-Human Primate 
     To evaluate the durability of serum TTR protein suppression by SNALP-18328, AD-18328 was formulated in SNALP and administered by IV infusion to non-human primates. At various timepoints post-dose, serum TTR protein levels were quantified. 
     Cynomolgus monkeys ( Macaca fascicularis ) (n=5 animals/group for SNALP-18328 groups and n=3 animals/group for SNALP-1955 and PBS groups) were administered a 15-minute IV infusion of SNALP-18328 (0.3, 1 or 3 mg/kg), SNALP-1955 (3 mg/kg) with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase), or PBS. At Days 0, 1, 2, 3, 4, 5, 7, 10, and 14 of the dosing phase, serum samples were collected and stored at −80° C. until analysis. 
     Western blot analysis was used to evaluate TTR protein levels in serum samples. Serum samples from each group were pooled and diluted 1:1 with Laemmli sample buffer (β-mercaptoethanol was added at a 1:20 dilution). The samples were heated at 95° C. for 10 minutes. 12.5 μl of each sample was loaded in each lane of a 10-20% Criterion (Biorad, Hercules, Calif.) prep gel and separated by SDS-PAGE at 120V for 1.5 hrs, then transferred to a nitrocellulose membrane using a semi-dry system at 15V for 1 hour. The membrane was blocked overnight at 4° C. in LiCOR (Lincoln, Nebr.) blocking buffer diluted 1:1 with 1× PBS. The blot was probed first with primary antibodies (goat anti-TTR from Santa Cruz (Santa Cruz, Calif.) at a dilution of 1:1000 diluted in LiCOR blocking buffer/PBS on a rocker for 1 hr at room temperature. Blots were washed 4× with PBS+0.2% Tween 20 (10 minutes per wash). The fluorescent labeled secondary antibodies (anti-goat 680 nm from Invitrogen (Carlsbad, Calif.) were added at a dilution of 1:10,000 in LiCOR blocking buffer/PBS and the blot was incubated for 1 hour at room temperature. After incubation, blots were washed 4× with PBS+0.2% Tween 20 followed by one wash with 1× PBS. The Li-COR&#39;s Odyssey Infrared Imaging System was used to detect the protein bands. TTR monomer migrates at 15 kDa. 
     The results are shown in  FIG. 8 . Serum TTR protein levels showed a dose-dependent reduction with 1 or 3 mg/kg SNALP-18328, as compared to pre-dose (Day 0) levels. The duration of suppression, following a single IV administration of SNALP-18328 is at least 14 days after 1 or 3 mg/kg SNALP-18328 treatment. 
     These results demonstrate that a single IV administration of SNALP-18328 produces durable suppression of TTR protein in the circulation in the non-human primate ( Macaca fascicularis ), with significant reduction of TTR protein at 14 days post-dose. 
     Example 9 
     In Vivo Reduction of Mutant (V30M) TTR in Peripheral Tissues by SNALP-18328 in the Transgenic Mouse 
     To evaluate the efficacy of SNALP-18328 in reducing TTR in peripheral tissues, hTTR V30M/HSF-1 knock-out mice were evaluated with immunohistochemical staining for TTR. Two-month old hTTR V30M/HSF-1 knock-out mice (Maeda, S., (2003), Use of genetically altered mice to study the role of serum amyloid P component in amyloid deposition.  Amyloid  Suppl. 1, 17-20) were administered an IV bolus of 3 mg/kg SNALP-18328 (12 animals), 3 mg/kg SNALP-1955 (with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase, 4 animals), or PBS (4 animals) once every two weeks for a total of four doses on days 0, 14, 28, and 42. TTR liver mRNA levels and TTR-immunoreactivity in multiple peripheral tissues were evaluated at 8 weeks post-first dose on day 56. 
     Mice were anesthetised with 1 mg/kg medetomidine, and given a lethal dose of ketamine. Tissues and organs of interest were collected. For immunohistochemistry, esophagus (E), stomach (S), intestine (duodenum (I1) and colon (I4)), nerve (N) and dorsal root ganglia (D) were fixed in neutral buffered formalin and embedded in paraffin. For TTR detection, rabbit anti-human TTR primary antibody (1:1000, DAKO, Denmark), and anti-rabbit biotin-conjugated secondary antibody (1:20 Sigma, USA) were followed by extravidin labelling (1:20, Sigma, USA) in order to stain for the TTR protein. The reaction was developed with 3-amino-9-ethyl carbaxole, AEC (Sigma, USA). Semi-quantitative analysis of immunohistochemical slides was performed using Scion image quant program that measures the area occupied by the substrate reaction color and normalizes this value to the total image area. Mean values of % occupied area are displayed with the corresponding standard deviation. Each animal tissue was evaluated in four different areas. The presence of human TTR in parasympathetic ganglia of the stomach and intestine was studied by double immunofluorescent staining with rabbit anti-human TTR (1:1000, DAKO, Denmark) and mouse anti-PGP9.5 (1:40, Serotec, USA) as the primary antibodies; secondary antibodies were, respectively: anti-rabbit Alexa Fluor 488 (Molecular probes, UK)and goat anti-mouse Alexa Fluor 568 (Molecular probes, UK). Slides were mounted with vectashield (Vector) and visualized in a Zeiss Cell Observer System microscope (Carl Zeiss, Germany) equipped with filters for FITC and rhodamine. 
     The results are graphed in  FIG. 9 . In contrast with PBS and SNALP-1955 treated animals, SNALP-18328 treated animals had a significant reduction of TTR-immunoreactivity in all tissues examined (esophagus (E), stomach (S), intestine (duodenum (I1) and colon (I4)), nerve (N) and dorsal root ganglia (D). 
     These results demonstrate that SNALP-18328 administration to hTTR V30M/HSF-1 knock-out mice causes a significant reduction of TTR protein in peripheral tissues and organs, including esophagus, stomach, intestine (duodenum and colon), nerve, and dorsal root ganglion. 
     Example 10 
     In Vivo Reduction of Wild-Type TTR mRNA in the Non-Human Primate Liver by XTC-SNALP-18328 
     To evaluate the efficacy of the novel lipid nanoparticle formulation XTC-SNALP for delivery of siRNA in non-human primate, TTR siRNA AD-18328 was formulated in XTC-SNALP (XTC-SNALP-18328) and administered by 15-minute IV infusion, and liver TTR mRNA was quantified. Cynomolgus monkeys ( Macaca fascicularis ) were administered 15-minute IV infusions of XTC-SNALP-18328 (0.03, 0.1, 0.3 or 1 mg/kg) or XTC-SNALP-1955 (1 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase). At forty-eight hours post-dosing, monkeys were anesthetized with sodium pentobarbital and exsanguinated. Liver tissue for TTR mRNA determination was collected, flash-frozen, and stored at −80° C. until processing. Methods used for TTR mRNA quantitation in liver tissue were similar to those described in Example 5 above. 
     The results are shown in  FIG. 10 . XTC-SNALP -18328 reduced TTR mRNA levels in the liver in a dose-dependent manner, compared to the negative control XTC-SNALP -1955. The mRNA ED50 was determined to be ˜0.1 mg/kg XTC-SNALP -18328. 
     These results demonstrate that XTC-SNALP-18328 is effective in suppressing wild-type TTR mRNA in non-human primate liver when administered by IV infusion. 
     Example 11 
     In Vivo Reduction of Wild-Type TTR mRNA in the Non-Human Primate Liver by LNP09-18328 and LNP11-18328 
     To evaluate the efficacy of two novel lipid nanoparticle formulations, LNP09 and LNP11, for delivery of siRNA in non-human primate, TTR siRNA AD-18328 was formulated in LNP09 (LNP09-18328) or LNP11 (LNP11-18328), and administered by 15-minute IV infusion, and liver TTR mRNA and serum TTR protein levels were assayed. Cynomolgus monkeys ( Macaca fascicularis ) were administered 15-minute IV infusions of LNP09-18328 (0.03, 0.1, or 0.3 mg/kg), LNP11-18328 (0.03, 0.1, or 0.3 mg/kg), or PBS. Liver biopsy samples were collected at 48 hrs post-dosing, flash-frozen, and stored at -80° C. until processing. Serum was collected before dosing (pre-bleed), and on Days 1, 2, 4, 7, 14, 21 and 28 post-dosing and stored at −80° C. until processing. Methods used for TTR mRNA quantitation in liver tissue and serum TTR protein evaluation were similar to those described in Examples 5 and 8 above. 
     The results are shown in  FIG. 11A  for mRNA, and in  FIG. 11B  and  FIG. 11C  for protein. LNP09-18328 and LNP11-18328 treated animals showed a dose-dependent decrease in TTR mRNA levels in the liver, reaching a maximum reduction at 0.3 mg/kg of ˜85% (LNP09-18328) and ˜90% (LNP11-18328) mRNA relative to the PBS control. The mRNA ED50 was determined to be ˜0.02 mg/kg for both LNP09-18328 and LNP11-18328. At Day 7 post-dosing, serum samples also exhibited a dose-dependent reduction of TTR protein for 0.1 and 0.3 mg/kg LNP09-18328 and LNP11-18328, compared to PBS control levels.  FIG. 11C  shows a decrease in TTR protein levels with a 0.3 mg/kg dose of LNP09-18328 that persisted over at least 28 days post-dosing, as compared to the PBS control group and as compared with the pre-bleed samples. 
     These results demonstrate that LNP09-18328 and LNP11-18328 are effective in suppressing wild-type TTR mRNA in non-human primate liver and wild-type TTR protein in the circulation, when administered by IV infusion. Furthermore, the suppression with LN09-18328 is durable, persisting for at least 28 days following the IV infusion. 
     Example 12 
     Synthesis of TTR Tiled Sequences 
     A set of TTR duplexes (“tiled duplexes”)were designed that targeted the TTR gene near the target region of AD-18328, which targets the human TTR gene starting at nucleotide 628 of NM_000371.3. 
     In the examples below, the numbering representing the position of the 5′ base of an siRNA on the transcript is based on NM_000371.3 ( FIG. 12 ; SEQ ID NO:1331). In the examples shown above, the numbering for siRNA targeting human siRNA was based on NM_000371.2 ( FIG. 13A ). NM_000371.3 extends the sequence of the 5′ UTR by 110 bases compared to NM_000371.2, as shown in  FIG. 14 . Thus, as an example, the starting position of AD-18328 is 628 on NM_000371.3 and 518 on NM_000371.2 ( FIG. 14 ). 
     TTR tiled sequences were synthesized on MerMade 192 synthesizer at lumol scale. For all the sequences in the list, ‘endolight’ chemistry was applied as detailed below.
         All pyrimidines (cytosine and uridine) in the sense strand contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)   In the antisense strand, pyrimidines adjacent to(towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides   A two base dTdT extension at 3′ end of both sense and anti sense sequences was introduced   The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software       

     Synthesis, Cleavage and Deprotection: 
     The synthesis of TTR sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. The synthesis of the sequences was performed at lum scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator. The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. The crude sequences were precipitated using acetone: ethanol (80:20) mix and the pellet were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity. 
     Purification and Desalting: 
     TTR tiled sequences were purified on AKTA explorer purification system using Source 15Q column. A column temperature of 65 C was maintained during purification. Sample injection and collection was performed in 96 well (1.8 mL -deep well) plates. A single peak corresponding to the full length sequence was collected in the eluent. The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted TTR sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The single strands were then submitted for annealing. 
     TTR Single Strands and Duplexes: 
     A detailed list of TTR tiled duplexes and corresponding single strands (sense and antisense) are shown in the table below (Table 13). 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 TTR tiled duplexes and corresponding single strands 
               
               
                 Strand: s = sense; as = antisense; 
               
               
                 Position: position of 5′ base on transcript 
               
               
                 (NM_000371.3, SEQ ID NO: 1331). 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 SEQ 
               
               
                 Duplex # 
                 Position 
                 Oligo # 
                 Strand 
                 Sequence (5′ to 3″) 
                 ID NO: 
               
               
                   
               
               
                 AD-18323 
                 618 
                 A-32335 
                 S 
                 GGGAuuucAuGuAAccAAGdTdT 
                 1332 
               
               
                   
                   
                 A-32336 
                 AS 
                 CUUGGUuAcAUGAAAUCCCdTdT 
                 1333 
               
               
                   
               
               
                 AD-18324 
                 619 
                 A-32337 
                 S 
                 GGAuuucAuGuAAccAAGAdTdT 
                 1334 
               
               
                   
                   
                 A-32338 
                 AS 
                 UCUUGGUuAcAUGAAAUCCdTdT 
                 1335 
               
               
                   
               
               
                 AD-23000 
                 620 
                 A-42927 
                 S 
                 GAuuucAuGuAAccAAGAGdTdT 
                 1336 
               
               
                   
                   
                 A-42928 
                 AS 
                 CUCUUGGUuAcAUGAAAUCdTdT 
                 1337 
               
               
                   
               
               
                 AD-23001 
                 621 
                 A-42929 
                 S 
                 AuuucAuGuAAccAAGAGudTdT 
                 1338 
               
               
                   
                   
                 A-42930 
                 AS 
                 ACUCUUGGUuAcAUGAAAUdTdT 
                 1339 
               
               
                   
               
               
                 AD-23002 
                 622 
                 A-42931 
                 S 
                 uuucAuGuAAccAAGAGuAdTdT 
                 1340 
               
               
                   
                   
                 A-42932 
                 AS 
                 uACUCUUGGUuAcAUGAAAdTdT 
                 1341 
               
               
                   
               
               
                 AD-23003 
                 623 
                 A-42933 
                 S 
                 uucAuGuAAccAAGAGuAudTdT 
                 1342 
               
               
                   
                   
                 A-42934 
                 AS 
                 AuACUCUUGGUuAcAUGAAdTdT 
                 1343 
               
               
                   
               
               
                 AD-18325 
                 624 
                 A-32339 
                 S 
                 ucAuGuAAccAAGAGuAuudTdT 
                 1344 
               
               
                   
                   
                 A-32340 
                 AS 
                 AAuACUCUUGGUuAcAUGAdTdT 
                 1345 
               
               
                   
               
               
                 AD-23004 
                 625 
                 A-42935 
                 S 
                 cAuGuAAccAAGAGuAuucdTdT 
                 1346 
               
               
                   
                   
                 A-42936 
                 AS 
                 GAAuACUCUUGGUuAcAUGdTdT 
                 1347 
               
               
                   
               
               
                 AD-18326 
                 626 
                 A-32341 
                 S 
                 AuGuAAccAAGAGuAuuccdTdT 
                 1348 
               
               
                   
                   
                 A-32342 
                 AS 
                 GGAAuACUCUUGGUuAcAUdTdT 
                 1349 
               
               
                   
               
               
                 AD-18327 
                 627 
                 A-32343 
                 S 
                 uGuAAccAAGAGuAuuccAdTdT 
                 1350 
               
               
                   
                   
                 A-32344 
                 AS 
                 UGGAAuACUCUUGGUuAcAdTdT 
                 1351 
               
               
                   
               
               
                 AD-23005 
                 628 
                 A-42937 
                 S 
                 uAAccAAGAGuAuuccAuudTdT 
                 1352 
               
               
                   
                   
                 A-42938 
                 AS 
                 AAUGGAAuACUCUUGGUuAdTdT 
                 1353 
               
               
                   
               
               
                 AD-23006 
                 629 
                 A-42939 
                 S 
                 AAccAAGAGuAuuccAuuudTdT 
                 1354 
               
               
                   
                   
                 A-42940 
                 AS 
                 AAAUGGAAuACUCUUGGUUdTdT 
                 1355 
               
               
                   
               
               
                 AD-23007 
                 631 
                 A-42941 
                 S 
                 AccAAGAGuAuuccAuuuudTdT 
                 1356 
               
               
                   
                   
                 A-42942 
                 AS 
                 AAAAUGGAAuACUCUUGGUdTdT 
                 1357 
               
               
                   
               
               
                 AD-23008 
                 632 
                 A-42943 
                 S 
                 ccAAGAGuAuuccAuuuuudTdT 
                 1358 
               
               
                   
                   
                 A-42944 
                 AS 
                 AAAAAUGGAAuACUCUUGGdTdT 
                 1359 
               
               
                   
               
               
                 AD-23009 
                 633 
                 A-42945 
                 S 
                 cAAGAGuAuuccAuuuuuAdTdT 
                 1360 
               
               
                   
                   
                 A-42946 
                 AS 
                 uAAAAAUGGAAuACUCUUGdTdT 
                 1361 
               
               
                   
               
               
                 AD-23010 
                 634 
                 A-42947 
                 S 
                 AAGAGuAuuccAuuuuuAcdTdT 
                 1362 
               
               
                   
                   
                 A-42948 
                 AS 
                 GuAAAAAUGGAAuACUCUUdTdT 
                 1363 
               
               
                   
               
               
                 AD-23011 
                 635 
                 A-42949 
                 S 
                 AGAGuAuuccAuuuuuAcudTdT 
                 1364 
               
               
                   
                   
                 A-42950 
                 AS 
                 AGuAAAAAUGGAAuACUCUdTdT 
                 1365 
               
               
                   
               
               
                 AD-23012 
                 636 
                 A-42951 
                 S 
                 GAGuAuuccAuuuuuAcuAdTdT 
                 1366 
               
               
                   
                   
                 A-42952 
                 AS 
                 uAGuAAAAAUGGAAuACUCdTdT 
                 1367 
               
               
                   
               
               
                 AD-23013 
                 637 
                 A-42953 
                 S 
                 AGuAuuccAuuuuuAcuAAdTdT 
                 1368 
               
               
                   
                   
                 A-42954 
                 AS 
                 UuAGuAAAAAUGGAAuACUdTdT 
                 1369 
               
               
                   
               
               
                 AD-23014 
                 638 
                 A-42955 
                 S 
                 GuAuuccAuuuuuAcuAAAdTdT 
                 1370 
               
               
                   
                   
                 A-42956 
                 AS 
                 UUuAGuAAAAAUGGAAuACdTdT 
                 1371 
               
               
                   
               
               
                 AD-23015 
                 639 
                 A-42957 
                 S 
                 uAuuccAuuuuuAcuAAAGdTdT 
                 1372 
               
               
                   
                   
                 A-42958 
                 AS 
                 CUUuAGuAAAAAUGGAAuAdTdT 
                 1373 
               
               
                   
               
               
                 AD-23016 
                 640 
                 A-42959 
                 S 
                 AuuccAuuuuuAcuAAAGcdTdT 
                 1374 
               
               
                   
                   
                 A-42960 
                 AS 
                 GCUUuAGuAAAAAUGGAAUdTdT 
                 1375 
               
               
                   
               
               
                 AD-23017 
                 641 
                 A-42961 
                 S 
                 uuccAuuuuuAcuAAAGcAdTdT 
                 1376 
               
               
                   
                   
                 A-42962 
                 AS 
                 UGCUUuAGuAAAAAUGGAAdTdT 
                 1377 
               
               
                   
               
               
                 AD-23018 
                 642 
                 A-42963 
                 S 
                 uccAuuuuuAcuAAAGcAGdTdT 
                 1378 
               
               
                   
                   
                 A-42964 
                 AS 
                 CUGCUUuAGuAAAAAUGGAdTdT 
                 1379 
               
               
                   
               
               
                 AD-23019 
                 643 
                 A-42965 
                 S 
                 ccAuuuuuAcuAAAGcAGudTdT 
                 1380 
               
               
                   
                   
                 A-42966 
                 AS 
                 ACUGCUUuAGuAAAAAUGGdTdT 
                 1381 
               
               
                   
               
               
                 AD-23020 
                 644 
                 A-42967 
                 S 
                 cAuuuuuAcuAAAGcAGuGdTdT 
                 1382 
               
               
                   
                   
                 A-42968 
                 AS 
                 cACUGCUUuAGuAAAAAUGdTdT 
                 1383 
               
               
                   
               
               
                 AD-23021 
                 645 
                 A-42969 
                 S 
                 AuuuuuAcuAAAGcAGuGudTdT 
                 1384 
               
               
                   
                   
                 A-42970 
                 AS 
                 AcACUGCUUuAGuAAAAAUdTdT 
                 1385 
               
               
                   
               
               
                 AD-23022 
                 646 
                 A-42971 
                 S 
                 uuuuuAcuAAAGcAGuGuudTdT 
                 1386 
               
               
                   
                   
                 A-42972 
                 AS 
                 AAcACUGCUUuAGuAAAAAdTdT 
                 1387 
               
               
                   
               
               
                 AD-23023 
                 647 
                 A-42973 
                 S 
                 uuuuAcuAAAGcAGuGuuudTdT 
                 1388 
               
               
                   
                   
                 A-42974 
                 AS 
                 AAAcACUGCUUuAGuAAAAdTdT 
                 1389 
               
               
                   
               
               
                 AD-23024 
                 648 
                 A-42975 
                 S 
                 uuuAcuAAAGcAGuGuuuudTdT 
                 1390 
               
               
                   
                   
                 A-42976 
                 AS 
                 AAAAcACUGCUUuAGuAAAdTdT 
                 1391 
               
               
                   
               
               
                 AD-23025 
                 649 
                 A-42977 
                 S 
                 uuAcuAAAGcAGuGuuuucdTdT 
                 1392 
               
               
                   
                   
                 A-42978 
                 AS 
                 GAAAAcACUGCUUuAGuAAdTdT 
                 1393 
               
               
                   
               
               
                 AD-23026 
                 650 
                 A-42979 
                 S 
                 uAcuAAAGcAGuGuuuucAdTdT 
                 1394 
               
               
                   
                   
                 A-42980 
                 AS 
                 UGAAAAcACUGCUUuAGuAdTdT 
                 1395 
               
               
                   
               
               
                 AD-23027 
                 651 
                 A-42981 
                 S 
                 AcuAAAGcAGuGuuuucAcdTdT 
                 1396 
               
               
                   
                   
                 A-42982 
                 AS 
                 GUGAAAAcACUGCUUuAGUdTdT 
                 1397 
               
               
                   
               
               
                 AD-23028 
                 652 
                 A-42983 
                 S 
                 cuAAAGcAGuGuuuucAccdTdT 
                 1398 
               
               
                   
                   
                 A-42984 
                 AS 
                 GGUGAAAAcACUGCUUuAGdTdT 
                 1399 
               
               
                   
               
               
                 AD-18330 
                 653 
                 A-32349 
                 S 
                 uAAAGcAGuGuuuucAccudTdT 
                 1400 
               
               
                   
                   
                 A-32350 
                 AS 
                 AGGUGAAAAcACUGCUUuAdTdT 
                 1401 
               
               
                   
               
               
                 AD-23029 
                 654 
                 A-42985 
                 S 
                 AAAGcAGuGuuuucAccucdTdT 
                 1402 
               
               
                   
                   
                 A-42986 
                 AS 
                 GAGGUGAAAAcACUGCUUUdTdT 
                 1403 
               
               
                   
               
               
                 AD-23030 
                 655 
                 A-42987 
                 S 
                 AAGcAGuGuuuucAccucAdTdT 
                 1404 
               
               
                   
                   
                 A-42988 
                 AS 
                 UGAGGUGAAAAcACUGCUUdTdT 
                 1405 
               
               
                   
               
               
                 AD-23031 
                 656 
                 A-42989 
                 S 
                 AGcAGuGuuuucAccucAudTdT 
                 1406 
               
               
                   
                   
                 A-42990 
                 AS 
                 AUGAGGUGAAAAcACUGCUdTdT 
                 1407 
               
               
                   
               
               
                 AD-18328 
                 628 
                 A-32345 
                 S 
                 GuAAccAAGAGuAuuccAudTdT 
                 1408 
               
               
                   
                   
                 A-32346 
                 AS 
                 AUGGAAuACUCUUGGUuACdTdT 
                 1409 
               
               
                   
               
            
           
         
       
     
     Example 13 
     In Vitro Screening of TTR Tiled siRNAs 
     Tiled TTR duplexes were assayed in Hep3B cells for inhibition of endogenous TTR expression using real time PCR assays. 
     Cell culture and transfection: Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in Eagle&#39;s Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of each siRNA in individual wells of a 96-well plate. To this 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax was added per well (Invitrogen, Carlsbad Calif. cat #13778-150) and the mixture was incubated at room temperature for 15 minutes. 80 μl of complete growth media described above, but without antibiotic containing 2.0×10 4  Hep3B cells were then added. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 0.1 or 10 nM final duplex concentration. 
     Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystems, Foster City Calif., part #: AM1830): Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 μl of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 μl of water. 
     cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif., Cat #4368813): A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold. 
     Real time PCR: 2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914 M1) and 10 μl Roche Probes Master Mix (Roche Cat # 04887301001) per well in a LightCycler 480 384 well plate (Roche cat # 0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex was tested in two independent transfections and each transfection was assayed in duplicate. 
     Real time data were analyzed using the MCt method. Each sample was normalized to GAPDH expression and knockdown was assessed relative to cells transfected with the non-targeting duplex AD-1955. Table 14 shows the knockdown of TTR using the siRNAs. Data are expressed as the percent of message remaining relative to cells targeted with AD-1955. 
     Many but not all tiled TTR-dsRNAs, targeting TTR near the target of AD-18328, reduced TTR mRNA by at least 70% when transfected into Hep3B cells at 0.1 nM. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Inhibition of TTR by tiled dsRNA targeting 
               
               
                 TTR near target of AD-18328. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 % message 
                 % SD 
                 % message 
                 % SD 
               
               
                 Duplex # 
                 remaining 0.1 nM 
                 0.1 nM 
                 remaining 10 nM 
                 10 nM 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 AD-18323 
                 6.7 
                 1.90 
                 1.7 
                 0.02 
               
               
                 AD-18324 
                 1.8 
                 0.58 
                 0.9 
                 0.10 
               
               
                 AD-23000 
                 5.5 
                 0.93 
                 2.1 
                 0.87 
               
               
                 AD-23001 
                 15.2 
                 4.89 
                 4.9 
                 1.74 
               
               
                 AD-23002 
                 3.1 
                 1.12 
                 1.4 
                 0.55 
               
               
                 AD-23003 
                 17.3 
                 3.13 
                 1.7 
                 0.06 
               
               
                 AD-18325 
                 1.5 
                 0.27 
                 1.4 
                 0.66 
               
               
                 AD-23004 
                 9.0 
                 0.15 
                 10.5 
                 0.96 
               
               
                 AD-18326 
                 22.0 
                 1.85 
                 7.6 
                 0.78 
               
               
                 AD-18327 
                 11.6 
                 2.64 
                 9.6 
                 1.67 
               
               
                 AD-18328 
                 1.1 
                 0.70 
                 0.6 
                 0.16 
               
               
                 AD-23005 
                 0.8 
                 0.31 
                 0.6 
                 0.21 
               
               
                 AD-23006 
                 1.5 
                 0.46 
                 1.2 
                 0.43 
               
               
                 AD-23007 
                 2.4 
                 0.91 
                 1.9 
                 0.46 
               
               
                 AD-23008 
                 0.6 
                 0.10 
                 0.8 
                 0.26 
               
               
                 AD-23009 
                 1.0 
                 0.13 
                 0.9 
                 0.22 
               
               
                 AD-23010 
                 60.1 
                 15.66 
                 66.2 
                 22.71 
               
               
                 AD-23011 
                 56.5 
                 16.99 
                 53.6 
                 4.70 
               
               
                 AD-23012 
                 7.7 
                 2.36 
                 7.7 
                 3.25 
               
               
                 AD-23013 
                 7.0 
                 0.64 
                 8.0 
                 1.06 
               
               
                 AD-23014 
                 0.7 
                 0.01 
                 0.6 
                 0.10 
               
               
                 AD-23015 
                 15.4 
                 0.25 
                 16.5 
                 7.07 
               
               
                 AD-23016 
                 27.1 
                 0.37 
                 6.7 
                 1.80 
               
               
                 AD-23017 
                 4.5 
                 1.26 
                 1.4 
                 0.40 
               
               
                 AD-23018 
                 44.6 
                 9.45 
                 7.5 
                 1.09 
               
               
                 AD-23019 
                 2.2 
                 0.68 
                 0.8 
                 0.10 
               
               
                 AD-23020 
                 52.7 
                 6.45 
                 29.7 
                 1.17 
               
               
                 AD-23021 
                 95.4 
                 16.16 
                 45.0 
                 3.00 
               
               
                 AD-23022 
                 70.1 
                 3.01 
                 60.8 
                 12.11 
               
               
                 AD-23023 
                 2.7 
                 1.12 
                 1.8 
                 0.07 
               
               
                 AD-23024 
                 1.7 
                 0.30 
                 1.8 
                 0.33 
               
               
                 AD-23025 
                 64.2 
                 13.21 
                 10.5 
                 1.34 
               
               
                 AD-23026 
                 1.9 
                 0.15 
                 1.9 
                 0.78 
               
               
                 AD-23027 
                 2.5 
                 0.21 
                 1.6 
                 0.49 
               
               
                 AD-23028 
                 6.7 
                 4.41 
                 1.2 
                 0.50 
               
               
                 AD-18330 
                 6.0 
                 0.56 
                 5.7 
                 1.15 
               
               
                 AD-23029 
                 4.5 
                 0.47 
                 1.6 
                 0.10 
               
               
                 AD-23030 
                 3.9 
                 0.25 
                 3.3 
                 0.84 
               
               
                 AD-23031 
                 3.4 
                 0.78 
                 1.7 
                 0.02 
               
               
                   
               
            
           
         
       
     
     Example 14 
     Evaluation of Infusion Duration on Efficacy of a Single Intravenous Administration of SNALP-18534 in Sprague-Dawley Rats 
     Objectives 
     To determine the effect of infusion duration on efficacy of a single IV infusion of SNALP-18534 on liver TTR mRNA levels in Sprague-Dawley rats. 
     
       
         
           
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 Abbreviations and definitions used 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 SNALP-18534 
                 Rodent transthyretin specific siRNA formulated 
               
               
                   
                 in SNALP 
               
               
                 SNALP-1955 
                 Non-mammalian luciferase specific siRNA formulated 
               
               
                   
                 in SNALP 
               
               
                   
               
            
           
         
       
     
     The sequences of the sense and antisense strands of AD-18534 are reproduced below from the tables above: 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                   
                 SEQ 
               
               
                   
                 Oligo 
                 Po- 
                 Sequence 
                 ID 
               
               
                 Strand 
                 # 
                 sition 
                 5′ to 3′ 
                 NO: 
               
               
                   
               
             
            
               
                 s 
                 A-32755 
                 532 
                 cAGuGuucuuGcucuAuAAdTdT 
                 1289 
               
               
                   
               
               
                 as 
                 A-32756 
                 550 
                 UuAuAGAGcAAGAAcACUGdTdT 
                 1290 
               
               
                   
               
            
           
         
       
     
     Study Materials 
     Test Article(s) 
     SNALP-18534 is comprised of an siRNA targeting rodent TTR mRNA (AD-18534), formulated in stable nucleic acid lipid particles (SNALP) for delivery to target tissues. The SNALP formulation (lipid particle) consists of a novel aminolipid (DLinDMA), a PEGylated lipid (mPEG2000-C-DMA), a neutral lipid (DPPC) and cholesterol. The ratio of lipid:nucleic acid in the SNALP formulation is approximately 5.8:1 (w:w). SNALP-1955 contains an siRNA targeting the non-mammalian luciferase mRNA, is formulated with the identical lipid particle as SNALP-18534, and serves as a non-pharmacologically active control. Dose levels are expressed as mg/kg based on the weight of siRNA content. 
     Study Design &amp; Procedures 
     Animals and Test Article Administration: 
     The study was comprised of 9 groups of Sprague-Dawley rats (4 males/ group). The animals were allowed to have at least a 2 day acclimation period before the study and all animals were 7 weeks old at the initiation of dosing. The dose administered was calculated based upon body weight data collected prior to dosing on Day 1. The test and control articles were administered as a single 15-minute, 1-hour, 2-hour, or 3-hour IV infusion via the tail vein using a 24G¾″ cannula sealed with a Baxter Injection Site septum connected via 27G Terumo butterfly needle to a Baxter AS40A Syringe Pump. The dose volume was 3 ml/kg, the infusion rate was 12 ml/kg/hr, and animals were freely moving in the cages during dosing. Rats were divided into nine treatment groups and administered a single IV infusion of SNALP-18534, SNALP-1955, or PBS as shown in Table 16: 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Test Animal Dosage Groups 
               
            
           
           
               
               
               
               
               
            
               
                 Group 
                 N 
                 Test Article 
                 Infusion Duration 
                 Dose 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 A 
                 4 
                 PBS 
                 15 
                 minute 
                 — 
               
               
                 B 
                 4 
                 PBS 
                 3 
                 hour 
                 — 
               
               
                 C 
                 4 
                 SNALP -1955 
                 1 
                 hour 
                 1 mg/kg 
               
               
                 D 
                 4 
                 SNALP -1955 
                 2 
                 hour 
                 1 mg/kg 
               
               
                 E 
                 4 
                 SNALP -1955 
                 3 
                 hour 
                 1 mg/kg 
               
               
                 F 
                 4 
                 SNALP-18534 
                 15 
                 minute 
                 1 mg/kg 
               
               
                 G 
                 4 
                 SNALP-18534 
                 1 
                 hour 
                 1 mg/kg 
               
               
                 H 
                 4 
                 SNALP-18534 
                 2 
                 hour 
                 1 mg/kg 
               
               
                 I 
                 4 
                 SNALP-18534 
                 3 
                 hour 
                 1 mg/kg 
               
               
                   
               
            
           
         
       
     
     Tissue Collection and RNA Isolation: 
     On Day 0, animals were anesthetized by isofluorane inhalation and pre-dosing blood samples were collected into serum separator tubes by retro-orbital bleed. The blood samples were allowed to clot at room temperature for approximately 30 minutes prior to centrifugation at 4° C. Serum samples were then stored at −80° C. until analysis was performed. On Day 3, animals in all nine treatment groups were given a lethal dose of ketamine/xylazine. Blood was collected via caudal vena cava into serum separation tubes, and then allowed to clot at room temperature for approximately 30 minutes prior to centrifugation at 4° C. Serum samples were stored at −80° C. until analysis was performed. Liver tissue was harvested and snap frozen on dry ice. Frozen liver tissue was ground and tissue lysates were prepared for liver mRNA quantitation. 
     TTR mRNA Quantitation: 
     TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer&#39;s instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. 
     To obtain the relative level of TTR mRNA expression, group mean values for SNALP-1955 and SNALP-18534 treated groups with 15-minute, 1 hour and 2 hour infusion durations were then normalized to the mean value for the PBS treated group with 15-minute infusion whereas group mean values for SNALP-1955 and SNALP-18534 treated groups with 3 hour infusion duration were then normalized to the mean value for the PBS treated group with 3 hour infusion duration. 
     Results 
     As shown in  FIG. 16 , a single IV infusion of 1 mg/kg SNALP-18534 with different infusion durations of 15 minutes to 3 hours results in comparable inhibition of liver TTR mRNA levels measured two days after dosing. A single IV infusion of 1 mg/kg SNALP-18534 also showed durable TTR downregulation over 29 days following a single 15 minute IV infusion, as compared to SNALP-1955 control (data not shown). Compared to the PBS-treated group, a single 15-minute, 1-hour, 2-hour, or 3-hour IV infusion of SNALP-18534 at 1 mg/kg significantly reduced relative TTR mRNA expression levels by 94% (p&lt;0.001), 94% (p&lt;0.001), 92% (p&lt;0.001) and 93% (p&lt;0.001), respectively. Specificity of SNALP-18534 activity is demonstrated by lack of significant target inhibition by SNALP-1955 administration via 1-hour, 2-hour, or 3-hour IV infusion at the same dose level. 
     Conclusions 
     This study demonstrates that varying the infusion duration from 15 minutes to up to 3 hours does not affect the efficacy of a single IV administration of 1 mg/kg SNALP-18534 in rats, as assessed by reduction of TTR mRNA levels in the liver. 
     Example 15 
     In Vivo Reduction of Wild-Type TTR mRNA in the Rat Liver by LNP07-18534 and LNP08-18534 
     To evaluate the efficacy of 2 novel lipid nanoparticle formulations, LNP07 and LNP08, for delivery of siRNA in the rat, the rodent-specific TTR siRNA, AD-18534, was formulated in LNP07 (LNP07-18534) or LNP08 (LNP08-18534), and administered by 15-minute IV infusion, and liver TTR mRNA was quantified. Sprague-Dawley rats (4 animals per group) were administered 15-minute IV infusions of LNP07-18534 (0.03, 0.1, 0.3 or 1 mg/kg), LNP08-18534 (0.01, 0.03 or 0.1 mg/kg), or LNP07-1955 (1 mg/kg) or LNP08-1955 (0.1 mg/kg) containing the negative control siRNA AD-1955 which targets the non-mammalian gene luciferase. Forty-eight hours later, animals were euthanized and liver tissue was collected, flash-frozen and stored at −80° C. until processing. 
     For TTR mRNA quantitation, frozen liver tissue was ground into powder, and lysates were prepared. TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer&#39;s instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group means of the normalized values were then further normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression. 
     The results are shown in  FIG. 17 . LNP07-18534 reduced TTR mRNA levels in the liver in a dose-dependent manner, with 94% suppression of TTR mRNA at 1 mg/kg. The effect was specific, since the negative control LNP07-1955 at 1 mg/kg did not significantly affect TTR mRNA levels compared to the PBS control. The mRNA ED50 was determined to be ˜0.05 mg/kg LNP07-18534. LNP08-18534 reduced TTR mRNA levels in the liver in a dose-dependent manner, with 86% suppression of TTR mRNA at 0.1 mg/kg. The effect was specific, since the negative control LNP08-1955 at 0.1 mg/kg did not significantly affect TTR mRNA levels compared to the PBS control. The mRNA ED50 was determined to be ˜0.02 mg/kg LNP08-18534. 
     These results demonstrate that LNP07-18534 and LNP08-18534 are effective in suppressing wild-type TTR mRNA in the rat liver when administered by IV infusion, and that LNP07 and LNP08 are effective formulations for delivering siRNA to the liver. 
     Example 16 
     Reduction of TTR Liver mRNA by a Single Intravenous Administration of LNP09-18534 or LNP11-18534 in Sprague-Dawley Rats 
     Objective: 
     To evaluate the efficacy of two novel lipid nanoparticle (LNP) formulations for delivery of the rodent TTR-specific siRNA, AD-18534 in the Sprague-Dawley rat for reducing endogenous (wild type) liver TTR mRNA levels. Rats were intravenously dosed via a 15 minute infusion with either 0.01, 0.03, 0.1, or 0.3 mg/kg LNP09-18534, LNP11-18534, or phosphate buffered saline (PBS) and TTR liver mRNA levels were assayed at 48 hrs post-treatment. 
     Material and Methods: 
     LNP09 formulation: (XTC/DSPC/Chol/PEG 2000 -C14)=50/10/38.5/1.5 mol %; Lipid:siRNA ˜11:1. LNP11 formulation: (MC3/DSPC/Chol/PEG 2000 -C14)=50/10/38.5/1.5 mol %; Lipid:siRNA ˜11.1 
     Tissue collection and RNA isolation: On Day 3, animals in all treatment groups were given a lethal dose of ketamine/xylazine. Blood was collected via caudal vena cava into serum separation tubes, and then allowed to clot at room temperature for approximately 30 minutes prior to centrifugation at 4° C. Serum samples were stored at −80° C. until for future analysis. Liver tissues were harvested and snap frozen on dry ice. Frozen liver tissue was ground and tissue lysates were prepared for liver mRNA quantitation. 
     TTR mRNA Quantitation: TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer&#39;s instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group mean values were then normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression. 
     Results: 
     As shown in  FIG. 18 , in contrast with PBS treated animals, LNP09-18534 and LNP11-18534 treated animals had a significant dose-dependent decrease in TTR mRNA levels in the liver, reaching maximum reduction of ˜90% mRNA reduction for both LNP09 and LNP11 formulated groups, relative to PBC control group at 0.3 mg/kg, and a dose achieving 50% reduction (ED 50 ) of &lt;0.03 mg/kg for LNP11-18534 and &lt;0.1 mg/kg for LNP09-18534. 
     Conclusions 
     This study demonstrates that a single 15 minute IV infusion of LNP09-18534 or LNP11-18534 in Sprague-Dawley rats results in a dose-dependent reduction of liver TTR mRNA. These data demonstrate the efficacy of LNP09-18328 and LNP11-18328 in reducing endogenously expressed (wild type) TTR mRNA with ED50 levels of &lt;0.03 and &lt;0.1 mg/kg for LNP11-18534 and LNP09-18534, respectively. 
     Example 17 
     Inhibition of TTR in Humans 
     A human subject is treated with a dsRNA targeted to a TTR gene to inhibit expression of the TTR gene to treat a condition. 
     A subject in need of treatment is selected or identified. The subject can have a liver disorder, transthyretin amyloidosis, and/or a transplanted liver. 
     The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject&#39;s home through the subject&#39;s own use of a self-testing kit. 
     At time zero, a suitable first dose of an anti-TTR siRNA is administered to the subject. The dsRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject&#39;s condition is evaluated, e.g., by measuring liver function. This measurement can be accompanied by a measurement of TTR expression in said subject, and/or the products of the successful siRNA-targeting of TTR mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject&#39;s needs. 
     After treatment, the subject&#39;s tumor growth rate is lowered relative to the rate existing prior to the treatment, or relative to the rate measured in a similarly afflicted but untreated subject.