Methods and compositions for treating transthyretin (TTR)- associated diseases

Abstract

The invention relates to methods of treating subjects that would benefit from reduction in expression of transthyretin (TTR), such as subjects having a TTR-associated disease, disorder, or condition, e.g., transthyretin-mediated amyloidosis, using double-stranded ribonucleic acid (dsRNA) compositions targeting the TTR gene. The invention also provides methods for preventing at least one symptom in a subject having a TTR-associated disease, disorder, or condition, e.g., transthyretin- mediated amyloidosis.

Description

[0001] Attorney Docket No.: 121301-25120

[0002] Alnylam Reference No.: ALN-542-WO

[0003] METHODS AND COMPOSITIONS FOR TREATING TRANSTHYRETIN (TTR)- ASSOCIATED DISEASES

[0004] RELATED APPLICATIONS

[0005] This application claims the benefit of priority of U.S. Provisional Application No.

[0006] 63 / 762,342, filed February 24, 2025, and U.S. Provisional Application No. 63 / 785,288, filed April 8, 2025. The entire contents of each of the foregoing applications are incorporated herein by reference.

[0007] SEQUENCE LISTING

[0008] This application contains a sequence listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 18, 2026, is named 121301-2512O.xml and is 58,781 bytes in size.

[0009] BACKGROUND OF THE INVENTION

[0010] Transthyretin (TTR) (also known as prealbumin) is found in serum and cerebrospinal fluid (CSF). TTR transports retinol-binding protein (RBP) and thyroxine (T4) and also acts as a carrier of retinol (vitamin A) through 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 P-peptide, and neuropeptide Y (Liz, M.A. et al. (2010) IUBMB Life, 62(6) :429-435).

[0011] 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 ellipsoid. Antiparallel beta-sheet interactions link monomers into dimers. A short loop from each monomer forms the main dimer-dimer interaction. These two pairs of loops separate the opposed, convex betasheets of the dimers to form an internal channel.

[0012] The liver is the major site of TTR expression. Other significant sites of expression include the choroid plexus, retina (particularly the retinal pigment epithelium) and pancreas.

[0013] Transthyretin is one of at least 27 distinct types of proteins that is a precursor protein in the formation of amyloid fibrils (Guan, J. et al. Am J Physiol Heart Circ Physiol, 2012 Feb 1;

[0014] 302(3):H544-52). Extracellular deposition of amyloid fibrils in organs and tissues is the hallmark of amyloidosis. Amyloid fibrils are composed of misfolded protein aggregates, which may result from either excess production of or specific mutations in precursor proteins. The amyloidogenic potential of TTR may be related to its extensive beta sheet structure; X-ray crystallographic studies indicate that certain amyloidogenic mutations destabilize the tetrameric structure of the protein (Saraiva M.J.M. (2002) Expert Reviews in Molecular Medicine, 4(12): 1-11).

[0015] Amyloidosis is 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

[0016] 1

[0017] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0018] Alnylam Reference No.: ALN-542-WO name starts with “A” for amyloid and is followed by an abbreviation of the precursor protein, e.g., ATTR for amyloidogenic transthyretin.

[0019] Transthyretin amyloidosis (ATTR) is classified as hereditary transthyretin amyloidosis (hATTR), and wild-type (non-hereditary) transthyretin amyloidosis (wtATTR). While wild-type TTR amyloidosis (wtATTR) is an acquired condition that mainly affects the heart, develops with age as the natural TTR protein becomes unstable and prone to misfold, and commonly affects men over the age of 60, hATTR is a severe systemic disease of adults caused by mutations in the TTR gene and transmitted in an autosomal dominant manner with incomplete penetrance. The mutations result in an abnormal TTR protein that is unstable and readily misfolds, forming aggregates which deposit as amyloid in various organs and tissues in the body. hATTR provides a wide range of clinical presentations that differ for age of onset, organ involvement, and severity of disease. hATTR has traditionally been described according to the predominant clinical features, typically either a polyneuropathy (hATTR-PN), formerly referred to as Familial Amyloid Polyneuropathy (FAP), or a cardiomyopathy (hATTR-CM), termed Familial Amyloid Cardiomyopathy (FAC), although most patients show symptoms and signs of both nerve and heart involvement. A cardiomyopathy is also characteristic of a wild-type form of the disease (wtATTR).

[0020] There are over 120 different mutations in the TTR gene. The V30M mutation is the most prevalent TTR mutation as a cause of hATTR (Lobato, L. (2003) J Nephrol, 16:438-442). The classical clinical presentation of the V30M mutation is characterized by a length-dependent neuropathy mainly involving the small fibers. When fully developed, the disease generally discloses a sensory-motor polyneuropathy with autonomic involvement (postural hypotension, and gastrointestinal, bladder and erectile dysfunction). The V122I 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).

[0021] Accordingly, there is a need in the art for effective treatments for transthyretin-mediated amyloidosis, e.g., hereditary transthyretin-mediated amyloidosis with polyneuropathy (hATTR-PN), and transthyretin-mediated amyloidosis with cardiomyopathy (ATTR-CM).

[0022] SUMMARY OF THE INVENTION

[0023] The invention provides methods and compositions for inhibiting the expression of a transthyretin (TTR) gene, for treating or preventing at least one symptoms in a subject having transthyretin-mediated amyloidosis. The methods include administering to the subject a fixed dose of an RNAi agent, e.g., a double stranded RNAi agent, or a pharmaceutically acceptable salt thereof, targeting a TTR gene.

[0024] Accordingly, in one aspect, the present invention provides a method for treating polyneuropathy and / or cardiomyopathy in a subject having transthyretin-mediated amyloidosis (ATTR), the method comprising administering to the subject a fixed dose of about 200-400 mg of a double stranded ribonucleic acid (dsRNA) agent, or a pharmaceutically acceptable salt thereof,

[0025] 2

[0026] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0027] Alnylam Reference No.: ALN-542-WO

[0028] targeting a TTR gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the modified nucleotide sequence 5’- csasagagUfaUfUfCfcauuuuuacu-3’ of SEQ ID NO: 17, and the antisense strand comprises the modified nucleotide sequence 5’ - asGfsuaaAfaauggaaUfaCfiicuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; and s is a phosphorothioate linkage; and wherein a ligand is conjugated to the 3 ’ end of the sense strand as shown in the following schematic

[0029]

[0030] thereby treating polyneuropathy and / or cardiomyopathy in the subject.

[0031] In one embodiment, the ATTR is hereditary ATTR (hATTR). In another embodiment, the ATTR is wild-type ATTR (wtATTR).

[0032] In one aspect, the present invention provides a method for treating polyneuropathy in a subject having hereditary transthyretin-mediated amyloidosis, the method comprising administering to the subject a fixed dose of about 200-400 mg of a double stranded ribonucleic acid (dsRNA) agent, or a pharmaceutically acceptable salt thereof, targeting a TTR gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the modified nucleotide sequence 5’ - csasagagUfaUfUfCfcauuuuuacu-3’ of SEQ ID NO: 17, and the antisense strand comprises the modified nucleotide sequence 5’-asGfsuaaAfaauggaaUfaCfiicuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; and s is a phosphorothioate linkage; and wherein a ligand is conjugated to the 3’ end of the sense strand as shown in the following schematic

[0033] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0034] Alnylam Reference No.: ALN-542-WO

[0035]

[0036] thereby treating polyneuropathy in the subject.

[0037] In another aspect, the present invention provides a method for treating cardiomyopathy in a subject having transthyretin-mediated amyloidosis (ATTR), the method comprising administering to the subject a fixed dose of about 200-400 mg of a double stranded ribonucleic acid (dsRNA) agent, or a pharmaceutically acceptable salt thereof, targeting a TTR gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the modified nucleotide sequence 5’ - csasagagUfaUfUfCfcauuuuuacu-3 ’ of SEQ ID NO: 17, and the antisense strand comprises the modified nucleotide sequence 5’-asGfsuaaAfaauggaaUfaCfiicuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; and s is a phosphorothioate linkage; and wherein a ligand is conjugated to the 3’ end of the sense strand as shown in the following schematic

[0038]

[0039] thereby treating cardiomyopathy in the subject.

[0040] In one embodiment, the ATTR is hereditary ATTR (hATTR). In another embodiment, the ATTR is wild-type ATTR (wtATTR).

[0041] 4

[0042] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0043] Alnylam Reference No.: ALN-542-WO In some embodiments, the subject is a human subject. In some embodiments, the human subject is 18-85 years of age.

[0044] In some embodiments, the human subject has a Neuropathy Impairment Score (NIS) of 5-130, a polyneuropathy disability (PND) sore of < 3b (or Illb), and / or Kamofsky Performance Scale (KPS) > 60%.

[0045] In some embodiments, the human subject is a male and has an end-diastolic interventricular septal wall thickness >12 mm, e.g., as determined by echocardiography. In some embodiments, the human subject is a female and has an end-diastolic interventricular septal wall thickness >11 mm, e.g., as determined by echocardiography.

[0046] In some embodiments, the subject has a Grade 2 or 3 cardiac uptake by Technetium scintigraphy.

[0047] In some embodiments, the subject has amyloid deposits in cardiac tissue.

[0048] In some embodiments, the subject has amyloid deposits of TTR protein in non-cardiac tissue, and (i) the subject has a Grade 2 or 3 cardiac uptake by Technetium scintigraphy, or (ii) the subject has amyloid deposits in cardiac tissue. In some embodiments, the non-cardiac tissue is selected from the group consisting of fat pad aspirate, salivary gland, and median nerve connective sheath.

[0049] In some embodiments, the subject has a medical history of heart failure with at least one prior hospitalization for heart failure, or clinical evidence of heart failure manifested by one or more symptoms of volume overload or elevated intracardiac pressures. In some embodiments, the hospitalization for heart failure is not due to arrhythmia or a conduction system disturbance treated with a permanent pacemaker. In some embodiments, the one or more symptoms of volume overload or elevated intracardiac pressures comprise elevated jugular venous pressure, shortness of breath, signs of pulmonary congestion on X-ray or auscultation, and / or peripheral edema.

[0050] In some embodiments, the subject has an NT-proBNP level of >300 ng / L and <8500 ng / L.In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 250-350 mg, about 275-325 mg, about 280-320 mg, about 290-310 mg, about 295-305 mg. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 275-325 mg.

[0051] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 200 mg, about 250, about 275 mg, about 280 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 320 mg, about 325 mg, about 350 mg, or about 400 mg. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 300 mg.

[0052] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or every 12 months. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject every 6 months.

[0053] 5

[0054] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0055] Alnylam Reference No.: ALN-542-WO In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 275-325 mg every six months.

[0056] In some embodiments, the dsRNA agent, or salt thereof, is administered to the subject at a dose of about 300 mg every six months.

[0057] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject intravenously, intramuscularly, or subcutaneously. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered via a pre-fdled syringe. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered via an auto-injector.

[0058] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously at a dose of about 275-325 mg every six months.

[0059] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously at a dose of about 300 mg every six months.

[0060] In some embodiments, the methods further comprise measuring the level of TTR mRNA expression or TTR protein expression in a sample from the subject.

[0061] In some embodiments, the methods further comprise measuring the level of vitamin A or RBP4 protein in a sample from the subject.

[0062] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject causes a decrease in TTR enzymatic activity, a decrease in TTR protein accumulation, a decrease in vitamin A level, and / or a decrease in RBP4 level in the subject.

[0063] In some embodiments, the TTR mRNA level in the subject is reduced to at least about 50%, 55%, 60%, 65%, or 70% of baseline level 6 months after the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof.

[0064] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject improves at least one indicia selected from the group consisting of Neuropathy Impairment Score (NIS), modified Neuropathy Impairment Score (mNIS+7), Norfolk Quality of Life Diabetic Neuropathy (Norfolk QoL-DN) questionnaire, modified Body Mass Index (mBMI), Rasch built Overall Disability Scale (R ODS) questionnaire, 10-meter walk test (10 MWT), Composite Autonomic Symptom Score (COMPASS-31), EuroQoL-5 Dimensions-5 Levels (EQ-5D-5L) questionnaire and the EuroQoL-Visual Analog Scale (EQ VAS), Familial Amyloidotic Polyneuropathy (FAP) stage, and Polyneuropathy Disability (PND) score, compared to a baseline as determined before the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof.

[0065] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject results in an improvement or a stabilization of a cardiac marker, an echocardiogram parameter, and / or a Technetium scintigraphy cardiac parameter, compared to a

[0066] 6

[0067] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0068] Alnylam Reference No.: ALN-542-WO baseline, as determined before the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof.

[0069] In some embodiments, the cardiac marker is selected from the group consisting of N-terminal prohormone B-type natriuretic peptide (NT-proBNP), Troponin I, Troponin T, and Neurofilament light chain (NfL).

[0070] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces the composite of all-cause mortality and recurrent cardiovascular (CV) events.

[0071] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces the all-cause mortality.

[0072] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces cardiovascular hospitalizations and / or urgent heart failure visits.

[0073] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject delays the onset of cardiovascular events or all-cause mortality.

[0074] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces recurrent cardiovascular events.

[0075] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces or prevents oral diuretic intensification / initiation (ODI).

[0076] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject stabilizes or improves the ATTR amyloidosis disease stage.

[0077] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject improves at least one indicia selected from the group consisting of Kansas City Cardiomyopathy Questionnaire (KCCQ) and New York Heart Association (NYHA) Class.

[0078] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces an amyloid TTR deposit in the subject.

[0079] In another aspect, the present invention provides a kit for performing the methods as described herein, comprising a) the dsRNA agent, or a pharmaceutically acceptable salt thereof, and b) instructions for use, and c) optionally, means for administering the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject. In some embodiments, the means for administering the dsRNA agent, or a pharmaceutically acceptable salt thereof, is a pre-filled syringe. In other embodiments, the means for administering the dsRNA agent, or a pharmaceutically acceptable salt thereof, is an auto-injector.

[0080] 7

[0081] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0082] Alnylam Reference No.: ALN-542-WO

[0083] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically depicts the Phase 3 study design of AD-649264 (ALN-TTRscO4) in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy (hATTR-PN).aThe vutrisiran group is used as an in-study comparator to ALN-TTRscO4 for serum TTR reduction and as a reference group to validate the use of the external placebo control group from the APOLLO study; however, for the primary and for secondary and exploratory clinical efficacy endpoints, ALN-TTRscO4 is compared against the external placebo control group of the APOLLO study.

[0084] FIG. 2 schematically depicts the Phase 3 study design of AD-649264 (ALN-TTRscO4) in patients with transthyretin-mediated amyloidosis with cardiomyopathy (ATTR-CM).aBackground therapies (e.g., TTR stabilizers) are allowed.bUsing an event-driven trial design, the primary analysis will be conducted after a pre-specified number of events has been reached, but no earlier than 24 months after the last patient is randomized. Therefore, the length of the DB period will vary for each individual patient, ranging from a minimum of 24 months to a maximum of 5 years, with an anticipated average duration of 32 months.cUpon entry into the OLE Period (OLE Day 1), all eligible patients receive open-label doses of 300 mg q6M ALN-TTRscO4 administered as subcutaneous injections.dPatients are eligible to enter the OLE Period after the DB Period has ended for the study or after they have completed 5 years in the DB Period, whichever comes first.eThe Safety Follow-up Period is 24 months after last dose of study drug for women of childbearing potential and 6 months for patients who discontinue study drug to start on TTR lowering therapy.

[0085] DETAILED DESCRIPTION OF THE INVENTION

[0086] The invention provides methods and compositions for inhibiting the expression of a transthyretin (TTR) gene, for treating or preventing at least one symptom in a subject having transthyretin-mediated amyloidosis. The methods include administering to the subject a fixed dose of an RNAi agent, e.g., a double stranded RNAi agent, or a pharmaceutically acceptable salt thereof, targeting a TTR gene.

[0087] The present invention provides iRNA compositions, which effect the RNA-induced silencing complex (RlSC)-mediated cleavage of RNA transcripts of a TTR gene. The TTR gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of a TTR gene, and for treating a subject who would benefit from inhibiting or reducing the expression of a TTR gene, e.g., a subject suffering or prone to suffering from a TTR-associated disease disorder, or condition, such as a subject suffering or prone to suffering from such as transthyretin-mediated amyloidosis.

[0088] I. Definitions

[0089] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are

[0090] 8

[0091] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0092] Alnylam Reference No.: ALN-542-WO

[0093] recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

[0094] The articles “a” and “an” are used herein to refer to one or to more than one (z. e. , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

[0095] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0096] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.

[0097] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as 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.

[0098] As used herein, “transthyretin” (“TTR”) refers to the well-known gene and protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter of retinol-binding protein (RBP), thyroxine (T4) and retinol, and it also acts as a protease. The liver secretes TTR into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR is also expressed in the pancreas and the retinal pigment epithelium. The greatest clinical relevance of TTR is that both normal (wild type) and mutant TTR protein can form amyloid fibrils that aggregate into extracellular deposits, causing amyloidosis. See, e.g., Saraiva M.J.M. (2002) Expert Reviews in Molecular Medicine, 4(12): 1-11 for a review. The molecular cloning and nucleotide sequence of rat transthyretin, as well as 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. (1974) J Mol Biol 88, 1-12.

[0099] The sequence of a human TTR mRNA transcript may be found at National Center for Biotechnology Information (NCBI) RefSeq accession number NM_000371.4 (SEQ ID NO: 1 ; reverse complement, SEQ ID NO:2). The sequence of mouse TTR mRNA may be found at RefSeq accession number NM_013697.2 (SEQ ID NO:3; reverse complement, SEQ ID NO:4). The sequence of rat TTR mRNA may be found at RefSeq accession number NM_012681.1 (SEQ ID NO:5; reverse complement, SEQ ID NO:6). The sequence of Macaca fascicularis TTR mRNA may be found at RefSeq accession number NM_001283593.1 (SEQ ID NO:7; reverse complement, SEQ ID NO:8). The sequence of Macaca mulatta TTR mRNA may be found at RefSeq accession number NM_001261679.1 (SEQ ID NO:9; reverse complement, SEQ ID NO: 10).

[0100] Additional examples of TTR mRNA sequences are readily available through publicly available databases, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site.

[0101] Further information on TTR can be found, for example, at

[0102] www .ncbi .nlm .nih ,gov / gene / ?term=TTR.

[0103] 9

[0104] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0105] Alnylam Reference No.: ALN-542-WO

[0106] The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of fding this application.

[0107] The term TTR, as used herein, also refers to variations of the TTR gene including variants provided in the SNP database. Numerous sequence variations within the TTR gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., www.ncbi.nlm ,nih.gov / snp / ?term=TTR, the entire contents of which is incorporated herein by reference as of the date of filing this application.

[0108] As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TTR gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TTR gene.

[0109] As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

[0110] The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, 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-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of TTR gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

[0111] In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double -stranded RNA and is referred to herein as a “double stranded RNAi agent,” “doublestranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i. e. , a TTR gene . In some embodiments of the invention, a double -stranded RNA (dsRNA) 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.

[0112] In general, the majority of nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and / or a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified intemucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to

[0113] 10

[0114] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0115] Alnylam Reference No.: ALN-542-WO

[0116] intemucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

[0117] 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.

[0118] As used herein, the term “region of complementarity” refers 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 complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5’- and / or 3 ’-terminus of the iRNA.

[0119] 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.

[0120] A “pharmaceutically acceptable salt” of the dsRNA of the invention being used in the methods of the invention includes any salt which is pharmaceutically acceptable, e.g., a sodium salt of the dsRNA agent. In one embodiment, the pharmaceutically acceptable salt of the dsRNA of the invention being used in the methods of the invention has the following structure:

[0121] S'i u.v

[0122] " &

[0123] &

[0124]

[0125] The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.

[0126] The phrase “inhibiting expression of a TTR gene,” as used herein, includes inhibition of expression of any TTR gene (such as, e.g., a mouse TTR gene, a rat TTR gene, a monkey TTR gene,

[0127] 11

[0128] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0129] Alnylam Reference No.: ALN-542-WO

[0130] or a human TTR gene) as well as variants or mutants of a TTR gene that encode a TTR protein. Thus, the TTR gene may be a wild-type TTR gene, a mutant TTR gene, or a transgenic TTR gene in the context of a genetically manipulated cell, group of cells, or organism.

[0131] “Inhibiting expression of a TTR gene” includes any level of inhibition of a TTR gene, e.g, at least partial suppression of the expression of a TTR gene, such as an inhibition by at least about 20%. In certain embodiments, inhibition is by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0132] The expression of a TTR gene may be assessed based on the level of any variable associated with TTR gene expression, e.g., TTR mRNA level or TTR protein level. The expression of a TTR gene may also be assessed indirectly based on, for example, the levels of circulating RBP4 protein, retinol, and / or vitamin A in a sample, such as a serum sample.

[0133] Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with 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).

[0134] In one embodiment, at least partial suppression of the expression of a TTR gene, is assessed by a reduction of the amount of TTR mRNA which can be isolated from, or detected, in a first cell or group of cells in which a TTR gene is transcribed and which has or have been treated such that the expression of a TTR gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).

[0135] The degree of inhibition may be expressed in terms of:

[0136] (mRNA in control cells)- (mRNA in treated cells)

[0137] - • 100%

[0138] (mRNA in control cells)

[0139] Inhibition of the expression of a TTR protein may be manifested by a reduction in the level of the TTR protein that is expressed by a cell or group of cells or in a subject sample (e.g., the level of protein in a blood sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells, or the change in the level of protein in a subject sample, e.g., blood or serum derived therefrom.

[0140] The level of TTR mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of TTR in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the TTR gene. RNA may be extracted from cells using RNA extraction

[0141] 12

[0142] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0143] Alnylam Reference No.: ALN-542-WO

[0144] techniques including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasyTM RNA preparation kits (Qiagen®) or PAXgeneTM (PreAnalytixTM, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis.

[0145] In some embodiments, the level of expression of TTR is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific TTR. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0146] Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to TTR mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of TTR mRNA.

[0147] An alternative method for determining the level of expression of TTR in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Patent No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli etal . (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh etal . (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Eizardi et al . (1988) Bio / Technology 6: 1197), rolling circle replication (Eizardi et al ., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of TTR is determined by quantitative Anorogenic RT-PCR (i.e., the TaqManTM System).

[0148] The expression levels of TTR mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by

[0149] 13

[0150] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0151] Alnylam Reference No.: ALN-542-WO

[0152] reference. The determination of TTR expression level may also comprise using nucleic acid probes in solution.

[0153] In some embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR).

[0154] The level of TTR protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluore scent assays, electrochemiluminescence assays, and the like.

[0155] In some embodiments, the efficacy of the methods of the invention are assessed by a decrease in TTR mRNA or protein level (e.g, in a liver biopsy). In certain embodiments, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the TTR gene or protein expression. In other embodiments, a blood sample serves as the subject sample for monitoring the reduction in the TTR protein expression. A reduction in the expression of TTR may also be assessed indirectly by measuring a decrease in the circulating level of RBP4 protein, retinol, and / or vitamin A in a sample, such as a serum sample.

[0156] In some embodiments, the efficacy of the methods of the invention can be monitored by detecting or monitoring a reduction in a symptom of a TTR-associate disorder, e.g., reduction in sensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and comeal lattice dystrophy. It is well within the ability of one skilled in the art to monitor efficacy of the methods by measuring any one of such parameters, or any combination of parameters.

[0157] In some embodiments of the methods of the invention, the iRNA is administered to a subject such that the iRNA is delivered to a specific site within the subject. The inhibition of expression of TTR may be assessed using measurements of the level or change in the level of TTR mRNA or TTR protein in a sample derived from fluid or tissue from the specific site within the subject (e.g., liver or blood).

[0158] The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0159] 14

[0160] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0161] Alnylam Reference No.: ALN-542-WO

[0162] Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and / or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

[0163] In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and / or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005 / 0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and / or are known in the art.

[0164] As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose).

[0165] In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in TTR expression; a human at risk for a disease, disorder or condition that would benefit from reduction in TTR expression; a human having a disease, disorder or condition that would benefit from reduction in TTR expression; and / or human being treated for a disease, disorder or condition that would benefit from reduction in TTR expression as described herein.

[0166] 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 associated disease, 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 diagnostic criteria for a TTR-associated disease.

[0167] As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result, such as reducing at least one sign or symptom of a TTR-associated disorder, e.g., transthyretin -mediated amyloidosis (ATTR), e.g., either hereditary ATTR (hATTR) or non-hereditary ATTR (wtATTR), in a subject. Treatment also includes a reduction of one or more sign or symptoms associated with

[0168] 15

[0169] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0170] Alnylam Reference No.: ALN-542-WO unwanted TTR expression; diminishing the extent of unwanted TTR activation or stabilization; amelioration or palliation of unwanted TTR activation or stabilization. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

[0171] The term “lower” in the context of a TTR-associated disease refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more. In certain embodiments, a decrease is at least 20%. ’’Lower” in the context of the level of TTR in a subject is preferably down to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, “lower” is the decrease in the difference between the level of a marker or symptom for a subject suffering from a disease and a level accepted within the range of normal for an individual. The term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from a TTR-associated disorder towards or to a level in a normal subject not suffering from a TTR-associated disorder. As used herein, if a disease is associated with an elevated value for a symptom, “normal” is considered to be the upper limit of normal. If a disease is associated with a decreased value for a symptom, “normal” is considered to be the lower limit of normal.

[0172] As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, may be treated or ameliorated by a reduction in expression of a TTR gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of a TTR-associated disorder, e.g., transthyretin-mediated amyloidosis (ATTR), e.g., either hereditary ATTR (hATTR) or non-hereditary ATTR (wtATTR), senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / Central Nervous System (CNS) amyloidosis. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

[0173] A “TTR-associated disease,” 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 abnormal cleavage of the TTR protein, instability of TTR tetramers, by abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. A “TTR-associated disease” includes any type of transthyretin-mediated amyloidosis (ATTR amyloidosis) wherein TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposits, e.g., either hereditary ATTR (h-ATTR) amyloidosis or non-hereditary ATTR (wtATTR) amyloidosis. TTR-associated diseases also include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / Central Nervous System (CNS) amyloidosis,

[0174] 16

[0175] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0176] Alnylam Reference No.: ALN-542-WO

[0177] amyloidotic vitreous opacities, carpal tunnel syndrome. Symptoms of TTR amyloidosis include polyneuropathy, cardiomyopathy, sensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, carpal tunnel syndrome, autonomic insufficiency, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and comeal lattice dystrophy.

[0178] "Therapeutically effective amount," as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a TTR-associated disease, disorder, or condition, is sufficient to effective treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective 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, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0179] “Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a TTR-associated disease, disorder, or condition, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The "prophylactically effective amount" may vary depending on the iRNA, 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 other individual characteristics of the patient to be treated.

[0180] A "therapeutically-effective amount" or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. iRNA 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.

[0181] A “fixed dose” (e.g., a dose in mg) means that one dose of an iRNA agent is used for all subjects regardless of any specific subject-related factors, such as weight. A fixed dose is different from a weight-based dose (e.g., a dose in mg / kg) which refers to a dose of the iRNA agent that will change depending on the subject’s weight.

[0182] In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject as a fixed dose of about 200-400 mg, about 250-350 mg, about 275-325 mg, about 280-320 mg, about 290-310 mg, about 295-305 mg. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a fixed dose of , e.g., a fixed dose of about 200 mg, about 250, about 275 mg, about 280 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 320 mg, about 325 mg, about 350 mg, or about 400 mg. Values and ranges intermediate to the foregoing recited values are also intended to be part of this invention.

[0183] 17

[0184] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0185] Alnylam Reference No.: ALN-542-WO

[0186] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0187] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid fdler, diluent, excipient, manufacturing aid (e.g, lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0188] The term “lower” in the context of the level of TTR gene expression or TTR protein production in a subject, or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of detection for the detection method. In certain embodiments, the expression of the target is normalized, i.e., decreased towards or to a level accepted as within the range of normal for an individual without such disorder, e.g., normalization of body weight, blood pressure, or a serum lipid level. As used here, “lower” in a subject can refer to lowering of gene expression or protein production in a cell in a subject does not require lowering of expression in all cells or tissues of a subject. For example, as used herein, lowering in a subject can include lowering of gene expression or protein production in the liver of a subject.

[0189] The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids,

[0190] 18

[0191] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0192] Alnylam Reference No.: ALN-542-WO

[0193] lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject.

[0194] II. Methods of the Invention

[0195] The present invention provides methods for inhibiting the expression of a transthyretin (TTR) gene, and methods for treating or preventing at least one symptom in a subject having transthyretin-mediated amyloidosis.

[0196] The methods include administering to the subject a fixed dose of about 200 mg to about 400 mg of a dsRNA agent, or a pharmaceutically acceptable salt thereof, of the invention. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a fixed dose of about 250 mg to about 350 mg, about 275 mg to about 325 mg, about 280 mg to about 320 mg, about 290 mg to about 310 mg, or about 295 mg to about 305 mg. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a fixed dose of, e.g., a fixed dose of about 200 mg, about 250, about 275 mg, about 280 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 320 mg, about 325 mg, about 350 mg, or about 400 mg.

[0197] In one aspect, the invention provides methods of treating a subject having transthyretin-mediated amyloidosis (ATTR). The methods include administering to the subject a therapeutically effective amount of dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR, thereby treating the subject.

[0198] The invention also provides methods of treating polyneuropathy and / or cardiomyopathy in a subject having transthyretin-mediated amyloidosis (ATTR). The methods include administering to the subject a therapeutically effective amount of dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR, thereby treating polyneuropathy and / or cardiomyopathy in the subject.

[0199] The invention further provides methods of treating polyneuropathy in a subject having transthyretin-mediated amyloidosis (ATTR). The methods include administering to the subject a therapeutically effective amount of dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR, thereby treating the subject.

[0200] In another aspect, the invention provides methods of treating polyneuropathy in a subject having hereditary transthyretin-mediated amyloidosis (hATTR). The methods include administering to the subject a therapeutically effective amount of dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR, thereby treating the subject.

[0201] 19

[0202] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0203] Alnylam Reference No.: ALN-542-WO In a further aspect, the invention provides methods of treating cardiomyopathy in a subject having transthyretin-mediated amyloidosis (ATTR). The methods include administering to the subject a therapeutically effective amount of dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR, thereby treating the subject.

[0204] In some embodiments, the ATTR is hereditary ATTR (h-ATTR). In some embodiments, the ATTR is non-hereditary ATTR (wtATTR).

[0205] As used herein, the term “polyneuropathy” refers to a condition in which a person’s peripheral nerves or nerves located outside of the brain and spinal cord are damaged. Polyneuropathy often causes weakness, numbness and pain, usually in the hands and feet. It can also affect other areas and body functions including digestion, urination and circulation. Polyneuropathy can result from traumatic injuries, infections, metabolic problems, inherited causes and exposure to toxins. There are two major categories of polyneuropathy: acute and chronic.

[0206] As used herein, the term “cardiomyopathy” refers to conditions that affect the myocardium (heart muscle). Cardiomyopathy can make the heart stiffen, enlarged or thickened and can cause scar tissue. As a result, the heart can’t pump blood effectively to the rest of the body. In time, heart can weaken and cardiomyopathy can lead to heart failure. There might be no signs or symptoms in the early stages of cardiomyopathy. But as the condition advances, signs and symptoms usually appear, including breathlessness with activity or even at rest, swelling of the legs, ankles and feet, bloating of the abdomen due to fluid buildup, cough while lying down, difficulty lying flat to sleep, fatigue, heartbeats that feel rapid, pounding or fluttering, chest discomfort or pressure, dizziness, lightheadedness and fainting. The most common types of cardiomyopathy are dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular dysplasia (ARVD), restrictive cardiomyopathy, and transthyretin-mediated amyloidosis with cardiomyopathy (ATTR-CM).

[0207] In one aspect, the invention provides methods of preventing at least one symptom in a subject having a disorder that would benefit from reduction in TTR expression, e.g., a TTR-associated disease, e.g., transthyretin-mediated amyloidosis (ATTR). The methods include administering to the subject a prophylactically effective amount of dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR, thereby preventing at least one symptom in the subject.

[0208] Subjects

[0209] Polyneuropathy

[0210] In some embodiments, the methods treat polyneuropathy in a subject having hereditary transthyretin-mediated amyloidosis (hATTR).

[0211] In some embodiments, the subject is a human subject. In some embodiments, the human subject is 18-85 years of age.

[0212] 20

[0213] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0214] Alnylam Reference No.: ALN-542-WO

[0215] In some embodiments, the human subject has a Neuropathy Impairment Score (NIS) of 5-130, a polyneuropathy disability (PND) sore of < 3b (or Illb), and / or Kamofsky Performance Scale (KPS) > 60%.

[0216] In some embodiments, the subject does not have primary amyloidosis or leptomeningeal amyloidosis. In some embodiments, the subject does not have a New York Heart Association heart failure classification >2 (e.g, a New York Heart Association Class III heart failure, and / or a New York Heart Association Class IV heart failure).

[0217] In some embodiments, the subject does not have a level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >2.5 x upper limit of normal (ULN), or the subject has a level of ALT or AST <2.5 x ULN.

[0218] In some embodiments, the subject does not have a level of total bilirubin >1.5 ULN, or the subject has a level of total bilirubin <1.5 ULN.

[0219] In some embodiments, the subject does not have an international normalized ratio >2.0, or the subject has an international normalized ratio <2.0.

[0220] In some embodiments, the subject does not have a platelet count <50,000 / pL, or the subject has a platelet count >50,000 / pL.

[0221] In some embodiments, the subject does not have an absolute neutrophil count (ANC) <1500 cells / mm3, or the subject has an absolute neutrophil count (ANC) >1500 cells / mm3.

[0222] In some embodiments, the subject does not have an estimated glomerular fdtration rate (eGFR) <30 mL / min / 1 ,73m2, or the subject has an eGFR of >30 mL / min / 1.73m2.

[0223] In some embodiments, the subject does not have known causes of sensorimotor or autonomic neuropathy (e.g., autoimmune disease, monoclonal gammopathy).

[0224] In some embodiments, the subject does not have acute coronary syndrome.

[0225] In some embodiments, the subject does not have uncontrolled clinically significant cardiac arrhythmia or unstable angina.

[0226] In some embodiments, the subject does not have type 1 diabetes. In some embodiments, the subject does not have type 2 diabetes mellitus.

[0227] In some embodiments, the subject does not have untreated hypo- or hyper-thyroidism.

[0228] In some embodiments, the subject does not have an active infection requiring systemic antiviral, antiparasitic, or antimicrobial therapy.

[0229] Cardiomyopathy

[0230] In some embodiments, the methods treat cardiomyopathy in a subject having transthyretin-mediated amyloidosis (e.g., hereditary ATTR or wild type ATTR).

[0231] In some embodiments, the subject is a human subject. In some embodiments, the subject has a hereditary ATTR. In some embodiments, the subject has a wild-type ATTR.

[0232] In some embodiments, the human subject is 18-85 years of age. In some embodiments, the subject is <75 years of age. In some embodiments, the subject is > 75 years of age.

[0233] 21

[0234] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0235] Alnylam Reference No.: ALN-542-WO

[0236] In some embodiments, the subject has an NT-proBNP level of >300 ng / L and <8500 ng / L. In some embodiments, the subject has an NT-proBNP level of <1800 ng / L. In some embodiments, the subject has an NT-proBNP level of >1800 ng / L and <3000 ng / L. In some embodiments, the subject has an NT-proBNP level of >3000 ng / L.

[0237] In some embodiments, the subject is a male and has an end-diastolic interventricular septal wall thickness >12 mm. In some embodiments, the subject is a female and has an end-diastolic interventricular septal wall thickness >11 mm. In some embodiments, the end-diastolic interventricular septal wall thickness is determined by echocardiography.

[0238] In some embodiments, the subject has a Grade 2 or 3 cardiac uptake by Technetium scintigraphy. The Perugini grading scale is a semi-quantitative method of scoring cardiac uptake following injection of a radiotracer, for example,99mTc— 3,3-diphosphono-l,2-propanodicarboxylic acid (DPD-Tc),99mTc-Pyrophosphate (PYP-Tc) or99mTc-hydroxymethylene diphosphonate (HMDP) scintigraphy in the investigation of cardiac amyloidosis (e.g, ATTR amyloidosis). The grading scale visually compares tracer uptake in the myocardium and ribs, and the classification is shown below:

[0239] Grade 0: No cardiac uptake and normal bone uptake.

[0240] Grade 1: Cardiac uptake less intense than bone signal.

[0241] Grade 2: Cardiac uptake with intensity similar to or greater than bone signal.

[0242] Grade 3: Cardiac uptake with much attenuated or absent bone signal.

[0243] In the context of cardiac amyloidosis imaging, a Perugini Grade 2 or 3 cardiac uptake, meaning myocardial uptake equal to or greater than bone uptake, is strongly suggestive of ATTR cardiac amyloidosis.

[0244] In some embodiments, the subject has amyloid deposits in cardiac tissue, e.g., with TTR protein identified by immunohistochemistry or mass spectrometry.

[0245] In some embodiments, the subject has amyloid deposits in non-cardiac tissue (e.g., fat pad aspirate, salivary gland, or median nerve connective sheath), and (i) the subject has a Grade 2 or 3 cardiac uptake by Technetium scintigraphy, or (ii) the subject has amyloid deposits of in cardiac tissue, e.g., with TTR protein identified by immunohistochemistry or mass spectrometry.

[0246] In some embodiments, the subject has a medical history of heart failure with at least one prior hospitalization for heart failure, or clinical evidence of heart failure manifested by one or more symptoms of volume overload or elevated intracardiac pressures. In some embodiments, the hospitalization for heart failure is not due to arrhythmia or a conduction system disturbance treated with a permanent pacemaker. In some embodiments, the one or more symptoms of volume overload or elevated intracardiac pressures comprise elevated jugular venous pressure, shortness of breath, signs of pulmonary congestion on X-ray or auscultation, and / or peripheral edema.

[0247] In some embodiments, the subject does not have primary amyloidosis or leptomeningeal amyloidosis.

[0248] 22

[0249] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0250] Alnylam Reference No.: ALN-542-WO

[0251] In some embodiments, the subject does not have a New York Heart Association Class IV heart failure. In some embodiments, the subject does not have a New York Heart Association Class III heart failure, and wherein the subject does not have stage 3 ATTR Amyloidosis.

[0252] In some embodiments, the subject does not have a polyneuropathy disability (PND) score of Illa, Illb, or IV.

[0253] In some embodiments, the subject does not have a level of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) >2. Ox upper limit of normal (ULN), or wherein the subject has a level of ALT or AST <2.0x ULN.

[0254] In some embodiments, the subject does not have a level of total bilirubin >2.0 ULN, or wherein the subject has a level of total bilirubin <2.0 ULN.

[0255] In some embodiments, the subject does not have an international normalized ratio >1.5, or wherein the subject has an international normalized ratio <1.5.

[0256] In some embodiments, the subject does not have an estimated glomerular fdtration rate (eGFR) <30 mL / min / 1.73m2, or wherein the subject has an eGFR of >30 mL / min / 1.73m2.

[0257] In some embodiments, the subject does not have non-TTR cardiomyopathy, hypertensive cardiomyopathy, cardiomyopathy due to valvular heart disease, or cardiomyopathy due to ischemic heart disease.

[0258] In some embodiments, the subject does not have unstable congestive heart failure.

[0259] In some embodiments, the subject does not have acute coronary syndrome or unstable angina. In some embodiments, the subject does not have a history of sustained ventricular tachycardia or aborted ventricular fibrillation due to ATTR amyloidosis.

[0260] In some embodiments, the subject does not have a history of atrioventricular nodal or sinoatrial nodal dysfunction.

[0261] In some embodiments, the subject does not have persistent elevation of systolic (>170 mmHg) or diastolic (>100 mmHg) blood pressure.

[0262] In some embodiments, the subject does not have untreated hypo- or hyperthyroidism. In some embodiments, the subject does not have an active infection requiring systemic antiviral, antiparasitic, or antimicrobial therapy. In some embodiments, the subject does not have prior heart, liver, or other organ transplant or implantation of left-ventricular assist device (LVAD).

[0263] In addition, the present invention provides methods of inhibiting expression of TTR in a cell, such as a cell in a subject. The methods include contacting the cell with an RNAi agent or pharmaceutical composition comprising an iRNA agent of the invention. In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

[0264] A cell suitable for treatment using the methods of the invention may be any cell that expresses a TTR gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat

[0265] 23

[0266] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0267] Alnylam Reference No.: ALN-542-WO

[0268] cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

[0269] TTR expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%. In preferred embodiments, TTR expression is inhibited by at least 20%.

[0270] In one embodiment, the in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the TTR gene of the mammal to be treated.

[0271] In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same are administered by intravenous infusion or injection. In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same are administered by subcutaneous injection, e.g., using a pre-fdled syringe, or an auto-injector.

[0272] In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA, or a pharmaceutically acceptable salt thereof, in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of TTR, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

[0273] In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA, or a pharmaceutically acceptable salt thereof, to the liver.

[0274] An iRNA, or a pharmaceutically acceptable salt thereof, of the invention may be present in a pharmaceutical composition, such as in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

[0275] Alternatively, the dsRNA agent, or a pharmaceutically acceptable salt thereof, of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

[0276] 24

[0277] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0278] Alnylam Reference No.: ALN-542-WO

[0279] The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

[0280] In one aspect, the present invention also provides methods for inhibiting the expression of a TTR gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a TTR gene in a cell of the mammal, thereby inhibiting expression of the TTR gene in the cell.

[0281] Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, enzymatic activity, described herein. For example, a reduction in the expression of TTR may be determined by determining the mRNA expression level of TTR using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of TTR using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques. In certain embodiments, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the TTR gene or protein expression. In other embodiments, a blood sample serves as the subject sample for monitoring the reduction in the TTR protein expression. A reduction in the expression of TTR may also be assessed indirectly by measuring a decrease in the level s of circulating RBP4 protein, retinol, and / or vitamin A in a sample, such as a serum sample.

[0282] The present invention further provides methods of treatment in a subject in need thereof, e.g., a subject diagnosed with a TTR-associated disorder, such as transthyretin-mediated amyloidosis (ATTR), e.g., hereditary ATTR (hATTR) or non-hereditary ATTR (wtATTR).

[0283] The present invention further provides methods of prophylaxis in a subject in need thereof. The treatment methods of the invention include administering a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention to a subject, e.g., a subject that would benefit from a reduction of TTR expression, in a prophylactically effective amount of a dsRNA, or a pharmaceutically acceptable salt thereof, targeting a TTR gene.

[0284] Treatment of a subject that would benefit from a reduction and / or inhibition of TTR gene expression includes therapeutic treatment (e.g., a subject is having a TTR-associated disorder) and prophylactic treatment (e.g., the subject is not having a TTR-associated disorder or a subject may be at risk of developing a TTR-associated disorder).

[0285] In some embodiments, the TTR-associated disorder is selected from the group consisting of transthyretin-mediated amyloidosis (ATTR), e.g., hereditary ATTR (hATTR) or non-hereditary ATTR (wtATTR); senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / Central Nervous System (CNS) amyloidosis.

[0286] 25

[0287] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0288] Alnylam Reference No.: ALN-542-WO

[0289] In one embodiment, the subject has a TTR-associated amyloidosis and the method reduces an amyloid TTR deposit in said subject. In one embodiment, the ATTR is hereditary ATTR (hATTR). In one embodiment, the ATTR is non-hereditary ATTR (wt ATTR).

[0290] In one embodiment, the TTR-associated disease is hereditary transthyretin-mediated amyloidosis and the method treat polyneuropathy in the subject.

[0291] In one embodiment, the TTR-associated disease is transthyretin-mediated amyloidosis and the method treat cardiomyopathy in the subject.

[0292] In one embodiment, administration of the dsRNA agent or pharmaceutical composition to the subject improves at least one indicia of neurological impairment, quality of life, ongoing nerve damage, or cardiovascular impairment in the subject.

[0293] In one embodiment, the methods of the invention improve at least one indicia of neurological impairment in the subject. “Improving at least one indicia of neurological impairment” in the subject refers to the ability of the methods of the invention to slow, reduce, or arrest neurological impairment, or improve any symptom associated with neurological impairment. 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 associated with neurological impairment.

[0294] In some embodiments, the indicia of neurological impairment is a Neuropathy Impairment (NIS) score.

[0295] NIS refers to a scoring system that measures weakness, sensation, and reflexes, especially with respect to peripheral neuropathy. The NIS score evaluates a standard group of muscles for weakness (1 is 25% weak, 2 is 50% weak, 3 is 75% weak, 3.25 is movement against gravity, 3.5 is movement with gravity eliminated, 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 touchpressure, 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 corrected for age, gender, and physical fitness.

[0296] In one embodiment, the methods of the invention reduce a NIS by at least 5%. In other embodiments, the methods of the invention result in a reduction of NIS by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or by at least 50%. In other embodiments, the methods arrest an increasing NIS score, e.g., the method results in a 0% increase of the NIS score. In yet other embodiments, the methods of the invention slow the rate at which an NIS score increases, e.g., the rate of increase of an NIS score in a subject treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention as compared to the rate of increase of an NIS score in a subject that is not treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention.

[0297] Methods for determining an NIS in a human subject are well known to one of skill in 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; 11(1):21-32.

[0298] In some embodiments, the indicia of neurological impairment is a Modified Neuropathy Impairment score (mNIS+7).

[0299] 26

[0300] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0301] Alnylam Reference No.: ALN-542-WO

[0302] As known to one of ordinary skill in the art, mNIS+7 refers to a clinical exam-based assessment of neurologic impairment (NIS) combined with electrophysiologic measures of small and large nerve fiber function (NCS and QST), and measurement of autonomic function (postural blood pressure). The mNIS+7 score is a modification of the NIS+7 score (which represents NIS plus seven tests). NIS+7 analyzes weakness and muscle stretch reflexes. Five of the seven tests include attributes of nerve conduction. These attributes are the peroneal nerve compound muscle action potential amplitude, motor nerve conduction velocity and motor nerve distal latency (MNDL), tibial 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 threshold and heart rate decrease with deep breathing.

[0303] The mNIS+7 score modifies NIS+7 to take into account the use of Smart Somatotopic Quantitative Sensation Testing, new autonomic assessments, and the use of compound muscle action potential of amplitudes of the ulnar, peroneal, and tibial nerves, and sensory nerve action potentials of the ulnar and sural nerves (Suanprasert, N. et al., (2014) J. Neurol. Sci., 344(1-2): pgs. 121-128).

[0304] In one embodiment, the methods of the invention reduce an mNIS+7 score by at least 5%. In other embodiments, the methods of the invention result in a reduction of an mNIS+7 score by at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, or by at least 50%. In other embodiments, the methods arrest an increasing mNIS+7, e.g., the methods result in a 0% increase of the mNIS+7. In yet other embodiments, the methods of the invention slow the rate at which an NIS+7 score increases, e.g., the rate of increase of an NIS+7 score in a subject treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention as compared to the rate of increase of an NIS+7 score in a subject that is not treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention.

[0305] In another embodiment, the methods of the invention improve at least one indicia of quality of life in the subject. “Improving at least one indicia of quality of life” in the subject refers to the ability of the methods of the invention to slow, reduce, or arrest quality of life worsening or improve quality of life. Any suitable measure of quality of life can be used to determine whether a subject has reduced, slowed, or arrested, quality of life worsening or improved quality of life.

[0306] Exemplary indicia of quality of life include, but are not limited to, a Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, a median body mass index (mBMI) score, a Rasch-built Overall Disability Scale (R-ODS) score, a 10-meter walk test score, a Composite Autonomic Symptom Score (COMPASS-31), an EuroQoL-5 Dimensions-5 Levels (EQ-5D-5L) questionnaire and a EuroQoL-Visual Analog Scale (EQ VAS) score a Kansas City Cardiomyopathy Questionnaire (KCCQ), and a New York Heart Association (NYHA) Class.

[0307] One suitable measurement of quality of life is the Norfolk Quality of Life -Diabetic Neuropathy (Norfolk QOL-DN) questionnaire. The Norfolk QOL-DN is a validated comprehensive questionnaire designed to capture the entire spectrum of DN related to large fiber, small fiber, and autonomic neuropathy not captured in existing instruments.

[0308] 27

[0309] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0310] Alnylam Reference No.: ALN-542-WO

[0311] In one embodiment, the methods of the invention improve a subject’s Norfolk QOL-DN score from baseline, e.g., a change of about -2.0, -2.5, -3.0, -3.5, -4.0, -4.5, -5.0, -5.5, -6.0, -6.7, -7.0, -7.5, -8.0, -8.5, -9.0, -9.5, or about -10.0. In other embodiments, the methods arrest an increasing Norfolk QOL-DN score, e.g., the methods result in a 0% decrease of the Norfolk QOL-DN score. In yet other embodiments, the methods of the invention slow the rate at which a Norfolk QOL-DN score increases, e.g., the rate of increase of a Norfolk QOL-DN score in a subject treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention as compared to the rate of increase of a Norfolk QOL-DN score in a subject that is not treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention.

[0312] In one embodiment, the indicia of quality of life is the Rasch-built Overall Disability Scale (R-ODS), which is a patient questionnaire designed to capture activity and social participation limitations in patients.

[0313] In one embodiment, the methods of the invention improve a subject’s R-ODS score from baseline, e.g., an increase of at least 0.1, for example at least 0.2, at least 0.3, at least 0.4, or at least 0.5, points of the subject’s R-ODS score. In other embodiments, the methods arrest a decreasing R-ODS score, e.g., the methods result in a 0% decrease of the R-ODS score. In yet other embodiments, the methods of the invention slow the rate at which an R-ODS score decreases, e.g., the rate of decrease of an R-ODS score in a subject treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention as compared to the rate of decrease of an R-ODS score in a subject that is not treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention.

[0314] Other quality of 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 decrease of about 2, 5, 7, 10, 12, 15, 20, or about 25. In other embodiments, the methods arrest an increasing mBMI index score, e.g., the methods result in a 0% increase of the mBMI score. In yet other embodiments, the methods of the invention slow the rate at which mBMI score increases, e.g., the rate of increase of a mBMI score in a subject treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention as compared to the rate of increase of a mBMI score in a subject that is not treated with a dsRNA, or a pharmaceutically acceptable salt thereof, of the invention.

[0315] Another quality of life indicia includes assessment of exercise capacity. One suitable measure is the 10-meter walk test (I0-MWT) which measures gait speed. In one embodiment, the methods of the invention provide to the subject an increase from baseline in the 10-meter walk test by at least about 10 minutes, e.g., about 0.025, 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.

[0316] In one embodiment, the indicia of quality of life is the Kansas City Cardiomyopathy Questionnaire (KCCQ). The KCCQ is a 23-item self-administered questionnaire quantifying 6 domains (symptoms, physical function, quality of life, social limitation, self-efficacy, and symptom

[0317] 28

[0318] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0319] Alnylam Reference No.: ALN-542-WO

[0320] stability) and 2 summary scores (clinical and overall summary). Scores are transformed to a range of 0-100, in which higher scores reflect better health status. The KCCQ is developed to independently measure the patient’s perception of health status, which includes heart failure (HF) symptoms, impact on physical and social function, and how their HF impacts their quality of life within a 2-week recall period (Green CP, et al., J Am Coll Cardiol. 2000 Apr;35(5): 1245-55).

[0321] In one embodiment, the indicia of quality of life is the Composite Autonomic Symptom Score (COMPASS-31). The COMPASS-31 is a validated, 31-item, self-administered questionnaire designed to evaluate the severity and extent of autonomic symptoms across six key domains: orthostatic intolerance, vasomotor, secretomotor, gastrointestinal, bladder, and pupillomotor. This tool provides a weighted total score from 0 to 100, where higher scores indicate greater autonomic dysfunction. It is widely used in research and clinical practice to evaluate changes in autonomic symptoms.

[0322] In one embodiment, the indicia of quality of life is the EuroQol 5-Dimension 5-level (EQ-5D-5L) questionnaire and the EQ visual analogue scale (EQ VAS). The EuroQol 5-Dimension 5-level (EQ-5D-5L) questionnaire is a globally used and multiply validated tool to assess health-related quality of life. The EQ-5D-5L essentially consists of 2 pages: the EQ-5D descriptive system and the EQ visual analogue scale (EQ VAS). The descriptive system comprises five dimensions: mobility, self-care, usual activities, pain / discomfort and anxiety / depression. Each dimension has 5 levels: no problems, slight problems, moderate problems, severe problems and extreme problems. The patient is asked to indicate his / her health state by ticking the box next to the most appropriate statement in each of the five dimensions. This decision results in a 1-digit number that expresses the level selected for that dimension. The digits for the five dimensions can be combined into a 5 -digit number that describes the patient’s health state. The five dimensions measuring health status can be converted to a single utility value (EQ-Index score).

[0323] In one embodiment, the methods of the invention stabilize or improve a New York Heart Association (NYHA) classification. The New York Heart Association (NYHA) classification helps to classify heart failure patients based on their symptoms into four classes. Class I: No limitation of physical activity. Ordinary physical activity does not cause undue fatigue, palpitation or shortness of breath. Class II: Slight limitation of physical activity. Comfortable at rest. Ordinary physical activity results in fatigue, palpitation, shortness of breath or chest pain. Class III: Marked limitation of physical activity. Comfortable at rest. Less than ordinary activity causes fatigue, palpitation, shortness of breath or chest pain. Class IV: Symptoms of heart failure at rest. Any physical activity causes further discomfort.

[0324] In one embodiment, the methods of the invention stabilize or improve a polyneuropathy disability (PND) score and familial amyloidotic polyneuropathy (FAP) stage. PND Score is determined as follows: PND I: preserved walking, sensory disturbances; PND II: impaired walking but can walk without stick or crutch; PND Illa: walk with 1 stick or crutch; PND Illb: walk with 2 sticks or crutches; PND IV: confined to wheelchair or bedridden. FAP stage is as follows: FAP I:

[0325] 29

[0326] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0327] Alnylam Reference No.: ALN-542-WO unimpaired ambulation; FAP II: assistance with ambulation required; FAP III: wheelchair bound or bedridden.

[0328] In some embodiments, the methods of the invention improve at least one indicia selected from the group consisting of Neuropathy Impairment Score (NIS), modified Neuropathy Impairment Score (mNIS+7), Norfolk Quality of Life Diabetic Neuropathy (Norfolk QoL-DN) questionnaire, modified Body Mass Index (mBMI), Rasch built Overall Disability Scale (R ODS) questionnaire, 10-meter walk test (10 MWT), Composite Autonomic Symptom Score (COMPASS-31), EuroQoL-5 Dimensions-5 Levels (EQ-5D-5L) questionnaire and the EuroQoL-Visual Analog Scale (EQ VAS), a Kansas City Cardiomyopathy Questionnaire (KCCQ), and a New York Heart Association (NYHA) Class, Familial Amyloidotic Polyneuropathy (FAP) stage, and Polyneuropathy Disability (PND) score, compared to a baseline, as determined before administration of the dsRNA agent or pharmaceutical composition.

[0329] In some embodiments, the methods of the invention result in an improvement or a stabilization of a cardiac marker, an echocardiogram parameter, and / or an Technetium scintigraphy cardiac parameter, compared to a baseline, as determined before administration of the dsRNA agent or pharmaceutical composition.

[0330] In some embodiments, the cardiac marker is selected from the group consisting of N-terminal prohormone B-type natriuretic peptide (NT-proBNP), Troponin I, Troponin T, and Neurofilament light chain (NfL).

[0331] In some embodiments, the cardiac biomarkers NT-proBNP and troponin I are used to assess cardiac stress and heart failure (HF) severity. These biomarkers have been shown to be prognostic of outcomes in HF, including in ATTR amyloidosis (Damy T, et al. Amyloid. 2016 Sep;23(3): 194-202; Kristen AV, et al., PLoS One. 2017;12(4):e0173086; 2017; Merlini G. et al., Leukemia. 2016 Oct;30(10): 1979-86).

[0332] In some embodiments, the cardiac biomarker, neurofdament light chain (NfL) is assessed. NfL is a biomarker for polyneuropathy signaling neuroaxonal injury. NfL levels are elevated in patients with hATTR amyloidosis and decrease with patisiran therapy (Ticau S. et al., Neurology. 2021 Jan 19;96(3):e412-e22).

[0333] In some embodiments, the echocardiogram parameter is selected from the group consisting of global longitudinal strain, mean left ventricular wall thickness, left ventricular dimensions, left ventricular ejection fraction, right ventricular dimensions, right ventricular ejection fraction, left atrial diameter, left ventricular strain, right ventricular strain, diastolic function, E / e’ ratio, pulmonary artery pressure, and tricuspid annular plane systolic excursion (TAPSE).

[0334] In one embodiment, the methods of the invention stabilize or improve the ATTR amyloidosis disease stage or NAC stage. The National Amyloidosis Centre (NAC) staging system for TTR amyloidosis with cardiomyopathy (both hATTR and wtATTR) uses N-terminal pro-B-type natriuretic peptide (NT-proBNP) and estimated glomerular filtration rate (eGFR) to stratify patients into stages I, II, III, and IV, with higher stages indicating more advanced disease and poorer prognosis (Gillmore

[0335] 30

[0336] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0337] Alnylam Reference No.: ALN-542-WO JD., et al., Eur Heart J. 2018 Aug 7;39(30):2799-806). Stage I was defined as NT-proBNP <3000 ng / L and eGFR >45 ml / min / 1.73m2. Stage III was defined as NT-proBNP >3000 ng / L and eGFR<45 ml / min / 1.73m2, and the remainder were Stage II.

[0338] In some embodiments, the methods of the invention reduce or prevent oral diuretic intensification / initiation (GDI). GDI is assessed as an indication of worsening of heart failure severity. GDI is defined as any post-randomization, sustained increase in dose of loop diuretic (e.g., azosemide, bumetanide, furosemide, piretanide, torsemide) for at least 7 days, or initiation of loop diuretic. GDI is associated with higher risk of subsequent CV events and death, and may therefore serve as an early marker for heart failure worsening (Fontana et al., J Am Coll Cardiol. 2024a Nov 18; Fontana et al., J Am Coll Cardiol. 2024b Dec 16).

[0339] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces the composite of all-cause mortality and recurrent cardiovascular (CV) events (e.g., CV hospitalizations and urgent heart failure visits). As used herein, the term “hospitalization” refer to a non-elective admission to an acute setting resulting in a stay of greater than 24 hours duration, or an emergency department ward visit of greater than 24 hours duration. As used herein, the term “urgent heart failure (HF) visits” refers to a visit that occurs outside of an inpatient hospitalization (e.g., emergency department or ward, e.g., of less than 24 hours duration, urgent care clinic, day clinic). In an urgent heart failure visit, a patient has signs, symptoms, and / or test results indicative of worsening heart failure for which the patient receives treatment similar to that received in a heart failure hospitalization, e.g., intravenous diuretic therapy. Augmentation of oral diuretic therapy alone does not qualify as an urgent heart failure visit (Hicks KA, et al.

[0340] Circulation. 2018 Feb 27; 137(9): 961-72). In some embodiments, the methods of the present invention result in a reduction in the composite of all-cause mortality and recurrent cardiovascular (CV) events by at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0341] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces CV hospitalizations and urgent heart failure visits. In some embodiments, the methods of the present invention result in a reduction in the CV hospitalizations and urgent heart failure visits by at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0342] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject delays the onset of cardiovascular (CV) events (e.g., CV hospitalizations and urgent heart failure visits) or all -cause mortality. In some embodiments, the methods of the present invention result in a delay in the onset of cardiovascular (CV) events (e.g., CV hospitalizations and urgent heart failure visits) or all -cause mortality by at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0343] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces the all-cause mortality. In some embodiments, the

[0344] 31

[0345] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0346] Alnylam Reference No.: ALN-542-WO

[0347] methods of the present invention result in a reduction in the all-cause mortality by at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0348] In some embodiments, the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces recurrent cardiovascular events. In some embodiments, the methods of the present invention result in a reduction in the recurrent cardiovascular events by at least 10%, e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0349] In one embodiment, the subject is suffering from familial amyloidotic cardiomyopathy (FAC). In another embodiment, the subject is suffering from FAC with a mixed phenotype, i.e., a subject having both cardiac and neurological impairments. In yet another embodiment, the subject is suffering from FAP with a mixed phenotype, i.e., a subject having both neurological and cardiac impairments. In one embodiment, the subject is suffering from FAP that has been treated with an orthotopic liver transplantation (OLT).

[0350] In another embodiment, the subject is suffering from senile systemic amyloidosis (SSA). In other embodiments of the methods of the invention, the subject is suffering from familial amyloidotic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Normal-sequence TTR causes cardiac amyloidosis in people who are elderly and is termed senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA often is accompanied by microscopic deposits in many other organs. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of clinically significant TTR amyloidosis (also called ATTR (amyloidosis-transthyretin type)). More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.

[0351] In some embodiments, the subject is suffering from transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP). Such subjects may suffer from ocular manifestations, such as vitreous opacity and glaucoma. It is known to one of skill in the art that amyloidogenic transthyretin (ATTR) synthesized by retinal pigment epithelium (RPE) plays important roles in the progression of ocular amyloidosis. Previous studies have shown that panretinal laser photocoagulation, which 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 amyloidosis (see, e.g., Kawaji, T., etal., Ophthalmology. (2010) 117: 552-555). Another TTR-associated disease is hyperthyroxinemia, also known as “dystransthyretinemic hyperthyroxinemia” or “dysprealbuminemic hyperthyroxinemia”. This type of hyperthyroxinemia may 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.

[0352] The dsRNA agent, or a pharmaceutically acceptable salt thereof, can be administered by any known methods in the art. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject intravenously, intramuscularly, or subcutaneously.

[0353] 32

[0354] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0355] Alnylam Reference No.: ALN-542-WO

[0356] The dsRNA agent, or a pharmaceutically acceptable salt thereof, can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis.

[0357] Before administration of a full dose of the dsRNA agent, or a pharmaceutically acceptable salt thereof, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

[0358] Alternatively, the dsRNA agent, or a pharmaceutically acceptable salt thereof, can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of dsRNA agent, or a pharmaceutically acceptable salt thereof, to a subject. The injections may be repeated over a period of time.

[0359] The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of dsRNA agent, or a pharmaceutically acceptable salt thereof, on a regular basis, such as every other day or to once a year.

[0360] In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered about once per week, once every 7-10 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once per month, once every 2 months, once every 3 months (once per quarter), once every 4 months, once every 5 months, or once every 6 months, or every 12 months.

[0361] In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered about once every 3 months (once per quarter). In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered about once every 4 months. In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered about once every 5 months. In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered about once every 6 months. In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered about every 9 months. In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered about every 12 months.

[0362] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 200-400 mg, about 250-350 mg, about 275-325 mg, about 280-320 mg, about 290-310 mg, about 295-305 mg every six months. In certain embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject as a fixed dose of about 275-325 mg every six months.

[0363] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a fixed dose of about 200 mg, about 250, about 275 mg, about 280 mg,

[0364] 33

[0365] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0366] Alnylam Reference No.: ALN-542-WO about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 320 mg, about 325 mg, about 350 mg, or about 400 mg every six months. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 300 mg every six months.

[0367] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously at a dose of about 275-325 mg every six months.

[0368] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously at a dose of about 300 mg every six months. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously via a pre-fdled syringe. In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously via an autoinjector.

[0369] In one embodiment, the method includes administering a composition featured herein such that expression of the target TTR gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target TTR gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

[0370] Administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and / or markers of such diseases or disorders in a patient. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0371] Administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, can reduce TTR levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more. In one embodiment, administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, can reduce TTR levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 20%.

[0372] Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well

[0373] 34

[0374] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0375] Alnylam Reference No.: ALN-542-WO

[0376] within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of the dsRNA agent , or a pharmaceutically acceptable salt thereof, or pharmaceutical composition thereof, "effective against" a TTR-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the disorder and the related causes.

[0377] In some embodiments, the efficacy of the methods of the invention can be measured by monitoring the severity of a symptom of a TTR-associated disorder (such as transthyretin-mediated amyloidosis), e.g., polyneuropathy, cardiomyopathy, reduction in sensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, carpal tunnel syndrome, autonomic insufficiency, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and corneal lattice dystrophy.

[0378] A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art.

[0379] In some embodiments, the methods further comprise determining the level of vitamin A, RBP4 and / or TTR in a sample(s) from the subject.

[0380] The invention further provides administering to the subject an additional therapeutic agent for treating a subject that would benefit from reduction and / or inhibition of TTR expression, e.g., a subject having a TTR-associated disease disorder, or condition. The additional therapeutic agent can be any known pharmaceuticals and / or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. In some embodiments, the additional therapeutic agent is selected from the group consisting of an agent which inhibits the expression and / or activity of transthyretin (TTR), a TTR stabilizer, a non-steroidal anti-inflammatory agent (NSAIDS), e.g., diflunisal, and diuretics, a synthetic retinoid fenretinide, an anti-VEGF therapy, a corticosteroid, insulin, a glucagon-like peptide 1 agonist, a sulfonylurea, a seglitinide, a biguanide, a thiazolidinedione, an alpha-glucosidase inhibitor, an SGLT2 inhibitor, a DPP -4 inhibitor, an HMG-CoA reductase inhibitor, and a combination of any of the foregoing.

[0381] A “TTR stabilizer” or a “therapeutic agent that stabilizes TTR” or “that stabilizes a TTR tetramer” is an agent that reduces or prevents the dissociation of the subunits of a TTR tetramer, e.g., into monomers. In some embodiments, the agent reduces the formation of TTR amyloid plaques, e.g.,

[0382] 35

[0383] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0384] Alnylam Reference No.: ALN-542-WO

[0385] by reducing the level of TTR monomers or proteolytic fragments of TTR monomers that form TTR amyloid plaques. Such agents include, but are not limited to, tafamidis, diflunisal, and AGIO.

[0386] The iRNA agent and an additional therapeutic agent and / or treatment may be administered at the same time and / or in the same combination, e.g., subcutaneously, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and / or by another method known in the art or described herein.

[0387] III. Delivery of an iRNA of the Invention

[0388] The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a TTR-associated disorder) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising a dsRNA, or a pharmaceutically acceptable salt thereof, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA.

[0389] One or more injections may be used to deliver the desired fixed dose of iRNA to the subject. The injections may be repeated over a period of time. In some embodiments, the subcutaneous injection is achieved using a pre-filled syringe. In some embodiments, the subcutaneous injection is achieved using an auto-injector.

[0390] The administration may be repeated on a regular basis. In certain embodiments, the iRNA is administered about once per month to about once per quarter, i. e. , about every three months, or about once per quarter to about twice per year, i. e. , about once every six months. In certain embodiments, the iRNA is administered once per month. In other embodiments, the iRNA is administered every three months (once per quarter). In yet another embodiment, the iRNA is administered every six months (biannually). In other embodiments, the iRNA is administer every twelve months (annually).

[0391] In some embodiments, the fixed dose is administered to the subject at an interval of once every month to every two months. In some embodiments, the fixed dose is administered to the subject at an interval of once every three to six months. In some embodiments, the fixed dose is administered to the subject at an interval of once every six to twelve months.

[0392] In some embodiments, the fixed dose is administered to the subject at an interval of once every month. In some embodiments, the fixed dose is administered to the subject at an interval of once every two months. In some embodiments, the fixed dose is administered to the subject at an interval of once every three months. In some embodiments, the fixed dose is administered to the subject at an interval of once every four months. In some embodiments, the fixed dose is administered to the subject at an interval of once every five months. In some embodiments, the fixed dose is administered to the subject at an interval of once every six months. In some embodiments, the fixed dose is administered to the subject at an interval of once every twelve months.

[0393] 36

[0394] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0395] Alnylam Reference No.: ALN-542-WO

[0396] In some embodiments, the method comprises administering to the subject a fixed dose of about 200-400 mg, about 250-350 mg, about 275-325 mg, about 280-320 mg, about 290-310 mg, about 295-305 mg, e.g., a fixed dose of about 200 mg, about 250, about 275 mg, about 280 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 320 mg, about 325 mg, about 350 mg, or about 400 mg, of a dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR.

[0397] In some embodiments, the method comprises administering to the subject subcutaneously a fixed dose of about 200-400 mg, about 250-350 mg, about 275-325 mg, about 280-320 mg, about 290-310 mg, about 295-305 mg, e.g., a fixed dose of about 200 mg, about 250, about 275 mg, about 280 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 320 mg, about 325 mg, about 350 mg, or about 400 mg, of a dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR.

[0398] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 275-325 mg every six months.

[0399] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously at a dose of about 275-325 mg every six months.

[0400] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 300 mg every six months.

[0401] In some embodiments, the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously at a dose of about 300 mg every six months.

[0402] IV. iRNAs for Use in the Methods of the Invention

[0403] Suitable double stranded RNAi agents for use in the methods of the invention include the dsRNA agent AD-649264.

[0404] AD-649264 comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the unmodified nucleotide sequence 5’-CAAGAGUAUUCCAUUUUUACU-3’ of SEQ ID NO: 11 and the antisense strand comprises the unmodified nucleotide sequence 5’-AGUAAAAAUGGAAUACUCUUGGU-3’ of SEQ ID NO: 12.

[0405] In some embodiments, the sense strand of AD-649264 comprises the modified nucleotide sequence 5’- csasagagUfaUfUfCfcauuuuuacu -3’ of SEQ ID NO: 17 and the antisense strand of AD-649264 comprises the modified nucleotide sequence 5’- asGfsuaaAfaauggaaUfaCfucuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; and s is a phosphorothioate linkage; and wherein a ligand is conjugated to the 3’ end of the sense strand as shown in the following schematic

[0406] 37

[0407] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0408] Alnylam Reference No.: ALN-542-WO

[0409]

[0410] wherein X is O. In some embodiments, the sense strand of AD-649264 comprises the modified nucleotide sequence 5’- csasagagUfaUfUfCfcauuuuuacuL96 -3’ of SEQ ID NO: 13 and the antisense strand of AD-649264 comprises the modified nucleotide sequence 5’ - asGfsuaaAfaauggaaUfaCfiicuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-0Me) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; s is a phosphorothioate linkage; and L96 is:

[0411] >

[0412]

[0413] In some embodiments, the L96 is conjugated to the 3’ end of the sense strand as shown in the following schematic

[0414]

[0415] wherein X is O.

[0416] 38

[0417] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0418] Alnylam Reference No.: ALN-542-WO

[0419] Suitable double stranded RNAi agents for use in the methods of the invention also include a pharmaceutically acceptable salt form of the dsRNA agent AD-649264. Any salt that is pharmaceutically acceptable, e.g., a sodium salt of the dsRNA agent, may be used. In one embodiment, the pharmaceutically acceptable salt of the dsRNA of the invention being used in the methods of the invention has the following structure:

[0420] 21 Na

[0421] " " " " " " >" > & & < " > "

[0422]

[0423] Additional dsRNA agents that may be used in the methods of the invention are described in International PCT Publication No. WO 2023 / 014677, the entire contents of which are incorporated herein by reference.

[0424] V. Pharmaceutical Compositions of the Invention

[0425] The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA of the invention are useful for treating a disease or disorder associated with the expression or activity of a TTR gene, e.g., a TTR-associated disease.

[0426] Such pharmaceutical compositions are formulated based on the mode of delivery.

[0427] The pharmaceutical compositions comprising RNAi agents of the invention may be, for example, solutions with or without a buffer, or compositions containing pharmaceutically acceptable carriers. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micellar formulations, emulsions, and gene therapy vectors.

[0428] In the methods of the invention, the RNAi agent may be administered in a solution. In some embodiments, the solution is a sterile solution. An RNAi agent may be administered in an unbuffered

[0429] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0430] Alnylam Reference No.: ALN-542-WO

[0431] solution, e.g., in saline or in water. Alternatively, the RNAi agent may also be administered in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administering to a subject.

[0432] In some embodiments, the pharmaceutical composition comprises a sterile formulation in a phosphate buffer. In some embodiments, the pharmaceutical composition has a pH of 7.0. In some embodiments, the pharmaceutical composition comprises a sterile formulation in a 5 mM phosphate buffer with a pH of 7.0.

[0433] In some embodiments, the pharmaceutical composition comprises a dsRNA agent (e.g., AD-649264), or a pharmaceutically acceptable salt thereof.

[0434] In some embodiments, the pharmaceutical composition comprises, per vial (e.g., a 0.5 mb vial), about 100 mg of a dsRNA agent (e.g., AD-649264), or about 106 mg of a pharmaceutically acceptable salt thereof, e.g., sodium form of AD-649264.

[0435] In some embodiments, the pharmaceutical composition comprises about 200 mg / mL of a dsRNA agent (e.g., AD-649264), or about 212 mg / mL of a pharmaceutically acceptable salt thereof, e.g., sodium form of AD-649264.

[0436] In some embodiments, the pharmaceutical composition comprises about 300 mg / mL of a dsRNA agent (e.g., AD-649264), or about 318 mg / mL of a pharmaceutically acceptable salt thereof, e.g., sodium form of AD-649264.

[0437] In some embodiments, the pharmaceutical composition comprises a dsRNA agent (e.g., AD-649264), or a pharmaceutically acceptable salt thereof, and a buffer, e.g., sodium dihydrogen phosphate dihydrate, and / or di-sodium hydrogen phosphate anhydrous.

[0438] In some embodiments, the pharmaceutical composition comprises, per vial (e.g., a 0.5 mL vial), about 100 mg of a dsRNA agent (e.g., AD-649264), or about 106 mg of a pharmaceutically acceptable salt thereof, e.g., sodium form of AD-649264, about 0.184 mg of sodium dihydrogen phosphate dihydrate, and about 0.188 mg of di-sodium hydrogen phosphate anhydrous.

[0439] In some embodiments, the pharmaceutical composition comprises about 200 mg / mL of a dsRNA agent (e.g., AD-649264), or about 212 mg / mL of a pharmaceutically acceptable salt thereof, e.g., sodium form of AD-649264, about 0.367 mg / mL of sodium dihydrogen phosphate dihydrate, and about 0.376 mg / mL of di-sodium hydrogen phosphate anhydrous.

[0440] In some embodiments, the pharmaceutical composition further comprises a diluent, e.g., water, and / or an agent for adjusting the pH, e.g., phosphoric acid, and / or sodium hydroxide. In some embodiments, compendial grade phosphoric acid at 0.3N or sodium hydroxide at 0.25N may be used as needed to adjust the pH of the composition.

[0441] In some embodiments, the pharmaceutical composition comprises, per vial (e.g., a 0.5 mL vial), about 100 mg of a dsRNA agent (e.g., AD-649264), or about 106 mg of a pharmaceutically acceptable salt thereof, e.g., sodium form of AD-649264, about 0.184 mg of sodium dihydrogen

[0442] 40

[0443] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0444] Alnylam Reference No.: ALN-542-WO

[0445] phosphate dihydrate, about 0.188 mg of di-sodium hydrogen phosphate anhydrous, water, phosphoric acid and sodium hydroxide.

[0446] In some embodiments, the pharmaceutical composition comprises about 200 mg / mL of a dsRNA agent (e.g., AD-649264), or about 212 mg / mL of a pharmaceutically acceptable salt thereof, e.g., sodium form of AD-649264, about 0.367 mg / mL of sodium dihydrogen phosphate dihydrate, about 0.376 mg / mL of di-sodium hydrogen phosphate anhydrous, water, phosphoric acid and sodium hydroxide.

[0447] In some embodiments, the buffer solution further comprises an agent for controlling the osmolarity of the solution, such that the osmolarity is kept at a desired value, e.g., at the physiologic values of the human plasma. Solutes which can be added to the buffer solution to control the osmolarity include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolarity of the solution is a salt. In certain embodiments, the agent for controlling the osmolarity of the solution is sodium chloride or potassium chloride.

[0448] In some embodiments, the pharmaceutical compositions of the invention are pyrogen free or non-pyrogenic.

[0449] The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by atransdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

[0450] One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a TTR gene. In some embodiments, a fixed dose of about 200 mg to about 400 mg of the iRNA agents is administered to the subject. In some embodiments, the method comprises administering to the subject a fixed dose of about 200-400 mg, about 250-350 mg, about 275-325 mg, about 280-320 mg, about 290-310 mg, about 295-305 mg, e.g., a fixed dose of about 200 mg, about 250, about 275 mg, about 280 mg, about 290 mg, about 295 mg, about 300 mg, about 305 mg, about 310 mg, about 320 mg, about 325 mg, about 350 mg, or about 400 mg, of a dsRNA agent, or a pharmaceutically acceptable salt thereof, that inhibits expression of TTR.

[0451] A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every month, every two months, every three months, every four months, every five months, every six months, once every 3-6 months, or once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter to about once per six months to about once per twelve months.

[0452] 41

[0453] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0454] Alnylam Reference No.: ALN-542-WO

[0455] After an initial treatment regimen, the treatments can be administered on a less frequent basis. Duration of treatment can be determined based on the severity of disease.

[0456] The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to mutations present in the subject, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a prophylactically or therapeutically effective amount, as appropriate, of a composition can include a single treatment or a series of treatments.

[0457] The RNAi agent can be delivered in a manner to target a particular tissue (e.g., hepatocytes). Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Formulations include those that target the liver.

[0458] The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers.

[0459] VII. Kits

[0460] The present invention also provides kits for performing any of the methods of the invention. Such kits include one or more RNAi agent(s) and instructions for use, e.g., instructions for administering a fixed dose of a double stranded RNAi agent(s).

[0461] The double stranded RNAi agent may be in a vial, or a pre-filled syringe, or an auto-injector. The kits may optionally further comprise means for administering the double stranded RNAi agent (e.g., an injection device, such as a pre-filled syringe, or an auto-injector), or means for measuring the inhibition of TTR (e.g., means for measuring the inhibition of TTRmRNA, TTR protein, and / or TTR activity). Such means for measuring the inhibition of TTR may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The kits of the invention may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount.

[0462] The present invention also provides vials comprising the dsRNA agent, or a pharmaceutically acceptable salt thereof, of the invention or the pharmaceutical composition of the invention. The present invention further provides syringes comprising the dsRNA agent, or a pharmaceutically acceptable salt thereof, of the invention or the pharmaceutical composition of the invention.

[0463] 42

[0464] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0465] Alnylam Reference No.: ALN-542-WO

[0466] In some embodiments, the RNAi agent (e.g., AD-649264), or a pharmaceutically acceptable salt thereof, described herein is administered (e.g., subcutaneously) in an auto-injector, or a syringe, such as a pre-fdled syringe to a subject in need thereof. Pre-fdled syringes are designed to fit into specialized syringes, which can be used to administer the RNAi agent, or a pharmaceutically acceptable salt thereof, described herein. Auto-injectors are devices that house a pre-filled syringe and automates the injection process, and can also be used to administer the RNAi agent, or a pharmaceutically acceptable salt thereof, described herein. Pre-filled syringes and auto-injectors offer several advantages including convenience, affordability, accuracy, sterility, and safety (Makwana et al., Int J Pharm Investig. 2011 Oct-Dec; 1(4): 200-206; incorporated in its entirety herein by reference). Pre-filled syringes and auto-injectors also assure that patients receive accurate dosages. This is especially advantageous for patients who need to self-inject medication, but have no medical training. In some embodiments, the RNAi agent (e.g., AD-649264) stored inside of a pre-filled syringe or an auto-injector is in a sterile solution.

[0467] In some embodiments the pre-filled syringe is made of glass. In some embodiments the prefilled syringe is made of plastic.

[0468] In some embodiments, the kit further comprises instructions, for example, for administering the RNAi agent (e.g., AD-649264), or a pharmaceutically acceptable salt thereof, in an auto-injector or a syringe, such as a pre-filled syringe. For example, the instructions may be performed under the supervision of a drug investigator. These instructions simply embody the disclosure provided herein.

[0469] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0470] 43

[0471] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0472] Alnylam Reference No.: ALN-542-WO

[0473] EXAMPLES

[0474] Example 1. A Phase 3, Global, Randomized, Open-Label Study to Evaluate the Efficacy and Safety of ALN-TTRscO4 in Patients with Hereditary Transthyretin-Mediated Amyloidosis with Polyneuropathy

[0475] This Example describes a global Phase 3, randomized, open-label study designed to evaluate the efficacy, safety, and pharmacokinetic (PK) / pharmacodynamic (PD) profiles of AD-649264 (ALN-TTRscO4) in adult patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy (hATTRPN).

[0476] Approximately 125 patients are planned for enrollment and randomization at a 4: 1 ratio to ALN-TTRscO4 or vutrisiran, a reference group. For the global population outside of Japan, randomization is stratified by transthyretin (TTR) genotype (V30M versus non-V30M) and baseline neuropathy impairment score (NIS) (<50 versus >50). Patients in Japan are randomized separately from the global population and without stratification. To match the cardiac disease severity with the APOLLO study population, the study plans to enroll no more than 15% of patients with N-terminal prohormone B-type natriuretic peptide (NT proBNP) values greater than 3000 ng / L at baseline. For the primary endpoint and most secondary and exploratory efficacy endpoints, ALN-TTRscO4 is compared to the placebo control group from the APOLLO study of patisiran. In addition, an in-study vutrisiran group will serve as a reference group to validate the use of the external control for the primary and secondary efficacy analyses. The in-study vutrisiran reference group validates the use of the external control for the primary and secondary efficacy analyses by allowing descriptive comparison of the clinical efficacy endpoints between treatment groups within this study. The comparator allows for a descriptive comparison of the vutrisiran arm with its established profile. In addition, comparison of ALN-TTRscO4 and within study vutrisiran is performed for the analysis of serum TTR reduction. The objectives and endpoints are described in Table 3 below.

[0477] The study consists of a Screening Period of up to 42 days, an 18-month Treatment Period (during which patients are receiving ALN-TTRscO4 or vutrisiran), and an up to 18-month Treatment Extension Period (during which all patients are receiving ALN-TTRscO4), and a Follow-up Period of up to 1 year (2 years for female patients of childbearing age) occurs after the last dose of study drug (see FIG. 1).

[0478] Patients enrolled in this study are randomized to receive ALN-TTRscO4 (300 mg subcutaneous [SC] once every 6 months [q6M]) or vutrisiran (25 mg SC once every 3 months [q3M]) during the Treatment Period. At the end of the Treatment Period, patients receiving vutrisiran are transitioned to ALN-TTRscO4 (300 mg SC q6M) during the 18-month Treatment Extension Period, while patients who received ALN-TTRscO4 continue their treatment.

[0479] This study includes adult patients (age 18 or age of legal consent, whichever is older) up to 85 years of age with a documented TTR mutation and a confirmed diagnosis of symptomatic hATTR

[0480] 44

[0481] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0482] Alnylam Reference No.: ALN-542-WO PN, with a NIS of 5 to 130 (inclusive), a polyneuropathy disability (PND) score of <3b, and Kamofsky Performance Scale (KPS) >60%.

[0483] During the Treatment Period, patients undergo assessments for efficacy and / or safety.

[0484] Primary and secondary efficacy evaluations include mNIS+7, Norfolk Quality of Life Diabetic Neuropathy (Norfolk QoL-DN) questionnaire, modified Body Mass Index (mBMI), Rasch built Overall Disability Scale (R ODS) questionnaire, 10-meter walk test (10 MWT), and percent TTR reduction. The placebo group of the APOLLO study was chosen as an external control for the primary and most secondary and exploratory efficacy analyses. The in study vutrisiran group was chosen as a control for the secondary and exploratory endpoints of reduction of serum TTR. It also serves as an in-study reference group enabling descriptive comparison of the clinical efficacy endpoints between treatment groups and helping to validate the use of an external control. These analyses are outlined in Table 3. The Treatment Extension Period is designed to allow the evaluation of the safety, PD, and efficacy of the dosing regimen for a total of up to 36 months of treatment).

[0485] Table 3: Objectives and Endpoints

[0486]

[0487] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0488] Alnylam Reference No.: ALN-542-WO

[0489]

[0490] Abbreviations: 10-MWT=10-meter walk test; ADA=anti-drug antibodies; AE=adverse event; APOLLO=Study ALN-TTR02-004; COMPASS-3 l=Composite Autonomic Symptom Score-31; EQ-5D-5L=EuroQoL-5 Dimensions-5 Levels; EQ-VAS=EuroQoL-Visual Analog Scale; FAP=Familial Amyloidotic Polyneuropathy; hATTR-PN=hereditary transthyretin-mediated amyloidosis with polyneuropathy; mBMI=modified body mass index; mNIS+7=Modified Neuropathy Impairment Score +7; NIS=Neuropathy Impairment Score;

[0491] NfL=Neurofilament light chain; Norfolk QoL-DN=Norfolk Quality of Life-Diabetic Neuropathy;

[0492] NT-proBNP=N-terminal prohormone B-type natriuretic peptide; PD=pharmacodynamics;

[0493] PK=pharmacokinetics; PND=Polyneuropathy Disability; R-ODS=Rasch-built Overall Disability Scale; TTR=transthy retin.

[0494] ALN-TTRscO4 is administered at a dose of 300 mg q6M SC, defined as administration every 24 weeks in this study.

[0495] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0496] Alnylam Reference No.: ALN-542-WO Study drug is administered using a single-use prefilled syringe and a needle safety device. For ALN-TTRscO4, the prefilled syringe delivers a 300 mg dose with a volume of 1.5 mL. For vutrisiran, the prefilled syringe delivers a 25 mg dose with a volume of 0.5 mL. The prefilled syringe is a singleuse injection device, and after injection, the needle safety device will engage to cover the exposed needle.

[0497] All patients are instructed to take the recommended daily allowance of vitamin A until the end of their Follow-up Period.

[0498] The duration of treatment in this study is up to a maximum of 36 months inclusive of the 18 month Treatment Period (ALN-TTRscO4 or vutrisiran) and the up to 18 month Treatment Extension Period (ALN-TTRscO4 only).

[0499] The estimated total time on study for each patient is up to 50 months (62 months for female patients of childbearing age), including up to 1.5 months of screening, up to a maximum of 36 months of treatment (Treatment Period and Treatment Extension Period), and 12 months of safety follow-up after the last dose of study drug (24 months in female patients of childbearing age) or until the patient starts a TTR lowering treatment as a part of clinical care, whichever comes first.

[0500] The primary endpoint and key secondary endpoint include change from baseline in mNIS+7 score and Norfolk QoL-DN total score, respectively, at Month 9. mNIS+7 is a sensitive measure developed to provide a comprehensive evaluation of polyneuropathy in patients with hATTR amyloidosis used previously as primary endpoint in the APOLLO study of patisiran and the HELIOS-A study of vutrisiran. The Norfolk QoL-DN is a patient-reported score validated in patients with hATTR-PN. Other secondary clinical efficacy endpoints such as mBMI, R-ODS questionnaire, and 10-MWT are included to comprehensively describe the impact of ALN-TTRscO4 treatment across a range of important disease manifestations.

[0501] For the primary and secondary clinical efficacy endpoint, the ALN-TTRscO4 group is compared to the placebo group from the APOLLO study at Month 9 and Month 18. For the secondary endpoint of TTR percent reduction, the ALN-TTRscO4 group is compared to the in-study vutrisiran group at Week 6, through Month 9, and through Month 18.

[0502] For the mNIS+7 change from baseline at Month 9, the observed mean (standard deviation [SD]) was 15.2 [17.2] points for the placebo group from the APOLLO study. Assuming that the treatment effect of ALN-TTRscO4 is the same as vutrisiran (HELIOS A) with a mean [SD] change of -1.4 [13.2] points from baseline and assuming 5% drop out rate at Month 9, there is > 95% power to establish the superiority over placebo using a 2-sided t-test with a significance level of 0.05.

[0503] For change in Norfolk-QoL DN total score from baseline at Month 9, the observed mean [SD] was 11.5 [19.2] points for the placebo group from the APOLLO study. Assuming that the treatment effect of ALN-TTRscO4 is the same as vutrisiran (HELIOS A) with a mean [SD] change of -4.3 [18.4] points from baseline and assuming 5% drop out rate at Month 9, there is >95% power to establish the superiority over placebo using 2-sided t test with a significance level of 0.05.

[0504] 47

[0505] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0506] Alnylam Reference No.: ALN-542-WO

[0507] Inclusion Criteria

[0508] Patients are eligible to be included in the study if all the following criteria apply:

[0509] Age and Sex

[0510] I. Male or female age 18 (or age of legal consent, whichever is older) to 85 years of age Patient and Disease Characteristics

[0511] 8. Have a diagnosis of hATTR amyloidosis with polyneuropathy with documented TTR mutation

[0512] 9. Have a neuropathy impairment score (NIS) of 5 to 130 (inclusive; this criterion must be met at the Baseline Visit 2)

[0513] 10. Have a Polyneuropathy Disability (PND) score of <3b (this criterion must be met at the Baseline Visit 3)

[0514] II. Have a Kamofsky Performance Status (KPS) of >60%

[0515] Informed Consent

[0516] 12. Patient is able to understand and is willing and able to comply with the study requirements and to provide written informed consent

[0517] Exclusion Criteria

[0518] Patients are excluded from the study if any of the following criteria apply:

[0519] Disease-specific Conditions

[0520] 1. Has had a liver transplant or is likely, in the opinion of the Investigator, to undergo liver transplantation during the Treatment Period of the study

[0521] 2. Has known other (non-hATTR) forms of amyloidosis or clinical evidence of leptomeningeal amyloidosis

[0522] 3. Has a New Y ork Heart Association heart failure classification >2

[0523] Laboratory Assessments

[0524] 4. Has any of the following laboratory parameter assessments at screening:

[0525] a. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) >2.5 upper limit of normal (ULN) reference range

[0526] b. Total bilirubin >1.5 ULN (>2 ULN in patients with Gilbert's Syndrome)

[0527] c. International normalized ratio (INR) >2.0 (patients on anticoagulant therapy with an INR of <3.5 will be allowed)

[0528] 5. Platelet count <50,000 / pL

[0529] 6. Absolute neutrophil count (ANC) <1500 cells / mm3

[0530] 7. Estimated glomerular filtration rate (eGFR) <30 mL / min / 1.73m2(using the Modification of Diet in Renal Disease [MDRD] formula)

[0531] 8. Has vitamin B12 levels below the lower limit of normal

[0532] 48

[0533] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0534] Alnylam Reference No.: ALN-542-WO 9. Has known human immunodeficiency virus infection; or evidence of acute or chronic hepatitis C virus or hepatitis B virus infection

[0535] Prior / Concomitant Therapy

[0536] 10. Received prior TTR-lowering treatment or participated in a gene therapy trial for ATTR amyloidosis

[0537] 11. Is currently taking tafamidis, acoramidis, doxycycline, or tauroursodeoxycholic acid; if previously on any of these agents, must have completed a 14-day wash-out prior to dosing (Day 1)

[0538] 12. Is currently taking diflunisal; if previously on this agent, must have at least a 3-day wash-out prior to dosing (Day 1)

[0539] 13. Received prior anti-TTR antibody treatment

[0540] 14. Anticipated to start an approved TTR stabilizing therapy (eg, tafamidis or acoramidis) within the first 18 months following randomization

[0541] 15. Received an investigational agent within the last 30 days (or 5 half-lives of the investigational drug, whichever is longer), or are currently in follow-up of another clinical study. Any agent that has received health agency authorization (including for emergency use) by local or regional authorities is not considered investigational.

[0542] Medical Conditions

[0543] 16. Has other known causes of sensorimotor or autonomic neuropathy (eg, autoimmune disease, monoclonal gammopathy) that the treating physician believes to be contributing to the neuropathy

[0544] 17. Had acute coronary syndrome within the past 3 months

[0545] 18. Has uncontrolled clinically significant cardiac arrhythmia or unstable angina

[0546] 19. Has known type 1 diabetes

[0547] 20. Has had type 2 diabetes mellitus for >5 years

[0548] 21. Has untreated hypo- or hyperthyroidism

[0549] 22. Has had a major surgery within the past 3 months or has a major surgery planned during the study through Month 18

[0550] 23. Has an active infection requiring systemic antiviral, antiparasitic, or antimicrobial therapy that will not be completed prior to dosing (Day 1)

[0551] 24. Has other medical conditions or comorbidities (e.g., malignancy, neuropsychiatric disorder, etc.) which, in the opinion of the Investigator, could interfere with study compliance or data interpretation

[0552] 25. Anticipated survival is less than 2 years, in the opinion of the Investigator

[0553] 26. History of intolerance to SC injection(s) or significant abdominal scarring that could potentially hinder study drug administration or evaluation of local tolerability

[0554] 49

[0555] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0556] Alnylam Reference No.: ALN-542-WO 27. Has a history of multiple drug allergies or history of allergic reactions to any component of or excipient in the study drug.

[0557] Contraception, Pregnancy, and Breastfeeding

[0558] 28. Is not willing to comply with the contraceptive requirements during the study period.

[0559] 29. Patient is pregnant or breastfeeding.

[0560] Alcohol Use

[0561] 30. Unwilling or unable to limit alcohol consumption throughout the course of the study. Alcohol intake of >2 units / day is excluded during the study (unit: 1 glass of wine [approximately 125 mb] = 1 measure of spirits [approximately 1 fluid ounce] = A pint of beer [approximately 284 mL]).

[0562] 31. History of alcohol use disorder, within the last 12 months before screening, in the opinion of the Investigator.

[0563] 32. History of illicit drug abuse within the past 5 years that in the opinion of the Investigator would interfere with compliance with study procedures or Follow-up visits.

[0564] Efficacy Analysis

[0565] Primary Endpoint

[0566] The primary endpoint of change in mNIS+7 from baseline at Month 9 is compared between the ALN-TTRscO4 group in this study and the placebo group from the APOLLO study. The treatment effect is estimated based on the least squares (LS) means using an analysis of covariance (ANCOVA) model with baseline mNIS+7 score as a covariate and factors including treatment group (ALN-TTRscO4 vs placebo), genotype (V30M vs non-V30M) and age of disease onset (<50 vs >50 years). Multiple imputation for missing data is done separately for each treatment group using a regression procedure based on baseline covariates.

[0567] Secondary Endpoints

[0568] For the secondary clinical endpoints (except for TTR), the treatment comparison is made between ALN-TTRscO4 group in this study and the placebo group in the APOLLO study at Month 9, or Month 18. For the TTR percent reduction endpoint at Week 6, through Month 9, and through Month 18, the ALN-TTRscO4 group is tested for superiority against the vutrisiran group in this study.

[0569] To control the overall type I error, the secondary endpoints are tested in the following hierarchical order:

[0570] o Norfolk QoL-DN total score change from baseline at Month 9

[0571] o Percent reduction in serum TTR levels through 9 months

[0572] o mBMI [kg / m2] change from baseline at Month 9

[0573] o R-ODS change from baseline at Month 9

[0574] o 10-MWT gait speed change from baseline at Month 9

[0575] 50

[0576] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0577] Alnylam Reference No.: ALN-542-WO For the secondary clinical endpoints of change in Norfolk QoL-DN total score and mBMI from baseline at Month 9, the analysis are based on an ANCOVA model similar to the model described for the analysis of change in mNIS+7 from baseline at Month 9, while adjusting for baseline value of the endpoint being modeled and including baseline NIS score (<50 vs >50) as an additional factor in the model. For these 2 endpoints, data that are missing are multiply imputed separately for each treatment group using a regression procedure based on baseline covariates.

[0578] For all the secondary clinical endpoints evaluated at Month 18 (mNIS+7 score, Norfolk QoL-DN total score, mBMI, R-ODS, and 10-MWT gait speed), change from baseline Month 18 analyses are based on a mixed-effects model for repeated measures, adjusting for a covariate (baseline value for the endpoint being modeled), categorical factors (treatment group, visit [Month 9 versus Month 18], genotype, age of disease onset, baseline NIS score), and an interaction term (treatment group by visit).

[0579] The TTR percent reduction at Week 6, through Month 9, and through Month 18, Wilcoxon rank sum test is used to establish superiority of ALN-TTRscO4 compared to vutrisiran in this study. The TTR percent reduction through Month 9 and through Month 18 are derived as the average TTR percent reduction from Week 6 to Month 9 and from Week 6 to Month 18, respectively, for both ALN-TTRscO4 and in-study vutrisiran. A Hodges-Lehmann method is used to estimate the 95% confidence interval for the median difference between the ALN-TTRscO4 and vutrisiran groups in this study.

[0580] In the APOLLO study, mBMI was not assessed at Month 9 or Month 18. The average values of Day 189 and Day 357 are derived as Month 9, and the Day 546 value is substituted as Month 18.

[0581] For the clinical efficacy endpoints, the vutrisiran group is summarized descriptively. Safety data will be summarized descriptively.

[0582] Example 2. A Phase 3, Global, Randomized, Open-Label Study to Evaluate the Efficacy and Safety of ALN-TTRscO4 in Patients with Transthyretin-Mediated Amyloidosis with Cardiomyopathy

[0583] This Example describes a global Phase 3, randomized, open-label study designed to evaluate the efficacy, safety, and pharmacokinetic (PK) / pharmacodynamic (PD) profiles of AD-649264 (ALN-TTRscO4) in adult patients with transthyretin-mediated amyloidosis (hereditary or wild-type) with cardiomyopathy (ATTR CM).

[0584] Patients enrolled in this study are randomized to receive ALN-TTRscO4 (300 mg subcutaneous once every 6 months [q6M]) or control during the treatment period. ALN-TTRscO4 is administered using a single-use prefilled syringe and a needle safety device. The prefilled syringe is filled with ALN-TTRscO4 (300 mg dose). The prefilled syringe is a single-use injection device, and after injection, the needle safety device will engage to cover the exposed needle. Patients undergo assessments for efficacy and / or safety using the methods described herein.

[0585] 51

[0586] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0587] Alnylam Reference No.: ALN-542-WO The primary objective of the study is to evaluate the efficacy of ALN-TTRscO4 compared to placebo in reducing all-cause mortality and cardiovascular (CV) events in patients with ATTR amyloidosis with cardiomyopathy. Secondary objectives of the study are to evaluate the efficacy of ALN-TTRscO4 compared with placebo on delaying time to first CV event (CV hospitalization or urgent heart failure [HF] visit) or all-cause mortality, reducing all-cause mortality, reducing CV events, and improving health status and health-related quality of life (Kansas City Cardiomyopathy Questionnaire Overall Summary [KCCQ-OS]). The objectives and endpoints are described in Table 4 below.

[0588] Table 4: Objectives and Endpoints

[0589]

[0590] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0591] Alnylam Reference No.: ALN-542-WO

[0592]

[0593] Abbreviations: ADA=anti-drug antibodies; AE=adverse event; ATTR amyloidosis=transthyretin-mediated amyloidosis; CV=cardiovascular; E / e’ ratio=measurement that combines the early filling velocity (E) with the early relaxation velocity (e1) to estimate the left ventricular (LV) filling pressure; HF=heart failure; KCCQ-OS=Kansas City Cardiomyopathy Questionnaire-Overall Summary; LV=left ventricular;

[0594] NAC=National Amyloidosis Centre; NT-proBNP=N-terminal prohormone B-type natriuretic peptide; NYHA=New York Heart Association; PD=pharmacodynamic(s); PK=pharmacokinetic(s);

[0595] TTR=transthyretin.

[0596] Approximately 1250 patients with ATTR amyloidosis with cardiomyopathy are planned for enrollment.

[0597] The study consists of 4 periods: (1) a Screening Period of up to 45 days (during which patients will undergo screening assessments to determine eligibility), (2) a double-blind (DB) period (during which the patients are randomized to receive 300 mg of ALN-TTRscO4 or placebo via subcutaneous injection once every 6 months (q6M)), (3) an Open-label Extension (OLE) Period of up to 2 years (during which all patients receive 300 mg of ALN-TTRscO4 via SC injection q6M), and (4) a safety follow-up period of up to 1 year (or 2 years for female patients of childbearing age) occurs after the last dose of study drug (see FIG. 2).

[0598] Patients are randomized 2: 1 to receive either ALN-TTRscO4 or placebo. Randomization is stratified as follows:

[0599] Patients who are not using TTR stabilizing therapies at baseline are stratified by the baseline N-terminal prohormone B-type natriuretic peptide (NT-proBNP) group: <1800 ng / L versus >1800 ng / L and <3000 ng / L versus >3000 ng / L)

[0600] Patients who are using TTR stabilizing therapies at baseline are stratified by the baseline NT-proBNP, the genotype, and the age:

[0601] • Baseline NT-proBNP group (<1800 ng / L versus >1800 ng / L and <3000 ng / L versus >3000 ng / L)

[0602] • Genotype (hATTR versus wtATTR)

[0603] • Age (<75 versus >75 years old)

[0604] The proportion of patients with baseline NT-proBNP values >3000 ng / L is limited to approximately 15% of the baseline TTR stabilizer use and monotherapy (patients not on TTR stabilizer at baseline) groups, each.

[0605] Patients may receive ALN-TTRscO4 on the study until the end of the OLE Period or until 1 of the following occurs: 1) they meet any of the study discontinuation criteria; 2) ALN-TTRscO4

[0606] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0607] Alnylam Reference No.: ALN-542-WO becomes commercially available in the patient’s country of residence, ALN-TTRscO4 is accessible to the patient, and the patient has completed their OLE Month 12 Visit; 3) they start TTR lowering therapy as part of clinical care or another clinical study; or 4) the ALN-TTRscO4 development program is discontinued.

[0608] Use of commercially available TTR stabilizers that are approved in the country of use are allowed.

[0609] All patients are instructed to take the recommended daily allowance of vitamin A while on study and until the end of their Follow-up Period.

[0610] Safety assessments are performed throughout the study. Safety assessments include collection of AEs, including serious AEs (SAEs); clinical laboratory safety tests (hematology, urinalysis, coagulation, serum chemistry [including liver function tests (LFTs)]); measurement of anti -drug antibodies (ADA); vital sign measurements (blood pressure, pulse rate, body temperature, and respiratory rate); and physical examinations including body weight. The assessment of AEs; reviewing / recording of hospitalizations, urgent HF visits, and procedures; vital status; and concomitant medications are monitored at clinic visits and during check-in contacts.

[0611] All patients who discontinue study drug during the DB Period are encouraged to remain on the study to complete the remaining assessments, including information on vital status, cardiac transplant procedures, left-ventricular assist device (LVAD) placement, and hospitalizations at a minimum. Assessment of AEs and concomitant medications should also be collected if at all possible. In the scenario where a patient discontinues study drug during the DB or OLE Period, time spent participating in the remainder of the DB or OLE Period, respectively, would be counted towards the fulfillment of the Safety Follow-up Period, which starts with the last dose of study drug. If a patient has left the study and withdrawn consent from the study, vital status data should be collected indirectly (from registries, electronic medical records, or other relevant sources [e.g., physicians]), where available and allowable by local law.

[0612] The planned duration of treatment for each patient ranges from 4 to 7 years, inclusive of an average DB Period of approximately 32 months (range: 2 to 5 years) and OLE Period of up to 24 months.

[0613] The estimated total time on the study for each patient ranges from 5 to 8 years, including a Screening Period of up to 45 days, an average DB Period of approximately 32 months (range: 2 to 5 years), an OLE Period of up to 24 months, and a Safety Follow-up Period of up to 12 months (24 months for female patients of childbearing age) after the patient’s last dose of study drug.

[0614] The primary endpoint is the composite outcome of all-cause mortality and recurrent CV events (CV hospitalizations and urgent HF visits). This endpoint provides definitive evidence of a clinically meaningful impact on the study population. The choice of primary endpoint concurs with the recommendations of the Committee for Medicinal Products for Human Use Guideline on clinical investigation of medicinal products for the treatment of chronic HF. The proposed approach is similar

[0615] 54

[0616] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0617] Alnylam Reference No.: ALN-542-WO to that taken in previous Phase 3 studies in ATTR amyloidosis with cardiomyopathy: the ATTR-ACT study of tafamidis conducted a primary analysis that hierarchically assessed all-cause mortality followed by frequency of CV hospitalizations, and the HELIOS-B study of vutrisiran used a primary endpoint of the composite outcome of all-cause mortality and CV events. [Fontana 2025; Maurer 2018] Cardiac death is expected to be the predominant cause of mortality in this population. Because non-CV death is a competing risk for CV-related death, all-cause rather than CV mortality is selected as a component of the composite outcome to avoid confounding. Also, ALN-TTRscO4 treatment may impact certain non-CV causes of death because ATTR amyloidosis is a systemic disease, as suggested by the impact of vutrisiran on all -cause mortality observed in HELIOS-B.

[0618] As a key secondary objective, the study evaluates the time to first CV event or all-cause mortality, as a marker of delay in worsening of cardiomyopathy. The study also evaluates the efficacy of ALN-TTRscO4 on reducing all-cause mortality and reducing CV events, 2 important components of the primary composite endpoint. In addition, the study evaluates the impact of ALN-TTRscO4 on KCCQ-OS score as a measure of patient-reported health status and health-related quality of life. KCCQ-OS is a common assessment in HF interventional studies and an independent predictor of prognosis (Heidenreich PA., et al., J Am Coll Cardiol. 2006 Feb 21 ;47(4) : 752-6) . In the Phase 3 HELIOS-B study of vutrisiran, a slowing of decline in KCCQ-OS was demonstrated with treatment (Fontana M. et al., NEnglJMed. 2025 Jan 2;392(l):33-44).

[0619] The study is placebo (e.g., phosphate-buffered saline) controlled. The use of placebo allows for an absolute assessment of efficacy and safety of ALN-TTRscO4 in a patient population at risk of cardiac events. In a DB study, a placebo control minimizes the potential for unintentional bias that can influence the outcomes of primary and secondary endpoints.

[0620] ALN-TTRscO4 is supplied as a sterile solution in phosphate buffered solution for SC injection.. The control drug for this study is a placebo (sodium chloride 0.9% w / v with 5 mM phosphate buffered saline for SC administration). ALN-TTRscO4 is administered using a single-use prefilled syringe and a needle safety device. The prefilled syringe is filled with either ALN-TTRscO4 (300 mg dose) or placebo with a volume of 1.5 mb. The prefilled syringe is a single-use injection device, and after injection, the needle safety device will engage to cover the exposed needle.

[0621] ALN-TTRscO4 is administered at a dose of 300 mg q6M SC in the Phase 3 study.

[0622] Furthermore, the q6M SC dosing regimen is infrequent and minimally invasive, which decreases treatment burden on patients and caregivers, potentially improving patient experience and long-term treatment compliance.

[0623] Inclusion Criteria

[0624] Patients are eligible to be included in the study if all the following criteria apply:

[0625] Age and Sex

[0626] 1. Age 18 (or age of legal consent, whichever is older) to 85 year, inclusive

[0627] 55

[0628] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0629] Alnylam Reference No.: ALN-542-WO

[0630] Patient and Disease Characteristics

[0631] 2. Documented diagnosis of ATTR amyloidosis with cardiomyopathy, classified as either hATTR amyloidosis with cardiomyopathy or wtATTR amyloidosis with cardiomyopathy: a. Hereditary ATTR amyloidosis with cardiomyopathy diagnosed based on meeting all of the following criteria:

[0632] i. Documentation of a TTR pathogenic mutation consistent with hATTR amyloidosis.

[0633] ii. Evidence of cardiac involvement by echocardiography with an end-diastolic interventricular septal wall thickness >12 mm for males and >11 mm for females.

[0634] iii. Technetium (99mTc) scintigraphy (99mTc-3,3-diphosphono-l,2- propanodicarboxylic acid [DPD-Tc],99mTc-pyrophosphate [PYP-Tc], or "Tc- hydroxymethylene diphosphonate [HMDP]) with Grade 2 or 3 cardiac uptake, if monoclonal gammopathy of undetermined significance (MGUS) had been excluded, OR amyloid deposits in cardiac tissue with TTR protein identification by immunohistochemistry (IHC) or mass spectrometry.

[0635] iv. If the patient has evidence of a MGUS based on serum and urine protein electrophoresis and serum free light chains, the following is required: documentation of TTR protein in noncardiac tissue (e.g., fat pad aspirate, salivary gland, median nerve connective sheath) with IHC or mass spectrometry AND Grade 2 or 3 cardiac uptake on99mTc scintigraphy per item 2aiii above OR documentation of TTR protein in cardiac tissue with IHC or mass spectrometry.

[0636] b. Wild-type ATTR amyloidosis with cardiomyopathy diagnosed based on meeting all of the following criteria:

[0637] i. Documentation of absence of pathogenic TTR mutation.

[0638] ii. Evidence of cardiac involvement by echocardiography with an end-diastolic interventricular septal wall thickness >12 mm for males and >11 mm for females.

[0639] iii. Technetium (99mTc) scintigraphy (DPD-Tc, PYP-Tc, or HMDP) with Grade 2 or 3 cardiac uptake, if MGUS had been excluded, OR amyloid deposits in cardiac tissue with TTR protein identification by IHC or mass spectrometry.

[0640] iv. If the patient has evidence of a MGUS based on serum and urine protein electrophoresis and serum free light chains, the following is required: documentation of 11 R protein in noncardiac tissue (e.g, fat pad aspirate, salivary gland, median

[0641] 56

[0642] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0643] Alnylam Reference No.: ALN-542-WO nerve connective sheath) with IHC or mass spectrometry AND Grade 2 or 3 cardiac uptake on99mTc scintigraphy per item 2biii above OR documentation of 11 R protein in cardiac tissue with IHC or mass spectrometry.

[0644] 3. Medical history of HF with at least 1 prior hospitalization for HF (not due to arrhythmia or a conduction system disturbance treated with a permanent pacemaker) OR clinical evidence of HF (with or without hospitalization) manifested by signs and symptoms of volume overload or elevated intracardiac pressures (e.g., elevated jugular venous pressure, shortness of breath or signs of pulmonary congestion on X-ray or auscultation, peripheral edema) that currently requires treatment with a diuretic.

[0645] 4. Patients may be receiving approved TTR stabilizers for ATTR amyloidosis (e.g., tafamidis, acoramidis) and may be receiving background therapy for HF; however, the doses of these medications (e.g., diuretics) must be stable for at least 30 days prior to screening.

[0646] 5. Patient is clinically stable, with no CV-related hospitalizations within 6 weeks prior to randomization.

[0647] 6. Screening NT-proBNP >300 ng / L and <8500 ng / L; in patients with permanent or persistent atrial fibrillation, Screening NT-proBNP >600 ng / L and <8500 ng / L.

[0648] Informed Consent

[0649] 7. Patient is able to understand and is willing and able to comply with the study requirements and to provide written informed consent

[0650] Exclusion Criteria

[0651] Patients are excluded from the study if any of the following criteria apply:

[0652] Disease-specific Conditions

[0653] 8. Has known primary amyloidosis (AL amyloidosis) or leptomeningeal amyloidosis

[0654] 9. New York Heart Association (NYHA) Class IV HF; or NYHA Class III heart failure AND ATTR Amyloidosis Disease Stage 3 (defined as NT-proBNP >3000 ng / L and estimated glomerular filtration rate [eGFR] <45 mL / min).

[0655] 10. Has a polyneuropathy disability (PND) Score Illa, Illb, or IV (requires cane or stick to walk due to polyneuropathy, or is wheelchair bound) at the Screening visit.

[0656] Laboratory Assessments

[0657] 11. Has any of the following laboratory parameter assessments at screening:

[0658] a. Alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST) >2.0 upper limit of normal (ULN) reference range

[0659] b. Total bilirubin >2.0 ULN

[0660] c. International normalized ratio (INR) >1.5 (unless patients were on anticoagulant therapy in which case excluded if INR >3.5)

[0661] 57

[0662] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0663] Alnylam Reference No.: ALN-542-WO 12. Has an estimated glomerular filtration rate (eGFR) <30 mL / min / 1.73m2(calculation based on the Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI] equation)

[0664] 13. Has known human immunodeficiency virus infection; or evidence of current or chronic hepatitis C virus or hepatitis B virus infection

[0665] Prior / Concomitant Therapy

[0666] 14. Received prior or currently receiving TTR-lowering therapy (e.g., revusiran, patisiran, inotersen, eplontersen, or vutrisiran or gene therapy targeting TTR)

[0667] 15. Patients for whom the Investigator actively plans or anticipates commencing treatment with a TTR-lowering therapy either during the Screening Period or the first 24 months following randomization, taking into consideration clinical status, patient preference and / or commercial availability.

[0668] 16. Current or prior anti-TTR antibody treatment (i.e., TTR depleter).

[0669] 17. Is currently taking diflunisal; if previously on this agent, must have at least a 30-day wash-out prior to dosing (Day 1).

[0670] 18. Current or future participation in another investigational device or drug study, scheduled to occur during this study, or has been treated with an investigational agent or device within 30 days (or 5 half-lives of the investigational drug, whichever is longer) prior to dosing (Day 1). In the case of investigational TTR stabilizer drugs, washout for 3 months prior to dosing (Day 1) is required.

[0671] Medical Conditions

[0672] 19. Other non-TTR cardiomyopathy, hypertensive cardiomyopathy, cardiomyopathy due to valvular heart disease, or cardiomyopathy due to ischemic heart disease (e.g., prior myocardial infarction with documented history of cardiac enzymes and electrocardiogram changes) that may be a significant contributor or the predominant cause of the patient’s HF.

[0673] 20. Unstable congestive heart failure (CHF) (including patients who require adjustment of existing diuretics or addition of new diuretics at time of screening for purposes of achieving optimal management of CHF).

[0674] 21. Had acute coronary syndrome or unstable angina within the past 3 months.

[0675] 22. Has history of sustained ventricular tachycardia or aborted ventricular fibrillation due to ATTR amyloidosis.

[0676] 23. Has history of atrioventricular nodal or sinoatrial nodal dysfunction for which a pacemaker is indicated but will not be placed.

[0677] 24. Has persistent elevation of systolic (>170 mmHg) or diastolic (>100 mmHg) blood pressure that was considered uncontrolled by physician.

[0678] 25. Has untreated hypo- or hyperthyroidism.

[0679] 58

[0680] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0681] Alnylam Reference No.: ALN-542-WO 26. Has an active infection requiring systemic antiviral, antiparasitic, or antimicrobial therapy that will not be completed prior to dosing (Day 1).

[0682] 27. Prior or anticipated (during the first 12 months after randomization) heart, liver, or other organ transplant or implantation of LVAD.

[0683] 28. History of multiple drug allergies or history of allergic reaction to any component of or excipient in the study drug.

[0684] 29. History of intolerance to SC injection(s).

[0685] 30. Has other medical conditions or comorbidities (e.g., malignancy, neuropsychiatric disorder, etc) which, in the opinion of the Investigator, would interfere with study compliance or data interpretation.

[0686] 31. Has a life expectancy of <2 years due to any non-CV condition, based on Investigator's clinical judgement.

[0687] Contraception, Pregnancy, and Breastfeeding

[0688] 32. Is not willing to comply with the contraceptive requirements during the study period.

[0689] 33. Patient is pregnant or breastfeeding.

[0690] Alcohol Use

[0691] 34. Unwilling or unable to limit alcohol consumption throughout the course of the study. Alcohol intake of >2 units / day is excluded during the study (unit: 1 glass of wine [approximately 125 m ] = 1 measure of spirits [approximately 1 fluid ounce] = A pint of beer [approximately 284 mL]).

[0692] 35. History of alcohol use disorder, within the last 12 months before screening, in the opinion of the Investigator.

[0693] 36. History of illicit drug abuse within the past 5 years that in the opinion of the Investigator would interfere with compliance with study procedures or Follow-up visits.

[0694] Screening Assessments

[0695] The Screening Period is 45 days. Patient demographic data and medical history / disease history are obtained. Any changes to medical history occurring between the screening assessment and Day 1 will be updated before study drug administration. Documented technetium scintigraphy and / or tissue biopsy testing for amyloidosis performed prior to study enrollment should be collected and recorded as part of medical history.

[0696] Information on prior medications, hospitalization, and interventional cardiac procedures through 1 year prior to first dose should be collected and recorded.

[0697] The Kamofsky Performance Status (KPS) is evaluated to assess general state of health and functioning. Polyneuropathy disability (PND) score is evaluated as well at screening.

[0698] 59

[0699] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0700] Alnylam Reference No.: ALN-542-WO Efficacy Assessments

[0701] Vital Status Check

[0702] Vital status checks are performed. Vital status refers to determination of whether the patient is alive or deceased. Given that heart transplantation or LVAD implantation procedures is considered deaths for the purposes of the primary analysis, vital status checks should include checking for the occurrence of these procedures.

[0703] Deaths, Hospitalizations, and Urgent Heart Failure Visits

[0704] All deaths, hospitalizations, urgent HF visits, and interventional cardiac procedures are recorded throughout the study as part of SAE and AE monitoring.

[0705] A hospitalization is defined as a non-elective admission to an acute setting resulting in a stay of greater than 24 hours duration [or an emergency department ward visit of greater than 24 hours duration] .

[0706] An urgent HF visit occurs outside of an inpatient hospitalization (e.g., emergency department or ward [of less than 24 hours duration], urgent care clinic, day clinic). In an urgent HF visit, a patient has signs, symptoms, and / or test results indicative of worsening HF for which the patient receives treatment similar to that received in a HF hospitalization, eg, intravenous diuretic therapy.

[0707] Augmentation of oral diuretic therapy alone does not qualify as an urgent HF visit (Hicks KA, et al. Circulation. 2018 Feb 27; 137(9): 961-72).

[0708] Kansas City Cardiomyopathy Questionnaire (KCCQ)

[0709] The KCCQ is a 23-item self-administered questionnaire developed to independently measure the patient’s perception of health status, which includes HF symptoms, impact on physical and social function, and how their HF impacts their quality of life within a 2-week recall period (Green CP, et al., J Am Coll Cardiol. 2000 Apr;35(5): 1245-55). The KCCQ quantifies 6 domains (symptoms, physical function, quality of life, social limitation, self-efficacy, and symptom stability) and 2 summary scores (clinical and overall summary).

[0710] Cardiac Assessments

[0711] Manifestations of cardiac amyloid involvement are assessed via echocardiogram, cardiac biomarkers, ATTR amyloidosis disease stage, and NYHA class as described below.

[0712] Cardiac Biomarkers

[0713] The cardiac biomarkers NT-proBNP and troponin I are used to assess cardiac stress and HF severity. These biomarkers have been shown to be prognostic of outcomes in HF, including in ATTR amyloidosis (Damy T, et al. Amyloid. 2016 Sep;23(3): 194-202; Kristen AV, et al., PLoS One.

[0714] 2017;12(4):e0173086; 2017; Merlini G. et al., Leukemia. 2016 Oct;30(10): 1979-86). Blood samples are drawn to measure cardiac biomarker levels.

[0715] 60

[0716] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0717] Alnylam Reference No.: ALN-542-WO

[0718] Echocardiogram

[0719] Echocardiographic parameters are used for assessment of cardiac structure and function. The 12-lead ECGs reporting rhythm, ventricular rate, RR interval, PR interval, QRS duration, and QT interval are obtained.

[0720] ATTR Amyloidosis Disease Stage

[0721] The ATTR amyloidosis disease staging used for this protocol stratifies patients with ATTR amyloidosis with cardiomyopathy (both hATTR and wtATTR) into prognostic categories using the serum biomarkers NT-proBNP and eGFR. (Gillmore JD., et al., Eur Heart J. 2018 Aug 7;39(30):2799-806).

[0722] Patients are categorized as follows:

[0723] • Stage 1 (lower risk): NT-proBNP <3000 ng / L and eGFR >45 mL / min / 1.73 m2

[0724] • Stage 2 (intermediate risk): all other patients not meeting criteria for Stages 1 or 3 • Stage 3 (higher risk): NT-proBNP >3000 ng / L and eGFR <45 mL / min / 1.73 m2

[0725] New York Heart Association (NYHA) Class

[0726] NYHA class is a clinical assessment of symptoms resulting from HF.

[0727] European Quality of Life-5 Dimensions 5-Levels (EQ-5D-5L)

[0728] General health-related quality of life is assessed through the use of the EQ-5D-5L, a standardized instrument of 5 questions and a Visual Analogue Scale for use as a measure of patient-reported health outcomes.

[0729] Neurofilament Light Chain

[0730] Neurofilament light chain (NfL) is a biomarker for polyneuropathy signaling neuroaxonal injury. NfL levels are elevated in patients with hATTR amyloidosis and decrease with patisiran therapy (Ticau S. et al., Neurology. 2021 Jan 19;96(3):e412-e22). Plasma NfL are assessed in this study to evaluate the effects of ALN-TTRscO4 on possible concurrent polyneuropathy in patients with ATTR amyloidosis with cardiomyopathy.

[0731] Oral Diuretic Intensification / Initiation (ODI)

[0732] GDI is assessed as an indication of worsening of HF severity. ODI is defined as any postrandomization, sustained increase in dose of loop diuretic (e.g., azosemide, bumetanide, furosemide, piretanide, torsemide) or initiation of loop diuretic. ODI, defined as an increase in dose sustained for at least 7 days, is associated with higher risk of subsequent CV events and death, and may therefore

[0733] 61

[0734] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0735] Alnylam Reference No.: ALN-542-WO serve as an early marker for HF worsening (Fontana et al., J Am Coll Cardiol. 2024a Nov 18;

[0736] Fontana et al., J Am Coll Cardiol. 2024b Dec 16). Changes in the dose of loop diuretic are evaluated as part of concomitant medications review.

[0737] Table 1. Abbreviations of nucleotide monomers used in nucleic acid sequences. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5'-3'-phosphodiester bonds; and it is understood that when the nucleotide contains a 2’-fluoro modification, then the fluoro replaces the hydroxy at that position in the parent nucleotide ( / . e. , it is a 2’-deoxy-2’-fluoronucleotide). It is to be further understood that the nucleotide abbreviations in the table omit the 3 ’-phosphate (z.e., they are 3 ’-OH) when placed at the 3 ’-terminal position of an oligonucleotide.

[0738]

[0739] 62

[0740] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0741] Alnylam Reference No.: ALN-542-WO

[0742]

[0743] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0744] Alnylam Reference No.: ALN-542-WO

[0745]

[0746] MEI 52705184v.1Attorney Docket No.: 121301-25120

[0747] Alnylam Reference No.: ALN-542-WO

[0748]

[0749] MEI 52705184v.1

Claims

Attorney Docket No.: 121301-25120Alnylam Reference No.: ALN-542-WO We claim:

1. A method for treating polyneuropathy and / or cardiomyopathy in a subject having transthyretin-mediated amyloidosis (ATTR), the method comprising administering to the subject a fixed dose of about 200-400 mg of a double stranded ribonucleic acid (dsRNA) agent, or a pharmaceutically acceptable salt thereof, targeting a TTR gene,wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the modified nucleotide sequence 5’-csasagagUfaUfUfCfcauuuuuacu-3’ of SEQ ID NO: 17, and the antisense strand comprises the modified nucleotide sequence 5’- asGfsuaaAfaauggaaUfaCfiicuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; and s is a phosphorothioate linkage; andwherein a ligand is conjugated to the 3’ end of the sense strand as shown in the following schematicthereby treating polyneuropathy and / or cardiomyopathy in the subject.

2. The method of claim 1, wherein the ATTR is hereditary ATTR (hATTR).

3. The method of claim 1, wherein the ATTR is wild-type ATTR (wtATTR).

4. A method for treating polyneuropathy in a subject having hereditary transthyretin-mediated amyloidosis (hATTR), the method comprising administering to the subject a fixed dose of about 200-400 mg of a double stranded ribonucleic acid (dsRNA) agent, or a pharmaceutically acceptable salt thereof, targeting a TTR gene,wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the modified nucleotide sequence 5’-MEl 52705184v.1Attorney Docket No.: 121301-25120Alnylam Reference No.: ALN-542-WO csasagagUfaUfUfCfcauuuuuacu-3’ of SEQ ID NO: 17, and the antisense strand comprises the modified nucleotide sequence 5’- asGfsuaaAfaauggaaUfaCfucuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; and s is a phosphorothioate linkage; andwherein a ligand is conjugated to the 3’ end of the sense strand as shown in the following schematicthereby treating polyneuropathy in the subject.

5. A method for treating cardiomyopathy in a subject having transthyretin-mediated amyloidosis, the method comprising administering to the subject a fixed dose of about 200-400 mg of a double stranded ribonucleic acid (dsRNA) agent, or a pharmaceutically acceptable salt thereof, targeting a TTR gene,wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the modified nucleotide sequence 5’-csasagagUfaUfUfCfcauuuuuacu-3’ of SEQ ID NO: 17, and the antisense strand comprises the modified nucleotide sequence 5’- asGfsuaaAfaauggaaUfaCfucuugsgsu -3’ of SEQ ID NO: 19, wherein a, g, c and u are 2'-O-methyl (2'-OMe) A, G, C, and U; Af, Cf, Gf and Uf are 2’-fluoro (2’-F) A, C, G and U; and s is a phosphorothioate linkage; andwherein a ligand is conjugated to the 3’ end of the sense strand as shown in the following schematicMEI 52705184v.1Attorney Docket No.: 121301-25120Alnylam Reference No.: ALN-542-WOthereby treating cardiomyopathy in the subject.

6. The method of claim 5, wherein the ATTR is hereditary ATTR (hATTR).

7. The method of claim 5, wherein the ATTR is wild-type ATTR (wtATTR).

8. The method of any one of claims 1-7, wherein the subject is a human subject.

9. The method of claim 8, wherein the human subject is 18-85 years of age.

10. The method of any one of claims 1-4, 8 and 9, wherein the human subject has a Neuropathy Impairment Score (NIS) of 5-130, a polyneuropathy disability (PND) sore of < 3b, and / or a Kamofsky Performance Scale (KPS) > 60%.

11. The method of any one of claims 1-3 and 5-9, wherein the human subject is a male and has an end-diastolic interventricular septal wall thickness >12 mm.

12. The method of any one of claims 1-3 and 5-9, wherein the human subject is a female and has an end-diastolic interventricular septal wall thickness >11 mm.

13. The method of any one of claims 1-3, 5-9, 11 and 12, wherein the human subject has a Grade 2 or 3 cardiac uptake by Technetium scintigraphy.

14. The method of any one of claims 1-3, 5-9, and 11-13, wherein the human subject has amyloid deposits in cardiac tissue.MEI 52705184v.1Attorney Docket No.: 121301-25120Alnylam Reference No.: ALN-542-WO 15. The method of any one of claims 1-3, 5-9, and 11-14, wherein the human subject has amyloid deposits of TTR protein in non-cardiac tissue, and(i) wherein the subject has a Grade 2 or 3 cardiac uptake by Technetium scintigraphy, or (ii) wherein the subject has amyloid deposits of in cardiac tissue.

16. The method of claim 15, wherein the non-cardiac tissue is selected from the group consisting of fat pad aspirate, salivary gland, and median nerve connective sheath.

17. The method of any one of claims 1-3, 5-9, and 11-16, wherein the human subject has a medical history of heart failure with at least one prior hospitalization for heart failure, or clinical evidence of heart failure manifested by one or more symptoms of volume overload or elevated intracardiac pressures.

18. The method of claim 17, wherein the hospitalization for heart failure is not due to arrhythmia or a conduction system disturbance treated with a permanent pacemaker.

19. The method of claim 17, wherein the one or more symptoms of volume overload or elevated intracardiac pressures comprise elevated jugular venous pressure, shortness of breath, signs of pulmonary congestion on X-ray or auscultation, and / or peripheral edema.

20. The method of any one of claims 1-3, 5-9, and 11-19, wherein the subject has an NT-proBNP level of >300 ng / L and <8500 ng / L.

21. The method of any one of claims 1-20, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 275-325 mg.

22. The method of any one of claims 1-21, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or every 12 months.

23. The method of any one of claims 1-22, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 275-325 mg every six months.

24. The method of any one of claims 1-23, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject at a dose of about 300 mg every six months.69MEI 52705184v.1Attorney Docket No.: 121301-25120Alnylam Reference No.: ALN-542-WO25. The method of any one of claims 1-24, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject intravenously, intramuscularly, or subcutaneously.

26. The method of any one of claims 1-25, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously.

27. The method of any one of claims 1-26, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject subcutaneously at a dose of about 300 mg every six months.

28. The method of any one of claims 1-27, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject via an auto-injector.

29. The method of any one of claims 1-27, wherein the dsRNA agent, or a pharmaceutically acceptable salt thereof, is administered to the subject via a pre-fdled syringe.

30. The method of any one of claims 1-29, further comprising administering an additional therapeutic to the subject.

31. The method of any one of claims 1-30, further comprising measuring the level of TTR mRNA expression or TTR protein expression in a sample from the subject.

32. The method of any one of claims 1-31, further comprising measuring the level of vitamin A or RBP4 protein in a sample from the subject.

33. The method of any one of claims 1-32, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject causes a decrease in TTR enzymatic activity, a decrease in TTR protein accumulation, a decrease in vitamin A level, and / or a decrease in RBP4 level in the subject.

34. The method of any one of claims 1-33, wherein the TTR mRNA level in the subject is reduced to at least about 50%, 55%, 60%, 65%, or 70% of baseline level 6 months after the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof.70MEI 52705184v.1Attorney Docket No.: 121301-25120Alnylam Reference No.: ALN-542-WO 35. The method of any one of claims 1-34, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject improves at least one indicia selected from the group consisting of Neuropathy Impairment Score (NIS), modified Neuropathy Impairment Score (mNIS+7), Norfolk Quality of Life Diabetic Neuropathy (Norfolk QoL-DN) questionnaire, modified Body Mass Index (mBMI), Rasch built Overall Disability Scale (R ODS) questionnaire, 10-meter walk test (10 MWT), Composite Autonomic Symptom Score (COMPASS-31), EuroQoL-5 Dimensions-5 Levels (EQ-5D-5L) questionnaire and the EuroQoL-Visual Analog Scale (EQ VAS), a Kansas City Cardiomyopathy Questionnaire (KCCQ), and a New York Heart Association (NYHA) Class, Familial Amyloidotic Polyneuropathy (FAP) stage, and Polyneuropathy Disability (PND) score, compared to a baseline as determined before the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof.

36. The method of any one of claims 1-35, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject results in an improvement or a stabilization of a cardiac marker, an echocardiogram parameter, and / or a Technetium scintigraphy cardiac parameter, compared to a baseline, as determined before the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof.

37. The method of claim 36, wherein the cardiac marker is selected from the group consisting of N-terminal prohormone B-type natriuretic peptide (NT-proBNP), Troponin I, Troponin T, and Neurofilament light chain (NfL).

38. The method of any one of claims 1-37, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces the composite of all -cause mortality and recurrent cardiovascular (CV) events.

39. The method of any one of claims 1-38, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces the all-cause mortality.

40. The method of any one of claims 1-39, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces cardiovascular hospitalizations and / or urgent heart failure visits.

41. The method of any one of claims 1-40, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject delays the onset of cardiovascular events or all-cause mortality.71MEI 52705184v.1Attorney Docket No.: 121301-25120Alnylam Reference No.: ALN-542-WO 42. The method of any one of claims 1-41, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces recurrent cardiovascular events.

43. The method of any one of claims 1-42, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces or prevents oral diuretic intensification / initiation (ODI) .

44. The method of any one of claims 1-43, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject stabilizes or improves the ATTR amyloidosis disease stage.

45. The method of any one of claims 1-44, wherein the administration of the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject reduces an amyloid TTR deposit in the subject.

46. A kit for performing the method of any one of claims 1-45, comprisinga) the dsRNA agent, or a pharmaceutically acceptable salt thereof, andb) instructions for use.

47. The kit of claim 46, further comprising means for administering the dsRNA agent, or a pharmaceutically acceptable salt thereof, to the subject.

48. The kit of claim 47, wherein the means for administering the dsRNA agent, or a pharmaceutically acceptable salt thereof, is a pre-filled syringe.

49. The kit of claim 47, wherein the means for administering the dsRNA agent, or a pharmaceutically acceptable salt thereof, is an auto-injector.72MEI 52705184v.1

Images