IL296106A - Compositions and methods for inhibiting expression of transthyretin (ttr) - Google Patents

Description

COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF TRANSTHYRETIN (TTR) Related Applications The present application claims the benefi tof priority to U.S. Provisional Application No. 62/985,950, filed on March 6, 2020, the entire contents of which are incorporat edherein by reference.

Sequence Listing The instant application contains a Sequence Listing which has been submitted electronica inlly ASCII forma tand is hereby incorporat edby reference in its entirety. Said ASCII copy, created on February 26, 2021, is named 121301_12320_SL.txt and is 44,455 bytes in size.

Background Transthyretin (TTR) (also known as prealbumin) is found in serum and cerebrospinal fluid (CSF). TTR transports retinol-bindi ngprotein (RBP) and thyroxine (T4) and also acts as a carrier of retinol (vitamin A) through its association with RBP in the blood and the CSF. Transthyretin is named for its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apoA-I (the major HDL apolipoprotein), amyloid -peptide, and neuropeptide Y. See Liz, M.A. et al. (2010) lUBMBLife, 62(6):429-435.

TTR is a tetramer of four identical 127-amino acid subunits (monomers) that are rich in beta sheet structure. Each monomer has two 4-stranded beta sheets and the shape of a prolate ellipsoi d.

Antiparall beta-sheetel interactions link monomers into dimers. A short loop from each monome formr s the main dimer-dimer interaction. These two pairs of loops separate the opposed, convex beta-sheet ofs the dimers to form an internal channel.

The liver is the major site of TTR expression. Other significant sites of expression include the choro idplexus, retina (particularl they retinal pigment epithelium )and pancreas.

Transthyretin is one of at leas 27t distinct types of proteins that is a precursor protein in the formation of amyloid fibrils. See Guan, J. et al. (Nov. 4, 2011) Current perspectives on cardiac amyloidosi Ams, J Physiol Heart Circ Physiol, doi: 10.1152/ajpheart.00815.2011. Extracellular deposition of amyloid fibrils in organs and tissues is the hallmark of amyloidosi Amyls. oid fibrils are compose dof misfolded protein aggregates ,which may result from either excess production of or specific mutations in precurso rproteins. The amyloidogenic potential of TTR may be related to its extensive beta sheet structure; X-ray crystallographi studiec s indicate that certain amyloidogenic mutations destabilize the tetrameric structure of the protein. See, e.g., Saraiva M.J.M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11.

Amyloidosi iss a general term for the group of amyloid diseases that are characterized by amyloid deposits. Amyloid diseases are classified based on their precursor protein; for example, the name starts with "A" for amyloid and is followed by an abbreviation of the precurso rprotein, e.g., ATTR for amloidogenic transthyretin. Ibid. 1 There are numerous TTR-associate dised ases ,most of which are amyloid diseases. Normal- sequenc eTTR is associated with cardiac amyloidos inis peopl ewho are elderly and is termed senil e systemic amyloido sis(SSA) (also called senile cardiac amyloido sis(SCA) or cardiac amyloidosis).

SSA often is accompanie byd microscopi depositsc in many othe rorgans. TTR amyloidosi manis fest s in various forms. When the peripheral nervous system is affected more prominentl y,the disease is termed familial amyloidoti polyc neuropat hy(FAP). When the heart is primaril yinvolved but the nervous system is not, the disease is called familial amyloidot cardiic omyopathy (FAC). A third major type of TTR amyloido sisis leptomeningea amyll oidosi alsos, known as leptomeningea orl meningocerebrovascul amyloar idosi cents, ral nervous system (CNS) amyloidosi ors, amyloidosi VIIs form. Mutation ins TTR may also cause amyloidotic vitreous opacitie s,carpal tunnel syndrome, and euthyroid hyperthyroxinemia, which is a non-amyloidoti diseasec thought to be secondary to an increased association of thyroxine with TTR due to a mutant TTR molecule with increased affinity for thyroxine. See, e.g., Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.

Abnorma TTRl alleles may be either inherited or acquired through somatic mutations. Guan, J. et al. (Nov. 4, 2011) Current perspectives on cardiac amyloidosi s,Am J Physiol Heart Circ Physiol, doi:10.1152/ajpheart.00815.2011. Transthyretin associated ATTR is the most frequent form of hereditary systemic amyloidosi s.Lobato, L. (2003) J. Nephrol., 16:438-442. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of ATTR. More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidos is.

TTR mutations usually give rise to systemic amyloi depositid on, with particul arinvolvement of the peripheral nervous system, although some mutations are associated with cardiomyopathy or vitreous opacities. Ibid.

The V30M mutation is the most prevalent TTR mutation. See, e.g., Lobato, L. (2003) J Nephrol, 16:438-442. The V1221 mutation is carried by 3.9% of the African American population and is the most common cause of FAC. Jacobson D.R., et al. (1997) N. Engl. J. Med. 336 (7): 466-73. It is estimated that SSA affect smore than 25% of the populatio overn age 80. Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87 (7): 2843-5.

Accordingly, there is a need in the art for effective treatments for TTR-associated diseases.

Summary of the Invention The present invention provides compositio nsand methods for inhibiting expression of TTR and methods of treating or preventing a transthyretin- (TTR-) associate diseased in a human subjec tusing double stranded RNAi agents ,targeting the TTR gene.

The invention provides a double stranded RNAi agent comprising a sense strand and an antisense strand ,wherein: eac hthe sense strand and the antisense strand are independentl upy to 30 nucleotides in length; the sense strand comprises the modified nucleotid sequence e5’- usgsggauUfuCfAfUfguaaccaa‘ga-3 (SEQ ID NO: 6); and 2 the antisense strand comprises the modified nucleotid sequee nce 5’- usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3’ (SEQ ID NO: 7), wherein a, c, g, andu are 2'-O-methyladenosine-3’-phosphate, 2'-O-methylcytidine-3’- phosphate, 2'-O-methylguanosine-3’-phosphate, and 2'-O-methyluridine-3’-phosphat respectie, vely; Af, Cf, Gf, and Uf are 2‘-fluoroadenosine-3‘ -phosphate, 2‘-fluorocytidine-3‘ -phosphate, 2’- fluoroguanosine-3’-phosphat e,and 2’-fluorouridine’-3-phosphat e,respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-Isomer; and s is a phosphorothioa linteker.

In certai nembodiments, the sense strand of the double stranded RNAi agent is conjugat edto at least one ligand. In certain embodiments, the ligand is one or more GalNAc derivatives attached through a bivalent or tri valent branched linker. In certain embodiments, the ligand is In certai nembodiments, the ligand is attache tod the 3' end of the sense strand.

In certai nembodiments, the double stranded RNAi agent is conjugat edto the ligand as shown in the followin schematg ic: 3' wherein X is O or S.

In certai nembodiments, the sense strand is 21 nucleotide ins lengt hand the antisense strand is 23 nucleotide ins length.

The invention provides a use of a double stranded RNAi agen tin a method of treating a human subject suffering from a TTR-associated disease, comprising administration of a fixed dose of about 25 mg to about 1000 mg of a double stranded RNAi agent, wherein: eac hthe sense strand and the antisense strand are independentl upy to 30 nucleotides in length; 3 the sense strand comprises the modified nucleotid sequence e5’- usgsggauUfuCfAfUfguaaccaaga-3’ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotid sequee nce 5’- usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3’ (SEQ ID NO: 7), wherein a, c, g, andu are 2'-O-methyladenosine-3’ -phosphate, 2'-O-methylcytidine-3’- phosphate, 2'-O-methylguanosine-3’ -phosphate, and 2'-O-methyluridine-3’-phosphat respectie, vely; Af, Cf, Of, and Uf are 2‘-fluoroadenosine-3‘ -phosphate, 2‘-fluorocytidine-3‘ -phosphate, 2’- fluoroguanosine-3’-phosphat e,and 2’-fluorouridine’-3-phosphat e,respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-Isomer; and s is a phosphorothioa linteker.

The invention also provides a use of a double stranded RNAi agen tin a method of inhibiting expression of TTR in a human subject who does not meet diagnosti cric teria of a TTR-associate d disease ,comprising administration of a fixed dose of about 25 mg to about 1000 mg of a double stranded RNAi agent ,wherein: eac hthe sense strand and the antisense strand are independentl upy to 30 nucleotides in length; the sense strand comprises the modified nucleotid sequence e5’- usgsggauUfuCfAfUfguaaccaaga-3‘ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotid sequee nce 5’- usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3’ (SEQ ID NO: 7), wherein a, c, g, andu are 2'-O-methyladenosine-3’-phosphate, 2'-O-methylcytidine-3’- phosphate, 2'-O-methylguanosine-3’ -phosphate, and 2'-O-methyluridine-3’-phosphat respectie, vely; Af, Cf, Gf, and Uf are 2‘-fluoroadenosine-3‘ -phosphate, 2‘-fluorocytidine-3‘ -phosphate, 2’- fluoroguanosine-3’-phosphat e,and 2’-fluorouridine’-3-phosphat e,respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-Isomer; and s is a phosphorothioa linteker.

In certai nembodiments, the sense strand of the double stranded RNAi agent is conjugat edto at least one ligand. In certain embodiments, the ligand is one or more GalNAc derivatives attached through a bivalent or tri valent branched linker. In certai nembodiments, the ligand is HO Z°H 4 In certai nembodiments, the ligand is attached to the 3' end of the sense strand. In certain embodiments, the double stranded RNAi agen tis conjugat edto the ligand as shown in the following schematic 3' wherein X is O or S.

In certai nembodiments, the sense strand is 21 nucleotide ins lengt hand the antisense strand is 23 nucleotide ins length.

In certain embodiments, the uses of the invention comprise improving at least one indici aof neurological impairement, qualit ofy life, nerve damage, cardiovascula health.r In certain embodiments, the indici aassessed is a neurologic impaal irment, for example, using a Neuropathy Impairment (NIS) score or a modified NIS (mNIS+7) score. In certai nembodiments, the indici ais a qualit ofy life indicia assessed, for example, using a SF-36® health survey score, a Norfolk Qualit yof Life-Diabetic Neuropath y(Norfolk QOL-DN) score, a NIS-W score, a Rasch-built Overall Disabilit yScale (R-ODS) score, a composit eautonom icsymptom score (COMPASS-31), a median body mass index (mBMI) score, a 6-minute wal ktest (6MWT) score, and a 10-meter walk test score. In certain embodiments, the indicia is nerve damage assessed, for example, a change in the level of one or more proteins selected from the group neurofilament light chai n(NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N- CDase, such as a human blood sample, or serum or plasm aderived therefrom. In certai nembodiments, the indici aof nerve damage is a change from baseline in the level of neurofilament light chai n(NfL) protein level In. certain embodiments, the indici aof cardiovascular impairment is cardiovascular hospitalizatio usinn, g Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS) with an increased score indicative of better healt stah tus, change from baseline in mean left !ventricular (LV) wall thickness by echocardiographic assessment ,change from baseline in globa longl itudin alstrain by echocardiographic assessment ,and change from baseline in N-terminal prohormone B-type Natriuretic Peptide (NTproBNP).

In certai nembodiments, the human subject carries a TTR gene mutation that is associated with the development of a TTR-associated disease, e.g., senile systemic amyloidosi (SSA),s systemic familial amyloidosi famis, lial amyloidoti polyc neuropat hy(FAP), familial amyloidoti cardic omyopat hy(FAC), leptomeningeal/Cent Nervousral System (CNS) amyloidosi ands, hyperthyroxinemia.

In certai nembodiments, the human subject has a transthyretin-mediated amyloidos (ATTRis amyloidos is)and the use of the double stranded RNAi agent reduces an amyloid TTR deposit in the human subject .In certai nembodiments, the ATTR amyloido sisis hereditary ATTR (h-ATTR) amyloidosi Ins. certain embodiments, the ATTR amyloidosi iss non-heriditary ATTR (wt ATTR) amyloidosis.

In certai nembodiments, the double stranded RNAi agent is administered to the human subject by subcutaneousl ory intravenously. In certain embodiments, the subcutaneous administration is self administration. In certain embodiments, the self-administration is via a pre-filled syringe or auto- injector device.

In certai nembodiments, the use further comprises assessing the level of TTR mRNA expression or TTR protein expression in a sample derived from the human subject, such as a human blood sample, or serum or plasm aderived therefrom.

In certai nembodiments, the double stranded RNAi agent is administered to the human subject once every month, once every othe rmonth, once every three months, once every four months, once every five months, or once every six months. In certain embodiments, the fixed dose of the double stranded RNAi agent is administere dto the human subject once about every three months In. certain embodiments, the fixed dose of the double stranded RNAi agent is administered to the human subject once about every six months.

In certai nembodiments, the double stranded RNAi agent is chronica admilly nistered to the human subject.

In certai nembodiments, the double stranded RNAi agent is administered to the human subject about once per quarter to about once per year. In certai nembodiments, the double stranded RNAi agent is administered to the human subject about once per quarter, about once every six months, or about once per year.

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

In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg to about 600 mg. In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg or about 600 mg about once 6 every six months to about once per year. In certain embodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg or about 600 mg about once every six months or about once per year.

In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 700 mg to about 1000 mg or about 700 mg to about 900 mg. In certain embodiments, the double stranded RNAi agen tis administered to the human subject at a fixed dose of about 700 mg, about 800 mg, about 900mg, or about 1000 mg about once per year.

In certai nembodiments, the use further comprises administering to the human subject an additional therapeuti cagent, e.g., a TTR tetramer stabilize orr a non-steroida antil -inflammator agenty .

The present invention also provides kits for performing any of the methods of the invention.

The kits may include the double stranded RNAi agent; and a label comprising instructions for use.

The present invention is further illustrated by the following detailed description and drawings.

Brief Description of the Drawings Figure 1 is a graph depicting relative serum TTR protein levels in V30M transgenic mice (n=3 per group) after administration of a singl e1 mg/kg dose of the indicated double stranded RNAi agent s on Day 0.

Figure 2 is a graph depicting relative serum TTR protein levels in cynomolgus monkeys (n=3 per group) after administration of a singl e1 mg/kg dose or 3 mg/kg dose of the indicated double stranded RNAi agents on Day 0. Results shown are from three independent studies.

Detailed Description of the Invention The present invention provides methods of inhibiting expression of TTR, including inhibiting TTR expression in a human subjec twho does not meet diagnosti cric teria of a TTR-associated disease and methods of treating a human subject with a Transthyretin- (TTR-) associated disease using double stranded RNAi agents ,targeting the TTR gene wherein the sense strand comprises the modified nucleotid sequee nce 5’-usgsggauUfuCfAfUfguaaccaaga-3’ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5’-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3’ (SEQ ID no: ר.

The following detailed description discloses how to make and use compositio nscontaining iRNA agents to selectively inhibit the expression of a TTR gene, as well as compositions, uses, and methods for treating subject shaving diseases and disorders that would benefi tfrom inhibition or reduction of the expression of a TTR gene.

I. Definitions In order that the present invention may be more readily understood, certain terms are first defined. In addition, it shoul bed noted that whenever a valu eor range of values of a parameter are 7 recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The article "sa" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatica objectl of the article. By way of example, "an element" means one element or more than one element ,e.g., a pluralit ofy elements.

The term "including" is used herein to mean, and is used interchangeabl wityh, the phrase "including, but not limited to".

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.

The term "about" is used herein to mean within the typical ranges of tolerances in the art, e.g., acceptable variation in time between doses, acceptable variation in dosage unit amount. For example, "about" can be understood as within about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is present before a series of numbers or a range, it is understood that "about" can modify each of the numbers in the series or range.

The term "at least" , "no less than", or "or more" prior to a number or series of numbers is understood to include the number adjacent to the term "at least", and all subsequent number sor integers that coul logicald bely included, as clear from context For. example, the number of nucleotides in a nucleic acid molecu lemust 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 indicated property. When at least is present before a series of number sor a range ,it is understood that "at leas"t can modify each of the number sin the series or range.

As used herein, "no more than" or "less than" is understood as the valu eadjacent to the phrase and logical lower values or integers, as logical from context to, zero. For example, a duplex with an overhang of "no more than 2 nucleotides" has a 2, 1, or 0 nucleotid overhang.e When "no more than" is present before a series of numbers or a range ,it is understood that "no more than" can modify each of the number sin the series or range.

As used herein, methods of detection can include determination that the amount of analyt e present is belo wthe level of detection of the method.

As used herein, a "transthyreti"n ("TTR") refers to the well-known gene and protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter of retinol- binding protein (RBP), thyroxine (T4) and retinol and, it also acts as a protease. The liver secretes TTR into the bloo d,and the choro idplexus secretes TTR into the cerebrospinal fluid. TTR is also expressed in the pancreas and the retinal pigment epithelium. The greatest clinic relalevanc ofe TTR is that both normal (wild type) and mutant TTR protein can form amyloid fibrils that aggregate into extracellula r deposits, causin gamyloidosi s.See, e.g., Saraiva M.J.M. (2002) Expert Reviews in Molecular Medicine, 4(12): 1-11 for a review. The molecula clonir ngand nucleotide sequenc eof rat transthyretin, as wel las the distribution of mRNA expression, was described by Dickson, P.W. et al. (1985) J. Biol. Chem. 260(13)8214-8219. The X-ray crystal structure of human TTR was described in Blake, C.C. et al. 8 (1974) J Mol Biol 88, 1-12. The sequenc eof a human TTR mRNA transcript can be found at National Center for Biotechnology Information (NCBI) RefSeq accession number NM_000371 (e.g., SEQ ID NOs:l and 5). The sequenc eof mouse TTR mRNA can be found at RefSeq accession number NM_013697.2, and the sequence of rat TTR mRNA can be found at RefSeq accession number NM_012681.1. Additional example sof TTR mRNA sequences are readil yavailable using publicl y available database e.g.,s, GenBank, UniProt, and OMIM.

A "TTR-associate dised ase," as used herein, is intended to include any disease associated with the TTR gene or protein. Such a disease may be caused, for example, by excess production of the TTR protein, by TTR gene mutations, by abnorm alcleavage of the TTR protein, instabilit ofy TTR tetramers, by abnorm alinteractions between TTR and othe rproteins or other endogenous or exogenous substances. A "TTR-associated disease" includes any type of transthyretin-mediated amyloidosi s (ATTR amyloidosis) wherein TTR plays a role in the formation of abnorm alextracellul aggregatar es or amyloid deposits, e.g., either herititary ATTR (h-ATTR) amyloido sisor non-heriditary ATTR (ATTR) amyloidosi s.TTR-associate dised ases include senile systemic amyloidos (SSAis ), systemic familial amyloidosi famis, lial amyloidoti polyc neuropat hy(FAP), familial amyloidoti cardic omyopat hy(FAC), leptomeningeal/Cent Nervousral System (CNS) amyloidosi amyls, oidoti vitrec ous opacities carpal, tunnel syndrome, and hyperthyroxinemia. Symptoms of TTR amyloido sisinclude sensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs), autonom icneuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostati hypotensic on), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficienc y,cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), crania l nerve dysfunction, and corneal lattic dystrophy.e As used herein, the term "strand comprising a sequence" refers to an oligonucleot ide comprising a chai nof nucleotides that is described by the sequence referred to using the standard nucleotid nomene clature.

The terms "iRNA," "RNAi agent," "iRNA agent,", "RNA interference agen"t as used interchangeabl herein,y refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specifi degradationc of mRNA through a process known as RNA interference (RNAi). The iRNA modulates e.g.,, inhibits, the expression of a TTR gene in a cell e.g.,, a cel witl hin a subject, such as a mammalian subject.

As used herein, an "iRNA" for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a "double stranded RNAi agent," "double stranded RNA (dsRNA) molecul" e,"dsRNA agent," or "dsRNA". The term "dsRNA" refers to a complex of ribonucl eicacid molecule havings, a duplex structure comprising two anti-paralle andl substantiall y complementary nucleic acid strands ,referred to as having "sense" and "antisense" orientations with respect to a target RNA, i.e., a TTR gene. A double stranded RNAi agent triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi. 9 As used herein, the term "modified nucleotid" erefers to a nucleotid havine g, independentl y,a modified sugar moiety, a modified internucleot idelinkage, or a modified nucleobase. Thus, the term modified nucleotid encompasse essubstitutions, additions or removal of, e.g., a functional group or atom ,to internucleosi lidenkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclose hereind or known in the art. Any such modifications, as used in a siRNA type molecule are, encompassed by "RNAi agen"t for the purposes of this specification and claims.

The duplex region may be of any lengt hthat permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 21 to 36 base pairs in length, e.g., about 21- base pairs in length, for example, about 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In one embodiment, an RNAi agent of the invention is a dsRNA agent ,each strand of which comprises 21-23 nucleotides that interacts with a TTR mRNA sequence to direct the cleavage of the targe tmRNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclea knownse as Dicer (Shar pet al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristi twoc base 3’ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporat edinto an RNA-induced silencing complex (RISC) where one or more helicas esunwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the targe tto induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, an RNAi agent of the invention is a dsRNA of 24- nucleotide thats interact wis th a TTR mRNA sequence to direct the cleavage of the targe tRNA.

As used herein, the term "nucleotid overhange " refers to at least one unpaired nucleotid thate protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand ,or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at leas onet nucleotide; alternatively, the overhang can compris eat least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleosid analog,e including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand ,the antisense strand or any combinati onthereof. Furthermore, the nucleotide(s) of an overhang can be present on the '-end, 3'-end or both ends of either an antisense or sense strand of a dsRNA. In one embodiment of the dsRNA, at least one strand comprise sa 3’ overhang of at leas t1 nucleotide In. another embodiment, at least one strand comprises a 3’ overhang of at leas 2t nucleotides, e.g., 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In othe rembodiments, at leas onet strand of the RNAi agen tcomprises a 5’ overhang of at least 1 nucleotide. In certain embodiments, at leas onet strand comprises a 5’ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides. In still othe rembodiments, both the 3’ and the 5’ end of one strand of the RNAi agen tcomprise an overhang of at leas 1t nucleotide.

In one embodiment, the antisense strand of a dsRNA has a 1-9 nucleotide, e.g., 0-3, 1-3, 2-4, 2- , 4-9, 5-9, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide overhang, at the 3’-end or the 5’-end. In one embodiment, the sense strand of a dsRNA has a 1-9 nucleotide e.g.,, a 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotide overhang, at the 3’-end or the 5’-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

"Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of the double stranded RNAi agent ,i.e., no nucleotid overhang.e A "blunt ended" RNAi agent is a dsRNA that is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecul e.The RNAi agents of the invention include RNAi agents with nucleotid overhangse at one end (i.e., agent s with one overhang and one blunt end) or with nucleotide overhangs at both ends.

The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a TTR mRNA.

As used herein, the term "region of complementari" tyrefers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a TTR nucleotide sequence, as defined herein. Where the region of complementari isty not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecul e.Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotides of the ’ - or 3’-terminus of the iRNA. In one embodiment, a double stranded RNAi agent of the invention include aa nucleotide mismatch in the antisense strand. In another embodiment, a double stranded RNAi agent of the invention includes a nucleotide mismatch in the sense strand. In one embodiment , the nucleotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucleotides from the 3’-terminus of the iRNA. In anothe embodir ment the, nucleotid mise match is, for example, in the 3’-terminal nucleoti de of the iRNA.

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

As used herein, the term "cleavage region" refers to a region that is locate immd ediately adjacen tot the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three base son either end of, and immediately adjacen to,t the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediatel yadjacent to, the cleavage site. In some embodiments, the cleavage site specifical occursly at the site bound by nucleotides 10 and 11 of the antisense strand ,and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unles sotherwise indicated, the term "complementary," when used to describe a first nucleotid sequence ein relation to a secon dnucleotid sequence,e refers to the abilit ofy an oligonucleoti orde polynucleot compide rising the first nucleotid sequee nce to hybridize and form a duplex structure under certain conditions with an oligonucleoti orde polynucleot compide rising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can be, for example, "stringent conditions", where stringent conditions can include: 400 mM NaCl 40, mM PIPES pH 6.4, 1 mM EDTA, 50 oC or 70 oC for 12-16 hours followed by washing (see, e.g., "Molecul ar Clonin g:A Laboratory Manual, Sambrook, et al. (1989) Col dSpring Harbor Laboratory Press). Other 11 conditions, such as physiological relevantly conditions as can be encountered inside an organism, can apply. The skilled person wil lbe able to determine the set of conditions most appropriate for a test of complementari ofty two sequence sin accordance with the ultimate application of the hybridized nucleotides.

Complementa rysequence swithin an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleoti orde polynucleot compide rising a first nucleotid sequee nce to an oligonucleoti orde polynucleot compide rising a second nucleotid sequence eover the entire lengt hof one or both nucleotide sequences .Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequenc eis referred to as "substantiall compley mentary" with respect to a secon dsequence herein, the two sequences can be full ycomplementary, or they can form one or more, but generall noty more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs ,whil eretaining the abilit toy hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression, in vitro or in vivo.. However, where two oligonucleotide ares designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shal notl be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleoti 21de nucleotides in lengt hand anothe olir gonucleoti 23de nucleotides in length, wherein the longer oligonucleoti comprisesde a sequenc eof 21 nucleotide thats is fully complementa tory the shorter oligonucleoti cande, yet be referred to as "full ycomplementary" for the purposes described herein.

"Complementa"ry sequences, as used herein, can also include, or be formed entirely from, non- Watson-Crick base pairs or base pairs formed from non-natura andl modified nucleotides, in so far as the above requirements with respect to their abilit toy hybridize are fulfilled. Such non-Watson-Cri ck base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms "complementary," "fully complementary" and "substantially complementary" herein can be used with respect to the base matchin betwg een the sense strand and the antisense strand of a dsRNA, or between two oligonucleotides or polynucleotides such, as the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleot thatide is "substantially complementa tory at least part of’ a messenger RNA (mRNA) refers to a polynucleot thatide is substantially complementa tory a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a TTR gene). For example, a polynucleotide is complementa tory at least a part of a TTR mRNA if the sequenc eis substantiall compley mentary to a non-interrupted portion of an mRNA encoding a TTR gene.

Accordingly, in some embodiments, the antisense polynucleotide discls osed herein are full y complementary to the targe tTTR sequence. In othe rembodiments, the antisense polynucleoti des disclosed herein are fully complementary to SEQ ID NO:8 (5’- UGGGAUUUCAUGUAACCAAGA - 3’). In one embodiment, the antisense polynucleot sequeide nce is 5’- UCUUGGUUACAUGAAAUCCCAUC -3’ (SEQ ID NO:9), wherein the U at position 7 of the antisense strand can be a T. 12 As used herein, a "reference level" is understood as a predetermined level to which a level obtained from an assay, e.g., a biomarke lever l, e.g., a protein biomarke lever l, is compared. In certain embodiments, a reference level can be a control level determined for a healthy population, e.g., a population that does not have a disease or condition associated with a changed level of the biomarker and does not have a predisposition, e.g., genetic predisposition, to a disease or condition associate witd h a changed level of the biomarker. In certain embodiments, the population shoul bed matched for certain criteria ,e.g., age, sex. In certai nembodiments, the reference level of the biomarke isr a level from the same subjec tat an earlier time, e.g., before the development of symptomatic disease or before the start of treatment. Typically sampl, es are obtained from the subject at clinical rellyevant intervals e.g.,, at intervals sufficiently separated in time that a change in the biomarke couldr be observed ,e.g., at leas at three month interval, at least a six month interval or, at leas at nine month interval. When more than two samples are obtained from a subject over time, it is understood that any of the prior samples can act as a reference level.

As used herein, a "change as compared to a reference level" and the like is understood as a statisticall ory clinical signily ficant change in the biomarke lever l, e.g., the change in the protein biomarker level as, compare dto the reference level is, greater than the typical standard deviation of the assay method. Moreover, the change shoul bed clinical relevly ant. The change as compare dto a reference level can be determined as a percent change. For example, if a reference level is 100 pg/ml for biomarker X, and the level of biomarker X in the subject is 150 pg/ml, the level is increased by 50% calcula byted ((150 pg/ml -100 pg/ml)/100 pg/ml) X 100% = 50%. If the level of biomarker X in the subject is 300 pg/ml, the level is increased by 300%. If the level of biomarker X in the subject is 50 pg/ml, the level is decreased by 50%. In certain embodiments, the change as compared to a reference level is increased by at least 50%. In certain embodiments, the change as compare dto a reference leve l is increased by at least 100%, at leas 200%,t or at leas 300%.t In certain embodiments, the change as compared to a reference sampl eis decreased by at least 25%. In certai nembodiments, the change as compared to a reference sampl eis decreased by at least 50%.

A "biologic samal ple from a subjec"t or a "sampl efrom a subjec"t as used herein, includes one or more fluids, cells or, tissues isolated from a subject. Examples of biologic fluidsal include blood, serum, serosal fluids, plasma, cerebrospinal fluid, ocula fluidsr ,lymph, urine, saliva, and the like.

Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from particul arorgans, parts of organs, or fluids or cell wits hin those organs. In certain embodiments, samples may be liver tissue or be derived from the liver. In some embodiments, a "biologic sampal le from a subjec"t can refer to blood or blood derived serum or plasm afrom the subject. In some embodiments, the fluid is substantially free of cells e.g.,, is free of cells.

As used herein, a "clinical relevantly difference" is understood as at leas greatt er difference than typical interobserver variatio forn the assessment, wherein the observer may be a trained healt careh professional a caregi, ver, or the patient, performing the same assessment on the same individual at around the same time, e.g., within a week, such as within consecutive days. It is understood that certain patien tobservations are subjective and shoul bed nearly identical when performed by different 13 observers within a short time frame, e.g., body weight, heart rate . Other qualitative measures, such as some aspect sof mNIS+7 (e.g., response to touch-pressure, vibration, joint position and motion) and Norfolk-Quali ofty Life (e.g., pain level, hot or cold hands and feet, steadines swhile standing) may vary more from day to day, and from one observer to another. Therefore ,composite scores are used to aggregate observations and larger variations between observers are expected without any indication of clinical relevantly change. Assays for biomarker levels have known level sof variations within and between samples. Determination of a clinical relevantly difference is within the ability of one of skil lin the art, e.g., a healt careh professional experienced in treating patients with TTR associated diseases , clinic laboal rato professry ional.

As used herein, "chronica admilly nistered" is understood as administration for an indefinite interval e.g.,, for the remainder of the life of the subject, until liver transplant.

As used herein, a "therapeuti cagent that stabilizes TTR" or "that stabilizes a TTR tetramer" is an agent that reduces or prevents the dissociatio ofn the subunits of a TTR tetramer ,e.g., into monomers. In some embodiments, the agent reduces the formation of TTR amyloid plaque s,e.g., by reducing the level of TTR monomers or proteolyti frac gment sof TTR monomer thats form TTR amyloid plaques. Such agents include, but are not limited to, tafamidis ,diflunisal, and AGIO.

As used herein, the term "administering a therapeutic agent" is understood as providing a therapeuti cagent to a subject. In embodiments, the therapeuti cagent is provided at an appropriate dosage and by a route of administration for the agent as provided, for example, by the label of the therapeuti cagent.

II. Methods for Treating a TTR-Associated Disease The present invention provides double stranded RNAi agents and their use in methods for treating a TTR-associated disease in a human subject, such as a transthyretin-mediated amyloidosi s (ATTR amyloidosis e.g.,), hereditary ATTR (h-ATTR) amyloido sisor non-heriditar ATTRy (wt ATTR) amyloidosis or; for inhibiting expression of TTR in a subject that does not yet meet the diagnostic criteria of a TTR-associated disease, but who is at risk for developing a TTR-associate diseasd e, e.g., a subject with a TTR mutation associate wid th TTR amyloidosi as, subject with some indicia of TTR amyloido siswho does not yet meet the diagnostic criteria of TTR amyloidosi as, subjec twith altered biomarker levels associate wid th TTR amyloidosi s.The methods include administering to the subject a therapeuticall effectiy ve amoun oft an RNAi agent of the invention.

In one aspect, the present invention provides methods of treating a human subject suffering from a TTR-associated disease or at risk for developing a TTR-associated disease. The methods include administering to the human subjec ta fixed dose of about 25 mg to about 1000 mg of a double stranded RNAi agent wherein the sense strand comprises the modified nucleotide sequenc e5’- usgsggauUfuCfAfUfguaaccaaga-3‘ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotid sequee nce 5’-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3’ (SEQ ID NO: 7), 14 wherein a, c, g, andu are 2'-O-methyladenosine-3’-phosphate, 2'-O-methylcytidine-3’- phosphate, 2'-O-methylguanosine-3’-phosphate, and 2'-O-methyluridine-3’-phosphat respectie, vely; Af, Cf, Gf, and Uf are 2‘-fluoroadenosine-3‘ -phosphate, 2‘-fluorocytidine-3‘ -phosphate, 2’- fluoroguanosine-3’-phosphat e,and 2’-fluorouridine’-3-phosphat e,respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-Isomer; and s is a phosphorothioa linteker.

In another aspect, the present invention provides methods of improving at least one indici aof neurological impairement or quali tyof life in a human subjec tsuffering from a TTR-associate disead se or at risk for developing a TTR-associate disead se. The methods include administerin gto the human subject a fixed dose of about 25 mg to about 1000 mg of a double stranded RNAi agent wherein the sense strand comprises the modified nucleotid sequee nce 5’-usgsggauUfuCfAfUfguaaccaaga-3’ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequenc e5’- usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3’ (SEQ ID NO: 7).

In another aspect, the present invention provides methods of reducing, slowing, or arresting a Neuropath yImpairment Score (NIS) or a modified NIS (mNIS+7) in a human subject suffering from a TTR-associate diseased or at risk for developing a TTR-associated disease. The methods include administering to the human subjec ta fixed dose of about 25 mg to about 1000 mg of a double stranded RNAi agent wherein the sense strand comprises the modified nucleotid sequence e5’- usgsggauUfuCfAfUfguaaccaaga-3‘ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotid sequee nce 5’-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3’ (SEQ ID NO: 7).

In another aspect, the present invention provides methods of increasin ag 6-minute wal ktest (6MWT) in a human subject suffering from a TTR-associated disease or at risk for developing a TTR- associate disead se. The methods include administering to the human subject a fixed dose of about 25 mg to about 1000 mg of a double stranded RNAi agent wherein the sense strand comprises the modified nucleotid sequee nce 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).

In certai nembodiments, the double stranded RNAi agent is administered to the human subject about once per quarter to about once per year. In certai nembodiments, the double stranded RNAi agent is administered to the human subject about once per quarter, about once every six months, or about once per year.

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

In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg to about 600 mg. In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg or about 600 mg about once every six months to about once per year. In certain embodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg or about 600 mg about once every six months or about once per year.

In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 700 mg to about 1000 mg or about 700 mg to about 900 mg. In certain embodiments, the double stranded RNAi agen tis administered to the human subject at a fixed dose of about 700 mg, about 800 mg, about 900mg, or about 1000 mg about once per year.

In an embodiment, the subject is a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in TTR gene expression; a human at risk for a disease, disorder or condition that would benefit from reduction in TTR gene expression, e.g., a human who does not meet the diagnosti cric teria of a TTR-associated disease, but who demonstrates at least one sign or symptom of a TTR-associated disease or has at least one risk factor of developing a TTR associate d disease; a human having a disease, disorder or condition that would benefit from reduction in TTR gene expression; or human being treated for a disease, disorder or condition that would benefit from reduction in TTR gene expression, as described herein.

In some embodiments, the human subject is suffering from a TTR-associated disease. In other embodiments, the subject is a subject at risk for developing a TTR-associated disease, e.g., a subject with a TTR gene mutation that is associated with the development of a TTR associate dised ase ,a subject with a family history of TTR-associated disease, or a subject who has signs or symptoms suggesting the development of TTR associated disease without meeting the diagnosti cric teria for a TTR-associate dised ase.

A "TTR-associate dised ase," as used herein, includes any disease caused by or associated with the formation of amyloid deposits in which the fibril precurosor sconsist of variant or wild-type TTR protein. Mutant and wild-type TTR give rise to various forms of amyloid deposition (amyloidosis ).

Amyloidosi involves thes formation and aggregation of misfolded proteins, resulting in extracellula r deposits that impair organ function. Climical syndromes associate wid th TTR aggregation include, for example, senile systemic amyloido sis(SSA); systemic familial amyloidosi famis; lial amyloidoti c polyneuropat hy(FAP); familial amyloidoti cardic omyopathy (FAC); and leptomeningea amyll oidosi s, also known as leptomeningeal or meningocerebrovascul amyloar idosi cents, ral nervous system (CNS) amyloidosi ors, amyloido sisVII form. 16 In one embodiment, the RNAi agents of the invention are administered to subject ssuffering from familial amyloidotic cardiomyopat hy(FAC). In anoth erembodiment, the RNAi agents of the invention are administered to subject ssuffering from FAC with a mixed phenotype, i.e., a subject having both cardiac and neurological impairements. In yet anothe embor diment, the RNAi agents of the invention are administered to subject ssuffering from FAP with a mixed phenotype, i.e., a subject having both neurologic andal cardiac impairements. In one embodiment, the RNAi agents of the invention are administered to subject ssuffering from FAP that has been treated with an orthotopic liver transplantati (OLonT).

In another embodiment, the RNAi agents of the invention are administered to subject ssuffering from senile systemic amyloidos (SSAis ). In othe rembodiments of the methods of the invention, RNAi agents of the invention are administered to subject ssuffering from familial amyloidoti cardiomyopathyc (FAC) and senil esystemic amyloido sis(SSA). Normal-sequence TTR cause scardiac amyloido sisin peopl ewho are elderly and is termed senil esystemic amyloido sis(SSA) (als ocalled senil ecardiac amyloido sis(SCA) or cardiac amyloidosis). SSA often is accompanie byd microscopic deposits in many othe rorgans. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of clinical signifily cant TTR amyloido sis(also call edATTR (amyloidosis-transthyret type)).in More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.

In some embodiments of the methods of the invention, RNAi agents of the invention are administered to subject ssuffering from transthyretin (TTR)-related familial amyloidoti polyneurc opathy (FAP). Such subject smay suffer from ocular manifestations, such as vitreous opacity and glaucoma. It is known to one of skil lin the art that amyloidogenic transthyretin (ATTR) synthesized by retinal pigment epithelium (RPE) plays important roles in the progression of ocular amyloidosi s.Previous studies have shown that panretinal laser photocoagulat whichion, reduced the RPE cells, prevented the progression of amyloid deposition in the vitreous, indicating that the effective suppression of ATTR expression in RPE may become a novel therapy for ocular amyloido sis(see, e.g., Kawaji T.,, et al., Ophthalmology. (2010) 117: 552-555). Another TTR-associated disease is hyperthyroxinemia, als o known as "dystransthyretinem ichyperthyroxinem"ia or "dysprealbuminemic hyperthyroxinem"ia. This type of hyperthyroxinem iamay be secondary to an increased association of thyroxine with TTR due to a mutant TTR molecul wite h increased affinity for thyroxine. See, e.g., Moses et al. (1982) J. Clin.

Invest., 86, 2025-2033.

The RNAi agents of the invention may be administered to a subject using any mode of administration known in the art, including, but not limited to subcutaneous, intravenou s,and intramuscular injection, and any combinations thereof.

In some embodiments, the agents are administered to the subject subcutaneously.

In some embodiments, a subject is administered a singl edose of an RNAi agent via subcutaneous injection, e.g., abdominal, thigh, or upper arm injection. In othe rembodiments, a subject is administered a spli tdose of an RNAi agent via subcutaneous injection. In one embodiment, the spli t dose of the RNAi agent is administered to the subject via subcutaneous injection at two different 17 anatomical locations on the subject. For example, the subjec tmay be subcutaneousl injey cted subcutaneously at a dose of 25 mg to 1000 mg. In some embodiments of the invention, the subcutaneous administration is self-administrati onvia, e.g., a pre-fille dsyringe or auto-inject orsyringe.

In some embodiments, a dose of the RNAi agent for subcutaneous administration is containe ind a volume of less than or equal to one ml of, e.g., a pharmaceutical acceptly able carrier. In some embodiments, the RNAi agent is in a non-pyrogenic formulation.

In some embodiments, the RNAi agent is administered to a subject in an amount effective to inhibit TTR expression in a cell within the subject. The amount effective to inhibit TTR expression in a cel witl hin a subject may be assessed using methods discussed below including, methods that involve assessment of the inhibition of TTR mRNA, TTR protein, or related variable suchs, as amyloid deposits.

In some embodiments, the RNAi agent is administered to a subject in a therapeuticall effey ctive amount.

"Therapeutical effectily ve amount," as used herein, is intended to include the amoun oft an RNAi agent that ,when administered to a patient for treating a TTR associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, maintaining, or slowing the progression of the existing disease as compared to an appropriate control or; diminishing, maintaining, or slowing one or more symptoms of disease as compared to an appropriate control The). "therapeuticall effey ctive amount" may vary depending on the RNAi agent ,how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by TTR expression, the types of preceding or concomitant treatments, if any, and other individual characteristi ofcs the patient to be treated. Diagnost iccriteria for TTR amyloidosis, polyneuropathi andes, cardiomyopathie ares discussed further below.

"Therapeutical effectily ve amount," as used herein, is intended to include the amount of an RNAi agent that ,when administered to a subject who does not yet meet the diagnositi ccriteria of a TTR-associate disease,d e.g., a subject who has not been diagnosed with hTTR amyloidosi s polyneuropathy; a subject who does not meet the diagnosti cric teria of Stage 1 FAP, but who may be predisposed to the disease ,e.g., a subject with a TTR mutation associate wid th TTR amyloidosi as, subject suffering from one or more of orthostati hypotension,c heart failure cardiac, arrhythmia, left ventricula wallr thickness, interventricular septal wall thickness, cardiac posterior wal thil cknes s diarrhea constipation,, erectile dysfunction, glaucom inta, ravitreal deposition, scalloped pupils ;carpal tunnel syndrome, lumbar spina lstenosis, and bicep tendon rupture; a subject with an elevate d neurofilament light chai n(NfL) level as compared to a reference sample, e.g., a Nfl level of at leas 37t pg/ml in serum, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Symptoms that may be ameliorat edinclude sensory neuropathy (e.g., paresthesia hypesthesi, a in distal limbs), autonom icneuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropat hy,carpal tunnel syndrome, autonomic insufficienc y,cardiomyopathy, vitreous opacitie s,renal insufficienc y, nephropathy, substantially reduced mBMI (modified Body Mass Index), crani alnerve dysfunction, corneal lattic dystrophy,e left !ventricular (LV) wall thickening by echocardiograph assessic ment, 18 increased global longitudinal strain by echocardiographic assessment ,increased N-terminal prohormone B-type Natriuretic Peptide (NTproBNP), and hospitalizati dueon to cardiac event. Diminishing the disease includes slowing the course of the disease or reducing the severity of later-developin diseag se.

The dose may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease ,and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and othe rindividual characteristic ofs the patient to be treated.

A "therapeutically-effect amountive " also includes an amoun oft an RNAi agen tthat produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. RNAi agents employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

As used herein, the phrase "therapeutical effectily ve amount" also includes an amount that provides a benefit in the treatment, prevention, or management of pathologic processesal or symptom(s) of pathologic procesal ses mediated by TTR expression. Symptoms of TTR amyloido sisinclude sensory neuropathy (e.g. paresthesia, hypesthesia in distal limbs), autonom icneuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostati hypotensic on), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonom icinsufficiency, cardiomyopathy, vitreous opacitie s,renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, corneal lattice dystrophy, left !ventricular (LV) wall thickening by echocardiographic assessment ,increased global longitudin alstrain by echocardiograph assessmic ent, increased N-terminal prohormone B-type Natriuretic Peptide (NTproBNP), and hospitalizati dueon to cardiac event.

In one embodiment, for example, when the subject has FAP, FAP with mixed phenotype, FAC with mixed phenotype, or FAP and has had an OLT, treatment of the subject with a dsRNA agent of the invention slows the progression of neuropathy. In anoth erembodiment, for example, when the subject has FAP, FAP with mixed phenotype, FAC with mixed phenotype SSA,, or FAP and has had an OLT, treatment of the subject with a dsRNA agent of the invention slow sthe progression of neuropathy and cardiomyopathy. In anothe embor diment, for example, when the subject has cardiac involvement, the method of the invention improve cardiac structure and function, including, for example, the methods reduce the mean left ventricula wallr thickness and longitudin alstrain, and reduce the expression level of the cardiac stress biomarker N-te, rminal pro b-type natriureti cpeptide (NT-proBNP).

Adminsitration of a therapeuticall or yprophylacticall effectiy ve amount of the RNAi agen tof the invention is also useful in methodsfor improving at leas onet indicia of neurological impairment or qualit ofy life in a subject suffering from or at risk of developing a TTR-associated disease.

For example, in one embodiment, the methods of the invention improve at least indicia of neurological impairment in the subject ."Improving at leas onet indici aof neurological impairment" in the subject refers to the ability of the methods of the invention to slow reduc, e, or arrest neurological impairment, or improve any symptom associated with neurologic imalpairment. Any suitable measure of neurological impairment can be used to determine whether a subject has reduced, slowed, or arrested, neurological impairment, or an improvement of a symptom associate wid th neurological impairment. 19 One suitable measure is a Neuropath yImpairment Score (NIS). NIS refers to a scoring system that measures weakness, sensation, and reflexes, especiall wiyth respect to peripheral neuropathy. The NIS score evaluate as standard group of muscles for weaknes s(1 is 25% weak, 2 is 50% weak, 3 is 75% weak, 3.25 is movement agains gravity,t 3.5 is movement with gravity eliminate d,3.75 is muscle flicker without movement, and 4 is paralyzed), a standard group of muscle stretch reflexes (0 is normal, 1 is decreased, 2 is absent) , and touch-pressure, vibration, joint position and motion, and pinprick (all graded on index finger and big toe: 0 is normal 1 ,is decreased, 2 is absent). Evaluations are correcte d for age, gender, and physical fitness.

In one embodiment, the methods of the invention reduce a NIS by at leas 5t points at 18 months from the start of dosing. In othe rembodiments, the methods of the invention result in a stabilization of NIS at 18 months from the star tof treatment with an RNAi agent provided herein. In othe r embodiments, the methods slow an increasing NIS score as compared to an appropriate control group showing the natura histol ry of the disease, e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependen tupon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment, the duration of treatment, prior treatments, and the specific TTR mutation present, if any.

Methods for determining a NIS in a human subject are well known to one of skil lin the art and can be found in, for example, Dyck, PJ et al., (1997) Neurology 1997. 49(1): pgs. 229-239); Dyck PJ. (1988) Muscle Nerve. Jan; 1 l(l):21-32.

Another suitable measurement of neurological impairment is a Modified Neuropathy Impairment Score (mNIS+7). As known to one of ordinary skil lin the art, mNIS+7 refers to a clinical exam-base dassessment of neurologic impairment (NIS) combine wid th electrophysiologic measures of smal andl large nerve fiber function (NCS and QST), and measurement of autonom icfunction (postural blood pressure). The mNIS+7 score is a modificatio ofn the NIS+7 score (which represents NIS plus seven tests). NIS+7 analyzes weaknes sand muscle stretch reflexes. Five of the seven tests include attributes of nerve conduction. These attribute sare the peroneal nerve compound muscle action potential amplitude, motor nerve conduction velocity and motor nerve distal latency (MNDL), tibia l MNDL, and sural sensory nerve action potential amplitudes. These values are corrected for variables of age, gender, height, and weight. The remaining two of the seven tests include vibratory detection threshol andd heart rate decrease with deep breathing.

The mNIS+7 score modifies NIS+7 to take into account the use of Smar tSomatotopic Quantitative Sensation Testing, new autonom icassessments ,and the use of compound muscle action potential of amplitudes of the ulnar, peroneal, and tibial nerves, and sensory nerve action potential ofs the ulna andr sural nerves (Suanpraser t,N. et al., (2014) J. Neurol. Sci., 344(1-2): pgs. 121-128).

In one embodiment, the methods of the invention reduce a mNIS+7 by at leas 5t points at 18 months from the start of dosing. In othe rembodiments, the methods of the invention result in a stabilization of mNIS+7 at 18 months from the start of treatment with an RNAi agent provided herein.

In othe rembodiments, the methods slow an increasin mNIg S+7 score as compare dto an appropriate control group showing the natura hisl tory of the disease, e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependent upon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment ,the duration of treatment, prior treatments, and the specific TTR mutation present, if any.

In another embodiment, the methods of the invention improve at least one indici aof quali tyof life in the subject ."Improving at least one indici aof qualit ofy life" in the subject refers to the abilit ofy the methods of the invention to slow or arrest quali tyof life worsening or improve qualit ofy life. Any suitable measure of quality of life can be used to determine whether a subject has slowed or arrested qualit ofy life worsening, or improved qualit ofy life.

For example, the SF-36® healt surveyh provides a self-reporting mult, i-item scal meae suring eight healt parah meters: physical functioning, role limitations due to physical healt problems,h bodily pain, general health, vitality (energy and fatigue), social functioning, role limitations due to emotional problems, and mental health (psychological distress and psychological well-being). Each scal ise directly transformed into a 0-100 scal one the assumption that eac hquestion carries equal weight. The lower the score the more disabilit y.The highe rthe score the less disability i.e., a score of zero is equivalent to maximum disability and a score of 100 is equivalent to no disability. The survey also provides a physical component summary and a mental compone ntsummary.

In one embodiment, the methods of the invention provide to the subject an improvement versus baseline in at least one of the SF- 36 physical healt relath ed parameter s(physical health, role-physical, bodil ypain or general health) or in at least one of the SF-36 mental healt relath ed parameters (vitality, social functioning, role-emotional or mental health). Such an improvement can take the form of an increas of,e for exampl eat least 2 or at leas 3t points ,on the scal fore any one or more parameter sat 9 months from the start of the dosing.

In othe rembodiments, the methods of the invention arrest a decreasing SF-36 parameter score at 9 months from the start of dosing for any one or more parameters, e.g., the methods result in no clinical signily ficant change of the SF-36 e.g., within the variation observed for individuals performing an SF-36 assessment. In yet othe rembodiments, the methods of the invention slow the rate at which a SF-36 score decreases at 9 months from the start of dosing, e.g., the rate of decrease of an SF-36 score in a subject treated with an RNAi agent of the invention as compared to the rate of decrease of an SF-36 score as compared to an appropriate control group showing the natural history of the disease, e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependent upon a number of factor incls uding, but not limited to, the severity of disease in the subject at the initiation of treatment, the duration of treatment, prior treatments, and the specific TTR mutation present, if any.

Another suitable measurement of qualit ofy life is the Norfolk Qualit yof Life-Diabetic Neuropath y(Norfolk QOL-DN) questionnair e.The Norfolk QOL-DN is a validated comprehensive questionna iredesigned to capture the entire spectrum of DN related to large fiber, small fiber, and autonomic neuropathy not captured in existing instruments. 21 In one embodiment, the methods of the invention improve a subjec’ts Norfolk QOL-DN score from baseline, e.g., a change of about -2.5, -3.0, -3.5, -4.0, -4.5, or -5.0 at 9 months from the start of treatment with an RNAi agent provided herein. In othe rembodiments, the methods arrest an increasing Norfolk QOL-DN score ,e.g., the methods result in no clinical signily ficant change of theNorfolk QOL- DN score ,e.g., within the variation observed between individual perfos rming a QOL-DN assessment.

In yet othe rembodiments, the methods of the invention slow the rate at which an QOL-DN score increase s,e.g., the rate of increase of a QOL-DN score in a subject treated with an RNAi agent of the invention as compared to the rate of increase of a QOL-DN score as compared to an appropriate control group showing the natural history of the disease, e.g., a placebo control group as provided, for exampl e in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependen tupon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment, the duration of treatment, prior treatments, and the specific TTR mutation present, if any.

Another suitable measurement of qualit ofy life is motor strength as assessed by, for example, a NIS-W score. A NIS-W score is a composite score that summates the weaknes sof head, trunk, and limb muscles. Using the NIS (W) (referring to the portion of the scal mease uring weakness), muscl e power is assessed as norma (0)l or complete paralysis (4) with intermediate grades; 1 representing a muscle that is deemed 25% weak by clinical strength testing, 2 as 50% weak, 3 as 75% weak, 3.25 as movement agains gravitt y, 3.50 as movement with gravity eliminated, and 3.75 as muscl eflicker.

In one embodiment, the methods of the invention provide to the subject an improvement versus baseline in an NIS-W score Such an improvement can take the form of a decrease of at least 5, 6, 7, 8, 9, or 10 points of the subjec’ts NIS-W score at 18 months from the start of treatment with an RNAi agent provided herein. In othe rembodiments, the methods arrest a decrease NIS-W score, e.g., the methods result in no clinical signily ficant increas ofe the NIS-W score, or a slowing in the rate of increas ofe NIS-W score as compared to an appropriate control group showing the natural history of the disease ,e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependent upon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment, the duratio nof treatment ,prior treatments, and the specific TTR mutation present, if any.

Yet anoth ersuitable indicia of quality of life is the Rasch-built Overal Disl abilit yScale (R- ODS), which is a a patient questionna iredesigned to capture activity and social participation limitations in patients. In one embodiment, the methods of the invention provide to the subject an improvement versus baseline in an R-ODS score. Such an improvement can take the form of an increas ofe at leas 2,t for example at least 2, 3, 4, or 5 points of the subject’s R-ODS score at 18 months from the start of treatment with an RNAi agent provided herein. In othe rembodiments, the methods arrest a decreasing R-ODS score ,e.g., the methods result in no clinical signily ficant decrease of the R-ODS score at 18 months from the start of treatment with an RNAi agent provided herein. In yet othe rembodiments, the methods of the invention slow the rate at which a R-ODS score decreases at 18 months from the start of treatment with an RNAi agent provided herein, e.g., the rate of decrease of a R-ODS score in a subject 22 treated with an RNAi agent of the invention as compare dto the rate of decrease of a R-ODS score as compared to an appropriate control group showing the natura histl ory of the disease, e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependent upon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment, the duration of treatment, prior treatments, and the specific TTR mutation present, if any.

The composit eautonom icsymptom score (COMPASS-31), a patient questionna irethat assesses symptoms of dysautonomi autonoma icwhich provides a symptom score from 0 to 100, is another suitable indici aof quality of life. In one embodiment, the methods of the invention provide to the subject an improvement versus baseline in a COMPASS-31 score. Such an improvement can take the form of an increas ofe at least 5, for example at least 5, 6, 7, 8, 9, or 10, point sof the subjec’ts COMPASS-31 score at 18 months from the start of treatment with an RNAi agent provided herein. In othe rembodiments, the methods arrest a decreasing COMPASS-31 score, e.g., the methods result in no clinical relevantly change of the COMPASS-31 score at 18 months from the start of treatment with an RNAi agent provided herein. In yet othe rembodiments, the methods of the invention slow the rate at which a COMPASS-31 score decreases, e.g., the rate of decrease of a COMPASS-31 score at 18 months from the start of treatment with an RNAi agen tprovided herein in a subject treated with an RNAi agent of the invention as compare dto the rate of decrease of a COMPASS-31 score as compare dto an appropriate control group showing the natural history of the disease, e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependen tupon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment, the duration of treatment, prior treatments, and the specific TTR mutation present, if any.

Other qualit ofy life indicia may include nutritional status (e.g., as assessed by change in median body mass index (mBMI). In one embodiment the, methods of the invention provide to the subject an improvement versus baseline in mBMI. Such an improvement can take the form of a mBMI score increas ofe at least 2, 3, 4, 5, or more at 18 months from the start of treatment with an RNAi agent provided herein. In othe rembodiments, the methods arrest a decreasing mBMI index score, e.g., the methods result in no clinical signily ficant change of the mBMI score at 18 months from the start of treatment with an RNAi agent provided herein. In yet othe rembodiments, the methods of the invention slow the rate at which mBMI score decreases, e.g., the rate of decrease of a mBMI score at 18 months from the start of treatment with an RNAi agen tprovided herein in a subject treated with an RNAi agent of the invention as compare dto the rate of decrease of a mBMI score in a subject as compared to an appropriate control group showing the natural history of the disease, e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21. It is understood that the rate of disease progression is dependen tupon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment, the duration of treatment, prior treatments, and the specific TTR mutation present, if any. 23 Another quality of life indicia includes assessment of exercise capacity. One suitable measure of exercise capacity is a 6-minute walk test (6MWT), which measures how far the subject can wal kin 6 minutes, i.e., the 6-minute walk distance (6MWD). In one embodiment, the methods of the invention provide to the subject an increase from baseline in the 6MWD by at least 10 meters, e.g., at least 10, 15, , or about 30 meters at 18 months from the start of treatment with an RNAi agent provided herein.

Another suitable measure is the 10-meter walk test which measures gait speed. In one embodiment, the methods of the invention provide to the subject an increas frome baseline in the 10- meter wal ktest by at least 0.025 meters/second, e.g., at leas 0.025,t 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0. 4.5, or about 5.0 meters/second at 18 months from the start of treatment with an RNAi agent provided herein.

In some embodiments, a change in a plasm abiomarker level is an indici aof a decreas ein ongoing nerve damage or progression of polyneuropathy in ATTR amyloidosi s.For example, a decrease in the level of neurofilament light chai n(NfL) at 9 months as compared to an NfL level at the start of treatment can be an indicia of a decrease in ongoing nerve damage or progression of polyneuropat hyin ATTR amyloidosi s.In certai nembodiments, a decrease in the level of othe rproteins, especial lyRSPO3, CCDC80, EDA2R, and NT-proBNP, either alone or in combinati onwith a decrease in NfL level at 9 months from the start of treatment as compared to its corresponding level at the start of treatment can be an indici aof a decrease in ongoing nerve damage or progression of polyneuropat hyin ATTR amyloidosi s.In certai nembodiments, an increas ine the level of N-CDase ,either alone or in combinati onwith the othe rmarkers listed above, at 9 months from the start of treatment as compare dto an its corresponding level at the start of treatment can be an indicia of a decrease in ongoing nerve damage or progression of polyneuropat hyin ATTR amyloidosi s.Further biomarkers that can act as indicia of a decrease in nerve damage or polyneuropat inhy ATTR amyloidosi suchs, as at 9 months from the initiation of treatment with an RNAi agent provided herein, are provided in Table 1. A decrease in ongoing nerve damage or progression of polyneuropat hyin ATTR amyloidosi iss correlated with a decrease in the proteins having a positive beta coefficient. A decrease in ongoing nerve damage or progression of polyneuropat hyin ATTR amyloido sisis correlated with an increas ine proteins having a negative beta coefficient. It is understood that the change in the biomarker level is a statistical ly significant change, i.e., a change larger than the inherent variabili ofty the assay.

In certai nembodiments, the methods of the invention provide an improvement in cardiovascular indicia e.g.,, increase in Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS), decreased left !ventricular (LV) wall thickening by echocardiographic assessment as compared to baseline, decreased global longitudinal strain by echocardiographic assessment as compared to baseline, decreased N-terminal prohormone B-type Natriuretic Peptide (NTproBNP) as compared to baseline, and decrease in hospitalizati dueon to cardiac event.

The methods of the present invention may also improve the prognosi sof the subject being treated. For example, the methods of the invention may provide to the subject a reduction in probability of a clinica worsenil ng event during the treatment period, or an increased longevity, or decreased hospitalizati ason compare dto an appropriate control group showing the natural history of the disease, 24 e.g., a placebo control group as provided, for example in Adams et al., N Engl J Med 2018;379:11-21.

In certai nembodiments, a reduction in the probabil itofy a clinic alworsening event during the treatment can include a decrease in all-cause mortali tyor rates of cardiovascular-relat hospited alizati ason assessed, e.g., according to the Finkelstein-Schoenfe metldhod, as compared to an appropriate control group, as provided, for example, in Maurer et al., N Engl J Med 2018:379:11-21. It is understood that the rate of disease progression is dependent upon a number of factors including, but not limited to, the severity of disease in the subject at the initiation of treatment ,the duration of treatment, prior treatments, and the specific TTR mutation present, if any.

The dose of an RNAi agent that is administered to a subject may be tailored to balance the risks and benefits of a particul ardose, for example, to achieve a desired level of inhibition of TTR gene expression (as assessed, e.g., based on TTR mRNA expression, TTR protein expression, or a reduction in an amyloid deposit, as defined above) or a desired therapeutic effect, while at the same time avoiding undesirable side effects.

In one embodiment, an iRNA agent of the invention is administered to a subject as a "fixed dose" (e.g., a dose in mg) means that one dose of an iRNA agent is used for all subject sregardless of any specific subject-relate factd ors, such as weight.

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

In certai nembodiments, the double stranded RNAi agent is administered to the human subject about once per quarter to about once per year. In certai nembodiments, the double stranded RNAi agent is administered to the human subject about once per quarter, about once every six months, or about once per year.

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

In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg to about 600 mg. In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg or about 600 mg about once every six months to about once per year. In certain embodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 400 mg or about 600 mg about once every six months or about once per year.

In certai nembodiments, the double stranded RNAi agent is administered to the human subject at a fixed dose of about 700 mg to about 1000 mg or about 700 mg to about 900 mg. In certain embodiments, the double stranded RNAi agen tis administered to the human subject at a fixed dose of about 700 mg, about 800 mg, about 900mg, or about 1000 mg about once per year.

In certai nembodiments, the administration is subcutaneous administration, e.g., self- administration via, e.g., a pre-fille dsyringe or auto-inject orsyringe. In some embodiments, a dose of the RNAi agent for subcutaneous administration is contain edin a volume of less than or equal to one ml of, e.g., a pharmaceutical accly eptable carrier.

Any of these schedules may optional bely repeated for one or more iterations .The number of iterations may depend on the achievement of a desired effect, e.g., the suppression of a TTR gene, retinol binding protein level vita, min A level, or the achievement of a therapeuti ceffect, e.g., reducing an amyloid deposit or reducing a symptom of a TTR-associate disead se. In certain embodiments, the iRNA agent is administered chronically, for an indefinit eperiod of time, e.g., throughout the life of the patient.

In some embodiments, the RNAi agent is administered with othe rtherapeuti cagents or other therapeuti cregimens. For example, othe ragents or other therapeuti cregimens suitable for treating a TTR-associate diseased may include a liver transplant a heart, transplant impl, antation of a pacemaker, an agent which can reduce monome TTRr levels in the body; Tafamidi s(Vyndaqel® or Vyndamax® or) AGIO, which kineticall stay bilizes the TTR tetramer preventing tetramer dissociation required for TTR amyloidogenesi nonsts; eroidal anti-inflammat drugsory (NSAIDS), e.g., diflunisal, and diuretics, which may be employed, for example, to reduce edema in TTR amyloido siswith cardiac involvement.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an RNAi agent. The maintenance dose or doses can be the same or lower than the initial dose, e.g., one-hal off the initia ldose. Followi ngtreatment, the patient can be monitore dfor chang esin his/her condition.

In some embodiments of the methods of the invention, expression of a TTR gene as assessed by serum or plasm aTTR levels is inhibited by at least 85%, in some embodiments at least 90%. It is understood that inhibition of TTR expression using the iRNA agents provided herein would inhibi t expression of TTR in the liver and not substantially in othe rtissues, e.g., TTR expression in the eye.

The term "inhibiting," as used herein, is used interchangeabl withy "reducing," "silencing," "downregulati"ng, "suppressing", and othe rsimilar terms, and includes any level of inhibition. In some embodiments, inhibiting includes a statistical signily ficant or clinical signily ficant inhibition.

The phrase "inhibiting expression of a TTR" is intended to refer to inhibition of expression of any TTR gene including variants or mutants of a TTR gene. Thus, the TTR gene may be a wild-type TTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise to amyloid deposition). 26 Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with TTR expression compare dwith a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control). As used herein, inhibition of TTR expression is typical lyassessed by determining a TTR level in an appropriate sample (e.g., historical control sample, level determined in a normal sample or clinic trialal) or before treatment of the subject with an iRNA agent, such as those provided herein or in PCT publications WO2010048228, WO2013075035, and WO2017023660, or othe ragent, e.g., antisense oligonucleot agent,ide dicer substrat eagent ,that inhibit s the expression of TTR, see, e.g., WO2011139917 and WO2015085158; and after treatment with an iRNA agent provided herein. It is understood that the iRNA agents provided herein are durabl bute slow acting. Therefore ,the level of knockdown is determined after sufficient time to reac hnadir, e.g., at least 3 weeks after first dose of the iRNA agent in a human subject, or when steady state of TTR knockdown has been achieved, e.g., after multiple doses with an iRNA agent provided herein.

Inhibition of the expression of a TTR gene may be manifeste dby a reduction of the amoun oft mRNA expressed by a first cell or group of cells (such cell mays be present, for example, in a sampl e derived from a subject) in which a TTR gene is transcribed and which has or have been treated (e.g., by contacti ngthe cel orl cell wis th an RNAi agen tof the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the expression of a TTR gene is inhibited, as compared to a second cel orl group of cell substas ntially identical to the first cel orl group of cell buts which has not or have not been so treated (control cell(s)). In some embodiments, the percent inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells using, the following formula: (mRNA in control cells - )(mRNA in treated cell s) ------------------------------------------------------------------•100% (mRNA in control cells) A similar calculati mayon be performed on serum TTR protein concentrations, e.g., in a blood sample obtained froma subject ,to determine percent inhibition of expression. If no TTR is detected in the serum or plasm asampl eafter treatment ,the amoun oft TTR present is considered to be the at the lower limit of detection of the assay used.

In some embodiments, the percent inhibition is determined using a validated and clinically acceptable method.

Alternatively, inhibition of the expression of a TTR gene may be assessed in terms of a reduction of a parameter that is functiona lllinkedy to TTR gene expression, e.g., TTR protein expression, retinol binding protein level, vitamin A level or, presence of amyloid deposits comprising TTR. TTR gene silencing may be determined in any cell expressing TTR, either constitutively or by genomic engineering, and by any assay known in the art. The liver is the major site of TTR gene expression. Other significant sites of expression include the retina and choro idplexus. 27 III. iRNAs of the Invention Suitable iRNAs for use in the methods of the present invention include double stranded ribonucl eicacid (dsRNA) molecul fores inhibiting the expression of a TTR gene in a cell such, as a cel l within a subject, e.g., a mammal such, as a human having a TTR-associated disease. The dsRNA includes an antisense strand having a region of complementari whichty is complementary to at least a part of an mRNA formed in the expression of a TTR gene. The region of complementarit isy about 21- nucleotide ors less in lengt h(e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, or 21 nucleotides in length). Upon contact with a cell expressing the TTR gene, the iRNA selectively inhibits the expression of the TTR gene (e.g., a human, a non-human primate, or a non-primate mammal TTR gene) by at least about 70% as assayed by, for example, real time PCR using the method provided in Exampl e4 of WO2013075035 when Hep3B cell ares transfected with 10 nM of the iRNA agent using the method provided therein.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementari thatty is substantially complementary, and generall fully y complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a TTR gene. The othe rstrand (the sense strand) includes a region that is complementary to the antisense strand ,such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequence sof a dsRNA can also be contain edas self- complementary regions of a singl enucleic acid molecule as, opposed to being on separate oligonucleotide. s Generally, the duplex structure is 21 to 30 base pairs in length. Similarly, the region of complementari toty the target sequenc eis 22 and 30 nucleotides in length.

A dsRNA can be synthesized by standard methods known in the art as further discussed below. iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecul aree prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution- phase or solid-phase organi csynthesi sor both. Organic synthesis offers the advantage that the oligonucleoti strande ds comprising unnatural or modified nucleotides can be easily prepared. Single - stranded oligonucleotide of thes invention can be prepared using solution-phase or solid-phase organi c synthesi sor both.

IV. Modified iRNAs of the Invention The iRNA agents for use in the methods of the invention include defined chemical modifications in the sense and antisense strand. When the lengt hof either strand is extended to provide an antisense strand longe thanr 23 nucleotides and a sense strand longer than 21 nucleotides, the nucleotides can include modifications including, but not limited to, sugar modifications, backbone modifications, and base modifications.Modificat ionsinclude, for example, end modifications, e.g., 5’- 28 end modifications (phosphorylation, conjugation, inverted linkages or) 3’-end modifications (conjugation, DNA nucleotides, inverted linkage s,etc.־); base modifications, e.g., replacement with stabilizing bases ,destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of base s(abas icnucleotides), or conjugat edbases; sugar modifications (e.g., at the 2’-position or 4’-position) or replacement of the sugar; or backbo nemodifications, including modification or replacement of the phosphodiest erlinkages .Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containi ngmodified backbon ores no natural internucleoside linkages. RNAs having modified backbon incles ude, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA has a phosphorus atom in its internucleoside backbone.

Modified RNA backbone incls ude, for example, phosphorothioa chites,ral phosphorothioates, phosphorodithioates phosphotries, ters, aminoalkylphosphotriest meters,hyl and other alkyl phosphonat includinges 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidat includinges 3'-amino phosphoramidate and aminoalkylphosphorami dates, thionophosphoramidat thionoalkyes, !phosphonates, thionoalkylphosphotrie andster boranophos, sphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those having inverted polarit whey rein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Modified RNA backbone thats do not include a phosphorus atom therein have backbones that are formed by short chai nalkyl or cycloalkyl internucleoside linkages, mixed heteroatom ands alkyl or cycloalkyl internucleoside linkages, or one or more short chai nheteroatom icor heterocycl ic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformace tylbackbones; methylene formacetyl and thioformacet backyl bones alke; ne containi ngbackbones; sulfamat backbones;e methyleneimino and methylenehydrazino backbones; sulfonate and sulfonami debackbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

In othe rembodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotid unitse are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomer iccompound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particul aran aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

Some embodiments featured in the disclosure include RNAs with phosphorothioa backboneste and oligonucleosides with heteroatom backbones and, in particul ar— CH2-NH—CH2-, — CH2—N(CH3)— 29 O—CH2—[known as a methylene (methylimino) or MMI backbone —], CH2-O—N(CH3)—CH2—, — CH2- N(CH3)—N(CH3)—CH2— and — N(CH3)—CH2—CH2— of the above-referenc edU.S. Patent No. ,489,677, and the amide backbones of the above-referenc edU.S. Patent No. 5,602,240. In some embodiments, the RNAs featured herein have morpholi nobackbo nestructures of the above-referenc ed U.S. Patent No. 5,034,506. The native phosphodiester backbo necan be represented as O-P(O)(OH)- OCH2-.

Modified RNAs can also conta inone or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl O-,; S- or N-alkynyl; or O-alkyl-O-alkyl wherein, the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C! to Cw alkyl or C2 to C10 alkenyl and alkynyl. Exemplar ysuitable modifications include O[(CH2)nO] mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In othe rembodiments, dsRNAs include one of the followi ngat the 2' position: C! to Cw lower alkyl, substituted lower alkyl alkar, yl, aralkyl, O-alkaryl or O-aralkyl SH,, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl heteroc, ycloalkaryl amin, oalkylamin polyalkylamo, ino,substituted silyl, an RNA cleaving group, a reporter group, an intercalat or,a group for improving the pharmacokine tic properties of an iRNA, or a group for improving the pharmacodynam propeic rties of an iRNA, and other substituent shaving simila propertr ies. In some embodiments, the modification includes a 2'-methoxyethoxy (2'-O—CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-M0E) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethylaminoethoxye thylor 2'-DMAEOE), i.e., 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 in these three families); 2’-alkoxyalkyl; and 2’-NMA (N-methylacetamide).

Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropo xy(2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Simila modir fications can also be made at othe rpositions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobut moiyl eties in place of the pentofuranosyl sugar.

The RNA of an iRNA of the invention can also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natura" lnucleobas es include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uraci l(U). Modified nucleobases include othe rsynthetic and natura nuclel obas suches as deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethy cytosine,l xanthine, hypoxanthine, 2-aminoadeni ne,6-methyl and othe ralkyl derivatives of adenine and guanine, 2-propyl and other alky l derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraci andl cytosine, 5-propynyl uraci land cytosine, 6-azo uracil cytosi, ne and thymine, 5-uraci l(pseudouracil), 4- thiouracil, 8-halo 8-ami, no, 8-thiol, 8-thioalkyl, 8-hydroxyl anal othe r8-substituted adenine sand guanines, 5-halo, particularl 5-bromo,y 5-trifluoromethyl and othe r5-substituted uracil ands cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- daazaadeni andne 3-deazaguani neand 3-deazaadenine. Certain of these nucleobases are particula rly useful for increasin theg binding affinity of the oligomeri compoundsc featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine 5-propynyluracil, and 5-propynylcytosine 5-methyl. cytosine substitutions have been shown to increas nucleice acid duplex stabilit byy 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularl wheny combined with 2'-O-methoxyethyl sugar modifications.

An RNAi agent of the disclosur cane also be modified to include one or more bicyclic sugar moities. A "bicycl icsugar" is a furanosyl ring modified by a ring formed by the bridging of two carbons, whether adjacent or non-adjacent atoms. A "bicycl icnucleoside" ("BNA") is a nucleoside having a sugar moiety comprising a ring formed by bridging comprising a bridge connecting two carbons, whether adjacent or non-adjacent, two carbon atoms of the sugar ring, thereby forming a bicycl icring system. In certai nembodiments, the bridge connect thes 4’-carbon and the 2’-carbon of the sugar ring, optionally via, the 2’-acycli oxygenc atom .Thus, in some embodiments an agent of the disclosur maye include one or more locked nuclei acic ds (LNA). A locked nuclei acic d is a nucleotide having a modified ribose moiety in which the ribose moiety comprise san extra bridge connecti ngthe 2' and 4' carbons. In othe rwords, an LNA is a nucleotid compe rising a bicyclic sugar moiety comprising a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3'-endo structural conformation.

The addition of locked nucleic acids to siRNAs has been shown to increas siRNe A stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane 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 polynucleoti desof the disclosure include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms.

In certai nembodiments, the antisense polynucleot agentside of the disclosure include one or more bicycl icnucleosides comprising a 4' to 2' bridge.

A locked nucleoside can be represented by the structure (omitting stereochemistry), wherein B is a nucleobase or modified nucleobase and L is the linking group that joins the 2’- carbon to the 4’-carbon of the ribose ring. 31 Examples of such 4' to 2' bridged bicycl icnucleosides, include but are not limited to 4'- (CH2)—0-2' (ENA); 4׳-(CH2)—S-2'; 4׳-(CH2)2—O-2' (ENA); 4׳-CH(CH3)—0-2׳ (also referred to as "constrained ethyl" or "cEt") and 4׳-CH(CH2OCH3)—O-2' (and analogs thereof; see, e.g., U.S. Pat.

No. 7,399,845); 4׳-C(CH3)(CH3)—O-2' (and analogs thereof; see e.g., US Patent No. 8,278,283); 4׳- CH2—N(OCH3)-2׳ (and analogs thereof; see e.g., US Patent No. 8,278,425); 4׳-CH2—O—N(CH3)-2׳ (see, e.g.,U.S. Patent Publication No. 2004/0171570); 4׳-CH2—N(R)—O-2', wherein R is H, C1-C12 alkyl or, a nitrogen protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4׳-CH2—C(H)(CH3)-2׳ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4׳-CH2—C(=CH2)-2׳ (and analogs thereof; see, e.g., US Patent No. 8,278,426). The entire contents of each of the foregoin gare hereby incorporat hereined by reference.

Additional representative US Patent sand US Patent Publications that teac hthe preparation of locke nucleicd acid nucleotides include, but are not limited to, the followin USg: Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 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; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporat ed herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for exampl ea-L-ribofuranose and -D-ribofuranos (seee WO 99/14226).

An RNAi agent of the disclosur cane also be modified to include one or more constraine ethyld nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge (i.e., L in the preceding structure). In one embodiment, a constraine ethyld nucleotide is in the S conformati onreferred to herein as "S-cEt." An iRNA of the invention may also include one or more "conformational restricly ted nucleotides" ("CRN"). CRN are nucleotide analogs with a linker connecting the C2’and C4’ carbons of ribose or the C3 and -C5׳ carbons of ribose . CRN lock the ribose ring into a stable conformati onand increas thee hybridization affinity to mRNA. The linker is of sufficien tlengt hto place the oxygen in an optimal position for stabilit yand affinity resulting in less ribose ring puckering.

One or more of the nucleotides of an iRNA of the invention may also include a hydroxymethyl substituted nucleotide. A "hydroxymethyl substituted nucleoti"de is an acycli 2c’-3’-seco-nucleoti de, also referred to as an "unlocked nucleic acid" ("UNA") modification.

Potentiall stabily izing modifications to the ends of RNA molecul escan include N- (acetylaminocaproyl)-4-hydroxyprol (Hyp-inolC6-NHAc), N-(caproyl-4-hydroxyproli (Hyp-C6),nol N- (acetyl-4-hydroxyprol inol(Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4- hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3’- phosphat e,inverted base dT(idT) and others .Disclosure of this modification can be found in WO 2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5’ phosphate or 5’ phosphat mimie c, e.g., a 5’-terminal phosphate or phosphate mimic on the antisense strand of an RNAi 32 agent. Suitable phosphat mime ics are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporat hereined by reference.

V. iRNAs Conjugated to Ligands Another modificatio ofn the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugat esthat enhance the activity, cellular distribution or cellula uptar ke of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal.,Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manohara etn al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, :1111-1118; Kabano etv al., FEES Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glyce orrol triethyl-ammoni um1,2-di-O-hexadecyl- rac-glycero-3-phosphon (Manoharanate et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl.

Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chai n(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholes moietteroly (Crooke et al., J.

Pharmacol. Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule cell, or cel type,l compartment, e.g., a cellula or rorgan compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Exemplary ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligand scan also include targeting groups, e.g., a cel orl tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cel typel such as a kidney cell.

A targeting group can be a thyrotropin, melanotropi lectn, in, glycoprotein, surfactant protein A, Mucin carbohydrat multe, ivalent lactose, monovalent galactose N-ac, etyl-galactosa mineN-ac, etyl - gulucoseamine multivalent mannose, multivalent fucose, glycosylat polyed aminoaci ds,multivalent galacto se,transferrin, bisphosphonate, poly glutamat e,polyaspartate a lipid,, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, ligands include monovale ornt multivalent galactose In. certai nembodiments, ligands include cholesterol.

Ligand-conjugat oligonucled eotide of thes invention may be synthesized by the use of an oligonucleoti thatde bears a pendant reactive functionalit suchy, as that derived from the attachment of a linking molecule onto the oligonucleoti (descride bed below) .This reactive oligonucleoti mayde be reacted directly with commerciall availy able ligand s,ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attache theretd o. 33 The oligonucleotide useds in the conjugat esof the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Any othe rmeans for such synthesi sknown in the art may additional orly alternatively be employed. It is also known to use similar techniqu esto prepare other oligonucleotides, such as the phosphorothioa andtes alkylated derivatives.

A. Carbohydrate Conjugates In some embodiments of the compositions and methods of the invention, an iRNA oligonucleoti furtherde comprises a carbohydrat Thee. carbohydrate conjugat ediRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound which is either a carbohydra te per se made up of one or more monosaccharid unitse having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen ,nitrogen or sulfur atom bonded to each carbon atom ;or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharid unites eac hhaving at least six carbon atoms (which can be linea r,branched or cyclic) wit, h an oxygen, nitrogen or sulfur atom bonded to eac hcarbon atom .Representative carbohydrates include the sugars (mono-, di-, tri- and oligosacchar containiides ngfrom about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysacchari suchdes as starches, glycogen, cellulose and polysacchari gums.de Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars ;di- and trisaccharides include sugars having two or three monosaccharid unitse (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosacchari Inde. anothe embor diment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of: 34 36 O Formula XVI, O Formula XVIII, 37 In one embodiment, the monosacchari is dean N-acetylgalactosa minesuch ,as Another representativ ecarbohydrate conjugate for use in the embodiments described herein includes, but is not limited to, 38 HO Z°H AcHN HO ZOH (Formula XXIII), when one of X or ¥ is an oligonucleotide, the othe ris a hydrogen.

In certai nembodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNA c derivative is attache tod an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agen tof the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In anothe embor diment, the double stranded RNAi agents of the invention compris ea pluralit (e.g.,y 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independentl atty ache tod a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chai nof nucleotides between the 3’-end of one strand and the 5’-end of the respective othe rstrand forming a hairpin loop comprising, a plurali ty of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently compris ea GalNAc or GalNAc derivative attache viad a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the carbohydrate conjugate further comprises one or more additiona l ligands as described above, such as, but not limited to, a PK modulato orr a cel permeatl ion peptide.

Additional carbohydrate conjugat essuitable for use in the present invention include those described in PCT Publicati onNos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporate hereind by reference.

B. Linkers In some embodiments, the conjugate or ligand described herein can be attache tod an iRNA oligonucleoti wideth various linkers that can be cleavable or non-cleavable.

The term "linker "or "linking group" means an organi moiec ty that connect twos parts of a compound, e.g., covalentl attyaches two parts of a compound. Linkers typical lycomprise a direct bond or an atom such as oxygen or sulfur ,a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chai nof 39 atoms, such as, but not limited to, substituted or unsubstituted alkyl subs, tituted or unsubstituted alkenyl subs, tituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalke nyl,heteroarylalkynyl, heterocyclylal hetekyl,rocyclylalke heterocyclnyl, ylalkynyl, aryl , heteroaryl heterocyclyl,, cycloalky cycloalkl, enyl, alkylarylalkyl alkyl, arylalken alkylarylyl, alkynyl, alkenylarylal alkenylarylalkenyl,kyl, alkenylarylalkynyl alkynylarylal, alkynylkyl, arylalke nyl, alkynylarylalkyn alkylheteroarylalkyl,yl, alkylheteroarylal kenyl,alkylheteroarylal kynyl, alkenylheteroarylalkyl, alkenylheteroarylal alkenylkenyl, heteroarylalkyny alkynl,ylheteroarylalkyl , alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylal alkylhetkyl, erocyclyla lkenyl, alkylhererocyclylal alkenylhetkynyl, erocyclyla alkenylheterocyclylalkenyl,lkyl, alkenylheterocyclylalkynyl, alkynylheterocyclylal alkynylheterocyclylalkenyl,kyl, alkynylheterocyclylalkynyl, alkylaryl alke, nylaryl alkyny, laryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroa whichryl, one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycli wherec; R8 is hydrogen, acyl, aliphati orc substituted aliphati c.In one embodiment, the linker is between about 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.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cel isl cleaved to release the two parts the linker is holding together. In one embodiment, the cleavable linking group is cleaved at leas aboutt 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cel orl under a first reference condition (which can, e.g., be selected to mimic or represent intracellul condiar tions than) in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavabl linkinge groups are susceptibl toe cleavage agents ,e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include : redox agents which are selected for particul arsubstrate sor which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptan press, ent in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment e.g.,, those that result in a pH of five or lower; enzymes that can hydrolyz e or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellul pHar is slightl lowey r, ranging from about 7.1-7.3.

Endosome haves a more acidic pH, in the rang eof 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a selected pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particul arenzyme. The type of cleavable linking group incorporat edinto a linker can depend on the cel tol be targeted. For 40 example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cell ares rich in esterases ,and therefore the linker wil lbe cleaved more efficiently in liver cells than in cel ltypes that are not esterase-rich. Other cell-types rich in esterases include cell ofs the lung, renal cortex, and testis.

Linkers that contai peptin de bonds can be used when targeting cel typesl rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluate byd testing the abilit ofy a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the abilit toy resist cleavage in the blood or when in contact with othe rnon-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cel andl the second is selected to be indicative of cleavage in othe rtissues or biological fluids , e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cel culture,l in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-fre ore culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate compounds are cleaved at leas aboutt 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cel (orl under in vitro conditions selected to mimic intracellul conditar ions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellul condiar tions). i. Redox cleavable linking groups In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductivel ycleavable linking group is a disulphide linking group (-S-S-). To determine if a candidate cleavab linkingle group is a suitable "reductivel ycleavable linking group," or for example is suitable for use with a particular iRNA moiety and particul artargetin g agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or othe rreducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cel l.The candidat escan also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the bloo d.In othe rembodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cel (orl under in vitro conditions selected to mimic intracellul conditar ions) as compared to blood (or under in vitro conditions selected to mimic extracellul conditiar ons). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellul mediaar and compare dto conditions chosen to mimic extracellul mediaar . ii. Phosphate-based cleavable linking groups In certai nembodiments, a cleavable linker comprises a phosphate-base clead vable linking group. A phosphate-base clead vable linking group is cleaved by agents that degrade or hydrolyze the phosphat group.e An example of an agent that cleaves phosphate groups in cell ares enzymes such as phosphata sesin cells. Examples of phosphate-based linking groups are -O- 41 P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S- P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O-, -S-P(O)(Rk)-S-, -O-P(S)( Rk)-S, wherein Rk at eac hoccurrence can be, independentl y, C1-C20 alkyl, C1-C20 haloalkyl C6-C10, aryl or, C7-C12 aralkyl. Exemplar yembodiments include - O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S-P(O)(OH)- S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-, - S-P(O)(H)-S-, and -O-P(S)(H)-S-. In one embodiment, a phosphate-base linkid ng group is -O- P(O)(OH)-O-. These candidat escan be evaluate usingd methods analogous to those described above.

Hi. Acid cleavable linking groups In certai nembodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles such, as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). An exemplary embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluate usind g methods analogous to those described above. iv. Ester-based linking groups In another embodiment, a cleavable linker comprises an ester-based cleavable linking group.

An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cell s.

Examples of ester-based cleavable linking groups include but are not limited to esters of alkylen e, alkenyle neand alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O-, or -OC(O)-. These candidat escan be evaluated using methods analogous to those described above. v. Peptide-based cleaving groups In yet anoth erembodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc?) and polypeptides .Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenyle neor alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavab linkingle groups have the general formula - NHCHRAC(O)NHCHRBC(O)-, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above. 42 In one embodiment, an iRNA of the invention is conjugat edto a carbohydrate through a linker.

Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositio nsand methods of the invention include, but are not limited to, (Formula XXVII), 43 (Formula XXVIII), (Formula XXIX), (Formula XXXI), when one of X or ¥ is an oligonucleotide, the othe ris a hydrogen.

In certai nembodiments of the compositions and methods of the invention, a ligand is one or more "GalNAc" (N-acetylgalactosam derivativesine) attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugat edto a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII) - (XXXV): 44 Femtila XXXII Fomala XXXIII Formul aXXXIV wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentl fory eac hoccurrence q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independentl fory each occurrence 0-20 and wherein the repeating unit can be the same or different; p2A p2B p3A p3B p4A p4B p5A p5B p5C p2A p2B p3A p3B p4A p4B p4A 56ך־י p5C each independentl fory eac hoccurrenc absente CO,, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH,O; q2a q2b q3a q3b q4a q4b, q5a q5b q5c independentl fory each occurrence absent alkylene,, substituted alkylene wherin one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R’)=C(R"), C=C or C(O); R2A, R2b, R3a, R3b, R4a, R4b, R5a, R5b, R5c are eac hindependentl fory each occurrence absent NH, , O, S, O ho-L H 1 CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=N-O, O _ _ R—R s—s or heterocyclyl; L2a, p2B, l3a؛ p3B, £،4a־ p4B, j^5a, j^5b a1K، j^5c 1ep1esent the ligand; i.e. eac hindependentl fory eac hoccurrence a monosacchari (suchde as GalNAc) disac, charide, trisacchari de,tetrasaccharid e, oligosacchar oride, polysaccharide andR; a is H or amino acid side chain.Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a targe t gene, such as those of formula (XXXVI): 45 Formula XXXVI -|5־A |_5A 5B-|5־B [_5B ____ t5C I 5C q5c י wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugati ngGalNA c derivatives include, but are not limited to, the structures recited above as formula II,s VII, XI, X, and XIII.

Representative U.S. patent sthat teac hthe preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; ,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; ,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; ,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; ,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of eac hof which are hereby incorporat hereined by reference.

It is not necessary for all positions in a given compound to be uniforml ymodified, and in fact more than one of the aforementioned modifications can be incorporat edin a single compound or even at a singl enucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

VI. Delivery of an iRNA of the Invention The delivery of an iRNA of the invention to a cel e.g.,l a cel witl hin a human subject (e.g., a subject in need thereof, such as a subject having a disease ,disorder or condition associated with contact activation pathway gene expression) can be achieved in a number of different ways . For example, delivery may be performed by contacti nga cel wil th an iRNA of the invention either in vitro or in vivo.

In vivo delivery may also be performed directly by administerin ga composition comprising an iRNA, e.g., a dsRNA, to a subject. Delivery may be performed, for example, by intravenous administration or subcutaneous administration. In certain embodiments, the iRNA agent is delivered by subcutaneous administration. In certain embodiments, the iRNA agent is administered by self-administrati onusing a pre-filled syringe or auto-inject ordevice.

In general any, method of delivering a nucleic acid molecu le(in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julia nRL. (1992) Trends Cell. Biol. 46 2(5): 139-144 and WO9402595, which are incorporat edherein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecu leinclude, for example, biological stability of the delivered molecul preventione, of non-specific effects, and accumulati ofon the delivered molecule in the target tissue.

VII. Pharmaceutical Compositions of the Invention The present invention also includes pharmaceutical compositions and formulations which include the iRNAs described herein for use in the methods of the invention. In one embodiment , provided herein are pharmaceutical compositions containi ngan iRNA, as described herein, and a pharmaceutical accly eptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associate wid th the expression or activity of a TTR gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parentera delil very, e.g., by subcutaneous (SC) or intravenous (IV) delivery. The pharmaceutical compositions of the invention may be administered in dosages sufficien tto inhibit expression of a TTR gene. In one embodiment, the iRNA agents of the invention, e.g., a dsRNA agent ,is formualted for subcutaneous administration in a pharmaceutical accly eptable carrier In othe rembodiments, a singl edose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administere dat 1 month intervals at, not more than 1, 2, 3, or 4 month intervals or, at quarterl (abouty every three months) intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositio nsof the invention is administered once per month .In othe rembodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered once every othe rmonth . In othe rembodiments, a single dose of the pharmaceutical compositions of the invention is administered quarterl y,i.e., about once every three months .In othe r embodiments, a singl edose of the pharmaceutical compositions of the invention is administered once every four months. In anothe embr odiment, a singl edose of the pharmaceutical compositions of the invention is administered once every five months. In othe rembodiments, a single dose of the pharmaceutical compositions of the invention is administered once every six months. In othe r embodiments, a singl edose of the pharmaceutical compositions of the invention is administered once every twelve months.

VIII. Kits The present invention also provides kits for performing any of the methods of the invention.

Such kits include one or more double stranded RNAi agent(s) and a label providing instructions for use of the double-stranded agent(s) for use in any of the methods if the invention. The kits may optionall y further compris emeans for contacti ngthe cel wil th the RNAi agent (e.g., an injection device or an infusion pump), or means for measuring the inhibition of TTR (e.g., means for measuring the inhibition of TTR mRNA or TTR protein). Such means for measuring the inhibition of TTR may comprise a 47 means for obtaining a sample from a subject, such as, e.g., a plasm asample. The kits of the invention may optional furtherly comprise means for administering the RNAi agent(s) to a subject or means for determining the therapeuticall effectiy ve or prophylacticall effectiy ve amount.

The RNAi agent may be provided in any convenient form, such as a solution in sterile water or othe rappropriate solutio n,e.g., PBS, normal saline, 5 mM phosphat buffer,e for resuspension and injection. For example, the RNAi agent may be provided as a 300 mg, 200 mg, 100 mg, or 50 mg vial with water or othe rappropriate solution in sterile water for injection. In certain embodiments, the RNAi agent is provided in a kit for self administration comprising a pre-filled syringe or autoinjector containi ng300mg, 200 mg, 100 mg, or 50 mg of the RNAi agent in an appropriate volume of excipien t for administratio n,optionall furtyher including instructions for use. In certain embodiments, the RNAi agent may be provided in multiple vial sor devices for administration of the dose by multiple injections to be given at about the same time, e.g, within one week, within one day, within one hour.

IX. Diagnosis of TTR amyloidosis polyneuropathy and assessment of disease burden TTR amyloido sisis a complex, multifactoria diseal se. An expanding list of criteria have been used to monitor the progression of TTR-FAP: neuropathy impairment score (NIS), NIS + 7, and modified NIS (mNIS) + 7 and mNIS + 71oniS. These diagnosti cric teria are wel lknown in the art and highlights of the criteria are provided below .As used herein, meeting the diagnosti cric teria of TTR- FAP is understood as meeting FAP stage 1 criteria, with or without the presence of a mutation associate wid th hereditary TTR-FAP. Progression of indicators of neuropathy is considered an increase of at least two points in modified neuropathy impairment score (mNIS) + 7.

Familial Amyloid Polyneuropathy (FAP) Stage Coutinho et al. developed a clinica staginl gsystem for the neuropathy symptoms of hATTR (formerly termed familial amyloid neuropathy). The scal rangese from 1 to 3, as follo ws(Ando et al.

Orphanct J Rare Dis. 2013;8:31): FAP Stage 1: Walking without assistance mi, ld neuropathy (sensory, autonomic, and motor )in lower limbs.

FAP Stage 2: Walking with assistance moderate, impairment in lower limbs, trunk, and upper limbs.

FAP Stage 3: wheelchair or bed-ridden, severe neuropathy.

A subjec twith no neuropathy is considered to be FAP Stage 0.

Neuropathy Impairment Scoring Methods Methods to assess neuropathies are known in the art. For example, in the Mayo Clinic Neurologi Examic nation Sheet and also in the weaknes ssubscores of Neuropath yImpairment Score (NIS), weakness (NIS-W) are scored in 25% decrements from 1 to 4 points and separately for major muscle groups of each side of the body (Dyck et al., Quantitating overal neuropathicl symptoms, impairments, and outcomes. In: Dyck PJ, Thomas PK, editors. Periphera lneuropathy. 4th ed.

Philadelphia: Elsevier; 2005. p. 1031-52). A broad group, especial lyof cranial proxima, l, and distal limb muscles, is evaluated in NIS-W with a maximum score of 192 points. A decreas eof the major 5 48 muscle stretch reflexes is usuall assy essed by neurologists and touch pressure, vibration, joint motion, and pin-prick sensation ofs feet and hands are scored in 25% decrements and from 1 to 4 in the Mayo Clinic Neurology Examination Sheet. To complete NIS of reflexes (NIS-R) and of sensation (NIS-S) Mayo Clinic record score sare transformed to NIS point score s(i.e., Mayo Clinic scores of 1 or 2 are given an NIS point score of 1 and Mayo Clinic scores of 3 or 4 are given an NIS score of 2). Therefore, the maximal NIS score sof the usual reflexes evaluated by neurologist (NISs -R) are 5 x 2 x 2 = 20 points and of the 4 modalities of sensation often evaluate byd neurologists (NIS-S) are 8 x 2 x 2 = 32 points. Therefore ,the maximum NIS score is: 192 + 20 + 32 =244 points. The NIS has been described in previous publications (Dyck et al. 2005 and Dyck et al., Neurol. 1997;49:229-39).

NIS + 7 has been used as the primary or co-primary outcome measure in the trials of diabetic sensorimotor polyneuropath TTRy, FAP, and othe rgeneralized sensorimotor polyneuropathies (N.

Suanprasert et al. J Neurol Sci 344 (2014) 121-128). NIS + 7 adequately assesses graded severities of muscle weaknes sand muscl estretch reflex abnormalit wiyth only minima lceiling effects for reflexes.

In NIS + 7, 5 of the 7 tests are attributes of nerve conduction — expressed either as normal deviates (Z scores) or points. The attributes included in NIS + 7 were chosen because their abnormal sensity itivel y detects diabetic sensorimotor polyneuropat hy(Dyck et al. Muscl eNerve 2003;27(2):202-10). The attributes included are the peroneal nerve compound muscle action potential (CMAP) amplitude, motor nerve conduction velocity (MNCV), and motor nerve distal latency (MNDL), tibia lMNDL and sural sensory nerve action potential (SNAP) amplitudes. Their measured values can be transforme dto normal deviates from percentile values correcting for applicabl varie able ofs age, gender, height, or weight as based on earlier studies of a large healthy subject reference cohort. Additional ly,these percentil valuese can be expressed as NIS points from obtained percentil valuese (i.e., N5th = 0 points; <5th- N 1st = 1 point and Assessment of weakness and reflex abnormal itassy, essment of sensation loss, autonomic dysfunction, and neurophysiologic test abnormalit areies not adequately assessed by NIS + 7 for use in trials of TTR FAP. In NIS + 7 sensation loss is not optimal lyassessed: 1) body distribution of sensation loss is not adequately taken into account, 2) large as compare dto smal fiberl sensory loss is over emphasized and 3) improved methods of testing and comparison to reference values are preferred over clinic alassessments .Also ,autonomic dysfunction is not adequately assessed by the use of only heart rate deep breathing (HRdb). The attributes of nerve conduction used to assess NIS + 7 are not ideal for the study of TTR FAP.

Modified neuropathy impairment score +7 (mNIS+7), and updated version of NIS + 7, is a composite score measuring motor strength, reflexes, sensation, nerve conduction, and autonomic function. Two versions of this composit emeasure were adapted from the NIS+7 to better reflect hATTR amyloido siswith polyneuropat hyand have been used as primary outcomes in inotersen and patisira n clinic trialal s. Key differences between these two versions, and the othe rneuropathy scoring systems, are summarized in the table below (from Adams et al., BMC Neurology, volum e17, Article number : 181 (2017)). In both scales, a lowe rscore represents better neurologic function (e.g. an increas ine score reflect sworsening of neurologic impairment). 49 Neuropathy Impairment Score Criteria NIS+7 mNIS+7Ionis MS I I NIS m-NlS+7 Total score 88 244 270 304 346.3 Assessment (score) Neurologic exam Neurologic Motor Neurologic exam Neurologic exam [lowe r exam Neurologic exam (192) strength/weakness (192) (192) limbs (192) only] (64) Neurologic exam Neurologic Neurologic exam Neurologic exam Reflexes [lowe r Neurologic exam (20) exam (20) (20) (20) limbs only] (8) QST - heat pain QST - heat pain and touch and touch pressure pressure at at multiple sites multiple sites (80) (80) Neurologic Sensation exam Neurologic Neurologic exam - [lowe r Neurologic exam (32) exam (32) (32) limbs Only] (16) Vibration detection threshold - - - (3.7) 2:5 - ulnar CMAP S5 - ulna CMAPr S5 - sural SNAP/ fibular nerve and SNAP, CMAP. tibia lmotor nerve distal and SNAP؛ Composite nerve latenc motory, nerve conduction - - peronealc CMAP, peroneal0 CMAP, conduction seore velocity motor, nerve distal tibial CMAP, tibial CMAP, sural latenc (18.6)y a sural SNAP (10) b SNAP (18.6)3 Heart rate Heart rate response to deep Postural blood Autonomic - - response:to deep pressure (2) function breathing (3.7)a breathin (3.7)g a 1. CMAP compound muscle action potential exa; m examination; mNTS+7 modified NIS+7; NIS Neuropathy Impairment Score; NIS-LL NIS based on examination of lower limbs only; QST quantitative sensory testing; SNAP sensory nerve action potential 2. 3Score expressed as normal deviates (0-3.72) based on healthy-subjec part ameters 3. ',Score graded according to defined categories: normal (95th percentile =) 0 points; mildly reduced (>95thto <99th percentile) = ! point; and very reduced (>99th percentile) = 2 points 4. cMay also be referred to as fibular Diagnosis of TTR amyloidosis cardiomyopathy (ATTR-CM) and assessment of disease burden Patients with hATTR amyloido sisand cardiomyopathy typically experience progressive symptoms of heart failure (HF) and cardiac arrhythmias, with death typical lyoccurring 2.5 to 5 years after diagnosis. Cardiac infiltration of the extracellul matar rix by TTR amyloid fibrils leads to a progressive increas ofe ventricula wallr thickness and a marked increas ine chambe stir ffness, resulting in impaired diastoli functic on. Systolic function is also impaired, typical lyreflected by abnormal longitudin alstrain despite a normal ejection fraction, which is preserved until late stages of the disease.

In patients with ATTR amyloidosi ands light-chai (AL)n cardiac amyloidosi boths, longitudin alstrain and N-terminal prohormone of brain natriureti cpeptide (NT-proBNP) have been shown to be independent predictors of survival.

Echocardiography is routinely used to assess cardiac structure and function; parameter spre- specified in the statistical analysi plans include mean left ventricula (LV)r wall thickness, LV mass , longitudin alstrain, and ejection fraction. Cardiac output, left atrial size, LV end-diastolic volume 50 (LVEDV), and LV end-systol icvolume (LVESV). Echocardiograms are routinely used for cardiac imaging. Myocardial strain can be assessed with speckl etracking using vendor-independe ntsoftware (TOMTEC, Munich, Germany). Analysis of NT-proBNP and troponi nI levels is routinely performed in clinic laboratal ories using commerciall availy able diagnostic tests, e.g., using chemiluminescence assays (Roche Diagnost icCobas, Indianapolis IN,, USA for NT-proBNP; Siemens Centaur XP, Camberley, Surrey, UK for troponi nI). Similarly, clinical practice routinely includes measurement of creatinine levels and estimated glomerul arfiltration rate (eGFR) based on creatinine level s,e.g., using the Modification of Diet in Renal Disease study formula.

A review providing screening and diagnostic methods for ATTR-CM was recently published by Witteles et al., 2019 (JACC: Heart Failure, 2019. 7:709-716) which provides information on methods of diagnos isincluding a list of "red flags" suggesting the presence of ATTR-CM and screening methods including echocardiography, electrocardiogra phy,cardiac magnetic resonance, the presence of systemic symptoms involving the peripheral or autonom icnervous system along with cardiac dysfunction including bilateral sensory motor polyneuropat hythat begins in the lower limbs and follo wsan ascending pattern, dysautonom iain the form of orthostati hypotension,c diarrhea/ constipation, and erectile dysfunction, and eye involvement such as glaucoma, intravitreal deposition, and scalloped pupils; carpal tunnel syndrome ,especial lybilateral carpal tunnel syndrome, lumbar spinal stenosis, and bicep tendon rupture. Other diagnostic methods include bone scintography with technetium (Tc)- labelled bisphosphonates localize tos TTR cardiac amyloid deposits for reasons that are not known.

Biopsy is also used to confirm the presence of TTR amyloido sisin heart.

Methods for assessment and classificat ionof cardiac function of the parameter sprovided above are known in the art. As used herein, the specific method of assessment or classificati ofon cardiac function may be any clinicall accy eptable standard to demonstrate sufficiently decreased cardiac function such that the standard of care includes a medical intervention, e.g., administration of a pharmacological agent ,surgery.

Serum Biomarkers as an Indicia of Nerve Damage and TTR Amyloidosis Progression The diagnosti cand monitoring methods set forth above are complex and often subjective.

Moreover, as TTR amyloidos isis rare, and the signs and symptoms above can be present in a number of othe rdiseases ,clinical validaly ted, non-invasive plasm abiomarkers may facilitate earlier diagnos isand aid monitoring of disease progression. In a study by Tica uet al., 2019 (see www.medrxiv.org/content/10.1101/19011155v2.full.pdf) plasm alevels of >1000 proteins were measured in patients with hATTR amyloido siswith polyneuropat hywho received either placebo or patisiran in the phase 3 APOLLO study (NCT01960348) and in a cohort of healthy individual s.The impact of treatment with patisiran, a lipid formulate RNAid agent that inhibits the hepati cexpression of TTR, on the time profile of each protein was determined by a linea mixer d model at 0, 9, and 18 months. Neurofilame ntlight chai n(NfL) protein was further assessed using an orthogonal quantitati ve approach. A significant change in the levels of 66 proteins was observed with patisiran vs placebo, with change in NfL, a marker of neuronal damage, most significant Analysis. of the changes in protein level s 51 demonstrated that the proteome of patients treated with patisira ntrended towards healthy individual ats 18 months. Plasma NfL levels in healthy controls were four-fold lower than in patients with TTR amyloido siswith polyneuropat hy(16.3 [SD 12.0] pg/mL vs 69.4 [SD 42.1] pg/mL, p< 10 16). Levels of NfL at 18 months increased with placebo (99.5 [SD 60.1] pg/mL) and decreased with patisiran treatment (48.8 [SD 29.9] pg/mL). At 18 months, improvement in modified Neuropathy Impairment Score+7 (mNIS+7) in patisiran-treat edpatients significantly correlat edwith a reduction in NfL level s (R=0.43, p<10 7). NfL reduction with patisiran treatment correlat edwith improvement in mNIS+7 suggests it may serve as a biomarker of nerve damage and polyneuropat hyin TTR amyloidosi Thiss. biomarker may enable earlier diagnos isof polyneuropat hyin patients with hATTR amyloidos andis facilitate monitoring of disease progression.

A decrease in the level of othe rproteins ,especiall RSPO3,y CCDC80, EDA2R, and NT- proBNP, were found to correlat wie th an improvement in mNIS+7, suggesting that, either alone or in combinati onwith eac hothe ror Nfl ,they may serve as a biomarker of nerve damage and polyneuropat hyin ATTR amyloidosi Ans. increas ine the level of N-CDase was found to correlat wite h an improvement in mNIS+7, suggesting that, either alone or in combinati onwith the other markers listed above, it may serve as a biomarker of nerve damage and polyneuropat hyin ATTR amyloidosis.

Further potential biomarkers are listed in the tabl belowe which were observed to change in response to treatment with patisiran. When a subject has an increas ine the level of a protein having a positive beta coefficient relative to a reference level indicative of progression of ATTR amyloidosis, and when a subject has a decrease in the level of a protein having a negative beta coefficient relative to a reference level indicative of progression of ATTR amyloidosi Changess. in the level of one or more of these markers can be indicia of improvement, stabilizatio orn, decrease in ongoing nerve damage and progression of polyneuropat hyin ATTR amyloidosis. 52 Table 1. Biomarkers with changed levels in response to patisiran treatment Protein -loglO (p-value) -!or10 (p-value) beta coeffident Protein beta coefficient 0.55 20.40 LDLR -0.20 5.85 M.

RSPO3 0.53 18.43 NOV 0.19 5.83 CCDC80 0.40 17.28 CD160 0.16 5.69 0.25 EDA2R 17.10 P13 0.19 5.61 -0.25 15.17 SMOC1 0.12 5.61 N-CPsse 0.62 13.15 TFPf-2 0.22 5.54 NT-9rQ^P. 11.43 0.46 LEP -0.38 5.49 WNT9A 0.20 10.78 TNFRSF19 0.16 5.47 SMOC2 0.14 10.33 ERB83 -0.07 5.44 PTN 0.39 9.20 DUA -0.14 5.43 HGF 0.17 9.02 IL-4RA 0.13 .33 0.14 6.13 REN -0.24 5.31 NELLI -0.16 8.98 CES2 -0.15 5.23 DCN CR2 0.10 8.90 0.15 5.23 ARSA -0.23 8.86 PSG1 0.21 5.20 KLK4 0.27 8.66 FGF-BP1 0.11 5.19 GPC1 8.57 0.18 OPN 0.18 .18 SFRP 0.21 8.41 TFF3 0.17 5.08 DRAXIN 0.20 8.11 ANGPT2 0.15 5.05 IL-18R1 -0.15 7.82 CCL24 -0.17 5.05 BNP 0.48 7.81 LAYN 0.13 5.02 GFR-alphal 0-17 7.69 FUCA1 -0.11 4.97 CXCL9 0.31 7.42 AXL 0.11 4.95 TIMD4 -0.21 7.02 TLR3 -0.10 4.94 RSPO1 0.16 6.94 SCARB2 0.12 4.90 6.94 MYOC 0.21 B4GAT1 -0.09 4.83 WFDC2 0.11 6.76 SORCS2 0.15 4.65 GUSB -0.27 6.71 CD300E 0.18 4.63 HSPB6 0.19 6.70 CD27 0.10 4.62 MFGE8 -0.21 6.68 SPON1 0.10 4.58 SMPD1 -0.15 6.59 IGFBP-2 0.15 4.58 FLT1 0.09 6.29 DNER -0.07 4.57 RARRES1 CTSD -0.13 6.06 0.09 4.57 CO209 -0.11 5.85 Dkk-4 0.15 4.49 Sequences for these biomarkers are provided herein as SEQ ID NOs: 17-34.

This invention is further illustrated by the followin examg ples which shoul notd be construe das limiting. The contents of all references and published patent sand patent applications cited throughout the application, as well the Sequence Listing are hereby incorporat edherein by reference. 53 EXAMPLES Exemplar ydouble straded RNAi agents for use in the methods of the invention are provided in Table 3 below .Tabl 2,e below, provides the abbreviation ofs the nucleotid monomere sand ligands used in nucleic acid sequenc erepresentation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiest erbonds unless otherwise indicated.

Table 2. Abbreviations of nucleotide monomers and ligands Abbreviation Nucleotide(s) and ligands A Adenosine- ’3 -phosphate Af 2 ’ -fluoroadenosine-3 ’ -phosphate Afs 2 ’ -fluoroadenosine-3 ’ -phosphorothioate As adenosine-3 ’ -phosphorothioate C cytidine-3 ’ -phosphate Cf 2 ’ -fluorocytidine-3 ’ -phosphate Cfs 2 ’ -fluorocytidine-3 ’ -phosphorothioate Cs cytidine-3 ’ -phosphorothioate G guanosine -3’ -phosphate Gf 2 ’ -fluoroguanosine-3 ’ -phosphate Gfs 2 ’ -fluoroguanosine-3 ’ -phosphorothioate Gs guanosine -3’ -phosphorothioate T ’ -methyluridine- ’3 -phosphate Tf 2 ’ -fluoro-5 -methyluridine-3 ’ -phosphate Tfs 2 ’ -fluoro-5 -methyluridine-3 ’ -phosphorothioate Ts 5-methyluridine- ’ 3-phosphorothioate U Uridine-3 ’ -phosphate Uf 2 ’ -fluorouridine-3 ’ -phosphate Ufs 2 ’ -fluorouridine -3 ’ -phosphorothioate Us uridine -3’-phosphorothioate N any nucleotide (G, A, C, T or U) a 2'-O-methyladenosine-3 ’ -phosphate as 2'-O-methyladenosine-3 ’ - phosphorothioate c 2'-O-methylcytidine-3 ’ -phosphate cs 2'-O-methylcytidine-3 ’ - phosphorothioate 2'-O-methylguanosine-3 ’ -phosphate g 2'-O-methylguanosine-3 ’ - phosphorothioate gs t 2 ’ -O-methyl-5-methylthymine-3 ’ -phosphate ts 2’-O-methyl-5-methylthymine-3’-phosphorothioate u 2'-O-methyluridine-3 ’ -phosphate 54 Abbreviation Nucleotide(s) and ligands us 2'-O-methyluridine-3 ’ -phosphorothioate s phosphorothioat linkae ge N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol L96 Hyp-(GalNAc-alkyl)3 HO /0H ——0 H H HO AcHN 0 1 H0'- H° <°H CL ho AcHN Q 0 00 ^־ HC^ J AcHN H H (Agn) Adenosine-glycol nucleic acid (GNA) (Cgn) Cytidine-glycol nucleic acid (GNA) (Ggn) Guanosine-glycol nucleic acid (GNA) thymidine-glycol nucleic acid (GNA) S-Isomer (Tgn) Table 3. Modified nucleotide sequences of sense and antisense strands of RNAi agents targeted to TTR SEQ ID Duplex ID Strand Modified Oligonucleotide Sequence (5’-3’) NO: AD-65492 sense usgsggauUfuCfAfUfguaaccaagaL96 10 antisense usCfsuugGfuuAfcaugAfaAfucccasusc 11 AD-87400 sense usgsggauUfuCfAfUfguaaccaagaL96 10 antisense usCfs(Tgn)ugGfuuAfcaugAfaAfucccasusc 12 AD-87401 sense usgsggauUfuCfAfUfguaaccaagaL96 10 antisense usCfsu(Tgn)gGfuuAfcaugAfaAfucccasusc 13 AD-87402 sense usgsggauUfuCfAfUfguaaccaagaL96 10 antisense usCfsuu(Ggn)GfuuAfcaugAfaAfucccasusc 14 AD-87403 sense usgsggauUfuCfAfUfguaaccaagaL96 10 antisense usCfsuug(Ggn)uuAfcaugAfaAfucccasusc AD-87404 sense usgsggauUfuCfAfUfguaaccaagaL96 10 antisense usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc 7 AD-87405 sense usgsggauUfuCfAfUfguaaccaagaL96 10 antisense usCfsuugGfu(Tgn)AfcaugAfaAfucccasusc 16 55 Example 1: TTR Protein Knockdown by RNAi agents in the V30M Transgenic Mouse The V30M mutation is a common amyloidogeni mutc ation in human TTR. Transgenic mice lacking mouse TTR and expressing human TTR with a V30M mutation were used in the study. Mice (n = 3 per group) were administered a singl esubcutaneous 1 mg/kg dose of RNAi agent AD-65492, previousl ydisclosed in WO2018112320, or othe rRNAi agents based on the sequenc eand chemistry of AD-65492 in which a chemical modificatio atn a single position of the antisense strand was changed to a GNA modification as shown in Table 2 above. Bloo sampld es were obtained on days 0 (pre-dose), 3, 7, 10, 14, 21, 35, and 49. Serum was prepared and human TTR level swere determined using ELISA assay (see, e.g., Coelho, et al. (2013) N Engl J Med 369:819). Relative TTR levels as compared to Day 0 are shown in Figure 1.

Incorporation of a GNA at position 7 or 8 in the antisense strand was wel ltolerated (AD-87404 and AD-87405). Simila kiner tics and maximum protein knockdow wern e simila tor the parent RNAi agent AD-65492. Durability of knockdow byn AD-87404 was simila tor AD-65492. Incorporati onof a GNA at positions 3-6 in the antisense strand was less well tolerated.

Example 2: TTR Protein Knockdown by RNAi agents in Non-Human Primate The sequenc eof the iRNA agents in Tabl 2e is fully cross-reactive with cynomolgus monkey TTR. A singl esubcutaneous dose of AD-65492 (1 mg/kg) or AD-87404 (1 mg/kg or 3 mg/k) was administered to cynomolgus monkey (n = 3 per group) on day 0 in three separate studies. Blood samples were collect variousled ony Days -7 (7 days predose) through Day 119 as shown in Figure 2. Serum was prepared and cynomolgus monkey TTR levels were determined using ELISA assay (see, e.g., Coelho, et al. (2013) N Engl J Med 369:819). Relative TTR levels as compare dto Day 0 are shown in Figure 2.

TTR knockdown was simila inr in monkeys administered 1 mg/kg of AD-65492 and 3 mg/kg of AD- 87404.

Example 3: Administration of a Single Dose of AD-87404 to Healthy Human Subjects In a Phase I, randomized, single-blind, placebo-controlled study, AD-87404 (Sense: 5’- usgsggauUfuCfAfUfguaacca aga- 3’ (SEQ ID NO: 6), wherein an L96 ligand is conjugat edto the 3’ end of the sense strand); Antisense: 5’- usCfsuugGf(Tgn)uAfcaugAfaAfucccas -usc 3’ (SEQ ID NO: 7)) are administered to healthy human voluntee rsas a single dose of 25mg, 75 mg, 100 mg, 200 mg, or 400 mg, with possibl edose groups of 600 mg, 700 mg, 900 mg, and 1000 mg. Groups are balanced for relevant demographic characteristics, e.g., age, sex, body weight.

Demographicall maty ched control subject sare also administered a single dose of a placebo.

Plasma samples are collect anded the level of TTR protein in the samples from the subject sin the placebo group and the subject sin all of the treatment groups is determined using an ELISA assay (see, e.g., Coelho, et al. (2013) N Engl J Med 369:819) at pre-determined intervals e.g.,, days 1, 2, 3, 8, , 22, 29, 43, 57, 90, and then, for active treatment group subjects, every twenty-eighth day until the level of TTR recovers to 80% of the pre-treatment level (up through, approximatel oney year post-dose).

Level and duratio nof knockdown are determined. 56 Subjects in both AD-87404 groups and the control group are also monitored for adverse events.

Exemplar yadverse events monitored in the study include, but are not limited to, injection site erythema, injection site pain, pruritus, cough, nausea, fatigue, and abdominal painand clinica llysignifican t chang esin physical exams ,ECG, vital signs, or clinic laboal rato parametry ers, e.g., renal function, hematologic parameters, and liver function (e.g., alani neaminotransfera (ALT),se aspartate aminotransferas (ASTe )).

The results of this study demonstrate that a single subcutaneous dose of AD-87404 potentl yand durably knocks down TTR protein levels in a dose dependent manner.

Multiple dose studies and multiple ascending dose studies are also contemplated with monitoring of TTR protein knockdown in serum and adverse events.

Equivalents: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalent tos the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. 57

Claims (51)

CLAIMED IS:

1. An RNAi agent comprising a sense strand and an antisense strand, wherein: each of the sense strand and the antisense strand are independently up to 30 nucleotides in 5 length; the sense strand comprises the modified nucleotide sequence 5’- usgsggauUfuCfAfUfguaaccaaga-3‘ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5’- usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3 ’ (SEQ ID NO: 7), 10 wherein a, c, g, andu 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, Of, and Uf are 2‘-fluoroadenosine-3‘ -phosphate, 2‘-fluorocytidine-3‘ -phosphate, 2’- fluoroguanosine-3’-phosphate, and 2’-fluorouridine-3’-phosphate, respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-Isomer; and 15 s is a phosphorothioate linker.

2. The RNAi agent of claim 1, wherein the sense strand of the double stranded RNAi agent is conjugated to at least one ligand. 20

3. The RNAi agent of claim 2, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

4. The RNAi agent of claim 3, wherein the ligand is 25

5. The RNAi ligand of any one of claims 2-4, wherein the ligand is attached to the 3' end of the sense strand. 58 WO 2021/178778 PCT/US2021/021049

6. The RNAi agent of claim 5, wherein the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic wherein X is O or S. 5

7. The RNAi agent of any one of claims 1-6, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

8. A use of an RNAi agent in a method of treating a human subject suffering from a TTR- 10 associated disease, comprising administering to the subject a fixed dose of 25 mg to 1000 mg of a double stranded RNAi agent, comprising a sense strand and an antisense strand, wherein: each of the sense strand and the antisense strand are independently up to 30 nucleotides in length; the sense strand comprises the modified nucleotide sequence 5’- 15 usgsggauUfuCfAfUfguaaccaaga-3‘ (SEQ ID NO: 6); and the antisense strand comprises the modified nucleotide sequence 5’- usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3 ’ (SEQ ID NO: 7), wherein a, c, g, andu are 2'-O-methyladenosine-3’ -phosphate, 2'-O-methylcytidine-3’- phosphate, 2'-O-methylguanosine-3’ -phosphate, and 2'-O-methyluridine-3’-phosphate, respectively; 20 Af, Cf, Of, and Uf are 2‘-fluoroadenosine-3‘ -phosphate, 2‘-fluorocytidine-3‘ -phosphate, 2’- fluoroguanosine-3’-phosphate, and 2’-fluorouridine-3’-phosphate, respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-Isomer; and s is a phosphorothioate linker. 25

9. A use of an RNAi agent in a method of inhibiting expression of TTR in a human subject who does not meet diagnostic criteria of a TTR-associated disease, comprising administering to the subject a fixed dose of 25 mg to 1000 mg of a double stranded RNAi agent, comprising a sense strand and an antisense strand, wherein: each the sense strand and the antisense strand are independently up to 30 nucleotides in length; 59 WO 2021/178778 PCT/US2021/021049 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), 5 wherein a, c, g, andu 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, Of, and Uf are 2‘-fluoroadenosine-3‘ -phosphate, 2‘-fluorocytidine-3‘ -phosphate, 2’- fluoroguanosine-3’-phosphate, and 2’-fluorouridine-3’-phosphate, respectively; (Tgn) is thymidine-glycol nucleic acid (GNA) S-Isomer; and 10 s is a phosphorothioate linker.

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 attached 15 through a bivalent or trivalent branched linker.

12. The use of claim 11, wherein the ligand is 20

13. The use of any one of claims 9-12, wherein the ligand is attached to the 3' end of the sense strand. 25

14. The use of claim 13, wherein the double stranded RNAi agent is conjugated to the ligand as shown in the following schematic 60 WO 2021/178778 PCT/US2021/021049 wherein X is O or S.

15. The use of any one of claims 8-14, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. 5

16. A use of any one of claims 8-15, wherein the method comprises improving at least one indicia of neurological impairement, quality of life, ongoing nerve damage, or cardiovascular impairment. 10

17. The use of claim 16, wherein the indicia is a neurological impairment indicia.

18. The use of claim 17, wherein the neurological impairment indicia is a change from baseline in an indicia selected from the group of Neuropathy Impairment (NIS) score, a modified NIS 15 (mNIS+7) score, a NIS-W score, a composite autonomic symptom score (COMPASS-31), a median body mass index (mBMI) score, a 6-minute walk test (6MWT) score, and a 10-meter walk test score.

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

20.20. The use of claim 19, wherein the quality of life indicia is a change from baseline in an indicia selected from the group of a SF-36® health survey score, a Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, and a Rasch-built Overall Disability Scale (R-ODS) score

21. The use of claim 16, wherein the indicia is ongoing nerve damage. 25

22. The use of claim 21, wherein the indicia of ongoing nerve damage is a change from baseline in a plasma protein level of one or more proteins selected from the group neurofilament light chain (NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N-CDase. 61 WO 2021/178778 PCT/US2021/021049

23. The use of claim 21, wherein the indicia of ongoing nerve damage is a change in plasma level of neurofilament light chain (NfL) protein level.

24. The use of claim 16, wherein the indicia is a cardiovascular impairment indicia. 5

25. The use of claim 24, wherein the indicia of cardiovascular impairment is cardiovascular hospitalization, a change from baseline using Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS) with an increased score indicative of better health status, change from baseline in mean left !ventricular (LV) wall thickness by echocardiographic assessment, change from baseline in 10 global longitudinal strain by echocardiographic assessment, and change from baseline in N-terminal prohormone B-type Natriuretic Peptide (NTproBNP)

26. The use of any one of claims 8-25, wherein the human subject carries a TTR gene mutation that is associated with the development of a TTR-associated disease. 15

27. The use of any one of claims 8-26, wherein the TTR-associated disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS) amyloidosis, hyperthyroxinemia, and cardiac amyloidosis. 20

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

29. The use of claim28, wherein the ATTR is hereditary ATTR (h-ATTR).

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

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

32. The use of claim31, wherein the subcutaneous administration is self administration. 35

33. The use of claim 32, wherein the self administration is via a pre-filled syringe or auto- injector syringe. 62 WO 2021/178778 PCT/US2021/021049

34. The use of any one of claims 8-33, further comprising assessing the level of TTR mRNA expression or TTR protein expression in a sample derived from the human subject.

35. The use of any one of claims 8-34, wherein the double stranded RNAi agent is 5 administered to the human subject once every three months to once a year.

36. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi agent is administered to the human subject once about every three months, once every six months, or once a year. 10

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

38. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi 15 agent is administered to the human subject at a fixed dose of 25 mg to 200 mg once every three months.

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

40. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi 20 agent is administered to the human subject at a fixed dose of 25 mg once every three months.

41. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi agent is administered to the human subject at a fixed dose of 75 mg once every three months. 25

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

43. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi 30 agent is administered to the human subject at a fixed dose of 200 mg once every three months.

44. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi agent is administered to the human subject at a fixed dose of 300 mg once every three months. 35 45. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg to 600 mg once every six months to once every 12 months. 63

45.WO 2021/178778 PCT/US2021/021049

46. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg to 600 mg once every six months or once every 12 months. 5

47. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg or 600 mg once every six months or once every 12 months.

48. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi 10 agent is administered to the human subject at a fixed dose of 700 mg to 1000 mg once every 12 months.

49. The use of any one of claims 8-35, wherein the fixed dose of the double stranded RNAi agent is administered to the human subject at a fixed dose of 700 mg, 800 mg, 900 mg, or 1000 mg once every 12 months. 15

50. The use of any one of claims 8-49, further comprising administering to the human subject an additional therapeutic agent.

51. The use of claim 50, wherein the additional therapeutic agent is a TTR tetramer 20 stabilizer or a non-steroidal anti-inflammatory agent. 64