Compositions and methods for inhibiting transthyretin expression

Abstract

To provide compositions and methods for treating pathological conditions and diseases, such as a TTR amyloidosis, in a mammal caused by expression of a transthyretin (TTR) gene.SOLUTION: There is provided a double-stranded ribonucleic acid (dsRNA) for inhibiting transthyretin (TTR) expression, where the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding transthyretin (TTR), where the region of complementarity is less than 30 nucleotides in length, and where the antisense strand comprises 15 or more contiguous nucleotides of a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to double-stranded ribonucleic acid (dsRNA) that targets the transthyretin (TTR) gene and methods of using dsRNA to inhibit expression of TTR.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61 / 106,956, filed October 20, 2008, U.S. Provisional Application No. 61 / 115,738, filed November 18, 2008, U.S. Provisional Application No. 61 / 156,670, filed March 2, 2009, U.S. Provisional Application No. 61 / 185,545, filed June 9, 2009, U.S. Provisional Application No. 61 / 242,783, filed September 15, 2009, and U.S. Provisional Application No. 61 / 244,794, filed September 22, 2009, all of which are incorporated herein by reference in their entirety for all purposes.

[0003] Reference to sequence listing This application contains a Sequence Listing that has been submitted electronically as a text file named ___________.txt, ________ bytes in size, created on __________, 2009. The Sequence Listing is incorporated by reference. [Background technology]

[0004] Transthyretin (TTR) is a secreted thyroid hormone-binding protein that binds and transports retinol-binding protein (RBP) / vitamin A in plasma and cerebrospinal fluid, and serum thyroxine (T4).

[0005] Both normal-sequence TTR and mutant-sequence TTR cause amyloidosis. Normal-sequence TTR causes cardiac amyloidosis in elderly people, which is called senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA)). SSA is often 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 primary site of TTR expression. Other important sites of expression include the choroid plexus, retina, and pancreas.

[0006] TTR amyloidosis manifests in various forms. When the peripheral nervous system is more significantly affected, the disease is called familial amyloidotic neuropathy (FAP). When the heart is primarily involved, but not the nervous system, the disease is called familial amyloidotic cardiomyopathy (FAC). The third major type of TTR amyloidosis is called meningeal / CNS (central nervous system) amyloidosis.

[0007] It has been shown that double-stranded RNA molecules (dsRNA) can block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi).International Publication WO99 / 32619 (Fire et al.) discloses the use of at least 25 nucleotides of dsRNA to inhibit the expression of gene in nematodes.In addition, dsRNA has been shown to degrade target RNA in other organisms, including plants (see, for example, International Publication WO99 / 53050, Waterhouse et al., and International Publication WO99 / 61631, Heifetz et al.), Drosophila (see, for example, Yang, D., et al., Curr.Biol.(2000)10:1191-1200) and mammals (see, for example, International Publication WO00 / 44895, Limmer, and German Patent DE101 00 586.5, Kreutzer et al.).

[0008] US Patent No. 20070207974 discloses functional and hyperfunctional siRNAs. US Patent No. 20090082300 discloses antisense molecules directed against TTR. US Patent No. 7,250,496 discloses microRNAs directed against TTR. Summary of the Invention

[0009] In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), the dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding transthyretin (TTR), the region of complementarity being 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 yet 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.

[0010] In one embodiment, the complementary region between the antisense strand of the dsRNA and the mRNA encoding transthyretin is 19 nucleotides in length.In another embodiment, the complementary region 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 yet another embodiment, each strand is 21 nucleotides in length.

[0011] In certain embodiments, the dsRNA for inhibiting transthyretin expression does not cleave 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 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 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.

[0012] In yet another related embodiment, the present invention provides the above-mentioned dsRNA for inhibiting the expression of transthyretin, wherein the dsRNA comprises one or more modified nucleotides.In a related embodiment, at least one modified nucleotide (or nucleotide) is selected from the group consisting of 2'-O-methyl modified nucleotides, nucleotides containing 5'-phosphorothioate groups, and terminal nucleotides linked to cholesteryl derivative groups or dodecanoic acid bisdecylamide groups.In another related embodiment, the modified nucleotide is selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and unnatural bases containing nucleotides.In some embodiments, the dsRNA comprises at least one 2'-O-methyl modified nucleotide.

[0013] In other embodiments, the above-mentioned dsRNA for inhibiting the expression of transthyretin is conjugated with ligand or formulated into lipid preparation.In some embodiments, the lipid preparation is formulated into LNP preparation, LNP01 preparation, XTC-SNALP preparation or SNALP preparation.In related embodiments, the XTC-SNALP preparation is as follows: 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC) is used with the ratio of XTC / DPPC / cholesterol / PEG-cDMA of 57.1 / 7.1 / 34.4 / 1.4 and the ratio of lipid: siRNA is about 7:1. In yet another related embodiment, the sense strand of the dsRNA consists of SEQ ID NO: 1009, the antisense strand consists of SEQ ID NO: 1010, and the dsRNA is formulated into an XTC-SNALP formulation as follows: 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC) is used 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 7:1. Alternatively, dsRNA such as those described above can be formulated into an LNP09 formulation as follows: XTC / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mole %. 2000 -C14 and a lipid: siRNA ratio of about 11:1 is used. In another variation, the dsRNA is formulated into the LNP11 formulation as follows: MC3 / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mole %. 2000-C14 and a lipid: siRNA ratio of about 11:1 are used. In yet another embodiment, the dsRNA is formulated into an LNP09 or LNP11 formulation and reduces TTR mRNA levels by about 85-90% at a dose of 0.3 mg / kg compared to a PBS control group. In yet another embodiment, the dsRNA is formulated into an LNP09 or LNP11 formulation and reduces TTR mRNA levels by about 50% at a dose of 0.1 mg / kg compared to a PBS control group. In yet another embodiment, the dsRNA is formulated into an LNP09 or LNP11 formulation and reduces TTR protein levels in a dose-dependent manner compared to a PBS control group, as measured by Western blot. In yet another embodiment, the dsRNA is formulated into a SNALP formulation as follows: DLinDMA is used in a ratio of DLinDMA / DPPC / cholesterol / PEG2000-cDMA of 57.1 / 7.1 / 34.4 / 1.4 with a lipid: siRNA ratio of approximately 7:1.

[0014] In one embodiment, the present invention provides dsRNA such as those described above for inhibiting the expression of transthyretin, wherein administering the dsRNA to cells results in about 95% inhibition of TTR mRNA expression as measured by real-time PCR assay, the cells are HepG2 cells or Hep3B cells, and the concentration of the dsRNA is 10nM.In a related embodiment, administering the dsRNA to cells results in about 74% inhibition of TTR mRNA expression as measured by branched DNA assay, the cells are HepG2 cells or Hep3B cells, and the concentration of the dsRNA is 10nM.In another related embodiment, the dsRNA has an IC50 of less than 10pM in HepG2 cells, and the concentration of the dsRNA is 10nM.In yet another related embodiment, the dsRNA has an ED50 of about 1mg / kg. In yet another related embodiment, administering this dsRNA reduces the TTR mRNA in the liver of cynomolgus monkeys by about 80%, and the concentration of this dsRNA is 3mg / kg.In yet another related embodiment, administering this dsRNA does not result in the immunostimulatory activity in human peripheral blood mononuclear cells (PBMCs), as measured by IFN-α and TNF-α ELISA assay.In yet another related embodiment, administering this dsRNA reduces the liver TTR mRNA level by about 97% or the serum TTR protein level by about 90%, and the concentration of this dsRNA is 6mg / kg.In yet another related embodiment, administering this dsRNA reduces the liver TTR mRNA level and / or serum TTR protein level for up to 22 days, and the concentration of this dsRNA is 6mg / kg or 3mg / kg. In yet another related embodiment, the dsRNA, when administered to a subject in need thereof at 1 mg / kg or 3 mg / kg, suppresses serum TTR protein levels until 14 days after treatment.In yet another related embodiment, the dsRNA, at a concentration of 0.1 nM, reduces the expression of TTR in Hep3B cells by 98.9%, as measured by real-time PCR.In yet another related embodiment, the dsRNA, at a concentration of 10 nM, reduces the expression of TTR in Hep3B cells by 99.4%, as measured by real-time PCR.

[0015] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises a region that is complementary to a portion of an mRNA encoding transthyretin (TTR), wherein the region of complementarity is less than 30 nucleotides in length, and 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.

[0016] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein the dsRNA comprises an antisense strand comprising a region complementary to 15 to 30 nucleotides of nucleotides 618 to 648 of SEQ ID NO: 1331, and the antisense strand base pairs with the guanine at position 628 of SEQ ID NO: 1331.

[0017] In some embodiments, the present invention provides a cell that contains any of the dsRNAs described in the Summary of the Invention above.In some other embodiments, the present invention provides a vector that comprises the nucleotide sequence that encodes at least one strand of any of the dsRNAs described in the Summary of the Invention above.In some embodiments, the vector is in a cell.

[0018] In another embodiment, the present invention provides a pharmaceutical composition for inhibiting the expression of TTR gene, comprising any of the dsRNAs described in the Summary of the Invention above and a pharmaceutically acceptable carrier.In a related embodiment, the present invention provides a pharmaceutical composition for inhibiting the expression of TTR gene, comprising a dsRNA and a SNALP formulation, wherein the dsRNA comprises an antisense strand less than 30 nucleotides in length and comprises 15 or more consecutive nucleotides of SEQ ID NO:170, SEQ ID NO:450, SEQ ID NO:730, or SEQ ID NO:1010, and the SNALP formulation comprises DlinDMA, DPPC, cholesterol, and PEG2000-cDMA in the ratio of 57.1 / 7.1 / 34.4 / 1.4, respectively.

[0019] In yet another embodiment, the present invention provides a method for inhibiting expression of TTR in a cell, the method comprising: (a) contacting the cell with any of the dsRNAs described in the Summary of the Invention above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the TTR gene, thereby inhibiting expression of the TTR gene in the cell.

[0020] In yet another embodiment, the present invention provides a method for treating a disorder mediated by TTR expression, comprising administering a therapeutically effective amount of any of the dsRNAs described in the Summary of the Invention to a human in need of such treatment. In a related embodiment, 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 suffers from transthyretin amyloidosis and / or liver disease. In a related embodiment, the human being is further provided with a liver transplant. In yet another embodiment, administration of the dsRNA reduces TTR mRNA in the human liver by about 80%, and 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 being, as measured by IFN-α and TNF-α ELISA assays. In yet another related embodiment, administering the dsRNA reduces liver TTR mRNA level by about 97% or serum TTR protein level by about 90%, and the concentration of the dsRNA is 6mg / kg.In yet another related embodiment, administering the dsRNA reduces liver TTR mRNA level and / or serum TTR protein level for up to 22 days, and the concentration of the dsRNA is 6mg / kg or 3mg / kg.In yet another related embodiment, the dsRNA is formulated into the LNP09 formulation as follows: XTC / DSPC / Chol / PEG in a 50 / 10 / 38.5 / 1.5 mole% ratio. 2000 -C14 and a lipid: siRNA ratio of about 11:1 is used. In yet another related embodiment, the dsRNA is formulated into an LNP11 formulation as follows: MC3 / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mole %. 2000-C14 and a lipid:siRNA ratio of about 11:1 are used. In yet another related embodiment, the dsRNA is formulated into an LNP09 or LNP11 formulation and reduces TTR mRNA levels by about 85-90% at a dose of 0.3 mg / kg compared to a PBS control group. In yet another related embodiment, the dsRNA is formulated into an LNP09 or LNP11 formulation and reduces TTR mRNA levels by about 50% at a dose of 0.1 mg / kg compared to a PBS control group. In yet another related embodiment, the dsRNA is formulated into an LNP09 or LNP11 formulation and reduces TTR protein levels in a dose-dependent manner compared to a PBS control group, as measured by Western blot. In yet another related embodiment, administration of the dsRNA suppresses serum TTR protein levels by 14 days after treatment when administered to humans at 1 mg / kg or 3 mg / kg. In yet another related embodiment, the dsRNA is formulated into a SNALP formulation as follows: DLinDMA is used in a ratio of DLinDMA / DPPC / cholesterol / PEG2000-cDMA of 57.1 / 7.1 / 34.4 / 1.4, and a lipid: siRNA ratio of approximately 7:1.

[0021] In another embodiment, the present invention provides the use of dsRNA for treating disorders mediated by TTR expression, comprising administering a therapeutically effective amount of any of the dsRNAs described in the Summary of the Invention to a person in need of such treatment.In a related embodiment, the dsRNA is administered to the person 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 person at about 1.0 mg / kg.In another related embodiment, the person suffers from transthyretin amyloidosis and / or liver disease.In yet another embodiment of the use provided by the present invention, the treated person is further provided with liver transplantation.

[0022] In yet another embodiment, the present invention provides the use of a dsRNA in a method for inhibiting expression of TTR in a cell, the method comprising: (a) contacting the cell with a dsRNA as described in the Summary of the Invention above; and (b) maintaining the cell produced in step (a) for a time sufficient to result in degradation of mRNA transcripts of the TTR gene, thereby inhibiting expression of the TTR gene in the cell.

[0023] 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 drawings, and from the claims. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a graph of TNFα and IFNα levels in cultured human PBMCs after transfection with TTR siRNA. [Figure 2] 2A and 2B are dose-response curves for AD-18324 and AD-18328, respectively, in HepG2 cells. [Figure 3] Dose-response curve for AD-18246 in HepG2 cells. [Figure 4A] Figure 1 shows the inhibition of hepatic mRNA and plasma protein levels in transgenic H129-mTTR-KO / iNOS-KO / hTTR mice by intravenous bolus administration of TTR-dsRNA (AD-18324, AD-18328, and AD-18246) formulated in LNP01. [Figure 4B] Figure 1 shows the inhibition of hepatic mRNA and plasma protein levels in transgenic H129-mTTR-KO / iNOS-KO / hTTR mice by intravenous bolus administration of TTR-dsRNA (AD-18324, AD-18328, and AD-18246) formulated in LNP01. [Figure 5]1 is a graph summarizing the measurement of TTR mRNA levels in the liver of non-human primates following a 15-minute intravenous infusion of TTR-dsRNA (AD-18324 and AD-18328) formulated in SNALP. [Figure 6A] Figure 1 shows the inhibition of human V30M TTR liver mRNA and serum protein levels in transgenic mice by intravenous bolus administration of SNALP-18328. Group means were determined, normalized to the PBS control group, and then plotted. Error bars indicate standard deviation. The percentage reduction in group means compared to PBS is shown for the SNALP-1955 and SNALP-18328 groups. (***p<0.001, one-way ANOVA and Dunn's post-hoc test) [Figure 6B] Figure 1 shows the inhibition of human V30M TTR liver mRNA and serum protein levels in transgenic mice by intravenous bolus administration of SNALP-18328. Group means were determined, normalized to the PBS control group, and then plotted. Error bars indicate standard deviation. The percentage reduction in group means compared to PBS is shown for the SNALP-1955 and SNALP-18328 groups. (***p<0.001, one-way ANOVA and Dunn's post-hoc test) [Figure 7A] Figure 1 shows the persistence of reduction in human V30M TTR liver mRNA and serum protein levels 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 in normalized TTR mRNA levels compared to SNALP-1955 at each time point was calculated and shown for the SNALP-18328 group. (***p<0.001, one-way ANOVA and Dunn's post-hoc test) [Figure 7B]Figure 1 shows the persistence of reduction in human V30M TTR liver mRNA and serum protein levels 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 in normalized TTR mRNA levels compared to SNALP-1955 at each time point was calculated and shown for the SNALP-18328 group. (***p<0.001, one-way ANOVA and Dunn's post-hoc test) [Figure 8] 1 shows the time course of serum protein levels of TTR in non-human primates over 14 days following a single 15-minute intravenous infusion of SNALP-18328. [Figure 9] Figure 1 shows reduced TTR-immunoreactivity in various tissues of human V30M TTR / HSF-1 knockout mice after intravenous bolus administration of SNALP-18328. E: esophagus; S: stomach; I1: intestine / duodenum; I4: intestine / colon; N: nerve; D: dorsal root ganglion. [Figure 10] 1 shows measurements of TTR mRNA levels in non-human primate liver following a single 15-minute intravenous infusion of XTC-SNALP-18328. [Figure 11A] 1 shows measurements of TTR mRNA in the liver and serum protein levels of non-human primates after a 15-minute intravenous infusion of LNP09-18328 or LNP11-18328, respectively. [Figure 11B] 1 shows measurements of TTR mRNA in the liver and serum protein levels of non-human primates after a 15-minute intravenous infusion of LNP09-18328 or LNP11-18328, respectively. [Figure 11C] 1 shows the time course of TTR serum protein levels over 28 days following a 15 minute intravenous infusion of 0.3 mg / kg LNP09-18328 compared to a PBS control group. [Figure 12] The sequence of human TTR mRNA (Reference sequence NM_000371.3, SEQ ID NO: 1331) is shown. [Figure 13]Figure 13A shows the sequence of human TTR mRNA (Reference Sequence NM_000371.2, SEQ ID NO: 1329). Figure 13B shows the sequence of rat TTR mRNA (Reference Sequence NM_012681.1, SEQ ID NO: 1330). [Figure 14] Nucleotide alignment of NM_000371.3, NM_000371.2, and AD-18328 is shown. [Figure 15] Illustrates the symptoms and mutations in TTR associated with familial amyloidotic neuropathy, familial amyloidotic cardiomyopathy, and CNS amyloidosis. [Figure 16] Figure 1 shows the reduction of TTR mRNA levels in the liver with SNALP-18534 at different TTR 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. After 48 hours, rats were euthanized and livers were harvested. TTR and GAPDH mRNA levels were measured from liver lysates using the Quantigene bDNA assay. The ratio of TTR mRNA levels 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 deviation. (***p<0.001, compared to PBS, one-way ANOVA and Bonferroni post-hoc test) [Figure 17] 1 shows measurements of TTR mRNA levels in rat liver following a 15-minute intravenous infusion of LNP07-18534 or LNP08-18534. [Figure 18]Figure 1 shows in vivo inhibition of endogenous TTR mRNA levels in the liver of Sprague-Dawley rats after a 15-minute intravenous infusion of LNP09-18534 or LNP11-18534. Groups of animals (n=4 / group) were administered 0.01, 0.03, 0.1, or 0.3 mg / kg of LNP09-18534, LNP-11-18534, or PBS intravenously via a 15-minute infusion. After 48 hours, animals were euthanized and livers were harvested. TTR and GAPDH mRNA levels were measured from liver biopsy lysates using the Quantigene bDNA assay. The ratio of TTR mRNA to GAPDH mRNA levels was calculated for each animal. Group means were determined, normalized to the PBS control group, and then plotted. Error bars indicate standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides dsRNA and methods of using dsRNA to inhibit expression of the TTR gene in cells or mammals, where the dsRNA targets the TTR gene. The present invention also provides compositions and methods for treating conditions and diseases in mammals caused by expression of the TTR gene, such as TTR amyloidosis. The dsRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi).

[0026] The dsRNA of the compositions featured herein comprises an RNA strand (antisense strand) having a region less than 30 nucleotides in length, generally 19-24 nucleotides in length, that is substantially complementary to at least a portion of the mRNA transcript of the TTR gene. The use of these dsRNAs enables targeted degradation of the mRNA of genes implicated in pathologies associated with TTR expression in mammals. In particular, very low dosages of TTR dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of TTR gene expression. Using cell-based assays, the inventors have demonstrated that dsRNAs targeting TTR can specifically and efficiently mediate RNAi, resulting in significant inhibition of TTR gene expression. Therefore, methods and compositions containing these dsRNAs are useful for treating pathological processes that may be mediated by downregulating TTR, such as in the treatment of liver disease or TTR amyloidosis, e.g., FAP.

[0027] The method and composition containing TTR dsRNA are useful for treating pathological processes mediated by the expression of TTR, such as TTR amyloidosis.In one embodiment, the method for treating disorders mediated by the expression of TTR comprises administering a therapeutically effective amount of dsRNA targeting TTR to a person who needs such treatment.In one embodiment, the dsRNA is administered to a person 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.

[0028] The following detailed description discloses methods for making and using compositions containing dsRNA to inhibit expression of the TTR gene, as well as compositions and methods for treating diseases and disorders caused by expression of this gene. Pharmaceutical compositions featured in the present invention comprise, together with a pharmaceutically acceptable carrier, dsRNA having an antisense strand that is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and that includes a region of complementarity that is substantially complementary to at least a portion of an RNA transcript of the TTR gene. Pharmaceutical compositions featured in the present invention also include dsRNA having an antisense strand that is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and that includes a region of complementarity that is substantially complementary to at least a portion of an RNA transcript of the TTR gene.

[0029] The sense strand of the dsRNA can comprise 15, 16, 17, 18, 19, 20, 21, or more consecutive nucleotides of SEQ ID NO: 169, SEQ ID NO: 449, SEQ ID NO: 729, or SEQ ID NO: 1009. The antisense strand of the dsRNA can comprise 15, 16, 17, 18, 19, 20, 21, or more consecutive nucleotides of SEQ ID NO: 170, SEQ ID NO: 450, SEQ ID NO: 730, or SEQ ID NO: 1010. In one embodiment, the sense strand of the dsRNA can consist of SEQ ID NO: 449 or a fragment thereof, and the antisense strand can consist of SEQ ID NO: 450 or a fragment thereof. In one embodiment, the sense strand of the dsRNA can consist of SEQ ID NO: 729 or a fragment thereof, and the antisense strand can consist of SEQ ID NO: 730 or a fragment thereof. In one embodiment, the sense strand of the dsRNA can consist of SEQ ID NO: 1009 or a fragment thereof, and the antisense strand can consist of SEQ ID NO: 1010 or a fragment thereof.

[0030] In one embodiment, the dsRNA can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In one embodiment, the modified nucleotides can include 2'-O-methyl modified nucleotides, nucleotides containing 5'-phosphorothioate groups, and / or terminal nucleotides linked to a cholesteryl derivative group or a dodecanoic acid bisdecylamide group. In one embodiment, the modified nucleotides can include 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and / or nucleotides with unnatural bases.

[0031] In one embodiment, the region of complementarity of the dsRNA is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more nucleotides in length. In one embodiment, the region of complementarity comprises 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides of SEQ ID NO: 169.

[0032] In one embodiment, each strand of the dsRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length. In one embodiment, the dsRNA comprises a sense strand or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotide fragments 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 fragments thereof selected from Tables 3A, 3B, 4, 6A, 6B, 7 and 16.

[0033] In one embodiment, administration of dsRNA to cells results in about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more inhibition of TTR mRNA expression as measured by real-time PCR assay. In one embodiment, administration of dsRNA to cells results in about 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% or more inhibition of TTR mRNA expression as measured by real-time PCR assay. In one embodiment, administration of dsRNA to cells 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 one embodiment, administration of dsRNA to cells results in about 40%-45%, 45%-50%, 50%-55%, 55%-60%, 60%-65%, 65%-70%, 70%-75%, 75%-80%, 80%-85%, 85%-90%, 90%-95% or more inhibition of TTR mRNA expression, as measured by a branched DNA assay.

[0034] In one embodiment, the 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 one embodiment, the dsRNA has an ED50 of about 0.01, 0.1, 1, 5, or 10 mg / kg.

[0035] In one embodiment, administration of dsRNA can reduce TTR mRNA in cynomolgus monkeys by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or more. In one embodiment, administration of 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 one embodiment, administration of the dsRNA reduces liver TTR mRNA levels and / or serum TTR protein levels for 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.

[0036] In one embodiment, the dsRNA is formulated into an 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 compared to a PBC control group. In one embodiment, the dsRNA is formulated into an LNP formulation and reduces TTR protein levels by about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95% or more compared to a PBC control group, as measured by Western blot. In one embodiment, the dsRNA, when administered 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 to a subject in need thereof, suppresses serum TTR protein levels for 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, or 25 days after treatment.

[0037] Thus, in some embodiments, the present invention features pharmaceutical compositions containing TTR dsRNA and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the TTR gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of the TTR gene.

[0038] I. Definition For convenience, the meanings 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 control.

[0039] "G", "C", "A", and "U" generally represent nucleotides containing guanine, cytosine, adenine, and uracil as bases, respectively. "T" and "dT" are used interchangeably herein and refer to deoxyribonucleotides whose nucleic acid base is thymine, for example, deoxyribothymine. However, it will be understood that the terms "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to modified nucleotides, as further detailed below, or alternative replacement moieties. Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be substituted by other moieties without substantially changing the base pairing properties of oligonucleotides containing nucleotides bearing such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine may be substituted for, for example, inosine-containing nucleotides in the nucleotide sequences of the invention, and sequences containing such substituted moieties are embodiments of the invention.

[0040] 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 the human TTR mRNA transcript can be found at NM_000371. The sequence of the mouse TTR mRNA can be found at NM_013697.2. The sequence of the rat TTR mRNA can be found at NM_012681.1.

[0041] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the TTR gene, including mRNA that is the product of RNA processing of the primary transcript.

[0042] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0043] As used herein, 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 to form a double-stranded structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions, as understood by those of skill in the art. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing. Other conditions, such as physiologically relevant conditions encountered in organisms, may be applied. Those skilled in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate use of the hybridized nucleotides.

[0044] This includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of the first and second nucleotide sequences. Such sequences can be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences may be fully complementary, or may form one or more, but generally not more than four, three, or two mismatched base pairs upon hybridization, while retaining the ability to hybridize under conditions most suitable for their final use. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA comprising an oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may still be referred to as "perfectly complementary" for purposes described herein.

[0045] Also, as used herein, "complementary" sequences may contain or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, GU wobble or Hoogsteen base pairs.

[0046] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used to refer to base matches between the sense and antisense strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as understood from the context in which they are used.

[0047] As used herein, a polynucleotide that is "substantially complementary to at least a portion of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRTA of interest (e.g., an mRTA encoding TTR), including the 5'UTR, open reading frame (ORF), or 3'UTR. For example, a polynucleotide is complementary to at least a portion of an mRTA of TTR if the sequence is substantially complementary to an uninterrupted portion of the mRTA encoding TTR.

[0048] As used herein, the term " double-stranded RNA " or " dsRNA " refers to the complex of ribonucleic acid molecules, which has a double-stranded structure and comprises two nucleic acid strands, which are antiparallel and substantially complementary as defined above.Generally, the majority of nucleotides in each strand are ribonucleotides, but as described in detail herein, each or both strands can also comprise at least one non-ribonucleotide, for example, deoxyribonucleotide and / or modified nucleotide.In addition, as used herein, " dsRNA " can comprise extensive modifications at multiple nucleotides, and can comprise chemical modifications to ribonucleotides, including any kind of modifications disclosed herein or known in the art.Any such modifications used in siRNA type molecules are encompassed by " dsRNA " for the purposes of this specification and claims.

[0049] The two strands that form a double-stranded structure may be different parts of a single longer RNA molecule, or they may be separate RNA molecules.When the two strands are part of a single longer molecule and are therefore connected by an uninterrupted nucleotide chain between the 3'-end of one strand that forms a double-stranded structure and the 5'-end of the other strand, the connecting RNA strand is called a "hairpin loop".When the two strands are covalently connected by means other than an uninterrupted nucleotide chain between the 3'-end of one strand that forms a double-stranded structure and the 5'-end of the other strand, the connecting structure is called a "linker".The RNA strands may have the same or different number of nucleotides.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA, minus any overhangs that exist in the double strand.In addition to the double-stranded structure, dsRNA may contain one or more nucleotide overhangs.In addition, the term "siRNA" is used herein to refer to dsRNA as described above.

[0050] As used herein, " nucleotide overhang " refers to an unpaired nucleotide, or a nucleotide that protrudes from the double-stranded structure of dsRNA when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa. " Blunt " or " blunt end " means that there is no unpaired nucleotide at the end of dsRNA, i.e., there is no nucleotide overhang. " Blunt end " dsRNA is a dsRNA that is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule.

[0051] The term " antisense strand " refers to the strand of dsRNA, which comprises the region that is substantially complementary to target sequence.As used herein, the term " complementary region " refers to the region of antisense strand that is substantially complementary to sequence, for example, the target sequence defined herein.When complementary region is not completely complementary to target sequence, this mismatch is most tolerated in this terminal region, and when present, it is generally in terminal region, for example, 6, 5, 4, 3 or 2 nucleotides at 5' and / or 3' end.

[0052] As used herein, the term "sense strand" refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.

[0053] As used herein, the term "SNALP" refers to a stable nucleic acid-lipid particle. SNALP refers to a small lipid vesicle that contains a small aqueous interior coating containing a nucleic acid, such as dsRNA or a plasmid into which the dsRNA is transcribed. SNALPs are described, for example, in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and U.S. Patent Application No. USSN61 / 045,228, filed April 15, 2008. These applications are incorporated herein by reference.

[0054] When referring to dsRNA, "introducing into cells" means promoting cellular uptake or absorption, as understood by those skilled in the art.The absorption or uptake of dsRNA can occur through unassisted diffusion process or cellular active process, or by auxiliary agent or device.The meaning of this term is not limited to cells in vitro; dsRNA can also be "introduced into cells" that are part of a living organism.In such cases, introducing into cells includes delivery to the organism.For example, for in vivo delivery, dsRNA can be injected into tissue site or administered systemically.Introducing into cells in vitro includes methods known in the art, such as electroporation and lipofection.Additional approaches are described herein or known in the art.

[0055] The terms "silence," "inhibit expression," "downregulate expression," and "suppress expression," as they refer to the TTR gene, refer herein to at least partial suppression of expression of the TTR gene, as manifested by a decrease in the amount of mRNA that can be isolated and / or detected from a first cell or group of cells that is transcribed and treated to inhibit expression of the TTR gene, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been so treated (control cells). The degree of inhibition is usually expressed as follows:

number

[0056] Alternatively, the degree of inhibition can be expressed in terms of the parameter that is functionally related to the expression of TTR gene, such as the amount of protein that is coded by TTR gene secreted by cell, or the number of cells that show a certain phenotype, such as apoptosis.In principle, the expression of TTR gene can be silenced in any cell that expresses the target, either constitutively or by genome engineering, and by any suitable assay.However, when reference is required to determine whether a certain dsRNA inhibits the expression of TTR gene to a certain degree and therefore falls within the scope of the present invention, the assay provided in the following examples serves as such reference.

[0057] For example, in certain cases, expression of the TTR gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of a double-stranded oligonucleotide featured in the present invention. In some embodiments, the TTR gene is suppressed by at least about 60%, 70%, or 80% by administration of a double-stranded oligonucleotide featured in the present invention. In some embodiments, the TTR gene is suppressed by at least about 85%, 90%, or 95% by administration of a double-stranded oligonucleotide featured in the present invention.

[0058] As used herein in the context of TTR expression, the terms "treat," "treatment," and the like refer to the alleviation or alleviation of a pathological process mediated by TTR expression. In the context of the present invention, when referring to any of the other conditions listed herein below (other than a pathological process mediated by TTR expression), the terms "treat," "treatment," and the like refer to the alleviation or alleviation of at least one symptom associated with such a condition, or the slowing or reversal of the progression of such a condition, such as the slowing of the progression of TTR amyloidosis, such as FAP. Symptoms of TTR amyloidosis include neuropathy (e.g., sensory impairment, distal hypoesthesia), autonomic neuropathy (e.g., gastrointestinal disorders such as gastric ulcers or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic failure, cardiomyopathy, vitreous opacities, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial neuropathy, and corneal lattice degeneration.

[0059] 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 a pathological process mediated by TTR expression, or an overt symptom of a pathological process mediated by TTR expression. The specific amount that is therapeutically effective can be readily determined by an ordinary practitioner and may vary depending on factors known in the art, such as the type of pathological process mediated by TTR expression, the patient's medical history and age, the stage of the pathological process mediated by TTR expression, and the administration of other drugs that combat pathological processes mediated by TTR expression.

[0060] As used herein, "pharmaceutical composition" comprises a pharmacologically effective amount of dsRNA and a pharmaceutically acceptable carrier.As used herein, "pharmacologically effective amount", "therapeutically effective amount", or simply "effective amount" refers to the amount of RNA that is effective to produce the desired pharmacological, therapeutic, or inhibitory results.For example, if a clinical treatment is considered effective when there is at least a 25% reduction in measurable parameters related to a disease or disorder, the therapeutically effective amount of a drug for treating the disease or disorder is the amount required to cause at least a 25% reduction in the parameter.For example, a therapeutically effective amount of a dsRNA targeting TTR can reduce the serum level of TTR by at least 25%.In another example, a therapeutically effective amount of a dsRNA targeting TTR can improve liver function or kidney function by at least 25%.

[0061] The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, with cornstarch and alginic acid being suitable disintegrating agents. Binders can include starch and gelatin, while lubricants, if present, will generally be magnesium stearate, stearic acid, or talc. If desired, tablets can be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.

[0062] As used herein, a "transformed cell" is a cell into which a vector has been introduced so that a dsRNA molecule can be expressed.

[0063] II. double-stranded ribonucleic acid (dsRNA) As described in more detail herein, the present invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting expression of the TTR gene in cells or mammals, e.g., in humans suffering from amyloidosis, wherein the dsRNA comprises an antisense strand having a region of complementarity complementary to at least a portion of the mRNA formed upon expression of the TTR gene, the region of complementarity being less than 30 nucleotides in length, generally 19-24 nucleotides in length, and wherein the dsRNA, upon contact with cells expressing the TTR gene, inhibits expression of the TTR gene by at least 30%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods such as Western blot. Expression of the TTR gene may be reduced by at least 30%, as measured by assays described in the Examples below. For example, expression of the TTR gene in cell cultures, such as Hep3B cells, may be assayed by measuring TTR mRNA levels, e.g., by bDNA or TaqMan assays, or by measuring protein levels, e.g., by ELISA assays. The dsRNA of the present invention may further comprise one or more single-stranded nucleotide overhangs.

[0064] The dsRNA can be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc., as further described below. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary, generally completely complementary, to a target sequence derived from the sequence of the mRNA formed during expression of the TTR gene, and the other strand (the sense strand) contains a region complementary to the antisense strand, such that the two strands hybridize to form a double-stranded structure when combined under suitable conditions. Generally, the double-stranded structure is 15 to 30, or 25 to 30, or 18 to 25, or 19 to 24, or 19 to 21, or 19, 20, or 21 base pairs in length. In one embodiment, the double-stranded structure 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 lengths of the duplexes may be the same or different.

[0065] Each strand of the dsRNA of the present invention is generally 15 to 30, or 18 to 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each strand is 25 to 30 nucleotides in length. Each strand of the duplex may be the same length or different lengths. When two different siRNAs are used in combination, the length of each strand of each siRNA may be the same or different.

[0066] The dsRNA of the present invention can comprise one or more single-stranded overhangs 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 an overhang of 1 to 10 nucleotides at each of the 3'-end and 5'-end of the sense strand.In a further embodiment, the sense strand of the dsRNA has an overhang of 1 to 10 nucleotides at each of the 3'-end and 5'-end of the antisense strand.

[0067] dsRNA with at least one nucleotide overhang can have unexpectedly better inhibitory properties than its blunt-ended counterpart.In some embodiments, the presence of only one nucleotide overhang enhances the interference activity of dsRNA without affecting its overall stability.The dsRNA with only one overhang has been found to be particularly stable and effective in vivo and in various cells, cell culture medium, blood, and serum.Generally, the single-stranded overhang is located at the 3'-end of the antisense strand, or alternatively, at the 3'-end of the sense strand.The dsRNA can also have a blunt end, which is generally located at the 5'-end of the antisense strand.Such dsRNA can have improved stability and inhibitory activity, and therefore allows for low dosage, i.e., administration of less than 5 mg per kg of recipient body weight per day.Generally, the antisense strand of the dsRNA has a nucleotide overhang at its 3'-end, and its 5'-end is blunt. In another embodiment, one or more of the nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0068] In one embodiment, TTR gene is human TTR gene.In certain embodiments, the sense strand of said dsRNA is one of the sense sequences from table 3A, 3B, 4, 6A, 6B or 7, and said antisense strand is one of the sense sequences from table 3A, 3B, 4, 6A, 6B or 7.Alternative antisense agents that target any of the target sequences provided in table 3A, 3B, 4, 6A, 6B or 7 can be easily determined by using target sequence and adjacent TTR sequence.

[0069] Those skilled in the art are well aware that dsRNAs with a double-stranded structure of 20-23, and especially 21, base pairs have been praised as being 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 similarly effective. Due to the nature of the oligonucleotide sequences provided in Tables 3A, 3B, 4, 6A, 6B, or 7 in the above embodiments, the dsRNAs featured in the present invention can comprise at least one strand of the length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences in Tables 3A, 3B, 4, 6A, 6B, or 7, minus a few nucleotides at one or both ends, can be similarly effective compared to the above dsRNAs. Thus, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences in Tables 3, 4, 6, or 7, whose ability to inhibit TTR gene expression in the assays described herein below differs by no more than 5, 10, 15, 20, 25, or 30% from a dsRNA containing the entire sequence, are contemplated by the present invention. Furthermore, dsRNAs that cleave within a desired TTR target sequence can be readily generated using the corresponding TTR antisense sequence and complementary sense sequence.

[0070] In addition, the dsRNA provided in Table 3A, 3B, 4, 6A, 6B, or 7 identifies a site in TTR that is susceptible to RNAi-based cleavage.Therefore, 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 at any point within the mRNA that is complementary to the antisense strand of the first dsRNA.Such a second dsRNA generally consists of at least 15 consecutive nucleotides from one of the sequences provided in Table 3A, 3B, 4, 6A, 6B, or 7, linked to additional nucleotide sequences taken from the region adjacent to the selected sequence in the TTR gene.

[0071] The dsRNA featured in the present invention may contain one or more mismatches with target sequence.In one embodiment, the dsRNA featured in the present invention contains three or less mismatches.When the antisense strand of dsRNA contains mismatches with target sequence, it is preferable that the range of mismatches is not located at the center of the complementary region.When the antisense strand of dsRNA contains mismatches with target sequence, it is preferable that the mismatches are limited to 5 nucleotides from either end, for example, 5, 4, 3, 2 or 1 nucleotide from either the 5' or 3' end of the complementary region.For example, for a 23-nucleotide dsRNA strand that is complementary to a region of TTR gene, the dsRNA generally does not contain any mismatches within the central 13 nucleotides.The method described in the present invention can be used to determine whether the dsRNA that contains mismatches with target sequence is effective in inhibiting the expression of TTR gene. Consideration of the effectiveness of mismatched dsRNA in inhibiting expression of the TTR gene is important, especially when specific regions of complementarity within the TTR gene are known to have polymorphic sequence diversity within the population.

[0072] qualification In another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids featured in the present invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, SLet et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Specific examples of dsRNA compounds useful in the present invention include dsRNAs that contain modified backbones or do not contain natural internucleoside linkages. As defined herein, dsRNAs with 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 referred to in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered oligonucleosides.

[0073] Modified dsRNA backbones include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates, including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate, including 3'-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate with normal 3'-5' bond, their analogs with 2'-5' bond, and those with reverse polarity, where adjacent pairs of nucleoside units are bonded from 3'-5' to 5'-3' or from 2'-5' to 5'-2'.Also include various salts, mixed salts, and free acid forms.

[0074] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include U.S. Patent 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, Nos. 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 incorporated herein by reference.

[0075] Modified dsRNA backbones that do not contain phosphorus atoms include backbones formed by single-stranded alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more single-stranded heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 components.

[0076] Representative U.S. patents that teach the preparation of the above oligonucleosides include U.S. Patent 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, Nos. 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 incorporated herein by reference.

[0077] In other suitable dsRNA mimics, both the sugar and internucleoside bond of nucleotide unit, i.e., backbone, are replaced with novel groups. Base unit is maintained to hybridize with appropriate nucleic acid target compound. One such oligomeric compound, dsRNA mimic, that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of dsRNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleic acid bases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide part of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, US Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0078] Other embodiments of the invention are dsRNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- (known as the methylene(methylimino) or MMI backbone), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the natural phosphodiester backbone is represented as --O--P--O--CH2--) of the aforementioned U.S. Pat. No. 5,489,677, and the amide backbones of the aforementioned U.S. Pat. No. 5,602,240.

[0079] Modified dsRNAs can also contain one or more substituted sugar moieties.Preferred dsRNAs include 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2 is particularly preferred, where n and m are from 1 to about 10. Other preferred dsRNAs include at the 2'-position: C1 to C 10The modified dsRNA may be one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intervening agent, group for improving the pharmacokinetic properties of dsRNA, or group for improving the pharmacodynamic properties of dsRNA, and other substituents with similar properties. Preferred modifications include 2'-methoxyethoxy (2'-O--CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., alkoxy-alkoxy group. Preferred further modifications include the 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH2)2ON(CH3)2 group, described herein below in the Examples, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, also described herein below in the Examples.

[0080] Other preferred modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on a dsRNA, particularly the 3' position of the sugar on the 3'-terminal nucleotide or in a 2'-5'-linked dsRNA, and the 5' position on a 5'-terminal nucleotide. dsRNAs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include 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, and 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, some of which are commonly owned by the present application, each of which is incorporated herein by reference in its entirety.

[0081] dsRNA may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include purine bases, adenine (A) and guanine (G), and pyrimidine bases, thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, 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-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), ... 5-hydroxymethylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethylcytosine, Other synthetic and naturally occurring nucleobases include uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and 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-deazaadenine, 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, JL, ed. John Wiley & Sons, 1990, those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed in Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, ST 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 present invention.These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp.276-278), and is an exemplary base substitution, and is particularly preferred when combined with 2'-O-methoxyethyl sugar modification.

[0082] Representative U.S. patents that teach the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include the above-described U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205, 5,130,300, 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 incorporated herein by reference, and U.S. Patent No. 5,750,692, which is also incorporated herein by reference.

[0083] conjugate Another modification of the dsRNA of the invention involves chemically linking to the dsRNA one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the dsRNA.Such moieties include cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., Proc. Natl. Acid. Sci. USA, 1994, 86, 6553-6556), cholic acid (Manoharan et al., Bior. Med. Chem. Let., 1994, 4, 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bior. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Be al., EMBO J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al. al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or lipid moiety such as octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0084] Representative United States patents that teach the preparation of such dsRNA conjugates include U.S. Patent 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, 5,4 No. 14,077, No. 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4,605,735, No. 4,667,025, No. 4,76 No. 2,779, No. 4,789,737, No. 4,824,941, No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5,082,830, No. 5,112 ,963, No. 5,214,136, No. 5,082,830, No. 5,112,963, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5,262, No. 536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. 5,371,241, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,4 75, 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 incorporated herein by reference.

[0085] Not all positions of a compound need be uniformly modified; indeed, one or more of the above modifications can be incorporated into a single compound, or even into a single nucleoside within a dsRNA.The present invention also includes dsRNA compounds that are chimeric compounds.In the context of the present invention, a "chimeric" dsRNA compound or "chimera" refers to a dsRNA compound, particularly a dsRNA that contains two or more chemically distinct regions, each of which consists 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 that is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to target nucleic acid.Another region of dsRNA can serve as a substrate for enzymes that can cleave RNA:DNA or RNA:RNA hybrids.As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H thus results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Therefore, when chimeric dsRNAs are used, comparable results can often be obtained with shorter dsRNAs compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region.

[0086] The cleavage of RNA target can be routinely detected by gel electrophoresis, and if necessary, by related nucleic acid hybridization techniques known in the art.The cleavage site of dsRNA in the target mRNA can generally be determined by methods known to those skilled in the art, for example, by the 5'-RACE method described in Soutschek et al., Nature; 2004, Vol.432, pp.173-178 (incorporated herein by reference for all purposes).In one embodiment, by using the 5'-RACE method described by Soutschek et al., it was confirmed that ALN-18328 cleaves 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 one embodiment, it was confirmed that ALN-18328 does not cleave 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.

[0087] In certain cases, the dsRNA can be modified by non-ligand group.Many non-ligand molecules have been conjugated to dsRNA to enhance the activity, cellular distribution or cellular uptake of dsRNA, and the procedures for such conjugation are available in scientific literature.Such non-ligand moieties include cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol 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), phospholipids, such as 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), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such dsRNA conjugates are listed above.Typical conjugation protocol involves the synthesis of dsRNA with amino linker at one or more positions of sequence.Then, amino group is reacted with the molecule to be conjugated using suitable coupling or activation reagent.Conjugation reaction can be carried out either with the dsRNA still bound to solid support or after cleavage of dsRNA in solution phase.Usually, dsRNA conjugate is purified by HPLC to obtain pure conjugate.

[0088] dsRNA encoded by the vector In another embodiment, TTR dsRNA molecules are expressed from transcription units inserted into DNA or RNA vectors (see, for example, 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. Patent No. 6,054,299). These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, and can be introduced and inherited as transgenes that are integrated into the host genome. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0089] Each strand of dsRNA can be transcribed using the promoter on two separate expression vectors and then co-transfected into target cells.Alternatively, each strand of dsRNA can be transcribed using the promoter located on the same expression plasmid.In one embodiment, dsRNA is expressed as an inverted repeat that is joined by linker polynucleotide sequence, so that dsRNA has stem-and-loop structure.

[0090] Recombinant dsRNA expression vector is generally DNA plasmid or virus vector.The virus vector of dsRNA expression can be based on but not limited to adeno-associated virus (referring to Muzyczka et al., Curr.Topics Micro.Immunol.(1992)158:97-129 for general information), adenovirus (referring to, for example, Berkner et al., BioTechniques(1998)6:616), Rosenfeld et al.(1991,Science252:431-434) and Rosenfeld et al.(1992),Cell68:143-155)) or alphavirus and other known in the art.Retroviruses have been used to introduce various genes into many different cell types in vitro and / or in vivo, including epithelial cells (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 87:6141-6145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1992, Proc. Natl. Acad. Sci. USA 89:1014-1016; 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. al.,1992,Human Gene Therapy 3:641-647, Dai et al.,1992,Proc.Natl.Acad.Sci.USA 89:10892-10895, Hwu et al. (See, e.g., Comette et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application No. WO 89 / 07136; PCT Application No. WO 89 / 02468; PCT Application No. WO 89 / 05345; and PCT Application No. WO 92 / 07573.) Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of cells 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 adenovirus vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rats, hamsters, dogs, and chimpanzees) (Hsu et al., 1992, J. Infectious Disease, 166:769), which also has the advantage of not requiring mitotically active cells for infection.

[0091] Any viral vector that can accept the coding sequence of the dsRNA molecule to be expressed can be used, for example, the vector derived from adenovirus (AV), adeno-associated virus (AAV), retrovirus (for example, lentivirus (LV), rhabdovirus, murine leukemia virus), herpes virus, etc. The tropism of viral vector can be modified by using the pseudotype of the vector with envelope protein or other surface antigens from other viruses, or by appropriately replacing the capsid protein of different viruses.

[0092] For example, lentiviral vectors featured in the present invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors featured in the present invention can be engineered to target different cells by engineering the vector to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is referred to as AAV2 / 2. The serotype 2 capsid gene in an AAV2 / 2 vector can be replaced with a serotype 5 capsid gene to produce an AAV2 / 5 vector. Techniques for constructing AAV vectors expressing different capsid protein serotypes are within the skill of the art; see, e.g., Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0093] The selection of recombinant viral vector suitable for use in the present invention, the method for inserting nucleic acid sequence into said vector for expressing dsRNA, and the method for delivering said viral vector to target cell are within the skill of the art.For example, see Dornburg R (1995), Gene Therap.2:301-310, Eglitis MA (1988), Biotechniques 6:608-614, Miller AD (1990), Hum Gene Therap.1:5-14, Anderson WF (1998), Nature 392:25-30, and Rubinson DA et al., Nat.Genet.33:401-406, the disclosure of which is incorporated herein by reference in its entirety.

[0094] Viral vectors can be derived from AV and AAV. In one embodiment, the dsRNA featured in the present invention is expressed as two separate complementary single-stranded RNA molecules from a recombinant AAV vector, for example, with U6 or H1 RNA promoter or cytomegalovirus (CMV) promoter.

[0095] AV vectors suitable for expressing the dsRNA of the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

[0096] AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J.Virol. 61:3096-3101, Fisher KJ et al. (1996), J.Virol, 70:520-532, Samulski R et al. (1989), J.Virol. 63:3822-3826, U.S. Patent No. 5,252,479, U.S. Patent No. 5,139,941, International Patent Application No. WO 94 / 13788, and International Patent Application No. WO 93 / 24641, the entire disclosures of which are incorporated herein by reference.

[0097] The promoter that drives the expression of dsRNA in any of the DNA plasmids or viral vectors featured in the present invention can be eukaryotic RNA polymerase I (for example, ribosomal RNA promoter), RNA polymerase II (for example, CMV early promoter, or actin promoter, or U1 snRNA promoter), or generally RNA polymerase III promoter (for example, U6 snRNA or 7SK RNA promoter), or prokaryotic promoter, for example, T7 promoter, provided that expression plasmid also encodes the T7 RNA polymerase that is required for transcription from T7 promoter.This promoter can also direct the expression of transgene into pancreas (for example, see the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc.Natl.Acad.Sci.USA 83:2511-2515)).

[0098] Furthermore, transgene expression can be precisely regulated by using inducible regulatory sequences and expression systems, such as regulatory sequences sensitive to specific physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Suitable inducible expression systems for controlling transgene expression in cells or mammals include regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the dsRNA transgene. Those skilled in the art will be able to select appropriate regulatory / promoter sequences based on the intended use of the dsRNA transgene.

[0099] 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 transient expression of dsRNA molecules.Such vectors can be repeatedly administered as needed.Once expressed, dsRNA binds to target RNA and regulates its function or expression.The delivery of dsRNA expressing vectors can be systemically, such as by intravenous or intramuscular administration, by administering to target cells explanted from a patient and then reintroducing them into the patient, or by any other means that allows introduction into desired target cells.

[0100] dsRNA-expressing DNA plasmids are typically transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdown targeting different regions of a single TTR gene or multiple TTR genes over a period of one week or more are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated using a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection of ex vivo cells can be ensured using a marker that provides transfected cells with resistance to certain environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0101] In addition, TTR-specific dsRNA molecules can be inserted into vectors and used as gene therapy vectors for human patients.Gene therapy vectors can be delivered to subjects by, for example, intravenous injection, local administration (see U.S. Patent No. 5,328,470), or stereotaxic injection (see, for example, Chen et al. (1994) Proc.Natl.Acad.Sci.USA 91:3054-3057).The pharmaceutical preparation of gene therapy vectors can comprise the gene therapy vector in an acceptable diluent, or can comprise a slow-release matrix in which gene delivery vehicle is embedded.Alternatively, when complete gene delivery vectors can be produced intact from recombinant cells, for example, retroviral vectors, pharmaceutical preparations can comprise one or more cells that produce gene delivery systems.

[0102] III. Pharmaceutical Compositions Containing dsRNA In one embodiment, the present invention provides a pharmaceutical composition comprising the dsRNA described herein and a pharmaceutically acceptable carrier.The pharmaceutical composition comprising the dsRNA is useful for treating diseases or disorders related to the expression or activity of the TTR gene, such as pathological processes mediated by the expression of TTR.Such pharmaceutical compositions are formulated based on the mode of delivery.One example is a composition formulated for systemic administration via parenteral administration, for example, by intravenous (IV) delivery.Another example is a composition formulated for direct delivery to the brain parenchyma, for example, by injection into the brain by continuous pump infusion.

[0103] The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of the TTR gene.

[0104] Generally, a suitable dosage of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram of recipient body weight per day, generally in the range of 1 to 50 mg per kilogram of 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.

[0105] In one embodiment, the dosage is 0.01-0.2mg / kg.For example, the dsRNA can be administered at the dosage of 0.01mg / kg, 0.02mg / kg, 0.3mg / kg, 0.04mg / kg, 0.05mg / kg, 0.06mg / kg, 0.07mg / kg, 0.08mg / kg, 0.09mg / kg, 0.10mg / kg, 0.11mg / kg, 0.12mg / kg, 0.13mg / kg, 0.14mg / kg, 0.15mg / kg, 0.16mg / kg, 0.17mg / kg, 0.18mg / kg, 0.19mg / kg or 0.20mg / kg.

[0106] In one embodiment, the dosage is 0.005mg / kg to 1.628mg / kg.For example, the dsRNA can be administered at the dose of 0.0059mg / kg, 0.0295mg / kg, 0.0590mg / kg, 0.163mg / kg, 0.543mg / kg, 0.5900mg / kg or 1.628mg / kg.

[0107] In one embodiment, the dosage is 0.2mg / kg to 1.5mg / kg.For example, the dsRNA can be administered at the dosage of 0.2mg / kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 0.6mg / kg, 0.7mg / kg, 0.8mg / kg, 0.9mg / kg, 1mg / kg, 1.1mg / kg, 1.2mg / kg, 1.3mg / kg, 1.4mg / kg or 1.5mg / kg.

[0108] The pharmaceutical composition can be administered once a day, or the dsRNA can be administered as 2, 3 or more sub-doses at appropriate intervals throughout the day, and can also be administered by continuous infusion or controlled-release preparation delivery.In this case, the dsRNA contained in each sub-dosage must be correspondingly smaller so as to achieve the total daily dosage.For example, the dosage unit can be formulated for delivery over several days, using conventional sustained-release preparations, which provide the sustained release of the dsRNA over several days.Sustained-release preparations are well known in the art, and are particularly useful for the delivery of drugs to specific sites, such as the sites that can be used with the drugs of the present invention.In this embodiment, the dosage unit comprises a corresponding number of daily doses.

[0109] The effect of a single dose on TTR levels is long-lasting, so that subsequent doses are administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart, or no more than 5, 6, 7, 8, 9, or 10 weeks apart.

[0110] Those skilled in the art will understand that certain factors, including but not limited to, the severity of disease or disorder, previous treatment, overall health and / or age of subject and other existing diseases, can affect the dosage and timing required to effectively treat subject.In addition, the treatment of subject with therapeutically effective amount of composition can comprise single treatment or a series of treatments.Effective dosage and in vivo half-life of each dsRNA encompassed by the present invention can be estimated by conventional method or by using suitable animal model in vivo test as described elsewhere herein.

[0111] With the progress of mouse genetics, many mouse models have been produced for the study of various human diseases, such as the pathological process mediated by the expression of TTR.This model is used for the in vivo test of dsRNA and to determine the therapeutically effective dose.Such a suitable mouse model is, for example, the mouse that contains the plasmid that expresses human TTR.Another suitable mouse model is the transgenic mouse that carries the transgene that expresses human TTR.

[0112] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compositions featured in the present invention generally lies within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the present invention, a therapeutically effective dose can be initially estimated from cell culture assays. Dosages can be formulated in animal models to achieve a circulating plasma concentration range of the compound, or, if appropriate, the polypeptide product of the target sequence (e.g., achieving a reduction in the concentration of the polypeptide), that includes the IC50 (i.e., the concentration of the test compound that 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. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0113] The dsRNA featured in the present invention can be administered in combination with other known drugs that are effective in treating the pathological process that is mediated by the expression of target gene.In any case, the administering physician can adjust the amount and timing of dsRNA administration based on the results obtained by using the standard efficacy measurement known in the art or as described herein.

[0114] Administration The present invention also includes pharmaceutical compositions and preparations that contain the dsRNA compounds of the present invention.Pharmaceutical compositions of the present invention can be administered in many ways, depending on whether local or systemic treatment is desired and the area to be treated.Administration can be topical, including by nebulizer, for example, by inhalation or insufflation of powder or spray, intratracheal, intranasal, epidermal and transdermal, oral or parenteral.Parenteral administration includes intravenous, intraarterial, subcutaneous, intracavity or intramuscular injection or infusion, or intracranial, for example, intraparenchymal, intrathecal or intraventricular administration.

[0115] The dsRNA can be delivered in a manner that targets a particular tissue, such as the liver (eg, hepatocytes of the liver).

[0116] The present invention comprises pharmaceutical compositions that can be delivered by direct injection into brain.This injection can be by stereotaxic injection into specific brain region (for example, substantia nigra, cortex, hippocampus, striatum or globus pallidus), and this dsRNA can be delivered to multiple regions of central nervous system (for example, multiple regions of brain and / or spinal cord).This dsRNA can be delivered to diffuse region of brain (for example, diffuse delivery to the cortex of brain).

[0117] In one embodiment, dsRNA targeting TTR can be delivered via a cannula or other delivery device with one end implanted in tissues such as the brain (for example, the substantia nigra, cortex, hippocampus, striatum, or globus pallidus of the brain).The cannula can be connected to a reservoir of the dsRNA composition.Inflow or delivery can be mediated by a pump, for example, an osmotic pump or a minipump such as the Alzet pump (Durect, Cupertino, CA).In one embodiment, the pump and reservoir are implanted in an area away from the tissue, for example, in the abdomen, and delivery is achieved by a conduit leading from the pump or reservoir to the release site.The infusion of the dsRNA composition into the brain can be for several hours or for several days, for example, 1, 2, 3, 5, or 7 days or more.Devices for delivery to the brain are described, for example, in U.S. Patent Nos. 6,093,180 and 5,814,014.

[0118] 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, etc. may be necessary or desirable. Coated condoms, gloves, etc. may also be useful. Suitable topical formulations include those in which the dsRNA featured in the present invention is mixed 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., dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine), cationic (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The dsRNA of the present invention can be encapsulated in liposome or can be complexed with it, particularly cationic liposome.Alternatively, dsRNA can be complexed with lipid, particularly cationic lipid.Suitable fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicapric acid, tricapric acid, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine or C 1~10 These include, but are not limited to, alkyl esters (e.g., isopropyl myristate (IPM)), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0119] Liposomal formulation In addition to microemulsions, many other organized surfactant structures have been studied and used for drug formulation. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted great interest from the perspective of drug delivery due to their specificity and duration of action. As used in the present invention, the term "liposome" refers to a vesicle of amphiphilic lipids arranged in one or more spherical bilayers.

[0120] Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes cannot fuse with the cell wall as efficiently, but are taken up by macrophages in vivo.

[0121] To cross intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. It is therefore desirable to use liposomes that are highly deformable and can pass through such micropores.

[0122] Additional advantages of liposomes include that liposomes derived from natural phospholipids are biocompatible and biodegradable, liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs, and 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, NY, volume 1, p. 245). Important considerations in preparing liposome formulations are the surface charge of the lipid, the size of the vesicles, and the aqueous volume of the liposomes.

[0123] Liposomes are useful for transporting and delivering active ingredients to the site of action. Because liposome membranes are structurally similar to biological membranes, when liposomes are applied to tissues, they begin to fuse with cell membranes, and as the fusion between liposomes and cells progresses, the contents of the liposomes are released into the cells, where the active agent can act.

[0124] Liposomal formulations have been the focus of extensive research as a delivery mode for many drugs. There is growing evidence that liposomes offer several advantages over other formulations for topical administration. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide range of drugs, both hydrophilic and hydrophobic, to the skin.

[0125] Several reports have detailed the ability of liposomes to deliver drugs, including high molecular weight DNA, to the skin. Painkillers, antibodies, hormones, and compounds containing high molecular weight DNA have been administered to the skin. Most applications have resulted in targeting of the upper epidermis.

[0126] Liposomes are broadly divided into two classes. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA / liposome complexes bind to the negatively charged cell surface and are internalized into endosomes. The acidic pH within the endosome causes the liposomes to rupture, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0127] pH-sensitive or negatively charged liposomes trap DNA rather than complexing with it. Because both DNA and lipids are similarly charged, repulsion occurs rather than complexation. Nevertheless, some DNA is trapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cultured cell monolayers. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0128] One major type of liposome composition includes phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean phosphatidylcholine and egg phosphatidylcholine. Another type is formed from a mixture of phospholipids, phosphatidylcholine, and / or cholesterol.

[0129] Several studies have evaluated the topical delivery of liposomal preparations to the skin. Applying liposomes containing interferon to the skin of guinea pigs has resulted in the alleviation of cutaneous herpes sores, while the delivery of interferon via other means (e.g., as a solution or emulsion) has been ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Furthermore, further studies have tested the effectiveness of administering interferon as part of a liposomal preparation compared to administering interferon using an aqueous system, and concluded that liposomal preparations are superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0130] Nonionic liposomal systems have also been investigated to determine the feasibility of delivering drugs to the skin, particularly in systems containing nonionic surfactants and cholesterol. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A to the dermis of mouse skin. Results showed that such nonionic liposomal systems were effective in promoting the deposition of cyclosporine A into different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4, 6, 466).

[0131] Liposomes also include "sterically stabilized" liposomes, which, as used herein, refers to liposomes containing one or more specialized lipids that, when incorporated into the liposome, result in improved circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the lipid moiety forming the liposome vesicle is (A) monosialoganglioside G M1or (B) are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that the increased circulation half-life of sterically stabilized liposomes, at least for those containing gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to 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).

[0132] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) describe the use of monosialoganglioside G M1 reported the ability of (1) sphingomyelin, and (2) ganglioside G to improve the blood half-life of liposomes. These findings are further explained by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and International Publication No. WO 88 / 04924, both by Allen et al., report the ability of (1) sphingomyelin, and (2) ganglioside G to improve the blood half-life of liposomes. M1 Alternatively, liposomes containing galactocerebroside sulfate are disclosed. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in International Publication WO 97 / 13499 (Lim et al.).

[0133] Many liposomes containing lipids derivatized with one or more hydrophilic polymers and their preparation methods are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describe liposomes containing 2C1215G, a nonionic surfactant containing a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) point out that hydrophilic coating of polystyrene particles with polymer glycols significantly increases their blood half-life. Synthetic phospholipids modified by the attachment of carboxylic acid groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Patent Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments showing that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have a significantly increased blood circulation half-life. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended these observations to other PEG-derivatized phospholipids, such as DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes with covalently bound PEG moieties on their outer surface are described in European Patent EP 0 445 131 B1 and International Publication WO 90 / 04384 (Fisher). Liposomal compositions containing 1-20 mole percent PE derivatized with PEG, and methods for their use, have been 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 EP 0 496 813 B1). Liposomes containing many other lipid-polymer conjugates are disclosed in International Publication No. WO 91 / 05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.), and International Publication No. WO 94 / 20073 (Zalipsky et al.).Liposomes containing PEG-modified ceramide lipids are described in International Publication WO 96 / 10391 (Choi et al.). U.S. Patent No. 5,540,935 (Miyazaki et al.) and U.S. Patent No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes whose surfaces can be further derivatized with functional moieties.

[0134] Many liposomes containing nucleic acids are known in the art. International Publication WO 96 / 40062 by Thierry et al. discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. Patent No. 5,264,221 by Tagawa et al. discloses protein-bound liposomes, and claims that the contents of such liposomes can include dsRNA. U.S. Patent No. 5,665,710 by Rahman et al. describes a specific method for encapsulating oligodeoxynucleotides in liposomes. International Publication WO 97 / 04787 by Love et al. discloses liposomes containing dsRNA that target raf gene.

[0135] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets, which are highly deformable and can easily penetrate pores smaller than the droplets. Transfersomes are adaptable to the environment in which they are used, for example, self-optimizing (adapting to the shape of skin pores), self-repairing, often reaching their target without fragmentation, and often self-loading. To create transfersomes, a surface edge activator, usually a surfactant, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0136] Surfactants find a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head group") provides the most useful means for classifying the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0137] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceuticals and cosmetics and are usable over a wide range of pH values. Generally, their HLB values ​​range from 2 to approximately 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. Polyoxyethylene surfactants are the most common members of the nonionic surfactant class.

[0138] If the surfactant molecule carries a negative charge when 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 alkyl sulfates and soaps.

[0139] 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. Quaternary ammonium salts are the most commonly used members of this class.

[0140] 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.

[0141] The use of surfactants in drugs, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0142] nucleic acid lipid particles In one embodiment, the TTR dsRNA featured in the present invention is fully encapsulated in a lipid formulation to form SPLP, pSPLP, SNALP, or other nucleic acid-lipid particles. As used herein, the term "SNALP" refers to stable nucleic acid-lipid particles, including SPLP. As used herein, the term "SPLP" refers to nucleic acid-lipid particles containing plasmid DNA encapsulated within lipid vesicles. SNALP and SPLP typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). SNALP and SPLP exhibit long circulation life after intravenous (iv) injection and accumulate at distant sites (e.g., sites physically distant from the administration site), making them very useful for systemic application. SPLPs include "pSPLP," which includes the complex of an encapsulated condensing agent and nucleic acid described in PCT Publication WO 00 / 03683. The particles of the present invention typically have an average 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, and most typically about 70 to about 90 nm, and are substantially non-toxic. Furthermore, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, and 6,815,432, and PCT Publication WO 96 / 40964.

[0143] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) will be within the range of about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1.

[0144] Examples of cationic lipids include N,N-dioleyl-N,N-dimethyl chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethyl chloride (DOTAP), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy (Dilinoleylcarba 1,2-Dilinoleyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 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), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-di Oxolane (DLin-K-DMA), or their analogs, (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-yl 4-(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 about 20 mol% to about 50 mol%, or about 40 mol% of the total lipid present in the particle.

[0145] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid siRNA nanoparticles. The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0146] In one embodiment, the lipid siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, 10% PEG-C-DOMG (mol percent), with a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0147] Non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine (DOPE). The lipid may be an anionic or neutral lipid, including, but not limited to, 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid, when cholesterol is included, may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0148] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol%, or about 2 mol% of the total lipid present in the particles.

[0149] In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, for example, in an amount of about 10 mol % to about 60 mol %, or about 48 mol %, of the total lipid present in the particle.

[0150] LNP01 In one embodiment, lipid-siRNA nanoparticles (i.e., LNP01 particles) can be prepared using the lipidoid ND98-4HCl (MW1487) (Formula 1), cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids). Stock solutions 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 mixed in a molar ratio of, for example, 42:48:10. The combined lipid solution can then be mixed with an aqueous siRNA solution (e.g., in sodium acetate (pH 5)) to a final ethanol concentration of approximately 35-45% and a final sodium acetate concentration of approximately 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cutoff) using, for example, a thermobarrel extruder such as the Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged, for example, with 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. [ka] LNP01 formulations are described, for example, in International Application Publication No. WO 2008 / 042973, which is incorporated herein by reference.

[0151] Further exemplary lipid siRNA formulations are as follows: [Table 1-1] [Table 1-2]

[0152] LNP09 formulations and formulations containing XTC are described, for example, in U.S. Provisional Application No. 61 / 239,686, filed September 3, 2009, which is incorporated herein by reference. LNP11 formulations and formulations containing MC3 are described, for example, in U.S. Provisional Application No. 61 / 244,834, filed September 22, 2009, which is incorporated herein by reference.

[0153] Formulations prepared by either standard or non-extrusion methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. They should be whitish, translucent solutions without aggregates or sediment. The particle size and size distribution of lipid nanoparticles can be measured by light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be approximately 20-300 nm in size, such as 40-100 nm. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as in the entrapped fraction, is estimated using a dye exclusion assay. Samples of the formulated siRNA are incubated with Ribogreen (Molecular The siRNA can be incubated with a dye that binds to RNA, such as surfactant-containing dyes (Probes). The total siRNA in the formulation can be determined by comparing the signal from the sample containing the surfactant with a standard curve. The captured fraction is determined by subtracting the siRNA "free" content (measured by the signal in the absence of surfactant) from the total siRNA content. The percentage of captured siRNA is typically greater than 85%. For SNALP formulations, 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 preferred range is typically at least about 50 nm to at least about 110 nm, at least about 60 nm to at least about 100 nm, or at least about 80 nm to at least about 90 nm.

[0154] Oral administration compositions and preparations include powder or granules, microparticles, nanoparticles, suspensions or solutions in water or hydrophobic medium, capsules, gel capsules, sachets, tablets or minitablets.Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.In some embodiments, oral preparations are those in which the dsRNA of the present invention is administered in combination with one or more penetration enhancers, surfactants and chelating agents.Suitable surfactants include fatty acids and / or their esters or salts, bile acids and / or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycolic 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, dicapric acid, tricapric acid, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, a combination of penetration enhancers is used, such as a fatty acid / salt combined with a bile acid / salt. An exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Additional penetration enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The dsRNA featured in the present invention can be orally delivered in granular form, including spray-dried particles, or can be complexed to form micro- or nanoparticles.dsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethylene glycols (PEG), and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methyl cyanoacrylate), poly(ethyl cyanoacrylate), poly(butyl cyanoacrylate), poly(isobutyl cyanoacrylate), poly(isohexyl cyanoacrylate), DEAE-methacrylate, DEAE-hexyl acrylate, DEA E-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations for dsRNA and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. Patent Publication No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference in its entirety.

[0155] 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.

[0156] The pharmaceutical compositions of the present invention include, but are not limited to, solution, emulsion and liposome-containing formulations.These compositions can be made from various components, including, but not limited to, preformed liquid, self-emulsifying solid and self-emulsifying semi-solid.The formulation that targets the liver is particularly preferred when treating liver damage such as liver cancer.

[0157] The pharmaceutical preparation of the present invention can be conveniently presented in unit dosage form, and can be prepared by the conventional technique well known in pharmaceutical industry.This technique includes the step of combining active ingredient with one or more pharmaceutical carriers or one or more excipients.Generally, this preparation is prepared by uniformly and intimately combining active ingredient with liquid carrier or finely divided solid carrier, or both, and then if necessary, shaping this product.

[0158] The compositions of the present invention can be formulated into any of many possible dosage forms, including, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, hydrophobic, or mixed media. Aqueous suspensions can further contain substances that increase the viscosity of the suspension, including, for example, sodium carboxymethylcellulose, sorbitol, and / or dextran. Suspensions can also contain stabilizers.

[0159] emulsion The composition of the present invention can be prepared and formulated as emulsion.Emulsion is typically a multiphase system in which one liquid is dispersed in another liquid, usually in the form of droplets with a diameter of more than 0.1 μm (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 2, p.335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often two-phase systems containing two immiscible liquid phases intimately mixed and dispersed with each other. Generally, emulsions can be either water-in-oil (w / o) or oil-in-water (o / w) types. When the aqueous phase is finely divided and dispersed as minute droplets in the majority oil phase, the resulting composition is called a water-in-oil (w / o) emulsion. Alternatively, when the oil phase is finely divided and dispersed as minute droplets in the majority aqueous phase, the resulting composition is called an oil-in-water (o / w) emulsion. Emulsions can contain additional components in addition to the dispersed phase, as well as active drugs, which can be present as a solution in either the aqueous or oil phase or as a separate phase. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes, and antioxidants, may also be present in emulsions as needed.Pharmaceutical emulsions may also be multiple emulsions, containing more than two phases, such as 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 offer certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o / w emulsion surround small water droplets constitute w / o / w emulsions. Similarly, a system of oil droplets enclosed within globules of water stabilized in a continuous phase of oil provides an o / w / o emulsion.

[0160] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of an emulsion is well dispersed in the external or continuous phase, and this form is maintained by means of an emulsifier or the viscosity of the formulation. Either phase of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Other methods of emulsion stabilization require the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly 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, NY, volume 1, p. 199).

[0161] Synthetic surfactants, also known as surface active agents, have found widespread application in emulsion formulations and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, NY, 1988, volume 1, p. 199). Surfactants are typically amphiphilic, containing hydrophilic and hydrophobic moieties. The ratio of a surfactant's hydrophilic to hydrophobic properties, referred to as its hydrophilic / lipophilic balance (HLB), is a valuable tool in classifying and selecting surfactants during formulation preparation. Surfactants can be divided 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, NY, volume 1, p. 285).

[0162] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin, and acacia. Absorption bases are hydrophilic, meaning they can absorb water to form water-in-oil emulsions while still retaining their semisolid consistency, 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, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments, and non-polar solids such as carbon or glyceryl tristearate.

[0163] A wide variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion, including 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, NY, volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199).

[0164] Hydrophilic colloids, or hydrocolloids, include naturally occurring gums and synthetic polymers, such as polysaccharides (e.g., acacia, agar, alginate, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethyl cellulose and carboxypropyl cellulose), and synthetic polymers (e.g., carbomer, cellulose ethers, and carboxyvinyl polymers), which disperse or swell in water to form colloidal solutions that form strong interfacial films around droplets of the dispersed phase, stabilizing the emulsion by increasing the viscosity of the external phase.

[0165] Because emulsions often contain many components, such as carbohydrates, proteins, sterols, and phosphatides, which can readily support microbial growth, preservatives are often incorporated into these formulations. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration. Antioxidants used can be free radical scavengers, such as tocopherol, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents, such as ascorbic acid and sodium metabisulfite, as well as antioxidant synergists, such as citric acid, tartaric acid, and lecithin.

[0166] The application of emulsion formulations via the dermal, oral, and parenteral routes, as well as methods for their preparation, have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger, and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Emulsion formulations for oral delivery are widely used due to their ease of formulation and effectiveness in terms of absorption and bioavailability (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 199). Mineral oil-based laxatives, oil-soluble vitamins, and high-fat nutritional preparations are among the materials commonly administered orally as o / w emulsions.

[0167] In one embodiment of the present invention, the composition of dsRNA and nucleic acid is formulated as microemulsion.Microemulsion can be defined as a system of water, oil and amphiphilic substance, 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, NY, volume 1, p.245).Usually, microemulsion is the system that is prepared by first dispersing oil in aqueous surfactant solution, and then adding sufficient amount of fourth component, generally medium-chain alcohol, to form a transparent system. Thus, microemulsions have also been described as thermodynamically stable, isotropic, transparent dispersions of two immiscible liquids stabilized by an interfacial film 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 are generally prepared through the combination of three to five components, including oil, water, surfactant, cosurfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the nature of the oil and surfactant used, as well as the structure and geometrical folding of the polar head and hydrocarbon tail of the surfactant molecule (Schott, in: Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0168] The phenomenological approach using phase diagrams has been extensively studied, providing those skilled in the art with comprehensive knowledge of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p.335).Compared with traditional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in the formulation of spontaneously formed thermodynamically stable droplets.

[0169] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, nonionic surfactants, Brij 96, polyoxyethylene oleyl ether, 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), and decaglycerol decaoleate (DAO750), either alone or in combination with cosurfactants. The cosurfactant, typically a short-chain alcohol such as ethanol, 1-propanol, or 1-butanol, serves to increase interfacial fluidity by penetrating the surfactant film and thereby forming an irregular film due to the voids created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants, and alcohol-free self-emulsifying microemulsion systems are known in the art.The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and ethylene glycol derivatives.The oil phase can 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 oils.

[0170] Microemulsions are particularly interesting from the standpoint of drug solubilization and enhanced drug absorption. 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 offer the advantages of improved drug solubilization, drug protection from enzymatic hydrolysis, potential enhancement of drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical efficacy, and reduced toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often, microemulsions can form spontaneously when their components are combined at ambient temperature. This can be particularly advantageous when formulating heat-labile drugs, peptides, or dsRNA. Microemulsions are also effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to promote improved systemic absorption of dsRNA and nucleic acids from the gastrointestinal tract and improve local cellular uptake of dsRNA and nucleic acids.

[0171] In addition, the microemulsion of the present invention can contain additional components and additives, such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers, to improve the properties of the formulation and enhance the absorption of the dsRNA and nucleic acids of the present invention.The penetration enhancers used in the microemulsion of the present invention can be classified into one of five major 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 described above.

[0172] penetration enhancers In one embodiment, the present invention uses various penetration enhancers to achieve the efficient delivery of nucleic acid, especially dsRNA, to animal skin.Most drugs exist in solution in both ionized and non-ionized forms.However, usually only lipophilic or lipophilic drugs can easily cross cell membrane.It has been discovered that even non-lipophilic drugs can cross cell membrane when the membrane to be crossed is treated with penetration enhancer.In addition to aiding the diffusion of non-lipophilic drugs across cell membrane, penetration enhancer also enhances the permeability of lipophilic drugs.

[0173] Penetration enhancers can 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 the foregoing classes of penetration enhancers is described in more detail below.

[0174] Surfactant: in the context of the present invention, surfactant (or " surface active agent ") is a chemical substance that, when dissolved in aqueous solution, reduces the surface tension of the solution or the interfacial tension between this aqueous solution and other liquids, resulting in the enhanced absorption of dsRNA through mucous membranes.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).

[0175] Fatty acids: Various fatty acids and their derivatives that 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, dicapric acid, tricapric acid, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, and their C 1-10 These include alkyl esters (e.g., methyl, isopropyl, and t-butyl), and their mono- and diglycerides (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).

[0176] Bile salts: The physiological role of bile includes promoting the distribution and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman'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), glycolic acid (sodium glucholate), glycolic 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'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. al., J. Pharm. Sci., 1990, 79, 579-583).

[0177] Chelating agent: The chelating agent used in the present invention can be defined as a compound that removes metal ions from solution by forming a complex with the metal ions, resulting in the enhanced absorption of dsRNA through mucous membranes.With regard to the use as a penetration enhancer in the present invention, chelating agent has the additional advantage of also functioning as a DNase inhibitor, since most characterized DNA nucleases require divalent metal ions for catalytic activity and are inhibited by chelating agents (Jarrett, J.Chromatogr., 1993,618,315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetic acid (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl 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).

[0178] Non-chelating non-surfactant: as used herein, non-chelating non-surfactant penetration enhancer compounds can be defined as compounds that show little activity as chelating agent or surfactant, but still enhance the absorption of dsRNA through digestive mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7,1-33).This class of penetration enhancer includes, for example, unsaturated cyclic urea, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,page 92), and non-steroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J.Pharm.Pharmacol., 1987,39,621-626).

[0179] Carrier In addition, certain compositions of the present invention incorporate a carrier compound into the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or an analog thereof, that is inert (i.e., has no biological activity itself) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of the biologically active nucleic acid, for example, by degrading the biologically active nucleic acid or promoting its removal from the circulation. Co-administration of nucleic acid and carrier compound typically involves an excess of the latter substance, which can result in a significant reduction in the amount of nucleic acid recovered in the liver, kidney, or other extracirculatory reservoirs, possibly due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of partial phosphorothioate dsRNA in liver tissue can be reduced when it is co-administered 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 & Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0180] 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. Excipients can be liquid or solid and are selected with the intended mode of administration in mind so as to provide the desired dosage, consistency, etc. when combined with the nucleic acids and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, and the like), disintegrants (e.g., starch, sodium starch glycolate, and the like), and wetting agents (e.g., sodium lauryl sulfate, and the like).

[0181] In addition, any pharmaceutically acceptable organic or inorganic excipient suitable for oral (non-parenteral) administration that does not adversely react with nucleic acids can be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc.

[0182] Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohol, or solutions of nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents, and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for oral (non-parenteral) administration that do not adversely react with nucleic acids can be used.

[0183] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0184] Other components The compositions of the present invention can further contain other auxiliary components conventionally found in pharmaceutical compositions at their art-established usage levels.Thus, for example, the compositions may further contain compatible pharmaceutically active materials, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials useful for physically formulating the compositions of the present invention into various dosage forms, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers.However, when added, such materials should not excessively interfere with the biological activity of the components of the compositions of the present invention.The preparations can be sterilized and, if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances, which do not adversely interact with one or more nucleic acids of the preparation.

[0185] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0186] In some embodiments, pharmaceutical compositions featured in the present invention include (a) one or more dsRNA compounds and (b) one or more anti-cytokine biologics that function via non-RNAi mechanisms. Examples of such biologics include those targeting IL1β (e.g., ankinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab).

[0187] The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine 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, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

[0188] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of the compositions featured in the present invention generally lies within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods featured in the present invention, a therapeutically effective dose can be initially estimated from cell culture assays. Dosages can be formulated in animal models to achieve a circulating plasma concentration range of the compound, or, if appropriate, the polypeptide product of the target sequence (e.g., achieving a reduction in the concentration of the polypeptide), that includes the IC50 (i.e., the concentration of the test compound that 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. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0189] In addition to their administration, as mentioned above, the dsRNA featured in the present invention can be administered in combination with other known drugs that are effective in treating the pathological process mediated by the expression of TTR.In any case, the administering physician can adjust the amount and timing of dsRNA administration based on the results obtained by using standard efficacy measurement known in the art or as described herein.

[0190] Methods for treating diseases caused by expression of the TTR gene The present invention particularly relates to the use of the dsRNA that targets TTR and the composition that contains at least one such dsRNA for treating TTR-mediated disorders or diseases.For example, the dsRNA that targets TTR gene can be useful for treating TTR amyloidosis, such as familial amyloidotic neuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), meningeal / CNS amyloidosis, amyloidosis type VII (also known as meningeal or cerebrovascular amyloidosis), hyperthyroxinemia, and cardiac amyloidosis (also known as senile systemic amyloidosis (SSA) and senile cardiac amyloidosis (SCA)).

[0191] Figure 15 illustrates the conditions and mutations in TTR associated with familial amyloidotic neuropathy, familial amyloidotic cardiomyopathy, and CNS amyloidosis. The present invention includes compositions and methods for the treatment of these diseases and conditions, and is directed to these mutant forms of TTR.

[0192] dsRNAs targeting the TTR gene are also used to treat conditions and disorders such as TTR amyloidosis. Symptoms associated with such amyloidosis include, for example, stroke, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic failure, cardiomyopathy, gastrointestinal disorders (e.g., gastric ulcers, diarrhea, constipation, or malabsorption), weight loss, hepatomegaly, lymphadenopathy, goiter, vitreous opacity, renal failure (including proteinuria and renal dysfunction), nephropathy, cranial neuropathy, lattice degeneration of the cornea, and congestive heart failure with generalized weakness and respiratory distress due to fluid retention.

[0193] Due to the inhibitory effect on the expression of TTR, the composition according to the present invention or a pharmaceutical composition prepared therefrom can improve the quality of life.

[0194] The present invention further relates to the use of dsRNA or pharmaceutical compositions thereof for treating, for example, TTR amyloidosis, in combination with other pharmaceuticals and / or other therapeutic methods, e.g., known pharmaceuticals and / or known therapeutic methods, such as those currently used to treat these disorders. In one example, a dsRNA targeting TTR can be administered in combination with liver transplantation. In another example, a dsRNA targeting TTR can be administered in combination with pharmaceuticals or therapeutic methods for treating symptoms of TTR disease, such as diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), or dialysis therapy for managing kidney function.

[0195] The dsRNA and 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.

[0196] The present invention provides a method for administering dsRNA targeting TTR to patients with diseases or disorders mediated by the expression of TTR, such as TTR amyloidosis (FAP).The administration of this dsRNA can stabilize and improve the function of the peripheral nervous system in patients with, for example, FAP.Patient can be administered a therapeutic amount of dsRNA, for example, 0.1mg / kg, 0.2mg / kg, 0.5mg / kg, 1.0mg / kg, 1.5mg / kg, 2.0mg / kg or 2.5mg / kg.The dsRNA can be administered for a certain period, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 60 minutes, 120 minutes or 180 minutes.For example, it can be administered periodically, for example, every two weeks (i.e., every two weeks) for a period of 1 month, 2 months, 3 months, 4 months or more.After the initial treatment regimen, the treatment can be administered less frequently. For example, it can be administered every other week for 3 months, and then repeatedly administered once a month for 6 months or more.The administration of this dsRNA can reduce the TTR level in the blood or urine of patient by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more.

[0197] Before administering the full amount of dsRNA, patients can be administered a small dose, such as 5% of the total dose, and monitored for side effects such as allergic reactions or changes in liver function. For example, in patients monitored for changes in liver function, a low incidence of changes in LFTs (liver function tests) (e.g., a 10-20% incidence of LFTs) is acceptable (e.g., a reversible, 3-fold increase in ALT (alanine aminotransferase) and / or AST (aspartate aminotransferase) levels).

[0198] Many TTR-related diseases and disorders are hereditary. Therefore, patients who require TTR dsRNA can be identified by obtaining a family history. Healthcare providers, such as doctors, nurses, or family members, can obtain a family history before prescribing or administering TTR dsRNA. DNA testing can also be performed on patients to identify mutations in the TTR gene before administering TTR dsRNA to the patient.

[0199] The patient can have biopsy carried out before receiving TTR dsRNA.The biopsy can be on tissue such as gastric mucosa, peripheral nerve, skin, abdominal fat, liver or kidney, and the biopsy can show amyloid plaque, which indicates the damage mediated by TTR.When amyloid plaque is confirmed, the patient will be administered TTR dsRNA.

[0200] Methods for inhibiting expression of the TTR gene In yet another aspect, the present invention provides a method for inhibiting expression of a TTR gene in a mammal, the method comprising administering to the mammal a composition featured in the present invention such that expression of the target TTR gene is silenced.

[0201] When the organism to be treated is a mammal, such as a human, the composition can be administered by any means known in the art, including, but not limited to, oral or parenteral routes, including intracranial (e.g., intraventricular, intracerebral parenchymal, and subarachnoid), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In some embodiments, the composition is administered by intravenous infusion or injection.

[0202] Unless otherwise defined, all technical terms and chemical terms used herein have the same meaning as those commonly understood by those skilled in the art in the field to which the present invention belongs.When practicing or testing the dsRNA and method of the present invention, similar or equivalent methods and materials as described herein can be used, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.In case of discrepancy, this specification, including definitions, shall prevail.In addition, materials, methods and examples are only illustrative and are not limiting. [Example]

[0203] Example Example 1. dsRNA synthesis Reagent Sources Unless the source of a reagent is specifically given herein, such reagents may be obtained from any supplier of molecular biology reagents of standard quality / purity for molecular biology applications.

[0204] siRNA synthesis Single-stranded RNA was produced by solid-phase synthesis on a 1 μmole scale 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 the solid support. RNA and RNA containing 2'-O-methyl nucleotides were produced by solid-phase synthesis using the corresponding phosphoramidites and 2'-O-methyl phosphoramidites (Proligo Biochemie GmbH, Hamburg, Germany), respectively. These building blocks were incorporated into selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry, as described in "Current protocols in nucleic acid chemistry," Beaucage, S. Lett. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA." The iodine oxidizer solution was replaced with a solution of Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%) to introduce phosphorothioate linkages. Further auxiliary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).

[0205] Crude oligoribonucleotides were deprotected and purified by anion-exchange HPLC according to established procedures. Yield and concentration were determined by UV absorption of the respective RNA solutions at 260 nm using a spectrophotometer (DU 640B, Beckman Coulter GmbH, Unterschleissheim, Germany). Double-stranded RNA was generated by mixing equimolar solutions of complementary strands in annealing buffer (20 mM sodium phosphate (pH 6.8), 100 mM sodium chloride), heating in a water bath at 85–90°C for 3 minutes, and cooling to room temperature over 3–4 hours. The annealed RNA solution was stored at -20°C until use.

[0206] For the synthesis of 3'-cholesterol-conjugated siRNA (referred to herein as -Chol-3'), a solid support appropriately modified for RNA synthesis was used. The modified solid support was prepared as follows.

[0207] Diethyl-2-azabutane-1,4-dicarboxylate AA [ka] A 4.7M aqueous solution of sodium hydroxide (50 mL) was added to a stirred, ice-cooled solution of ethyl glycine hydrochloride (32.19 g, 0.23 mol) in water (50 mL). Ethyl acrylate (23.1 g, 0.23 mol) was then added, and the mixture was stirred at room temperature until the completion of the reaction was confirmed by TLC. After 19 hours, the solution was partitioned with dichloromethane (3 x 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was distilled to give AA (28.8 g, 61%).

[0208] 3-{ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB [ka] Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled on ice. Diisopropylcarbodiimide (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0°C. Then, diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylaminopyridine (0.305 g, 2.5 mmol) were added. The solution was allowed to warm to room temperature and stirred for an additional 6 hours. Completion of the reaction was confirmed by TLC. The reaction mixture was concentrated under vacuum, and ethyl acetate was added to precipitate diisopropylurea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% saturated sodium bicarbonate, and water. The combined organic layers were dried over sodium sulfate and concentrated to give the crude product, which was purified by column chromatography (50% EtOAC / hexanes) to give 11.87 g (88%) of AB.

[0209] 3-[(6-amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC [ka] 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 stirred for 1 hour. The reaction mixture was concentrated in vacuo, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by converting it to its hydrochloride salt.

[0210] 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 [ka] 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. Diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added to the suspension. Cholesteryl chloroformate (6.675 g, 14.8 mmol) was added to the resulting solution. 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%).

[0211] 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 [ka] 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 minutes. The temperature was maintained below 5°C during the addition. Stirring was continued at 0°C for 30 minutes, and 1 mL of glacial acetic acid was added, followed immediately by the addition of 4 g of NaH2PO4H2O in 40 mL of water. The resulting mixture was extracted twice with 100 mL of dichloromethane, and the combined organic extracts were washed twice with 10 mL of phosphate buffer, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0°C, and washed with 50 mL of dichloromethane. The extract was extracted three times with cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid and extracted five times with 40 mL of chloroform each, then combined, dried, and evaporated to dryness. The residue was purified by column chromatography using 25% ethyl acetate / hexane to give 1.9 g of the β-ketoester (39%).

[0212] [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 [ka] Methanol (2 mL) was added dropwise over 1 h to a refluxing mixture of β-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 for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added and the mixture was extracted with ethyl acetate (3 × 40 mL). The combined ethyl acetate layers were dried over anhydrous sodium sulfate and concentrated in vacuo to give the product, which was purified by column chromatography (10% MeOH / CHCl3) (89%).

[0213] (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 [ka] Diol AF (1.25 g, 1.994 mmol) was evaporated to dryness in vacuo with pyridine (2 × 5 mL). Anhydrous pyridine (10 mL) and 4,4'-dimethoxytrityl chloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was allowed to proceed overnight at room temperature. Methanol was added to quench the reaction. The reaction mixture was concentrated in vacuo, and dichloromethane (50 mL) was added to the residue. The organic layer was washed with 1 M saturated aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. Residual pyridine was removed by evaporation of toluene. The crude product (1.75 g, 95%) was purified by column chromatography (2% MeOH / chloroform, Rf = 0.5 in 5% MeOH / CHCl3).

[0214] Succinic acid mono-(4-[bis-(4-methoxyphenyl)-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 [ka] 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 overnight in vacuum at 40 ° C. 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 for 16 hours under an argon atmosphere. It was then diluted with dichloromethane (40 mL) and washed with ice-cold aqueous citric acid solution (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 directly in the next step.

[0215] Cholesterol-derivatized CPG AI [ka] Succinic acid AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane and acetonitrile (3:2, 3 mL). To the solution, DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL) and 2,2'-dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile and dichloroethane (3:1, 1.25 mL) were sequentially added. To the resulting solution, triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 mL) was added. The reaction mixture turned bright orange. The solution was briefly stirred (5 min) using a wrist-action shaker. Long-chain alkylamine-CPG (LCAA-CPG) (1.5 g, 61 mmol) was added. The suspension was stirred for 2 h. The CPG was filtered through a sintered funnel and washed successively with acetonitrile, dichloromethane, and ether. Unreacted amino groups were blocked using acetic anhydride / pyridine. The achieved CPG loading was determined by taking UV measurements (37 mM / g).

[0216] Synthesis of siRNAs bearing a 5'-12-dodecanoic acid bisdecylamide group (referred to herein as "5'-C32-") or a 5'-cholesteryl derivative group (referred to herein as "5'-Chol-") was carried out as described in International Publication WO 2004 / 065601, except that for cholesteryl derivatives, an oxidation step was performed using Beaucage reagent to introduce a phosphorothioate bond at the 5' end of the nucleic acid oligomer.

[0217] The nucleic acid sequences are set forth below using standard nucleotide nomenclature, specifically the abbreviations in Table 1. [Table 2]

[0218] Example 2A. siRNA Design for TTR Transcripts siRNA design was performed to identify siRNAs targeting the gene transthyretin from humans (symbol TTR) and rats (symbol Ttr). The design used the TTR transcripts NM_000371.2 (sequence number 1329) (human) and NM_012681.1 (sequence number 1330) (rat) from the NCBI Refseq collection. 100% identical siRNA duplexes were designed to their respective TTR genes.

[0219] siRNA design and specificity prediction The predicted specificity of all possible 19-mers was determined for each sequence. TTR siRNAs were used in a comprehensive search against the human and rat transcriptomes (defined as the set of NM_ and XM_ records in the NCBI Refseq set) using the FASTA algorithm. The Python script "Off-Target Fasta.py" was then used to analyze the alignment and generate a score based on the position and number of mismatches between the siRNA and any potential "off-target" transcripts. The off-target score is weighted to emphasize differences in the "seeded" region of the siRNA, 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 penalized: a mismatch in the seeded region at positions 2-9 of the oligo is penalized 2.8, a mismatch at the putative cleavage sites 10 and 11 is penalized 1.2, and a mismatch at positions 12-19 is penalized 1. Mismatches at position 1 are not considered. The off-target score for each oligo-transcript pair was then calculated by summing the mismatch penalties. The minimum off-target score from all oligo-transcript pairs was then determined and used for subsequent classification of the oligos. Both siRNA strands were assigned to specificity categories according to their calculated scores. A score greater than 3 was considered highly specific, a score equal to 3 was considered specific, and a score between 2.2 and 2.8 was considered moderately specific. When selecting which oligos to synthesize, the off-target scores of the antisense strands were ranked in descending order, and the best 144 (minimum off-target score) oligo pairs from humans and the best 26 pairs from rats were selected.

[0220] siRNA sequence selection A total of 140 sense and 140 antisense siRNA oligos derived from human TTR were synthesized and duplexed. A total of 26 sense and 26 antisense siRNA oligos derived from rat TTR were synthesized and duplexed. The duplexes contained in the oligos are shown in Tables 2-4 (human TTR) and Tables 5-7 (rat TTR).

[0221] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0222] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7]

[0223] [Table 5-1] [Table 5-2] Table 5-3 Table 5-4 Table 5-5 Table 5-6

[0224] Table 6-1 Table 6-2 Table 6-3 Table 6-4 Table 6-5 Table 6-6

[0225] Table 7-1

[0226] Table 8-1 Table 8-2

[0227] Table 9-1 [Table 9-2]

[0228] [Table 10-1] [Table 10-2]

[0229] Synthesis of TTR sequence The TTR sequence was synthesized on a MerMade 192 synthesizer on a 1 μmole scale. For all sequences in the sequence listing, "endo-write" chemistry was applied as detailed below. All pyrimidines (cytosine and uridine) in the sense strand were replaced with the corresponding 2'-O-methyl bases (2'-O-methyl C and 2'-O-methyl U). In the antisense strand, pyrimidines adjacent to the ribo-A nucleosides (towards the 5' position) were replaced with their corresponding 2-O-methyl nucleosides. A two-base dTdT extension was introduced at the 3' end of both the sense and antisense sequences. -Sequence files were converted to text files to make them compatible for loading in MerMade192 synthesis software.

[0230] Synthesis of the TTR sequence was performed using immobilized oligonucleotide synthesis with phosphoramidite chemistry. The synthesis of the above sequence was carried out on a 1 μm scale in a 96-well plate. The amidite solution was prepared at a 0.1 M concentration, and ethylthiotetrazole (0.6 M in acetonitrile) was used as the activator.

[0231] The synthesized sequences were cleaved and deprotected in a 96-well plate using methylamine in the first step and triethylamine 3HF in the second step. The crude sequences thus obtained were precipitated using a mixture of acetone and ethanol, and the pellets were resuspended in 0.5 M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS, and the resulting mass data confirmed the sequence identity. A selected set of samples was also analyzed by IEX chromatography.

[0232] The next step in the process was purification. All sequences were purified on an AKTA explorer purification system using a Source 15Q column. A single peak corresponding to the full-length sequence was collected in the eluent and subsequently analyzed for purity by ion exchange chromatography.

[0233] The purified sequences were desalted on a Sephadex G25 column using an AKTA purifier. The desalted TTR sequences were analyzed for concentration and purity. The single strands were then annealed to form TTR-dsRNA.

[0234] Example 2B: In vitro screening of TTR siRNA for mRNA suppression Human TTR-targeting dsRNA (Table 2) was 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) was synthesized and assayed for inhibition of endogenous TTR expression in H.4.II.E cells using bDNA assays. Results from single-dose assays were used to select a subset of TTR dsRNA duplexes for dose-response experiments to calculate IC50s. IC50 results were used to select TTR dsRNAs for further testing.

[0235] Cell culture and transfection: Hepatocyte cell lines HepG2, Hep3B, and H.4.II.E cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in a 5% CO atmosphere in Dulbecco's modified Eagle'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 Eagle's minimum essential medium. Reverse transcription was performed by adding 5 μL of Opti-MEM to 5 μL of siRNA duplexes per well in a 96-well plate, along with 10 μL of Opti-MEM plus 0.2 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, cat #13778-150) per well, and incubating at room temperature for 15 minutes. 4 × 10 4 (HepG2), 2 × 10 4 (Hep3B), or 2 × 10 4 80 μL of complete growth medium containing (H.4.II.E) cells without antibiotics was added. Cells were incubated for 24 hours before RNA purification. Single-dose experiments were performed at a final 2x concentration of 10 nM, and dose-response experiments were performed at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, and 0.00001 nM.

[0236] Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystems, Foster City CA, Part Number: AM1830): Cells were harvested and lysed in 140 μL of lysis / binding solution, then mixed for 1 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed remained constant throughout). 20 μL of magnetic beads were added to the cell lysate and mixed for 5 minutes. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the magnetic beads were washed with Wash Solution 1 (added with isopropanol) and mixed for 1 minute. The beads were recaptured and the supernatant was removed. The beads were then washed with 150 μL of Wash Solution 2 (added with ethanol), captured, and the 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 10–15 minutes. After mixing, 100 μL of RNA renaturation solution was added and mixed for 3 minutes. The supernatant was removed, and the magnetic beads were washed again with 150 μL of Wash Solution 2, mixed for 1 minute, and the supernatant was removed completely. The magnetic beads were mixed for 2 minutes and allowed to dry before the RNA was eluted with 50 μL of water.

[0237] cDNA synthesis using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat# 4368813): A master mix of 2 μL of 10× buffer, 0.8 μL of 25× dNTPs, 2 μL of random primers, 1 μL of reverse transcriptase, 1 μL of RNase inhibitor, and 3.2 μL of HO per reaction was added to 10 μL of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and a 4°C hold.

[0238] Real-time PCR: Two microliters of cDNA was added per well of a MicroAmp Optical 96-well plate (Applied Biosystems cat# 4326659) to 1 μL of 18S TaqMan probe (Applied Biosystems cat# 4319413E), 1 μL of TTR TaqMan probe (Applied Biosystems cat# HS00174914 M1), and 10 μL of TaqMan Universal PCR Master Mix (Applied Biosystems cat# 4324018). Real-time PCR was performed on an ABI 7000 Prism or ABI 7900HT real-time PCR system (Applied Biosystems) using the ΔΔCt (RQ) assay. All reactions were performed in triplicate.

[0239] Real-time data were analyzed using the ΔΔCt 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 targeting a non-mammalian luciferase gene) to calculate fold changes.

[0240] Branched DNA Assay - QuantiGene 1.0 (Panomics, Fremont, CA. Cat#: QG0004) - Used to screen for rodent-specific duplexes H.4.II.E cells (ATCC) were transfected with 10 nM siRNA. After removing the medium, H.4.II.E cells were lysed with 100 μl of diluted lysis mixture (a mixture of 1 volume of lysis mixture, 2 volumes of nuclease-free water, and 10 μl of proteinase K per mL for a final concentration of 20 mg / mL) and then incubated at 65°C for 35 minutes. Next, 80 μL of the working probe set (a mixture of TTR or GAPDH probes) and 20 μl of cell lysate were added to the capture plate. The capture plate was incubated overnight (approximately 16–20 hours) at 53°C ± 1°C. The capture plate was washed three times with 1x wash buffer (a mixture of nuclease-free water, buffer component 1, and wash buffer component 2) and then dried by centrifugation at 1000 rpm for 1 minute. 100 μL of amplification reagent was added to the capture plate, which was then sealed and incubated at 46°C ± 1°C for 1 hour. The washing and drying steps were repeated after 1 hour of incubation, and 100 μL of labeling solution reagent was added. The plate was then washed, dried, and 100 μL of substrate (a mixture of lithium lauryl sulfate and substrate solution) was added. The capture plate was placed in an incubator at 46°C ± 1°C for 30 minutes. The capture plate was then removed from the incubator and incubated at room temperature for 30 minutes. Finally, the capture plate was read using a Victor Luminometer (Perkin Elmer, Waltham, MA).

[0241] Branched DNA Assay - QuantiGene 2.0 (Panomics cat#: QS0011): Used to screen all other duplexes After 24 hours of incubation at the indicated dose, the medium was removed, and the cells were lysed in 100 μL of 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) and then incubated at 65°C for 35 minutes. Next, 20 μL of the working probe set (TTR probe for the gene target and GAPDH for the endogenous control) and 80 μL of cell lysate were added to the capture plate. The capture plate was incubated at 55°C ± 1°C (approximately 16-20 hours). The next day, the capture plate was washed three times with 1x wash buffer (nuclease-free water, buffer component 1, and wash buffer component 2) and then dried by centrifugation at 240 g for 1 minute. 100 μL of pre-amplification working reagent was added to the capture plate, which was then sealed with aluminum foil and incubated at 55°C ± 1°C for 1 hour. After 1 hour of incubation, the wash step was repeated, and then 100 μL of amplification working reagent was added. After 1 hour, the wash and dry step was repeated, and 100 μL of labeled probe was added. The capture plate was incubated at 50°C ± 1°C for 1 hour. The plate was then washed with 1x wash buffer, dried, and then 100 μL of substrate was added to the capture plate. After 5-15 minutes of incubation, the capture plate was read using a SpectraMax Luminometer (Molecular Devices, Sunnyvale, CA).

[0242] bDNA Data Analysis: The bDNA data were analyzed by (i) subtracting the average background from each triplicate sample, (ii) averaging the resulting triplicate GAPDH (control probe) and TTR (experimental probe) values, and then (iii) obtaining the ratio: (experimental probe-background) / (control probe-background).

[0243] result A summary of single dose and IC50 results for TTR-dsRNA (TTR siRNA) is shown in Table 8 below. Single dose results are expressed as a percentage of TTR mRNA relative to the control, assayed in HepG2 cells. IC50s were determined in HepG2 and / or Hep3B cells, as indicated.

[0244] [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0245] The dose-response data used to determine the IC50 for five TTR-dsRNAs (AD-18258, AD-18274, AD-18324, AD-18328, and AD-18339) are detailed in Table 9 below. All five siRNAs were confirmed to have IC50s in pM. The IC50 data for the dsRNAs in Table 8 is a summary of the data presented in Table 9 below.

[0246] [Table 12] A summary of single-dose results for rodent-specific TTR-dsRNA (TTR siRNA) is presented below in Table 10. Single-dose results are expressed as a percentage of TTR mRNA relative to control and were assayed in rat H.4.II.E cells after transfection with rodent-specific TTR siRNA at 10 nM. These results demonstrate that several rodent-specific TTR siRNAs are effective in silencing endogenous rat TTR mRNA in vitro.

[0247] [Table 13]

[0248] Example 3. In vitro assay of TTR siRNA for induction of TNF-α and IFN-α secretion To assess their potential for immune stimulation, TTR siRNA was assayed in vitro for induction of TNF-α and IFN-α secretion.

[0249] Human PBMCs were isolated from freshly collected buffy coats (Research Blood Components, Inc., Boston, MA) obtained from healthy donors by standard Ficoll-Hypaque density centrifugation. Freshly isolated cells (1 × 10 5 / well / 100 μL) were seeded into 96-well plates and cultured in RPMI 1640 GlutaMax medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic / antimycotic (Invitrogen).

[0250] PBMCs were transfected with siRNA using DOTAP transfection reagent (Roche Applied Science). DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing siRNA. The siRNA / DOTAP complexes were incubated as specified by the manufacturer's instructions and then added to PBMCs (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 the positive control siRNA. AD-5048 corresponds to a sequence targeting human apolipoprotein B (Soutschek et al., 2004) and induces the secretion of both IFN-α and TNF-α in this assay. AD-1955, which does not induce the secretion of IFN-α or TNF-α in this assay, was used as the 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.

[0251] Cytokines in culture supernatants were detected and quantified using commercially available ELISA kits for IFN-α (BMS216INST) and TNF-α (BMS223INST), both from Bender MedSystems (Vienna, Austria). Cytokine induction by TTR siRNA is expressed as the percentage of IFN-α or TNF-α produced relative to the positive control siRNA AD-5048.

[0252] The IFN-α and TNF-α stimulation results for a number of TTR siRNAs are shown in Figure 1 (means of quadruplicate wells ± standard deviation) and Table 11 below (percentages compared to AD-5048). TTR siRNAs were not assessed to induce significant TNF-α or IFN-α secretion by cultured human PBMCs. [Table 14]

[0253] Five TTR-targeting lead dsRNAs (TTR siRNAs) were selected based on IC50 values ​​in the pM range in the human hepatocyte cell lines HepG2 and Hep3B and in 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 allow better translation of cross-species toxicological data and have broader applicability to human patients, duplexes with 100% identity in orthologous genes from rat, cynomolgus monkey, and human and that do not target regions with known polymorphisms are generally preferred. Five lead compounds were selected based on IC50 values ​​in hepatocyte cell lines in the pM range, in the absence of immunostimulatory activity, specificity for the human TTR transcript, and the absence of known polymorphisms (mutations) within the mRNA region targeted by the duplex. In the case of TTR, no 19-base oligos with perfect identity were found in humans, rats, and cynomolgus monkeys. A summary of these data is presented in Table 12, which also includes information on known TTR mutations within the region targeted by the duplex and cross-species reactivity.

[0254] [Table 15]

[0255] 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 selected for in vivo evaluation. These duplexes demonstrated potent dose-dependent silencing in liver cell lines (e.g., HepG2) in vivo. Figures 2A and 2B show the dose response in HepG2 cells after transfection with AD-18324 (Figure 2A) or AD-18328 (Figure 2B), where the dose is expressed in nM on the x-axis and the response is expressed as the fraction of TTR mRNA remaining compared to the control on the y-axis. In HepG2 cells, the IC50 values ​​of AD-18324 and AD-18328 were confirmed to be 2 pM and 3 pM, respectively. The TTR target site for both lead dsRNA candidates is within the 3' untranslated region of TTR mRNA, a region with no reported mutations in the literature.

[0256] The sequences of each strand of the two lead candidates from the table are reproduced below. Strand: s = sense, as = antisense Location: 5' base in the transcript of NM_000371.2 [Table 16A] In addition, a rodent cross-reactive TTR dsRNA, AD-18246, was selected for further in vivo evaluation. AD-18246 targets a sequence starting at position 88 of the open reading frame, which contains three mutations reported in the literature. The dose-response curve for AD-18246 in HepG2 cells is shown in Figure 3. AD-18246 was substantially less potent than AD-18324 and AD-18328, and the IC50 of AD-18246 was determined to be 265 pM.

[0257] AD-18324, AD-18328, and AD-18246 were formulated into LNP01 and then administered to transgenic mice. Three- to five-month-old H129-mTTR-KO / iNOS-KO / hTTR transgenic mice (mouse transthyretin knockout / inducible nitric oxide synthase knockout / human transthyretin transgenic) were intravenously (IV) administered via the tail vein with 200 μL of transthyretin-specific siRNA (AD-18324, AD-18328, or AD-18246) formulated in LNP01, control siRNA targeting the non-mammalian luciferase gene (AD-1955), or PBS 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 lipid-like formulation consisting of ND98, cholesterol, and PEG-Ceramide C16.

[0258] Approximately 40 hours later, mice were anesthetized with 200 μL of ketamine and then bled by cutting the right tail artery. Whole blood was isolated, and plasma was isolated and stored at −80°C until assayed. Liver tissue was collected, flash-frozen, and stored at −80°C until processing.

[0259] Treatment efficacy was assessed by (i) measuring TTR mRNA in liver 48 hours after dosing, and (ii) measuring TTR protein in plasma before and after dosing. TTR liver mRNA levels were assayed using the branched DNA assay - QuantiGene 2.0 (Panomics cat#: QS0011). Briefly, mouse liver samples were pulverized and tissue lysates were prepared. The liver 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) was incubated at 65°C for 35 minutes. Then, 20 μL of the working probe set (TTR probe for the gene target and GAPDH for the endogenous control) and 80 μL of cell lysate were added to the capture plate. The capture plate was incubated at 55°C ± 1°C (approximately 16–20 hours). The next day, the capture plate was washed three times with 1x wash buffer (nuclease-free water, buffer component 1, and wash buffer component 2) and then dried by centrifugation at 240g for 1 minute. 100µL of pre-amplification working reagent was added to the capture plate, which was sealed with aluminum foil and incubated at 55°C ± 1°C for 1 hour. After 1 hour of incubation, the wash step was repeated, followed by the addition of 100µL of amplification working reagent. After 1 hour, the wash and dry steps were repeated, followed by the addition of 100µL of labeled probe. The capture plate was incubated at 50°C ± 1°C for 1 hour. The plate was then washed with 1x wash buffer, dried, and 100µL of substrate was added to the capture plate. After a 5-15 minute incubation, the capture plate was read using a SpectraMax Luminometer. The bDNA data were analyzed by subtracting the average background from each triplicate sample, averaging the resulting triplicate GAPDH (control probe) and TTR (experimental probe) values, and then computing the ratio: (experimental probe-background) / (control probe-background).

[0260] TTR plasma levels were assayed using the commercially available "AssayMax Human Prealbumin ELISA Kit" (AssayPro, St. Charles, MO, Catalog #EP3010-1) according to the manufacturer's guidelines. Briefly, mouse plasma was diluted 1:10,000 in 1x mixed diluent and added to a plate precoated with the standard kit, incubated at room temperature for 2 hours, and then washed five times with the kit's wash buffer. Fifty microliters of biotinylated prealbumin antibody was added to each well, incubated at room temperature for 1 hour, and then washed five times with the wash buffer. Fifty microliters of streptavidin-peroxidase conjugate was added to each well, incubated at room temperature for 30 minutes, and then washed as described above. The reaction was stopped by adding 50 μL / well of stop solution, followed by adding 50 μL / well of chromogenic substrate and incubating at room temperature for 10 minutes. Absorbance at 450 nm was read on a microplate reader (Molecular Devices, Sunnyvale, Calif.) and data were analyzed using the Softmax 4.6 software package (Molecular Devices).

[0261] LNP01-18324 and LNP01-18328 were found to reduce liver TTR mRNA (Figure 4A) and plasma TTR protein (Figure 4B) levels in a dose-dependent manner following IV bolus administration. The mRNA ED50 for LNP01-18328 was determined to be approximately 1 mg / kg, while the ED50 for LNP01-18324 was confirmed to be approximately 2 mg / kg. The effects of LNP01-18324 and LNP01-18328 were specific, as the control LNP01-1955 at 6 mg / kg did not significantly affect liver TTR mRNA levels compared to the PBS group. LNP01-18324 and LNP01-18328 reduced plasma TTR protein levels with similar efficacy to that of TTR mRNA levels compared to the PBS group. At 3 mg / kg, LNP01-18246 reduced hepatic TTR mRNA levels to a lesser extent than LNP01-18324 or LNP01-18328 at 3 mg / kg.

[0262] These results demonstrate that LNP01-18324 and LNP01-18328, administered by IV bolus, substantially reduced human TTR mRNA expressed by the liver of transgenic mice, resulting in a reduction in circulating human TTR protein.

[0263] Example 5. In vivo reduction of wild-type TTR mRNA in non-human primate liver by SNALP-18324 and SNALP-18328 To evaluate the efficacy of TTR siRNA AD-18324 and AD-18328 on liver TTR mRNA levels in non-human primates, the siRNA was formulated into SNALP and administered via 15-minute IV infusion. Cynomolgus monkeys (Macaca fascicularis) (2-5 kg, 3 animals per group) were administered 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, targeting the non-mammalian luciferase gene) via 15-minute IV infusion. 48 hours after administration, the monkeys were anesthetized with sodium pentobarbital and exsanguinated. Liver tissues for TTR mRNA determination were collected, flash-frozen, and stored at -80°C until processing.

[0264] TTR mRNA levels in liver were assayed using a custom branched DNA assay and QuantiGene 1.0 technology. Briefly, monkey liver samples were pulverized and tissue lysates were prepared. The liver 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) was incubated at 65°C for 35 minutes. 20 μL of the working probe set (TTR probe for the gene target and GAPDH for the endogenous control) and 80 μL of cell lysate were then added to the capture plate. The capture plate was incubated at 55°C ± 1°C (approximately 16-20 hours). The next day, the capture plate was washed three times with 1x wash buffer (nuclease-free water, buffer component 1, and wash buffer component 2) and then dried by centrifugation at 240g for 1 minute. 100 μL of pre-amplification working reagent was added to the capture plate, which was then sealed with aluminum foil and incubated at 55°C ± 1°C for 1 hour. After the 1-hour incubation, the wash step was repeated, followed by the addition of 100 μL of amplification working reagent. After 1 hour, the wash and dry steps were repeated, followed by the addition of 100 μL of labeled probe. The capture plate was incubated at 50°C ± 1°C for 1 hour. The plate was then washed with 1x wash buffer, dried, and 100 μL of substrate was then added to the capture plate. After a 5-15 minute incubation, the capture plate was read using a SpectraMax Luminometer. The bDNA data were analyzed by (i) subtracting the average background from each triplicate sample, (ii) averaging the resulting GAPDH (control probe) and TTR (experimental probe) values, and (iii) obtaining the ratio: (experimental probe - background) / (control probe - background).

[0265] The results are shown in Figure 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 ED50 for SNALP-18328 and SNALP-18324 was determined to be approximately 0.3 and approximately 1 mg / kg, respectively.

[0266] These results demonstrate that SNALP-18324 and SNALP-18328, when administered by IV infusion, are effective in suppressing wild-type TTR mRNA in the liver of non-human primates.

[0267] Example 6. In vivo reduction of mutant (V30M) TTR mRNA and protein by SNALP-18328 in transgenic mice To evaluate the efficacy of the TTR siRNA AD-18328 on mutant (V30M) TTR mRNA in the liver and mutant (V30M) TTR protein in serum, AD-18328 was formulated into SNALP and administered via an IV bolus to V30M hTTR transgenic mice. Eight- to 12-week-old V30M hTTR transgenic mice (five animals per group) were intravenously administered 200 μL of SNALP-18328 (0.03, 0.3, or 3 mg / kg), SNALP-1955 (3 mg / kg with negative control siRNA AD-1955, targeting the non-mammalian luciferase gene), or PBS. Mice used were Mus musculus strain H129-hTTR KO from the Institute of Molecular and Cellular Biology, Porto, Portugal. Briefly, hTTR H129 transgenic mice were crossed with H129 endogenous TTR KO mice (null mice in the TTR background) to generate H129-hTTR transgenic 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.).

[0268] Forty-eight hours after injection, animals in all five treatment groups received 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.

[0269] For TTR mRNA quantification, frozen liver tissue was ground into powder and lysates were prepared. TTR mRNA levels relative to GAPDH mRNA levels were determined in the lysates using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, CA). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean TTR mRNA levels were normalized to the mean GAPDH mRNA levels for each sample. The group means of the normalized values ​​were then further normalized to the mean values ​​for the PBS-treated group to obtain the relative levels of TTR mRNA expression.

[0270] For TTR protein quantification, serum was assayed using the Assaymax PreAlbumin ELISA kit from AssayPro (St. Charles, MO) according to the manufacturer's protocol.

[0271] The results for liver mRNA and serum protein are shown in Figures 6A and 6B, respectively. V30M hTTR transgenic mice treated with SNALP-18328 had a dose-dependent and significant reduction in liver TTR mRNA levels compared to the PBS control group, with a maximum reduction of 97% (p<0.001) at 3 mg / kg of SNALP-18328 and a 50% reduction (ED50) at approximately 0.15 mg / kg of SNALP-18328. Serum TTR protein was also suppressed in a dose-dependent manner, with a maximum reduction of 99% (p<0.01) of serum TTR protein (compared to pre-dose levels) at 3 mg / kg of SNALP-18328, consistent with the reduction in TTR mRNA levels. SNALP-1955 at 3 mg / kg had no statistically significant effect on either TTR mRNA or protein levels compared to PBS.

[0272] These results demonstrate that when administered IV, SNALP-18328 is active in suppressing mutant V30M TTR mRNA in the liver of transgenic mice, resulting in a reduction in circulating mutant V30M TTR protein.

[0273] Example 7. Duration of TTR mRNA and protein suppression by SNALP-18328 in transgenic mice To assess the durability of TTR mRNA and protein suppression by SNALP-18328, AD-18328 was formulated into SNALP and administered as an IV bolus to V30M hTTR transgenic mice. Liver TTR mRNA and serum TTR protein levels were quantified at various time points after dosing. Eight- to 12-week-old V30M hTTR transgenic mice (four animals per group) were intravenously administered 200 μL of SNALP-18328 (1 mg / kg) or SNALP-1955 (1 mg / kg, containing the negative control siRNA AD-1955, targeting the non-mammalian luciferase gene). Mice used were the Mus musculus strain H129-hTTR KO from the Institute of Molecular and Cellular Biology, Porto, Portugal. Briefly, hTTR H129 transgenic mice were crossed with H129 endogenous TTR KO mice (null mice in a TTR null background) to generate H129-hTTR transgenic 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). On days 3, 8, 15, or 22 after dosing, animals in both treatment groups received 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.

[0274] For TTR mRNA quantification, frozen liver tissue was ground into powder and lysates were prepared. TTR mRNA levels relative to GAPDH mRNA levels were determined in the lysates using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, CA). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean TTR mRNA levels were normalized to the mean GAPDH mRNA levels for each sample. The group means of the normalized values ​​were then further normalized to the mean values ​​for the PBS-treated group to obtain the relative levels of TTR mRNA expression.

[0275] For TTR protein quantification, serum was assayed using the Assaymax PreAlbumin ELISA kit from AssayPro (St. Charles, MO) according to the manufacturer's protocol.

[0276] The results for liver mRNA and serum protein are shown in Figures 7A and 7B, respectively. In hTTR V30M transgenic mice, a single IV bolus administration of SNALP-18328 resulted in sustained inhibition of liver TTR mRNA and serum TTR protein levels. Compared with the control group (1 mg / mL SNALP-1955), a single IV administration of 1 mg / kg SNALP-18328 significantly reduced relative TTR mRNA levels by 96% (p<0.001), 90% (p<0.001), 82% (p<0.001), and 73% (p<0.001) on days 3, 8, 15, and 22, respectively, and did not return to baseline levels at the end of the study (day 22). Protein levels were also reduced with a maximal reduction in serum TTR of 97% (p<0.001) (compared to SNALP-1955) on day 3 of dosing. On days 8, 15, and 22 of dosing, TTR protein levels were suppressed by 72% (p<0.05), 32% (p<0.05), and 40% (p<0.001), respectively, compared to SNALP-1955. These results demonstrate that a single IV administration of SNALP-18328 produces sustained suppression of target liver mRNA and serum protein levels in V30M hTTR transgenic mice, resulting in significant reductions in liver TTR mRNA and serum TTR protein on day 22 of dosing.

[0277] Example 8. Duration of serum TTR protein suppression by SNALP-18328 in non-human primates To assess the durability of serum TTR protein suppression by SNALP-18328, SNALP-18328 was formulated into SNALP and administered by IV infusion to non-human primates. Serum TTR protein levels were quantified at various time points after dosing.

[0278] Cynomolgus monkeys (Macaca fascicularis) (n=5 animals / group for the SNALP-18328 group and n=3 animals / group for the SNALP-1955 and PBS groups) were administered SNALP-18328 (0.3, 1, or 3 mg / kg), SNALP-1955 (3 mg / kg with negative control siRNA AD-1955, targeting a non-mammalian luciferase gene), or PBS via a 15-minute IV infusion. Serum samples were collected on days 0, 1, 2, 3, 4, 5, 7, 10, and 14 of the administration period and stored at -80°C until analysis.

[0279] Western blot analysis was used to assess 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 into each lane of a 10-20% Criterion (Biorad, Hercules, CA) preparative gel and separated by SDS-PAGE at 120V for 1.5 hours. Subsequently, the samples were transferred to nitrocellulose membranes using a semi-dry system at 15V for 1 hour. The membranes were blocked overnight at 4°C in LiCOR (Lincoln, NE) blocking buffer diluted 1:1 with 1x PBS. The blot was first probed with a primary antibody (goat anti-TTR from Santa Cruz, Santa Cruz, CA) at a 1:1000 dilution in LiCOR blocking buffer / PBS for 1 hour at room temperature on a rocker. The blot was washed four times with PBS + 0.2% Tween 20 (10 minutes per wash). A fluorescently labeled secondary antibody (goat anti-680 nm from Invitrogen, Carlsbad, CA) was added at a 1:10,000 dilution in LiCOR blocking buffer / PBS, and the blot was incubated for 1 hour at room temperature. After incubation, the blot was washed four times with PBS + 0.2% Tween 20, followed by one wash with 1 L PBS. Protein bands were detected using a Li-COR Odyssey Infrared Imaging System. TTR monomer migrates at 15 kDa.

[0280] The results are shown in Figure 8. Serum TTR protein levels showed a dose-dependent reduction at 1 or 3 mg / kg SNALP-18328 when compared to pre-dose (day 0) levels. Following a single IV administration of SNALP-18328, the suppression persisted for at least 14 days following treatment with 1 or 3 mg / kg SNALP-18328.

[0281] These results demonstrate that a single IV administration of SNALP-18328 produces sustained suppression of circulating TTR protein in non-human primates (cynomolgus monkeys (Macaca fascicularis)), resulting in a significant reduction in TTR protein by 14 days post-dose.

[0282] Example 9: In vivo reduction of mutant (V30M) TTR in peripheral tissues by SNALP-18328 in transgenic mice To evaluate the efficacy of SNALP-18328 in lowering TTR in peripheral tissues, hTTR V30M / HSF-1 knockout mice were evaluated by immunohistochemical staining for TTR. Two-month-old hTTR V30M / HSF-1 knockout 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 control siRNA AD-1955 targeting the non-mammalian luciferase gene, 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 assessed on day 56, 8 weeks after the first dose.

[0283] Mice were anesthetized with 1 mg / kg medetomidine and administered a lethal dose of ketamine. Tissues and organs of interest were collected. For immunohistochemistry, the esophagus (E), stomach (S), intestine (duodenum (I1) and colon (I4)), nerve (N), and dorsal root ganglion (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 used, followed by extravidin-conjugated antibody (1:20, Sigma, USA) to stain the TTR protein. The reaction was developed using 3-amino-9-ethylcarbazole (AEC) (Sigma, USA). Semiquantitative analysis of immunohistochemistry slides was performed using the Scion image quant program, which measures the area occupied by the substrate reaction color and normalizes this value to the total image area. The mean percentage of occupied area is shown with the corresponding standard deviation. Each animal tissue was evaluated in four different regions. The presence of human TTR in gastric and intestinal parasympathetic ganglia was examined by double immunofluorescence staining with rabbit anti-human TTR (1:1000, DAKO, Denmark) and mouse anti-PGP9.5 (1:40, Serotec, USA) as primary antibodies, and anti-rabbit Alexa Fluor 488 (Molecular probes, UK) and goat anti-mouse Alexa Fluor 568 (Molecular probes, UK), respectively. 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.

[0284] The results are depicted graphically in Figure 9. Compared to animals treated with PBS and SNALP-1955, animals treated with SNALP-18328 had significantly reduced TTR immunoreactivity in all tissues examined (esophagus (E), stomach (S), intestine (duodenum (I1) and colon (I4)), nerve (N), and dorsal root ganglion (D)).

[0285] These results demonstrate that administration of SNALP-18328 to hTTR V30M / HSF-1 knockout mice results in a significant reduction of TTR protein in peripheral tissues and organs, including the esophagus, stomach, intestine (duodenum and colon), nerves, and dorsal root ganglia.

[0286] Example 10. In vivo reduction of wild-type TTR mRNA in non-human primate liver by XTC-SNALP-18328 To evaluate the efficacy of the novel lipid nanoparticle formulation XTC-SNALP for siRNA delivery in non-human primates, TTR siRNA AD-18328 was formulated in XTC-SNALP (XTC-SNALP-18328) and administered via 15-minute IV infusion, followed by quantification of liver TTR mRNA. Cynomolgus monkeys (Macaca fascicularis) were administered XTC-SNALP-18328 (0.03, 0.1, 0.3, or 1 mg / kg) or XTC-SNALP-1955 (1 mg / kg, containing control siRNA AD-1955 targeting the non-mammalian luciferase gene) via 15-minute IV infusion. Forty-eight hours after dosing, the 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. The method used for quantification of TTR mRNA in liver tissue was similar to that described in Example 5 above.

[0287] The results are shown in Figure 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 approximately 0.1 mg / kg XTC-SNALP-18328.

[0288] These results demonstrate that XTC-SNALP-18328, when administered by IV infusion, is effective in suppressing wild-type TTR mRNA in the liver of non-human primates.

[0289] Example 11: In vivo reduction of wild-type TTR mRNA in non-human primate liver by LNP09-18328 and LNP11-18328 To evaluate the efficacy of two novel lipid nanoparticle formulations, LNP09 and LNP11, for siRNA delivery in non-human primates, TTR siRNA AD-18328 was formulated in LNP09 (LNP09-18328) or LNP11 (LNP11-18328) and administered via 15-minute IV infusion. Liver TTR mRNA and serum TTR protein levels were assayed. Cynomolgus monkeys (Macaca fascicularis) were administered LNP09-18328 (0.03, 0.1, or 0.3 mg / kg), LNP11-18328 (0.03, 0.1, or 0.3 mg / kg), or PBS via 15-minute IV infusion. Liver biopsies were collected 48 hours after dosing, flash-frozen, and stored at -80°C until processing. Serum was collected prior to dosing (before blood sampling) and on days 1, 2, 4, 7, 14, 21, and 28 after dosing and stored at -80°C until processing. Methods used for quantification of TTR mRNA in liver tissue and serum TTR protein assessment were similar to those described in Examples 5 and 8 above.

[0290] Results are shown in Figure 11A for mRNA and Figures 11B and 11C for protein. Animals treated with LNP09-18328 and LNP11-18328 showed a dose-dependent reduction in TTR mRNA levels in the liver, reaching a maximum reduction of approximately 85% (LNP09-18328) and approximately 90% (LNP11-18328) of mRNA at 0.3 mg / kg compared to the PBS control. The mRNA ED50 was determined to be approximately 0.02 mg / kg for both LNP09-18328 and LNP11-18328. At day 7 after dosing, serum samples also showed a dose-dependent reduction in TTR protein for 0.1 and 0.3 mg / kg LNP09-18328 and LNP11-18328 compared to PBS control levels. FIG. 11C shows that the reduction in TTR protein levels with a 0.3 mg / kg dose of LNP09-18328 persists for at least 28 days after dosing compared to the PBS control group and compared to pre-bleed samples.

[0291] These results demonstrate that LNP09-18328 and LNP11-18328, when administered by IV infusion, are effective in suppressing wild-type TTR mRNA in the liver and wild-type TTR protein in the blood circulation of non-human primates. Furthermore, suppression by LN09-18328 is sustained and persists for at least 28 days after IV infusion.

[0292] Example 12. Synthesis of TTR-Arranged Sequences A set of TTR duplexes ("aligned duplexes") was designed that are targeted to the TTR gene near the target region of AD-18328, which targets the human TTR gene starting at nucleotide 628 of NM_000371.3.

[0293] In the examples below, the numbering indicating the base at the 5' position of the siRNA on the transcript is based on NM_000371.3 (Figure 12, SEQ ID NO: 1331). In the examples above, the numbering of siRNAs targeting human siRNAs is based on NM_000371.2 (Figure 13A). NM_000371.3 extends the sequence of the 5'UTR by 110 bases compared to NM_000371.2, as shown in Figure 14. Thus, as an example, the start position of AD-18328 is 628 on NM_000371.3 and 518 on NM_000371.2 (Figure 14).

[0294] TTR-aligned sequences were synthesized on a MerMade 192 synthesizer on a 1 μmole scale. For all sequences in the sequence listing, "endo-write" 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 the ribo-A nucleosides (towards the 5' position) were replaced with their corresponding 2-O-methyl nucleosides. A two-base dTdT extension was introduced at the 3' end of both the sense and antisense sequences. -Sequence files were converted to text files to make them compatible for loading in MerMade 192 synthesis software.

[0295] Synthesis, cleavage, and deprotection: Synthesis of the TTR sequences was performed using immobilized oligonucleotide synthesis with phosphoramidite chemistry. Sequence synthesis was performed at a 1 μm scale in 96-well plates. Amidite solutions were prepared at 0.1 M concentration, and ethylthiotetrazole (0.6 M in acetonitrile) was used as the activator. The synthesized sequences were cleaved and deprotected in 96-well plates using methylamine in the first step and a fluoride reagent in the second step. Crude sequences were precipitated using a mixture of acetone:ethanol (80:20), and the pellet was resuspended in 0.2 M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm UV identity for quantification, and a selected set of samples by IEX chromatography to determine purity.

[0296] Purification and desalting: The TTR-aligned sequences were purified on an AKTA explorer purification system using a Source 15Q column. A column temperature of 65°C was maintained throughout the purification. Sample injection and collection were performed in a 96-well (1.8 mL deep-well) plate. A single peak corresponding to the full-length sequence was collected in the eluent. The purified sequences were desalted onto a Sephadex G25 column using an 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.

[0297] Single- and double-stranded TTR: A detailed list of TTR aligned duplexes and corresponding single strands (sense and antisense) is shown in the table below (Table 13). [Table 17-1] [Table 17-2] [Table 17-3]

[0298] Example 13. In vitro screening of TTR-flanked siRNAs The TTR-flanked duplexes were assayed in Hep3B cells for inhibition of endogenous TTR expression using a real-time PCR assay.

[0299] Cell Culture and Transfection: Hep3B cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in a 5% CO atmosphere in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transcription was performed 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 (Invitrogen, Carlsbad, CA, cat# 13778-150) was added per well, and the mixture was incubated at room temperature for 15 minutes. Then, as above, 2.0 × 10 4 80 μL of complete growth medium containing Hep3B cells without antibiotics was added. Cells were incubated for 24 hours before RNA purification. Experiments were performed at final duplicate concentrations of 0.1 or 10 nM.

[0300] Total RNA isolation using the MagMAX-96 Total RNA Isolation Kit (Applied Biosystems, Foster City, CA, part number: AM1830): Cells were harvested and lysed in 140 μL of lysis / binding solution, then mixed for 1 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout). Twenty microliters of magnetic beads and lysis / binding enhancer mixture was added to the cell lysate and mixed for 5 minutes. The magnetic beads were captured using a magnetic stand, and the supernatant was removed without disturbing the beads. After removing the supernatant, the magnetic beads were washed with Wash Solution 1 (added with isopropanol) and mixed for 1 minute. The beads were captured again, and the supernatant was removed. The beads were then washed with 150 μL of Wash Solution 2 (added with ethanol), captured, and the supernatant was removed. Next, 50 μL of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was added to the beads and mixed for 10–15 minutes. After mixing, 100 μL of RNA renaturation solution was added and mixed for 3 minutes. The supernatant was removed, and the magnetic beads were washed again with 150 μL of wash solution 2, mixed for 1 minute, and the supernatant was completely removed. The magnetic beads were mixed for 2 minutes and allowed to dry before the RNA was eluted with 50 μL of water.

[0301] cDNA synthesis using the ABI High-Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat# 4368813): A master mix of 2 μL of 10× buffer, 0.8 μL of 25× dNTPs, 2 μL of random primers, 1 μL of reverse transcriptase, 1 μL of RNase inhibitor, and 3.2 μL of HO per reaction was added to 10 μL of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the following steps: 25°C for 10 minutes, 37°C for 120 minutes, 85°C for 5 seconds, and a 4°C hold.

[0302] Real-time PCR: 2 μL of cDNA was added to a master mix containing 0.5 μL of GAPDH TaqMan probe (Applied Biosystems Cat#4326317E), 0.5 μL of TTR TaqMan probe (Applied Biosystems Cat#HS00174914 M1), and 10 μL of Roche Probes Master Mix (Roche Cat#04887301001) per well in a LightCycler 480 384-well plate (Roche cat#0472974001). Real-time PCR was performed in a LightCycler 480 real-time PCR machine (Roche). Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate.

[0303] Real-time data was analyzed using the ΔΔCt method. Each sample was normalized to GAPDH expression, and knockdown was assessed relative to cells transfected with non-targeting duplex AD-1955. Table 14 shows the knockdown of TTR using siRNA. Data are expressed as the percentage of message remaining relative to cells targeted with AD-1955.

[0304] Many, but not all, of the arrayed TTR-dsRNAs targeting TTR, the approximate target of AD-18328, reduced TTR mRNA by at least 70% when transfected into Hep3B cells at 0.1 nM. [Table 18-1] [Table 18-2]

[0305] Example 14. Evaluation of the duration of infusion on the efficacy of a single intravenous dose of SNALP-18534 in Sprague-Dawley rats the purpose To determine the effect of infusion duration on the efficacy of a single IV infusion of SNALP-18534 on hepatic TTR mRNA levels in Sprague-Dawley rats. [Table 19] The sequences of the sense and antisense strands of AD-18534 from the table above are reproduced below. [Table 20A] research materials Test substance SNALP-18534 consists of siRNA targeting rodent TTR mRNA (AD-18534) formulated into stable nucleic acid-lipid particles (SNALP) for delivery to target tissues. The SNALP formulation (lipid particle) consists of a novel amino lipid (DLinDMA), a PEGylated lipid (mPEG2000-C-DMA), a neutral lipid (DPPC), and cholesterol. The lipid:nucleic acid ratio in the SNALP formulation is approximately 5.8:1 (w:w). SNALP-1955 contains siRNA targeting non-mammalian luciferase mRNA formulated in the same 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 the siRNA content.

[0306] Study Design & Procedures Animals and test substance administration: The study consisted of nine groups of Sprague-Dawley rats (four males per group). Animals were allowed at least two days of acclimation before the study, and all animals were 7 weeks of age at the start of dosing. Administered doses were calculated based on body weight data collected before dosing on Day 1. Test and control substances were administered as a single 15-minute, 1-hour, 2-hour, or 3-hour IV infusion via the tail vein using a 24G 3 / 4" cannula sealed with a Baxter injection site septum connected via a 27G Terumo butterfly needle to a Baxter AS40A syringe pump. The dose volume was 3 mL / kg, the infusion rate was 12 mL / kg / hour, and animals were free to move about in their 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 21]

[0307] Tissue collection and RNA isolation: On day 0, animals were anesthetized with isoflurane inhalation, and predose blood samples were collected into serum separator tubes by retro-orbital bleed. Blood samples were allowed to clot at room temperature for approximately 30 minutes before centrifugation at 4°C. Serum samples were then stored at -80°C until analysis. On day 3, animals in all nine treatment groups received a lethal dose of ketamine / xylazine. Blood was collected via the retrocaval vena cava into serum separator tubes and allowed to clot at room temperature for approximately 30 minutes before centrifugation at 4°C. Serum samples were stored at -80°C until analysis. Liver tissue was harvested and flash-frozen on dry ice. Frozen liver tissue was pulverized, and tissue lysates were prepared for liver mRNA quantification.

[0308] TTR mRNA quantification: TTR mRNA levels relative to GAPDH mRNA levels were determined in lysates using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, CA). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean TTR mRNA levels were normalized to the mean GAPDH mRNA levels for each sample.

[0309] To obtain relative levels of TTR mRNA expression, the group mean values ​​for the SNALP-1955 and SNALP-18534 treatment groups at 15 minute, 1 hour, and 2 hour infusion times were then normalized to the mean value for the PBS treatment group at 15 minute infusion time, while the group mean values ​​for the SNALP-1955 and SNALP-18534 treatment groups at 3 hour infusion time were then normalized to the mean value for the PBS treatment group at 3 hour infusion time.

[0310] result As shown in Figure 16, a single IV infusion of 1 mg / kg SNALP-18534 at different infusion times, from 15 minutes to 3 hours, resulted in comparable inhibition of liver TTR mRNA levels measured 2 days after dosing. A single IV infusion of 1 mg / kg SNALP-18534 also demonstrated sustained TTR downregulation over 29 days from a single 15-minute IV infusion compared with the SNALP-1955 control (data not shown). Compared with 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<0.001), 94% (p<0.001), 92% (p<0.001), and 93% (p<0.001), respectively. The specificity of SNALP-18534 activity is demonstrated by the lack of significant target inhibition by SNALP-1955 administered by 1, 2, or 3 hour IV infusion at the same dose level.

[0311] conclusion This study demonstrates that different infusion times, from 15 minutes up to 3 hours, do not affect the efficacy of a single IV dose of 1 mg / kg SNALP-18534 in rats, as assessed by reduction in TTR mRNA levels in the liver.

[0312] Example 15. In vivo reduction of wild-type TTR mRNA in rat liver by LNP07-18534 and LNP08-18534 To evaluate the efficacy of two novel lipid nanoparticle formulations, LNP07 and LNP08, for siRNA delivery in rats, a rodent-specific TTR siRNA, AD-18534, was formulated in LNP07 (LNP07-18534) or LNP08 (LNP08-18534) and administered by 15-minute IV infusion, and hepatic TTR mRNA was quantified. Sprague-Dawley rats (4 animals per group) were administered a 15-minute IV infusion 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 targeting the non-mammalian luciferase gene. Forty-eight hours later, animals were anesthetized, and liver tissue was collected, flash-frozen, and stored at -80°C until processing.

[0313] For TTR mRNA quantification, frozen liver tissue was ground into powder and lysates were prepared. TTR mRNA levels relative to GAPDH mRNA levels were determined in the lysates using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, CA). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean TTR mRNA levels were normalized to the mean GAPDH mRNA levels for each sample. The group means of the normalized values ​​were then further normalized to the mean values ​​for the PBS-treated group to obtain the relative levels of TTR mRNA expression.

[0314] The results are shown in Figure 17. LNP07-18534 reduced TTR mRNA levels in the liver in a dose-dependent manner, achieving 94% suppression of TTR mRNA at 1 mg / kg. The effect was specific, as the negative control LNP07-1955 at 1 mg / kg had no significant effect on TTR mRNA levels compared to the PBS control. The mRNA ED50 was determined to be approximately 0.05 mg / kg for LNP07-18534. LNP08-18534 reduced TTR mRNA levels in the liver in a dose-dependent manner, achieving 86% suppression of TTR mRNA at 0.1 mg / kg. The effect was specific, as the negative control LNP08-1955 at 0.1 mg / kg had no significant effect on TTR mRNA levels compared to the PBS control. The mRNA ED50 was determined to be approximately 0.02 mg / kg of LNP08-18534.

[0315] These results demonstrate that LNP07-18534 and LNP08-18534 are effective in suppressing wild-type TTR mRNA in rat liver when administered by IV infusion, and that LNP07 and LNP08 are effective formulations for delivering siRNA to the liver.

[0316] Example 16: Reduction of TTR liver mRNA by a single intravenous administration of LNP09-18534 or LNP11-18534 in Sprague-Dawley rats the purpose: To evaluate the efficacy of two novel lipid nanoparticle (LNP) formulations for delivery of the rodent TTR-specific siRNA AD-18534 in Sprague-Dawley rats to reduce endogenous (wild-type) liver TTR mRNA levels. Rats were administered 0.01, 0.03, 0.1, or 0.3 mg / kg of either LNP09-18534, LNP11-18534, or phosphate-buffered saline (PBS) intravenously via a 15-minute infusion, and TTR liver mRNA levels were assayed 48 hours after treatment.

[0317] material and method: LNP09 formulation: (XTC / DSPC / Chol / PEG 2000 -C14) = 50 / 10 / 38.5 / 1.5 mol%, lipid: siRNA approximately 11:1. LNP11 formulation: (MC3 / DSPC / Chol / PEG 2000 -C14) = 50 / 10 / 38.5 / 1.5 mol%, lipid: siRNA ca. 11.1 Tissue collection and RNA isolation: On day 3, animals in all treatment groups received a lethal dose of ketamine / xylazine. Blood was collected via the retrocaval vena cava into serum separator tubes and then allowed to clot at room temperature for approximately 30 minutes before centrifugation at 4°C. Serum samples were stored at -80°C until analysis. Liver tissue was harvested and flash-frozen on dry ice. Frozen liver tissue was pulverized, and tissue lysates were prepared for liver mRNA quantification.

[0318] TTR mRNA quantification: TTR mRNA levels relative to GAPDH mRNA levels were determined in lysates using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, CA). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean TTR mRNA levels were normalized to the mean GAPDH mRNA levels for each sample. Group means were then normalized to the mean for the PBS-treated group to obtain relative levels of TTR mRNA expression.

[0319] result: As shown in Figure 18, compared to PBS-treated animals, animals treated with LNP09-18534 and LNP11-18534 had a significant dose-dependent reduction in TTR mRNA levels in the liver, reaching a maximum reduction of approximately 90% of mRNA at 0.3 mg / kg for both the LNP09 and LNP11-formulated groups compared to the PBC control group, with doses below 0.03 mg / kg for LNP11-18534 and 0.1 mg / kg for LNP09-18534 achieving a 50% reduction (ED 50 ) was reached.

[0320] conclusion This study demonstrates that a single 15-minute IV infusion of LNP09-18534 or LNP11-18534 results in a dose-dependent reduction of hepatic TTR mRNA in Sprague-Dawley rats. These data demonstrate the efficacy of LNP09-18328 and LNP11-18328 in reducing endogenously expressed (wild-type) TTR mRNA, with ED50 levels of less than 0.03 mg / kg and less than 0.1 mg / kg for LNP11-18534 and LNP09-18534, respectively.

[0321] Example 17: Inhibition of TTR in humans Human subjects are treated with dsRNA targeted to the TTR gene to inhibit expression of the TTR gene to treat the condition.

[0322] A subject in need of treatment is selected or identified. The subject may have liver disease, transthyretin amyloidosis, and / or a transplanted liver.

[0323] The subject's identification may be performed in a clinical setting or elsewhere, for example, by the subject themselves in their own home through the use of a self-test kit.

[0324] At time zero, the first dose of suitable anti-TTR siRNA is administered to the subject.The dsRNA is formulated as described herein.After the first administration, at a certain period, for example, 7 days, 14 days, and 21 days later, the condition of the subject is evaluated, for example, by measuring liver function.This measurement can be accompanied by measuring the expression of TTR in the subject and / or the product of successful siRNA targeting of TTR mRNA.Other relevant criteria can also be measured.The number and strength of the dose are adjusted according to the needs of the subject.

[0325] Following treatment, the subject's tumor growth rate is reduced compared to the rate existing before treatment or compared to the rate measured in a similarly affected but untreated subject.

Claims

1. 1. A pharmaceutical composition comprising a salt of double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR) in a lipid formulation, the dsRNA comprises a sense strand and an antisense strand that form a double-stranded region; the antisense strand comprises the nucleotide sequence of SEQ ID NO: 170: 5'-AUGGAAUACUCUUGGUUAC-3', and the sense strand comprises the nucleotide sequence of SEQ ID NO: 169: 5'-GUAACCAAGAGUAUUCCAU-3'; The lipid formulation comprises a cationic lipid, a non-cationic lipid, and a lipid that inhibits particle aggregation; Pharmaceutical compositions.

2. 2. The pharmaceutical composition of claim 1, wherein the antisense strand consists of the nucleotide sequence of SEQ ID NO: 450: 5'-AUGGAAUACUCUUGGUUACNN-3', and the sense strand consists of the nucleotide sequence of SEQ ID NO: 449: 5'-GUAACCAAGAGUAUUCCAUNN-3'.

3. 2. The pharmaceutical composition of claim 1, wherein the antisense strand consists of the nucleotide sequence of SEQ ID NO: 730: 5'-AUGGAAUACUCUUGGUUACdTdT-3', and the sense strand consists of the nucleotide sequence of SEQ ID NO: 729: 5'-GUAACCAAGAGUAUUCCAUdTdT-3'.

4. 2. The pharmaceutical composition of claim 1, wherein the antisense strand consists of the nucleotide sequence of SEQ ID NO: 1010: 5'-AUGGAAuACUCUUGGUuACdTdT-3', and the sense strand consists of the nucleotide sequence of SEQ ID NO: 1009: 5'-GuAAccAAGAGuAuuccAudTdT-3', where A is adenine, C is cytosine, G is guanine, and U is uracil; u and c are 2'O-methyl U and 2'O-methyl C, respectively, and dT is 2'-deoxythymidine.

5. 3. The pharmaceutical composition of claim 1, wherein the dsRNA comprises at least one modified nucleotide, and the at least one modified nucleotide is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative group or a dodecanoic acid bisdecylamide group, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base containing nucleotide.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the cationic lipid is (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3).

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the non-cationic lipid is distearoylphosphatidylcholine (DSPC).

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the lipid that prevents particle aggregation is a polyethylene glycol (PEG)-lipid.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the lipid formulation further comprises cholesterol.

10. A salt of double-stranded ribonucleic acid (dsRNA) for inhibiting expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises the nucleotide sequence of SEQ ID NO: 1009, 5'-GuAAccAAGAGuAuuccAudTdT-3', and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 1010, 5'-AUGGAAuACUCUUGGUuACdTdT-3', wherein A is adenine, C is cytosine, G is guanine, and U is uracil; u and c are 2'O-methyl U and 2'O-methyl C, respectively, and dT is 2'-deoxythymidine.

11. 11. The salt of claim 10, wherein the dsRNA is formulated in a lipid formulation containing a cationic lipid, a non-cationic lipid, and a lipid that prevents particle aggregation.

12. The salt of dsRNA according to claim 11, wherein the cationic lipid is (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3).

13. 12. The salt of claim 11, wherein the non-cationic lipid is distearoylphosphatidylcholine (DSPC).

14. 12. The salt of dsRNA of claim 11, wherein the lipid that prevents particle aggregation is a polyethylene glycol (PEG)-lipid.

15. The salt of dsRNA according to any one of claims 11 to 14, wherein the lipid formulation further comprises cholesterol.

16. A pharmaceutical composition comprising the salt of the dsRNA according to any one of claims 10 to 15.

17. A pharmaceutical composition according to any one of claims 1 to 9 and 16 or a salt of a dsRNA according to any one of claims 10 to 15 for use in treating a disorder mediated by expression of FAP in a human, (a) the human has transthyretin amyloidosis; and / or (b) the human has liver disease; A pharmaceutical composition or a salt of a dsRNA.

18. 18. The pharmaceutical composition or salt of dsRNA of claim 17, which is administered intravenously.

19. 18. The pharmaceutical composition or salt of dsRNA of claim 17, wherein the human is further receiving an additional therapeutic method for treating TTR amyloidosis, the additional therapeutic method being selected from the group consisting of a diuretic, an angiotensin-converting enzyme inhibitor, an angiotensin receptor blocker, dialysis therapy, and liver transplantation.

20. 1. An in vitro method for inhibiting expression of FAP in a cell, comprising: (a) contacting the cells with the pharmaceutical composition of any one of claims 1 to 9 and 16 or the salt of the dsRNA of any one of claims 10 to 15; (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of mRNA transcripts of the FAP gene, thereby inhibiting expression of the FAP gene in the cells.

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