Compositions and methods for inhibiting transthyretin (TTR) expression

Abstract

The present invention provides compositions and methods for treating transthyretin (TTR)-related diseases using RNAi agents, eg, double-stranded RNAi agents, that target the TTR gene.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Application No. 62 / 985,950, filed March 6, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on February 26, 2021, is named 121301_12320_SL.txt, and is 44,455 bytes in size. [Background technology]

[0003] background Transthyretin (TTR) (also known as prealbumin) is found in serum and cerebrospinal fluid (CSF). TTR transports retinol-binding protein (RBP) and thyroxine (T4) and also acts as a carrier of retinol (vitamin A) through binding to RBP in the blood and CSF. Transthyretin is named for its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apoA-I (the major HDL apolipoprotein), amyloid β-peptide, and neuropeptide Y. See Liz, MA et al. (2010) IUBMB Life, 62(6):429-435.

[0004] TTR is a tetramer of four identical 127-amino acid subunits (monomers) rich in beta-sheet structure. Each monomer has two four-stranded beta-sheets and is prolate ellipsoidal in shape. Antiparallel beta-sheet interactions link the monomers into dimers. A short loop from each monomer forms the major dimer-dimer interaction. Two pairs of these loops separate the opposing, convex beta-sheets of the dimer, forming an internal channel.

[0005] The liver is the major site of TTR expression. Other prominent sites of expression include the choroid plexus, retina (especially the retinal pigment epithelium), and pancreas.

[0006] Transthyretin is one of at least 27 distinct types of proteins that are precursor proteins in the formation of amyloid fibrils. See Guan, J. et al. (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi:10.1152 / ajpheart.00815.2011. Extracellular deposition of amyloid fibrils in organs and tissues is a hallmark of amyloidosis. Amyloid fibrils consist of misfolded protein aggregates that may result from overproduction of precursor proteins or specific mutations. The amyloidogenic potential of TTR may be related to its extensive beta-sheet structure; X-ray crystallographic studies indicate that certain amyloidogenic mutations destabilize the tetrameric structure of the protein. See, e.g., Saraiva MJM (2002) Expert Reviews in Molecular Medicine, 4(12):1-11.

[0007] Amyloidosis is a general term for a group of amyloid diseases characterized by amyloid deposits. Amyloid diseases are classified by precursor protein; for example, the name begins with "A" for amyloid, followed by the abbreviation of the precursor protein, e.g., ATTR for amyloidogenic transthyretin. Ibid.

[0008] There are numerous TTR-related diseases, most of which are amyloid diseases. Normal-sequence TTR is associated with cardiac amyloidosis in the elderly, also known as senile systemic amyloidosis (SSA), also known as senile cardiac amyloidosis (SCA) or cardiac amyloidosis. SSA is often accompanied by microscopic deposits in many other organs. TTR amyloidosis manifests in a variety of forms. When the peripheral nervous system is more prominently affected, the disease is called familial amyloid polyneuropathy (FAP). When the heart is primarily involved but not the nervous system, the disease is called familial amyloid cardiomyopathy (FAC). The third most common type of TTR amyloidosis is leptomeningeal amyloidosis, also known as leptomeningeal or meningeal cerebrovascular amyloidosis, central nervous system (CNS) amyloidosis, or amyloidosis form VII. Mutations in TTR can also cause amyloid vitreous opacities, carpal tunnel syndrome, and euthyroid hyperthyroxinemia, a non-amyloidotic disorder thought to be secondary to increased binding of thyroxine to TTR due to mutant TTR molecules with increased affinity for thyroxine (see, e.g., Moses et al. (1982) J. Clin. Invest., 86, 2025-2033).

[0009] Abnormal TTR alleles can be either inherited or acquired through somatic mutation. Guan, J. et al. (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi:10.1152 / ajpheart.00815.2011. Transthyretin-associated ATTR is the most frequent form of hereditary systemic amyloidosis. Lobato, L. (2003) J. Nephrol., 16:438-442. TTR mutations accelerate the TTR amyloidogenic process and are the most important risk factor for the development of ATTR. More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis. TTR mutations usually result in systemic amyloid deposition with specific involvement of the peripheral nervous system, but some mutations are associated with cardiomyopathy or vitreous opacities. Ibid.

[0010] The V30M mutation is the most common TTR mutation. See, e.g., Lobato, L. (2003) J Nephrol, 16:438-442. The V122I mutation is carried by 3.9% of the African American population and is the most common cause of FAC. Jacobson, DR et al. (1997) N. Engl. J. Med. 336 (7): 466-73. SSA is estimated to affect more than 25% of the population over 80 years of age. Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. USA 87 (7): 2843-5. Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, there is a need in the art for effective treatments for TTR-related diseases. [Means for solving the problem]

[0012] Summary of the Invention The present invention relates to compositions for inhibiting the expression of TTR using double-stranded RNAi agents that target the TTR gene, as well as methods for treating or preventing transthyretin (TTR)-related diseases in human subjects.

[0013] The present invention provides a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: each sense strand and antisense strand independently is at most 30 nucleotides in length; the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is the S-isomer of thymidine glycol nucleic acid (GNA); and s is a phosphorothioate linker.

[0014] In some embodiments, the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. In some embodiments, the ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker. In some embodiments, the ligand is [ka] is.

[0015] In certain embodiments, the ligand is attached to the 3' end of the sense strand.

[0016] In certain embodiments, the double-stranded RNAi agent is attached to a ligand as shown in the following formula: [ka] wherein X is O or S.

[0017] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0018] The present invention relates to the use of a double-stranded RNAi agent in a method of treating a human subject having a TTR-related disease, comprising administering a fixed dose of about 25 mg to about 1000 mg of the double-stranded RNAi agent, wherein: each sense strand and antisense strand independently being at most 30 nucleotides in length; the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is the S-isomer of thymidine glycol nucleic acid (GNA); and s is a phosphorothioate linker; Provide use.

[0019] The present invention also relates to the use of a double-stranded RNAi agent in a method for inhibiting expression of TTR in a human subject who does not meet diagnostic criteria for a TTR-related disorder, comprising administering a fixed dose of about 25 mg to about 1000 mg of the double-stranded RNAi agent, wherein: each sense strand and antisense strand independently being at most 30 nucleotides in length; the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is the S-isomer of thymidine glycol nucleic acid (GNA); and s is a phosphorothioate linker; Use is also provided.

[0020] In some embodiments, the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. In some embodiments, the ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker. In some embodiments, the ligand is [ka] is.

[0021] In some embodiments, the ligand is attached to the 3' end of the sense strand. In some embodiments, the double-stranded RNAi agent is attached to the ligand as shown in the following formula: [ka] wherein X is O or S.

[0022] In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0023] In some embodiments, the use of the present invention includes improvement in at least one indicator of neurological impairment, quality of life, nerve damage, or cardiovascular symptoms. In some embodiments, the indicator assessed is nerve dysfunction, for example, using the Neuropathic Impairment Score (NIS) score or the modified NIS (mNIS+7) score. In some embodiments, the indicator is a quality of life indicator, for example, assessed using the SF-36® Health Survey score, Norfolk Quality of Life - Diabetic Neuropathy (Norfolk QOL-DN) score, NIS-W score, Rasch-Constructed Global Disability Scale (R-ODS) score, Composite Autonomic Symptom Score (COMPASS-31), central body mass index (mBMI) score, 6-minute walk test (6MWT) score, and 10-meter walk test score. In one embodiment, the indicator is neuronal damage, as assessed by changes in the levels of one or more proteins selected from the group consisting of neurofilament light chain (NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N-CDase, for example, in a human blood sample or serum or plasma derived therefrom. In one embodiment, the indicator of neuronal damage is a change from baseline in neurofilament light chain (NfL) protein levels. In one embodiment, the indicator of cardiovascular dysfunction is cardiovascular hospitalization using the Kansas City Cardiomyopathy Questionnaire - Overall Summary (KCCQ-OS), with an increase in the score indicating good health, a change from baseline in mean left ventricular (LV) wall thickness by echocardiographic assessment, a change from baseline in global longitudinal strain by echocardiographic assessment, and a change from baseline in N-terminal prohormone B-type natriuretic peptide (NT-proBNP).

[0024] In certain embodiments, the human subject has a TTR gene mutation associated with the development of a TTR-related disease, such as senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, and hyperthyroxinemia.

[0025] In some embodiments, a human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the use of double-stranded RNAi agent reduces amyloid TTR deposition in the human subject.In some embodiments, the ATTR amyloidosis is hereditary ATTR (h-ATTR) amyloidosis.In some embodiments, the ATTR amyloidosis is non-hereditary ATTR (wt ATTR) amyloidosis.

[0026] In some embodiments, double-stranded RNAi agent is administered to human subjects subcutaneously or intravenously.In some embodiments, subcutaneous administration is self-administration.In some embodiments, self-administration is via pre-filled syringe or automatic injection device.

[0027] In certain embodiments, the use further comprises assessing TTR mRNA expression or TTR protein expression levels in a sample from a human subject, such as a human blood sample or serum or plasma derived therefrom.

[0028] In some embodiments, double-stranded RNAi agent is administered to human subject once a month, once every two months, once every three months, once every four months, once every five months or once every six months.In some embodiments, fixed dose double-stranded RNAi agent is administered to human subject once about every three months.In some embodiments, fixed dose double-stranded RNAi agent is administered to human subject once about every six months.

[0029] In certain embodiments, the double-stranded RNAi agent is administered chronically to a human subject.

[0030] In some embodiments, the double-stranded RNAi agent is administered to a human subject about once a quarter to about once a year. In some embodiments, the double-stranded RNAi agent is administered to a human subject about once a quarter, about once every six months, or about once a year.

[0031] In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg to about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 50 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 100 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg; about 25 mg to about 200 mg; about 75 mg to about 200 mg; about 25 mg; about 50 mg; about 75 mg; about 100 mg; about 200 mg; or about 300 mg once per quarter, i.e., about once every three months.

[0032] In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg to about 600 mg. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg or about 600 mg about once every six months to about once a year. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg or about 600 mg about once every six months or about once a year.

[0033] In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 700 mg to about 1000 mg or about 700 mg to about 900 mg. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 700 mg, about 800 mg, about 900 mg, or about 1000 mg about once a year.

[0034] In certain embodiments, the use further comprises administering to the human subject an additional therapeutic agent, for example, a TTR tetramer stabilizer or a nonsteroidal anti-inflammatory agent.

[0035] The invention also provides kits for carrying out any of the methods of the invention. The kits may include a double-stranded RNAi agent; and a label containing instructions for use.

[0036] The present invention is further illustrated by the following detailed description and drawings. [Brief explanation of the drawings]

[0037] [Figure 1] 1 is a graph showing relative serum TTR protein levels in V30M transgenic mice (n=3 / group) following a single administration of a 1 mg / kg dose of the indicated double-stranded RNAi agent on day 0.

[0038] [Figure 2] 1 is a graph showing relative serum TTR protein levels in cynomolgus monkeys (n=3 / group) following a single administration of a 1 mg / kg dose or a 3 mg / kg dose of the indicated double-stranded RNAi agent on day 0. Results shown are from three independent studies. DETAILED DESCRIPTION OF THE INVENTION

[0039] Detailed Description of the Invention The present invention provides methods for inhibiting the expression of TTR, including inhibiting TTR expression in human subjects who do not meet diagnostic criteria for a TTR-related disease, as well as methods for treating human subjects with a transthyretin (TTR)-related disease, comprising the use of a double-stranded RNAi agent that targets the TTR gene, wherein the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7).

[0040] The following detailed description describes how to make and use compositions containing iRNA agents to selectively inhibit expression of the TTR gene, as well as compositions, uses and methods for treating subjects with diseases and disorders that would benefit from inhibiting or reducing expression of the TTR gene.

[0041] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. Furthermore, it should be noted that when a value or range of values ​​for a parameter is recited, all recited values ​​and values ​​between the values ​​are intended to be part of the present invention.

[0042] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object. By way of example, "an element" means one element or more than one element, for example, a plurality of elements.

[0043] The term "including" is used to mean, and is used interchangeably with, the term "including but not limited to."

[0044] The term "or" is used to mean, and is used interchangeably with, the term "and / or," unless context indicates otherwise.

[0045] The term "about" is used to mean within the typical range of acceptable error in the art, for example, allowable variation in administration intervals, allowable variation in dosage unit amounts. For example, "about" can be understood as within about 2 standard deviations from the mean. In some embodiments, about means +10%. In some embodiments, about means +5%. When "about" is before a series of numerical values ​​or ranges, it is understood that "about" can modify each of the series of numerical values ​​or ranges.

[0046] The terms "at least," "not less than," or "greater than or equal to" before or after a numerical value or a range of numerical values ​​should be understood to include the number adjacent to the term "at least" and all subsequent numerical values ​​or integers that are logically encompassed by the context. For example, the number of nucleotides in a nucleic acid molecule should be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" precedes a series of numerical values ​​or a range, it is understood that "at least" can modify each of the numerical values ​​in the series or range.

[0047] As used herein, "less than" or "less than" is understood as a logically smaller value or integer up to 0, as is clear from the value adjacent to the term and the context. For example, a duplex having an overhang of "two or fewer nucleotides" has 2, 1, or 0 nucleotide overhangs. When "less than" is after a series of numbers or ranges, it is understood that "less than" can modify each of the series of numbers or ranges.

[0048] As used herein, a detection method can include determining that the amount of analyte present is below the detection level of the method.

[0049] As used herein, "transthyretin" ("TTR") refers to the well-known gene and protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter of retinol-binding protein (RBP), thyroxine (T4), and retinol, and also acts as a protease. The liver secretes TTR into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR is also expressed in the pancreas and retinal pigment epithelium. The greatest clinical relevance of TTR is that both normal (wild-type) and mutant TTR proteins can aggregate to form amyloid fibrils, which become extracellular deposits that cause amyloidosis. See, e.g., Saraiva MJM (2002) Expert Reviews in Molecular Medicine, 4(12):1-11 for review. The molecular cloning and nucleotide sequence of rat transthyretin and the distribution of mRNA expression were found by Dickson, PW et al. (1985) J. Biol. Chem. 260(13)8214-8219. The X-ray crystal structure of human TTR is described in Blake, CC et al. (1974) J Mol Biol 88, 1-12. The sequence of human TTR mRNA transcripts can be found in the National Center for Biotechnology Information (NCBI) RefSeq accession number NM_000371 (e.g., SEQ ID NOs: 1 and 5). The sequence of mouse TTR mRNA can be found in RefSeq accession number NM_013697.2 and the sequence of rat TTR mRNA can be found in RefSeq accession number NM_012681.1. Further examples of TTR mRNA sequences are readily available using publicly available databases, such as GenBank, UniProt, and OMIM.

[0050] As used herein, the term "TTR-related disease" is intended to include any disease associated with the TTR gene or protein. Such diseases may be caused, for example, by overproduction of TTR protein, TTR gene mutations, abnormal truncation of TTR protein, instability of the TTR tetramer, or abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. "TTR-related disease" also includes any type of transthyretin-mediated amyloidosis (ATTR amyloidosis) in which TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposits, such as hereditary ATTR (h-ATTR) amyloidosis or non-hereditary ATTR (ATTR) amyloidosis. TTR-related diseases include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, amyloid vitreous opacities, carpal tunnel syndrome, and hyperthyroxinemia. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesias, distal limb hypoesthesia), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcers or orthostatic hypotension), motor neuropathy, epilepsy, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacities, renal dysfunction, nephropathy, significantly reduced mBMI (modified body mass index), cranial nerve dysfunction, and lattice corneal degeneration.

[0051] As used herein, the term "a strand comprising a sequence" refers to an oligonucleotide comprising a strand of nucleotides described by a sequence referenced using standard nucleotide nomenclature.

[0052] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," as used interchangeably herein, refer to agents that include RNA as defined herein and mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). iRNA regulates, e.g., inhibits, TTR gene expression in cells, e.g., cells within a subject, such as a mammalian subject.

[0053] As used herein, " iRNA " refers to double-stranded RNA, and is herein referred to as " double-stranded RNAi agent ", " double-stranded RNA (dsRNA) molecule ", " dsRNA agent " or " dsRNA " for use in the compositions, uses and methods of the present invention. The term " dsRNA " refers to a complex of ribonucleic acid molecules with a duplex structure, comprising two antiparallel and substantially complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations with respect to target RNA, i.e., TTR gene. Double-stranded RNAi agents initiate the degradation of target RNA, for example, mRNA, through a post-transcriptional gene silencing mechanism, herein referred to as RNA interference or RNAi.

[0054] As used herein, the term "modified nucleotide" refers to a nucleotide that has, independently, a modified sugar moiety, a modified internucleotide bond, or a modified nucleobase. Thus, the term modified nucleotide encompasses, for example, the substitution, addition, or removal of a functional group or atom in an internucleoside bond, a sugar moiety, or a nucleobase. Modifications suitable for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. Any such modifications, such as those used in siRNA-type molecules, are included in the "RNAi agent" for the purposes of this specification and claims.

[0055] The duplex region can be any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can range from about 21 to 36 base pairs in length, e.g., about 21 to 30 base pairs in length, e.g., about 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length. In certain embodiments, the RNAi agents of the invention are dsRNA agents, each strand of which contains 21 to 23 nucleotides that interact with the TTR mRNA sequence to direct cleavage of the target mRNA. Without intending to be bound by theory, long double-stranded RNAs introduced into cells are broken down into siRNAs by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes dsRNA into 19-23 base pair small interfering RNAs with characteristic two-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). After binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with the TTR mRNA sequence to direct cleavage of the target RNA.

[0056] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide extending from a double-stranded iRNA, such as a dsRNA. For example, a nucleotide overhang occurs when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA contains an overhang of at least one nucleotide; alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. A nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, including a deoxynucleotide / nucleoside. The overhang may be located on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotide may be located at the 5'-end, 3'-end, or both ends of the antisense or sense strand of the dsRNA. In some embodiments of dsRNA, at least one strand contains a 3'-overhang of at least one nucleotide. In other embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In yet other embodiments, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

[0057] In some embodiments, the antisense strand of dsRNA has an overhang of 1 to 9 nucleotides at the 3'-end or 5'-end, for example, 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 9, 5 to 9, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides. In some embodiments, the sense strand of dsRNA has an overhang of 1 to 9 nucleotides at the 3'-end or 5'-end, for example, 1, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides. In other embodiments, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphate.

[0058] " Blunt" or "blunt end" means that there is no unpaired nucleotide at the end of the double-stranded RNAi agent, i.e., there is no nucleotide overhang. " Blunt-end" RNAi agent refers to the dsRNA that is double-stranded throughout its entire length, i.e., there is no nucleotide overhang at either end of the molecule. The RNAi agent of the present invention includes the RNAi agent that has nucleotide overhang at one end (i.e., the agent that has one overhang and one blunt end) or has nucleotide overhang at both ends.

[0059] The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., a dsRNA, that contains a region that is substantially complementary to a target sequence, e.g., TTR mRNA. As used herein, the term "region of complementarity" refers to a region of the antisense strand that is substantially complementary to a sequence defined herein, e.g., a target sequence, e.g., a TTR nucleotide sequence. When the region of complementarity is not completely complementary to the target sequence, mismatches can occur in the internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotide of the 5' or 3' end of the iRNA. In certain embodiments, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the antisense strand. In other embodiments, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the sense strand. In certain embodiments, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotide of the 3' end of the iRNA. In other embodiments, the nucleotide mismatch is, for example, the 3'-terminal nucleotide of the iRNA.

[0060] As used herein, the term "sense strand" or "passenger strand" refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as defined herein.

[0061] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region includes 3 bases on either end of the cleavage site and adjacent to it. In some embodiments, the cleavage region includes 2 bases on either end of the cleavage site and adjacent to it. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0062] As used herein and unless otherwise specified, the term "complementarity," when used to describe a first nucleotide sequence relative to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to hybridize to a second nucleotide sequence and form a duplex structure under certain conditions, as understood by those of skill in the art. Such conditions can be, for example, "stringent conditions," where stringent conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered inside an organism, can be applied. Those skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences depending on the ultimate application of the hybridized nucleotides.

[0063] Complementary sequences within an iRNA, e.g., a dsRNA, described herein include base pairing across the entire length of one or both nucleotide sequences, an oligonucleotide or polynucleotide comprising a first nucleotide sequence, and an oligonucleotide or polynucleotide comprising a second nucleotide sequence. Such sequences may be referred to herein as "fully complementary" with respect to each other. However, when a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be fully complementary but may form one or more, but generally no more than five, four, three, or two mismatched base pairs upon hybridization of up to 30 base pairs duplex while retaining the ability to hybridize in vitro or in vivo under conditions most relevant to the end application, e.g., inhibition of gene expression. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches in determining complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide comprises a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be referred to as "fully complementary" for purposes of this description.

[0064] As used herein, a "complementary" sequence may contain or be formed entirely from non-Watson-Crick base pairs or base pairs formed from unnatural and modified nucleotides, so long as the sequence meets the above requirements for hybridization ability. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0065] The terms "complementary," "fully complementary," and "substantially complementary" herein may be used in reference to base matching between two oligonucleotides or polynucleotides, such as between the sense and antisense strands of a dsRNA or the antisense strand and a target sequence of an iRNA agent, as understood from the context in which they are used.

[0066] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding the TTR gene). For example, a polynucleotide is complementary to at least a portion of a TTR mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding the TTR gene.

[0067] Thus, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target TTR sequence. In other embodiments, the antisense polynucleotides disclosed herein are fully complementary to SEQ ID NO: 8 (5'-UGGGAUUUCAUGUAACCAAGA-3'). In some embodiments, the antisense polynucleotide sequence is 5'-UCUUGGUUACAUGAAAUCCCAUC-3' (SEQ ID NO: 9), where the U at position 7 of the antisense strand can be a T.

[0068] As used herein, the term "control level" refers to a predetermined level to which a level obtained from an assay, e.g., a biomarker level, e.g., a protein biomarker level, is compared. In some embodiments, the control level may be determined for a healthy population, e.g., a population that does not have a disease or condition associated with a change in biomarker level and does not have a predisposition, e.g., a genetic predisposition, to a disease or condition associated with a change in biomarker level. In some embodiments, the populations should be matched for certain criteria, e.g., age, sex. In some embodiments, the control level of a biomarker is a level obtained in the same subject at an earlier time, e.g., before the onset of symptomatic disease or before the start of treatment. Typically, samples are obtained from a subject at clinically relevant intervals, e.g., at intervals sufficiently distant to observe changes in the biomarker, e.g., at least 3 months apart, at least 6 months apart, or at least 9 months apart. It is understood that if more than two samples are obtained from a subject over time, any of the preceding samples can serve as the control level.

[0069] As used herein, "change compared to a control level" or the like is understood to refer to a statistically or clinically significant change in a biomarker level, e.g., a change in a protein biomarker level compared to a control level that is greater than the typical standard deviation of the assay method. Furthermore, the change must be clinically relevant. The change compared to a control level can be determined as a percent change. For example, if the control level for biomarker X is 100 pg / ml and the level of biomarker X in a subject is 150 pg / ml, the level will increase by 50%, as calculated by ((150 pg / ml - 100 pg / ml) / 100 pg / ml) x 100% = 50%. If the level of biomarker X in a subject is 300 pg / ml, the level will increase by 300%. If the level of biomarker X in a subject is 50 pg / ml, the level will decrease by 50%. In certain embodiments, the change compared to the control level is an increase of at least 50%. In some embodiments, the change compared to the control level is an increase of at least 100%, at least 200%, or at least 300%. In some embodiments, the change compared to the control level is a decrease of at least 25%. In some embodiments, the change compared to the control level is a decrease of at least 50%.

[0070] As used herein, a "biological sample from a subject" or a "sample from a subject" includes one or more bodily fluids, cells, or tissues isolated from a subject. Examples of biological fluids include blood, serum, serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, etc. A tissue sample can include a sample from a tissue, organ, or localized area. For example, a sample can be derived from a specific organ, part of an organ, or bodily fluid or cells within an organ. In some embodiments, a sample can be derived from liver tissue or liver. In some embodiments, a "biological sample from a subject" can refer to blood or blood-derived serum or plasma from a subject. In some embodiments, the bodily fluid is substantially cell-free, e.g., acellular.

[0071] As used herein, a "clinically relevant difference" is understood as a difference greater than typical interobserver variability for an assessment, where the observer can be a trained healthcare professional, caregiver, or patient who performs the same assessment on the same individual at approximately the same time, e.g., within a week, e.g., on consecutive days. Some patient observations are subjective and should be approximately identical when performed by different observers within a short period of time, e.g., weight, heart rate. Other qualitative measures, such as some aspects of the mNIS+7 (e.g., response to touch, vibration, joint position and movement) and Norfolk Quality of Life (e.g., pain level, warmth or coldness of extremities, standing stability), may vary from day to day and between observers. Therefore, composite scores are used to aggregate observations, and large interobserver variability is expected without any indication of clinically relevant changes. Assays of biomarker levels have known levels of variability within and between samples. Determining clinically relevant differences is within the capabilities of those skilled in the art, e.g., healthcare professionals and clinical laboratory specialists with experience treating patients with TTR-related disorders.

[0072] As used herein, "chronically administered" is understood as administration at indefinite intervals, for example, for the life of the subject, up until liver transplantation.

[0073] As used herein, a "therapeutic agent that stabilizes TTR" or "stabilizes the TTR tetramer" refers to an agent that reduces or prevents the dissociation of subunits of the TTR tetramer, e.g., into monomers. In certain embodiments, the agent reduces the formation of TTR amyloid plaques, e.g., by reducing the levels of TTR monomers or proteolytic fragments of TTR monomers that form TTR amyloid plaques. Such agents include, but are not limited to, tafamidis, diflunisal, and AG10.

[0074] As used herein, the term "administering a therapeutic agent" is understood to mean providing a therapeutic agent to a subject. In some embodiments, the therapeutic agent is provided at an appropriate dosage and route of administration, e.g., as provided on the label of the therapeutic agent.

[0075] II. Methods of Treating TTR-Related Disorders The present invention provides double-stranded RNAi agents and their use for treating TTR-related diseases, such as transthyretin-mediated amyloidosis (ATTR amyloidosis) in human subjects, e.g., hereditary ATTR (h-ATTR) amyloidosis or non-hereditary ATTR (wt ATTR) amyloidosis; or for inhibiting TTR expression in subjects who do not yet meet the diagnostic criteria for a TTR-related disease but are at risk of developing a TTR-related disease, such as subjects with a TTR mutation associated with TTR amyloidosis, subjects with some indicators of TTR amyloidosis who do not yet meet the diagnostic criteria for TTR amyloidosis, or subjects with altered levels of biomarkers associated with TTR amyloidosis. The method comprises administering a therapeutically effective amount of the RNAi agent of the present invention to the subject.

[0076] In one embodiment, the present invention provides a method of treating a human subject having or at risk of developing a TTR-related disorder, the method comprising administering to the human subject a fixed dose of about 25 mg to about 1000 mg of a double-stranded RNAi agent; wherein the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is the S-isomer of thymidine glycol nucleic acid (GNA); and s is a phosphorothioate linker.

[0077] In another aspect, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in a human subject having or at risk of developing a TTR-related disorder, comprising administering to the human subject a fixed dose of about 25 mg to about 1000 mg of a double-stranded RNAi agent, wherein the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7).

[0078] In another aspect, the present invention provides a method for reducing, slowing, or halting the Neuropathic Impairment Score (NIS) or modified NIS (mNIS+7) in a human subject having or at risk of developing a TTR-related disorder. The method comprises administering to the human subject a fixed dose of about 25 mg to about 1000 mg of a double-stranded RNAi agent, wherein the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7).

[0079] In another aspect, the present invention provides a method for increasing the 6-minute walk test (6MWT) in a human subject having or at risk of developing a TTR-related disorder. The method comprises administering to the human subject a fixed dose of about 25 mg to about 1000 mg of a double-stranded RNAi agent, wherein the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7).

[0080] In some embodiments, the double-stranded RNAi agent is administered to a human subject about once a quarter to about once a year. In some embodiments, the double-stranded RNAi agent is administered to a human subject about once a quarter, about once every six months, or about once a year.

[0081] In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg to about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 50 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 100 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg; about 25 mg to about 200 mg; about 50 mg to about 300 mg; about 25 mg; about 50 mg; about 100 mg; about 200 mg; or about 300 mg once per quarter, i.e., about once every three months.

[0082] In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg to about 600 mg. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg or about 600 mg about once every six months to about once a year. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg or about 600 mg about once every six months or about once a year.

[0083] In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 700 mg to about 1000 mg or about 700 mg to about 900 mg. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 700 mg, about 800 mg, about 900 mg, or about 1000 mg about once a year.

[0084] In certain embodiments, the subject is a human being treated or evaluated for a disease, disorder, or condition described herein that would benefit from reduced TTR gene expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced TTR gene expression, e.g., a human not meeting diagnostic criteria for a TTR-related disease but who exhibits at least one sign or symptom of a TTR-related disease or has at least one risk factor for developing a TTR-related disease; a human having a disease, disorder, or condition that would benefit from reduced TTR gene expression; or a human being being treated for a disease, disorder, or condition that would benefit from reduced TTR gene expression.

[0085] In some embodiments, the human subject has a TTR-related disease. In other embodiments, the subject is at risk of developing a TTR-related disease, such as a subject with a TTR gene mutation associated with the development of a TTR-related disease, a subject with a family history of a TTR-related disease, or a subject with signs or symptoms suggestive of the development of a TTR-related disease without meeting the diagnostic criteria for a TTR-related disease.

[0086] As used herein, "TTR-related disease" includes any disease caused by or associated with the formation of amyloid deposits whose fibril precursors consist of variant or wild-type TTR proteins. Mutant and wild-type TTRs produce various forms of amyloid deposits (amyloidosis). Amyloidosis involves the formation and aggregation of misfolded proteins, resulting in extracellular deposits that impair organ function. Clinical syndromes associated with TTR aggregation include, for example, senile systemic amyloidosis (SSA); systemic familial amyloidosis; familial amyloid polyneuropathy (FAP); familial amyloid cardiomyopathy (FAC); and leptomeningeal amyloidosis, also known as leptomeningeal or meningeal cerebrovascular amyloidosis, central nervous system (CNS) amyloidosis, or amyloidosis type VII.

[0087] In some embodiments, the RNAi agent of the present invention is administered to the subject with familial amyloid cardiomyopathy (FAC).In other embodiments, the RNAi agent of the present invention is administered to the subject with mixed phenotype FAC, that is, the subject with both cardiac and neurological disorders.In still other embodiments, the RNAi agent of the present invention is administered to the subject with mixed phenotype FAP, that is, the subject with both neurological and cardiac dysfunction.In some embodiments, the RNAi agent of the present invention is administered to the subject with FAP who is being treated with orthotopic liver transplantation (OLT).

[0088] In another embodiment, the RNAi agent of the present invention is administered to a subject with senile systemic amyloidosis (SSA). In another embodiment of the method of the present invention, the RNAi agent of the present invention is administered to a subject with familial amyloid cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Normal sequence TTR is associated with cardiac amyloidosis in the elderly, also known as senile systemic amyloidosis (SSA) (also known as senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA is often accompanied by microscopic deposits in many other organs. TTR mutations accelerate the TTR amyloid formation process and are the most important risk factor for the development of TTR amyloidosis (also known as ATTR (amyloidosis-transthyretin type)). More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.

[0089] In one embodiment of the method of the present invention, the RNAi agent of the present invention is administered to a subject with transthyretin (TTR)-associated familial amyloidotic polyneuropathy (FAP). Such subjects may have ocular symptoms such as vitreous opacity and glaucoma. Those skilled in the art know that amyloidogenic transthyretin (ATTR) synthesized by the retinal pigment epithelium (RPE) plays an important role in the progression of ocular amyloidosis. Previous studies have shown that panretinal photocoagulation, which reduces RPE cells, prevents the progression of amyloid deposition in the vitreous, indicating that effective suppression of ATTR expression in the RPE may be a novel treatment for ocular amyloidosis (see, for example, Kawaji, T., et al., Ophthalmology. (2010) 117: 552-555). Another TTR-related disease is hyperthyroxinemia, also known as "abnormal transthyretin hyperthyroxinemia" or "abnormal prealbumin hyperthyroxinemia." This type of hyperthyroxinemia may be secondary to increased binding of thyroxine to TTR due to mutant TTR molecules with increased affinity for thyroxine (see, e.g., Moses et al. (1982) J. Clin. Invest., 86, 2025-2033).

[0090] The RNAi agents of the present invention can be administered to a subject using any method of administration known in the art, including, but not limited to, subcutaneous, intravenous, and intramuscular injection, and any combination thereof.

[0091] In some embodiments, the agent is administered to the subject subcutaneously.

[0092] In some embodiments, a subject is administered a single dose of an RNAi agent by subcutaneous injection, for example, in the abdomen, thigh, or upper arm. In other embodiments, a subject is administered divided doses of an RNAi agent by subcutaneous injection. In some embodiments, the divided doses of the RNAi agent are administered to the subject by subcutaneous injection at two different anatomical sites of the subject. For example, a subject may be subcutaneously injected with a subcutaneous dose of 25 mg to 1000 mg. In some embodiments of the present invention, subcutaneous administration is self-administered, for example, by a prefilled syringe or an autoinjector syringe. In some embodiments, the dose of the RNAi agent for subcutaneous administration is contained in a volume of 1 ml or less, for example, in a pharmaceutically acceptable carrier. In some embodiments, the RNAi agent is a non-pyrogenic formulation.

[0093] In some embodiments, the RNAi agent is administered to a subject in an amount effective to inhibit TTR expression in cells within the subject. An effective amount to inhibit TTR expression in cells within the subject can be assessed using the methods described below, including methods involving assessment of inhibition of relevant variables such as TTR mRNA, TTR protein, or amyloid deposits.

[0094] In certain embodiments, the RNAi agent is administered to the subject in a therapeutically effective amount.

[0095] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a patient for the treatment of a TTR-related disease, is sufficient to treat the disease (e.g., by reducing, maintaining, or slowing the progression of an existing disease compared to a suitable control; or by reducing, maintaining, or slowing one or more symptoms of the disease compared to a suitable control). A "therapeutically effective amount" may vary depending on the RNAi agent, the method of administration of the agent, the disease and its severity and medical history, age, weight, family history, genetic makeup, the stage of pathological progression mediated by TTR expression, the type of prior or concurrent treatment, if any, and other individual characteristics of the patient being treated. Diagnostic criteria for TTR amyloidosis, polyneuropathy, and cardiomyopathy are described further below.

[0096] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject who does not yet meet diagnostic criteria for a TTR-related disease, e.g., a subject who has not yet been diagnosed with hTTR amyloidosis polyneuropathy; a subject who does not meet diagnostic criteria for stage 1 FAP but may be predisposed to the disease, e.g., a subject with a TTR mutation associated with TTR amyloidosis, orthostatic hypotension, heart failure, cardiac arrhythmia, left ventricular wall thickness, interventricular septal wall thickness, posterior cardiac wall dilation, diarrhea, constipation, erectile dysfunction, glaucoma, intravitreal deposits, scalloped pupil; a subject with elevated neurofilament light chain (NfL) levels compared to a control sample, e.g., an NfL level of at least 37 pg / ml in serum, is sufficient to prevent or alleviate a disease or one or more symptoms of a disease. Symptoms that may be alleviated include sensory neuropathy (e.g., paresthesia, distal limb hypoesthesia), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer or orthostatic hypotension), motor neuropathy, epilepsy, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacities, renal dysfunction, nephropathy, significantly reduced mBMI (modified body mass index), cranial nerve dysfunction, lattice corneal degeneration, echocardiographically assessed left ventricular (LV) wall thickening, echocardiographically assessed increased global longitudinal strain, increased N-terminal prohormone B-type natriuretic peptide (NTproBNP), and hospitalization for cardiac events. Disease reduction includes slowing the course of the disease or reducing the severity of later-onset disease. Dosages may vary depending on the RNAi agent, the method of administration of the agent, the degree of risk and medical history of the disease, age, weight, family history, genetic makeup, type of previous or concurrent treatment, if any, and other individual characteristics of the patient being treated.

[0097] A "therapeutically effective amount" also includes the amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio that is acceptable for any treatment. The RNAi agents used in the methods of the invention are administered in amounts sufficient to produce a reasonable benefit / risk ratio that is acceptable for such treatment.

[0098] As used herein, the term "therapeutically effective amount" also includes an amount that provides benefit in the treatment, prevention, or management of a pathological process or symptoms of a pathological process mediated by TTR expression. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, distal limb hypoesthesia), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcers or orthostatic hypotension), motor neuropathy, epilepsy, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacities, renal dysfunction, nephropathy, significantly reduced mBMI (modified body mass index), cranial nerve dysfunction, lattice corneal degeneration, left ventricular (LV) wall thickening by echocardiography, increased global longitudinal strain by echocardiography, increased N-terminal prohormone B-type natriuretic peptide (NTproBNP), and hospitalization for cardiac events.

[0099] In some embodiments, for example, when subject has FAP, mixed phenotype FAP, mixed phenotype FAC or FAP and has OLT, the treatment of subject with the dsRNA agent of the present invention slows down the progression of neuropathy.In other embodiments, for example, when subject has FAP, mixed phenotype FAP, mixed phenotype FAC, SSA or FAP and has OLT, the treatment of subject with the dsRNA agent of the present invention slows down the progression of neuropathy and cardiomyopathy.In other embodiments, for example, when subject has cardiac complications, the method of the present invention improves cardiac structure and function, for example, the method reduces the mean left ventricular wall thickness and longitudinal strain and reduces the expression level of cardiac stress biomarker, N-terminal pro-b-type natriuretic peptide (NT-proBNP).

[0100] Administration of a therapeutically or prophylactically effective amount of a RNAi agent of the invention is also useful in methods of improving at least one indicator of neurological dysfunction or quality of life in a subject having or at risk of developing a TTR-related disorder.

[0101] For example, in some embodiments, the methods of the present invention improve at least one indicator of neurological dysfunction in a subject. "Improving at least one indicator of neurological dysfunction" in a subject refers to the ability of the methods of the present invention to improve neurological dysfunction or any symptoms associated with neurological dysfunction. Any suitable measure of neurological dysfunction can be used to determine whether a subject's neurological dysfunction is reduced, slowed, or stopped, or whether symptoms associated with neurological dysfunction are improved.

[0102] One suitable measure is the Neuropathy Impairment Score (NIS). The NIS is a scoring system that measures weakness, sensation, and reflexes, particularly in relation to peripheral neuropathy. The NIS score assesses norms for muscle weakness (1 = 25% weakness, 2 = 50% weakness, 3 = 75% weakness, 3.25 = movement against gravity, 3.5 = movement with gravity loss, 3.75 = muscle flicker without movement, and 4 = paralysis), muscle stretch reflex norms (0 = normal, 1 = decreased, 2 = absent), and touch, vibration, joint position and movement, and pinprick (all assessed on the index finger and big toe: 0 = normal, 1 = decreased, 2 = absent). The scores are adjusted for age, sex, and physical fitness.

[0103] In one embodiment, the method of the present invention reduces the NIS by at least 5 points 18 months after initiation of administration. In another embodiment, the method of the present invention results in stabilization of the NIS 18 months after initiation of treatment with the RNAi agent provided herein. In another embodiment, the method slows the increase in the NIS score compared to an appropriate control group representing the natural history of the disease, for example, a placebo control group, such as those provided in Adams et al., N Engl J Med 2018;379:11-21. The rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the start of treatment, the duration of treatment, previous treatments, and the specific TTR mutations, if any, present.

[0104] Methods for determining NIS in human subjects are well known to those skilled in the art and can be found, for example, in Dyck, PJ et al., (1997) Neurology 1997. 49(1): pgs. 229-239; Dyck PJ. (1988) Muscle Nerve. Jan; 11(1):21-32.

[0105] Another suitable measure of neurological dysfunction is the modified Neuropathy Impairment Score (mNIS+7). As known to those skilled in the art, the mNIS+7 refers to a clinical test-based assessment of neurological dysfunction (NIS) combined with electrophysiological measures of small and large nerve fiber function (NCS and QST) and measures of autonomic function (postural blood pressure). The mNIS+7 score is a revised version of the NIS+7 score (representing the NIS+7 test). The NIS+7 analyzes muscle weakness and muscle stretch reflexes. Five of the seven tests include neurological status measures: peroneal nerve compound action potential amplitude, motor nerve conduction velocity and motor nerve distal latency (MNDL), tibial MNDL, and sural sensory nerve action potential amplitude. These values ​​are adjusted for age, sex, height, and weight variables. The remaining two of the seven tests are vibration detection threshold and heart rate reduction during deep breathing.

[0106] The mNIS+7 score is a novel autonomic assessment that considers the use of Smart Somatotopic Quantitative Sensation Testing, which uses compound muscle action potentials of the ulnar, peroneal, and tibial nerves and sensory nerve action potentials of the ulnar and sural nerves (Suanprasert, N. et al., (2014) J. Neurol. Sci., 344(1-2):pgs. 121-128).

[0107] In one embodiment, the methods of the present invention result in a reduction in mNIS+7 of at least 5 points 18 months after initiation of administration. In another embodiment, the methods of the present invention result in a stabilization of mNIS+7 18 months after initiation of treatment with the RNAi agents provided herein. In another embodiment, the methods slow the increase in mNIS+7 score compared to an appropriate control group representative of the natural history of the disease, e.g., a placebo control group, such as those provided in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the start of treatment, the duration of treatment, previous treatments, and the specific TTR mutations, if any, present.

[0108] In other embodiments, the methods of the invention improve at least one indicator of quality of life in a subject. "Improving at least one indicator of quality of life" in a subject refers to the ability of the methods of the invention to slow or stop the deterioration of quality of life or improve quality of life. Any suitable measure of quality of life can be used to determine whether the deterioration of quality of life in a subject has slowed or stopped or whether quality of life has improved.

[0109] For example, the SF-36® Health Survey is a self-report, multi-item scale that measures eight health parameters: physical functioning, role limitations due to physical health problems, bodily pain, general health, vitality (energy and fatigue), social functioning, role limitations due to emotional problems, and mental health (mental distress and emotional well-being). Each scale is directly converted to a 0-100 scale, assuming equal weighting for each question. Lower scores indicate greater disability. Higher scores indicate less disability, i.e., a score of 0 equates to maximal disability and a score of 100 equates to no disability. The survey also provides a physical summary score and a mental summary score.

[0110] In some embodiments, the methods of the present invention provide a subject with an improvement over baseline in at least one of the SF-36 physical health-related parameters (physical health, role-physical, bodily pain, or general health status) or at least one of the SF-36 mental health-related parameters (vitality, social functioning, role-emotional, or mental health). Such improvement can take the form of, for example, an increase of at least 2 or at least 3 points on the scale of one or more parameters at 9 months after starting medication.

[0111] In another embodiment, the methods of the present invention stop the decline of any one or more parameters of the SF-36 parameter score 9 months after the start of dosing, e.g., the method does not result in a clinically significant change in SF-36, e.g., within the observed variability of the individual performing the SF-36 assessment. In yet another embodiment, the methods of the present invention slow the rate of decline in the SF-36 score 9 months after the start of dosing, e.g., the rate of decline in the SF-36 score of a subject treated with an RNAi agent of the present invention compared to the rate of decline in the SF-36 score of an appropriate control group representing the natural history of the disease, e.g., a placebo control group, as provided in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the start of treatment, the duration of treatment, previous treatments, and the specific TTR mutation, if any, present.

[0112] Another suitable measure of quality of life is the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) questionnaire. The Norfolk QOL-DN is a validated, comprehensive questionnaire designed to capture the full spectrum of DN associated with large fiber, small fiber, and autonomic neuropathies that are not captured by existing instruments.

[0113] In some embodiments, the methods of the present invention improve the subject's Norfolk QOL-DN score from baseline, e.g., a change of about -2.5, -3.0, -3.5, -4.0, -4.5, or -5.0 at 9 months after initiation of treatment with the RNAi agent provided herein. In other embodiments, the methods halt the increase in the Norfolk QOL-DN score, e.g., the methods do not result in a clinically significant change in the Norfolk QOL-DN score, e.g., within the observed variability of individuals performing the QOL-DN assessment. In yet other embodiments, the methods of the present invention slow the rate of increase in the QOL-DN score of a subject treated with an RNAi agent of the present invention, compared to the rate of increase of an appropriate control group representing the natural history of the disease, e.g., a placebo control group, e.g., as provided in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the start of treatment, the duration of treatment, previous treatments, and the specific TTR mutation, if any, present.

[0114] Another suitable measure of quality of life is motor strength, as measured, for example, by the NIS-W score. The NIS-W score is a composite score summing head, trunk, and limb muscle weakness. Using the NIS(W) (see the section of the scale that measures weakness), muscle strength is assessed as normal (0) or complete paralysis (4), with intermediate grades: 1 indicating 25% muscle weakness as determined by clinical strength testing, 2 indicating 50% weakness, 3 indicating 75% weakness, 3.25 indicating movement against gravity, 3.50 indicating movement without gravity, and 3.75 indicating muscle flicker.

[0115] In some embodiments, the method of the present invention provides a subject with an improvement in the NIS-W score compared to baseline. Such improvement can be in the form of a reduction of at least 5, 6, 7, 8, 9, or 10 points in the subject's NIS-W score 18 months after starting treatment with the RNAi agent provided herein. In other embodiments, the method stops the decrease in the NIS-W score, for example, the method does not result in a clinically significant increase or slowing of the NIS-W score compared to the increase in the NIS-W score of an appropriate control group that represents the natural history of the disease, for example, the placebo control group provided in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the disease at the start of treatment of the subject, the duration of treatment, previous treatment, and the specific TTR mutation, if any, present.

[0116] Yet another suitable indicator of quality of life is the Rasch-constructed Global Disability Scale (R-ODS), a patient questionnaire designed to capture a patient's activity and social engagement limitations. In certain embodiments, the methods of the invention provide a subject with an improvement in their R-ODS score relative to baseline. Such improvement can take the form of an increase of at least 2, e.g., at least 2, 3, 4, or 5 points, in the subject's R-ODS score 18 months after initiation of treatment with an RNAi agent provided herein. In other embodiments, the methods halt the decline in the R-ODS score, e.g., the methods do not result in a clinically significant decline in the R-ODS score 18 months after initiation of treatment with an RNAi agent provided herein. In yet another embodiment, the methods of the present invention slow the rate of decline in R-ODS scores in subjects treated with the RNAi agents of the present invention 18 months after initiation of treatment with the RNAi agents provided herein, compared to the rate of decline in R-ODS scores in an appropriate control group representative of the natural history of the disease, e.g., a placebo control group, as provided in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the time treatment begins, the duration of treatment, previous treatments, and the particular TTR mutation, if any, present.

[0117] The Composite Autonomic Symptom Score (COMPASS-31), a patient questionnaire assessing symptoms of autonomic neuropathy that provides a symptom score from 0 to 100, is another suitable indicator of quality of life. In certain embodiments, the methods of the invention provide a subject with an improvement in their COMPASS-31 score relative to baseline. Such improvement can take the form of an increase of at least 5, e.g., at least 5, 6, 7, 8, 9, or 10 points, in the subject's COMPASS-31 score 18 months after initiation of treatment with an RNAi agent provided herein. In other embodiments, the methods halt the decline in COMPASS-31 score, e.g., the methods do not result in a clinically relevant change in COMPASS-31 score 18 months after initiation of treatment with an RNAi agent provided herein. In yet another embodiment, the methods of the present invention slow the rate of decrease in the COMPASS-31 score of a subject treated with an RNAi agent of the present invention, e.g., compared to the rate of decrease in the COMPASS-31 score of a suitable control group representative of the natural history of the disease, e.g., the placebo control group provided in Adams et al., N Engl J Med 2018;379:11-21, 18 months after initiation of treatment with the RNAi agent provided herein. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the time treatment is initiated, the duration of treatment, previous treatments, and the particular TTR mutation, if any, present.

[0118] Other quality of life indicators can include nutritional status (e.g., as assessed by change in central body mass index (mBMI)). In certain embodiments, the methods of the present invention provide a subject with an improvement in mBMI relative to baseline. Such improvement can take the form of an mBMI score of at least 2, 3, 4, 5, or more at 18 months after initiation of treatment with an RNAi agent provided herein. In other embodiments, the methods halt the decrease in mBMI index score, e.g., the methods do not result in a clinically significant change in mBMI score at 18 months after initiation of treatment with an RNAi agent provided herein. In yet other embodiments, the methods of the present invention slow the rate of decrease in mBMI score in a subject treated with an RNAi agent of the present invention, e.g., compared to the rate of decrease in mBMI score in a suitable control group representative of the natural history of the disease, e.g., a placebo control group, e.g., as provided in Adams et al., N Engl J Med 2018;379:11-21, at 18 months after initiation of treatment with an RNAi agent provided herein. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the time treatment begins, the duration of treatment, previous treatments, and the particular TTR mutation, if any, present.

[0119] Another quality of life indicator is an assessment of exercise tolerance. One suitable measure of exercise tolerance is the 6-minute walk test (6MWT), which measures how far a subject can walk in 6 minutes, i.e., 6-minute walk distance (6MWD). In certain embodiments, the methods of the invention provide a subject with an improvement in 6MWD from baseline of at least 10 meters, e.g., at least 10 meters, 15 meters, 20 meters, or about 30 meters, 18 months after initiation of treatment with an RNAi agent provided herein.

[0120] Another suitable measure is the 10-meter walk test, which measures walking speed. In one embodiment, the methods of the invention provide a subject with an improvement from baseline in the 10-meter walk test of at least 0.025 meters / second, for example, at least 0.025 meters, 0.03 meters, 0.04 meters, 0.05 meters, 0.06 meters, 0.07 meters, 0.08 meters, 0.09 meters, 1.0 meters, 1.5 meters, 2.0 meters, 2.5 meters, 3.0 meters, 3.5 meters, 4.0 meters, 4.5 meters, or about 5.0 meters / second, at 18 months after initiating treatment with an RNAi agent provided herein.

[0121] In some embodiments, the change in plasma biomarker level indicates the reduction of ongoing nerve damage or the progression of polyneuropathy in ATTR amyloidosis.For example, the decrease in neurofilament light chain (NfL) level at 9 months compared with the NfL level at the start of treatment can indicate the reduction of ongoing nerve damage or the progression of polyneuropathy in ATTR amyloidosis.In some embodiments, the decrease in other protein levels, particularly RSPO3, CCDC80, EDA2R and NT-proBNP levels, compared with the corresponding levels at the start of treatment at 9 months, alone or in combination with the decrease in NfL levels, can indicate the reduction of ongoing nerve damage or the progression of polyneuropathy in ATTR amyloidosis.In some embodiments, the increase in N-CDase levels, compared with the corresponding levels at the start of treatment at 9 months, alone or in combination with the above other markers, can indicate the reduction of ongoing nerve damage or the progression of polyneuropathy in ATTR amyloidosis. Additional biomarkers that may serve as indicators of a reduction in nerve damage or polyneuropathy in ATTR amyloidosis, such as at 9 months after treatment with the RNAi agents provided herein, are provided in Table 1. A reduction in the progression of ongoing nerve damage or polyneuropathy in ATTR amyloidosis correlates with a decrease in proteins with a positive beta coefficient. A reduction in the progression of ongoing nerve damage or polyneuropathy in ATTR amyloidosis correlates with an increase in proteins with a negative beta coefficient. It should be understood that changes in biomarker levels are statistically significant, i.e., changes greater than the inherent variability of the assay.

[0122] In certain embodiments, the methods of the present invention provide improved cardiovascular indices, such as an increase in Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS), a decrease in left ventricular (LV) wall thickening by echocardiography compared to baseline, a decrease in global longitudinal strain by echocardiography compared to baseline, a decrease in N-terminal prohormone B-type natriuretic peptide (NTproBNP) compared to baseline, and a decrease in hospitalizations due to cardiac events.

[0123] The methods of the present invention can also improve the prognosis of patients being treated. For example, the methods of the present invention can provide a subject with a reduced probability of clinical deterioration events during treatment, or an extended lifespan or reduced hospitalization, compared to a suitable control group representing the natural history of the disease, such as a placebo control group, as provided in Adams et al., N Engl J Med 2018;379:11-21. In some embodiments, the reduced probability of clinical deterioration events during treatment can include a reduced all-cause mortality rate or cardiovascular-related hospitalization rate, as assessed by, for example, the Finkelstein-Schoenfeld method, compared to a suitable control group, as provided in Maurer et al., N Engl J Med 2018:379:11-21. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the severity of the subject's disease at the start of treatment, the duration of treatment, previous treatments, and the specific TTR mutations, if any, present.

[0124] The dose of an RNAi agent administered to a subject can be adjusted to balance the risks and benefits of a particular dose, for example, to achieve a desired level of inhibition of TTR gene expression (e.g., as assessed based on TTR mRNA expression, TTR protein expression, or by reduction in amyloid deposits, as described above) or a desired therapeutic effect, while simultaneously avoiding undesirable side effects.

[0125] In certain embodiments, an iRNA agent of the invention is administered to a subject at a "fixed dose" (e.g., a mg dose), meaning that one dose of an iRNA agent is used for the entire subject, regardless of any particular subject-related factors, such as body weight.

[0126] In certain embodiments, the RNAi agent is administered at a fixed dose of about 25 mg to about 1000 mg, e.g., about 25 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg.

[0127] In some embodiments, the double-stranded RNAi agent is administered to a human subject about once a quarter to about once a year. In some embodiments, the double-stranded RNAi agent is administered to a human subject about once a quarter, about once every six months, or about once a year.

[0128] In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg to about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 50 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 100 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 200 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg; about 25 mg to about 200 mg; about 75 mg to about 200 mg; about 25 mg; about 50 mg; about 100 mg; about 200 mg; or about 300 mg once per quarter, i.e., about once every three months.

[0129] In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg to about 600 mg. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg or about 600 mg about once every six months to about once a year. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 400 mg or about 600 mg about once every six months or about once a year.

[0130] In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 700 mg to about 1000 mg or about 700 mg to about 900 mg. In some embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 700 mg, about 800 mg, about 900 mg, or about 1000 mg about once a year.

[0131] In some embodiments, administration is subcutaneous administration, for example, self-administration, for example, by pre-filled syringe or auto-injector syringe.In some embodiments, the dose of RNAi agent for subcutaneous administration is contained in, for example, pharmaceutically acceptable carrier, for example, with a volume of 1 ml or less.

[0132] Any of these schedules can be repeated one or more times as desired. The number of repetitions can depend on the desired effect, such as suppressing the TTR gene, achieving retinol-binding protein levels, vitamin A levels, or achieving a therapeutic effect, such as reducing amyloid deposits or reducing TTR-related disease symptoms. In certain embodiments, the iRNA agent can be administered chronically for an indefinite period, such as for the patient's lifetime.

[0133] In some embodiments, RNAi agent can be administered together with other therapeutic agents or other therapeutic regimens.For example, other drugs or other therapeutic regimens suitable for treating TTR-related diseases can include liver transplantation, heart transplantation, pacemaker implantation, drugs that can reduce the level of monomeric TTR in the body; Tafamidis (Vyndaqel (registered trademark) or Vyndamax (registered trademark)) or AG10, which kinetically stabilizes TTR tetramers and prevents the tetramer dissociation required for TTR amyloid formation; Non-steroidal anti-inflammatory drugs (NSAIDS), such as diflunisal, and diuretics, which can be used to reduce edema in, for example, TTR amyloidosis with cardiac complications.

[0134] In some embodiments, the subject is administered an initial dose of RNAi agent and one or more maintenance doses.The one or more maintenance doses can be the same as or lower than the initial dose, for example, half of the initial dose.After treatment, the patient can be monitored for changes in condition.

[0135] In certain embodiments of the methods of the invention, expression of the TTR gene, as assessed by serum or plasma TTR levels, is inhibited by at least 85%, and in some embodiments, by at least 90%. It is understood that inhibition of TTR expression using the iRNA agents provided herein inhibits TTR expression in the liver and does not substantially inhibit TTR expression in other tissues, such as the eye.

[0136] As used herein, the term "inhibition" is used interchangeably with "reduction," "silencing," "downregulation," "suppression," and other similar terms, and includes any level of inhibition. In some embodiments, inhibition includes statistically significant or clinically significant inhibition.

[0137] The term "inhibiting TTR expression" is intended to refer to inhibition of expression of any TTR gene, including variants or mutants of the TTR gene. Thus, the TTR gene may be a wild-type TTR gene or a mutant TTR gene (e.g., a mutant TTR gene that causes amyloid deposition).

[0138] Inhibition can be assessed by a decrease in the absolute or relative level of one or more variables associated with TTR expression compared to a control level. The control level can be any type of control level available in the art, such as a pre-administration baseline level or a level determined in a similar subject, cell, or sample that is untreated or treated with a control (e.g., a buffer-only control or an inactive agent control). As used herein, inhibition of TTR expression can typically be assessed by determining TTR levels in an appropriate sample (e.g., a historical control sample, a normal sample, or a level determined in a clinical trial) or before treating a subject with an iRNA agent such as those provided herein or in PCT publications WO2010048228, WO2013075035, and WO2017023660, or other agents, such as antisense oligonucleotide agents, dicer substrate agents that inhibit TTR expression (see, e.g., WO2011139917 and WO2015085158); and after treatment with an iRNA agent provided herein. It is understood that the iRNA agents provided herein are sustained, but sustained-release. Thus, the level of knockdown is determined after sufficient time for a nadir to be reached, e.g., at least 3 weeks after the first administration of the iRNA agent in a human subject, or after a steady state of TTR knockdown is achieved, e.g., after multiple administrations of an iRNA agent provided herein.

[0139] Inhibition of TTR gene expression can be manifested by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from a subject) that has been treated (e.g., by contacting one or more cells with an RNAi agent of the present invention or by administering an RNAi agent of the present invention to a subject in which cells are or were present) such that the TTR gene is transcribed and expression of the TTR gene is inhibited, compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but is not treated. In one embodiment, percent inhibition is assessed by expressing the mRNA level in treated cells as a percentage of the mRNA level in control cells using the following formula:

number

[0140] Similar calculations can be performed on serum TTR protein concentrations, for example, from blood samples obtained from subjects, to determine percent inhibition of expression. If no TTR is detected in post-treatment serum or plasma samples, the amount of TTR present is considered the lower limit of detection of the assay used.

[0141] In certain embodiments, percent inhibition is determined using a validated and clinically accepted method.

[0142] Alternatively, the inhibition of TTR gene expression can be evaluated in terms of a decrease in parameters functionally related to TTR gene expression, such as TTR protein expression, retinol-binding protein level, vitamin A level, or the presence of amyloid deposits containing TTR.TTR gene silencing can be determined in any cell that expresses TTR constitutively or by genome engineering, by any assay known in the art.The liver is the main site of TTR gene expression.Other prominent expression sites are the retina and choroid plexus.

[0143] III. iRNA of the Invention Suitable iRNAs for use in the methods of the present invention include double-stranded ribonucleic acid (dsRNA) molecules for inhibiting expression of the TTR gene in cells, such as cells in a subject, e.g., a mammal, such as a human, with a TTR-related disorder. The dsRNA includes an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed by expression of the TTR gene. The region of complementarity is about 21 to 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, or 21 nucleotides in length). Upon contact with cells expressing the TTR gene, the iRNA selectively inhibits TTR gene (e.g., human, non-human primate, or non-primate mammalian TTR gene) expression by at least about 70% when Hep3B cells are transfected with 10 nM of the iRNA agent using the methods provided herein, as assayed by real-time PCR, e.g., using the method provided in Example 4 of WO2013075035.

[0144] dsRNA comprises two complementary RNA strands, which hybridize under the conditions in which dsRNA is used to form a duplex structure.One strand (antisense strand) of dsRNA comprises a complementary region, which is substantially complementary and generally completely complementary to target sequence.Target sequence can be derived from the sequence of mRNA formed during the expression of TTR gene.The other strand (sense strand) comprises a region that is complementary to antisense strand, so that when combined under appropriate conditions, the two strands hybridize and form a duplex structure.As described elsewhere herein and known in the art, the complementary sequence of dsRNA can be contained as a self-complementary region in a single nucleic acid molecule, instead of being in separate oligonucleotides.

[0145] Generally, the duplex structure is 21 to 30 base pairs long. Similarly, the region complementary to the target sequence is 22 and 30 nucleotides long.

[0146] dsRNA can be synthesized by standard methods known in the art, as described further below.

[0147] The iRNA compounds of the present invention can be produced using a two-step method. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compounds can be produced using solution phase or solid phase organic synthesis, or both. Organic synthesis has the advantage that it can easily produce oligonucleotide strands containing unnatural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be produced using solution phase or solid phase organic synthesis, or both.

[0148] IV. Modified iRNAs of the Invention The iRNA agents used in the methods of the present invention contain defined chemical modifications in the sense and antisense strands. When the length of either strand is extended to provide an antisense strand longer than 23 nucleotides and a sense strand longer than 21 nucleotides, the nucleotides may contain modifications, including, but not limited to, sugar modifications, backbone modifications, and base modifications. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkage, etc.); base modifications, such as substitution with a stabilizing base, a destabilizing base, or a base that base-pairs with an expanded repertoire of partners, base removal (abasic nucleotide) or conjugated base; sugar modifications (e.g., 2'- or 4'-position) or sugar substitution; or backbone modifications, including modification or substitution of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or that do not contain natural internucleoside linkages. RNAs with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification and as sometimes referenced in the literature, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In certain embodiments, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0149] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reversed polarity, in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included.

[0150] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0151] In other embodiments, suitable RNA mimics are intended for use in iRNA, in which both the sugar and internucleoside linkages of nucleotide units, i.e., the backbone, are replaced with novel groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. Some such oligomeric compounds, which are RNA mimics and have been shown to have excellent hybridization properties, are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are maintained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone.

[0152] Some embodiments of interest in the present invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular, --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- (known as methylene (methylimino) or MMI backbones), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- of the above-cited U.S. Patent 5,489,677, and amide backbones of the above-cited U.S. Patent 5,602,240. In some embodiments, RNAs of interest herein have the morpholino backbone structure of the above-cited U.S. Patent 5,034,506. A natural phosphodiester backbone can be represented as O-P(O)(OH)-OCH2-.

[0153] Modified RNAs can also contain one or more substituted sugar moieties. iRNAs, e.g., dsRNAs, of note herein, can have at the 2' position OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl (wherein alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6). 10 Alkyl or C2-C 10 An example of a suitable modification is O[(CH2) n O] m CH3, O(CH2).n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises a C1-C 10 The modification may include one of the following: lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacokinetic properties of iRNA or group that improves the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-(2-methoxyethyl) or 2'-MOE, also known as 2'-O-CHCHOCH) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Other exemplary modifications include the 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH)ON(CH), group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH-O-CH-N(CH). Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers of these families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0154] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions in the RNA of an iRNA, particularly the 3' position of the sugar of the 3'-terminal nucleotide or 2'-5'-linked dsRNA and the 5' position of 5'-terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0155] The RNA of the iRNA of the present invention may also contain nucleobase (often simply referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxythymidine (dT), 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. Other synthetic and natural nucleobases include pseudouracil, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are an exemplary base substitution, particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0156] The RNAi agents of the present invention can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified with a ring formed by a bridge between two carbon atoms of adjacent or non-adjacent atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety containing a ring formed by a bridge that includes two carbon atoms, adjacent or non-adjacent, of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridge optionally connects the 4'-carbon and 2'-carbon of the sugar ring through a 2'-acyclic oxygen atom. Thus, in some embodiments, the agents of the present invention can include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety in which the ribose moiety includes an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" ribose into a 3'-endo conformation.The addition of locked nucleic acid to siRNA is believed to increase siRNA stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).Examples of bicyclic nucleosides for use in the polynucleotide of the present invention include, but are not limited to, nucleosides that comprise a bridge between the 4' and 2' ribosyl ring atoms.In some embodiments, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides that comprise a 4'→2' bridge.

[0157] Locked nucleosides have the structure (stereochemistry omitted): [ka] where B is a nucleobase or modified nucleobase and L is a linking group connecting the 2'-carbon to the 4'-carbon of the ribose ring.

[0158] Examples of such 4'→2' bridged bicyclic nucleosides include 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Patent 7,399,845); 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt"); and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see, e.g., U.S. Patent 7,399,845); '-C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Patent 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Patent 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Publication 2004 / 0171570); 4'-CH2-N(R)-O-2' (where R is H, C1-C 12 Examples of suitable protecting groups include, but are not limited to, 4'-CH2-C(H)(CH3)-2' (which is an alkyl or nitrogen protecting group) (see, e.g., U.S. Patent 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Patent 8,278,426), the entire contents of each of which are incorporated herein by reference.

[0159] Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Patents 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,1 25; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US2008 / 0039618; and US2009 / 0012281 (the entire contents of each of which are incorporated herein by reference).

[0160] Any of the above bicyclic nucleosides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0161] The RNAi agents of the present invention can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge (i.e., L in the structure above). In some embodiments, the constrained ethyl nucleotide is in the S conformation, referred to herein as "S-cEt."

[0162] The iRNA of the present invention may also contain one or more "conformation-restricting nucleotides" ("CRNs"). A CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation, increasing hybridization affinity to mRNA. The linker is long enough to position the oxygen in an optimal position for stability and affinity, resulting in reduced ribose ring puckering.

[0163] One or more of the nucleotides of the iRNA of the invention can also comprise a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as an "unlocked nucleic acid" ("UNA") modification.

[0164] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6 amino), 2-docosanoyl-uridine-3'-phosphate, inverted base dT (idT), and others. Disclosure of this modification can be found in WO2011 / 005861.

[0165] Another modification of the nucleotides of the iRNA of the present invention is a 5' phosphate or 5' phosphate mimic, such as a 5' terminal phosphate or phosphate mimic, of the antisense strand of the RNAi agent. Suitable phosphate mimics can be found, for example, in US Patent Publication 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0166] V. Ligand-Conjugated iRNA Another modification of the RNA of the iRNA of the invention includes chemically linking to the RNA one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include lipid moieties, e.g., cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 4:1053-1060), and the like. 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moieties (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237) or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0167] In some embodiments, the ligand changes the distribution, targeting or life span of the iRNA agent that is incorporated.In some embodiments, the ligand provides enhanced affinity to a selected target, for example, a molecule, a cell or cell type, a compartment, for example, a cell or organ compartment, tissue, organ or region in the body, compared to, for example, a species that lacks such a ligand.Examples of ligands do not participate in the duplex pairing of double-stranded nucleic acid.

[0168] The ligand can also comprise a targeting group, such as a cell or tissue targeting agent, for example, a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type, such as kidney cells. The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, monovalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimic. In some embodiments, the ligand comprises monovalent or polyvalent galactose. In some embodiments, the ligand comprises cholesterol.

[0169] Ligand-conjugated oligonucleotides of the invention can be synthesized using an oligonucleotide bearing a pendant reactive functional group, such as that resulting from the attachment of a linking molecule to the oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with a commercially available ligand, a synthetic ligand bearing any of a variety of protecting groups, or a ligand having a linking moiety attached.

[0170] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared through the well-known technique of solid phase synthesis. Any other means of such synthesis known in the art can additionally or alternatively be used. The use of similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives, is also known.

[0171] A. Carbohydrate Conjugates In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for compositions suitable for in vivo delivery of nucleic acids and in vivo therapy, as described herein. As used herein, "carbohydrate" refers to a compound that is a carbohydrate itself, consisting of one or more monosaccharide units (which may be linear, branched, or cyclic) having at least six carbon atoms with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound that has, as a moiety, one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include sugars of C5 or greater (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include sugars with two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0172] In certain embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide, hi other embodiments, the carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: [ka] [ka] [ka] [ka] [ka]

[0173] In some embodiments, the monosaccharide is [ka] and other N-acetylgalactosamines.

[0174] Other exemplary carbohydrate conjugates for use in the embodiments described herein include: [ka] (In the formula, when one of X or Y is an oligonucleotide, the other is hydrogen.) Including, but not limited to:

[0175] In some embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a monovalent linker. In some embodiments, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a bivalent linker. In yet other embodiments of the invention, GalNAc or GalNAc derivatives are linked to iRNA agents of the invention via a trivalent linker.

[0176] In some embodiments, the double-stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative linked to the iRNA agent. In other embodiments, the double-stranded RNAi agents of the invention comprise multiple (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently linked to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0177] In certain embodiments, for example, the two strands of an iRNA agent of the invention are part of a single larger molecule connected by an uninterrupted stretch of nucleotides between the 3' end of one strand and the 5' end of each other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop can contain a GalNAc or GalNAc derivative independently linked via a monovalent linker. Hairpin loops can also be formed by an extended overhang on one strand of the duplex.

[0178] In certain embodiments, the carbohydrate conjugate further comprises one or more additional ligands such as, but not limited to, a PK modulator or a cell-penetrating peptide as described above.

[0179] Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publications WO2014 / 179620 and WO2014 / 179627, the contents of each of which are incorporated herein by reference in their entirety.

[0180] B. Linker In certain embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers, which can be cleavable or non-cleavable.

[0181] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, for example, that covalently bonds two parts of a compound. The linker is typically a direct bond or an atom or unit such as oxygen or sulfur, e.g., NR, C(O), C(O)NH, SO, SO, SONH or a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylhetero ...alkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkynylarylalkynyl and alkylaryl, alkenylheteroarylalkynyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl (one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic); where R is hydrogen, acyl, aliphatic, or substituted aliphatic.In some embodiments, the linker is about 1 to 24 atoms, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 6 to 18, 7 to 18, 8 to 18 atoms, 7 to 17, 8 to 17, 6 to 16, 7 to 16, or 8 to 16 atoms.

[0182] A cleavable tether is one that is sufficiently stable outside a cell but that, upon entering a target cell, cleaves to release the two moieties that the linker joins together. In some embodiments, the cleavable tether is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or more times, or at least about 100 times faster in a target cell or under first control conditions (which can be selected, e.g., to mimic or represent intracellular conditions) than in the subject's blood or under second control conditions (which can be selected to mimic or represent conditions found in blood or serum).

[0183] Cleavable linking groups are sensitive to cleaving factors, such as pH, redox potential, or the presence of degradative molecules. Generally, cleaving factors are more prevalent or found at higher levels or activity within cells than in serum or blood. Examples of such degradative factors include redox factors that are selective for specific substrates or have no substrate specificity, including, for example, oxidizing or reducing enzymes or reducing factors such as mercaptans present in cells that can degrade redox-cleavable linking groups by reduction; esterases; endosomes or factors that can create an acidic environment, such as a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as a general acid, peptidases (which may be substrate-specific), and phosphatases.

[0184] Cleavable linking groups, such as disulfide bonds, can be pH-sensitive. Human serum has a pH of 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, at about 5.0. Some linkers have cleavable linking groups that are cleaved at a selected pH, thereby releasing the cationic lipid from the ligand within the cell or to a desired compartment of the cell.

[0185] The linker may contain a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the target cell. For example, a liver targeting ligand may be linked to a cationic lipid via a linker containing an ester group. Liver cells are rich in esterase, and therefore the linker is cleaved more efficiently from liver cells than from cell types that are not rich in esterase. Other cell types that are rich in esterase include lung, renal cortex and testicular cells.

[0186] Linkers containing peptide bonds can be used when the targeted cell type is rich in peptidases, such as hepatocytes and synovial cells.

[0187] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradable agent (or condition) to cleave the candidate linking group. It may also be desirable to test the ability of the candidate cleavable linking group to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between first and second conditions (where the first is selected to be indicative of cleavage in target cells and the second is selected to be indicative of cleavage in other tissues or body fluids, e.g., blood or serum) can be determined. Evaluations can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial evaluations in cell-free or culture conditions, followed by further confirmation by whole animal evaluations. In certain embodiments, useful candidate compounds are cleaved at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0188] i. Redox-cleavable linker In some embodiments, the cleavable linker is a redox-cleavable linker that is cleaved by reduction or oxidation. An example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is suitable for use with a suitable "reductively cleavable linker," for example, a particular iRNA moiety and a particular targeting agent, the methods described herein can be performed. For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, e.g., target cells. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In one example, the candidate compound is cleaved at most about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0189] ii. Phosphate-based cleavable linkers In some embodiments, the cleavable linker comprises a phosphate-based cleavable linker. The phosphate-based cleavable linker is cleaved by an agent that decomposes or hydrolyzes the phosphate group. An example of an agent that cleaves the phosphate group in a cell is an enzyme such as a phosphatase in the cell. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S, (where Rk in each occurrence is independently C-C 20 Alkyl, C1-C 20 Haloalkyl, C6-C 10 Aryl or C7-C 12 aralkyl). Example embodiments include -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. In certain embodiments, the phosphate-based linking group is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0190] iii. Acid-cleavable linking group In some embodiments, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In some embodiments, the acid-cleavable linker is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less) or by factors such as enzymes that can act as general acids. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). An exemplary embodiment is when the carbon bonded to the oxygen (alkoxy group) of the ester is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0191] iv. Ester-based linking groups In other embodiments, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved by enzymes such as cellular esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0192] v. Peptide-based cleaving groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved by enzymes such as cellular peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids that result in oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (—C(O)NH—). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids that result in peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that result in peptides and proteins, and do not include amide functional groups altogether. Peptide-based cleavable linkers have the general formula —NHCHRAC(O)NHCHRBC(O)—, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0193] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the present invention include, but are not limited to: [ka] [ka] (In the formula, when one of X or Y is an oligonucleotide, the other is hydrogen.)

[0194] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0195] In certain embodiments, the dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formulas (XXXII)-(XXXV): [ka] [During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C are, independently in each occurrence, 0 to 20, where the repeat units may be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is independently at each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is, independently at each occurrence, absent, alkylene, or substituted alkylene, where one or more of the methylenes is selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5Cis, independently in each occurrence, absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C is a ligand; i.e., independently in each occurrence, a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R aは H or an amino acid side chain. Formula (XXXVI): [ka] [In the formula, L 5A , L 5B and L 5C is a monosaccharide such as a GalNAc derivative. Trivalent conjugated GalNAc derivatives such as are particularly useful for use in RNAi agents for the inhibition of target gene expression.

[0196] Examples of suitable bivalent and trivalent branched linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures shown above as Formulas II, VII, XI, X, and XIII.

[0197] Representative U.S. patents that teach the preparation of RNA conjugates include U.S. Patents 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718;5,608,046;4,587,044;4,605,735;4,667,025;4,762,779;4,789,737;4,824,941;4,835,263;4,876,335;4,904,582;4,958,013;5,082,830;5,112,963;5,214,136; ,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241, 5,391,723;5,416,203, 5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5, 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022 (the entire contents of each of which are incorporated herein by reference).

[0198] Not all positions in a compound need be uniformly modified, and in fact more than one of the above modifications can be incorporated into a single compound or nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0199] VI. Delivery of iRNA of the Invention Delivery of the iRNA of the present invention to cells, for example, cells in a human subject (e.g., a subject in need of treatment, such as a subject with a disease, disorder, or condition associated with contact activation pathway gene expression) can be achieved by several different methods. For example, delivery can be achieved by contacting the iRNA of the present invention with cells in vitro or in vivo. In vivo delivery can also be achieved by directly administering a composition containing the iRNA, e.g., dsRNA, to a subject. Delivery can be achieved, for example, by intravenous or subcutaneous administration. In some embodiments, the iRNA agent is delivered by subcutaneous administration. In some embodiments, the iRNA agent is administered by self-administration using a pre-filled syringe or an auto-injection device.

[0200] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO9402595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to consider for iRNA molecule delivery include, for example, the biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue.

[0201] VII. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNAs described herein for use in the methods of the invention. In certain embodiments, provided herein are pharmaceutical compositions comprising the iRNAs described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions comprising iRNAs are useful for treating diseases or disorders associated with TTR gene expression or activity. Such pharmaceutical compositions are formulated based on the delivery method. One example is a composition formulated for systemic administration via parenteral delivery, e.g., subcutaneous (SC) or intravenous (IV) delivery. Pharmaceutical compositions of the invention can be administered at a dosage sufficient to inhibit TTR gene expression. In certain embodiments, an iRNA agent, e.g., a dsRNA agent, of the invention is formulated for subcutaneous administration in a pharmaceutically acceptable carrier.

[0202] In other embodiments, a single dose of the pharmaceutical composition can be continuous, such that subsequent administrations are at monthly intervals, at most one, two, three, or four month intervals, or quarterly (approximately every three months). In one embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered monthly. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once every other month. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered quarterly, i.e., approximately once every three months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every four months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every five months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every six months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every 12 months.

[0203] VIII. Kit The present invention also provides kits for carrying out any of the methods of the present invention. Such kits include one or more double-stranded RNAi agents and a label providing instructions for using the double-stranded agents for use in any of the methods of the present invention. The kits can optionally further include a means for contacting a cell with the RNAi agent (e.g., an injection device or infusion pump) or a means for measuring TTR inhibition (e.g., a means for measuring TTR mRNA or TTR protein inhibition). Such a means for measuring TTR inhibition can include a means for obtaining a sample, such as a plasma sample, from a subject. The kits of the present invention can optionally further include a means for administering an RNAi agent to a subject or a means for determining a therapeutically or prophylactically effective amount.

[0204] RNAi agent can be provided in any conventional form, such as in sterile water solution or other suitable solution for resuspension and injection, for example, in PBS, physiological saline, 5mM phosphate buffer solution.For example, RNAi agent can be provided as 300mg, 200mg, 100mg or 50mg vial with water or other suitable solution in sterile water for injection.In some embodiments, RNAi agent is provided in a kit for self-administration, which comprises a pre-filled syringe or automatic injection device that contains 300mg, 200mg, 100mg or 50mg of RNAi agent in the appropriate volume of additive for administration, and optionally further comprises instructions for use.In some embodiments, RNAi agent is provided in multiple vials or device for administration by multiple injections that are given at approximately the same time, for example, within 1 week, within 1 day, within 1 hour.

[0205] IX. Diagnosis and Assessment of Disease Burden in TTR Amyloidosis Polyneuropathy TTR amyloidosis is a complex, multifactorial disease. The list of criteria used to monitor TTR-FAP progression is growing: Neuropathy Impairment Score (NIS), NIS+7 and modified NIS (mNIS)+7 and mNIS+7. IonisThese diagnostic criteria are well known in the art, and the main criteria are provided below. As used herein, meeting the diagnostic criteria for TTR-FAP is understood as meeting FAP stage 1 criteria with or without the presence of a mutation associated with hereditary TTR-FAP. Progression of neuropathy indicators is considered an increase of at least 2 points in the modified Neuropathy Impairment Score (mNIS) +7.

[0206] Familial amyloid polyneuropathy (FAP) stages Coutinho et al. developed a clinical staging system for the neuropathic manifestations of hATTR (formerly called familial amyloidotic neuropathy). The scale ranges from 1 to 3 as follows (Ando et al. Orphanet J Rare Dis. 2013;8:31): FAP Stage 1: Walking without assistance, mild neuropathy in the lower extremities (sensory, autonomic and motor). FAP Stage 2: Moderate impairment of walking with assistance, lower limbs, trunk, and upper limbs. FAP Stage 3: Wheelchair or bedridden, severe neuropathy. Subjects without neuropathy are considered FAP stage 0.

[0207] Neuropathic Dysfunction Scoring Method Methods for assessing neuropathy are known in the art. For example, on the Mayo Clinic Neurologic Examination Sheet and also on the weakness subscore of the Neuropathy Impairment Score (NIS), weakness (NIS-W) is scored separately for major muscle groups on each side of the body, with a 25% reduction from 1 to 4 points (Dyck et al., Quantitating overall neuropathic symptoms, impairments, and outcomes. In: Dyck PJ, Thomas PK, editors. Peripheral neuropathy. 4th ed. Philadelphia: Elsevier; 2005. pp. 1031-1032). A broad group of muscles, particularly the cranial, proximal, and distal limbs, is scored with a maximum score of 192 points on the NIS-W. Decreased stretch reflexes in the five major muscles are usually assessed by a neurologist, and sensations of touch, vibration, joint movement, and pinprick sensation in the feet and hands are scored 25% reduced from 1 to 4 on the Mayo Clinic Neurology Examination Sheet. For the complete NIS for reflexes (NIS-R) and sensations (NIS-S), Mayo Clinic recorded scores were converted to NIS point scores (i.e., a Mayo Clinic score of 1 or 2 is an NIS point score of 1, and a Mayo Clinic score of 3 or 4 is an NIS score of 2). Therefore, the highest NIS score for normal reflexes (NIS-R) assessed by neurologists is 5 × 2 × 2 = 20 points, and the highest score for the four sensory modalities often assessed by neurologists (NIS-S) is 8 × 2 × 2 = 32 points. Therefore, the highest NIS score is 192 + 20 + 32 = 244 points. The NIS has been described in previous publications (Dyck et al. 2005 and Dyck et al., Neurol. 1997;49:229-39).

[0208] The NIS+7 has been used as a primary or coprimary endpoint measure in clinical trials of diabetic sensorimotor polyneuropathy, TTR FAP, and other generalized sensorimotor polyneuropathies (N. Suanprasert et al. J Neurol Sci 344 (2014) 121-128). The NIS+7 adequately assesses the graded severity of muscle weakness and muscle stretch reflex abnormalities with only a minimal ceiling effect. Five of the seven tests in the NIS+7 are neurological status measures—expressed as a normalized deviation (Z score) or points. The measures included in the NIS+7 were selected because their abnormalities are sensitive enough to detect diabetic sensorimotor polyneuropathy (Dyck et al. Muscle Nerve 2003;27(2):202-10). Included traits are peroneal nerve compound action potential (CMAP) amplitude, motor nerve conduction velocity (MNCV) and motor nerve distal latency (MNDL), tibial MNDL, and sural sensory nerve action potential (SNAP) amplitude. These measurements can be converted from percentiles to normal deviations, correcting for applicable variables such as age, sex, height, or weight, based on previous studies of large healthy subject control cohorts. Furthermore, these percentiles can be expressed as NIS points from the resulting percentile (i.e., N5th = 0 points; ≦5th-N1st = 1 point and ≦1st = 2 points (and similarly when the abnormality is at the upper end of the normal distribution)).

[0209] Assessment of weakness and reflex abnormalities, assessment of sensory loss, autonomic dysfunction, and neurophysiological abnormalities are not adequately assessed with the NIS+7 for use in clinical trials of TTR FAP. Sensory loss is not optimally assessed with the NIS+7: 1) the body distribution of sensory loss is not adequately considered, 2) large-fiber sensory loss is overemphasized compared with small-fiber sensory loss, and 3) improved testing methods and comparison with control values ​​are preferred for clinical assessment. Additionally, autonomic dysfunction is not adequately assessed by using only heart rate during deep breathing (HRdb). The nerve conduction characteristics used to assess the NIS+7 are not ideal for testing TTR FAP.

[0210] The modified Neuropathy Dysfunction Score +7 (mNIS+7) and updated NIS+7 are composite scores measuring motor strength, reflexes, sensation, nerve conduction, and autonomic function. Two versions of this composite scale were adapted from the NIS+7 to better reflect hATTR amyloidosis with polyneuropathy and have been used as primary endpoints in clinical trials of inotersen and patisiran. Important differences between these two versions and other neuropathy scoring systems are summarized in the table below (from Adams et al., BMC Neurology, volume 17, Article number: 181 (2017)). On both scales, lower scores indicate better neurologic function (e.g., increasing scores reflect worsening neurologic dysfunction).

[0211] Neuropathic Dysfunction Score Criteria [Table 1]

[0212] Diagnosis and assessment of disease burden in TTR amyloidosis cardiomyopathy (ATTR-CM) Patients with hATTR amyloidosis and cardiomyopathy typically experience symptomatic progression of heart failure (HF) and cardiac arrhythmias, typically resulting in death 2.5–5 years after diagnosis. Cardiac infiltration of extracellular matrix by TTR amyloid fibrils leads to progressive increases in ventricular wall thickening and marked increases in ventricular stiffness, resulting in diastolic dysfunction. Systolic function is also impaired, typically reflected by abnormal longitudinal strain despite a normal ejection fraction that is maintained until late in the disease. In patients with ATTR amyloidosis and light-chain (AL) cardiac amyloidosis, both longitudinal strain and N-terminal prohormone of brain natriuretic peptide (NT-proBNP) have been shown to be independent predictors of survival.

[0213] Echocardiography is routinely used to assess cardiac structure and function; parameters prespecified in the statistical analysis plan include mean left ventricular (LV) wall thickness, LV mass, longitudinal strain, and ejection fraction. Cardiac output, left atrial size, LV end-diastolic volume (LVEDV), and LV end-systolic volume (LVESV). Echocardiograms are routinely used for cardiac imaging. Myocardial strain can be assessed by speckle tracking using vendor-independent software (TOMTEC, Munich, Germany). Analysis of NT-proBNP and troponin I levels is routinely performed in clinical laboratories using commercially available diagnostic tests, e.g., chemiluminescence assays (Roche Diagnostic Cobas, Indianapolis, IN, USA for NT-proBNP; and Siemens Centaur XP, Camberley, Surrey, UK for troponin I). Similarly, clinical practice routinely includes measurements of creatinine levels and estimated glomerular filtration rates (eGFR) based on creatinine levels, using, for example, the Improved Test of Diet in Renal Disease formula.

[0214] A review providing information on diagnostic and diagnostic methods for ATTR-CM was recently published by Witteles et al., 2019 (JACC: Heart Failure, 2019. 7:709-716), which provides information on diagnostic and screening methods, including a list of "red flags" that suggest the presence of ATTR-CM. These include echocardiography, electrocardiography, cardiac magnetic resonance imaging, autonomic neuropathy in the form of bilateral sensorimotor polyneuropathy beginning in the lower extremities and continuing in an ascending pattern, orthostatic hypotension, diarrhea / constipation, and erectile dysfunction, and ocular complications such as glaucoma, intravitreal deposits, and scalloped pupils; and the presence of systemic symptoms involving the peripheral or autonomic nervous system accompanied by cardiac dysfunction, including carpal tunnel syndrome, particularly bilateral carpal tunnel syndrome, lumbar spinal stenosis, and biceps tendon rupture. Other diagnostic methods include bone scintigraphy with technetium (Tc)-labeled bisphosphonates, which localize to TTR cardiac amyloid deposits for unknown reasons. Biopsies are also used to confirm the presence of TTR amyloidosis in the heart.

[0215] The methods for assessing and classifying cardiac function parameters provided above are known in the art. The particular method of assessing or classifying cardiac function used herein may be any clinically accepted standard for indicating sufficiently impaired cardiac function such that standard of care includes medical intervention, e.g., drug administration, surgery.

[0216] Serum biomarkers as indicators of neuronal damage and progression of TTR amyloidosis These diagnostic and monitoring methods are complex and often subjective. Furthermore, because TTR amyloidosis is rare and the signs and symptoms may be present in several other diseases, clinically validated, non-invasive plasma biomarkers could facilitate early diagnosis and aid in monitoring disease progression. In a study by Ticau et al., 2019 (see www.medrxiv.org / content / 10.1101 / 19011155v2.full.pdf), plasma levels of over 1,000 proteins were measured in a cohort of patients with hATTR amyloidosis with polyneuropathy and healthy controls who received placebo or patisiran in the Phase 3 APOLLO trial (NCT01960348). The effect of treatment with patisiran, a lipid-formulated RNAi agent that inhibits hepatic expression of TTR, on the time profile of each protein was determined using a linear mixed-model model at 0, 9, and 18 months. Neurofilament light chain (NfL) protein was further evaluated using an orthogonal quantitative approach. Significant changes in the levels of 66 proteins were observed with patisiran versus placebo, with the most pronounced change in NfL, a marker of neuronal damage. Analysis of changes in protein levels showed a trend toward healthy individuals in patisiran-treated patients at 18 months. Plasma NfL levels in healthy controls were four-fold lower than in patients with TTR amyloidosis with polyneuropathy (16.3 [SD 12.0] pg / mL vs. 69.4 [SD 42.1] pg / mL, p<10 -16 At 18 months, NfL levels increased with placebo (99.5 [SD 60.1] pg / mL) and decreased with patisiran treatment (48.8 [SD 29.9] pg / mL). At 18 months, improvement in modified Neuropathy Impairment Score +7 (mNIS+7) in patisiran-treated patients was significantly correlated with the decrease in NfL levels (R = 0.43, p < 10 -7The reduction in NfL correlated with improvement in mNIS+7 with patisiran treatment suggests that this biomarker may serve as a biomarker for nerve damage and polyneuropathy in TTR amyloidosis. This biomarker may enable early diagnosis of polyneuropathy in patients with hATTR amyloidosis and facilitate monitoring of disease progression.

[0217] Decreased levels of other proteins, particularly RSPO3, CCDC80, EDA2R, and NT-proBNP, were found to correlate with mNIS+7 improvement, suggesting that they may serve as biomarkers of nerve damage and polyneuropathy in ATTR amyloidosis, alone or in combination with each other or Nfl. Increased levels of N-CDase were found to correlate with mNIS+7 improvement, suggesting that they may serve as biomarkers of nerve damage and polyneuropathy in ATTR amyloidosis, alone or in combination with the other markers listed above.

[0218] Further potential biomarkers that were observed to change in response to patisiran treatment are listed in the table below. An increase in the subject's protein level with a positive beta coefficient compared to the control level is indicative of progression of ATTR amyloidosis, and a decrease in the subject's protein level with a negative beta coefficient compared to the control level is indicative of progression of ATTR amyloidosis. A change in the level of one or more of these markers may be indicative of an improvement, stabilization, or reduction in the progression of ongoing nerve damage and polyneuropathy in ATTR amyloidosis.

[0219] [Table 2] The sequences of these biomarkers are provided herein as SEQ ID NOs: 17-34.

[0220] The present invention is further illustrated by the following examples, which should not be construed as limiting. All references and published patents and patent applications cited throughout this application, as well as the sequence listing, are hereby incorporated by reference. [Example]

[0221] Examples of double-stranded RNAi agents for use in the methods of the invention are shown in Table 3 below. Table 2 below provides a comparison of the nucleotide monomer and ligand abbreviations used in nucleic acid sequence descriptions. It is understood that these monomers, when present in an oligonucleotide, are linked to each other by 5'-3'-phosphodiester bonds unless otherwise specified.

[0222] [Table 3]

[0223] [Table 4]

[0224] Example 1: TTR protein knockdown by RNAi agents in V30M transgenic mice The V30M mutation is a common amyloidogenic mutation in human TTR. Transgenic mice lacking mouse TTR and expressing human TTR with the V30M mutation were used for this study. Mice (n=3 / group) were administered a single subcutaneous 1 mg / kg dose of the RNAi agent AD-65492 previously disclosed in WO2018112320 or other RNAi agents based on the sequence and chemistry of AD-65492, but with a single chemical modification in the antisense strand changed to a GNA modification, as shown in Table 2 above. Blood samples were collected on days 0 (pre-administration), 3, 7, 10, 14, 21, 35, and 49. Serum was obtained, and human TTR levels were determined using an ELISA assay (see, e.g., Coelho, et al. (2013) N Engl J Med 369:819). Relative TTR levels compared to day 0 are shown in Figure 1. Incorporation of GNA into positions 7 or 8 of the antisense strand was well tolerated (AD-87404 and AD-87405). Kinetics and maximum protein knockdown were similar to those of the parent RNAi agent, AD-65492. Durability of knockdown with AD-87404 was similar to that of AD-65492. Incorporation of GNA into positions 3-6 of the antisense strand was not well tolerated.

[0225] Example 2: TTR protein knockdown by RNAi agents in non-human primates The iRNA agent sequences in Table 2 were fully cross-reactive with cynomolgus monkey TTR. A single subcutaneous dose of AD-65492 (1 mg / kg) or AD-87404 (1 mg / kg or 3 mg / kg) was administered to cynomolgus monkeys (n=3 / group) on day 0 in three separate studies. Blood samples were collected prospectively from day -7 (7 days before administration) through day 119, as shown in Figure 2. Serum was obtained, and cynomolgus monkey TTR levels were determined using an ELISA assay (see, e.g., Coelho, et al. (2013) N Engl J Med 369:819). Relative TTR levels compared to day 0 are shown in Figure 2. TTR knockdown was similar in monkeys administered 1 mg / kg AD-65492 and 3 mg / kg AD-87404.

[0226] Example 3: Administration of a single dose of AD-87404 to healthy human subjects In a Phase 1, randomized, double-blind, placebo-controlled study, AD-87404 (sense: 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6), where the L96 ligand is conjugated to the 3' end of the sense strand; antisense: 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7)) will be administered to healthy human volunteers at single doses of 25 mg, 75 mg, 100 mg, 200 mg, or 400 mg, with dose groups of 600 mg, 700 mg, 900 mg, and 1000 mg available. Relevant demographic characteristics, such as age, sex, and weight, will be balanced between groups. Demographically matched control subjects also receive a single dose of placebo. Plasma samples are collected, and TTR protein levels are determined in samples from placebo subjects and all treatment group subjects at scheduled intervals, for example, on days 1, 2, 3, 8, 15, 22, 29, 43, 57, and 90, and then every 28 days for active treatment group subjects until TTR levels return to 80% of pre-treatment levels (up to approximately 1 year after maximum administration) using ELISA assays (e.g., Coelho, et al. (2013) N Engl J Med 369:819). The level and duration of knockdown are determined. Subjects in both the AD-87404 group and the control group will also be monitored for adverse events. Examples of adverse events monitored in the study include, but are not limited to, injection site erythema, injection site pain, itching, cough, nausea, fatigue, and abdominal pain, as well as clinically significant changes in physical examination, ECG, vital signs, or laboratory parameters, such as renal function, hematological parameters, and liver function (e.g., alanine aminotransferase (ALT), aspartate aminotransferase (AST)). The results of this study demonstrate that a single subcutaneous dose of AD-87404 potently and persistently knocks down TTR protein levels in a dose-dependent manner. Multiple dose studies and multiple ascending dose studies will also be performed along with monitoring of TTR protein knockdown in serum and adverse events.

[0227] Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed within the scope of the following claims. The present disclosure provides, for example: [Section 1] 1. An RNAi agent comprising a sense strand and an antisense strand, wherein: each of the sense and antisense strands is independently up to 30 nucleotides in length; the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is the S-isomer of thymidine glycol nucleic acid (GNA); and s is a phosphorothioate linker; RNAi agents. [Section 2] The RNAi agent of paragraph 1, wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. [Section 3] The RNAi agent of paragraph 2, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. [Section 4] The ligand [C1] JPEG0007802676000023.jpg56169, the RNAi agent of item 3. [Section 5] 5. The RNAi ligand according to any one of items 2 to 4, wherein the ligand binds to the 3' end of the sense strand. [Section 6] The double-stranded RNAi agent binds to a ligand according to the following formula: [Case 2] JPEG0007802676000024.jpg59169 (wherein X is O or S). , RNAi agents in section 5. [Section 7] 7. The RNAi agent of any one of items 1 to 6, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. [Section 8] 1. Use of an RNAi agent in a method for treating a human subject having a TTR-related disease, comprising administering to the subject a fixed dose of 25 mg to 1000 mg of a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: each of the sense and antisense strands is independently up to 30 nucleotides in length; the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7); wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is the S-isomer of thymidine glycol nucleic acid (GNA); and s is a phosphorothioate linker; use. [Section 9] 1. Use of an RNAi agent in a method for inhibiting expression of TTR in a human subject who does not meet diagnostic criteria for a TTR-related disease, comprising administering to the subject a fixed dose of 25 mg to 1000 mg of a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: each sense strand and antisense strand independently being at most 30 nucleotides in length; the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7); wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is the S-isomer of thymidine glycol nucleic acid (GNA); and s is a phosphorothioate linker; use. [Section 10] The use of paragraph 8 or 9, wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. [Section 11] 11. Use according to paragraph 10, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent branched linker. [Section 12] The ligand [C3] Use of item 11, which is JPEG0007802676000025.jpg56169. [Section 13] 13. The use of any of paragraphs 9 to 12, wherein the ligand is attached to the 3' end of the sense strand. [Section 14] The double-stranded RNAi agent binds to a ligand according to the following formula: [C4] JPEG0007802676000026.jpg59169 (wherein X is O or S). , the use of Section 13. [Section 15] 15. The use of any one of items 8 to 14, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. [Section 16] 16. The use of any of paragraphs 8 to 15, wherein the method comprises improving at least one indicator of neurological impairment, quality of life, ongoing neurological damage, or cardiovascular dysfunction. [Section 17] Use of paragraph 16, wherein the indicator is a neurological dysfunction indicator. [Section 18] The use of paragraph 17, wherein the neurological dysfunction index is a change from baseline in an index selected from the group consisting of the Neuropathic Impairment Score (NIS) score, modified NIS (mNIS+7) score, NIS-W score, Composite Autonomic Symptom Score (COMPASS-31), central body mass index (mBMI) score, 6-minute walk test (6MWT) score, and 10-meter walk test score. [Section 19] Use of item 16, where the indicator is a quality of life indicator. [Section 20] Use of paragraph 19, wherein the quality of life indicator is the change from baseline in an indicator selected from the group consisting of the SF-36® Health Survey score, the Norfolk Quality of Life - Diabetic Neuropathy (Norfolk QOL-DN) score, and the Rasch-Constructed Global Disability Scale (R-ODS) score. [Section 21] Use of paragraph 16, wherein the indicator is ongoing nerve damage. [Section 22] 22. The use of paragraph 21, wherein the indicator of ongoing neuronal damage is a change from baseline in plasma protein levels of one or more proteins selected from the group neurofilament light chain (NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N-CDase. [Section 23] 22. The use of paragraph 21, wherein the indicator of ongoing neuronal damage is a change in plasma levels of neurofilament light chain (NfL) protein levels. [Section 24] Use of paragraph 16, wherein the indicator is a cardiovascular dysfunction indicator. [Section 25] Indicators of cardiovascular dysfunction are cardiovascular hospitalizations, change from baseline using the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS) with an increase in score indicating good health, change from baseline in mean left ventricular (LV) wall thickness by echocardiographic assessment, change from baseline in global longitudinal strain by echocardiographic assessment, and change from baseline in N-terminal prohormone B-type natriuretic peptide (NTproBNP), item 24. [Section 26] 26. The use of any one of items 8 to 25, wherein the human subject has a TTR gene mutation associated with the development of a TTR-related disease. [Section 27] 27. The use of any of items 8 to 26, wherein the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, and cardiac amyloidosis. [Section 28] 26. The use of any of paragraphs 8 to 25, wherein the human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the method using the RNAi agent reduces amyloid TTR deposits in the human subject. [Section 29] Use of paragraph 28, wherein the ATTR is a hereditary ATTR (h-ATTR). [Section 30] Use of paragraph 28, wherein the ATTR is a non-inherited ATTR (wt ATTR). [Section 31] 31. The use of any of paragraphs 8 to 30, wherein the double-stranded RNAi agent is administered to a human subject by subcutaneous or intravenous administration. [Section 32] 32. Use of paragraph 31, wherein the subcutaneous administration is self-administration. [Section 33] 33. Use of paragraph 32, wherein self-administration is via a pre-filled syringe or an auto-injector syringe. [Section 34] The use of any of paragraphs 8 to 33, further comprising assessing TTR mRNA expression or TTR protein expression levels in a sample from a human subject. [Section 35] 35. The use of any of paragraphs 8 to 34, wherein the double-stranded RNAi agent is administered to the human subject once every three months to once a year. [Section 36] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose of the double-stranded RNAi agent is administered to the human subject about once every three months, once every six months, or once a year. [Section 37] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose of the double-stranded RNAi agent is administered to the human subject at a fixed dose of 25 mg to 300 mg once every three months. [Section 38] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose of the double-stranded RNAi agent is administered to the human subject at a fixed dose of 25 mg to 200 mg once every three months. [Section 39] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose of the double-stranded RNAi agent is administered to the human subject at a fixed dose of 75 mg to 200 mg once every three months. [Section 40] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to a human subject at a fixed dose of 25 mg once every three months. [Section 41] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to a human subject at a fixed dose of 75 mg once every three months. [Section 42] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 100 mg once every three months. [Section 43] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 200 mg once every three months. [Section 44] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to a human subject at a fixed dose of 300 mg once every three months. [Section 45] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg to 600 mg once every 6 months to once every 12 months. [Section 46] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg to 600 mg once every 6 months or once every 12 months. [Section 47] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg or 600 mg once every 6 months or once every 12 months. [Section 48] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to a human subject at a fixed dose of 700 mg to 1000 mg once every 12 months. [Section 49] 36. The use of any of paragraphs 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to a human subject at a fixed dose of 700 mg, 800 mg, 900 mg, or 1000 mg once every 12 months. [Section 50] 50. The use of any of paragraphs 8 to 49, further comprising administering to the human subject an additional therapeutic agent. [Section 51] 51. The use of paragraph 50, wherein the further therapeutic agent is a TTR tetramer stabilizer or a nonsteroidal anti-inflammatory agent.

Claims

1. 1. A double-stranded RNAi agent, or a salt thereof, for inhibiting transthyretin (TTR) expression in a cell, comprising a sense strand and an antisense strand forming a double-stranded region, wherein: the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length; the sense strand comprises the nucleotide sequence of SEQ ID NO:6, 5'-usgsggauUfuCfAfUfguaaccaaga-3'; and the antisense strand comprises the nucleotide sequence of SEQ ID NO: 7, 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3'; wherein a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, 2'-O-methylguanosine-3'-phosphate, and 2'-O-methyluridine-3'-phosphate, respectively; Af, Cf, Gf and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate and 2'-fluorouridine-3'-phosphate, respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker; A double-stranded RNAi agent, or a salt thereof.

2. 10. The double-stranded RNAi agent of claim 1, or a salt thereof, wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand.

3. 3. The double-stranded RNAi agent of claim 2, or a salt thereof, wherein the ligand is one or more GalNAc ligands attached via a bivalent or trivalent branched linker.

4. The ligand 【Chemistry 1】 4. The double-stranded RNAi agent of claim 3, wherein:

5. The double-stranded RNAi agent or salt thereof according to any one of claims 2 to 4, wherein the ligand is attached to the 3' end of the sense strand.

6. The double-stranded RNAi agent is conjugated to a ligand as shown in the following formula: 【Chemistry 2】 wherein X is O or S.

6. The double-stranded RNAi agent of claim 5, or a salt thereof.

7. 10. A pharmaceutical composition for treating a human subject having a TTR-related disease, comprising a fixed dose of 25 mg to 1000 mg of the double-stranded RNAi agent of any one of claims 1 to 6, or a salt thereof.

8. 10. A pharmaceutical composition for inhibiting expression of TTR in a human subject who does not meet diagnostic criteria for a TTR-related disease, the pharmaceutical composition comprising a fixed dose of 25 mg to 1000 mg of the double-stranded RNAi agent of any one of claims 1 to 6, or a salt thereof.

9. 9. The pharmaceutical composition of claim 7 or 8, which improves at least one indicator of neurological impairment, quality of life, ongoing neurological damage or cardiovascular dysfunction.

10. The pharmaceutical composition according to claim 9, wherein the indicator is an indicator of neurological dysfunction.

11. 11. The pharmaceutical composition of claim 10, wherein the neurological dysfunction index is a change from baseline in an index selected from the group consisting of Neuropathic Injury Score (NIS) score, modified NIS (mNIS+7) score, NIS-W score, Composite Autonomic Symptom Score (COMPASS-31), central body mass index (mBMI) score, 6-minute walk test (6MWT) score, and 10-meter walk test score.

12. The pharmaceutical composition according to claim 9, wherein the index is a quality of life index.

13. 13. The pharmaceutical composition of claim 12, wherein the quality of life index is the change from baseline in an index selected from the group consisting of SF-36® Health Survey score, Norfolk Quality of Life - Diabetic Neuropathy (Norfolk QOL-DN) score, and Rasch-constructed Global Disability Scale (R-ODS) score.

14. The pharmaceutical composition of claim 9, wherein the indicator is ongoing nerve damage.

15. 15. The pharmaceutical composition of claim 14, wherein the indicator of ongoing neuronal damage is a change from baseline in plasma protein levels of one or more proteins selected from the group neurofilament light chain (NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N-CDase.

16. 15. The pharmaceutical composition of claim 14, wherein the indicator of ongoing neuronal damage is a change in plasma levels of neurofilament light chain (NfL) protein.

17. The pharmaceutical composition according to claim 9, wherein the indicator is a cardiovascular dysfunction indicator.

18. 18. The pharmaceutical composition of claim 17, wherein the indicators of cardiovascular dysfunction are cardiovascular hospitalizations, change from baseline using the Kansas City Cardiomyopathy Questionnaire-Overall Summary (KCCQ-OS) with an increase in score indicating good health status, change from baseline in mean left ventricular (LV) wall thickness by echocardiographic assessment, change from baseline in global longitudinal strain by echocardiographic assessment, and change from baseline in N-terminal prohormone B-type natriuretic peptide (NTproBNP).

19. The pharmaceutical composition according to any one of claims 7 to 18, wherein the human subject has a TTR gene mutation associated with the development of a TTR-related disease.

20. 20. The pharmaceutical composition according to any one of claims 7 to 19, wherein the TTR-related disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, hyperthyroxinemia, and cardiac amyloidosis.

21. 19. The pharmaceutical composition of any one of claims 7 to 18, wherein the human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the pharmaceutical composition reduces amyloid TTR deposits in the human subject.

22. The pharmaceutical composition of claim 21, wherein the ATTR is hereditary ATTR (h-ATTR).

23. 22. The pharmaceutical composition of claim 21, wherein the ATTR is a non-inherited ATTR (wt ATTR).

24. 24. The pharmaceutical composition of any one of claims 7 to 23, wherein the double-stranded RNAi agent, or salt thereof, is administered to a human subject by subcutaneous or intravenous administration.

25. 25. The pharmaceutical composition of claim 24, wherein the subcutaneous administration is self-administration.

26. 26. The pharmaceutical composition of claim 25, wherein self-administration is via a pre-filled syringe or an auto-injector syringe.

27. 27. The pharmaceutical composition of any one of claims 7 to 26, wherein the double-stranded RNAi agent, or salt thereof, is administered to a human subject from once every three months to once a year.

28. The double-stranded RNAi agent, or a salt thereof, is: (a) about once every three months, once every six months, or once a year; (b) 25 mg to 300 mg in a fixed dose once every 3 months; (c) 25 mg to 200 mg in a fixed dose once every 3 months; (d) 75 mg to 200 mg in a fixed dose once every 3 months; (e) 25 mg fixed dose once every 3 months; (f) 75 mg fixed dose once every 3 months; (g) 100 mg fixed dose once every 3 months; (h) 200 mg fixed dose every 3 months; (i) 300 mg fixed dose every 3 months; (j) 400 mg to 600 mg in a fixed dose once every 6 months to once every 12 months; (k) 400 mg to 600 mg in a fixed dose once every 6 months or once every 12 months; (l) 400 mg or 600 mg fixed dose once every 6 months or once every 12 months; (m) 700 mg to 1000 mg in a fixed dose once every 12 months; or (n) 700 mg, 800 mg, 900 mg, or 1000 mg in a fixed dose once every 12 months; The pharmaceutical composition according to any one of claims 7 to 27, for administration to a human subject.

29. The pharmaceutical composition of any one of claims 7 to 28, further comprising an additional therapeutic agent.

30. 30. The pharmaceutical composition of claim 29, wherein the additional therapeutic agent is a TTR tetramer stabilizer or a nonsteroidal anti-inflammatory agent.

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