Compositions and methods for inhibiting trans tyretin expression

Abstract

This provides a dsRNA for inhibiting the expression of trans tyretin. [Solution] Disclosed are a double-stranded ribonucleic acid (dsRNA) that targets the trans tiretin (TTR) gene, and a method of using the dsRNA to inhibit TTR expression.

Description

[Technical Field]

[0001] This invention relates to a double-stranded ribonucleic acid (dsRNA) that targets the trans tiretin (TTR) gene, and to a method for using dsRNA to inhibit TTR expression.

[0002] Cross-reference of related applications This application claims the interests 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 listings This application includes a sequence listing submitted electronically as a text file named ___________.txt, with a size of ________ bytes, created in __________ in 2009. This sequence listing is incorporated by reference. [Background technology]

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

[0005] Both normal-sequence TTR and mutant TTR cause amyloidosis. Normal-sequence TTR causes cardiac amyloidosis in older adults, known as senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA)). SSA is often accompanied by microdeposits in many other organs. Mutations in TTR accelerate the process of TTR amyloidogenesis and are the most important risk factor for the development of clinically significant TTR amyloidosis (also known as ATTR (amyloidosis-trans-tiretin 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 significantly affected, the disease is called familial amyloid neuropathy (FAP). When the heart is primarily involved but the nervous system is not, the disease is called familial amyloid cardiomyopathy (FAC). The third major type of TTR amyloidosis is called meningeal / CNS amyloidosis.

[0007] Double-stranded RNA molecules (dsRNAs) have been shown to block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi). International Publication WO 99 / 32619 (Fire et al.) discloses the use of dsRNAs of at least 25 nucleotides in length to inhibit gene expression in nematodes. Furthermore, dsRNAs have been shown to degrade target RNA in other organisms, including plants (see, e.g., International Publication WO 99 / 53050, Waterhouse et al., and International Publication WO 99 / 61631, Heifetz et al.), fruit flies (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see International Publication WO 00 / 44895, Limmer, and German Patent DE 101 00 586.5, Kreutzer et al.).

[0008] U.S. Patent No. 20070207974 discloses functional and hyperfunctional siRNAs. U.S. Patent No. 20090082300 discloses antisense molecules targeting TTRs. U.S. Patent No. 7,250,496 discloses microRNAs targeting TTRs. [Overview of the project]

[0009] In one embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of trans tiretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding trans tiretin (TTR), the complementary region being less than 30 nucleotides long, and the antisense strand comprising 15 or more consecutive 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 consecutive nucleotides of SEQ ID NO: 169, SEQ ID NO: 449, SEQ ID NO: 729, or SEQ ID NO: 1009. In another related embodiment, the sense strand comprises SEQ ID NO: 449 and the antisense strand comprises SEQ ID NO: 450. In yet another related embodiment, the sense strand comprises SEQ ID NO: 729 and the antisense strand comprises SEQ ID NO: 730. In yet another related embodiment, the sense strand comprises SEQ ID NO: 1009 and the antisense strand comprises 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 trans tiletin is 19 nucleotides long. In another embodiment, the complementary region consists of sequence number 169. In yet another embodiment, each strand of the dsRNA is 19, 20, 21, 22, 23, or 24 nucleotides long. In yet another embodiment, each strand is 21 nucleotides long.

[0011] In one embodiment, the dsRNA for inhibiting trans tiretin expression does not cleave the 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 another embodiment, the dsRNA cleaves the 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 one embodiment, the dsRNA anneals to the 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 further related embodiments, the present invention provides the above-described dsRNA for inhibiting the expression of trans tyretin, wherein the dsRNA comprises one or more modified nucleotides. In related embodiments, at least one modified nucleotide (or nucleotide) is selected from the group consisting of 2'-O-methyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides bonded to a cholesteryl derivative group or a dodecanoic acid bisdecylamide group. 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, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural bases. In one embodiment, the dsRNA comprises at least one 2'-O-methyl-modified nucleotide.

[0013] In other embodiments, the dsRNA for inhibiting transtiretin expression is coupled to a ligand or formulated into a lipid formulation. In some embodiments, the lipid formulation is formulated into an LNP formulation, an LNP01 formulation, an XTC-SNALP formulation, or a SNALP formulation. In related embodiments, the XTC-SNALP formulation is 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 lipid:siRNA in a ratio of approximately 7:1. In further related embodiments, the sense strand of the dsRNA is SEQ ID NO: 1009, the antisense strand is 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 lipids:siRNA in a ratio of approximately 7:1. Alternatively, dsRNAs such as the above may be formulated into an LNP09 formulation as follows: XTC / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mol%. 2000 -C14 and lipid:siRNA in a ratio of approximately 11:1 are used. In another variation, the dsRNA is formulated into an LNP11 formulation as follows: MC3 / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mol%. 2000-C14 and a lipid:siRNA ratio of approximately 11:1 are used. In yet another embodiment, the dsRNA is formulated in an LNP09 or LNP11 formulation and reduces TTR mRNA levels by approximately 85-90% at a dose of 0.3 mg / kg compared to a PBS control group. In yet another embodiment, the dsRNA is formulated in an LNP09 or LNP11 formulation and reduces TTR mRNA levels by approximately 50% at a dose of 0.1 mg / kg compared to a PBS control group. In yet another embodiment, the dsRNA is formulated in 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 blotting. In another embodiment, the dsRNA is formulated into a SNALP preparation as follows: DlinDMA is used in a ratio of DLinDMA / DPPC / cholesterol / PEG2000-cDMA in the order of 57.1 / 7.1 / 34.4 / 1.4, and lipids in a ratio of approximately 7:1:siRNA.

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

[0015] In another embodiment, the present invention provides a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of transthyretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, and the antisense strand comprises a region complementary to a part of the mRNA encoding transthyretin (TTR), and the complementary region is less than 30 nucleotides in length, and the dsRNA comprises a sense strand selected from Table 3A, 3B, 4, 6A, 6B, 7, and 16, and an antisense strand selected from Table 3A, 3B, 4, 6A, 6B, 7, and 16.

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

[0017] In one embodiment, the present invention provides a cell containing any one of the dsRNAs described in the summary of the above invention. In some other embodiments, the present invention provides a vector comprising a nucleotide sequence encoding at least one strand of any one of the dsRNAs described in the summary of the above invention. In one embodiment, the vector is inside a cell.

[0018] In other embodiments, the present invention provides a pharmaceutical composition for inhibiting the expression of the TTR gene, comprising any one of the dsRNAs described in the summary of the invention above, and a pharmaceutically acceptable carrier. In related embodiments, the present invention provides a pharmaceutical composition for inhibiting the expression of the 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 at a ratio of 57.1 / 7.1 / 34.4 / 1.4, respectively.

[0019] In yet another embodiment, the present invention provides a method for inhibiting the expression of TTR in a cell, the method comprising: (a) contacting the cell with any one 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 the mRNA transcript of the TTR gene, thereby inhibiting the expression of the TTR gene in the cell.

[0020] In another embodiment, the present invention provides a method for treating a disorder mediated by TTR expression, comprising administering a therapeutically effective amount of one of the dsRNAs described in the above 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 another related embodiment, the dsRNA is administered to the person at about 1.0 mg / kg. In another related embodiment, the person to be treated suffers from trans tiretin amyloidosis and / or liver disease. In a related embodiment, the person is further provided with a liver transplant. In 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 another related embodiment, administration of the dsRNA does not result in immunostimulatory activity in the person, as measured by IFN-α and TNF-α ELISA assays. In another related embodiment, administration of the dsRNA reduces hepatic TTR mRNA levels by approximately 97% or serum TTR protein levels by approximately 90%, with a dsRNA concentration of 6 mg / kg. In another related embodiment, administration of the dsRNA reduces hepatic TTR mRNA levels and / or serum TTR protein levels for up to 22 days, with a dsRNA concentration of 6 mg / kg or 3 mg / kg. In another related embodiment, the dsRNA is formulated into an LNP09 formulation as follows: XTC / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mol%. 2000 -C14 and lipid:siRNA in a ratio of approximately 11:1 are used. In 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 mol%. 2000-C14 and lipid:siRNA in a ratio of approximately 11:1 are used. In yet another relevant embodiment, the dsRNA is formulated in an LNP09 or LNP11 formulation and reduces TTR mRNA levels by approximately 85-90% at a dose of 0.3 mg / kg compared to a PBS control group. In yet another relevant embodiment, the dsRNA is formulated in an LNP09 or LNP11 formulation and reduces TTR mRNA levels by approximately 50% at a dose of 0.1 mg / kg compared to a PBS control group. In yet another relevant embodiment, the dsRNA is formulated in 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 blotting. In yet another relevant embodiment, administration of the dsRNA suppresses serum TTR protein levels in humans up to 14 days post-treatment when administered 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 DLinDMA / DPPC / cholesterol / PEG2000-cDMA ratio 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 a use of dsRNA for treating a disorder mediated by TTR expression, comprising administering to a person in need of such treatment a therapeutically effective amount of one of the dsRNAs described in the above summary of the invention. In a relevant 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 relevant embodiment, the dsRNA is administered to the person at about 1.0 mg / kg. In another relevant embodiment, the person suffers from trans tyretin amyloidosis and / or liver disease. In yet another embodiment of the use provided by the present invention, the treated person is further provided with a liver transplant.

[0022] In another embodiment, the present invention provides the use of dsRNA in a method for inhibiting intracellular TTR expression, the method comprising (a) contacting the cells with the dsRNA described in the abstract of the invention above, and (b) maintaining the cells produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the TTR gene, thereby inhibiting intracellular TTR gene expression.

[0023] Details of one or more embodiments of the present invention are described below. Other features, purposes, and advantages of the present invention will become apparent from the description and drawings and from the claims. [Brief explanation of the drawing]

[0024] [Figure 1] This graph shows the TNFα and IFNα levels in cultured human PBMCs after transfusion of TTR using siRNA. [Figure 2] Figures 2A and 2B show the dose-response curves for AD-18324 and AD-18328, respectively, in HepG2 cells. [Figure 3] This is the dose-response curve for AD-18246 in HepG2 cells. [Figure 4A] Intravenous bolus administration of TTR-dsRNA (AD-18324, AD-18328, and AD-18246) formulated in LNP01 inhibits liver mRNA and plasma protein levels in transgenic H129-mTTR-KO / iNOS-KO / hTTR mice. [Figure 4B] Intravenous bolus administration of TTR-dsRNA (AD-18324, AD-18328, and AD-18246) formulated in LNP01 inhibits liver mRNA and plasma protein levels in transgenic H129-mTTR-KO / iNOS-KO / hTTR mice. [Figure 5]This graph summarizes the measurement of TTR mRNA levels in the livers of non-human primates after 15 minutes of intravenous infusion of TTR-dsRNA (AD-18324 and AD-18328) formulated in SNALP. [Figure 6A] This report shows the inhibition of human V30M TTR liver mRNA and serum protein levels in transgenic mice induced by intravenous bolus administration of SNALP-18328. Group means were determined, normalized relative to the PBS control group, and then plotted. Error bars indicate standard deviation. The percentage reduction in group mean 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] This report shows the inhibition of human V30M TTR liver mRNA and serum protein levels in transgenic mice induced by intravenous bolus administration of SNALP-18328. Group means were determined, normalized relative to the PBS control group, and then plotted. Error bars indicate standard deviation. The percentage reduction in group mean 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] This study demonstrates the persistence of the decline 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 percentage decrease 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]This study demonstrates the persistence of the decline 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 percentage decrease 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] This 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] This shows a decrease in 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] The following shows the measured mRNA levels of TTR in the livers of non-human primates after a single 15-minute intravenous infusion of XTC-SNALP-18328. [Figure 11A] The following shows the respective measurements of TTR mRNA and serum protein levels in the livers of non-human primates after a 15-minute intravenous infusion of LNP09-18328 or LNP11-18328. [Figure 11B] The following shows the respective measurements of TTR mRNA and serum protein levels in the livers of non-human primates after a 15-minute intravenous infusion of LNP09-18328 or LNP11-18328. [Figure 11C] This shows the time course of serum protein levels in TTR over 28 days following a 15-minute intravenous infusion of 0.3 mg / kg of LNP09-18328, compared to the PBS control group. [Figure 12] The mRNA sequence of human TTR (reference sequence NM_000371.3, sequence number 1331) is shown. [Figure 13]These are the mRNA sequences of human and rat TTR, respectively. Figure 13A shows the mRNA sequence of human TTR (reference sequence NM_000371.2, SEQ ID NO: 1329). Figure 13B shows the mRNA sequence of rat TTR (reference sequence NM_012681.1, SEQ ID NO: 1330). [Figure 14] The nucleotide alignments of NM_000371.3, NM_000371.2, and AD-18328 are shown. [Figure 15] The symptoms and variations in TTRs associated with familial amyloid neuropathy, familial amyloid cardiomyopathy, and CNS amyloidosis are illustrated. [Figure 16] This study shows the reduction in mRNA levels in the liver using SNALP-18534 at different infusion durations for TTR. Animal groups (n=4 / group) were administered 1 mg / kg of SNALP-18534 via infusions of 15 minutes, or 1, 2, or 3 hours. After 48 hours, rats were euthanized and livers were collected. mRNA levels of TTR and GAPDH were measured from liver lysates using a Quantigene bDNA assay. The ratio of TTR mRNA level to GAPDH mRNA level was calculated for each animal. Group means were determined, normalized to the PBS control group, and then plotted. Error bars indicate standard deviation. (***p<0.001, one-way ANOVA and Bonferroni post-hoc tests compared to PBS) [Figure 17] The following shows the measured mRNA levels of TTR in rat liver after 15 minutes of intravenous infusion of LNP07-18534 or LNP08-18534. [Figure 18]This study demonstrates in vivo inhibition of endogenous TTR mRNA levels in the liver of Sprague-Dawley rats after 15-minute intravenous infusion of LNP09-18534 or LNP11-18534. Animal groups (n=4 / group) were intravenously administered 0.01, 0.03, 0.1, or 0.3 mg / kg of LNP09-18534, LNP-11-18534, or PBS via 15-minute infusion. After 48 hours, animals were euthanized and livers were collected. TTR and GAPDH mRNA levels were measured from lysates of liver biopsies using a Quantigene bDNA assay. The ratio of TTR mRNA level to GAPDH mRNA level was calculated for each animal. Group means were determined, normalized against the PBS control group, and then plotted. Error bars indicate standard deviation. [Modes for carrying out the invention]

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

[0026] The dsRNAs of the compositions discussed herein comprise RNA strands (antisense strands) having a region less than 30 nucleotides in length, generally 19–24 nucleotides in length, and are substantially complementary to at least a portion of the mRNA transcript of the TTR gene. The use of these dsRNAs enables targeted degradation of mRNA of genes involved in pathologies related to TTR expression in mammals. In particular, very low doses 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 can be mediated by downregulating TTR, such as in the treatment of liver disease or TTR amyloidosis, e.g., FAP.

[0027] Methods and compositions containing dsRNA of TTR are useful for treating pathological processes mediated by TTR expression, such as TTR amyloidosis. In one embodiment, a method for treating a disorder mediated by TTR expression includes administering a therapeutically effective amount of TTR-targeted dsRNA to a person in need of such treatment. In one embodiment, the dsRNA is administered to a person at approximately 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 preparing and using compositions containing dsRNA for inhibiting the expression of the TTR gene, as well as compositions and methods for treating diseases and disorders caused by the expression of this gene. The pharmaceutical compositions covered by the present invention include, together with a pharmaceutically acceptable carrier, dsRNA having an antisense strand less than 30 nucleotides long, generally 19 to 24 nucleotides long, and containing a complementary region that is substantially complementary to at least a portion of the RNA transcript of the TTR gene. The pharmaceutical compositions covered by the present invention also include dsRNA having an antisense strand less than 30 nucleotides long, generally 19 to 24 nucleotides long, and containing a complementary region that is substantially complementary to at least a portion of the RNA transcript of the TTR gene.

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

[0030] In one embodiment, the dsRNA may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides. In one embodiment, the modified nucleotides may include 2'-O-methyl-modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and / or terminal nucleotides bonded to a cholesteryl derivative group or a dodecanoic acid bisdecylamide group. In one embodiment, the modified nucleotides may include 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramides, and / or non-natural bases having nucleotides.

[0031] In one embodiment, the complementary region of the dsRNA has a nucleotide length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more. In one embodiment, the complementary region contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or more consecutive nucleotides of sequence number 169.

[0032] In one embodiment, each strand of dsRNA has a nucleotide length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides. 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 inhibition of TTR mRNA expression of approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or more, as measured by a real-time PCR assay. In one embodiment, administration of dsRNA to cells results in approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or greater inhibition of TTR mRNA expression, as measured by 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 levels in cynomolgus monkeys by approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or more. In one embodiment, administration of dsRNA can reduce hepatic TTR mRNA levels by approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or more, or serum TTR protein levels by approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or more. In one embodiment, administration of dsRNA reduces hepatic 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 days or longer.

[0036] In one embodiment, dsRNA is formulated in an LNP formulation and reduces TTR mRNA levels by approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or more compared to a PBC control group at doses of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / kg. In one embodiment, dsRNA is formulated in an LNP formulation and reduces TTR protein levels by approximately 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, or more compared to a PBC control group, as measured by Western blotting. In one embodiment, when dsRNA is administered to a subject requiring it at a dose of 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, it 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] Accordingly, in some embodiments, pharmaceutical compositions containing TTR dsRNA and a drug-acceptable carrier, methods of using the composition to inhibit the expression of the TTR gene, and methods of using the pharmaceutical composition to treat diseases caused by the expression of the TTR gene are covered in the present invention.

[0038] I. Definition For convenience, the meanings of certain terms and phrases used in this specification, the examples, and the appended claims are provided below. In the event of any clear difference between the usage of terms in other parts of this specification and the definitions provided in this section, the definitions in this section shall prevail.

[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, where the nucleic acid base is thymine, e.g., deoxyribothymine. However, it will be understood that the terms "ribonucleotide," "nucleotide," or "deoxyribonucleotide" may also refer to modified nucleotides or alternative substitutional moieties, which will be further detailed below. Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil are substituted by other moieties without substantially altering the base-pairing ability of oligonucleotides containing nucleotides carrying such substitutional moieties. For example, nucleotides containing inosine as a base, but not limited to these, can base-pair with nucleotides containing adenine, cytosine, or uracil. Therefore, nucleotides containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of the present invention for, for example, nucleotides containing inosine. Sequences containing such substitutions are embodiments of the present invention.

[0040] As used herein, “trans tyretin” (“TTR”) refers to a gene within a cell. TTR is also known as ATTR, HsT2651, PALB, prealbumin, TBPA, and trans tyretin (prealbumin, amyloidosis type I). The mRNA transcript sequence of human TTR can be found at NM_000371. The mRNA sequence of mouse TTR can be found at NM_013697.2. The mRNA sequence of rat TTR 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 the transcription of a TTR gene, including mRNA, which is the product of RNA processing of the primary transcript.

[0042] As used herein, the term “sequence-containing chain” refers to an oligonucleotide containing a nucleotide chain, described by a sequence referred to using standard nucleotide nomenclature.

[0043] As used herein, unless otherwise specified, the term “complementary” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence means, as will be understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence to form a double-stranded structure under certain conditions. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 12–16 hours at 50°C or 70°C, followed by washing. Other conditions, such as physiologically appropriate conditions that may be encountered in living organisms, may be applied. Those skilled in the art will be able to determine a set of conditions that are most appropriate for testing the complementarity of the two sequences, depending on the final use of the hybridized nucleotides.

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

[0045] Furthermore, the “complementary” sequences used herein may include, or may be entirely formed from, non-Watson-Crick base pairs and / or non-natural and modified nucleotides, provided that the above requirements regarding their hybridizing ability are met. Such non-Watson-Crick base pairs include, but are not limited to, GU fluctuations or Hoogsteen-type base pairs.

[0046] The terms “complementary,” “fully complementary,” and “substantially complementary” as used herein may be used for base matching between the sense strand and antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as can be 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" messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a contiguous portion of the mRNA in question (e.g., the mRNA encoding the TTR), including the 5'UTR, open reading frame (ORF), or 3'UTR. For example, a polynucleotide is complementary to at least a portion of the mRNA of the TTR if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the TTR.

[0048] As used herein, the terms “double-stranded RNA” or “dsRNA” refer to a complex of ribonucleic acid molecules having a double-stranded structure comprising two nucleic acid strands that are antiparallel and substantially complementary as defined above. Generally, the majority of nucleotides in each strand are ribonucleotides, but each or both strands may also contain at least one non-ribonucleotide, e.g., deoxyribonucleotide and / or modified nucleotide, as described in detail herein. In addition, as used herein, “dsRNA” may include chemical modifications to ribonucleotides, including significant modifications at multiple nucleotides, and including all kinds of modifications disclosed herein or known in the art. Any such modifications used within an siRNA-type molecule are encompassed by “dsRNA” for the purposes of this specification and the claims.

[0049] The two strands forming a double-stranded structure may be different parts of a longer RNA molecule, or they may be separate RNA molecules. If the two strands are part of a longer molecule and are connected by an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the other, the connecting RNA strands are referred to as a “hairpin loop.” If the two strands are covalently connected by means other than an uninterrupted nucleotide chain between the 3' end of one strand and the 5' end of the other, the connecting structure is referred to as 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 the dsRNA, minus any overhangs present in the double-stranded structure. In addition to the double-stranded structure, dsRNA may contain one or more nucleotide overhangs. Furthermore, 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 a dsRNA when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. “Bluish” or “blunt-ended” means that there is no unpaired nucleotide at the end of the dsRNA, i.e., there is no nucleotide overhang. A “blunt-ended” dsRNA is a dsRNA that is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at any end of the molecule.

[0051] The term “antisense strand” refers to a strand of dsRNA that includes a region substantially complementary to the target sequence. As used herein, the term “complementary region” refers to a region of the antisense strand that is substantially complementary to the sequence, e.g., the target sequence as defined herein. If the complementary region is not perfectly complementary to the target sequence, the mismatch is most acceptable and, if present, generally within the terminal region, e.g., within 6, 5, 4, 3, or 2 nucleotides at the 5' and / or 3' ends.

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

[0053] As used herein, the term “SNALP” refers to a stable nucleic acid lipid particle. A SNALP represents a lipid vesicle coating a small aqueous interior containing nucleic acids, such as dsRNA or plasmids on which dsRNA is transcribed. SNALPs are described, for example, in U.S. Patent Applications Publications 20060240093, 20070135372, and U.S. Patent Application Publication No. USSN61 / 045,228, filed April 15, 2008. These applications are incorporated herein by reference.

[0054] When referring to dsRNA, “introduction into cells” means, as understood by those skilled in the art, facilitating uptake or absorption into cells. Absorption or uptake of dsRNA may occur through unassisted diffusion processes, active cellular processes, or by auxiliary agents or devices. 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, introduction into cells includes delivery to the organism. For example, in vivo delivery may involve injecting dsRNA into a tissue site or administering it systemically. In vitro introduction into cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein or known in the art.

[0055] Terms such as “to stop expression,” “to inhibit the expression of,” “to downregulate the expression of,” and “to suppress the expression of,” insofar as they refer to the TTR gene, in this specification, refer to at least partial suppression of TTR gene expression, which can be isolated and / or detected from a first cell or cell population that has been treated to transcribe the TTR gene and inhibit TTR gene expression, compared to a second cell or cell population (control cell) that is substantially identical to the first cell or cell population but has not been treated in the same way. The degree of inhibition is usually expressed as follows:

number

[0056] Alternatively, the degree of inhibition may be expressed in terms of parameters functionally linked to TTR gene expression, such as the amount of TTR gene-encoded protein secreted by cells, or a specific phenotype, such as a reduction in the number of cells exhibiting apoptosis. In principle, arrest of TTR gene expression can be determined in any cell expressing the target constitutively or by genomic engineering, and by any suitable assay. However, if a reference is needed to determine whether a certain dsRNA inhibits TTR gene expression to a certain extent and is therefore included in the present invention, the assays provided in the following examples serve as such references.

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

[0058] As used herein in the context of TTR expression, terms such as “treat” and “cure” refer to the reduction or mitigation of the pathological processes mediated by TTR expression. In the context of the present invention, to the extent that it relates to any of the other pathological conditions listed below herein (other than pathological processes mediated by TTR expression), terms such as “treat” and “cure” mean the reduction or mitigation of at least one symptom associated with such a pathological condition, or the delay or reversal of the progression of such a condition, such as the delay of the progression of TTR amyloidosis, including FAP. Symptoms of TTR amyloidosis include neuropathy (e.g., paresthesia, 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 dysfunction, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial neuropathy, and corneal lattice degeneration.

[0059] As used herein, the phrases “therapeutably effective dose” and “prophylactically effective dose” refer to a dose that provides therapeutic utility in treating, inhibiting, or controlling a pathological process mediated by TTR expression, or in the manifest symptoms of a pathological process mediated by TTR expression. The specific therapeutically effective dose can be readily determined by a typical 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 other TTR-mediated pathological processes.

[0060] As used herein, “pharmaceutical composition” comprises a pharmacologically effective amount of dsRNA and a drug-acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutably effective amount,” or simply “effective amount” refers to the amount of RNA effective in producing the desired pharmacological, therapeutic, or inhibitory outcome. For example, if a clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, then a therapeutically effective amount of drug to treat the disease or disorder is the amount required to bring about at least a 25% reduction in that parameter. For example, a therapeutically effective amount of dsRNA targeting TTR can reduce serum TTR levels by at least 25%. In another example, a therapeutically effective amount of dsRNA targeting TTR can improve liver 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 explicitly excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inactive diluents, disintegrants, binders, lubricants, sweeteners, flavorings, colorants, and preservatives. Suitable inactive diluents include sodium carbonate and calcium, sodium phosphate and calcium, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, will generally be magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to slow absorption in the gastrointestinal tract.

[0062] As used herein, "transformed cells" are cells into which a vector has been introduced, enabling them to express dsRNA molecules.

[0063] II. Double-stranded ribonucleic acid (dsRNA) As described in more detail herein, the present invention provides a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the TTR gene in cells or mammals, for example, in humans suffering from amyloidosis, wherein the dsRNA comprises an antisense strand having a complementary region complementary to at least a portion of the mRNA formed in TTR gene expression, the complementary region being less than 30 nucleotides long, generally 19 to 24 nucleotides long, and upon contact with cells expressing the TTR gene, the dsRNA inhibits the expression of the TTR gene by at least 30% when assayed, for example, by PCR or branched DNA (bDNA) based methods, or by protein-based methods such as Western blotting. The expression of the TTR gene can be reduced by at least 30% by assay, as described in the following examples. For example, the expression of the TTR gene in cell culture, such as Hep3B cells, can be assayed by measuring the mRNA level of TTR by bDNA or TaqMan assay, or by measuring the protein level by ELISA assay, etc. The dsRNA of the present invention may further contain overhangs of one or more single-stranded nucleotides.

[0064] The dsRNA can be synthesized by standard methods known in the art, such as those commercially available from Biosearch, Applied Biosystems, Inc., as will be 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 (antisense strand) contains a complementary region that is substantially complementary to, and generally fully complementary to, a target sequence derived from the mRNA sequence formed during TTR gene expression, and the other strand (sense strand) contains a region complementary to the antisense strand. As a result, the two strands, when combined under favorable conditions, hybridize to form a double-stranded structure. Generally, the double-stranded structure is 15–30, or 25–30, or 18–25, or 19–24, or 19–21, or 19, 20, or 21 base pairs long. In one embodiment, the double-stranded structure is 19 base pairs long. In another embodiment, the double strand is 21 base pairs long. When two different siRNAs are used in combination, the lengths of the double strands may be the same or different.

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

[0066] The dsRNA of the present invention may include one or more single-chain overhangs of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-chain nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotide overhangs at each of the 3' and 5' ends of the sense strand. In a further embodiment, the sense strand of the dsRNA has 1 to 10 nucleotide overhangs at each of the 3' and 5' ends of the antisense strand.

[0067] dsRNAs having at least one nucleotide overhang may exhibit unexpectedly superior inhibitory properties compared to their blunt-ended counterparts. In several embodiments, the presence of only one nucleotide overhang enhances the interference activity of the dsRNA without affecting its overall stability. dsRNAs with only one overhang have been found to be particularly stable and effective in vivo, as well as in various cells, cell culture media, blood, and serum. Generally, the single-chain overhang is located at the 3' end of the antisense strand, or alternatively, at the 3' end of the sense strand. The dsRNA may also have a blunt end, generally located at the 5' end of the antisense strand. Such dsRNAs may have improved stability and inhibitory activity, thus allowing for lower doses, i.e., less than 5 mg per kg of recipient body weight per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3' end and a blunt end at the 5' end. In another embodiment, one or more nucleotides within the overhang are substituted with nucleoside thiophosphates.

[0068] In one embodiment, the TTR gene is a human TTR gene. In a particular embodiment, the sense strand of the dsRNA is one of the sense sequences from Table 3A, 3B, 4, 6A, 6B, or 7, and the antisense strand is one of the sense sequences from Table 3A, 3B, 4, 6A, 6B, or 7. Alternative antisense agents targeting any site of the target sequence provided in Table 3A, 3B, 4, 6A, 6B, or 7 can be easily determined using the target sequence and adjacent TTR sequences.

[0069] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of 20–23, but especially 21, base pairs have been praised for their particular effectiveness in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877–6888). However, others have found that shorter or longer dsRNAs may be equally effective. In the embodiments described above, due to the nature of the oligonucleotide sequences provided in Table 3A, 3B, 4, 6A, 6B, or 7, the dsRNAs addressed in the present invention may include at least one strand of the lengths described herein. It can be reasonably anticipated that shorter dsRNAs having one of the sequences in Table 3A, 3B, 4, 6A, 6B, or 7, with a small number of nucleotides subtracted from one or both ends, may be equally effective compared to the dsRNAs described above. Therefore, dsRNAs having partial sequences of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one of the sequences in Tables 3, 4, 6, or 7 are intended to be dsRNAs whose ability to inhibit TTR gene expression in the assays described below herein differs from dsRNAs containing the complete sequence by 5, 10, 15, 20, 25, or 30% or less of the inhibition, as intended by the present invention. Furthermore, dsRNAs that cleave within a desired TTR target sequence can be easily constructed using a corresponding TTR antisense sequence and a complementary sense sequence.

[0070] In addition, the dsRNAs provided in Tables 3A, 3B, 4, 6A, 6B, or 7 identify sites within the TTR that are susceptible to RNAi-based cleavage. Thus, the present invention further features dsRNAs that target within sequences targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within a sequence of the first dsRNA if the second dsRNA cleaves the message at any site 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 Tables 3A, 3B, 4, 6A, 6B, or 7, which are ligated to a further nucleotide sequence taken from a region adjacent to a selected sequence within the TTR gene.

[0071] The dsRNAs discussed in this invention may contain one or more mismatches with the target sequence. In one embodiment, the dsRNAs discussed in this invention contain three or fewer mismatches. When the antisense strand of the dsRNA contains a mismatch with the target sequence, it is preferable that the range of the mismatch is not located in the center of the complementary region. When the antisense strand of the dsRNA contains a mismatch with the target sequence, it is preferable that the mismatch is 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 complementary to the region of the TTR gene, the dsRNA generally does not contain any mismatches within the central 13 nucleotides. Using the methods described in this invention, it is possible to determine whether a dsRNA containing a mismatch with the target sequence is effective in inhibiting the expression of the TTR gene. Considering the effectiveness of mismatched dsRNAs in inhibiting TTR gene expression is important, especially when specific complementary regions within the TTR gene are known to exhibit polymorphic sequence diversity within the population.

[0072] qualification In another embodiment, the dsRNA is chemically modified to enhance its stability. The nucleic acids addressed in this 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, S. Let al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which are incorporated herein by reference. Specific examples of dsRNA compounds useful in this invention include dsRNAs that contain a modified skeleton or that do not contain a natural internucleoside bond. As defined herein, dsRNAs having a modified skeleton include those that retain a phosphorus atom in the skeleton and those that do not. For the purposes of this specification and as is sometimes referred to in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside skeleton can also be considered oligonucleosides.

[0073] Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramides including 3'-aminophosphoramides and aminoalkyl phosphoramides, thionophosphoramides, thionoalkyl phosphonates, thionoalkyl phosphotryesters, as well as boranophosphates with normal 3'-5' linkages, their analogues with 2'-5' linkages, and those with reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0074] Representative U.S. patents teaching the preparation of the phosphorus-containing bond described above include U.S. Patents No. 3,687,808, No. 4,469,863, No. 4,476,301, No. 5,023,243, No. 5,177,195, No. 5,188,897, No. 5,264,423, No. 5,276,019, No. 5,278,302, No. 5,286,717, No. 5,321,131, No. 5,399,676, No. 5,405,939, and This includes, but is not limited to, patents 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, which are each incorporated herein by reference.

[0075] Modified dsRNA skeletons that do not contain a phosphorus atom have skeletons formed by single-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more single-chain heteroatoms or heterocyclic nucleoside bonds. These include those having morpholino bonds (partially formed from the sugar portion of a nucleoside), siloxane skeletons, sulfides, sulfoxides, and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamic acid skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and others having mixed components of N, O, S, and CH2.

[0076] Representative U.S. patents teaching the preparation of the above-mentioned oligonucleotides include U.S. Patents No. 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, and 5,489,677. This includes, but is not limited to, Patent 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, which are each incorporated herein by reference.

[0077] In other suitable dsRNA mimetic compounds, both the sugar and nucleoside bonds of the nucleotide units, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound that has been shown to have excellent hybridization properties is a dsRNA mimetic compound called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of dsRNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleic acid bases are retained and bonded directly or indirectly to the aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082, 5,714,331, and 5,719,262, which are incorporated herein by reference, respectively. Further teachings on 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 methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of U.S. Patent No. 5,489,677 above, wherein the natural phosphodiester backbone is represented as --O--P--O--CH2-, and the amide backbone of U.S. Patent No. 5,602,240 above.

[0079] Also, the modified dsRNA can also contain one or more substituted sugar moieties. Preferred dsRNAs contain at the 2'-position one of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 alkenyl and alkynyl. O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2)[[ID=J15]] n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2 are particularly preferred, where n and m are 1 to about 10. Other preferred dsRNAs have at the 2'-position C1-C 10The group comprises lower alkyl groups, substituted lower alkyl groups, alkali groups, aralkyl groups, O-alkaryl groups, or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl groups, heterocycloalkaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleavage groups, reporter groups, interveners, groups for improving the pharmacokinetic properties of dsRNA, or groups for improving the pharmacodynamic properties of dsRNA, as well as one of other substituents having similar properties. Preferred modifications include 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., an alkoxy-alkoxy group. Preferred further modifications include 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, as described in the following examples herein, and similarly, 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the following examples herein.

[0080] Other preferred modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can be made at other positions on the dsRNA, particularly at the 3' position of the sugar in the 3'-terminal nucleotide or 2'-5'-linked dsRNA, and at the 5' position of the 5'-terminal nucleotide. The dsRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. patents teaching the preparation of such modified sugar structures include U.S. Patents 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. This includes, but is not limited to, patents No. 5,597,909, No. 5,610,300, No. 5,627,053, No. 5,639,873, No. 5,646,265, No. 5,658,873, No. 5,670,633, and No. 5,700,920, some of which are jointly owned by this application, and each of them is incorporated herein by reference in whole.

[0081] dsRNA may also include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include purine bases, adenine (A) and guanine (G), as well as pyrimidine bases, thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases 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, and 5-uracil (pseudo This includes other synthetic and natural nucleic acid bases such as uracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 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, as well as 3-deazaguanine and 3-deazaadenine. Further nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, 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, STand Lebleu, B., ed., CRC Press, 1993.Certain of these nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds addressed 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 double helix 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, even more particularly preferred when combined with 2'-O-methoxyethyl sugar modification.

[0082] Representative U.S. patents teaching specific modified nucleic acid bases as described above, as well as the preparation of other modified nucleic acid bases, include U.S. Patent No. 3,687,808, and U.S. Patents No. 4,845,205, No. 5,130,30, No. 5,134,066, No. 5,175,273, No. 5,367,066, No. 5,432,272, No. 5,457,187, No. 5,459,255, and No. 5,484. This includes, but is not limited to, U.S. Patent Nos. 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, as is U.S. Patent No. 5,750,692.

[0083] conjugate Another modification of dsRNA according to the present invention involves the chemical binding of one or more moieties or conjugates to the dsRNA, which enhances the activity, cell distribution, or cell uptake of the dsRNA.These parts include cholesterol (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYAcad. 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.) 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. This includes, but is not limited to, lipid moieties such as al., Nucleosides & Nucleotides, 1995, 14, 969-973, adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0084] Representative U.S. patents teaching the preparation of such dsRNA conjugates include U.S. Patents 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, and 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 This includes, but is not limited to, patents No. 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, which are each incorporated herein by reference.

[0085] It is not necessary for all positions of a compound to be uniformly modified; in fact, one or more of the aforementioned 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” is a dsRNA compound, in particular a dsRNA containing two or more chemically distinct regions, each consisting 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 to the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to a target nucleic acid. Further regions of the dsRNA can act as substrates for enzymes that can cleave RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA double-stranded DNA. Activation of RNase H, therefore, leads to cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Thus, when chimeric dsRNAs are used, comparable results can often be obtained with shorter dsRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region.

[0086] Cleavage of RNA targets can be routinely detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art. The cleavage site in the target mRNA of dsRNA can generally be determined using methods known to those skilled in the art, e.g., the 5'-RACE method described by Soutschek et al., Nature; 2004, Vol. 432, pp. 173-178 (incorporated herein by reference for all purposes). In one embodiment, 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 the 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 may be modified with a non-ligand group. Many non-ligand molecules are coupled to dsRNA to enhance its activity, cell distribution, or cell uptake, and procedures for such coupling are available in the scientific literature.Such non-ligand portions 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. NYA Acad. Sci., 1992, 660:306, Manoharan et al., Bioorg. Med. Chem. Let., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969); or adamantane acetate (Manoharan et al., Tetrahedron These include lipid moieties such as Lett., 1995, 36:3651, palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such dsRNA-conjugates are listed above.A typical conjugation protocol involves the synthesis of dsRNA having aminolinkers at one or more positions in its sequence. The amino group is then reacted with the molecule to be conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be performed either with dsRNA still bound to a solid support or after cleavage of the dsRNA in solution. Typically, the dsRNA conjugate is purified by HPLC to obtain a pure conjugate.

[0088] dsRNA encoded by a vector In another embodiment, the TTR dsRNA molecule is expressed from a transcription unit inserted into a DNA or RNA vector (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A et al., International PCT Publication WO 00 / 22113; Conrad, International PCT Publication 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 integrated into the host genome. Transgenes can also be constructed to enable inheritance as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0089] Individual strands of dsRNA can be transcribed using promoters on two separate expression vectors and simultaneously transfected into target cells. Alternatively, each individual strand of dsRNA may be transcribed using promoters located on the same expression plasmid. In one embodiment, dsRNA is expressed as a reverse repeat joined by a linker polynucleotide sequence, such that the dsRNA has a stem-and-loop structure.

[0090] Recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. Viral vectors expressing dsRNA can be constructed based on, but are not limited to, adeno-associated viruses (see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129 for an overview), adenoviruses (see, e.g., Berkner, et al., BioTechniques (1998) 6:616, Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155), or alphaviruses, as well as other known in the art.Retroviruses have been used to introduce various genes in vitro and / or in vivo into many different cell types, 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 87:61416145, Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043, Ferry et al. 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 al., 1993, J.Immunol. 150:4104-4115, U.S. Patent Nos. 4,868,116, 4,980,286, PCT applications WO 89 / 07136, WO 89 / 02468, WO 89 / 05345, and WO 92 / 07573). Recombinant retroviral vectors that can express genes inserted into the genome of cells by transduction can be produced by transtransferring a 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 range of cells and tissues within 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 capable of accepting the coding sequence of the expressed dsRNA molecule can be used, examples of which include vectors derived from adenoviruses (AV), adeno-associated viruses (AAV), retroviruses (e.g., lentiviruses (LV), rhabdoviruses, mouse leukemia viruses), herpesviruses, etc. The directivity of the viral vector can be modified by pseudotyping the vector with an envelope protein or other surface antigens from other viruses, or by appropriately substituting the capsid protein of a different virus.

[0092] For example, the lentiviral vectors discussed in this invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mocola, etc. The AAV vectors discussed in this invention can be configured to target different cells by manipulating the vector to express different serotypes of capsid proteins. For example, an AAV vector expressing the serotype 2 capsid on a serotype 2 genome is referred to as AAV2 / 2. The AAV2 / 5 vector can be produced by substituting the serotype 2 capsid gene in the AAV2 / 2 vector with the serotype 5 capsid gene. Techniques for constructing AAV vectors expressing different capsid protein serotypes are within the scope of the art, and refer to, for example, 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 vectors suitable for use in the present invention, methods for inserting nucleic acid sequences into the vectors for dsRNA expression, and methods for delivering the viral vectors to target cells are within the scope of the art. See, for example, 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 entirety of which their disclosures are incorporated herein by reference.

[0094] The viral vectors may be derived from AV and AAV. In one embodiment, the dsRNA addressed in the present invention is expressed as two distinct and complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, a U6 or H1 RNA promoter, or a cytomegalovirus (CMV) promoter.

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

[0096] Suitable AAV vectors for expressing the dsRNAs addressed 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 WO 94 / 13788, and International Patent Application WO 93 / 24641, the entirety of which disclosures are incorporated herein by reference.

[0097] The promoters that drive dsRNA expression within either the DNA plasmid or viral vector discussed in this invention may be eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV initial promoter, or actin promoter, or U1 snRNA promoter), or generally RNA polymerase III promoters (e.g., U6 snRNA or 7SK RNA promoter), or prokaryotic promoters, such as the T7 promoter, provided that the expression plasmid also encodes the T7 RNA polymerase required for transcription from the T7 promoter. These promoters can also lead to the expression of transgenes into the pancreas (see, for example, the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

[0098] Furthermore, the expression of transgenes can be precisely regulated by using induced 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 induced expression systems for controlling the expression of transgenes in cells or mammals include regulation by ecdysone, estrogen, progesterone, tetracycline, dimerizing chemotherapeutics, 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.

[0099] Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below and survive within target cells. Alternatively, viral vectors that provide transient expression of dsRNA molecules can be used. Such vectors can be administered repeatedly as needed. Upon expression, dsRNA binds to target RNA and modulates its function or expression. Delivery of dsRNA-expressing vectors may be systemic, such as by intravenous or intramuscular administration, by administration to target cells explanted from the patient and subsequent reintroduction into the patient, or by any other means that enable introduction into desired target cells.

[0100] dsRNA-expressing DNA plasmids are typically translocated 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 translocations 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 in this invention. The success of vector introduction into host cells can be monitored using various known methods. For example, transient translocation can be indicated using a reporter such as a fluorescent marker such as green fluorescent protein (GFP). Stable translocation of ex vivo cells can be ensured by using markers that provide resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance, in the translocated cells.

[0101] Furthermore, dsRNA molecules specific to TTR can be inserted into a vector and used as a gene therapy vector for human patients. The gene therapy vector can be delivered to the target, for example, by intravenous injection, local administration (see U.S. Patent No. 5,328,470), or stereotactic injection (see, for example, Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector may contain the gene therapy vector in an acceptable diluent, or it may contain a sustained-release matrix in which the gene delivery medium is embedded. Alternatively, if a complete gene delivery vector can be produced intact from recombinant cells, for example, a retroviral vector, the pharmaceutical preparation may contain one or more cells that produce the gene delivery system.

[0102] Pharmaceutical composition containing III dsRNA In one embodiment, the present invention provides a pharmaceutical composition containing a dsRNA described herein and a pharmacopoeia-acceptable carrier. The pharmaceutical composition containing 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 TTR expression. Such pharmaceutical compositions are formulated based on the delivery method. 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 infusion into the brain by continuous pump infusion.

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

[0104] Generally, a suitable dose of dsRNA would be in the range of 0.01 to 200.0 milligrams per kilogram of body weight per day, typically ranging from 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered in single doses of 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.

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

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

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

[0108] The pharmaceutical composition may be administered once daily, or the dsRNA may be administered in two, three, or more sub-doses at appropriate intervals throughout the day, further delivered by continuous infusion or controlled-release formulation. In this case, the amount of dsRNA contained in each sub-dose must be correspondingly smaller to achieve the total daily dose. The dose unit may be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of the dsRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for the delivery of drugs to specific sites, such as sites where the drug of the present invention may be used. In this embodiment, the dose unit comprises a plurality of corresponding daily doses.

[0109] The effect of a single dose on TTR levels is long-lasting, and as a result, subsequent doses should not be administered at intervals of more than 3, 4, or 5 days, or more than 1, 2, 3, or 4 weeks, or more than 5, 6, 7, 8, 9, or 10 weeks.

[0110] Those skilled in the art will understand, but will not be limited, that certain factors, including the severity of the disease or disorder, previous treatments, overall health, and / or the age of the subject, as well as other pre-existing conditions, may influence the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of the composition may include a single treatment or a series of treatments. Estimation of the effective dosage and in vivo half-life for each dsRNA encompassed by the present invention can be made using conventional methods or based on in vivo studies using appropriate animal models as described elsewhere in this specification.

[0111] Advances in mouse genetics have led to the creation of numerous mouse models for studying various human diseases, including pathological processes mediated by TTR expression. These models are used for in vivo testing of dsRNAs and for determining therapeutically effective doses. A suitable mouse model is, for example, a mouse containing a plasmid expressing human TTR. Another suitable mouse model is a transgenic mouse carrying a transgene expressing human TTR.

[0112] Data obtained from cell culture assays and animal studies can be used to determine a set of dosage formulations for human use. The dosages of the compositions addressed in this invention are generally within the range of circulating concentrations containing little to no toxicity ED50. Doses may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods addressed in this invention, a therapeutically effective dose can first be estimated from a cell culture assay. The dosage can be formulated in animal models to achieve the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence (e.g., achieving a reduction in the concentration of the polypeptide), including the IC50 (i.e., the concentration of the test compound that achieves half-value inhibition of the symptom) determined in cell culture. Using such information, a more accurate determination of a useful dose in humans can be made. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0113] The dsRNAs discussed in this invention can be administered in combination with other known agents effective in treating pathological processes mediated by the expression of target genes. In any case, the administering physician may adjust the amount and timing of dsRNA administration based on results obtained using standard efficacy measures known in the art or described herein.

[0114] Administration The present invention also includes pharmaceutical compositions and formulations comprising the dsRNA compounds discussed herein. The pharmaceutical compositions of the present invention can be administered in many ways, depending on whether topical or systemic treatment is desired and the extent of treatment. Administration may be topical, including by sprayers, for example, intrapulmonary, intratracheal, intranasal, epidermal and transdermal, or oral or parenteral, by inhalation or blowing of powder or spray. Parenteral administration may include intravenous, intra-arterial, subcutaneous, intracavitary, or intramuscular injection or infusion, or intracranial, for example, intraparenchymal, subarachnoid, or intraventricular administration.

[0115] The dsRNA can be delivered in a manner that targets specific tissues, such as the liver (e.g., hepatocytes of the liver).

[0116] The present invention includes pharmaceutical compositions that can be delivered by direct injection into the brain. The injection may be by stereotactic injection into a specific region of the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus), and the dsRNA may be delivered to multiple regions of the central nervous system (e.g., multiple regions of the brain and / or spinal cord). The dsRNA may be delivered to a diffusion region of the brain (e.g., diffusion delivery to the cerebral cortex).

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

[0118] Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, greases, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, etc., may be required or preferred. Coated condoms, gloves, etc., may also be useful. Suitable topical formulations include those in which the dsRNA addressed in the present invention is mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl (DOPE) ethanolamine, dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine), negative (e.g., dimyristoylphosphatidylglycerol (DMPG)), and cationic (e.g., dioleoyltetramethylaminopropyl (DOTAP) and dioleoylphosphatidylethanolamine (DOTMA)). The dsRNAs discussed in this invention may be encapsulated within liposomes, or they may form complexes with them, particularly cationic liposomes. Alternatively, the dsRNAs may be complexed with lipids, particularly cationic lipids. 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-monocapric acid, 1-dodecyl azacycloheptan-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] Liposome formulations In addition to microemulsions, which are studied and used for drug formulation, there are many other organized surfactant structures. These include monolayers, micelles, bilayers, and vesicles. Vesicles such as liposomes have attracted considerable interest from the standpoint of drug delivery due to the specificity and duration of action they exhibit. As used in this invention, the term "liposome" refers to a vesicle of amphiphilic lipids arranged in one or more spherical bilayers.

[0120] Liposomes are monolayered or multilayered vesicles having a membrane formed from a lipophilic material and an aqueous portion. 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 do not fuse with the cell wall as efficiently, but are taken up in vivo by macrophages.

[0121] To traverse 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. Therefore, it is desirable to use liposomes that are highly deformable and capable of passing through such micropores.

[0122] Further advantages of liposomes include the biocompatibility and biodegradability of liposomes derived from natural phospholipids, their ability to encapsulate a wide range of water-soluble and lipid-soluble drugs, and their ability to protect encapsulated drugs from metabolism and degradation within their internal compartments (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, NY, volume 1, p. 245). Important considerations in the preparation of liposomal formulations include the surface charge of the lipids, the size of the vesicles, and the aqueous volume of the liposomes.

[0123] Liposomes are useful for the transport and delivery of active ingredients to the site of action. Because the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissue, they begin to fuse with the cell membrane, and as the fusion of liposomes and cells progresses, the contents of the liposomes are released into the cell, where the active drug can act.

[0124] Liposome formulations have become the focus of extensive research as a mode of delivery for many drugs. For topical administration, growing evidence suggests that liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with higher absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to deliver a wide range of drugs—both hydrophilic and hydrophobic—to the skin.

[0125] Several reports detail the ability of liposomes to deliver drugs containing high molecular weight DNA to the skin. Analgesics, antibodies, hormones, and compounds containing high molecular weight DNA have been administered to the skin. The majority of applications have resulted in targeting of the upper epidermal layer.

[0126] Liposomes are broadly classified into two classes. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form a stable complex. The positively charged DNA / liposome complex binds to the negatively charged cell surface and is taken up into an endosome. Due to the acidic pH inside the endosome, the liposome ruptures, releasing its contents into the cell's cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0127] pH-sensitive or negatively charged liposomes capture DNA rather than complex it. Since both DNA and lipids are similarly charged, repulsion occurs rather than complex formation. Nevertheless, a portion of the DNA is still captured within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding thymidine kinase genes to cell monolayers under culture. Exogenous gene expression was detected in target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0128] One major type of liposome composition contains phospholipids other than naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic membrane-fusion liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy phosphatidylcholine and egg phosphatidylcholine. Yet another type is formed from mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0129] Several studies have evaluated the topical delivery of liposomal formulations to the skin. Application of interferon-containing liposomes to guinea pig skin resulted in a reduction of cutaneous herpes, while delivery of interferon via other means (e.g., as a solution or emulsion) was ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Furthermore, a subsequent study tested the efficacy of interferon administered as part of a liposomal formulation compared to administration using an aqueous system, concluding that liposomal formulations are superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).

[0130] Furthermore, nonionic liposome systems have been investigated to determine the practicality of drug delivery to the skin, particularly in systems containing nonionic surfactants and cholesterol. Nonionic liposome 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. The results showed that such nonionic liposome systems were effective in promoting the deposition of cyclosporine A to different layers of the skin (Hu et al. STPPharma.Sci., 1994, 4, 6, 466).

[0131] Furthermore, liposomes include “stereostabilized” liposomes, which, as used herein, refer to liposomes containing one or more specified lipids, which, when incorporated into liposomes, result in improved circulating lifespan compared to liposomes lacking such specified lipids. An example of a stereostabilized liposome is one in which a portion of the lipid moiety forming the liposome vesicle contains (A) monosialoganglioside G M1(B) These contain one or more glycolipids, or are derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Although not bound by any particular theory, it is considered in the art that, for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivativeized lipids, the increased circulating half-life of these sterically stabilized liposomes is due to a decrease in intracellular uptake by 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. NYAcad. Sci., 1987, 507, 64) described monosialoganglioside G M1 The ability of galactocerebroside sulfate and phosphatidylinositol to improve the half-life of liposomes in the blood has been reported. These findings are described in detail by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Allen et al., U.S. Patent No. 4,837,028 and International Publication No. WO 88 / 04924, both describe (1) sphingomyelin and (2) ganglioside G 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-dimiristoylphosphatidylcholine are disclosed in International Publication No. WO 97 / 13499 (Lim et al.).

[0133] Many liposomes containing lipids derivatized with one or more hydrophilic polymers, and methods for preparing them, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describe liposomes containing the nonionic surfactant 2C1215G, which contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) point out that hydrophilic coating of polystyrene particles with polymer glycols results in a significant increase in blood half-life. Synthetic phospholipids modified by the attachment of carboxylic acid groups to polyalkylene glycols (e.g., PEG) have been described by Sears (U.S. Patents 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) describe experiments showing that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have a significantly increased circulating half-life in the blood. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivative phospholipids, such as DSPE-PEG, formed from a combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having a covalently bound PEG moiety on the outer surface are described in European Patent EP 0 445 131 B1 and International Publication WO 90 / 04384 (Fisher). Liposome compositions containing 1 to 20 mole percent of PE derivatized by PEG, and methods of use thereof, are described by Woodle et al. (U.S. Patents 5,013,556 and 5,356,633) and Martin et al. (U.S. Patent 5,213,804 and European Patent EP 0 496 813 B1). Liposomes containing many other lipid-polymer conjugates are disclosed in International Publication WO 91 / 05545 and U.S. Patent 5,225,212 (both by Martin et al.) and International Publication 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. Patents 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe PEG-containing liposomes in which the functional group moiety on the surface can be further derivatized.

[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-binding liposomes and claims that the contents of such liposomes may 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 targeting the raf gene.

[0135] Transfersomes are yet another type of liposome, highly deformable lipid aggregates, which are intriguing candidates for drug delivery media. Transfersomes may also be described as lipid droplets, which, due to their high deformability, can easily penetrate smaller pores. Transfersomes are adaptable to the environment in which they are used, for example, self-optimal (adapting to the shape of skin pores), self-repairing, often reaching the target without fragmentation, and often self-loading. To construct transfersomes, surface edge activators, usually surfactants, 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 serum albumin-containing solution.

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

[0137] When a surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have found a wide range of applications in pharmaceuticals and cosmetics and can be used across a wide range of pH values. Generally, their HLB values ​​range from 2 to about 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers also belong to this class. Polyoxyethylene surfactants are the most common components in the class of nonionic surfactants.

[0138] When a surfactant molecule retains a negative charge when dissolved or dispersed in water, it is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acylamides of amino acids, sulfuric acid esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates, and sulfosuccinates, as well as phosphates. The most important components of the class of anionic surfactants are alkyl sulfates and soaps.

[0139] When a surfactant molecule possesses a positive charge, it is classified as cationic when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used components in this class.

[0140] A surfactant is classified as amphoteric if its molecule has the ability to possess either a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

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

[0142] nucleic acid lipid particles In one embodiment, the dsRNA of the TTR addressed in the present invention is completely 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 are very useful for systemic application because they exhibit a long circulating lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site). SPLP includes "pSPLP," which includes complexes of encapsulated condensants and nucleic acids as 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 nm to about 90 nm, and are substantially non-toxic. Furthermore, the nucleic acid, when present in the nucleic acid lipid particles of the present invention, is resistant to degradation by nucleases in aqueous solution. Nucleic acid lipid particles and methods for preparing them are disclosed, for example, in U.S. Patents 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432, and PCT Publication WO 96 / 40964.

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

[0144] Cationic lipids include, for example, 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-dilinolelenyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy (Dilinoleylcarba (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleyloxy-2-Linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-Dilinoleylthio-3-trimethylaminopropane chloride (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-propanediol (propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-di Oxolane (DLin-K-DMA), or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-yl)ethylazanejyl)didodecane-2-ol (Tech G1), or a mixture thereof. Cationic lipids may constitute approximately 20 mol% to 50 mol%, or approximately 40 mol%, of the total lipids present in the particles.

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

[0147] Noncationic lipids include distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoyl phosphatidylcholine (POPC), palmitoyloleoyl phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine. These may be anionic or neutral lipids, including but not limited to 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. Noncationic lipids, if cholesterol is included, may constitute about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles.

[0148] Conjugated lipids that inhibit particle aggregation may include, for example, polyethylene glycol (PEG)-lipids, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates may include, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C8). The amount of conjugated lipids that inhibit particle aggregation may be 0 mol% to about 20 mol%, or about 2 mol%, of the total lipids present in the particles.

[0149] In some embodiments, the nucleic acid lipid particles further contain, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particles, which is cholesterol.

[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). The stock solutions in ethanol can be prepared as follows: ND98, 133 mg / mL; cholesterol, 25 mg / mL; PEG-Ceramide C16, 100 mg / mL. The stock solutions of ND98, cholesterol, and PEG-Ceramide C16 can then be mixed in a molar ratio, for example, 42:48:10. The mixed lipid solution can then be mixed with an aqueous siRNA solution (e.g., in sodium acetate (pH 5)) so that the final ethanol concentration is approximately 35-45% and the final sodium acetate concentration is 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 a thermobarrel extruder, such as a Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged with phosphate-buffered saline (PBS) at, for example, approximately pH 7, e.g., approximately pH 6.9, approximately pH 7.0, approximately pH 7.1, approximately pH 7.2, approximately pH 7.3, or approximately pH 7.4. [ka] For example, the LNP01 formulation is described in International Patent Application Publication 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] Formulations containing LNP09 and XTC are described, for example, in U.S. Provisional Application No. 61 / 239,686, filed on September 3, 2009, which is incorporated herein by reference. Formulations containing LNP11 and MC3 are described, for example, in U.S. Provisional Application No. 61 / 244,834, filed on September 22, 2009, which is incorporated herein by reference.

[0153] Formulations prepared by either standard methods 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 precipitates. The particle size and particle size distribution of lipid nanoparticles can be measured by light scattering using, for example, Malvern Zetasizer Nano ZS (Malvern (USA)). The particles should have a particle size of approximately 20–300 nm, such as 40–100 nm. The particle size distribution should be unimodal. The total siRNA concentration in the formulation and in the captured fraction is estimated using a dye exclusion assay. Samples of formulated siRNA are subjected to Ribogreen (Molecular) in the presence or absence of a surfactant that breaks down the formulation, such as 0.5% Triton-X100. The formulation can be incubated with RNA-binding dyes such as Probes. The total siRNA in the formulation can be determined by the signal from the surfactant-containing sample relative to a standard curve. The captured fraction is determined by subtracting the siRNA-free content (measured by the signal in the absence of the 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. Preferred ranges are 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] Compositions and formulations for oral administration include powders or granules, fine particles, nanoparticles, suspensions or solutions in water or a hydrophobic medium, capsules, gel capsules, sachets, tablets, or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing agents, or binders may be desirable. In some embodiments, the oral formulation is one in which the dsRNA addressed in the present invention is administered in combination with one or more osmotic 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, glutolic acid, glycolic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydrofusidate, 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-monocapric acid, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, or monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof (e.g., sodium). In some embodiments, combinations of osmotic enhancers are used, such as fatty acids / salts combined with bile acids / salts. One exemplary combination is lauric acid, capric acid, and the sodium salt of UDCA. Further osmotic enhancers include polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether. The dsRNAs addressed in this invention may be delivered orally in granular form containing spray-dried particles, or compounded to form microparticles or nanoparticles.dsRNA complexes include polyamino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxetanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethylene glycols (PEG), and starches; polyalkylcyanoacrylates; DEAE-derivativeized polyimines, pollulans, celluloses, and starches. Suitable compound agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermine, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P (TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcyanoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEA These include E-acrylamide, DEAE-albumin and DEAE-dextran, polymethyl acrylate, polyhexyl acrylate, poly(D,L-lactic acid), poly(DL-lactic acid-co-glycolic acid (PLGA), alginates, and polyethylene glycol (PEG). Oral formulations for dsRNA and their preparations are described in detail in U.S. Patent No. 6,887,906, U.S. Patent Publication No. 20030027780, and U.S. Patent No. 6,747,014, which are each incorporated herein by reference in their entirety.

[0155] Compositions and formulations for parenteral, intraparenchymal (into the brain), subarachnoid, intraventricular, or intrahepatic administration may contain sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives such as osmotic enhancers, carrier compounds, and other carriers or excipients that are acceptable as pharmaceutical agents.

[0156] The pharmaceutical compositions of the present invention include, but are not limited to, formulations containing solutions, emulsions, and liposomes. These compositions can be produced from, but are not limited to, various components including pre-formed liquids, self-emulsifying solids, and self-emulsifying semi-solids. Formulations that target the liver are particularly preferred when treating liver disorders such as liver cancer.

[0157] The pharmaceutical formulations of the present invention can be conveniently presented in unit dosage forms and can be prepared by conventional techniques well known in the pharmaceutical industry. Such techniques include the step of combining an active ingredient with one or more pharmaceutical carriers or one or more excipients. Generally, the formulations are prepared by uniformly and closely associating the active ingredient with a liquid carrier, a pulverized solid carrier, or both, and then molding the product if necessary.

[0158] The compositions of the present invention can be formulated into any of many possible dosage forms, including, but are 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 may further contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspensions may also contain stabilizers.

[0159] Emulsion The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically multiphase systems in which one liquid is dispersed in another liquid in the form of droplets, usually with a diameter greater 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). An emulsion is often a two-phase system containing two immiscible liquid phases that are closely mixed and dispersed with each other. Generally, emulsions can be either water in oil (w / o) or oil in water (o / w). When the aqueous phase is finely divided and dispersed as microdroplets in the predominantly 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 microdroplets in the predominantly aqueous phase, the resulting composition is called an oil in water (o / w) emulsion. In addition to the dispersed phases, emulsions may contain further components and active drugs that may exist as solutions in either the aqueous or oil phase, or as separate phases themselves. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants may also be present in the emulsion as needed.Furthermore, pharmaceutical emulsions may be multiple emulsions containing more than two phases, such as oil-in-water (o / w / o) and water-in-oil (w / o / w) emulsions. Such complex formulations often offer specific advantages that simple binary emulsions do not. A multiple emulsion in which individual oil droplets of an o / w emulsion surround small water droplets constitutes a w / o / w emulsion. Similarly, a system of oil droplets surrounded by small spheres of water stabilized in a continuous phase of oil provides an o / w / o emulsion.

[0160] Emulsions are characterized little to no by thermodynamic stability. Often, the dispersed or discontinuous phase of an emulsion is well dispersed in the outer or continuous phase, and this form is maintained by the emulsifier or the viscosity of the formulation. Any of the phases of an emulsion can be semi-solid or solid, as in the case of emulsion-type ointment bases and creams. Other means of stabilizing emulsions require the use of emulsifiers that can be incorporated into any of the phases of the emulsion. Emulsifiers can be broadly classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorbent 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 surfactants, have found broad applicability in emulsion formulations and are outlined 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 both hydrophilic and hydrophobic parts. The ratio of hydrophilic to hydrophobic parts of a surfactant is called the hydrophilic / lipophilic balance (HLB) and is a valuable tool in the classification and selection of surfactants during formulation preparation. Surfactants can be classified into different classes—nonionic, anionic, cationic, and amphoteric—based on the properties of their hydrophilic groups (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. Absorbent bases are hydrophilic, absorbing water to form w / o emulsions, but still retaining the consistency of their semi-solid forms, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids are also used as good emulsifiers, particularly in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides; non-expanding clays, such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal aluminum magnesium silicate; pigments; and non-polar solids, such as carbon or glyceryl tristearate.

[0163] Furthermore, a wide range of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, 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 include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbomer, cellulose ether, and carboxyvinyl polymer). These disperse or swell in water to form colloidal solutions that stabilize the emulsion by forming a strong interfacial thin film around droplets of the dispersed phase, thereby increasing the viscosity of the outer phase.

[0165] Because emulsions often contain many components such as carbohydrates, proteins, sterols, and phosphatides that can readily support microbial growth, these formulations often incorporate preservatives. Commonly used preservatives in emulsion formulations include methylparaben, propylparaben, quaternary ammonium salts, benzalkonium chloride, p-hydroxybenzoic acid esters, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration. Antioxidants used may include 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 cutaneous, oral, and parenteral routes, as well as methods for manufacturing them, are outlined 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 their 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, fat-soluble vitamins, and high-fat nutritional preparations are commonly included in materials administered orally as o / w emulsions.

[0167] In one embodiment of the present invention, a composition of dsRNA and nucleic acid is formulated as a microemulsion. A microemulsion can be defined as a system of water, oil, and an amphiphilic substance that 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). Typically, a microemulsion is prepared by first dispersing oil in an aqueous surfactant solution, and then adding a sufficient amount of a fourth component, generally an alcohol of intermediate chain length, to form a transparent system. Therefore, microemulsions are also described as thermodynamically stable, isotropic, transparent dispersions of two immiscible liquids stabilized by an interfacial thin 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 a combination of 3 to 5 components, including oil, water, surfactant, co-surfactant, and electrolyte. Whether a microemulsion is water-in-oil (w / o) or oil-in-water (o / w) depends on the properties of the oil and surfactant used, as well as the structure and geometric convolution 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] Phenomenological methods utilizing phase diagrams have been extensively studied, providing those skilled in the art with comprehensive knowledge of methods for formulating 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 to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in formulations 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 ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), and decaglycerol decaoleate (DAO750), either alone or in combination with co-surfactants. Co-surfactants are typically short-chain alcohols such as ethanol, 1-propanol, and 1-butanol, which increase interfacial fluidity by penetrating the thin film of surfactant and consequently forming an irregular thin film due to the voids created between the surfactant molecules. However, microemulsions can be prepared without the use of co-surfactants, and alcohol-free, self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerol, propylene glycol, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium-chain (C8-C12) mono, di, and triglycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolated C8-C10 glycerides, vegetable oils, and silicone oils.

[0170] Microemulsions are of particular interest from the standpoint of enhancing drug solubilization and absorption. To enhance the oral bioavailability of peptide-containing drugs, lipid-based microemulsions (both o / w and w / o types) have been proposed (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth.Find.Exp.Clin.Pharmacol., 1993, 13, 205). Microemulsions offer advantages such as improved drug solubilization, protection of drugs 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 beyond 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 attracted together at ambient temperature. This can be particularly advantageous when formulating thermally unstable drugs, peptides, or dsRNAs. Microemulsions are also effective for transdermal delivery of active components in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the present invention are expected to promote improved in vivo absorption of dsRNAs and nucleic acids from the gastrointestinal tract and improve local cellular uptake of dsRNAs and nucleic acids.

[0171] Furthermore, the microemulsion of the present invention may contain further 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. Penetration enhancers used in the microemulsion of the present invention can be classified as belonging to one of five major categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactant agents (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes is described above.

[0172] Penetration enhancer In one embodiment, the present invention utilizes various penetration enhancers to achieve efficient delivery of nucleic acids, particularly dsRNA, to animal skin. Most drugs exist in solution in both ionized and non-ionized forms. However, typically only lipid-soluble or lipophilic drugs readily cross cell membranes. It has been found that even non-lipophilic drugs can cross cell membranes if the membrane being crossed is treated with a penetration enhancer. In addition to assisting the cross-membrane diffusion of non-lipophilic drugs, penetration enhancers also enhance the permeability of lipophilic drugs.

[0173] Penetration enhancers can be classified into one of five major categories: surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactant agents (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the aforementioned classes of penetration enhancers is described in more detail below.

[0174] Surfactants: In relation to the present invention, surfactants (or "surface-activating agents") are chemical substances that, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, resulting in 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), as well as perfluoro compound 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-monoleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocapric acid, 1-dodecyl azacycloheptan-2-one, acylcarnitine, acylcholine, and their C 1-10 This includes alkyl esters (e.g., methyl, isopropyl, and t-butyl), as well as their mono and diglycerides (i.e., oleates, laurates, caprates, myristates, palmitates, stearates, linoleates, 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 roles of bile include promoting the dispersion 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 osmotic enhancers. Therefore, 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 pharmacoagulated sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glycolic acid (sodium glucose), glycolic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), tauro-24,25-dihydrofusidate sodium (STDHF), glycodihydrofusidate sodium, 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 agents: Chelating agents used in connection with the present invention can be defined as compounds that remove metal ions from a solution by forming a complex with them, resulting in enhanced absorption of dsRNA through the mucous membrane. With regard to their use as penetration enhancers in the present invention, since most characterized DNA nucleases require divalent metal ions for catalytic activity and are therefore inhibited by chelating agents, chelating agents have the further advantage of also functioning as DNase inhibitors (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include, but are not limited to, disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanilate), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of beta-diketones (enamine) (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 compounds: As used herein, non-chelating, non-surfactant osmotic enhancers can be defined as compounds that exhibit only slight activity as chelating agents or surfactants, but nevertheless enhance the absorption of dsRNA through the gastrointestinal mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of osmotic enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenyl azacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92), as well as nonsteroidal anti-inflammatory drugs such as diclofenac sodium, indomethacin, and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

[0179] Carrier Furthermore, certain compositions of the present invention incorporate a carrier compound in the formulation. As used herein, “carrier compound” or “carrier” may refer to a nucleic acid or analogue thereof that is inert (i.e., not biologically active itself) but is recognized as a nucleic acid by an in vivo process that reduces the bioavailability of biologically active nucleic acids, for example, by the degradation of biologically active nucleic acids or by promoting their removal from circulation. Co-administration of nucleic acids and carrier compounds typically results in an excess of the latter substance and may lead to a significant reduction in the amount of nucleic acid recovered in the liver, kidneys, or other extracirculation storage sites, possibly due to competition between the carrier compound and nucleic acid for a common receptor. For example, the recovery of partial phosphorothioate dsRNAs in liver tissue may be reduced when administered co-administered with polyinosinate, dextran sulfate, polycytidic acid, or 4'-acetamido-4 isothiocyanostilbene-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 carrier compounds, a "pharmaceutical carrier" or "excipient" is a pharmacodynamically acceptable solvent, suspension, or any other pharmacologically inert medium for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected with the planned mode of administration in mind so as to provide the desired dose, consistency, etc., when combined with the nucleic acid and other components of the given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylate, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, polyethylene glycol, sodium benzoate, sodium acetate), disintegrants (e.g., starch, sodium starch glycolate), and wetting agents (e.g., sodium lauryl sulfate).

[0181] Furthermore, organic or inorganic excipients that do not react harmfully with nucleic acids and are acceptable as agents suitable for oral (non-parenteral) administration can be used to formulate the compositions of the present invention. Suitable and acceptable carriers include, but are not limited to, water, salt solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0182] Nucleic acid formulations for topical administration may 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. These solutions may also contain buffers, diluents, and other suitable additives. Organic or inorganic excipients that do not react adversely with nucleic acids and are acceptable as agents suitable for oral (non-parenteral) administration may be used.

[0183] Acceptable excipients as suitable agents include, but are not limited to, water, saline solutions, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0184] Other components The compositions of the present invention may further contain other conventionally recognized auxiliary components in pharmaceutical compositions, at levels of use established in the art. For example, the compositions may further contain compatible, pharmacoactive materials such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents, or further materials useful for physically formulating the compositions of the present invention into various dosage forms, such as dyes, flavorings, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials should not excessively interfere with the biological activity of the components of the compositions of the present invention when added. The formulations may be sterilized and, if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts, buffers, colorants, flavorings, and / or aromatics that do not adversely interact with one or more nucleic acids of the formulation.

[0185] The aqueous suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. The suspension may also contain stabilizers.

[0186] In some embodiments, the pharmaceutical compositions covered by the present invention include (a) one or more dsRNA compounds and (b) one or more anticytokine bioagents that function by a non-RNAi mechanism. 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 effects of such compounds can be determined, for example, by standard pharmaceutical procedures in cell cultures or experimental animals to determine the LD50 (lethal dose in 50% of the population) and ED50 (therapeutably effective dose in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds exhibiting a high therapeutic index are preferred.

[0188] Data obtained from cell culture assays and animal studies can be used to determine a set of dosage formulations for human use. The dosages of the compositions addressed in this invention are generally within the range of circulating concentrations containing an ED50 that is little to no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods addressed in this invention, a therapeutically effective dose can first be estimated from a cell culture assay. The dose can be formulated in animal models to achieve the circulating plasma concentration range of the compound, or, where appropriate, the polypeptide product of the target sequence (e.g., to achieve a reduction in the concentration of the polypeptide), including the IC50 (i.e., the concentration of the test compound that achieves half-value inhibition of the symptom) determined in cell culture. Using such information, a useful dose in humans can be determined more accurately. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0189] In addition to those administrations, as described above, the dsRNAs addressed in this invention can be administered in combination with other known agents effective in treating pathological processes mediated by TTR expression. In any case, the administering physician may adjust the amount and timing of dsRNA administration based on results obtained using standard efficacy measures known in the art or described herein.

[0190] Methods for treating diseases caused by TTR gene expression The present invention relates, in particular, to dsRNAs targeting TTR and to the use of compositions containing at least one such dsRNA for the treatment of TTR-mediated disorders or diseases. For example, dsRNAs targeting the TTR gene may be useful for the treatment of TTR amyloidosis such as familial amyloid neuropathy (FAP), familial amyloid cardiomyopathy (FAC), meningeal / CNS amyloidosis, amyloidosis type VII (also known as meningeal or cerebrovascular amyloidosis), hyperthyroxinemia, and cardiac amyloidosis (also referred to as senile systemic amyloidosis (SSA) and senile cardiac amyloidosis (SCA)).

[0191] Figure 15 illustrates the symptoms and mutations in TTR associated with familial amyloid neuropathy, familial amyloid cardiomyopathy, and CNS amyloidosis. The present invention includes compositions and methods for the treatment of these diseases and symptoms, targeting these mutations of TTR.

[0192] dsRNAs targeting the TTR gene are also used to treat symptoms and disorders such as TTR amyloidosis. Symptoms associated with such amyloidosis include, for example, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, 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 nerve disorders, corneal lattice degeneration, and congestive heart failure with general weakness, as well as dyspnea due to fluid retention.

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

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

[0195] dsRNA and further therapeutic agents can be administered in similar combinations, for example, parenterally, or further therapeutic agents can be administered as part of a separate composition or by other methods described herein.

[0196] This invention relates to a method for administering a dsRNA targeting TTR to patients with TTR amyloidosis, such as FAP, a disease or disorder mediated by TTR expression. Administration of the dsRNA can stabilize and improve peripheral nervous system function, for example, in patients with FAP. Patients may receive therapeutic doses of dsRNA, such as 0.1 mg / kg, 0.2 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, or 2.5 mg / kg. The dsRNA can be administered over a set period, for example, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 60 minutes, 120 minutes, or 180 minutes. For example, it may be administered regularly, for example, every other week (i.e., every two weeks) for a period of one month, two months, three months, four months, or longer. After the initial treatment regimen, the treatment may be administered at a lower frequency. For example, after administration every other week for three months, it can be repeated once a month for six months or longer. Administration of the dsRNA can reduce blood or urine TTR levels in patients by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more.

[0197] Before administering the total dose of the dsRNA, patients may 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 LFT (liver function test) changes (e.g., 10-20% incidence of LFT) is acceptable (e.g., a reversible, triplicate increase in ALT (alanine aminotransferase) and / or AST (aspartate aminotransferase) levels).

[0198] Many TTR-related diseases and disorders are hereditary. Therefore, patients requiring 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 may also be performed on patients to identify mutations in the TTR gene before administering TTR dsRNA.

[0199] The patient may have a biopsy performed before receiving TTR dsRNA. This biopsy may be from tissue such as the gastric mucosa, peripheral nerves, skin, abdominal fat, liver, or kidney, and may show amyloid plaques indicating TTR-mediated damage. Upon confirmation of amyloid plaques, the patient is administered TTR dsRNA.

[0200] Method for inhibiting TTR gene expression In yet another embodiment, the present invention provides a method for inhibiting the expression of a TTR gene in a mammal. The method comprises administering a composition discussed in the present invention to the mammal such that the expression of a target TTR gene is stopped.

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

[0202] Unless otherwise defined, all technical and chemical terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention pertains. In practicing or testing the dsRNAs and methods discussed herein, methods and materials similar to or equivalent to those described herein may be used, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. In case of any conflict, including definitions, this specification shall prevail. Furthermore, materials, methods, and examples are illustrative and not limiting. [Examples]

[0203] Examples Example 1. dsRNA synthesis Reagent supply source If the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology in a quality / purity standard for molecular biology applications.

[0204] siRNA synthesis Single-chain RNA was synthesized on a 1 μmol scale by solid-phase synthesis 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 a solid support. RNA containing RNA and 2'-O-methylnucleotides was also synthesized by solid-phase synthesis using the corresponding phosphoramidite and 2'-O-methylphosphoramidite (Proligo Biochemie GmbH (Hamburg, Germany)), respectively. These components were incorporated into selected sites within the oligoribonucleotide chain sequence using standard nucleoside phosphoramidite chemical reactions, as described in Current protocols in nucleic acid chemistry, Beaucage, Slet al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA. The iodine oxidizing agent solution was replaced with a solution of Beaucage reagent (Chruachem Ltd (Glasgow, UK)) in acetonitrile (1%) to introduce a phosphorothioate bond. 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 each RNA solution at a wavelength of 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-azabutan-1,4-dicarboxylate AA [ka] A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added to a stirred, ice-cooled solution of glycine ethyl hydrochloride (32.19 g, 0.23 mol) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mol) was added, and the mixture was stirred at room temperature until completion of the reaction was confirmed by TLC. After 19 hours, the solution was partitioned with dichloromethane (3 × 100 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated. The residue was distilled to obtain AA (28.8 g, 61%).

[0208] 3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionate ethyl ester AB [ka] Fmoc-6-aminohexanoic 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-azabutan-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylaminopyridine (0.305 g, 2.5 mmol) were added. The solution was allowed to reach room temperature and stirred for a further 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. This 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 obtain the crude product, which was then purified by column chromatography (50% EtOAC / hexane) to obtain 11.87 g (88%) of AB.

[0209] 3-[(6-amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionate ethyl ester AC [ka] 3-{ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}propionate ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0°C. This solution was stirred for 1 hour. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. This crude product was purified by converting it to its hydrochloride salt.

[0210] 3-({6-[17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionate ethyl ester AD [ka] The hydrochloride salt of 3-[(6-amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionate ethyl ester AC (4.7 g, 14.8 mmol) was incorporated into dichloromethane. This suspension was cooled to 0°C on ice. Diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added to this 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-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylate ethyl ester AE [ka] Potassium t-butoxide (1.1 g, 9.8 mmol) was prepared as a slurry in 30 mL of dry toluene. This mixture was cooled to 0°C on ice, and 5 g (6.6 mmol) of diester AD was slowly added 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 4 g of NaH2PO4H2O in 40 mL of water. The resulting mixture was extracted twice with 100 mL of dichloromethane each time, and the combined organic extract was washed twice with 10 mL of phosphate buffer each time, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0°C, and 50 mL of each solution was added. The extract was extracted three times with cold carbonate buffer at pH 9.5. The aqueous extract was adjusted to pH 3 with phosphoric acid, and each was extracted five times with 40 mL of chloroform. The combined extract was dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethyl acetate / hexane to obtain 1.9 g of β-ketoester (39%).

[0212] [6-(3-hydroxy-4-hydroxymethyl-pyrrolidine-1-yl)-6-oxohexyl]-carbamate 17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester AF [ka] Methanol (2 mL) was added dropwise over 1 hour to a reflux 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 temperature for 1 hour. After cooling to room temperature, 12.5 mL of 1 N HCl was added, and the mixture was extracted with ethyl acetate (3 × 40 mL). The combined ethyl acetate layer was dried on anhydrous sodium sulfate and concentrated under vacuum to obtain the product, which was purified by column chromatography (10% MeOH / CHCl3) (89%).

[0213] (6-{3-[bis-(4-methoxyphenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidine-1-yl}-6-oxohexyl)-carbamate 17-(1,5-dimethylhexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester AG [ka] Diol AF (1.25 g, 1.994 mmol) was evaporated to dryness under vacuum using 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 carried out overnight at room temperature. Methanol was added to stop the reaction. The reaction mixture was concentrated under vacuum, and dichloromethane (50 mL) was added to the residue. This organic layer was washed with 1 M saturated sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. Toluene was evaporated to remove pyridine from the residue. The crude product was purified by column chromatography (2% MeOH / chloroform, 5% MeOH / CHCl3, Rf=0.5) (1.75 g, 95%).

[0214] Mono-(4-[bis-(4-methoxyphenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethylhexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthrene-3-yloxycarbonylamino]-hexanoyl}pyrrolidine-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 a 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 under an argon atmosphere for 16 hours. This was then diluted with dichloromethane (40 mL) and washed with ice-cold citric acid aqueous solution (5% by weight, 30 mL) and water (2 × 20 mL). The organic phase was dried on anhydrous sodium sulfate and concentrated to dryness. The residue was used directly in the next step.

[0215] Cholesterol-derivative CPG AI [ka] 0.254 g, 0.242 mmol succinate AH was dissolved in a mixture of dichloromethane / acetonitrile (3:2, 3 mL). To this 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 / dichloroethane (3:1, 1.25 mL) were added sequentially. Triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 mL) was added to the resulting solution. The reaction mixture changed color to a bright orange. This solution was briefly stirred using a wrist-action shaker (5 minutes). Long-chain alkylamine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was stirred for 2 hours. The CPG was filtered through a sintered funnel and sequentially washed with acetonitrile, dichloromethane, and ether. Unreacted amino groups were shielded using acetic anhydride / pyridine. The achieved CPG load was measured by taking UV measurements (37 mM / g).

[0216] The synthesis of siRNAs supporting a 5'-12-bisdecylamide dodecanoate 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 the cholesteryl derivative, the oxidation step was performed using Beaucage reagent to introduce a phosphorothioate bond to the 5' end of the nucleic acid oligomer.

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

[0218] Example 2A. siRNA design for TTR Transfer siRNA design was performed to identify siRNAs targeting the gene trans tiretin from human (symbol TTR) and rat (symbol Ttr). The design utilized TTR transcripts NM_000371.2 (sequence number 1329) (human) and NM_012681.1 (sequence number 1330) (rat) from NCBI Refseq collection. Double-stranded siRNAs with 100% identity to their respective TTR genes were designed.

[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 human and rat transcriptomes (defined as sets of NM_ and XM_ records in the NCBI Refseq set) using the FASTA algorithm. The alignment was then analyzed using the Python script "offtargetFasta.py" to obtain a score based on the location and number of mismatches between the siRNA and any potential "off-target" transcripts. The off-target score is weighted to highlight differences in the "seeding" region of the siRNA at positions 2–9 from the 5' end of the molecule. The off-target score is calculated as follows: Penalties are imposed for mismatches between the oligo and the transcript. A penalty of 2.8 is imposed for seeding region mismatches at positions 2–9 of the oligo, a penalty of 1.2 is imposed for mismatches at estimated cleavage sites 10 and 11, and a penalty of 1 is imposed for mismatches at positions 12–19. Mismatches at position 1 are not considered. Next, the off-target score for each oligo transcript pair is calculated by summing the mismatch penalties. Then, the minimum off-target score from all oligo transcript pairs is determined and used for subsequent classification of the oligos. Both siRNA strands were assigned to specificity categories according to the calculated scores. Scores greater than 3 are considered highly specific, scores equal to 3 are considered specific, and scores between 2.2 and 2.8 are considered moderately specific. When selecting which oligos should be synthesized, the off-target scores of the antisense strands were sorted 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 formed into double strands. A total of 26 sense and 26 antisense siRNA oligos derived from rat TTR were synthesized and formed into double strands. The double strands contained in the oligos are shown in Tables 2 to 4 (human TTR) and Tables 5 to 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

[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] TTR sequence synthesis TTR sequences were synthesized in a MerMade 192 synthesizer at a scale of 1 μmol. For all sequences in the sequence listing, the "endright" chemical reaction was applied as detailed below. All pyrimidines (cytosine and uridine) in the sense chain were substituted with their corresponding 2'-O-methyl bases (2'-O-methyl C and 2'-O-methyl U). In the antisense chain, the pyrimidines adjacent to the ribo-A nucleoside (towards the 5' position) were substituted with their corresponding 2-O-methyl nucleosides. • We introduced a two-base extension, dTdT, at the 3' end of both the sense and antisense sequences. • The array files were converted to text files to maintain compatibility with the load on the MerMade192 synthesis software.

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

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

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

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

[0234] Example 2B: In vitro screening of TTR siRNA for mRNA suppression For human TTRs targeting dsRNA (Table 2), qPCR (real-time PCR) and bDNA (branched DNA) assays were used to quantify TTR mRNA and inhibit endogenous TTR expression in HepG2 and Hep3B cells. Rodent TTRs targeting dsRNA (Table 5) were synthesized, and their inhibition of endogenous TTR expression in H.4.II.E cells was performed using bDNA assays. Results from single-dose assays were used to select a subset of TTR dsRNA double strands for dose-response experiments to calculate IC50. IC50 results were used to select TTR dsRNA for further testing.

[0235] Cell culture and translocation: 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% CO2 atmosphere in Dulbecco's Modified Eagle Medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC), before being released from the plate by trypsin treatment. 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 double strands per well in a 96-well plate, along with 0.2 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA, cat#13778-150) per well, in addition to 10 μL of Opti-MEM, and incubated at room temperature for 15 minutes. Then, 4 × 10⁶ 4 (HepG2), 2 × 10 4 (Hep3B), or 2 × 10 4 80 μL of antibiotic-free full growth medium containing (H.4.II.E) cells was added. The cells were incubated for 24 hours before RNA purification. A single dose experiment was performed at a final 2x concentration of 10 nM, and dose-response experiments were conducted 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 the MagMAX-96 Total RNA Isolation Kit (Applied Biosystems, Foster City CA, Part Number #: AM1830): Cells were collected, lysed in 140 μL of lysis / binding solution, and then mixed for 1 minute at 850 rpm using an Eppendorf thermomixer (the mixing rate remained constant throughout the process). 20 microliters of magnetic beads were added to the cell lysate and mixed for 5 minutes. The magnetic beads were captured using a magnetic stand, and suspended matter was removed without disturbing the beads. After removing the suspended matter, the magnetic beads were washed with washing solution 1 (with added isopropanol) and mixed for 1 minute. The beads were captured again, and suspended matter was removed. The beads were then washed with 150 μL of washing solution 2 (with added ethanol), captured, and suspended matter removed. Next, 50 μL of a DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was added to the beads, and they were mixed for 10–15 minutes. After mixing, 100 μL of RNA regeneration solution was added, and mixed for 3 minutes. After removing the suspended particles, the magnetic beads were washed again with 150 μL of washing solution 2, mixed for 1 minute, and the suspended particles were completely removed. The magnetic beads were mixed for 2 minutes and dried before eluting the RNA 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 consisting 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 H2O 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: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C, and holding at 4°C.

[0238] Real-time PCR: 2 μL of cDNA was added to each well in a MicroAmp Optical 96-well plate (Applied Biosystems cat#4326659) containing 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 using the ΔΔCt(RQ) assay on an ABI 7000 Prism or ABI 7900HT real-time PCR system (Applied Biosystems). All reactions were performed in triple replication.

[0239] Using the ΔΔCt method, real-time data were analyzed and magnification changes were calculated by normalizing assays performed on cells transfected with 10nM BlockIT fluorescent oligo (Invitrogen Cat#2013) or 10nM AD-1955 (a control double-stranded oligo targeting a non-mammalian luciferase gene).

[0240] Branched DNA assay - QuantiGene 1.0 (Panomics, Fremont, CA. cat#: QG0004) - Used to screen rodent-specific double-stranded DNA. H.4.II.E cells (ATCC) were transfused with 10 nM siRNA. After removing the culture medium, the H.4.II.E cells were lysed in 100 μl of diluted lysis mixture (1 volume of lysis mixture, 2 volumes of nuclease-free water, and 10 μl of proteinase K per mL to a final concentration of 20 mg / mL), and then incubated at 65°C for 35 minutes. Next, 80 μL of a working probe set (a mixture of TTR or GAPDH probes) and 20 μl of cell lysate were added to a 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 1× 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 and 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 double-stranded DNA. After incubation for 24 hours at the indicated dose, the medium was removed, and the cells were lysed in 100 μl of lysis mixture (1 volume of lysis mixture, 2 volumes of nuclease-free water, and 10 μl of proteinase K / mL to 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 gene targeting and GAPDH for endogenous control) and 80 μL of cell lysates were added to the capture plate. The capture plate was incubated at 55°C ± 1°C (approximately 16-20 hours). The following day, the capture plate was washed three times with 1× 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, sealed with aluminum foil, and incubated at 55°C ± 1°C for 1 hour. After incubation for 1 hour, the washing step was repeated, and then 100 μL of amplification reagent was added. After 1 hour, the washing and drying steps were 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 1× washing buffer, dried, and then 100 μL of substrate was added to the capture plate. After incubation for 5–15 minutes, the capture plate was read using a SpectraMax Luminometer (Molecular Devices, Sunnyvale, CA).

[0242] bDNA data analysis: bDNA data were analyzed by (i) subtracting the mean background from each of the three samples, (ii) averaging the resulting three GAPDH (control probe) and TTR (experimental probe) values, and then (iii) obtaining the ratio: (experimental probe - background) / (control probe - background).

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

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

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

[0246] [Table 12] Table 10 below summarizes the results of single-dose assays for rodent-specific TTR-dsRNA (TTR siRNA). Single-dose results are expressed as the percentage of TTR mRNA relative to the control, and were assayed in rat H.4.II.E cells after transfusion with 10 nM rodent-specific TTR siRNA. These results indicate that several rodent-specific TTR siRNAs are effective in suppressing 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 the potential for immune stimulation, TTR siRNA was assayed in vitro for the induction of TNF-α and IFN-α secretion.

[0249] Human PBMCs were isolated from freshly collected buffy coat (Research Blood Components, Inc., Boston, MA) obtained from healthy donors by standard Ficoll-Hypaque density centrifugation. (1 × 10⁻⁶ cells) 5 (100 μL per well) was seeded into 96-well plates and cultured in RPMI 1640 GlutaMax medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic / antifungal (Invitrogen).

[0250] siRNA was transfused into PBMCs using DOTAP transfusion 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 complex was incubated as specified in the manufacturer's instructions, then added to PBMCs (50 μL / well), and 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 and 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 the translocation reagent was 16.5 pmol per 1 μg of DOTAP.

[0251] In both cases, cytokines in the culture suspension were detected and quantified using commercially available ELISA kits for IFN-α (BMS216INST) and TNF-α (BMS223INST) 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 results of IFN-α and TNF-α stimulation of many TTR siRNAs are shown in Figure 1 (mean of quadruple wells ± standard deviation) and Table 11 below (percentage compared to AD-5048). Significant TNF-α or IFN-α secretion induced by cultured human PBMCs was not evaluated for TTR siRNAs. [Table 14]

[0253] Five lead dsRNAs (siRNAs of TTRs) targeting TTRs were selected in human hepatocyte lines HepG2 and Hep3B, and in the absence of immunostimulatory activity, based on IC50 in the pM range. Double-stranded leads without any mismatches are more likely to achieve significant knockdown of the target transcript than double-stranded leads with mismatches between the oligo and mRNA. Double-stranded leads that have 100% identity in orthologous genes from rat, cynomolgus monkeys, and humans, and do not target regions with known polymorphisms, are generally preferred to enable better translation of cross-species toxicological data and to have broad applicability to human patients. The five lead compounds were selected based on IC50 in hepatocyte lines in the pM range, in the absence of immunostimulatory activity, specificity to human TTR transcripts, and in the absence of known polymorphisms (mutations) within the mRNA region targeted by the double-stranded leads. In the case of TTRs, no 19-nucleotide oligos with complete identity were found in humans, rats, and cynomolgus monkeys. A summary of this data is presented in Table 12, which also includes information on known TTR mutations within regions targeted by double-strand and cross-species reactivity.

[0254] [Table 15]

[0255] Example 4. In vivo reduction of hepatic TTR mRNA and plasma TTR protein in transgenic mice using LNP01-18324, LNP01-18328, and LNP01-18246. Two TTR siRNAs, AD-18324 and AD-18328, were selected for in vivo evaluation. These double-stranded siRNAs showed robust dose-dependent arrest in vivo in hepatocyte lines (e.g., HepG2). Figures 2A and 2B show the dose-response in HepG2 cells after transduction 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 on the y-axis as the residual mRNA of the fractional TTR compared to the control. In HepG2 cells, the IC50s of AD-18324 and AD-18328 were confirmed to be 2 pM and 3 pM, respectively. The TTR target sites for both lead dsRNA candidates are located within the 3' untranslated region of the TTR mRNA, a region without mutations reported in the literature.

[0256] The sequences of the strands of the two candidate reads from the table are reproduced below. Chain: s = sense, as = antisense Position: Base at the 5' position in the transcript of NM_000371.2 [Table 16A] In addition, the 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, where three mutations have been reported in the literature. The dose-response curve for AD-18246 in HepG2 cells is shown in Figure 3. AD-18246 was found to be 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 genetically modified mice. H129-mTTR-KO / iNOS-KO / hTTR transgenic mice (mouse trans tiretin knockout / induced nitric oxide synthase knockout / human trans tiretin gene transfection) aged 3-5 months were administered intravenously (IV) via tail vein in 200 μL of LNP01 containing trans tiretin-specific siRNAs (AD-18324, AD-18328, or AD-18246), control siRNA (AD-1955) formulated in LNP01 targeting a non-mammalian luciferase gene, or PBS. The doses were 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 siRNAs AD-18246, and 6.0 mg / kg for siRNAs AD-1955. LNP01 is a lipid-like preparation consisting of ND98, cholesterol, and PEG-Ceramide C16.

[0258] Approximately 40 hours later, mice were anesthetized with 200 μL of ketamine, and blood was collected by severing the right caudal artery. Whole blood was isolated, and plasma was isolated and stored at -80°C until assayed. Liver tissue was collected, rapidly frozen, and stored at -80°C until processed.

[0259] The efficacy of the treatment was evaluated by (i) measurement of TTR mRNA in the liver 48 hours after administration, and (ii) measurement of TTR protein in plasma 48 hours after pre-collection and administration. TTR liver mRNA levels were assayed using branched DNA assay-QuantiGene2.0 (Panomics cat#:QS0011). Briefly, mouse liver samples were pulverized and tissue lysates were prepared. A liver lysate mixture (1 volume of lysate, 2 volumes of nuclease-free water, and 10 μl of proteinase K / mL to a final concentration of 20 mg / mL) was incubated at 65°C for 35 minutes. Then, 20 μL of a working probe set (TTR probe for gene targeting and GAPDH for endogenous control) and 80 μL of cell lysate were added to a capture plate. The capture plate was incubated at 55°C ± 1°C (approximately 16–20 hours). The following day, the capture plate was washed three times with 1× wash buffer (water without nuclease, buffer component 1, and wash buffer component 2), and then dried by centrifugation at 240 g for 1 minute. 100 μL of pre-amplification reagent was added to the capture plate, sealed with aluminum foil, and incubated at 55°C ± 1°C for 1 hour. After 1 hour of incubation, the washing step was repeated, and then 100 μL of amplification reagent was added. After 1 hour, the washing and drying steps were 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 1× wash buffer, dried, and 100 μL of substrate was added to the capture plate. After incubation for 5–15 minutes, the capture plate was read using a SpectraMax Luminometer. bDNA data were analyzed by subtracting the mean background from each of the three samples, averaging the resulting three GAPDH (control probe) and TTR (experimental probe) values, and then calculating the ratio: (experimental probe - background) / (control probe - background) using a computer.

[0260] TTR plasma levels were assayed using the commercially available "AssayMax Human Prealbumin ELISA Kit" (AssayPro, St. Charles, MO, Catalog#EP3010-1) in accordance with the manufacturer's guidelines. Briefly, mouse plasma was diluted 1:10,000 in 1× mixed diluent, added to a pre-coated plate with the standard kit, incubated at room temperature for 2 hours, and then washed 5 times with the kit's wash buffer. 50 microliters of biotinylated prealbumin antibody were added to each well, incubated at room temperature for 1 hour, and then washed 5 times with the wash buffer. 50 microliters of streptavidin-peroxidase conjugate were 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, and then the reaction was initiated by adding 50 μL / well of chromogenic substrate and incubating at room temperature for 10 minutes. Absorbance at 450 nm was read using a microplate reader (Molecular Devices, Sunnyvale, CA), and the data was analyzed using the Softmax 4.6 software package (Molecular Devices).

[0261] LNP01-18324 and LNP01-18328 were found to reduce hepatic TTR mRNA (Figure 4A) and plasma TTR protein (Figure 4B) levels in a dose-dependent manner with IV bolus administration. The mRNA ED50 of LNP01-18328 was determined to be approximately 1 mg / kg, while the ED50 of 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 hepatic 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 via IV bolus, substantially reduce human TTR mRNA expressed in transgenic mouse livers, resulting in a decrease in human TTR protein in circulation.

[0263] Example 5. In vivo reduction of wild-type TTR mRNA in the liver of non-human primates by SNALP-18324 and SNALP-18328. To evaluate the efficacy of the TTR siRNAs AD-18324 and AD-18328 at the mRNA level of hepatic TTR in non-human primates, the siRNAs were formulated in SNALP and administered by IV infusion over 15 minutes. 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, containing the negative control siRNA AD-1955, which targets the non-mammalian luciferase gene) by IV infusion over 15 minutes. Forty-eight hours after administration, the monkeys were anesthetized with pentobarbital sodium and induced hemorrhage. Liver tissue for TTR mRNA determination was collected, rapidly frozen, and stored at -80°C until processing.

[0264] The mRNA levels of TTR 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. A liver lysate mixture (1 volt lysate, 2 volts nuclease-free water, and 10 ul of proteinase K / mL to a final concentration of 20 mg / mL) was incubated at 65°C for 35 minutes. Then, 20 μL of a working probe set (TTR probe for gene targeting and GAPDH for endogenous control) and 80 μL of cell lysate were added to a capture plate. The capture plate was incubated at 55°C ± 1°C (approximately 16-20 hours). The following day, the capture plate was washed three times with 1× 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 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 washing step was repeated, and then 100 μL of amplification reagent was added. After 1 hour, the washing and drying steps were 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 1× washing buffer, dried, and then 100 μL of substrate was added to the capture plate. After incubation for 5–15 minutes, the capture plate was read using a SpectraMax Luminometer. bDNA data were analyzed by (i) subtracting the mean background from each triple sample, (ii) averaging the resulting GAPDH (control probe) and TTR (experimental probe) values, and then (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 1 mg / kg, respectively.

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

[0267] Example 6. In vivo reduction of mutant (V30M) TTR mRNA and protein by SNALP-18328 in transgenic mice. To evaluate the efficacy of TTR siRNA AD-18328 against mutant (V30M)TTR mRNA in the liver and mutant (V30M)TTR protein in the serum, AD-18328 was formulated into SNALP and administered as an IV bolus to V30M hTTR-generated mice. 8-12 week old V30M hTTR-generated mice (5 animals / group) were administered 200 μL intravenously (IV) of SNALP-18328 (0.03, 0.3, or 3 mg / kg), SNALP-1955 (3 mg / kg, containing the negative control siRNA AD-1955 targeting a non-mammalian luciferase gene), or PBS. The mice used were Mus musculus strain H129-hTTR KO from the Institute of Molecular and Cellular Biology, Porto, Portugal. In short, hTTR H129 gene-transfected mice were crossed with H129 endogenous TTR KO mice (null mice) to generate H129-hTTR gene-transfected mice in a background of TTR (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, all animals in all five treatment groups were administered a lethal dose of ketamine / xylazine. Serum samples were collected and stored at -80°C until analysis. Liver tissue was collected, rapidly frozen, and stored at -80°C until processing.

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

[0270] For the quantification of TTR proteins, 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-generated mice treated with SNALP-18328 showed a dose-dependent and significant reduction in liver TTR mRNA levels compared to the PBS control group, reaching 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) at 3 mg / kg of SNALP-18328 (compared to pre-treatment levels), which was consistent with the reduction in TTR mRNA levels. SNALP-1955 at 3 mg / kg did not have a statistically significant effect on either TTR mRNA or protein levels compared to PBS.

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

[0273] Example 7. Sustained suppression of TTR mRNA and protein by SNALP-18328 in genetically modified mice. To evaluate the persistence of TTR mRNA and protein repression by SNALP-18328, AD-18328 was formulated into SNALP and administered as an IV bolus to V30M hTTR-generated mice. Hepatic TTR mRNA levels and serum TTR protein levels were quantified at various time points after administration. 8-12 week old V30M hTTR-generated mice (4 animals / group) were administered intravenously (IV) 200 μL of either SNALP-18328 (1 mg / kg) or SNALP-1955 (1 mg / kg, containing the negative control siRNA AD-1955, which targets a non-mammalian luciferase gene). The mice used were Mus musculus strain H129-hTTR KO from the Institute of Molecular and Cellular Biology, Porto, Portugal. In short, hTTR H129 gene-transfected mice were crossed with H129 endogenous TTR KO mice (null mice) to generate H129-hTTR gene-transfected mice in a background of null mouse TTR (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 administration, animals in both treatment groups were given a lethal dose of ketamine / xylazine. Serum samples were collected and stored at -80°C until analysis. Liver tissue was collected, rapidly frozen, and stored at -80°C until processing.

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

[0275] For the quantification of TTR proteins, 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 of SNALP-18328 resulted in sustained inhibition of TTR mRNA levels in the liver and TTR protein levels in the serum. Compared to the control group (1 mg / mL SNALP-1955), a single IV dose of 1 mg / kg of SNALP-18328 significantly reduced relative TTR mRNA levels at days 3, 8, 15, and 22 after administration by 96% (p<0.001), 90% (p<0.001), 82% (p<0.001), and 73% (p<0.001), respectively, and did not return to baseline levels at the end of the study (day 22 after administration). Protein levels also decreased on day 3 after administration, with a maximum decrease of 97% (p<0.001) in serum TTR (compared to SNALP-1955). On days 8, 15, and 22 after administration, 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 dose of SNALP-18328 induces sustained suppression of target liver mRNA and serum protein levels in V30M hTTR-generated mice, resulting in a significant decrease in liver TTR mRNA and serum TTR protein at day 22 after administration.

[0277] Example 8. Sustained suppression of serum TTR protein by SNALP-18328 in non-human primates. To evaluate the sustained suppression of serum TTR protein by SNALP-18328, AD-18328 was formulated into SNALP and administered to non-human primates via IV infusion. Serum TTR protein levels were quantified at various time points after administration.

[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 non-mammalian luciferase genes), or PBS via 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 blotting 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 (with β-mercaptoethanol 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 separate lanes of a 10–20% Criterion (Biorad, Hercules, CA) preparation gel, separated by SDS-PAGE at 120 V for 1.5 hours, and then transferred to a nitrocellulose membrane using a semi-dry system at 15 V for 1 hour. The membrane was blocked overnight at 4°C in LiCOR (Lincoln, NE) blocking buffer diluted 1:1 with 1×PBS. First, the blot was probed with primary antibody (goat anti-TTR from Santa Cruz (Santa Cruz, CA)) using a 1:1000 dilution in LiCOR blocking buffer / PBS, and incubated on a rocker at room temperature for 1 hour. The blot was washed four times with PBS + 0.2% Tween 20 (10 minutes per wash). Fluorescently labeled secondary antibody (anti-goat 680nm from Invitrogen (Carlsbad, CA)) was added in a 1:10,000 dilution in LiCOR blocking buffer / PBS, and the blot was incubated at room temperature for 1 hour. After incubation, the blot was washed four times with PBS + 0.2% Tween 20, followed by one wash with PBS. Protein bands were detected using Li-COR's Odyssey Infrared Imaging System. TTR monomers migrate at 15 kDa.

[0280] The results are shown in Figure 8. Serum TTR protein levels showed a dose-dependent decrease with 1 or 3 mg / kg of SNALP-18328 compared to pre-treatment (day 0). The duration of suppression after a single IV dose of SNALP-18328 was at least 14 days from treatment with 1 or 3 mg / kg of SNALP-18328.

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

[0282] Example 9: In vivo reduction of mutant (V30M) TTR in peripheral tissues by SNALP-18328 in genetically modified mice. To evaluate the efficacy of SNALP-18328 in reducing 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) received an IV bolus of 3 mg / kg of SNALP-18328 (12 animals), 3 mg / kg of SNALP-1955 (4 animals with the control siRNA AD-1955 targeting a non-mammalian luciferase gene), or PBS (4 animals) every two weeks for a total of four doses on days 0, 14, 28, and 42. TTR liver mRNA levels and TTR immunoactivity in multiple peripheral tissues were evaluated 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. Target tissues and organs were collected. For immunohistochemistry, the esophagus (E), stomach (S), intestines (duodenum (I1) and colon (I4)), nerves (N), and dorsal root ganglia (D) were fixed in neutral buffered formalin and embedded in paraffin. For TTR detection, rabbit anti-human TTR primary antibody (1:1000, DAKO, Denmark) and anti-rabbit biotin-conjugated secondary antibody (1:20, Sigma, USA) were used, followed by extravidin-labeled solution (1:20, Sigma, USA) to stain the TTR protein. The reaction was induced using 3-amino-9-ethylcarbazole, AEC (Sigma, USA). Semi-quantitative analysis of immunohistochemistry slides was performed using the Scion image quant program, which measured the area occupied by the substrate reaction color and normalized this value relative to the total image area. The mean percentage of occupied area is shown along with the corresponding standard deviation. Each animal tissue was evaluated in four different regions. The presence of human TTR in the parasympathetic ganglia of the stomach and intestines 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 the secondary antibodies were anti-rabbit Alexa Fluor 488 (Molecular probes, UK) and goat anti-mouse Alexa Fluor 568 (Molecular probes, UK), respectively. Slides were visualized in a Zeiss Cell Observer System microscope (Carl Zeiss, Germany) mounted with vectashield (Vector) and equipped with filters for FITC and rhodamine.

[0284] The results are illustrated in Figure 9. Compared to animals treated with PBS and SNALP-1955, animals treated with SNALP-18328 showed a significant reduction in TTR immunoactivity in all tissues examined (esophagus (E), stomach (S), intestines (duodenum (I1) and colon (I4)), nerves (N), and dorsal root ganglia (D)).

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

[0286] Example 10. In vivo reduction of wild-type TTR mRNA in the liver of non-human primates using XTC-SNALP-18328. To evaluate the efficacy of a novel lipid nanoparticle formulation, XTC-SNALP, for siRNA delivery in non-human primates, TTR siRNA AD-18328 was formulated into XTC-SNALP (XTC-SNALP-18328) and administered by 15-minute IV infusion, followed by quantification of hepatic TTR mRNA. Cynomolgus monkeys (Macaca fascicularis) were administered either XTC-SNALP-18328 (0.03, 0.1, 0.3, or 1 mg / kg) or XTC-SNALP-1955 (1 mg / kg, containing the control siRNA AD-1955 targeting a non-mammalian luciferase gene) by 15-minute IV infusion. Forty-eight hours after administration, the monkeys were anesthetized with pentobarbital sodium and induced hemorrhage. Liver tissue for TTR mRNA determination was collected, rapidly frozen, and stored at -80°C until processing. The method used for quantifying TTR mRNA in liver tissue was the same as 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 for XTC-SNALP-18328.

[0288] These results demonstrate that XTC-SNALP-18328 is effective in suppressing wild-type TTR mRNA in non-human primate livers when administered by IV infusion.

[0289] Example 11: In vivo reduction of wild-type TTR mRNA in non-human primate liver using LNP09-18328 and LNP11-18328. In non-human primates, to evaluate the efficacy of two novel lipid nanoparticle formulations, LNP09 and LNP11, for siRNA delivery, TTR siRNA AD-18328 was formulated into LNP09 (LNP09-18328) or LNP11 (LNP11-18328) and administered by 15-minute IV infusion. Hepatic 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 by 15-minute IV infusion. Liver biopsy samples were collected 48 hours after administration, rapidly frozen, and stored at -80°C until processing. Serum was collected before administration (before blood collection) and on days 1, 2, 4, 7, 14, 21, and 28 after administration, and stored at -80°C until processing. The methods used for quantifying TTR mRNA in the evaluation of liver tissue and serum TTR proteins were the same as those described in Examples 5 and 8 above.

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

[0291] These results demonstrate that LNP09-18328 and LNP11-18328 are effective in suppressing wild-type TTR mRNA in non-human primate livers and wild-type TTR protein in circulation when administered via IV infusion. Furthermore, the suppression by LNP09-18328 is persistent and lasts for at least 28 days after IV infusion.

[0292] Example 12. Synthesis of an array in which TTRs are arranged. We designed a set of TTR double helix ("parallel double helix") that targets 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 example below, the numbering of the 5' base of the siRNA on the transcript is based on NM_000371.3 (Figure 12, Sequence ID 1331). In the example above, the numbering of the siRNA targeting human siRNA is based on NM_000371.2 (Figure 13A). NM_000371.3 extends the 5'UTR sequence up to 110 bases compared to NM_000371.2, as shown in Figure 14. Therefore, as an example, the start position of AD-18328 is 628 on NM_000371.3 and 518 on NM_000371.2 (Figure 14).

[0294] Sequences with TTRs were synthesized in a MerMade 192 synthesizer at a scale of 1 μmol. For all sequences in the sequence listing, the "endright" chemical reaction was applied as detailed below. All pyrimidines (cytosine and uridine) in the sense chain contained 2'-O-methyl bases (2'-O-methyl C and 2'-O-methyl U). In the antisense chain, the pyrimidines adjacent to the ribo-A nucleoside (towards the 5' position) were substituted with their corresponding 2-O-methyl nucleosides. • We introduced a two-base extension, dTdT, at the 3' end of both the sense and antisense sequences. • The array files were converted to text files to maintain compatibility with the load on MerMade 192 synthesis software.

[0295] Synthesis, cleavage, and deprotection: TTR sequences were synthesized using a phosphoramidite chemical reaction with immobilized oligonucleotide synthesis. Sequence synthesis was performed on a scale of 1 μm in a 96-well plate. Amidite solution was prepared at a concentration of 0.1 M, and ethylthiotetrazole (0.6 M in acetonitrile) was used as the activator. The synthesized sequences were cleaved and deprotected in a 96-well plate using methylamine in the first step and a fluoride reagent in the second step. The 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 identity under ultraviolet light for quantification, and by IEX chromatography to determine purity.

[0296] Purification and desalting: The sequences containing TTRs were purified using a Source 15Q column in an AKTA explorer purification system. The column temperature was maintained at 65°C during purification. Sample injection and collection were performed in a 96-well (1.8 mL deep well) plate. Single peaks corresponding to the full-length sequences were collected in the eluent. The purified sequences on a Sephadex G25 column were desalted using an AKTA purifier. The desalted TTR sequences were analyzed for concentration (by UV measurement with A260) and purity (by ion-exchange HPLC). Single chains were then submitted for annealing.

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

[0298] Example 13. In vitro screening of siRNAs with TTRs. Double-stranded TTRs were assayed in Hep3B cells using a real-time PCR assay to inhibit the expression of endogenous TTRs.

[0299] Cell Culture and Translocation: Hep3B cells (ATCC, Manassas, VA) were grown to near confluence at 37°C in a 5% CO2 atmosphere in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsin treatment. Reverse transcription was performed in individual wells of a 96-well plate by adding 5 μL of Opti-MEM to each siRNA. To this, 0.2 μL of Lipofectamine RNAiMax (Invitrogen, Carlsbad CA, cat#13778-150) was added per well in addition to 10 μL of Opti-MEM, and the mixture was incubated at room temperature for 15 minutes. Subsequently, 2.0 × 10⁶ cells were transcribed. 4 Hep3B cells were added to 80 μL of antibiotic-free full growth medium. The cells were incubated for 24 hours before RNA purification. Experiments were performed at a final dual concentration 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 collected and lysed in 140 μL of lysis / binding solution, then mixed for 1 minute at 850 rpm using an Eppendorf Thermomixer (the mixing rate remained constant throughout the process). A mixture of 20 microliters of electromagnetic beads and lysis / binding enhancer was added to the cell lysate and mixed for 5 minutes. The magnetic beads were captured using a magnetic stand, and suspended matter was removed without disturbing the beads. After removing the suspended matter, the magnetic beads were washed with washing solution 1 (with added isopropanol) and mixed for 1 minute. The beads were captured again, and suspended matter was removed. The beads were then washed with 150 μL of washing solution 2 (with added ethanol), captured, and suspended matter 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 regeneration solution was added and mixed for 3 minutes. Suspensions were removed, and the magnetic beads were washed again with 150 μL of washing solution 2, mixed for 1 minute, and the suspensions were completely removed. The magnetic beads were mixed for 2 minutes and dried before eluting the RNA with 50 μL of water.

[0301] cDNA synthesis using the ABI High Performance cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, Cat#4368813): 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 a master mix of 3.2 μL of H2O per reaction were 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: 10 minutes at 25°C, 120 minutes at 37°C, 5 seconds at 85°C, and holding at 4°C.

[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 using a LightCycler 480 real-time PCR machine (Roche). Each double-stranded DNA was tested in two independent translocations, and each translocation was assayed in duplicate.

[0303] Real-time data were analyzed using the ΔΔCt method. Each sample was normalized to GAPDH expression, and knockdown was evaluated by comparing it to cells transfected with non-target double-stranded AD-1955. Table 14 shows TTR knockdown using siRNA. The data is expressed as the percentage of cells targeted by AD-1955 in which the message persists.

[0304] Numerous, but not all, of the arranged TTR-dsRNAs targeting TTR, which is the primary target of AD-18328, were found at a level of 0.1 nM, and when transfused into Hep3B cells, they resulted in at least a 70% reduction in TTR mRNA. [Table 18-1] [Table 18-2]

[0305] Example 14. Evaluation of the efficacy of a single intravenous administration 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 sense strand and antisense strand sequences of AD-18534, as shown in the table above, are reproduced below. [Table 20A] research materials Test substance SNALP-18534 consists of an 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 aminolipid (DLinDMA), PEGylated lipid (mPEG2000-C-DMA), neutral lipid (DPPC), and cholesterol. The lipid:nucleic acid ratio in the SNALP formulation is approximately 5.8:1 (w:w). SNALP-1955 contains an siRNA targeting non-mammalian luciferase mRNA, formulated into the same lipid particles as SNALP-18534, and serves as a non-pharmacologically active control. Dose levels are expressed in mg / kg based on the weight of the siRNA content.

[0306] Research Design & Procedure Administration of animals and test substances: The study consisted of nine groups of Sprague-Dawley rats (four males per group). The animals were given a 2-day acclimatization period prior to the study, and all animals were 7 weeks old at the start of treatment. The administered dose was calculated based on body weight data collected before the first day of treatment. The test and control substances were administered via the tail vein as a single IV infusion over 15 minutes, 1 hour, 2 hours, or 3 hours using a 24G 3 / 4” cannula sealed with a Baxter injection site diaphragm, connected to a Baxter AS40A syringe pump via a 27G Terumo winged needle. The dose was 3 mL / kg, the infusion rate was 12 mL / kg / hour, and the animals were allowed to move freely in their cages during treatment. The rats were divided into nine treatment groups and administered as 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 by isoflurane inhalation, and pre-administration blood samples were collected in serum separators by retro-orbital bleed. Blood samples were allowed to coagulate 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, all nine treatment groups were administered a lethal dose of ketamine / xylazine. Blood was collected in serum separators via the posterior vena cava and then allowed to coagulate at room temperature for approximately 30 minutes before centrifugation at 4°C. Serum samples were then stored at -80°C until analysis. Liver tissue was harvested and flash-frozen on dry ice. The frozen liver tissue was pulverized, and tissue lysates were prepared for the quantification of liver mRNA.

[0308] Quantification of TTR mRNA: The mRNA levels of TTR were determined in lysates compared to GAPDH mRNA levels using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, CA). In short, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue lysates according to the manufacturer's instructions. The average TTR mRNA level was normalized to the average GAPDH mRNA level 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 infusion times of 15 minutes, 1 hour, and 2 hours were then normalized to the mean value for the PBS treatment group at a 15-minute infusion time, while the group mean values ​​for the SNALP-1955 and SNALP-18534 treatment groups at a 3-hour infusion time were then normalized to the mean value for the PBS treatment group at a 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 ranging from 15 minutes to 3 hours resulted in comparable inhibition of hepatic TTR mRNA levels as measured 2 days after administration. A single IV infusion of 1 mg / kg SNALP-18534 also showed sustained downregulation of TTR over 29 days from a 15-minute single IV infusion compared to the SNALP-1955 control (data not shown). Compared to the PBS-treated group, a single IV infusion of 15 minutes, 1 hour, 2 hours, or 3 hours 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 absence of significant target inhibition with SNALP-1955 administration via 1-hour, 2-hour, or 3-hour IV infusion at the same dose level.

[0311] conclusion This study demonstrates that different infusion times, ranging from 15 minutes to a maximum of 3 hours, do not affect the efficacy of a single IV dose of 1 mg / kg SNALP-18534 in rats, as assessed by a decrease 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. In rats, to evaluate the efficacy of two novel lipid nanoparticle formulations for siRNA delivery, LNP07 and LNP08, rodent-specific TTR siRNA, AD-18534, was formulated into LNP07 (LNP07-18534) or LNP08 (LNP08-18534) and administered by IV infusion over 15 minutes, and hepatic TTR mRNA was quantified. Sprague-Dawley rats (4 animals per group) were administered 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 a non-mammalian luciferase gene, via IV infusion over 15 minutes. 48 hours later, the animals were anesthetized, and liver tissue was collected, rapidly frozen, and stored at -80°C until processed.

[0313] To quantify TTR mRNA, frozen liver tissue was pulverized and lysates were prepared. TTR mRNA levels were determined in the lysates compared to GAPDH mRNA levels using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, CA). In short, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue lysates according to the manufacturer's instructions. The mean level of TTR mRNA was normalized relative to the mean level of GAPDH mRNA for each sample. The group mean of the normalized values ​​was then further normalized relative to the mean for the PBS-treated group to obtain the relative level 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 a 94% suppression of TTR mRNA at 1 mg / kg. The effect was specific, as the negative control LNP07-1955 at 1 mg / kg did not significantly affect TTR mRNA levels compared to the PBS control. mRNA ED50 was confirmed to be approximately that of LNP07-18534 at 0.05 mg / kg. LNP08-18534 reduced TTR mRNA levels in the liver in a dose-dependent manner, achieving an 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 did not significantly affect TTR mRNA levels compared to the PBS control. mRNA ED50 was confirmed to be LNP08-18534 at approximately 0.02 mg / kg.

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

[0316] Example 16: Decrease in TTR liver mRNA in Sprague-Dawley rats following single intravenous administration of LNP09-18534 or LNP11-18534. the purpose: To evaluate the efficacy of two novel lipid nanoparticle (LNP) formulations for the delivery of the rodent TTR-specific siRNA AD-18534 in Sprague-Dawley rats in reducing endogenous (wild-type) hepatic TTR mRNA levels. Rats were intravenously administered either 0.01, 0.03, 0.1, or 0.3 mg / kg of LNP09-18534, LNP11-18534, or phosphate-buffered saline (PBS) 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 preparation: (MC3 / DSPC / Chol / PEG 2000 -C14) = 50 / 10 / 38.5 / 1.5 mol%, Lipids: siRNA approximately 11.1 Tissue collection and RNA isolation: On day 3, all treatment groups of animals were administered a lethal dose of ketamine / xylazine. Blood was collected in serum separators via the posterior vena cava and then allowed to coagulate at room temperature for approximately 30 minutes before centrifugation at 4°C. Serum samples were stored at -80°C until analysis was performed. Liver tissue was harvested and flash-frozen on dry ice. The frozen liver tissue was pulverized, and tissue lysates were prepared for the quantification of liver mRNA.

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

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

[0320] conclusion This study demonstrates that a single 15-minute intravenous 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 at ED50 levels of <0.03 mg / kg and <0.1 mg / kg for LNP11-18534 and LNP09-18534, respectively.

[0321] Example 17: Inhibition of TTR in humans Human subjects are treated with dsRNAs targeting the TTR gene to inhibit TTR gene expression in order to treat the disease.

[0322] Select or identify subjects who require treatment. These subjects may have liver disease, trans-tyretin amyloidosis, and / or a transplanted liver.

[0323] The identification of the subject can be done in a clinical setting or elsewhere, for example, at the subject's home through the use of a self-test kit.

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

[0325] After treatment, the tumor growth rate in the target group decreases compared to the pre-treatment rate or the rate measured in similarly affected but untreated subjects.

Claims

1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of trans tiretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprises a region complementary to a portion of the mRNA encoding trans tiretin (TTR), the complementary region is less than 30 nucleotides long, and the antisense strand comprises 15 or more consecutive nucleotides of SEQ ID NO: 170, SEQ ID NO: 450, SEQ ID NO: 730, or SEQ ID NO: 1010.

2. The dsRNA according to claim 1, wherein the sense strand comprises 15 or more consecutive nucleotides of SEQ ID NO: 169, SEQ ID NO: 449, SEQ ID NO: 729, or SEQ ID NO: 1009.

3. The dsRNA according to claim 1, wherein the sense strand consists of sequence number 449 and the antisense strand consists of sequence number 450.

4. The dsRNA according to claim 1, wherein the sense strand consists of sequence number 729 and the antisense strand consists of sequence number 730.

5. The dsRNA according to claim 1, wherein the sense strand consists of sequence number 1009 and the antisense strand consists of sequence number 1010.

6. The dsRNA according to claim 1, wherein the dsRNA comprises a sense strand selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16, and an antisense strand selected from Tables 3A, 3B, 4, 6A, 6B, 7, and 16.

7. The dsRNA according to claim 1, 2, or 6, wherein the complementary region is 19 nucleotides long.

8. The complementary region comprises SEQ ID NO: 169, as per claim 1, 2, or 6, for the dsRNA.

9. The dsRNA according to claim 1, 2, or 6, wherein each strand of the dsRNA is 19, 20, 21, 22, 23, or 24 nucleotides long.

10. The dsRNA according to claim 1, 2, or 6, wherein each strand is 21 nucleotides long.

11. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein the dsRNA does not cleave the 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.

12. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein the dsRNA cleaves the 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.

13. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein the dsRNA is annealed 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.

14. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein the dsRNA comprises at least one modified nucleotide.

15. The dsRNA according to claim 14, wherein at least one of the modified nucleotides is selected from the group consisting of a 2'-O-methyl modified nucleotide, a nucleotide containing a 5'-phosphorothioate group, and a terminal nucleotide bonded to a cholesteryl derivative group or a dodecanoic acid bisdecylamide group.

16. The dsRNA according to claim 14, wherein the modified nucleotide is selected from the group of non-natural bases including 2'-deoxy-2'-fluoro-modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides.

17. The dsRNA according to claim 14, comprising at least one 2'-O-methyl modified nucleotide.

18. The dsRNA is the dsRNA according to claim 1, 2, 3, 4, 5, or 6, which is coupled to a ligand.

19. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein the dsRNA is formulated into a lipid preparation.

20. The dsRNA according to claim 19, wherein the dsRNA is formulated into an LNP preparation, an LNP01 preparation, an XTC-SNALP preparation, or an SNALP preparation.

21. The dsRNA according to claim 19, wherein the dsRNA is formulated into an XTC-SNALP formulation by using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC) with XTC / DPPC / cholesterol / PEG-cDMA in a ratio of 57.1 / 7.1 / 34.4 / 1.4 and lipid:siRNA in a ratio of approximately 7:

1.

22. The dsRNA according to claim 19, wherein the sense strand 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 by using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC) with XTC / DPPC / cholesterol / PEG-cDMA in a ratio of 57.1 / 7.1 / 34.4 / 1.4 and lipid:siRNA in a ratio of approximately 7:

1.

23. The aforementioned dsRNA is XTC / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mol%. ２０００ - The dsRNA according to claim 19, formulated into an LNP09 formulation by using C14 and lipid:siRNA in a ratio of approximately 11:

1.

24. The dsRNA is MC3 / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mol%. ２０００ - The dsRNA according to claim 19, which is formulated into an LNP11 formulation by using C14 and lipid:siRNA in a ratio of approximately 11:

1.

25. The dsRNA according to claim 19, wherein the dsRNA is formulated into an LNP09 formulation or an LNP11 formulation, and at a dose of 0.3 mg / kg, reduces the TTR mRNA level by approximately 85-90% compared to a PBS control group.

26. The dsRNA according to claim 19, wherein the dsRNA is formulated into an LNP09 formulation or an LNP11 formulation, and at a dose of 0.1 mg / kg, reduces the TTR mRNA level by approximately 50% compared to a PBS control group.

27. The dsRNA according to claim 19, wherein the dsRNA is formulated into an LNP09 formulation or an LNP11 formulation and reduces the TTR protein level in a dose-dependent manner compared to a PBS control group, as measured by Western blotting.

28. The dsRNA according to claim 19, wherein the dsRNA is formulated into an SNALP preparation by using DlinDMA in a ratio of 57.1 / 7.1 / 34.4 / 1.4 for DlinDMA / DPPC / cholesterol / PEG2000-cDMA and a ratio of approximately 7:1 for lipids:siRNA.

29. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein administration of the dsRNA to cells results in approximately 95% inhibition of TTR mRNA expression as measured by a real-time PCR assay, the cells being HepG2 cells or Hep3B cells, and the concentration of the dsRNA being 10 nM.

30. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein administration of the dsRNA to cells results in approximately 74% inhibition of TTR mRNA expression, as measured by a branched DNA assay, the cells are HepG2 cells or Hep3B cells, and the concentration of the dsRNA is 10 nM.

31. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein the dsRNA has an IC50 of less than 10 pM in HepG2 cells, and the concentration of the dsRNA is 10 nM.

32. The dsRNA is the dsRNA according to claim 1, 2, 3, 4, 5, or 6, having an ED50 of about 1 mg / kg.

33. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein administration of the dsRNA reduces TTR mRNA in the liver of cynomolgus monkeys by approximately 80%, and the concentration of the dsRNA is 3 mg / kg.

34. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein the administration of the dsRNA does not result in immunostimulatory activity in human peripheral blood mononuclear cells (PBMCs), as measured by IFN-α and TNF-α ELISA assays.

35. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein administration of the dsRNA reduces the mRNA level of hepatic TTR by about 97% or the protein level of serum TTR by about 90%, and the concentration of the dsRNA is 6 mg / kg.

36. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein administration of the dsRNA reduces the mRNA level of hepatic TTR and / or the protein level of serum TTR for up to 22 days, and the concentration of the dsRNA is 6 mg / kg or 3 mg / kg.

37. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, wherein when administered to a subject requiring it at a dose of 1 mg / kg or 3 mg / kg, it suppresses serum TTR protein levels up to 14 days after treatment.

38. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, which, when measured by real-time PCR, reduces TTR expression in Hep3B cells by 98.9% at a concentration of 0.1 nM.

39. The dsRNA according to claim 1, 2, 3, 4, 5, or 6, which, when measured by real-time PCR, reduces TTR expression in Hep3B cells by 99.4% at a concentration of 10 nM.

40. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of trans tiretin (TTR), wherein the dsRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding trans tiretin (TTR), the complementary region being less than 30 nucleotides in length, and the dsRNA comprising 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.

41. A double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of trans tiretin (TTR), wherein the dsRNA includes an antisense strand containing a region complementary to 15 to 30 nucleotides among nucleotides 618 to 648 of sequence number 1331, and the antisense strand forms a base pair with guanine at position 628 of sequence number 1331.

42. A cell containing the dsRNA described in claims 1 to 41.

43. A vector comprising a nucleotide sequence encoding at least one strand of dsRNA according to claims 1 to 41.

44. A cell comprising the vector of claim 43.

45. A pharmaceutical composition for inhibiting the expression of a TTR gene, comprising the dsRNA described in claims 1 to 41 and a drug-acceptable carrier.

46. A pharmaceutical composition for inhibiting the expression of the TTR gene, comprising dsRNA and a SNALP preparation, wherein the dsRNA comprises an antisense strand less than 30 nucleotides long and contains 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 preparation comprises DlinDMA, DPPC, cholesterol, and PEG2000-cDMA in a ratio of 57.1 / 7.1 / 34.4 / 1.4, respectively.

47. A method for inhibiting intracellular TTR expression, (a) Contacting the dsRNA described in claims 1 to 41 with the cells, (b) A method comprising maintaining the cells produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the TTR gene, thereby inhibiting the expression of the TTR gene within the cells.

48. A method for treating a disorder mediated by TTR expression, comprising administering a therapeutically effective amount of dsRNA according to claims 1 to 41 to a person in need of such treatment.

49. The method according to claim 48, wherein the dsRNA is administered to the human at a dose of approximately 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg / kg.

50. The method according to claims 48 to 49, wherein the dsRNA is administered to the human at a dose of approximately 1.0 mg / kg.

51. The method according to claims 48 to 50, wherein the human being suffers from trans tyretin amyloidosis.

52. The method according to claims 48 to 51, wherein the human being suffers from a liver disease.

53. The method according to claims 48 to 52, wherein the human is further provided with a liver transplant.

54. The method according to claims 48 to 53, wherein the administration of the dsRNA reduces TTR mRNA in human liver by approximately 80%, and the concentration of the dsRNA is 3 mg / kg.

55. The method according to claims 48 to 54, wherein the administration of the dsRNA does not produce immunostimulatory activity in the human, as measured by IFN-α and TNF-α ELISA assays.

56. The method according to claims 48 to 55, wherein the administration of the dsRNA reduces the mRNA level of hepatic TTR by about 97% or the protein level of serum TTR by about 90%, and the concentration of the dsRNA is 6 mg / kg.

57. The method according to claims 48 to 56, wherein the administration of the dsRNA reduces the mRNA level of hepatic TTR and / or the protein level of serum TTR for up to 22 days, and the concentration of the dsRNA is 6 mg / kg or 3 mg / kg.

58. The aforementioned dsRNA is XTC / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mol%. ２０００ The method according to claims 48 to 57, wherein the LNP09 formulation is obtained by using -C14 and lipid:siRNA in a ratio of approximately 11:

1.

59. The dsRNA is MC3 / DSPC / Chol / PEG in a ratio of 50 / 10 / 38.5 / 1.5 mol%. ２０００ The method according to claims 48 to 58, wherein the LNP11 formulation is obtained by using -C14 and lipid:siRNA in a ratio of approximately 11:

1.

60. The method according to claims 48 to 59, wherein the dsRNA is formulated into an LNP09 formulation or an LNP11 formulation, and at a dose of 0.3 mg / kg, the mRNA level of TTR is reduced by approximately 85 to 90% compared to the PBS control group.

61. The method according to claims 48 to 60, wherein the dsRNA is formulated into an LNP09 formulation or an LNP11 formulation, and at a dose of 0.1 mg / kg, the mRNA level of TTR is reduced by approximately 50% compared to the PBS control group.

62. The method according to claims 48 to 61, wherein the dsRNA is formulated into an LNP09 formulation or an LNP11 formulation and, as measured by Western blotting, reduces the protein level of TTR in a dose-dependent manner compared to a PBC control group.

63. The method according to claims 48 to 62, wherein the dsRNA, when administered to humans at a dose of 1 mg / kg or 3 mg / kg, suppresses serum TTR protein levels up to 14 days after treatment.

64. The method according to claims 48 to 63, wherein the dsRNA preparation is formulated into an SNALP preparation by using DlinDMA in a ratio of 57.1 / 7.1 / 34.4 / 1.4 with DLinDMA / DPPC / cholesterol / PEG2000-cDMA and lipid:siRNA in a ratio of approximately 7:

1.

65. Use of dsRNA for treating a disorder mediated by TTR expression, comprising administering a therapeutically effective amount of the dsRNA described in claims 1 to 41 to a person in need of such treatment.

66. The use according to claim 65, wherein the dsRNA is administered to the human at approximately 0.01, 0.1, 0.5, 1.0, 2.5, or 5.0 mg / kg.

67. The use according to claims 65 to 66, wherein the dsRNA is administered to the human at a dose of approximately 1.0 mg / kg.

68. The use according to claims 65 to 67, wherein the human being suffers from trans tyretin amyloidosis.

69. The use according to claims 65 to 68, wherein the human being suffers from liver disease.

70. The use according to claims 65-69, wherein the human is further provided with a liver transplant.

71. The use of dsRNA to inhibit intracellular TTR expression, (a) Contacting the dsRNA described in claims 1 to 41 with the cells, (b) Use comprising maintaining the cells produced in step (a) for a sufficient time to obtain degradation of the mRNA transcript of the TTR gene, thereby inhibiting the expression of the TTR gene within the cells.

Images