Compositions and methods for inhibiting transthyretin (TTR) expression

Abstract

This invention provides compositions for treating transthyretin (TTR)-related diseases and their uses. [Solution] An RNAi agent targeting the TTR gene, such as a double-stranded RNAi agent, is used. The sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. The ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker.

Description

Technical Field

[0001] Related applications This application claims the benefit of priority based on U.S. Provisional Application No. 62 / 985,950, filed on March 6, 2020, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application is electronically filed in ASCII format and includes a sequence listing which is incorporated herein by reference in its entirety. The ASCII copy was created on February 26, 2021, has the name 121301_12320_SL.txt, and is 44,455 bytes in size.

Background Art

[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 for retinol (vitamin A) through its binding to RBP in blood and CSF. Transthyretin is named after 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, M.A. 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 - strand beta - sheets and is in the shape of an oblong ellipsoid. Antiparallel beta - sheet interactions bind monomers into dimers. Short loops from each monomer form the major dimer - dimer interactions. Two pairs of these loops separate the opposing, convex beta - sheets of the dimers and form an internal channel.

[0005] The liver is the major site of TTR expression. Other notable sites of expression include the choroid plexus, retina (especially 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 characteristic of amyloidosis. Amyloid fibrils consist of misfolded protein aggregates that can result from overproduction of precursor proteins or specific mutations. The amyloid - forming ability of TTR may be related to its extensive beta - sheet structure; studies by X - ray crystallography show that certain amyloid - forming mutations destabilize the protein's tetrameric structure. See, e.g., Saraiva M.J.M. (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 their precursor proteins; for example, the name starts with "A" for amyloid and is followed by the abbreviation of the precursor protein, e.g., ATTR for amyloidogenic transthyretin. Ibid.

[0008] There are numerous TTR-associated diseases, the majority of which are amyloid disorders. Normal-sequence TTR is associated with cardiac amyloidosis in older adults, also known as senile systemic amyloidosis (SSA) (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 significantly affected, the disease is called familial amyloid polyneuropathy (FAP). When the heart is primarily involved but the nervous system is not, the disease is called familial amyloid cardiomyopathy (FAC). The third most common type of TTR amyloidosis is leptomeningeal amyloidosis, also known as leptomeningeal or meningovascular amyloidosis, central nervous system (CNS) amyloidosis, or amyloidosis VII form. Mutations in TTR can also cause amyloid vitreous opacity, carpal tunnel syndrome, and thyroid hyperthyroxinemia, a non-amyloid disorder thought to be secondary to increased thyroxine-TTR binding due to mutant TTR molecules with increased affinity for thyroxine. See, for example, Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.

[0009] Abnormal TTR alleles can be either hereditary 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 amyloidogenesis 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 cause systemic amyloid deposition with specific involvement of the peripheral nervous system, but some mutations are associated with cardiomyopathy or vitreous opacity. Ibid.

[0010] The V30M mutation is the most common TTR mutation. See, for example, Lobato, L. (2003) J Nephrol, 16:438-442. The V122I mutation is present in 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. [Overview of the project] [Problems that the invention aims to solve]

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

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

[0013] The present invention relates to a double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein: Each sense strand and antisense strand is independently up to 30 nucleotides long; The sense strand contains the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), Here, 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 a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker.

[0014] In one embodiment, the sense strand of a double-stranded RNAi agent is conjugated to at least one ligand. In one embodiment, the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker. In one embodiment, the ligand is [ka] That is the case.

[0015] In one embodiment, the ligand binds to the 3' end of the sense chain.

[0016] In one embodiment, the double-stranded RNAi agent binds to the ligand as shown in the following formula: [ka] [In the equation, X is either O or S.]

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

[0018] The present invention relates to the use of a double-stranded RNAi agent in a method for treating a human subject having a TTR-related disease, comprising administering a fixed dose of the double-stranded RNAi agent ranging from approximately 25 mg to approximately 1000 mg, wherein: Each sense strand and antisense strand is independently up to 30 nucleotides long; The sense strand contains the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), Here, 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 a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker. Provides usage.

[0019] The present invention also relates to the use of a double-stranded RNAi agent in a method for inhibiting TTR expression in human subjects who do not meet the diagnostic criteria for TTR-related disease, comprising administering a fixed dose of a double-stranded RNAi agent in a range of approximately 25 mg to approximately 1000 mg, wherein: Each sense strand and antisense strand is independently up to 30 nucleotides long; The sense strand contains the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), Here, 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 a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker. We also provide usage.

[0020] In one embodiment, the sense strand of a double-stranded RNAi agent is conjugated to at least one ligand. In one embodiment, the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker. In one embodiment, the ligand is [ka] That is the case.

[0021] In one embodiment, the ligand binds to the 3' end of the sense strand. In one embodiment, the double-stranded RNAi agent binds to the ligand as shown in the following formula: [ka] [In the equation, X is either O or S.]

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

[0023] In some embodiments, the use of the present invention includes improvement in at least one of the following indicators: neurological impairment, quality of life, nerve damage, and cardiovascular symptoms. In some embodiments, the indicator to be evaluated is neurological impairment, for example, using the Neuropathy Impairment (NIS) score or the Modified NIS (mNIS+7) score. In some embodiments, the indicator is a quality of life indicator, for example, using the SF-36® Health Survey score, the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, the NIS-W score, the Rush Construction Global Disability Scale (R-ODS) score, the Composite Autonomic Symptoms Score (COMPASS-31), the Central Body Mass Index (mBMI) score, the 6-Minute Walk Test (6MWT) score, and the 10-Meter Walk Test score. In one embodiment, the indicator is nerve injury, assessed by changes in the level of one or more proteins selected from the group of nerve filament light chains (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 nerve injury is the change from baseline in nerve filament light chain (NfL) protein levels. In one embodiment, the indicator of cardiovascular dysfunction is a cardiovascular hospitalization procedure, using the Kansas City Cardiomyopathy Questionnaire overall summary (KCCQ-OS) with an increase in a score indicating good health status, a change from baseline in mean left ventricular (LV) wall thickness as assessed by echocardiography, a change from baseline in longitudinal global strain as assessed by echocardiography, and a change from baseline in N-terminal prohormone type B natriuretic peptide (NTproBNP).

[0024] In one embodiment, a human subject has a TTR gene mutation associated with the development of TTR-related diseases, 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 one embodiment, the human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the use of double-stranded RNAi agents reduces amyloid TTR deposition in the human subject. In one embodiment, the ATTR amyloidosis is hereditary ATTR(h-ATTR) amyloidosis. In one embodiment, the ATTR amyloidosis is non-hereditary ATTR(wt ATTR) amyloidosis.

[0026] In one embodiment, the double-stranded RNAi agent is administered subcutaneously or intravenously to human subjects. In one embodiment, subcutaneous administration is self-administered. In one embodiment, self-administered is via a pre-filled syringe or an automated infusion device.

[0027] In one embodiment, use further includes evaluating TTR mRNA expression or TTR protein expression levels in human subject-derived samples such as human blood samples or serum or plasma derived therefrom.

[0028] In one embodiment, the double-stranded RNAi agent is administered to human subjects 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 one embodiment, a fixed dose of the double-stranded RNAi agent is administered to human subjects approximately once every three months. In one embodiment, a fixed dose of the double-stranded RNAi agent is administered to human subjects approximately once every six months.

[0029] In one embodiment, double-stranded RNAi agents are administered chronically to human subjects.

[0030] In one embodiment, the double-stranded RNAi agent is administered to human subjects approximately once every quarter to approximately once a year. In another embodiment, the double-stranded RNAi agent is administered to human subjects approximately once every quarter, approximately once every six months, or approximately once a year.

[0031] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg to approximately 300 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg to approximately 200 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 75 mg to approximately 200 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 50 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 75 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 100 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 200 mg. In one embodiment, the double-stranded RNAi agent is administered to human subjects in fixed doses of approximately 25 mg to 300 mg; approximately 25 mg to 200 mg; approximately 75 mg to 200 mg; approximately 25 mg; approximately 50 mg; approximately 75 mg; approximately 100 mg; approximately 200 mg; or approximately 300 mg once quarter, i.e., once every three months.

[0032] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg to approximately 600 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg or approximately 600 mg, approximately once every six months to approximately once a year. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg or approximately 600 mg, approximately once every six months or approximately once a year.

[0033] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 700 mg to approximately 1000 mg or approximately 700 mg to approximately 900 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 700 mg, approximately 800 mg, approximately 900 mg, or approximately 1000 mg approximately once a year.

[0034] In one embodiment, use further involves administering to a human subject an additional therapeutic agent, such as a TTR tetramer stabilizer or a nonsteroidal anti-inflammatory agent.

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

[0036] The present invention will be further described by the following detailed description and drawings. [Brief explanation of the drawing]

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

[0038] [Figure 2] This graph shows the relative serum TTR protein levels in cynomolgus monkeys (n=3 / group) after a single dose of a double-stranded RNAi agent at a dose of 1 mg / kg or 3 mg / kg on day 0. The results shown are from three independent studies. [Modes for carrying out the invention]

[0039] Detailed description of the invention The present invention provides a method for inhibiting TTR expression, including inhibiting TTR expression in human subjects that do not meet the diagnostic criteria for TTR-related disease, and a method for treating human subjects having 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 concerns how compositions comprising iRNA agents for selectively inhibiting TTR gene expression are prepared and used, as well as compositions, uses, and methods for treating subjects with diseases and disorders in which inhibition or reduction of TTR gene expression would be beneficial.

[0041] I. Definition To make the present invention easier to understand, certain terms are first defined. Furthermore, it should be noted that when parameter values ​​or ranges of values ​​are cited, the values ​​and values ​​between those values ​​are also intended to be part of the present invention.

[0042] The singular expression is used here to indicate that the grammatical object is one or more than one (i.e., at least one). For example, “element” means one element or more than one element, e.g., multiple elements.

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

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

[0045] The term "approximately" is used to mean a typical range of tolerance in this art, such as the permissible variation in dosing intervals or the permissible variation in dose units. For example, "approximately" can be understood as being within approximately two standard deviations from the mean. In one embodiment, approximately means +10%. In another embodiment, approximately means +5%. When "approximately" precedes a set of numbers or ranges, it is understood that "approximately" can modify each of those numbers or ranges.

[0046] The terms “at least,” “not less than,” or “greater than or equal to” preceding or following a number or range of numbers should be understood to include all subsequent numbers or integers that are logically encompassed by the number adjacent to the term “at least” and 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 range of numbers or a number, it is understood that “at least” can modify each of the numbers or ranges in that range.

[0047] As used herein, “less than or equal to” or “less than” is understood to mean a logically smaller value or integer up to 0, as is evident from the values ​​adjacent to the term and the context. For example, a double helix with an overhang of “2 or less nucleotides” has an overhang of 2, 1, or 0 nucleotides. When “less than or equal to” follows a set of numbers or ranges, it is understood that “less than or equal to” can modify each of those numbers or ranges.

[0048] The detection method used here may include determining that the amount of the sample present is below the detection level of the method.

[0049] The term "transthyretin" ("TTR") used herein refers to the well-known gene and protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter for retinol-binding protein (RBP), thyroxine (T4), and retinol, and also acts as a protease. The liver secretes TTR into the bloodstream, 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 are extracellular deposits that cause amyloidosis. See, for example, Saraiva MJM (2002) Expert Reviews in Molecular Medicine, 4(12):1-11 for a review. Molecular cloning, nucleotide sequencing, and mRNA expression distribution of rat transthyretin were detected by Dickson, PW et al. (1985) J. Biol. Chem. 260(13)8214-8219. The X-ray crystal structure of human TTR is described by Blake, CC et al. (1974) J Mol Biol 88, 1-12. Sequences of human TTR mRNA transcripts can be found at National Center for Biotechnology Information (NCBI) RefSeq accession number NM_000371 (e.g., SEQ ID NOs. 1 and 5). Sequences of mouse TTR mRNA can be found at RefSeq accession number NM_013697.2, and sequences of rat TTR mRNA can be found at RefSeq accession number NM_012681.1. Further examples of TTR mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.

[0050] As used herein, “TTR-related disorders” are intended to include all disorders associated with the TTR gene or protein. Such disorders may be caused, for example, by overproduction of the TTR protein, TTR gene mutations, abnormal cleavage of the TTR protein, instability of the TTR tetramer, or abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. “TTR-related disorders” include all types 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 disorders include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloid cardiomyopathy (FAC), leptomeningeal / central nervous system (CNS) amyloidosis, amyloid vitreous opacity, carpal tunnel syndrome, and hyperthyroxinemia. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, hypoesthesia of the distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, e.g., gastric ulcer or orthostatic hypotension), motor neuropathy, epilepsy, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal dysfunction, nephropathy, significantly reduced mBMI (modified body mass index), cranial nerve dysfunction, and lattice keratopathy.

[0051] The term "chain containing a sequence" as used herein refers to an oligonucleotide containing a nucleotide chain described by a sequence referenced using standard nucleotide nomenclature.

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

[0053] In the compositions, uses, and methods of the present invention, the "iRNA" used herein is double-stranded RNA, and is hereafter referred to as "double-stranded RNAi agent," "double-stranded RNA (dsRNA) molecule," "dsRNA agent," or "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure containing two antiparallel and substantially complementary nucleic acid strands referred to as having "sense" and "antisense" orientations with respect to the TTR gene. The double-stranded RNAi agent initiates the degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism referred hereafter to as RNA interference or RNAi.

[0054] As used herein, the term "modified nucleotide" refers independently to a nucleotide having a modified sugar moiety, a modified nucleotide-nucleotide bond, or a modified nucleic acid base. Therefore, the term "modified nucleotide" encompasses substitution, addition, or removal of, for example, functional groups or atoms of nucleoside bonds, sugar moieties, or nucleic acid bases. 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 "RNAi agents" for the purposes of this specification and the claims.

[0055] The double-stranded region can be of any length that allows for the specific degradation of the desired target RNA via the RISC pathway, and may be in the range of approximately 21–36 base pairs, e.g., approximately 21–30 base pairs, e.g., approximately 21–30, 21–29, 21–28, 21–27, 21–26, 21–25, 21–24, 21–23, or 21–22 base pairs. In one embodiment, the RNAi agent of the present invention is a dsRNA agent, each strand containing 21–23 nucleotides that interact with the TTR mRNA sequence to direct the cleavage of the target mRNA. While not intended to be constrained by theory, long double-stranded RNA introduced into cells is destroyed by a type III endonuclease known as Dicer into siRNA (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 2-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 double helix, 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 the 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 a TTR mRNA sequence to direct cleavage of the target RNA.

[0056] The term "nucleotide overhang" as used herein refers to at least one unpaired nucleotide protruding from a double-stranded iRNA, such as a dsRNA. For example, a nucleotide overhang exists 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; or 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 contain or consist of a nucleotide / nucleoside analog containing a deoxynucleotide / nucleoside. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotides of the overhang may be at the 5' end, 3' end, or both ends of the antisense or sense strand of the dsRNA. In one embodiment of the dsRNA, at least one strand contains a 3' overhang of at least one nucleotide. In another embodiment, at least one strand includes a 3' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In yet another embodiment, at least one strand of the RNAi agent includes a 5' overhang of at least one nucleotide. In one embodiment, at least one strand includes a 5' overhang of at least two nucleotides, for example, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent include an overhang of at least one nucleotide.

[0057] In one embodiment, the antisense strand of the dsRNA has an overhang of 1 to 9 nucleotides at its 3' 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 another embodiment, one or more nucleotides in the overhang are substituted with a nucleoside thiophosphate.

[0058] "Smooth" or "blunt-ended" means that a double-stranded RNAi agent has no unpaired nucleotides at its ends, i.e., no nucleotide overhangs. A "blunt-ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at either end of the molecule. The RNAi agents of the present invention include RNAi agents having a nucleotide overhang at one end (i.e., an agent with one overhang and one blunt end) or RNAi agents having nucleotide overhangs at both ends.

[0059] The terms “antisense strand” or “guide strand” refer to the strand of iRNA, e.g., dsRNA, that contains a region substantially complementary to the target sequence, e.g., TTR mRNA. The term “complementary region” as used herein refers to a region of the antisense strand that is substantially complementary to the sequence defined herein, e.g., the target sequence, e.g., TTR nucleotide sequence. When the complementary region is not perfectly complementary to the target sequence, the mismatch may be located in the interior or terminal regions of the molecule. Generally, the most acceptable mismatches are within 5, 4, 3, 2, or 1 nucleotide in the terminal region, e.g., the 5' or 3' end of the iRNA. In one embodiment, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the antisense strand. In another embodiment, the double-stranded RNAi agent of the present invention contains a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, the 3' terminal nucleotide of the iRNA.

[0060] The terms "sense strand" or "passenger strand" used herein refer to the iRNA strand that contains a region substantially complementary to the antisense strand region as defined herein.

[0061] The term "cleavage region" as used herein refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In one embodiment, the cleavage region contains three bases at either end of the cleavage site and adjacent to it. In another embodiment, the cleavage region contains two bases at either end of the cleavage site and adjacent to it. In yet another embodiment, the cleavage site occurs specifically at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region contains nucleotides 11, 12, and 13.

[0062] To the extent used herein and unless otherwise specified, the term “complementarity” means, as understood by those skilled in the art, the ability of an oligonucleotide or polynucleotide containing a first nucleotide sequence to hybridize with an oligonucleotide or polynucleotide containing a second nucleotide sequence under certain conditions to form a double-stranded structure, when used to describe a first nucleotide sequence relative to a second nucleotide sequence. Such conditions may be, for example, “stringent conditions,” where stringent conditions include washing with 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12–16 hours (see, for example, “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 within living organisms, may be applied. Those skilled in the art can determine the set of conditions best suited for testing the complementarity of two sequences, depending on the final application of the hybridized nucleotides.

[0063] The complementary sequences within iRNA, for example, dsRNA, described herein involve base pairing over the full length of one or both nucleotide sequences of an oligonucleotide or polynucleotide containing the first nucleotide sequence and an oligonucleotide or polynucleotide containing the second nucleotide sequence. Such sequences may be said herein to be “fully complementary” with respect to each other. However, when the first sequence is said herein to be “substantially complementary” with respect to the second sequence, even if the two sequences are fully complementary, they may form one or more mismatched base pairs by double-strand hybridization of up to 30 base pairs, while retaining the ability to hybridize in vitro or in vivo under conditions most relevant to the final application, e.g., inhibition of gene expression. However, when two oligonucleotides are designed to form one or more single-stranded overhangs by hybridization, such overhangs should not be considered mismatches for the purpose of determining complementarity. For example, a dsRNA containing one oligonucleotide of 21 nucleotides and the other oligonucleotide of 23 nucleotides, in which the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for the purposes described herein.

[0064] The “complementary” sequences used herein may include, or be entirely formed from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, as long as they meet the above requirements regarding their ability to hybridize. Such non-Watson-Crick base pairs include, but are not limited to, G:U fluctuations or Hoogsteen base pairings.

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

[0066] The polynucleotides used here that are "substantially complementary to at least a portion" of messenger RNA (mRNA) refer to polynucleotides that are substantially complementary to the continuous portion of the target mRNA (e.g., the mRNA encoding the TTR gene). For example, a polynucleotide is complementary to at least a portion of the TTR mRNA if its sequence is substantially complementary to the uninterrupted portion of the mRNA encoding the TTR gene.

[0067] Therefore, in one embodiment, the antisense polynucleotide disclosed herein is fully complementary to the target TTR sequence. In another embodiment, the antisense polynucleotide disclosed herein is fully complementary to SEQ ID NO: 8 (5'-UGGGAUUUCAUGUAACCAAGA-3'). In one embodiment, the antisense polynucleotide sequence is 5'-UCUUGGUUACAUGAAAUCCCAUC-3' (SEQ ID NO: 9), where the U at position 7 of the antisense strand may be T.

[0068] The “control level” used herein is understood as a predetermined level to which the level obtained from the assay, e.g., biomarker level, e.g., protein biomarker level, is compared. In one embodiment, the control level may be a control level determined for a healthy population, e.g., a population that does not have the disease or condition associated with the change in biomarker level and does not have a predisposition to the disease or condition associated with the change in biomarker level, e.g., a genetic predisposition. In one embodiment, the population should be matched for certain criteria, e.g., age, sex. In one embodiment, the control level of the biomarker is an early stage in the same subject, e.g., a level before the onset of symptomatic disease or before the initiation of treatment. Typically, samples are obtained from subjects at clinically relevant intervals, e.g., intervals sufficiently far apart for changes in the biomarker to be observed, e.g., at least 3 months, at least 6 months, or at least 9 months. If more than two samples are obtained from a subject over time, it is understood that any of the earlier samples may serve as a control level.

[0069] The term "change compared to control level" used here is understood as a statistically or clinically significant change in the biomarker level. For example, the change in the protein biomarker level compared to the control level is greater than the typical standard deviation of the assay method. Furthermore, the change must be clinically relevant. The change compared to the control level can be determined as a percentage change. For example, if the control level for biomarker X is 100 pg / ml and the level of biomarker X in the subject is 150 pg / ml, the level increases by 50%, calculated as ((150 pg / ml - 100 pg / ml) / 100 pg / ml) × 100% = 50%. If the level of biomarker X in the subject is 300 pg / ml, the level increases by 300%. If the level of biomarker X in the subject is 50 pg / ml, the level decreases by 50%. In one embodiment, the change compared to the control level is an increase of at least 50%. In one embodiment, the change compared to the control level is an increase of at least 100%, at least 200%, or at least 300%. In one embodiment, the change compared to the control level is a decrease of at least 25%. In one embodiment, the change compared to the control level is a decrease of at least 50%.

[0070] As used herein, “biological sample from subject” or “sample from subject” includes one or more bodily fluids, cells, or tissues isolated from the subject. Examples of biological bodily fluids include blood, serum, serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, and saliva. Tissue samples may include samples from tissue, organs, or localized areas. For example, a sample may originate from a specific organ, a part of an organ, or bodily fluids or cells within an organ. In one embodiment, a sample may originate from liver tissue or the liver. In one embodiment, “biological sample from subject” may mean blood or blood-derived serum or plasma from the subject. In one embodiment, the bodily fluids are substantially cell-free, e.g., acellular.

[0071] The “clinically relevant difference” used here is understood as a difference in assessment that is at least greater than typical inter-observer variability, where the observer may be a trained healthcare professional, caregiver, or patient performing 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 nearly identical when performed by different observers within a short period, e.g., weight, heart rate. Other qualitative measures, such as mNIS+7 (e.g., response to touch pressure, vibration, joint position and movement) and some aspects of Norfolk Quality of Life (e.g., pain level, warmth or coldness of extremities, standing stability), may vary from day to day and from observer to observer. Therefore, by using a composite score to aggregate observations, large inter-observer variability is predicted without any indication of clinically relevant change. The levels of variability in biomarker level assays are known to vary within and between samples. Determining clinically relevant differences is within the capabilities of those skilled in the art, e.g., healthcare professionals with experience in treating patients with TTR-related diseases, clinical laboratory specialists.

[0072] As used here, "chronic administration" is understood to mean administration at indefinite intervals, for example, throughout the subject's lifetime, up to the point of liver transplantation.

[0073] The terms "TTR-stabilizing" or "TTR tetramer-stabilizing therapeutic agent" used herein refer to agents that reduce or prevent the dissociation of TTR tetramer subunits, for example, into monomers. In one embodiment, the agent reduces the formation of TTR amyloid plaques by, for example, reducing the level 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] The term "administer therapeutic agent" as used herein is understood to mean providing the therapeutic agent to a target. In one embodiment, the therapeutic agent is provided, for example, on the label of the therapeutic agent, in an appropriate dosage and route of administration.

[0075] II. Treatment of TTR-related diseases The present invention provides a double-stranded RNAi agent and its use for a method of inhibiting TTR expression in human subjects for treating transthyretin-mediated amyloidosis (ATTR amyloidosis), such as hereditary ATTR(h-ATTR) amyloidosis or non-hereditary ATTR(wt ATTR) amyloidosis; or in subjects who do not yet meet the diagnostic criteria for TTR-related disease but are at risk of developing TTR-related disease, such as subjects with TTR mutations associated with TTR amyloidosis, subjects who do not yet meet the diagnostic criteria for TTR amyloidosis but have certain indicators of TTR amyloidosis, or subjects with altered biomarker levels 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 for treating human subjects who have or are at risk of developing TTR-related disease. The method comprises administering a fixed dose of a double-stranded RNAi agent, ranging from about 25 mg to about 1000 mg, to the human subject. Here, the sense strand contains the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and the antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7). Here, 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 a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker.

[0077] In another embodiment, the present invention provides a method for improving at least one indicator of neurological impairment or quality of life in human subjects having or at risk of developing TTR-related disease. The method comprises administering a fixed dose of a double-stranded RNAi agent to a human subject in a dose of about 25 mg to about 1000 mg, 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 embodiment, the present invention provides a method for reducing, slowing, or halting the neuropathic dysfunction score (NIS) or modified NIS (mNIS+7) in human subjects having or at risk of developing TTR-related disease. The method comprises administering a fixed dose of a double-stranded RNAi agent to a human subject, ranging from about 25 mg to about 1000 mg, 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 embodiment, the present invention provides a method for increasing the 6-minute walk test (6MWT) in human subjects having or at risk of developing TTR-related disease. The method comprises administering a fixed dose of a double-stranded RNAi agent to a human subject, ranging from about 25 mg to about 1000 mg, 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 one embodiment, the double-stranded RNAi agent is administered to human subjects approximately once every quarter to approximately once a year. In another embodiment, the double-stranded RNAi agent is administered to human subjects approximately once every quarter, approximately once every six months, or approximately once a year.

[0081] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg to approximately 300 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg to approximately 200 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 75 mg to approximately 200 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 50 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 75 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 100 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 200 mg. In one embodiment, the double-stranded RNAi agent is administered to human subjects in fixed doses of approximately 25 mg to 300 mg; approximately 25 mg to 200 mg; approximately 50 mg to 300 mg; approximately 25 mg; approximately 50 mg; approximately 100 mg; approximately 200 mg; or approximately 300 mg once quarter, i.e., once every three months.

[0082] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg to approximately 600 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg or approximately 600 mg, approximately once every six months to approximately once a year. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg or approximately 600 mg, approximately once every six months or approximately once a year.

[0083] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 700 mg to approximately 1000 mg or approximately 700 mg to approximately 900 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 700 mg, approximately 800 mg, approximately 900 mg, or approximately 1000 mg approximately once a year.

[0084] In one embodiment, the subjects are: a person being treated or evaluated for a disease, disorder or condition for which a reduction in TTR gene expression would be beneficial; a person at risk of a disease, disorder or condition for which a reduction in TTR gene expression would be beneficial, for example, a person who does not meet the diagnostic criteria for TTR-related disease but exhibits at least one sign or symptom of TTR-related disease or has at least one risk factor for developing TTR-related disease; a person having a disease, disorder or condition for which a reduction in TTR gene expression would be beneficial; or a person being treated for a disease, disorder or condition for which a reduction in TTR gene expression would be beneficial.

[0085] In one embodiment, the human subject has a TTR-related disease. In another embodiment, the subject is at risk of developing a TTR-related disease, for example, 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 developing a TTR-related disease without meeting the diagnostic criteria for a TTR-related disease.

[0086] As used herein, “TTR-related diseases” include all diseases caused by or associated with the formation of amyloid deposits, in which the fibrillary precursor consists of variant or wild-type TTR proteins. Variants and wild-type TTRs give rise to various forms of amyloid deposition (amyloidosis). Amyloidosis includes the formation and aggregation of misfolded proteins, resulting in extracellular deposition that impairs 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 vascular amyloidosis, central nervous system (CNS) amyloidosis, or amyloidosis type VII.

[0087] In one embodiment, the RNAi agent of the present invention is administered to a subject having familial amyloid cardiomyopathy (FAC). In another embodiment, the RNAi agent of the present invention is administered to a subject having a mixed phenotype of FAC, i.e., a subject having both cardiac and neurological impairments. In yet another embodiment, the RNAi agent of the present invention is administered to a subject having a mixed phenotype of FAP, i.e., a subject having both neurological and cardiac dysfunctions. In one embodiment, the RNAi agent of the present invention is administered to a subject having FAP treated with orthotopic liver transplantation (OLT).

[0088] In other embodiments, the RNAi agent of the present invention is administered to subjects with senile systemic amyloidosis (SSA). In other embodiments of the method of the present invention, the RNAi agent of the present invention is administered to subjects with familial amyloid cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Normal-sequenced TTR is associated with cardiac amyloidosis in elderly individuals, also known as senile cardiac amyloidosis (SCA) or cardiac amyloidosis. SSA is often accompanied by microscopic deposition in many other organs. TTR mutations accelerate the TTR amyloidogenesis 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 subjects having transthyretin (TTR)-related familial amyloid polyneuropathy (FAP). Such subjects may have ocular signs such as vitreous opacity and glaucoma. It is known to those skilled in the art that amyloidogenic transthyretin (ATTR), synthesized by the retinal pigment epithelium (RPE), plays a crucial role in the progression of ocular amyloidosis. Previous studies have shown that panretinal photocoagulation with reduced RPE cells prevents the progression of amyloid deposition in the vitreous humor, suggesting that effective suppression of ATTR expression in the RPE could be a novel treatment for ocular amyloidosis (see, e.g., Kawaji, T., et al., Ophthalmology. (2010) 117: 552-555). Another TTR-related disorder 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, for example, Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.

[0090] The RNAi agents of the present invention may be administered to subjects 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 one embodiment, the drug is administered subcutaneously to the subject.

[0092] In one embodiment, the subject is administered a single dose of RNAi by subcutaneous injection, for example, by injection into the abdomen, thigh, or upper arm. In another embodiment, the subject is administered a divided dose of RNAi by subcutaneous injection. In one embodiment, the divided dose of RNAi is administered to the subject by subcutaneous injection into two different anatomical sites. For example, the subject may receive a subcutaneous dose of 25 mg to 1000 mg. In one embodiment of the present invention, subcutaneous administration is self-administered, for example, by a pre-filled syringe or an autoinjector syringe. In one embodiment, the dose of RNAi for subcutaneous administration is contained in a volume of 1 ml or less, for example, a pharmaceutically acceptable carrier. In one embodiment, the RNAi is a non-pyrogenic formulation.

[0093] In one embodiment, the RNAi agent is administered to a subject in an effective dose that inhibits TTR expression in cells within the subject. The effective dose that inhibits TTR expression in cells within the subject may be evaluated using the following methods, which include methods that evaluate the inhibition of relevant variable values ​​such as TTR mRNA, TTR protein, or amyloid deposition.

[0094] In one embodiment, the RNAi agent is administered to the subject in a therapeutically effective dose.

[0095] The “therapeutic dose” as used herein is intended to include the amount of 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 the pre-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). The “therapeutic dose” may vary depending on the RNAi agent, the method of drug administration, the disease and its severity and history, age, weight, family history, genetic structure, 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 further described below.

[0096] The “therapeutic effective dose” used herein is intended to include an amount of RNAi agent sufficient to prevent or alleviate one or more symptoms of the disease when administered to subjects who do not yet meet the diagnostic criteria for TTR-related disease, e.g., subjects not yet diagnosed with hTTR amyloidosis polyneuropathy; subjects who do not meet the diagnostic criteria for Stage 1 FAP but may be predisposed to the disease, e.g., subjects with TTR mutations associated with TTR amyloidosis; subjects with orthostatic hypotension, heart failure, cardiac arrhythmias, left ventricular wall thickness, ventricular septal wall thickness, posterior cardiac wall dilation, diarrhea, constipation, erectile dysfunction, glaucoma, intravitreal deposition, scalloped pupils; subjects with one or more of the following conditions: carpal tunnel syndrome, lumbar spinal stenosis, and biceps tendon rupture; or subjects with elevated nerve filament light chain (NfL) levels compared to a control sample, e.g., serum NfL levels of at least 37 pg / ml. Symptoms that may be alleviated include sensory neuropathy (e.g., paresthesia, hypoesthesia of the distal extremities), autonomic neuropathy (e.g., gastrointestinal dysfunction, e.g., gastric ulcer or orthostatic hypotension), motor neuropathy, epilepsy, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal insufficiency, nephropathy, significantly reduced mBMI (modified body mass index), cranial nerve dysfunction, lattice keratopathy, left ventricular (LV) wall thickening on echocardiographic assessment, increased longitudinal global strain on echocardiographic assessment, increased N-terminal prohormone type B natriuretic peptide (NTproBNP), and hospitalization due to cardiac events. Disease reduction includes slowing the course of the disease or reducing the severity of subsequently developing conditions. The dosage may vary depending on the RNAi agent, the method of drug administration, the degree of disease risk and medical history, age, weight, family history, genetic makeup, the type of prior or concurrent treatment, if any, and other individual characteristics of the patient being treated.

[0097] The “therapeutic effective dose” also includes the amount of RNAi agent that produces a desired local or systemic effect with an acceptable and reasonable benefit / risk ratio for any treatment. The RNAi agent used in the method of the present invention is administered in an amount sufficient to produce an acceptable and reasonable benefit / risk ratio for such treatment.

[0098] The term “therapeutic dose” as used herein also includes the amount that provides benefit in treating, preventing, or managing pathological processes or symptoms of pathological processes mediated by TTR expression. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, hypoesthesia of distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, e.g., gastric ulcers or orthostatic hypotension), motor neuropathy, epilepsy, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal insufficiency, nephropathy, significantly reduced mBMI (modified body mass index), cranial nerve dysfunction, lattice keratopathy, left ventricular (LV) wall thickening on echocardiographic assessment, increased longitudinal global strain on echocardiographic assessment, increased N-terminal prohormone type B natriuretic peptide (NTproBNP), and hospitalization due to cardiac events.

[0099] In one embodiment, for example, when a subject has FAP, mixed phenotypic FAP, mixed phenotypic FAC, or FAP, and also has OLT, treatment of the subject with the dsRNA agent of the present invention slows the progression of neuropathy. In another embodiment, for example, when a subject has FAP, mixed phenotypic FAP, mixed phenotypic FAC, SSA, or FAP, and also has OLT, treatment of the subject with the dsRNA agent of the present invention slows the progression of neuropathy and cardiomyopathy. In yet another embodiment, for example, when a subject has cardiac complications, the method of the present invention improves cardiac structure and function, for example, the method reduces mean left ventricular wall thickness and longitudinal strain, and reduces the expression level of the cardiac stress biomarker, N-terminal pro-β natriuretic peptide (NT-proBNP).

[0100] Administration of a therapeutic or prophylactic dose of the RNAi agent of the present invention is also useful as a method to improve at least one indicator of neurological dysfunction or quality of life in subjects who have or are at risk of developing TTR-related disease.

[0101] For example, in one embodiment, the method of the present invention improves at least one indicator of neurological dysfunction in a subject. “Improvement of at least one indicator of neurological dysfunction” in a subject means the ability of the method of the present invention to improve any symptom that causes or is associated with neurological dysfunction. Any appropriate measure of neurological dysfunction can be used to determine whether the neurological dysfunction in the subject has been reduced, slowed, or stopped, or whether the symptoms associated with neurological dysfunction have 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 the standard range for muscle weakness (1 = 25% weakness, 2 = 50% weakness, 3 = 75% weakness, 3.25 = movement against gravity, 3.5 = movement without gravity, 3.75 = muscle flicker without movement, and 4 = paralysis), the standard range for muscle stretch reflexes (0 = normal, 1 = decreased, 2 = absent), and touch pressure, vibration, joint position and movement, and needle stick (all assessed using the index finger of the hand and the big toe: 0 = normal, 1 = decreased, 2 = absent). The assessment is 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 at 18 months from the start of administration. In another embodiment, the method of the present invention results in stabilization of the NIS at 18 months from the start of treatment with the RNAi agent provided herein. In yet another embodiment, the method slows the increase in the NIS score compared to a suitable control group representing the natural course of the disease, e.g., Adams et al., N Engl J Med 2018;379:11-21, e.g., a placebo control group. The rate of disease progression depends on several factors, including, but not limited to, the disease severity at the start of treatment, the duration of treatment, previous treatments, and any specific TTR mutations 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; ll(l):21-32.

[0105] Another appropriate measure of neurological dysfunction is the corrected neuropathic dysfunction score (mNIS+7). As is known to those skilled in the art, mNIS+7 refers to a clinical laboratory-based assessment of neurological dysfunction (NIS) combined with electrophysiological measures of small and large nerve fiber function (NCS and QST) and measurements of autonomic function (postural blood pressure). The mNIS+7 score is a revised version of the NIS+7 score (representing the NIS+7 test). NIS+7 analyzes muscle weakness and muscle stretch reflexes. Five of the seven tests include characteristics of neurological conditions. These characteristics are peroneal nerve composite 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 corrected for the variables of age, sex, height, and weight. 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 utilizes the Smart Somatotopic Quantitative Sensation Testing, which uses composite muscle action potentials of the ulnar, fibular, and tibial nerves, as well as 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 method of the present invention reduces mNIS+7 by at least 5 points at 18 months from the start of administration. In another embodiment, the method of the present invention results in stabilization of mNIS+7 at 18 months from the start of treatment with the RNAi agent provided herein. In yet another embodiment, the method slows the increase in the mNIS+7 score compared to a suitable control group representing the natural course of the disease, e.g., Adams et al., N Engl J Med 2018;379:11-21, e.g., a placebo control group. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the disease severity at the start of treatment, the duration of treatment, previous treatments, and any specific TTR mutations present.

[0108] In other embodiments, the method of the present invention improves at least one measure of quality of life in a subject. “Improvement of at least one measure of quality of life” in a subject means the ability of the method of the present invention to slow or halt quality of life deterioration or to improve quality of life. Any appropriate measure of quality of life can be used to determine whether quality of life deterioration in a subject has slowed or halted 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 status, vitality (energy and fatigue), social functioning, role limitations due to emotional problems, and mental health (mental distress and mental well-being). Each scale is directly converted to a 0-100 scale, assuming equal weight for each question. A lower score indicates greater impairment, while a higher score indicates less impairment; that is, a score of 0 is equivalent to the highest level of impairment, and a score of 100 is equivalent to no impairment. The survey also provides a physical summary score and a mental summary score.

[0110] In one embodiment, the method of the present invention provides an improvement over baseline in at least one SF-36 physical health-related parameter (physical health, role-physical, bodily pain, or general health status) or at least one SF-36 mental health-related parameter (vitality, social functioning, role-emotion, or mental health) in a subject. Such improvement may take the form of an increase of, for example, at least 2 or at least 3 points on any of the scales of one or more parameters at 9 months from the start of medication.

[0111] In other embodiments, the method of the present invention halts the decrease of any one or more parameters of the SF-36 parameter score at 9 months from the start of treatment, for example, the method does not result in a clinically significant change in SF-36, for example, within the variability of observations of individuals undergoing SF-36 assessment. In yet another embodiment, the method of the present invention slows the rate at which the SF-36 score decreases at 9 months from the start of treatment, for example, the rate at which the SF-36 score of subjects treated with the RNAi agent of the present invention decreases compared to a suitable control group showing the natural course of the disease, for example, provided in Adams et al., N Engl J Med 2018;379:11-21, for example, a placebo control group. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the disease severity at the start of treatment of the subject, the duration of treatment, previous treatments, and any specific TTR mutations present.

[0112] Another appropriate 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 fibril, small fibril, and autonomic neuropathy that are not captured by existing devices.

[0113] In one embodiment, the method of the present invention improves the subject's Norfolk QOL-DN score from baseline, for example, a change of approximately -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 another embodiment, the method halts the increase in the Norfolk QOL-DN score, for example, the method does not result in a clinically significant change in the Norfolk QOL-DN score, for example, within the variability of observations of individuals performing the QOL-DN assessment. In yet another embodiment, the method of the present invention slows the rate of increase in the subject's QOL-DN score treated with the RNAi agent of the present invention compared to the rate of increase in a suitable control group showing the natural course of the disease, for example, provided in Adams et al., N Engl J Med 2018;379:11-21, for example, a placebo control group. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the subject's disease severity at initiation of treatment, duration of treatment, previous treatments, and any specific TTR mutations present.

[0114] Another appropriate measure of quality of life is exercise intensity, measured by the NIS-W score. The NIS-W score is a composite score that sums the muscle weakness of the head, torso, and limbs. Using NIS(W) (see section on the scale for measuring weakness), muscle strength is assessed using intermediate grades, with normal (0) or complete paralysis (4); 1 for 25% muscle weakness as determined by clinical strength testing; 2 for 50% weakness; 3 for 75% weakness; 3.25 for movement against gravity; 3.50 for movement without gravity; and 3.75 for muscle flicker.

[0115] In one embodiment, the method of the present invention provides a subject with an improvement in the baseline NIS-W score. Such improvement may take the form of a decrease of at least 5, 6, 7, 8, 9, or 10 points in the subject's NIS-W score 18 months after initiation of treatment with the RNAi agent provided herein. In other embodiments, the method halts the decrease in the NIS-W score, for example, the method results in no clinically significant increase or slowing of the NIS-W score compared to an appropriate control group demonstrating the natural course of the disease, e.g., Adams et al., N Engl J Med 2018;379:11-21, e.g., a placebo control group. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the subject's disease severity at initiation of treatment, duration of treatment, prior treatments, and any specific TTR mutations present.

[0116] Another suitable indicator of quality of life is the Rush Construction Global Disability Scale (R-ODS), a patient questionnaire designed to capture limitations in activity and social engagement in patients. In one embodiment, the method of the present invention provides an improvement in the R-ODS score relative to baseline in a subject. Such improvement may 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 the RNAi agent provided herein. In other embodiments, the method halts the decrease in the R-ODS score; for example, the method does not result in a clinically significant decrease in the R-ODS score 18 months after initiation of treatment with the RNAi agent provided herein. In yet another embodiment, the method of the present invention slows the rate of decrease in the R-ODS score in subjects treated with the RNAi agent of the present invention, 18 months after the initiation of treatment with the RNAi agent provided herein, compared to the rate of decrease in the R-ODS score in a suitable control group showing the natural course of the disease, for example, provided in Adams et al., N Engl J Med 2018;379:11-21, for example, a placebo control group. It is understood that the rate of disease progression depends on several factors, including, but not limited to, the disease severity at the initiation of treatment in the subject, the duration of treatment, previous treatments, and any specific TTR mutations present.

[0117] The Composite Autonomic Symptom Score (COMPASS-31), a patient questionnaire that assesses symptoms of autonomic dysfunction and provides a symptom score from 0 to 100, is another suitable indicator of quality of life. In one embodiment, the method of the present invention provides a subject with an improvement in the COMPASS-31 score relative to baseline. Such improvement may 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 the RNAi agent provided herein. In another embodiment, the method halts the decrease in the COMPASS-31 score; for example, the method does not result in a clinically relevant change in the COMPASS-31 score 18 months after initiation of treatment with the RNAi agent provided herein. In yet another embodiment, the method of the present invention slows the rate of decline in the COMPASS-31 score of subjects treated with the RNAi agent of the present invention compared to the rate of decline in the COMPASS-31 score of a suitable control group showing the natural course of the disease, for example, a placebo control group provided in Adams et al., N Engl J Med 2018;379:11-21, 18 months after the 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 disease severity at the initiation of treatment, the duration of treatment, previous treatments, and any specific TTR mutations present.

[0118] Other quality of life indicators may include nutritional status (e.g., assessed by changes in central body mass index (mBMI)). In one embodiment, the method of the present invention provides a subject with an improvement in baseline mBMI. Such improvement may take the form of an mBMI score of at least 2, 3, 4, 5 or more months after initiation of treatment with the RNAi agent provided herein. In another embodiment, the method halts the decline in the mBMI index score; for example, the method does not result in a clinically significant change in the mBMI score after 18 months after initiation of treatment with the RNAi agent provided herein. In yet another embodiment, the method of the present invention slows the rate of decline in the mBMI score in subjects treated with the RNAi agent of the present invention compared to the rate of decline in the mBMI score in a suitable control group showing the natural course of the disease, e.g., Adams et al., N Engl J Med 2018;379:11-21, e.g., a placebo control group, after 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 disease severity at the start of treatment, the duration of treatment, previous treatments, and any specific TTR mutations present.

[0119] Another quality of life indicator is the assessment of exercise capacity. One suitable measure of exercise capacity is the 6-minute walk test (6MWT), which measures how far a subject can walk in 6 minutes, i.e., the 6-minute walk distance (6MWD). In one embodiment, the method of the present invention provides a subject with an improvement of at least 10 meters, e.g., at least 10 meters, 15 meters, 20 meters, or about 30 meters, in 6MWD 18 months after the start of treatment with the RNAi agent provided herein, compared to baseline.

[0120] Another suitable measure is the 10-meter walk test, which measures walking speed. In one embodiment, the method of the present invention provides a subject with an improvement of at least 0.025 meters / second from baseline in the 10-meter walk test, 18 months after the start of treatment with the RNAi agent provided herein, 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, in 10-meter walk tests.

[0121] In some embodiments, changes in plasma biomarker levels are indicators of reduced progression of progressive nerve damage or polyneuropathy in ATTR amyloidosis. For example, a decrease in nerve filament light chain (NfL) levels at 9 months compared to NfL levels at the start of treatment may be an indicator of reduced progression of progressive nerve damage or polyneuropathy in ATTR amyloidosis. In some embodiments, decreases in other proteins, particularly RSPO3, CCDC80, EDA2R, and NT-proBNP levels, at 9 months after the start of treatment, either alone or in combination with a decrease in NfL levels, compared to their corresponding levels at the start of treatment, may be indicators of reduced progression of progressive nerve damage or polyneuropathy in ATTR amyloidosis. In some embodiments, an increase in N-CDase levels, at 9 months after the start of treatment, either alone or in combination with the other markers mentioned above, compared to their corresponding levels at the start of treatment, may be an indicator of reduced progression of progressive nerve damage or polyneuropathy in ATTR amyloidosis. Table 1 provides further biomarkers that may serve as indicators of the reduction in nerve damage or polyneuropathy in ATTR amyloidosis, such as at 9 months after the initiation of RNAi therapy. 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., greater than the intrinsic variability of the assay.

[0122] In one embodiment, the method of the present invention provides improvements in cardiovascular indicators, such as an increase in the Kansas City Cardiomyopathy Questionnaire overall summary (KCCQ-OS), a decrease in left ventricular (LV) wall thickness as assessed by echocardiography compared to baseline, a decrease in longitudinal global strain as assessed by echocardiography compared to baseline, a decrease in N-terminal prohormone type B natriuretic peptide (NTproBNP) compared to baseline, and a decrease in hospitalizations due to cardiac events.

[0123] The methods of the present invention may also improve the prognosis of the treated patients. For example, the methods of the present invention may provide subjects with a reduced probability of clinical exacerbation events during the treatment period or an extended lifespan or reduced hospitalizations compared to a suitable control group that demonstrates the natural course of the disease, e.g., provided in Adams et al., N Engl J Med 2018;379:11-21, e.g., a placebo control group. In one embodiment, the reduced probability of clinical exacerbation events during treatment may include a reduction in all-cause mortality or cardiovascular-related hospitalization rates, e.g., as assessed by the Finkelstein-Schoenfeld method, compared to a suitable control group, e.g., 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 disease severity of the subject at the start of treatment, the duration of treatment, previous treatments, and any specific TTR mutations present.

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

[0125] In one embodiment, the iRNA agent of the present invention is administered to a subject in a "fixed dose" (e.g., mg dose), meaning that one dose of the iRNA agent is used for all subjects, regardless of any specific subject-related factors such as body weight.

[0126] In one embodiment, the RNAi agent is administered in a fixed dose of approximately 25 mg to approximately 1000 mg, for example, approximately 25 mg, approximately 50 mg, approximately 100 mg, approximately 200 mg, approximately 300 mg, approximately 400 mg, approximately 500 mg, approximately 600 mg, approximately 700 mg, approximately 800 mg, approximately 900 mg, or approximately 1000 mg.

[0127] In one embodiment, the double-stranded RNAi agent is administered to human subjects approximately once every quarter to approximately once a year. In another embodiment, the double-stranded RNAi agent is administered to human subjects approximately once every quarter, approximately once every six months, or approximately once a year.

[0128] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg to approximately 300 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg to approximately 200 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 75 mg to approximately 200 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 25 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 50 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 75 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 100 mg. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 200 mg. In one embodiment, the double-stranded RNAi agent is administered to human subjects in fixed doses of approximately 25 mg to 300 mg; approximately 25 mg to 200 mg; approximately 75 mg to 200 mg; approximately 25 mg; approximately 50 mg; approximately 100 mg; approximately 200 mg; or approximately 300 mg once quarter, i.e., once every three months.

[0129] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg to approximately 600 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg or approximately 600 mg, approximately once every six months to approximately once a year. In yet another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 400 mg or approximately 600 mg, approximately once every six months or approximately once a year.

[0130] In one embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 700 mg to approximately 1000 mg or approximately 700 mg to approximately 900 mg. In another embodiment, the double-stranded RNAi agent is administered to human subjects in a fixed dose of approximately 700 mg, approximately 800 mg, approximately 900 mg, or approximately 1000 mg approximately once a year.

[0131] In one embodiment, administration is subcutaneous, e.g., self-administration, via, for example, a pre-filled syringe or an auto-injection syringe. In one embodiment, the dose of the RNAi agent for subcutaneous administration is contained in, for example, a pharmaceutically acceptable carrier in a volume of 1 ml or less.

[0132] Any of these schedules may be repeated in one or more iterations as desired. The number of iterations may depend on the desired effect, e.g., suppression of the TTR gene, achievement of retinol-binding protein levels, vitamin A levels, or therapeutic effect, e.g., reduction of amyloid deposition or reduction of TTR-related disease symptoms. In one embodiment, the iRNA agent may be administered chronically for an indefinite period, e.g., over the patient's lifetime.

[0133] In one embodiment, RNAi agents may be administered in conjunction with other therapeutic agents or other therapeutic regimens. For example, other agents or other therapeutic regimens suitable for treating TTR-related diseases may include liver transplantation, heart transplantation, pacemaker implantation, agents that can reduce monomeric TTR levels in the body; tafamidis (Vyndaqel® or Vyndamax®) or AG10, which kinetically stabilize TTR tetramers by inhibiting tetramer dissociation necessary for TTR amyloid formation; nonsteroidal anti-inflammatory drugs (NSAIDS), such as diflunisal, and diuretics, for example, which can be used to reduce edema in TTR amyloidosis with cardiac complications.

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

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

[0136] The term "inhibition" as used herein is interchangeable with "reduction," "silencing," "downcontrol," "suppression," and other similar terms, and includes all levels of inhibition. In some embodiments, inhibition includes statistically significant or clinically significant inhibition.

[0137] The term "inhibition of TTR expression" is intended to refer to the inhibition of expression of any TTR gene, including variants or mutants of the TTR gene. Therefore, the TTR gene can be the wild-type TTR gene or a mutant TTR gene (e.g., amyloid-depositing mutant TTR gene).

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

[0139] Inhibition of TTR gene expression may manifest as a decrease in the amount of mRNA expressed by a first cell or cell group (such cells may be present, for example, in a sample derived from the subject) that is substantially the same as that of the first cell or cell group, but is not treated, compared to an untreated second cell or cell group (control cells), after the TTR gene has been transcribed and treated to inhibit TTR gene expression (e.g., by contact of one or more cells with the RNAi agent of the present invention or by administration of the RNAi agent of the present invention to a subject in which cells are present or have been present). In one embodiment, percentage inhibition is evaluated by expressing the mRNA level in treated cells as a percentage of the mRNA level in control cells, using the following formula:

number

[0140] A similar calculation can be performed, for example, using the serum TTR protein concentration in a blood sample obtained from the subject to determine the percentage inhibition of expression. If TTR is not detected in the post-treatment serum or plasma sample, the amount of TTR present is considered the detection limit of the assay used.

[0141] In one embodiment, the percentage inhibition is determined using a validated and clinically acceptable method.

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

[0143] III. iRNA of the present invention Suitable iRNAs for use in the methods of the present invention include double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the TTR gene in cells, such as cells in a mammal, such as a human with a TTR-related disease. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed by the expression of the TTR gene. The complementary region 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 the expression of the TTR gene (e.g., human, non-human primate, or non-primate mammalian TTR gene) by at least about 70% when Hep3B cells are transfected with 10 nM of the iRNA agent using the method provided herein and assayed by real-time PCR using the method provided in Example 4 of WO2013075035.

[0144] dsRNA is complementary and contains two RNA strands that hybridize to form a double-stranded structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a complementary region that is substantially complementary and generally fully complementary to the target sequence. The target sequence may be derived from the mRNA sequence formed during the expression of the TTR gene. The other strand (the sense strand) contains a region that is complementary to the antisense strand such that, when combined under appropriate conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere in this specification and as is known in the art, the complementary sequence of the dsRNA may be contained as a self-complementary region of a single nucleic acid molecule, rather than being found in separate oligonucleotides.

[0145] Generally, the double-strand structure is 21–30 base pairs long. Similarly, the regions complementary to the target sequence are 22 and 30 nucleotides long.

[0146] dsRNA can be synthesized by standard methods known in this field, as described below.

[0147] The iRNA compounds of the present invention can be produced using a two-step method. First, the individual strands of a double-stranded RNA molecule are prepared separately. Then, the elemental strands are annealed. The individual strands of the siRNA compound can be produced using solution-phase or solid-phase organic synthesis or both. Organic synthesis has the advantage of easily producing oligonucleotide chains containing non-natural 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 iRNA of the present invention The iRNA agents used in the methods of the present invention include defined chemical modifications in the sense and antisense strands. When either strand is elongated to provide an antisense strand longer than 23 nucleotides and a sense strand longer than 21 nucleotides, the nucleotides may include modifications including, but not limited to, sugar modifications, backchain modifications, and base modifications. Modifications include, for example, terminal modifications, e.g., 5'-terminal modifications (phosphorylation, conjugation, inverse bond) or 3'-terminal modifications (conjugation, DNA nucleotide, inverse bond, etc.); base modifications, e.g., substitution with a stabilizing base, destabilizing base, or a base that base pairs with an expanded repertoire of partners, base removal (debasing nucleotide), or conjugate base; sugar modifications (e.g., 2' or 4' position) or sugar substitution; or backchain modifications including modification or substitution of phosphodiester bonds. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that include a modified backchain or RNAs that do not include native nucleoside bonds. RNAs having a modified backchain include, among other things, those that do not have a phosphorus atom in the backchain. For the purposes of this specification and as sometimes referred to in the literature, modified RNAs that do not have a phosphorus atom in the internucleoside backbone can also be considered oligonucleosides. In one embodiment, the modified iRNA has a phosphorus atom in the internucleoside backbone.

[0149] Modified RNA backbone includes, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoamides and aminoalkyl phosphoramidates, thionophosphoamides, thionoalkyl phosphonates, thionoalkyl phosphotryesters and boranophosphates with normal 3'-5' binding, their 2'-5' binding analogs, and those with inverted polarity where adjacent pairs of nucleoside units are bound from 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 backchains that do not contain phosphorus atoms have backchains formed by short alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short heteroatoms or heterocyclic nucleoside bonds. These include morpholino bonds (partially formed from the sugar moiety of nucleosides); siloxane backchains; sulfide, sulfoxide, and sulfone backchains; formacetyl and thioformacetyl backchains; methyleneformacetyl and thioformacetyl backchains; alkene-containing backchains; sulfamate backchains; methyleneimino and methylenehydrazino backchains; sulfonate and sulfonamide backchains; amide backchains; and others having mixed N, O, S, and CH2 elemental moieties.

[0151] In other embodiments, suitable RNA mimetic is intended for use in iRNA, where the sugar and nucleoside bonds of the nucleotide units, i.e., both of the main chains, are replaced with novel groups. The base units are maintained for hybridization with suitable nucleic acid target compounds. Such oligomeric compounds, which are RNA mimetic that have been shown to have excellent hybridization properties, are called peptide nucleic acids (PNAs). In PNA compounds, the sugar main chain of RNA is replaced with an amide-containing main chain, particularly an aminoethylglycine main chain. The nucleic acid bases are maintained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the main chain.

[0152] One embodiment of the present invention includes RNA having a phosphorothioate backbone and oligonucleosides having heteroatom backbones and, in particular, the --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- and the amide backbone of the above-referenced U.S. Patent 5,602,240. In one embodiment, the RNA of interest herein has the morpholino backbone structure of the above-referenced U.S. Patent 5,034,506. Natural phosphodiester backbones can be represented as OP(O)(OH)-OCH2-.

[0153] Modified RNA may also contain one or more substituted sugar moieties. The iRNA of interest here, for example dsRNA, has OH at the 2' position;F;O-, S- or N-alkyl;O-, S- or N-alkenyl;O-, S- or N-alkynyl;or O-alkyl-O-alkyl (where alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C). 10 Alkyl or C2-C 10 It may contain one of the alkenyls and alkinyls. 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 has C1-C at the 2'-position 10 Lower alkyl, substituted lower alkyl, aralkyl, aralkyl, O-aralkyl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloaralkyl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group that improves the pharmacokinetic properties of the iRNA or a group that improves the pharmacodynamic properties of the iRNA and one of other substituents having similar properties. In certain embodiments, the modification is 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-MOE, 2'-O--CH2CH2OCH3) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), that is, it contains an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE as described in the examples below, that is, the O(CH2)2ON(CH3)2 group and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), that is, it contains 2'-O--CH2--O--CH2--N(CH3)2. 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'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also occur at other positions on the iRNA RNA, particularly at the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide of 2'-5' linked dsRNA. iRNA may also have sugar mimetic molecules such as cyclobutyl moieties instead of pentofuranosyl sugars.

[0155] The RNA of the iRNA of the present invention may also include nucleic acid base (often simply referred to as "bases" in the art) modifications or substitutions. The "unmodified" or "natural" nucleic acid bases used herein include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases 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. This includes other synthetic and native nucleic acid bases such as (pseudracil), 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 nucleic acid bases are particularly useful for increasing the binding affinity of the oligomeric compounds of interest in this 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 double-strand stability at 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 particularly exemplary base substitutions when combined with 2'-O-methoxyethyl sugar modifications.

[0156] The RNAi agent of the present invention may also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by 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 bridge-formed ring, which includes a bridge connecting two carbon atoms of adjacent or non-adjacent sugar rings, thereby forming a bicyclic ring system. In some embodiments, the bridge optionally connects the 4'-carbon and 2'-carbon of the sugar ring via a 2'-acyclic oxygen atom. Therefore, in some embodiments, the agent of the present invention may include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having 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 including a 4'-CH2-O-2' bridge. This structure efficiently "locks" ribose into the 3'-end conformation. The addition of locked nucleic acids to siRNA is thought to increase serum siRNA stability 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 polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between 4' and 2' ribosyl ring atoms. In one embodiment, the antisense polynucleotide agent of the present invention comprises one or more bicyclic nucleosides containing a 4'→2' bridge.

[0157] Locked nucleosides have a structure (stereochemistry is omitted). [ka] This is represented by , where B is a nucleic acid base or modified nucleic acid base, and L is a linking group that bonds 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 known as "restricted ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogues; see, for example, U.S. Patent 7,399,845); 4 '-C(CH3)(CH3)-O-2' (and its analogs; see, e.g., US Patents 8,278,283); 4'-CH2-N(OCH3)-2' (and its analogs; see, e.g., US Patents 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., US Patent Publication 2004 / 0171570); 4'-CH2-N(R)-O-2' (where R is H, C1-C 12 This includes, but is not limited to, alkyl or nitrogen protecting groups (see, for example, U.S. Patent 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, for example, Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and its analogues; see, for example, U.S. Patent 8,278,426). The entirety of each of the foregoing is incorporated herein by reference.

[0159] Further representative US patents and publications teaching the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: US 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 these are incorporated herein by reference).

[0160] Any of the aforementioned bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, for example, including α-L-ribofuranose and β-D-ribofuranose (see WO99 / 14226).

[0161] The RNAi agent of the present invention may also be modified to include one or more restricted ethyl nucleotides. The “restricted ethyl nucleotide” or “cEt” used herein is a locked nucleic acid containing a bicyclic sugar moiety with a 4'-CH(CH3)-O-2' bridge (i.e., L in the above-described structure). In one embodiment, the restricted ethyl nucleotide is the S conformation referred to herein as “S-cEt”.

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

[0163] One or more nucleotides of the iRNA of the present invention may include hydroxymethyl-substituted nucleotides. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also known as an "unlocked nucleic acid" ("UNA") modification.

[0164] Modifications that may stabilize the ends of RNA molecules may 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-C6amino), 2-docosanoyluridine-3'-phosphate, inverted base dT (idT), and others. Disclosure of these modifications can be found in WO2011 / 005861.

[0165] Other nucleotide modifications of the iRNA of the present invention include the 5' phosphate or 5' phosphate mimetic of the antisense strand of the RNAi agent, e.g., the 5' terminal phosphate or phosphate mimetic. Suitable phosphate mimetics can be found, for example, in U.S. Patent Publication 2012 / 0157511 (the entire contents of which are incorporated herein by reference).

[0166] V. iRNA conjugated to a ligand Other modifications of the iRNA of the present invention include chemical linkage to the RNA of one or more ligands, portions, or conjugates that enhance the activity, cell distribution, or cell uptake of the iRNA. These parts include lipid portions, such as cholesterol (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, such as 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), and thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), aliphatic chains, e.g., 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, e.g., di-hexadecyl-rac-glycerol or triethylammonium 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 chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973) or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), palmityl moiety (Mishra et al., Biochim.This includes, but is not limited to, octadecylamine or hexylamino-carbonyloxycholesterol moieties (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 alters the distribution, targeting, or lifespan of the incorporated iRNA agent. In some embodiments, the ligand provides enhanced affinity to selected targets, e.g., molecules, cells or cell types, compartments, e.g., cellular or organ compartments, tissues, organs, or regions within the body, compared to species lacking such ligands. Examples of ligands do not participate in the double-strand pairing of double-stranded nucleic acids.

[0168] Ligands may also include targeting groups, such as cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins that bind to specific cell types, such as kidney cells, or antibodies. Targeting groups can be thyroid-stimulating hormone, melanocyte-stimulating hormone, lectins, glycoproteins, surfactants, protein A, mucin carbohydrates, polyvalent lactose, monovalent galactose, N-acetyl-galactosamine, N-acetyl-glucoseamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimetic compounds. In some embodiments, the ligand comprises monovalent or polyvalent galactose. In some embodiments, the ligand comprises cholesterol.

[0169] The ligand-conjugated oligonucleotides of the present invention can be synthesized using oligonucleotides having pendant-reactive functional groups, such as those derived from the binding of a linking molecule to the oligonucleotide (see below). These reactive oligonucleotides can be directly reacted with commercially available ligands, synthetic ligands having a variety of protecting groups, or ligands to which the linking portion is bound.

[0170] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely produced by well-known solid-phase synthesis techniques. Any other means of such synthesis known in the art may be used in addition to or separately from these. The use of similar techniques for preparing 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 and in vivo treatment of nucleic acids, as described herein. As used herein, “carbohydrate” means a compound that is a carbohydrate itself, consisting of one or more monosaccharide units 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; or a compound that has as part a carbohydrate moiety consisting of 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. Typical 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. Certain monosaccharides contain one or more C5 (e.g., C5, C6, C7, or C8) sugars; disaccharides and trisaccharides contain two or three monosaccharide units (e.g., C5, C6, C7, or C8) sugars.

[0172] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is selected from the group consisting of the following: [ka] [ka] [ka] [ka] [ka]

[0173] For one reason, monosaccharides are, [ka] These include N-acetylgalactosamine.

[0174] Other representative carbohydrate conjugates for use in the embodiments described herein are: [ka] (In the formula, if either X or Y is an oligonucleotide, the other is hydrogen.) This includes, but is not limited to, the following:

[0175] In one embodiment of the present invention, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a monovalent linker. In another embodiment, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a divalent linker. In yet another embodiment of the present invention, GalNAc or a GalNAc derivative binds to the iRNA agent of the present invention via a trivalent linker.

[0176] In one embodiment, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative bound to an iRNA agent. In another embodiment, the double-stranded RNAi agent of the present invention comprises multiple (e.g., two, three, four, five, or six) GalNAc or GalNAc derivatives, each independently bound to multiple nucleotides of the double-stranded RNAi agent via multiple monovalent linkers.

[0177] In one embodiment, for example, the two strands of the iRNA agent of the present invention are part of a single large molecule connected by an uninterrupted strand of nucleotides between the 3' end of one strand and the 5' end of the other strand, forming a hairpin loop containing multiple unpaired nucleotides, each unpaired nucleotide in the hairpin loop may contain GalNAc or a GalNAc derivative, independently linked via a monovalent linker. The hairpin loop may also be formed by an elongation overhang of one of the strands of the double helix.

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

[0179] Further carbohydrate conjugates suitable for use in the present invention include those described in PCT Publications WO2014 / 179620 and WO2014 / 179627 (each of which is incorporated herein by reference in its entirety).

[0180] B. Linker In one embodiment, the conjugates or ligands described herein may be conjugated to iRNA oligonucleotides using various linkers that may be cleavable or non-cleavable.

[0181] The term "linker" or "linking group" refers to an organic part of a compound that connects two points, for example, two points of a compound that are covalently bonded together. Linkers are typically directly bonded to atoms, units such as oxygen or sulfur, e.g., NR8, C(O), C(O)NH, SO, SO2, SO2NH, or 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, alkenylheteroarylalkyl, alkenylheteroarylalkyl The following are chains of atoms, including but not limited to: yl heteroaryl alkenyls, alkenyl heteroaryl alkynyls, alkynyl heteroaryl alkyls, alkynyl heteroaryl alkenyls, alkynyl heteroaryl alkynyls, alkyl heterocyclyl alkyls, alkyl heterocyclyl alkenyls, alkyl heterocyclyl alkynyls, alkenyl heterocyclyl alkyls, alkenyl heterocyclyl alkenyls, alkenyl heterocyclyl alkynyls, alkynyl heterocyclyl alkyls, alkynyl heterocyclyl alkenyls, alkynyl heterocyclyl alkynyls, alkylaryls, alkenylaryls, alkynylaryls, alkylheteroaryls, alkenyl heteroaryls, and alkynyl heteroaryls (one or more methylene atoms may be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryls, substituted or unsubstituted heteroaryls, or substituted or unsubstituted heterocyclic structures); where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic.In one embodiment, the linker consists of approximately 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

[0182] The cleavable linking group is sufficiently stable outside the cell, but upon entering the target cell, it cleaves, releasing the two parts that the linker holds together. In one embodiment, the cleavable linking group is cleaved at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 or more, or at least about 100 times faster, than in the target cell or under first control conditions (which may be selected to mimic or represent intracellular conditions, for example) or under the target blood or second control conditions (which may be selected to mimic or represent conditions found in blood or serum).

[0183] Cleavable linkers are sensitive to cleavage factors, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage factors are more dominant or found at higher levels or activity within cells than in serum or blood. Examples of such degradable factors include, for example, cellular redox factors such as mercaptans that can degrade redox cleavable linkers by oxidation or reduction; redox factors that are selective for a particular substrate or lack substrate specificity; esterases; factors that can create an acidic environment, such as endosomes or those resulting in a pH of 5 or less; and enzymes, including peptidases (which may be substrate-specific) and phosphatases, that can hydrolyze or degrade acid-cleavable linkers by acting as general acids.

[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 approximately 7.1 to 7.3. Endosomes are more acidic, ranging from 5.5 to 6.0, and lysosomes are even more acidic, at approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a selected pH, thereby releasing cationic lipids from a ligand within the cell or into a desired compartment of the cell.

[0185] Linkers may contain cleavable linking groups that can be cleaved by specific enzymes. The type of cleavable linking group incorporated into a linker may depend on the target cell. For example, liver-targeting ligands may bind to cationic lipids via linkers containing ester groups. Hepatocytes are esterase-rich, and therefore linkers are cleaved more efficiently in hepatocytes than in cell types that are not esterase-rich. Other esterase-rich cell types include lung, renal cortical, and testicular cells.

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

[0187] Generally, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degrading factor (or condition) to cleave the candidate linker. It is also desirable to test the candidate cleavable linker's ability to withstand cleavage in the blood or in contact with other non-target tissues. Thus, the relative sensitivity to cleavage between first and second conditions (where the first is selected to be an indicator of cleavage in target cells, and the second is selected to be an indicator of cleavage in other tissues or fluids, e.g., blood or serum) can be determined. Evaluation can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform an initial evaluation in cell-free or culture conditions and then confirm it with evaluation in whole animals. In one embodiment, a useful candidate compound is cleaved at least about 2 times, 4 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or about 100 times 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 linking groups In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved by reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-SS-). The methods described herein can be employed to determine whether a candidate cleavable linking group is suitable for use as a suitable "reductively cleavable linking group" or, for example, with a specific iRNA moiety and a specific targeting agent. For example, a 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. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. In one example, a candidate compound is cleaved by up to about 10% in blood. In other embodiments, useful candidate compounds are degraded at least approximately 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or approximately 100 times 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 cleavage rate of candidate compounds can be determined using a standard enzyme reaction kinetics assay under conditions selected to mimic an intracellular medium and compared to conditions selected to mimic an extracellular medium.

[0189] ii. Phosphate-based cleavable linking groups In one embodiment, the cleavable linker includes a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by factors that degrade or hydrolyze the phosphate group. Examples of factors that cleave phosphate groups in cells include enzymes such as phosphatases in cells. Examples of phosphate-based linking groups include -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 (wherein Rk is independently C1-C in each case). 20 Alkyl, C1-C 20 Haloalkyl, C6-C 10 Aryl or C7-C 12 (It is aralkyl). Examples of 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 one embodiment, 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 one embodiment, a cleavable linker includes an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In one embodiment, an acid-cleavable linking group is cleaved by 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 a factor such as an enzyme that can act as a general acid. In cells, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may 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 linking group. The ester-based cleavable linking group is cleaved by enzymes such as cellular esterases and amidases. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene, and alkylylene groups. The ester-based cleavable linking group has 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 cleavage groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as cellular peptidases and proteases. The peptide-based cleavable linking group is a peptide bond formed between amino acids that yield oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not contain an amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids that yield peptides and proteins. The peptide-based cleavable group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids that yield peptides and proteins, and does not contain the entire amide functional group. The peptide-based cleavable linking group has the general formula -NHCHRAC(O)NHCHRBC(O)- (wherein RA and RB are the R groups of two adjacent amino acids). These candidates can be evaluated using methods similar to those described above.

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

[0194] In one embodiment of the present invention, the composition and method, the ligand, is one or more "GalNAc" (N-acetylgalactosamine) derivatives bound via a divalent or trivalent branched linker.

[0195] In one embodiment, the dsRNA of the present invention is conjugated to a bivalent or trivalent branched linker selected from the structural bases shown in any of formulas (XXXII) to (XXXV): [ka] [During the ceremony, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C are, in each case independently, between 0 and 20, where the iterative 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 In each case, these are independently absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O; Q 2A Q 2B Q 3A Q 3B Q 4A Q 4B Q 5A Q 5B Q 5C In each case independently, these are non-existent, alkylene, and substituted alkylene, where one or more methylene groups are O, S, S(O), SO2, and N(R). N ), C(R')=C(R''), C≡C or C(O) may be interrupted or terminated at one or more points; R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5CIn each case independently, non-existence, 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 heterocycline; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C is a ligand; that is, in each case independently, it is a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide; and R aは It is either H or an amino acid side chain. Formula (XXXVI): [ka] [In the formula, L 5A , L 5B and L 5C It is a monosaccharide such as a GalNAc derivative. Trivalent conjugate GalNAc derivatives such as these are particularly useful for use as RNAi agents to inhibit targeted gene expression.

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

[0197] Representative U.S. patents teaching 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;5,082,830;5,112,963;5 ,214,136;5,245,022;5,254,469;5,258,506;5,262,536;5,272,250;5,292,873;5,317,098;5,371,241, 5,391,723;5,416,203, 5,451,463;5,510,475;5,512,667;5,514,785;5,565,552;5,567,810;5,574,142;5, This includes, but is not limited to, 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 (each of which is incorporated herein by reference in its entirety).

[0198] It is not necessary for all positions of a compound to be uniformly modified; in fact, more than one of the above modifications can be incorporated into a single compound or a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0199] VI. Delivery of iRNA according to the present invention The delivery of the iRNA of the present invention to cells, for example, to cells within human subjects (e.g., subjects requiring treatment, such as subjects with diseases, disorders, or conditions associated with contact activation pathway gene expression), can be achieved by several different methods. For example, delivery can be achieved by contact between cells and the iRNA of the present invention in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition containing the iRNA, for example, dsRNA, to the subject. Delivery can be carried out, for example, by intravenous or subcutaneous administration. In one embodiment, the iRNA agent is delivered by subcutaneous administration. In one embodiment, the iRNA agent is administered by self-administration using a pre-filled syringe or an automated injection device.

[0200] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for the iRNA-based delivery of the present invention (see, for example, khtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO9402595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to be considered for iRNA molecule delivery include, for example, the biological stability of the delivery molecule, prevention of nonspecific effects, and accumulation of the delivery molecule in the target tissue.

[0201] VII. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations comprising the iRNA described herein for use in the methods of the present invention. In one embodiment, provided herein is a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier. The iRNA-containing pharmaceutical composition is useful for treating diseases or disorders associated with the expression or activity of TTR genes. Such pharmaceutical compositions are formulated based on a delivery method. One example is a composition formulated for systemic administration via non-enteral delivery, such as subcutaneous (SC) or intravenous (IV) delivery. The pharmaceutical compositions of the present invention can be administered in doses sufficient to inhibit the expression of TTR genes. In one embodiment, the iRNA agent of the present invention, for example, the dsRNA agent, is formulated for subcutaneous administration in a pharmaceutically acceptable carrier.

[0202] In other embodiments, a single dose of the pharmaceutical composition may be sustained so that subsequent administrations are at one-month intervals, or at most one-month, two-month, three-month, 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 once a month. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once every two months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered quarterly, i.e., 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 twelve months.

[0203] VIII. Kit The present invention also provides a kit for carrying out any of the methods of the present invention. Such a kit includes one or more double-stranded RNAi agents and a label providing instructions for the use of the double-stranded agents for use in any of the methods of the present invention. The kit may optionally further include means for bringing cells into contact with the RNAi agent (e.g., an injection device or infusion pump) or means for measuring TTR inhibition (e.g., means for measuring TTR mRNA or TTR protein inhibition). Such means for measuring TTR inhibition may include means for obtaining a sample from a subject, such as a plasma sample. The kit of the present invention may optionally further include means for administering the RNAi agent to a subject or means for determining a therapeutic or prophylactic dose.

[0204] RNAi agents may be provided in any conventional form, such as in a solution in sterile water or in other suitable solutions for resuspension and injection, e.g., in PBS, physiological saline, 5 mM phosphate buffer, etc. For example, RNAi agents may be provided as 300 mg, 200 mg, 100 mg, or 50 mg vials, together with water or other suitable solutions in sterile water for injection. In one embodiment, the RNAi agent is provided in a self-administration kit comprising a pre-filled syringe or automated injection device containing 300 mg, 200 mg, 100 mg, or 50 mg of the RNAi agent in an appropriate volume of additive for administration, and optionally further including instructions for use. In one embodiment, the RNAi agent is provided in multiple vials or devices for administration of multiple injection doses given at approximately the same time, e.g., within one week, within one day, or within one hour.

[0205] IX. Diagnosis and disease burden assessment of TTR amyloidosis polyneuropathy TTR amyloidosis is a complex, multifactorial disease. The list of criteria used to monitor TTR-FAP progression continues to grow: Neuropathy Dysfunction Score (NIS), NIS+7 and Modified NIS (mNIS)+7 and mNIS+7 IonisThese diagnostic criteria are well-known in this field, and the main parts of the criteria are provided below. Meeting the diagnostic criteria for TTR-FAP as used herein is understood to mean meeting the FAP Stage 1 criteria with or without the presence of hereditary TTR-FAP-associated mutations. Progression of neuropathy indicators is considered to be an increase of at least 2 points on the Adjusted Neuropathy Disorder Score (mNIS) +7.

[0206] Familial amyloid polyneuropathy (FAP) stage Coutinho et al. developed a clinical staging system for the neuropathic symptoms of hATTR (formerly known as familial amyloid 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 functional impairment in walking, lower limbs, trunk, and upper limbs with assistance. FAP Stage 3: Wheelchair-bound or bedridden, severe neuropathy. Subjects without neuropathy are considered to be FAP stage 0.

[0207] Neuropathic Dysfunction Scoring Method Methods for assessing neuropathy are known in this field. For example, in the Mayo Clinic Neurologic Examination Sheet and the neuropathic impairment score (NIS) weakness subscore, weakness (NIS-W) is scored separately from the major muscle groups on each side of the body, with a 25% decrease ranging 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. p. 1031-52). In particular, a wide range of cranial, proximal, and distal limb muscles are scored with the highest score of 192 points on the NIS-W. Reduced stretch reflexes in the five major muscles are usually assessed by a neurologist, and tactile pressure, vibration, joint movement, and needle-like sensation in the foot and hand are scored with a 25% decrease ranging from 1 to 4 on the Mayo Clinic Neurology Examination Sheet. For complete NIS of reflexes (NIS-R) and sensation (NIS-S), Mayo Clinic record scores are converted to NIS point scores (i.e., a Mayo Clinic score of 1 or 2 corresponds to an NIS point score of 1, and a Mayo Clinic score of 3 or 4 corresponds to an NIS score of 2). Therefore, the highest NIS score for normal reflexes (NIS-R) assessed by a neurologist is 5 × 2 × 2 = 20 points, and the highest score for the four modalities of sensation (NIS-S) often assessed by a neurologist is 8 × 2 × 2 = 32 points. Thus, the highest NIS score is 192 + 20 + 32 = 244 points. NIS is described in the aforementioned 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 co-primary endpoint scale in clinical trials for diabetic sensorimotor polyneuropathy, TTR FAP, and other generalized sensorimotor polyneuropathy (N. Suanprasert et al. J Neurol Sci 344 (2014) 121-128). The NIS+7 appropriately assesses the graded severity of muscle weakness and stretch reflex abnormalities with only minimal ceiling effect on reflexes. In the NIS+7, five of the seven tests are neurological characteristics - expressed as normal deviations (Z scores) or points. The characteristics included in the NIS+7 were selected to be sensitive to abnormalities in the detection of diabetic sensorimotor polyneuropathy (Dyck et al. Muscle Nerve 2003;27(2):202-10). The properties included are peroneal complex 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 percentile values ​​to normal deviations, correcting for applicable variables such as age, sex, height, or weight, based on previous studies of a large healthy control cohort. Furthermore, these percentile values ​​can be expressed as NIS points (i.e., N5th=0; ≤5th-N1st=1 and ≤1st=2 (and similarly, when the abnormality is above the normal distribution)).

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

[0210] The Adjusted Neuropathy Dysfunction Score +7 (mNIS+7) and the updated NIS+7 are composite scores that measure motor intensity, reflexes, sensation, nerve conduction, and autonomic nervous system function. These two versions of the composite scale have been adapted from NIS+7 to better reflect hATTR amyloidosis with polyneuropathy and are used as primary endpoints in the inotersen and patisiran clinical trials. Key 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)). In both scales, lower scores indicate better neurological function (e.g., higher scores reflect worsening neurological dysfunction).

[0211] Neuropathy Dysfunction Score Criteria [Table 1]

[0212] Diagnosis and assessment of disease burden of TTR amyloidosis cardiomyopathy (ATTR-CM) Patients with ATTR amyloidosis and cardiomyopathy typically experience progression of heart failure (HF) and cardiac arrhythmias and typically die 2.5–5 years after diagnosis. Extracellular matrix infiltration of the cardiac matrix by TTR amyloid fibrils leads to progressive increases in ventricular wall thickness and marked increases in ventricular stiffness, resulting in diastolic dysfunction. Systolic function is also impaired, typically reflected by abnormal long-axis strain despite normal ejection fraction being maintained into the later stages of the disease. In patients with ATTR amyloidosis and light chain (AL) cardiac amyloidosis, both long-axis strain and the 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 pre-specified in the statistical analysis plan include mean left ventricular (LV) wall thickness, LV mass, longitudinal strain and ejection fraction, cardiac output, left atrial size, terminal LV-diastolic volume (LVEDV), and terminal LV-systolic volume (LVESV). Echocardiography is routinely used for cardiac angiography. Myocardial strain can be assessed using speckle tracking with software from multiple vendors (TOMTEC, Munich, Germany). Analysis of NT-proBNP and troponin I levels is routinely performed in clinical laboratory settings 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 practices routinely include measuring creatinine levels and, for example, using dietary improvement test formulas in renal disease, estimating glomerular filtration rate (eGFR) based on creatinine levels.

[0214] A recent review by Witteles et al., 2019 (JACC: Heart Failure, 2019. 7:709-716) provides information on screening and diagnostic methods for ATTR-CM, including a list of “warnings” suggesting the presence of ATTR-CM, as well as screening methods including echocardiography, electrocardiogram recording, cardiac magnetic resonance, bilateral sensorimotor polyneuropathy beginning in the lower extremities and continuing in an ascending pattern, autonomic neuropathy in the form of orthostatic hypotension, diarrhea / constipation and erectile dysfunction, and ocular complications such as glaucoma, intravitreal deposition and scalloped pupils; and the presence of peripheral or autonomic systemic symptoms with 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 that localize to TTR cardiac amyloid deposition for unknown reasons. Biopsies are also used to confirm the presence of TTR amyloidosis in the heart.

[0215] The methods for evaluating and classifying cardiac function parameters provided above are known in this field. The specific methods for evaluating or classifying cardiac function used herein may be any clinically acceptable standards to indicate sufficiently impaired cardiac function, such as standard treatment including medical interventions, e.g., drug administration, surgery.

[0216] Serum biomarkers as indicators of nerve damage and TTR amyloidosis progression The diagnostic and monitoring methods described above are complex and often subjective. Furthermore, because TTR amyloidosis is rare and the above signs and symptoms can be present in several other diseases, clinically validated, non-invasive plasma biomarkers can 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 1000 proteins were measured in a cohort of patients with hATTR amyloidosis with polyneuropathy and healthy individuals who received placebo or patisiran in the Phase 3 APOLLO trial (NCT01960348). The effects of treatment with patisiran, a lipid-based RNAi agent that inhibits hepatic expression of TTR, on the time profile of each protein were determined at 0, 9, and 18 months using a linear mixed model. Neuronal filament light chain (NfL) proteins were further evaluated using an orthogonal quantitative approach. Significant changes in the levels of 66 proteins were observed with patisiran compared to placebo, with the most significant change being in NfL, a marker of neuronal damage. Analysis of protein level changes showed an enhanced tendency toward healthy individuals in patisiran-treated patients at 18 months. Plasma NfL levels in healthy controls were four times 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 in the placebo group (99.5 [SD 60.1] pg / mL) and decreased in the patisiran treatment group (48.8 [SD 29.9] pg / mL). At 18 months, improvement in the adjusted neuropathic dysfunction score +7 (mNIS+7) in patisiran-treated patients was significantly correlated with a decrease in NfL levels (R=0.43, p<10). -7) The decrease in NfL that correlates with the improvement of mNIS+7 in patisiran treatment suggests that it 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 the monitoring of disease progression.

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

[0218] Potential additional biomarkers that showed changes in response to patisiran treatment are listed in the table below. When the protein level with a positive beta coefficient for the subject increases compared to the control level, it is an indicator of the progression of ATTR amyloidosis, and when the protein level with a negative beta coefficient for the subject decreases compared to the control level, it is an indicator of the progression of ATTR amyloidosis. Changes in the levels of one or more of these markers may be indicators of improvement, stabilization, or decrease in the progression of ongoing nerve damage and polyneuropathy in ATTR amyloidosis.

[0219]

Table 2

[0220] The present invention will be further illustrated by the following embodiments, which should not be construed as limiting. All prior art referenced throughout this application, as well as published patents and patent applications and sequence listings, are incorporated herein by reference. [Examples]

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

[0222] [Table 3]

[0223] [Table 4]

[0224] Example 1: RNAi-mediated knockdown of TTR protein 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 in the study. Mice (n=3 / group) were administered a single subcutaneous dose of 1 mg / kg of either the RNAi agent AD-65492, previously disclosed in WO2018112320, or another RNAi agent based on the sequence and chemistry of AD-65492, with one chemical modification on the antisense strand changed to a GNA modification, as shown in Table 2 above. Blood samples were collected on day 0 (pre-administration), day 3, day 7, day 10, day 14, day 21, day 35, and day 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 at position 7 or 8 of the antisense strand was well-tolerated (AD-87404 and AD-87405). The reaction kinetics and peak protein knockdown were similar to those of the parent RNAi agent AD-65492. The persistence of knockdown by AD-87404 was similar to that of AD-65492. Incorporation of GNA at positions 3–6 of the antisense strand was not well-tolerated.

[0225] Example 2: RNAi-mediated knockdown of TTR protein in non-human primates The iRNA 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 from day 0-7 (7 days before administration) to 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 of AD-65492 and 3 mg / kg of AD-87404.

[0226] Example 3: Administration of a single dose of AD-87404 to healthy human subjects. In a Phase I, randomized, double-blind, placebo-controlled trial, AD-87404 (sense: 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6), where the L96 ligand is conjugated at the 3' end of the sense strand; antisense: 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7)) will be administered to healthy human volunteers in single doses of 25 mg, 75 mg, 100 mg, 200 mg, or 400 mg, along with, if possible, 600 mg, 700 mg, 900 mg, and 1000 mg dose groups. Relevant demographic characteristics, such as age, sex, and weight, will be balanced across groups. A demographically fit control group will also receive a single dose of placebo. Plasma samples are collected, and TTR protein levels in samples from the placebo group and all treatment groups are determined using ELISA assays at planned intervals, e.g., on days 1, 2, 3, 8, 15, 22, 29, 43, 57, and 90, and then every 28 days (up to approximately one year after maximum administration) until TTR levels recover to 80% of pre-treatment levels for the active treatment group (e.g., Coelho, et al. (2013) N Engl J Med 369:819). The level and duration of knockdown are then determined. Adverse events will be monitored in both the AD-87404 group and the control group. 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 findings, ECG, vital signs, or laboratory parameters, such as renal function, hematological parameters, and hepatic 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 sustainably knocks down TTR protein levels in a dose-dependent manner. Multiple dose studies and multiple ascending dose studies are also performed, along with monitoring of serum TTR protein knockdown and adverse events.

[0227] Equal parts: Those skilled in the art will recognize many equivalents of the specific embodiments and methods described herein, or can verify them through experiments not exceeding routine operations. Such equivalents are intended to be included within the scope of the following claims.

Claims

1. An RNAi agent comprising a sense strand and an antisense strand, wherein: Each of the sense strand and antisense strand is independently up to 30 nucleotides long; The sense strand contains the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), Here, 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 a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker. RNAi agent.

2. The RNAi agent according to claim 1, wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand.

3. The RNAi agent according to claim 2, wherein the ligand is one or more GalNAc derivatives to which a divalent or trivalent branched linker is bound.

4. Ligand 【Chemistry 1】 The RNAi agent according to claim 3.

5. An RNAi ligand according to any one of claims 2 to 4, wherein the ligand binds to the 3' end of the sense strand.

6. The double-stranded RNAi agent binds to the ligand as shown in the following formula: 【Chemistry 2】 [In the equation, X is either O or S.] , the RNAi agent according to claim 5.

7. An RNAi agent according to any one of claims 1 to 6, wherein the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

8. A method for treating human subjects with TTR-related disease, comprising administering to the subject a double-stranded RNAi agent containing a sense strand and an antisense strand in a fixed dose of 25 mg to 1000 mg, wherein: Each of the sense strand and antisense strand is independently up to 30 nucleotides long; The sense strand contains the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), Here, 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 a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker. use.

9. A method for inhibiting TTR expression in human subjects who do not meet the diagnostic criteria for TTR-related disease, comprising administering a double-stranded RNAi agent containing a sense strand and an antisense strand in a fixed dose of 25 mg to 1000 mg to the subject, wherein: Each sense strand and antisense strand is independently up to 30 nucleotides long; The sense strand contains the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); and The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7), Here, 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 a thymidine-glycol nucleic acid (GNA) S-isomer; and s is a phosphorothioate linker. use.

10. The use of claim 8 or 9, wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand.

11. The use of claim 10, wherein the ligand is one or more GalNAc derivatives bound via a divalent or trivalent branched linker.

12. Ligand 【Transformation 3】 The use of claim 11.

13. The use of any one of claims 9 to 12, wherein the ligand is bound to the 3' end of the sense chain.

14. The double-stranded RNAi agent binds to the ligand as shown in the following formula: 【Chemistry 4】 [In the equation, X is either O or S.] , use of claim 13.

15. The use of any one of claims 8 to 14, wherein the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long.

16. Use of any of claims 8 to 15, wherein the method includes improvement of at least one indicator of neurological impairment, quality of life, ongoing nerve damage, or cardiovascular dysfunction.

17. The use of claim 16, wherein the indicator is an indicator of neurological dysfunction.

18. The use of claim 17, wherein the neuropathic dysfunction index is the change from baseline in an index selected from the group of neuropathic dysfunction (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.

19. The use of claim 16, wherein the indicator is a quality of life indicator.

20. The use of claim 19, wherein the quality of life index is the change from baseline in an index selected from the group of SF-36® Health Survey score, Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score and Rush Construction Global Disability Scale (R-ODS) score.

21. Use of claim 16, wherein the indicator is ongoing nerve damage.

22. The use of claim 21, wherein the indicator of ongoing nerve damage is the change from baseline in plasma protein levels of one or more proteins selected from group nerve filament light chains (NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N-CDase.

23. The use of claim 21, wherein an indicator of ongoing nerve damage is a change in plasma levels of nerve filament light chain (NfL) protein.

24. The use of claim 16, wherein the indicator is an indicator of cardiovascular dysfunction.

25. Use of claim 24, wherein indicators of cardiovascular dysfunction are changes from baseline using the Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS) with cardiovascular hospitalization and an increase in scores indicating good health status, changes from baseline in mean left ventricular (LV) wall thickness as measured by echocardiography, changes from baseline in longitudinal global strain as measured by echocardiography, and changes from baseline in N-terminal prohormone type B natriuretic peptide (NTproBNP).

26. Use of any one of claims 8 to 25, wherein the human subject has a TTR gene mutation associated with the development of TTR-related disease.

27. The use of any of claims 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.

28. The use of any one of claims 8 to 25, wherein the human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis), and the method of using an RNAi agent reduces amyloid TTR deposition in the human subject.

29. Use of claim 28, wherein ATTR is hereditary ATTR (h-ATTR).

30. The use of claim 28, wherein the ATTR is a non-hereditary ATTR (wt ATTR).

31. The use of any one of claims 8 to 30, wherein the double-stranded RNAi agent is administered to human subjects by subcutaneous or intravenous administration.

32. The use of claim 31, wherein subcutaneous administration is self-administered.

33. The use of claim 32, wherein self-administration is via a pre-filled syringe or an autoinjector syringe.

34. The use of any one of claims 8 to 33 further comprises evaluating the level of TTR mRNA expression or TTR protein expression in a sample derived from a human subject.

35. The use of any of claims 8 to 34, wherein a double-stranded RNAi agent is administered to human subjects once every three months to once a year.

36. The use according to any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects approximately once every three months, once every six months, or once a year.

37. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 25 mg to 300 mg once every three months.

38. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 25 mg to 200 mg once every three months.

39. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 75 mg to 200 mg once every three months.

40. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 25 mg once every three months.

41. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 75 mg once every three months.

42. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 100 mg once every three months.

43. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 200 mg once every three months.

44. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 300 mg once every three months.

45. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 400 mg to 600 mg once every 6 months to once every 12 months.

46. The use according to any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 400 mg to 600 mg once every six months or once every twelve months.

47. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 400 mg or 600 mg once every six months or once every twelve months.

48. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 700 mg to 1000 mg once every 12 months.

49. The use of any one of claims 8 to 35, wherein a fixed dose of a double-stranded RNAi agent is administered to human subjects at a fixed dose of 700 mg, 800 mg, 900 mg, or 1000 mg once every 12 months.

50. Any use of claim 8 to 49 further comprises administering an additional therapeutic agent to a human subject.

51. The use of claim 50, wherein the further therapeutic agent is a TTR tetramer stabilizer or a nonsteroidal anti-inflammatory agent.

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