KR20220152270A - Compositions and methods for inhibiting the expression of transthyretin (TTR) - Google Patents

Abstract

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

Description

Compositions and methods for inhibiting the expression of transthyretin (TTR)

related application

This application claims priority to U.S. Provisional Application No. 62/985,950, filed March 6, 2020, the entirety of which is incorporated herein by reference.

sequence listing

This application contains a sequence listing submitted electronically in ASCII format, which is incorporated by reference in its entirety. Said ASCII copy, created on February 26, 2021, has the file name 121301_12320_SL.txt and is 44,455 bytes in size.

Transthyretin (TTR) (also known as proalbumin) 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 association with RBP in blood and CSF. Transthyretin is so named because of its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apoA-I (the major HDL apolipoprotein), amyloid β-peptide, and neuropeptide Y. See Liz, MA et al. (2010) IUBMB Life , 62(6):429-435.

TTR is a tetramer of four identical 127 amino acid subunits (monomers) enriched in beta sheet structures. Each monomer has the shape of a prolate ellipsoid with two four-stranded beta sheets. Antiparallel beta sheet interactions link monomers to dimers. A short loop from each monomer forms the main dimer-dimer interaction. These two pairs of loops separate the opposite convex beta sheets of the dimer to form an internal channel.

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

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

Amyloidosis is a general term for a group of amyloid diseases characterized by amyloid deposits. Amyloid diseases are classified based on their precursor proteins; For example, the name starts with "A" for amyloid followed by an ATTR for a precursor protein, eg, amyloidogenic transthyretin . Ibid.

There are numerous TTR-related diseases, most of which are amyloid diseases. Normal sequence TTR is associated with cardiac amyloidosis in the elderly and is called senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA is often accompanied by microscopic deposits in many other organs. TTR amyloidosis presents in various forms. When the peripheral nervous system is more prominently affected, the disease is called familial amyloidogenic polyneuropathy (FAP). When the heart is primarily involved but the nervous system is not involved, the disease is called familial amyloidogenic cardiomyopathy (FAC). The third major type of TTR amyloidosis is leptomeningeal amyloidosis, also known as leptomeningeal or meningocerebrovascular amyloidosis, central nervous system (CNS) amyloidosis or form amyloidosis VII. Mutations in TTR can also cause amyloidotic vitreous opacity, carpal tunnel syndrome, and normothyroid hyperthyroxinemia, which have been shown to be secondary to increased association of TTR with thyroxine due to mutant TTR molecules with increased affinity for thyroxine. It is believed to be a non-amyloidic disease. See, eg, Moses et al . (1982) J. Clin. Invest. , 86, 2025-2033.

Abnormal TTR alleles may be inherited or acquired through somatic mutation. Guan, J. et al . (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol , doi:10.1152/ajpheart.00815.2011. Transthyretin-associated ATTR is the most common form of hereditary systemic amyloidosis. Lobato, L. (2003) J. Nephrol. , 16:438-442. TTR mutation accelerates the process of TTR amyloidogenesis and is the most important risk factor for the development of ATTR. More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis. While some mutations are associated with cardiomyopathy or vitreous opacity, TTR mutations generally cause systemic amyloid deposits with particular involvement of the peripheral nervous system. Ibid.

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

Thus, there is a need in the art for effective treatments for TTR-related diseases.

Summary of the Invention

The present invention provides compositions and methods for inhibiting expression of TTR, and methods for treating or preventing transthyretin- (TTR-) related diseases in human subjects using double-stranded RNAi agents targeting the TTR gene. .

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

In certain embodiments, the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. In certain embodiments, the ligand is one or more GalNAc derivatives attached through a divalent or trivalent side chain linker. In certain embodiments, the ligand is

이다.

to be.

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

In certain embodiments, the double-stranded RNAi agent is conjugated to a ligand as shown in the scheme below;

여기서, X는 O 또는 S이다.

Here, X is O or S.

In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

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

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

In certain embodiments, the sense strand of a double-stranded RNAi agent is conjugated to at least one ligand. In certain embodiments, the ligand is one or more GalNAc derivatives attached through a divalent or trivalent side chain linker. In certain embodiments, the ligand is

이다.

to be.

In certain embodiments, the ligand is attached to the 3' end of the sense strand. In certain embodiments, the double-stranded RNAi agent is conjugated to a ligand as shown in the scheme below;

여기서, X는 O 또는 S이다.

Here, X is O or S.

In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In certain embodiments, uses of the present invention include improving at least one indicator of neurological impairment, quality of life, neurological impairment, or cardiovascular health. In certain embodiments, the assessed index is neurological impairment using, for example, a neuropathic impairment (NIS) score or a modified NIS (mNIS+7) score. In certain embodiments, the indicator is, for example, the SF-36® Health Survey score, the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, the NIS-W score, the Rasch-constructed total disability Quality of life indicators assessed using scale (R-ODS) score, composite autonomic symptom score (COMPASS-31), median body mass index (mBMI) score, 6-minute walk test (6MWT) score, and 10-meter walk test score to be. In certain embodiments, the indicator is an assessed neuronal injury, e.g., a change in the level of one or more proteins selected from the group of neurofilament light chain (NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N-CDase, e.g., Neuronal damage assessed by changes in human blood samples, or serum or plasma derived therefrom. In certain embodiments, the indicator of nerve damage is a change from baseline in neurofilament light chain (NfL) protein levels. In certain embodiments, the indicator of cardiovascular damage is better health, change from baseline in mean left ventricular (LV) wall thickness by echocardiographic assessment, change from baseline in global longitudinal tension by echocardiographic assessment, and N Cardiovascular admissions using the Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS) with an increased score representing change from baseline in terminal prohormone type B natriuretic peptide (NTproBNP).

In certain embodiments, the human subject has a TTR-related disease, eg, senile systemic amyloidosis (SSA), familial systemic amyloidosis, familial amyloidogenic polyneuropathy (FAP), leptomeningeal/central nervous system (CNS) amyloidosis, and hyperthyroxine. Carrying a TTR gene mutation associated with the development of hyperemia.

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

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

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

In certain embodiments, the double-stranded RNAi agent is administered to a human subject once monthly, once every other month, once every 3 months, once every 4 months, once every 5 months, or once every 6 months. In certain embodiments, a fixed dose of a double-stranded RNAi agent is administered to a human subject about once every 3 months. In certain embodiments, a fixed dose of a double-stranded RNAi agent is administered to a human subject about once every 6 months.

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

In certain embodiments, the double-stranded RNAi agent is administered to a human subject from about once per quarter to about once per year. In certain embodiments, the double-stranded RNAi agent is administered to a human subject about once per quarter, about once every 6 months, or about once per year.

In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg to about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 50 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 100 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is about 25 mg to about 300 mg; about 25 mg to about 200 mg; about 75 mg to about 200 mg; about 25 mg; about 50 mg; about 75 mg; about 100 mg; about 200 mg; or a fixed dose of about 300 mg is administered to a human subject once quarterly, ie, about once every 3 months.

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

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

In certain embodiments, the use further comprises administering to the human subject an additional therapeutic agent, eg, a TTR tetramer stabilizer or a non-steroidal anti-inflammatory agent.

The invention also provides kits for performing any of the methods of the invention. The kit includes a double-stranded RNAi agent; and a cover including instructions for use.

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

Figure 1 is a graph depicting relative serum TTR protein levels in V30M transgenic mice (n=3 per group) after administration of a single 1 mg/kg dose of the indicated double-stranded RNAi preparations on day 0.
Figure 2 is a graph depicting relative serum TTR protein levels in cynomolgus monkeys (n=3 per group) after administration of a single 1 mg/kg dose or 3 mg/kg dose of the indicated double-stranded RNAi preparations on day 0. . Results originate from three independent studies.

The present invention provides a method for inhibiting TTR expression, comprising inhibiting TTR expression in a human subject who does not meet criteria for diagnosis of a transthyretin- (TTR-) related disease, and a double-stranded RNAi agent targeting the TTR gene Provided is a method of treating a human subject having a transthyretin- (TTR-) related disease using the method, wherein the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6); The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7).

The detailed description below describes methods of making and using compositions containing RNAi agents to selectively inhibit expression of the TTR gene, and methods for treating subjects having diseases and disorders that would benefit from inhibition or reduction of expression of the TTR gene. Compositions, uses and methods for

I. Definition

In order that the present invention may be more readily understood, certain terms are first defined. Also, whenever a value or range of values for a parameter is recited, it should be noted that intermediate values and ranges of the recited values are also intended to be part of the invention.

The singular articles (“a” and “an”) are used herein to refer to one or more than one (ie, at least one) of the grammatical objects of that article. For example, “element” means one element or more than one element, eg, a plurality of elements.

The term "comprising" is used herein to mean the phrase "including but not limited to" and is used interchangeably with it.

The term “or” is used herein to mean the term “and/or” and is used interchangeably with it, unless expressly indicated otherwise.

The term "about" is used to mean an acceptable variation in dosage unit amount, eg, an acceptable variation in the time between doses, within the normal tolerance range in the art. For example, “about” can be understood as within about 2 standard deviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is preceded by a series of numbers or ranges, it is understood that “about” can modify each number in the series or range.

The terms "at least", "more than", or "more than" in front of a number or sequence of numbers are intended to include the number adjacent to the term "at least" and any subsequent number or whole number that may logically be included as is clear from the context. I understand. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. Where at least precedes a series of numbers or ranges, it is understood that “at least” can modify each number in the series or range.

As used herein, "less than" or "less than" is understood to be a value adjacent to a phrase and, in context, as a logically low value or integer up to and including zero. For example, a duplex with an overhang of “less than 2 nucleotides” has an overhang of 2, 1, or 0 nucleotides. When "less than" is preceded by a series of numbers or ranges, it is understood that "less than" can modify each number in the series or range.

As used herein, a detection method may include determining that the amount of an analyte present is below the detection level of the method.

“Transthyretin” (“TTR”) as used herein refers to well-known genes and proteins. TTR is also known as proalbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter for 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 retinal pigment epithelium. The greatest clinical relevance of TTR is that both normal (wild-type) and mutant TTR proteins can form amyloid fibrils that aggregate into extracellular deposits and cause amyloidosis. See, eg, Saraiva MJM (2002) Expert Reviews in Molecular Medicine , 4(12):1-11 for a review. The molecular cloning and distribution of nucleotide sequence and mRNA expression of rat transthyretin is described by Dickson, PW et al. (1985) J. Biol. Chem. 260(13)8214-8219. The X-ray crystal structure of human TTR has been described by Blake, CC et al. (1974) J Mol Biol 88 , 1-12. The sequence of the human TTR mRNA transcript can be found at the National Center for Biotechnology Information (NCBI) RefSeq Accession No. NM_000371 (eg , SEQ ID NOs: 1 and 5). The sequence of the mouse TTR mRNA is RefSeq Accession No. Nm_013697.2 and the sequence of rat TTR mRNA can be found under RefSeq Accession No. Nm_012681.1 Additional examples of TTR mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, and OMIM. reasonably available

As used herein, "TTR-related disease" is intended to include any disease associated with the TTR gene or protein. These disorders can be caused, for example, by overproduction of the TTR protein, mutations in the TTR gene, abnormal cleavage of the TTR protein, instability of the TTR tetramer, or abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. "TTR-related disease" means any type of transthyretin-mediated amyloidosis in which TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposits (ATTR amyloidosis), including hereditary ATTR (h-ATTR) amyloidosis or non-hereditary ATTR amyloidosis. ATTR (ATTR) amyloidosis. TTR-related diseases include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidogenic polyneuropathy (FAP), familial amyloidogenic cardiomyopathy (FAC), leptomeningeal/central nervous system (CNS) amyloidosis, and amyloidotic vitreous opacity. , carpal tunnel syndrome and hyperthyroxinemia. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, decreased sensation in the extremities), autonomic neuropathy (e.g., gastrointestinal dysfunction such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, and dementia. , myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction, and corneal lattice dystrophy.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a nucleotide chain described by a stated sequence using standard nucleotide nomenclature.

The terms "iRNA," "RNAi agent," "iRNA agent," "RNA interference agent," as used interchangeably herein, refer to an RNA-induced silencing complex (RISC) pathway that contains RNA as defined herein and refers to agents that mediate targeted cleavage of RNA transcripts via iRNAs direct sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). An iRNA regulates, eg, inhibits, the expression of a TTR gene in a cell, eg, in a cell in a subject, such as a mammalian subject.

As used herein, "iRNA" for use in the compositions, uses and methods of the present invention is a double-stranded RNA and is herein referred to as a "double-stranded RNA preparation", "double-stranded RNA (dsRNA) molecule", "dsRNA preparation" or "dsRNA". The term "dsRNA" refers to a target RNA, i.e., a complex of ribonucleic acid molecules having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having a "sense" and "antisense" orientation relative to the TTR gene. refers to Double-stranded RNAi agents trigger degradation of a target RNA, eg mRNA, through a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

As used herein, the term "modified nucleotide" independently refers to a nucleotide having a modified sugar moiety, modified internucleotide linkage, or modified nucleotide group. Thus, the term modified nucleotide includes substitution, addition or removal of, for example, functional groups or atoms to internucleoside linkages, sugar moieties or nucleobases. Modifications suitable for use in the formulations of the present invention include all types of modifications described herein or known in the art. Any of the above modifications, as used in siRNA type molecules, are encompassed by "RNAi agents" for purposes of this specification and claims.

The duplex region can be of any length that allows specific degradation of the target RNA of interest via the RISC pathway, and is about 21 to 36 base pairs in length, such as about 21-30 base pairs, such as about 21-36 base pairs in length. 30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In one embodiment, the RNAi agent of the invention is a dsRNA agent, each strand of which comprises 21-23 nucleotides, which interacts with the TTR mRNA sequence to direct cleavage of the target mRNA. Without wishing to be bound by theory, long double-stranded RNA introduced into cells is digested into siRNA by a type III endonuclease known as Dicer (Sharp et al . (2001) Genes Dev . 15:485). Dicer, a type III ribonuclease enzyme, processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two base 3' overhang (Bernstein, et al . ,( 2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC) in which one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen , et al . , (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, leading to silencing (Elbashir, et al . , (2001) Genes Dev . 15:188). In one embodiment, the RNAi agent of the invention is a dsRNA of 24-30 nucleotides, which interacts with the TTR mRNA sequence to direct cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide protruding from a duplex structure of an iRNA, eg, a dsRNA. For example, there is a nucleotide overhang when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand or vice versa. A dsRNA may contain an overhang of at least one nucleotide; Alternatively, the overhang may comprise at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides or more. Nucleotide overhangs may comprise or consist of nucleotide/nucleoside analogs, including deoxynucleotides/nucleosides. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotide(s) of the overhang may be present on the 5'-end, 3'-end or both ends of the antisense or sense strand of the dsRNA. In one embodiment of the dsRNA, at least one strand comprises a 3' overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides, eg, 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides. In another embodiment, at least one strand of the RNAi agent comprises a 5' overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5' overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 8 or 9 nucleotides. In yet another embodiment, both the 3' and 5' ends of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

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

"blunt" or "blunt ends" means that there are no unpaired nucleotides at the ends of the double-stranded RNAi preparation, i.e. there are no nucleotide overhangs. A "blunt ended" RNAi preparation is It is a dsRNA that is double-stranded over its entire length, that is, there is no nucleotide overhang at either end of the molecule.The RNAi preparation of the present invention has a nucleotide overhang at one end (i.e., has one overhang and one blunt end). preparations) or RNAi preparations with nucleotide overhangs at both ends.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, eg, a dsRNA, that includes a region substantially complementary to a target sequence, eg, TTR mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence as defined herein, eg, a target sequence, eg, a TTR nucleotide sequence. If the region of complementarity is not completely complementary to the target sequence, the mismatch may be in an internal or terminal region of the molecule. Generally, the most permissible mismatches are within 5, 4, 3, 2 or 1 nucleotides of the terminal region, eg the 5'- or 3'-end of the iRNA. In one embodiment, the double-stranded RNAi agent of the invention comprises a nucleotide mismatch within the antisense strand. In another embodiment, the double-stranded RNAi agent of the invention comprises a nucleotide mismatch in the sense strand. In one embodiment, the nucleotide mismatch is eg within 5, 4, 3, 2 or 1 nucleotides from the 3'-end of the iRNA. In another embodiment, the nucleotide mismatch is within, for example, the 3'-terminal nucleotide of the iRNA.

As used herein, the term "sense strand" or "passenger strand" refers to the strand of an iRNA comprising a region substantially complementary to a region of the antisense strand, as the terms are defined herein.

As used herein, the term "cleavage region" refers to the region immediately adjacent to the cleavage site. A cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on and immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage region comprises two bases on and immediately adjacent to either end of the cleavage site. In some embodiments, the cleavage site is specifically at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term "complementarity" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence includes the first nucleotide sequence as understood by one of ordinary skill in the art. It refers to the ability of an oligonucleotide or polynucleotide to form a duplex structure by hybridizing under certain conditions. The conditions may be, for example, “stringent conditions,” wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12 to 16 hours. °C, followed by washing (see, eg, “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may apply, such as physiologically relevant conditions as may be encountered inside an organism. One skilled in the art will be able to determine the most appropriate set of conditions for complementarity testing of two sequences depending on the ultimate application of the hybridized nucleotides.

A complementary sequence within an iRNA, e.g., within a dsRNA described herein, is an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. It involves base pairing of nucleotides. The sequences may be referred to herein as "perfect complementarity" to each other. However, when a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences may be completely complementary, or they may hybridize to a duplex of 30 base pairs or less, at least one; However, they are generally capable of forming no more than 5, 4, 3 , or 2 mismatched base pairs and hybridize under conditions most relevant to their ultimate application, e.g., inhibition of gene expression in vitro or in vivo. have the ability to However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, the overhangs are not considered as mismatches with respect to complementarity determination. For example, the longer oligonucleotide comprises one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is completely complementary to the shorter oligonucleotide. A dsRNA may still be referred to as “fully complementary” for purposes described herein.

As used herein, "complementary" sequences also include or from non-Watson-click base pairs or base pairs formed from non-natural and modified nucleotides, so long as the above requirements with respect to their ability to hybridize are met. can be completely formed. The non-Watson-click base pairing includes, but is not limited to, G:U wobble or Hoogstein base pairing.

As used herein, the terms “complementarity,” “complete complementarity,” and “substantially complementarity,” as understood from references to base matching between the sense strand and the antisense strand of a dsRNA or their use, include the antisense strand of an iRNA preparation and a target sequence, such as It can be used in connection with base matching between two oligonucleotides or polynucleotides.

As used herein, a polynucleotide that is “substantially complementary to at least a portion” of a messenger RNA (mRNA) is a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (eg, an mRNA encoding a TTR gene). refers to nucleotides. For example, a polynucleotide is complementary to at least a portion of a TTR mRNA if the sequence is substantially complementary to a non-interrupting portion of the mRNA encoding the TTR gene.

Thus, in some embodiments, an antisense polynucleotide described herein is fully complementary to a target TTR sequence. In another embodiment, the antisense polynucleotides described herein are fully complementary to SEQ ID NO: 8 (5'- UGGGAUUUCAUGUAACCAAGA -3'). In one embodiment, the antisense polynucleotide sequence is 5'- UCUUGGUUACAUGAAAUCCCAUC -3' (SEQ ID NO: 9), wherein U at position 7 of the antisense strand may be a T.

As used herein, a “reference level” is understood to be a predetermined level to which a level obtained in an assay, eg, a biomarker level, eg, a protein biomarker level, is compared. In certain embodiments, a reference level is a predisposition, eg, a genetic predisposition to a disease or condition associated with an altered level of a biomarker, without a healthy population, eg, a disease or condition associated with an altered level of the biomarker. It may be a control level determined for a population without. In certain implementations, populations must be matched for certain criteria, eg age, gender. In certain embodiments, the reference level of a biomarker is a level from the same subject at an earlier time, eg, before the onset of the symptomatic disease or before the start of treatment. Typically, samples are obtained at clinically relevant intervals, e.g., intervals sufficiently separated in time that changes in the biomarker can be observed, e.g., at least 3 months apart, at least 6 months apart, or at least 9 months apart. do. It is understood that if more than two samples are obtained from a subject over time, any of the previous samples may serve as the reference level.

As used herein, "change compared to reference level" and the like means a statistically or clinically significant change in biomarker level, e.g., a change in protein biomarker level compared to a reference level, typical of an assay method. It is understood to be greater than the standard deviation. Moreover, the change must be clinically relevant. The change compared to the reference level can be determined as a percent change. For example, if the reference level is 100 pg/ml for biomarker X and the level of biomarker X in the subject is 150 pg/ml, then the level is ((150 pg/ml -100 pg/ml)/100 pg /ml) X 100% = 50%. When the level of biomarker X in the subject is 300 pg/ml, the level is increased by 300%. When the level of biomarker X in the subject is 50 pg/ml, the level is reduced by 50%. In certain embodiments, the change compared to the reference level is increased by at least 50%. In certain embodiments, the change compared to the reference level is increased by at least 100%, at least 200% or at least 300%. In certain embodiments, the change compared to a reference sample is reduced by at least 25%. In certain embodiments, the change compared to a reference sample is reduced by at least 50%.

As used herein, a “biological sample from a subject” or “sample from a subject” includes one or more bodily fluids, cells or tissues isolated from a subject. Examples of biological body fluids include blood, serum and intestinal fluid, plasma, cerebrospinal fluid, eye fluid, lymph, urine, saliva and the like. A tissue sample may include a sample from a tissue, organ or localized area. For example, a sample may be from a particular organ, part of an organ, or a body fluid or cell within that organ. In certain embodiments, the sample may be liver tissue or may be derived from the liver. In some embodiments, a “biological sample from a subject” may refer to blood or blood derived serum or plasma from a subject. In some embodiments, a bodily fluid is substantially free of cells, e.g., free of cells.

As used herein, "clinically relevant difference" is understood to be a difference that is at least greater than the typical inter-observer variability for an assessment, wherein the observers are the same subject at approximately the same time, e.g., on consecutive days. It could be a trained healthcare professional, caregiver or patient performing the same evaluation within a week. It is understood that certain patient observations are subjective and should be approximately the same (eg weight, heart rate) when performed by other observers within a short time frame. Some aspects of the mNIS+7 (e.g., response to touch pressure, vibration, joint position and motion) and Norfolk-quality of life (e.g., level of pain, hot/cold hands and feet, standing Other qualitative measures, such as stability over time, may vary from day to day and from observer to observer. Thus, a composite score is used to aggregate observations, and greater variability between observers is expected without any indication of clinically relevant changes. Assays for biomarker levels have a known level of variation within and between samples. Determination of clinically relevant differences is well within the capabilities of those skilled in the art, eg, health care professionals, clinical laboratory professionals experienced in treating patients with TTR-related disorders.

As used herein, "chronically administered" is understood as administration for indefinite intervals, eg, for the remainder of the subject's life up to liver transplantation.

As used herein, "therapeutic agent stabilizing TTR" or "therapeutic agent stabilizing TTR tetramer" means to reduce or prevent dissociation of subunits of TTR tetramers, e.g., into monomers. It is a drug that In some embodiments, the agent reduces the formation of TTR amyloid plaques, for example 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 Ag10.

As used herein, the term "administration of a therapeutic agent" is understood as providing a therapeutic agent to a subject. In an embodiment, the therapeutic agent is provided at a dose appropriate for the agent and by route of administration, eg, as provided by the label of the therapeutic agent.

II. Methods for targeting TTR-related diseases

The present invention relates to double-stranded RNAi preparations and TTR-related diseases in human subjects, such as transthyretin-mediated amyloidosis (ATTR amyloidosis), such as hereditary ATTR (h-ATTR) amyloidosis or non-hereditary ATTR (wt ATTR) for treating amyloidosis; or subjects who do not yet meet the diagnostic criteria for a TTR-related disease but are at risk of developing a TTR-related disease, e.g., those who have a TTR mutation associated with TTR amyloidosis, a subset of TTR amyloidosis that does not yet meet the diagnostic criteria for TTR amyloidosis Provided are their use in a method for inhibiting the expression of TTR in a subject with symptoms, a subject with altered biomarker levels associated with TTR amyloidosis. The method comprises administering to a subject a therapeutically effective amount of an RNAi agent of the present invention.

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

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

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

In another aspect, the invention provides a method of increasing the 6 Minute Walk Test (6MWT) in a human subject suffering from or at risk of developing a TTR-related disorder. The method comprises administering to a human subject a fixed dose of about 25 mg to about 1000 mg of a double-stranded RNAi preparation, wherein the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6) contains; The antisense strand contains the modified nucleotide sequence 5'-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3' (SEQ ID NO: 7).

In certain embodiments, the double-stranded RNAi agent is administered to a human subject from about once per quarter to about once per year. In certain embodiments, the double-stranded RNAi agent is administered to a human subject about once per quarter, about once every 6 months, or about once per year.

In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg to about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 50 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 100 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is about 25 mg to about 300 mg; about 25 mg to about 200 mg; about 50 mg to about 300 mg; about 25 mg; about 50 mg; about 100 mg; about 200 mg; or a fixed dose of about 300 mg is administered to a human subject once quarterly, ie, about once every 3 months.

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

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

In one embodiment, the subject is a person being treated or evaluated for a disease, disorder or condition for which a reduction in TTR gene expression would benefit; A person at risk for a disease, disorder, or condition for which a decrease in TTR gene expression would benefit, e.g., who does not meet criteria for a diagnosis of a TTR-related disease but exhibits at least one sign or symptom of a TTR-related disease or who has a TTR-related disease people with at least one risk factor for; people with a disease, disorder or condition that would benefit from a reduction in TTR gene expression; or a person being treated for a disease, disorder or condition for which a reduction in TTR gene expression would benefit, as described herein.

In some embodiments, the human subject is suffering from a TTR-related disease. In another embodiment, the subject is at risk of developing a TTR-related disease, eg, a subject having a TTR gene mutation associated with developing a TTR-related disease, a subject with a family history of a TTR-related disease, or a diagnosis of a TTR-related disease. A subject who does not meet the criteria and has signs or symptoms suggestive of developing a TTR-related disease.

As used herein, "TTR-related disease" includes any disease caused by or associated with the formation of amyloid deposits in which fibrillar precursors consist of variant or wild-type TTR protein. Mutant and wild-type TTR cause various forms of amyloid deposits (amyloidosis). Amyloidosis involves the formation and aggregation of misfolded proteins resulting in extracellular deposits that impair organ function. Clinical conditions associated with TTR aggregation include, for example, senile systemic amyloidosis (SSA); systemic familial amyloidosis; Familial Amyloidosis Polyneuropathy (FAP); Familial Amyloid Cardiomyopathy (FAC); and leptomeningeal amyloidosis, also known as leptomeningeal or meningocerebrovascular amyloidosis, central nervous system (CNS) amyloidosis or form of amyloidosis VII.

In one embodiment, an RNAi agent of the invention is administered to a subject suffering from familial amyloidogenic cardiomyopathy (FAC). In another embodiment, an RNAi agent of the invention is administered to a subject suffering from FAC with a mixed phenotype, ie, a subject with both cardiac and nervous system damage. In yet another embodiment, an RNAi agent of the invention is administered to a subject suffering from FAP with a mixed phenotype, ie, a subject with both nervous system and cardiac damage. In one embodiment, an RNAi agent of the invention is administered to a subject suffering from FAP treated with orthotopic liver transplantation (OLT).

In another embodiment, an RNAi agent of the invention is administered to a subject suffering from senile systemic amyloidosis (SSA). In another embodiment of the methods of the invention, an RNAi agent of the invention is administered to a subject suffering from familial amyloidogenic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Normal sequence TTR causes cardiac amyloidosis in the elderly and is called senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA is often accompanied by microscopic deposits in many other organs. TTR mutations promote the process of TTR amyloidogenesis and are the most important risk factor for the development of clinically significant TTR amyloidosis (also called ATTR (transthyretin type amyloidosis)). More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.

In some embodiments of the methods of the invention, an RNAi agent of the invention is administered to a subject suffering from transthyretin (TTR) associated familial amyloidogenic polyneuropathy (FAP). Such subjects may suffer from ocular conditions such as vitreous opacity and glaucoma. It is known to those skilled in the art that amyloidogenic transthyretin (ATTR) synthesized by the retinal pigment epithelium (RPE) plays an important role in the development of ocular amyloidosis. Previous studies have shown that panretinal laser photocoagulation to reduce RPE cells prevents the progression of amyloid deposits in the vitreous, suggesting that effective inhibition of ATTR expression in RPE could represent a novel therapy for ocular amyloidosis (Ref. For example, Kawaji, T., et al., Ophthalmology. (2010) 117: 552-555). Another TTR-related disorder is hyperthyroxinemia, also known as “dystranslettinemia hyperthyroxinemia” or “dysproalbuminemia hyperthyroxinemia”. This type of hyperthyroxinemia may be secondary to increased association of thyroxine with TTR due to mutant TTR molecules having increased affinity for thyroxine. See, eg, Moses et al . (1982) J. Clin. Invest. , 86, 2025-2033.

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

In some embodiments, the formulation is administered subcutaneously to the subject.

In some embodiments, the subject is administered a single dose of the RNAi agent via subcutaneous injection, eg, abdominal, femoral, or brachial injection. In another embodiment, the subject receives divided doses of the RNAi agent via subcutaneous injection. In one embodiment, split doses of the RNAi agent are administered to the subject via subcutaneous injection at two different anatomical locations on the subject. For example, a subject can be injected subcutaneously at a dose of 25 mg to 1000 mg. In some embodiments of the invention, subcutaneous administration is self-administration, eg via a pre-filled syringe or auto-injecting syringe. In some embodiments, a dose of an RNAi agent for subcutaneous administration is contained in a volume of eg, 1 ml or less of a pharmaceutically acceptable carrier. In some embodiments, the RNAi agent is in a non-pyrogenic formulation.

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

In some embodiments, an RNAi agent is administered to a subject in a therapeutically effective amount.

As used herein, "therapeutically effective amount" is intended to include an amount of an RNAi agent sufficient to effect treatment of a disease when administered to a patient to treat a TTR-associated disease (e.g., compared to an appropriate control). reducing, maintaining or slowing the progression of an existing disease by reducing, maintaining or slowing down one or more symptoms of the disease compared to an appropriate control). "Therapeutically effective amount" refers to the RNAi agent, the manner in which the agent is administered, the disease and its severity and history, age, weight, family history, genetic makeup, stage of the pathological process mediated by TTR expression, preceding or concomitant use as the case may be. Depending on the type of treatment and other individual characteristics of the patient being treated, diagnostic criteria for TTR amyloidosis, polyneuropathy and cardiomyopathy are further discussed below.

As used herein, “therapeutically effective amount” refers to subjects who do not yet meet diagnostic criteria for a TTR-related disease, eg, subjects who have not been diagnosed with hTTR amyloidosis polyneuropathy; Subjects who do not meet the diagnostic criteria for stage 1 FAP but are predisposed to the disease, eg, those with a TTR mutation associated with TTR amyloidosis, orthostatic hypotension, heart failure, cardiac arrhythmias, left ventricular wall thickness, ventricular septal wall thickness, cardiac posterior wall thickness diarrhea, constipation, impotence, glaucoma, intravitreal deposition, scallop pupil; carpal tunnel syndrome, lumbar spinal canal stenosis and biceps tendon rupture; sufficient to prevent or ameliorate a disease or one or more symptoms of a disease when administered to a subject having an elevated neurofibrillary tangle (NfL) level compared to a reference sample, eg, an Nfl level of at least 37 pg/ml in serum. , the amount of the RNAi agent. Symptoms that can be ameliorated include sensory neuropathy (e.g., paresthesia, paresthesia of distal limbs), autonomic neuropathy (e.g., gastrointestinal insufficiency such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, Dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction, corneal lattice dystrophy, echocardiographic evaluation left ventricular (LV) wall thickening by echocardiography, assessment of increased total longitudinal tension by echocardiography, increased N-terminal prohormone type B natriuretic peptide (NTproBNP), and hospitalization for cardiac events. Reduction of a disease includes slowing the disease process or reducing the severity of a late onset disease. "Dosage" may vary depending on the RNAi agent, the way the agent is administered, the degree of disease risk, medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment as the case may be, and other individual characteristics of the patient to be treated. can

"Therapeutically 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. The RNAi agent used in the methods of the present invention is such It can be administered in an amount sufficient to produce a reasonable benefit/risk ratio applicable to treatment.

As used herein, the phrase “therapeutically effective amount” also includes an amount that provides benefit in the treatment, prevention, or management of a pathological process or symptom(s) of a pathological process mediated by TTR expression. Symptoms of TTR amyloidosis include sensory neuropathy (e.g. paresthesia, paresthesia of distal limbs), autonomic neuropathy (e.g. gastrointestinal dysfunction such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, and dementia. , myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic dysfunction, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction, corneal lattice dystrophy, by echocardiographic evaluation Left ventricular (LV) wall thickening, assessment of increased total longitudinal tension by echocardiographic evaluation, increased N-terminal prohormone type B natriuretic peptide (NTproBNP), and hospitalization for cardiac events.

In one embodiment, treatment of a subject with a dsRNA agent of the present invention prevents progression of neuropathy, e.g., if the subject has FAP, FAP with a mixed phenotype, FAC with a mixed phenotype, or FAP and has an OLT. slow down In another embodiment, treatment of a subject with a dsRNA agent of the present invention may treat neuropathy and myocardial slow the progression of the disease. In another embodiment, eg, where the subject has cardiac involvement, the methods of the present invention improve cardiac structure and function, eg, the method reduces mean left ventricular wall thickness and longitudinal strain and reduces cardiac Reduces the expression level of the stress biomarker, N-terminal pro b-type natriuretic peptide (NT-proBNP).

Administration of a therapeutically or prophylactically effective amount of an RNAi agent of the invention is also useful in methods of improving at least one indicator of neurological impairment or quality of life in a subject suffering from or at risk of developing a TTR-related disease.

For example, in one embodiment, a method of the invention improves an indicator of neurological impairment in at least a subject. "Improving at least one indicator of neurological impairment" in a subject refers to the ability of a method of the present invention to slow, reduce or halt neurological impairment, or ameliorate any symptoms associated with neurological impairment. refers to Any suitable measure of nervous system damage can be used to determine whether a subject has reduced, slowed, or halted improvement in nervous system damage, or symptoms associated with nervous system damage.

One suitable measure is the Neuropathic Impairment Score (NIS). NIS refers to a scoring system that measures weakness, sensation, and reflexes, particularly in relation to peripheral neuropathy. The NIS score is the standard group of muscles for weakness (1 = 25% weakness, 2 = 50% weakness, 3 = 75% weakness, 3.25 is movement against gravity, 3.5 is movement with gravity removed, 3.75 is no movement). no muscle blink, and 4 paralyzed), standard group of muscle stretch reflexes (0 normal, 1 reduced, 2 absent), touch pressure, vibration, joint position and movement, pin prick (all with index and big toe) Graded: 0 normal, 1 reduced, 2 absent). Assessments are corrected for age, sex and fitness.

In one embodiment, the methods of the invention reduce NIS by at least 5 points at 18 months from start of dosing. In another embodiment, the methods of the invention stabilize NIS at 18 months of initiation of treatment with an RNAi agent provided herein. In another embodiment, the method increases NIS scores compared to an appropriate control group indicative of a natural history of disease, eg, a placebo control group as provided in Adams et al., N Engl J Med 2018;379. slow down The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

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

Another suitable measure of neurological impairment is the modified neuropathic impairment score (mNIS+7). As known to those skilled in the art, mNIS+7 refers to a clinical trial-based assessment of neurological impairment (NIS) combined with electrophysiological measures of small and large nerve fiber function (NCS and QST) and measures of autonomic function (postural blood pressure). do. The mNIS+7 score is a variant of the NIS+7 score (representing NIS+7 tests). NIS+7 analyzes weakness and muscle stretch reflexes. Five of the seven tests involved nerve conduction properties. These characteristics are peroneal nerve complex muscle action potential amplitude, motor nerve conduction velocity and motor nerve distal latency (MNDL), tibial MNDL and splenic sensory nerve action potential amplitudes. These values are corrected for the variables of age, sex, height and weight. The other two of the seven trials involved vibration detection thresholds and heart rate reduction following deep breathing.

The mNIS+7 score is a modification of the NIS+7 using the Smart Somatotopic Quantitative Sensation Testing, the use of novel autonomic assessments, compound muscle action potentials of the amplitudes of the ulnar, peroneal and tibial nerves and ulnar and ulnar nerves. Consider using sensory nerve action potentials of the splenic nerve (see Suanprasert, N. et al . , (2014) J. Neurol. Sci ., 344(1-2): pgs. 121-128).

In one embodiment, the methods of the invention reduce mNIS+7 by at least 5 points at 18 months from start of dosing. In another embodiment, the methods of the invention stabilize mNIS+7 at 18 months after initiation of treatment with an RNAi agent provided herein. In another embodiment, the method comprises an increase in mNIS+ compared to a suitable control group indicative of a natural history of the disease, eg, a placebo control group as provided in Adams et al., N Engl J Med 2018;379. 7 Slow down the score. The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

In another embodiment, the methods of the present invention improve at least one indicator of quality of life in at least a subject. “Improving at least one indicator of quality of life” in a subject refers to the ability of a method of the present invention to slow or halt deterioration in quality of life or improve quality of life. Any suitable measure of quality of life can be used to determine whether a subject has a slow or stationary deterioration in quality of life or an improved quality of life.

For example, the SF-36® Health Survey covers physical functioning, role limitations due to physical health problems, body pain, general health, vitality (energy and fatigue), social functioning, role limitations due to emotional problems, and mental health (psychological It provides a self-reported multi-item scale that measures eight health parameters (e.g., pain and psychological well-being). Each scale is directly converted to a 0-100 scale under the assumption that each question has equal weight. The lower the score, the more dysfunction. Higher scores indicate less dysfunction, i.e., a score of zero corresponds to maximum dysfunction and a score of 100 equates to no dysfunction. The survey also provides a physical component summary and a mental component summary.

In one embodiment, the methods of the present invention provide a subject with at least one of the SF-36 physical health related parameters (physical health, role-physical, body pain or general health) or the SF-36 mental health related parameters (vitality, social function, role-emotional or mental health). The improvement may take the form of, for example, an increase of at least 2 or at least 3 points on a scale for any one or more parameters at 9 months from start of dosing.

In another embodiment, the method of the present invention stops a decreasing SF-36 parameter score at 9 months from the start of dosing for any one or more parameters, e.g., the method comprises an SF-36 assessment does not induce clinically significant changes in SF-36 within the variability observed for individuals performing In another embodiment, the method of the present invention is directed to a suitable control group showing the rate of decline in the SF-36 score at 9 months from the start of administration, a natural history of the disease, e.g., as described in Adams et al., N Engl J Med 2018;379:11-21) slows the rate of decrease in SF-36 score in subjects treated with an RNAi agent of the invention compared to the rate of decrease in SF-36 score compared to a placebo control group . The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

Another suitable measure of quality of life is the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) questionnaire. The Norfolk QOL-DN is a validated comprehensive questionnaire designed to capture the full spectrum of DN associated with large fibers, small fibers and autonomic neuropathy not captured by existing tools.

In one embodiment, the methods of the invention measure the subject's Norfolk QOL-DN score from baseline, e.g., about -2.5, -3.0, -3.5, -4.0 at 9 months from initiation of treatment with an RNAi agent provided herein. , -4.5, or -5.0 changes. In another embodiment, the method halts an increasing Norfolk QOL-DN score, eg, the method clinically monitors the Norfolk QOL-DN score within observed variability among individuals performing the QOL-DN assessment. does not cause significant change. In another embodiment, the methods of the present invention can be performed on a suitable control group showing the rate at which the QOL-DN score increases, eg, a natural history of the disease, eg, Adams et al., N Engl J Med 2018 ;379:11-21) slows the rate of increase in QOL-DN scores in subjects treated with the RNAi agents of the present invention relative to the rate of increase in QOL-DN scores compared to a placebo control group. The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

Another suitable measure of quality of life is exercise intensity, as assessed, for example, by the NIS-W score. The NIS-W score is a composite score summing the weakness of the muscles of the head, trunk and extremities. Using the NIS(W) (see the part of the scale that measures weakness) strength is rated as normal (0) or completely paralyzed (4) with a moderate rating; 1 represents muscles considered 25% weak in the clinical strength test, 2 is 50%, 3 is 75%, 3.25 is movement against gravity, 3.50 is movement with gravity removed, and 3.75 is muscle blink.

In one embodiment, the methods of the invention provide a subject with an improvement from baseline in NIS-W score. Such improvement may take the form of a reduction of at least 5, 6, 7, 8, 9 or 10 points in the subject's NIS-W score at 18 months of initiation of treatment with an RNAi agent provided herein. In another embodiment, the method halts the decrease in NIS-W score, eg, the method can be performed on a suitable control group showing natural history of the disease, eg, as described in Adams et al., N Engl J Med. 2018;379:11-21) does not induce a clinically significant increase in NIS-W score or a slowdown in the rate of increase in NIS-W score compared to a placebo control group. The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

Another relevant indicator of quality of life is the Overall Dysfunction Scale (R-ODS) constructed by Rasch, which is a patient questionnaire designed to capture the patient's activity and social participation limitations. In one embodiment, the methods of the invention provide a subject with an improvement from baseline in R-ODS score. Such improvement may take the form of an increase of at least 2, eg, at least 2, 3, 4 or 5 points in the subject's R-ODS score at 18 months of initiation of treatment with an RNAi agent provided herein. In another embodiment, the method stops a decreasing R-ODS score, e.g., the method does not result in a clinically significant decrease in R-ODS score at 18 months of initiation of treatment with an RNAi agent provided herein. don't In another embodiment, the method of the invention is performed on a suitable control group, e.g., showing the rate at which the R-ODS score declines, e.g., a natural history of the disease, at 18 months of initiation of treatment with an RNAi agent provided herein. For example, as provided in the literature (Adams et al., N Engl J Med 2018;379:11-21) compared to the rate of decrease in R-ODS scores compared to a placebo control group treated with an RNAi agent of the present invention Slow the rate of decline in the R-ODS score in the subject. The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

The Composite Autonomic Symptom Score (COMPASS-31), a patient questionnaire assessing symptoms of dysautonomia providing a symptom score from 0 to 100, is another suitable indicator of quality of life. In one embodiment, the methods of the invention provide a subject with an improvement from baseline in a COMPASS-31 score. Such improvement may take the form of an increase in the subject's COMPASS-31 score of at least 5, eg, at least 5, 6, 7, 8, 9 or 10 points, at 18 months of initiation of treatment with an RNAi agent provided herein. have. In another embodiment, the method stops a decreasing COMPASS-31 score, eg, the method does not result in a clinically relevant change in the COMPASS-31 score at 18 months of initiation of treatment with an RNAi agent provided herein. . In another embodiment, the method of the present invention can be performed on a suitable control group showing the rate at which the COMPASS-31 score decreases, eg, a natural history of the disease, such as Adams et al., N Engl J Med 2018 ;379:11-21) slows the rate of decrease in COMPSS-31 score at 18 months of initiation of treatment with an RNAi agent provided herein compared to the rate of decrease in COMPASS-31 score compared to a placebo control group . The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

Other quality-of-life indicators may include nutritional status as assessed, for example, by change in median body mass index (mBMI). In one embodiment, the methods of the invention provide a subject with an improvement from baseline in mBMI. Such improvement may take the form of an increase in mBMI score of at least 2, 3, 4, 5 or more at 18 months of initiation of treatment with an RNAi agent provided herein. In another embodiment, the method stops a decreasing mBMI index score, eg, the method does not result in a clinically significant change in mBMI score at 18 months of initiation of treatment with an RNAi agent provided herein. In another embodiment, the method of the present invention is performed on a suitable control group showing the rate at which the mBMI score decreases, eg, the natural history of the disease, eg, Adams et al., N Engl J Med 2018;379 : 11-21) mBMI score at 18 months of initiation of treatment with an RNAi agent provided herein in subjects treated with an RNAi agent of the present invention relative to the rate of decrease in mBMI score in subjects compared to a placebo control group as provided in 11-21). slow down the rate of decrease of The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

Another quality-of-life indicator includes assessment of exercise capacity. One suitable measure of exercise capacity is the 6-minute walking test (6MWT), which measures how far a subject can walk in 6 minutes, ie the 6-minute walking distance (6MWD). In one embodiment, the methods of the invention achieve an increase from baseline of at least 10 meters, e.g., at least 10, 15, 20, or about 30 meters, at 6MWD at 18 months of initiation of treatment with an RNAi agent provided herein. provided to the subject.

Another suitable measurement is the 10-meter walk test, which measures walking speed. In one embodiment, a method of the invention measures at least 0.025 meters/second from baseline, e.g., at least 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/sec.

In some embodiments, changes in plasma biomarker levels are indicative of ongoing nerve damage in ATTR amyloidosis or reduction in progression of polyneuropathy. For example, a decrease in neurofilament light chain (NfL) levels at 9 months compared to NfL levels at the start of treatment may be indicative of a reduction in ongoing nerve damage or polyneuropathy progression in ATTR amyloidosis. In certain embodiments, a decrease in the level of other proteins, particularly RSPO3, CCDC80, EDA2R and NT-proBNP, alone or in combination with a decrease in NfL levels at 9 months from the start of treatment compared to their corresponding levels at the start of treatment Thus, it may be an indicator of a reduction in ongoing nerve damage in ATTR amyloidosis or a reduction in the progression of polyneuropathy. In certain embodiments, an increase in N-CDase level, alone or in combination with the other markers listed above, at 9 months from the start of treatment compared to the corresponding level at the start of treatment is an increase in ongoing nerve damage or polyneuropathy in ATTR amyloidosis. It may be an indicator of a decrease in the progression of the condition. Additional biomarkers that can serve as indicators of reduction in nerve damage or polyneuropathy in ATTR amyloidosis, such as, for example, at 9 months from initiation of treatment with an RNAi agent provided herein, are provided in Table 1. Decreased progression of ongoing nerve damage or polyneuropathy in ATTR amyloidosis correlates with a decrease in proteins with positive beta coefficients. Decreased progression of ongoing nerve damage or polyneuropathy in ATTR amyloidosis correlates with increases in proteins with negative beta coefficients. Changes in biomarker levels are understood to be statistically significant changes, i.e., changes greater than the inherent variability of the test.

In certain embodiments, the methods of the present invention provide an improvement in a cardiovascular parameter, e.g., an increase in the Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS), a decrease in left ventricular (LV) wall thickening by echocardiographic evaluation compared to baseline, Reduced global longitudinal tension by echocardiographic assessment compared to baseline, decreased N-terminal prohormone type B natriuretic peptide (NTproBNP) compared to baseline, and reduced hospitalizations due to cardiac events.

The methods of the present invention may also improve the prognosis of the subject being treated. For example, the methods of the present invention can be performed using a suitable control group showing a natural history of the disease, such as a placebo control group as provided in the literature (Adams et al., N Engl J Med 2018;379:11-21). Provides the subject with a reduced likelihood of a clinically deteriorating event or reduced hospital admission during the treatment period compared to In certain embodiments, a reduction in the likelihood of a clinically deteriorating event during treatment is compared to an appropriate control group, eg, provided in the literature (Maurer et al., N Engl J Med 2018:379:11-21), For example, an assessment according to the Finkelstein-Schoenfeld method may include a reduction in all-cause mortality or cardiovascular-related hospitalizations. The rate of disease progression depends on a number of factors including, but not limited to, the severity of the subject's disease at initiation of treatment, the duration of treatment, previous treatment, and the specific TTR mutation present, if any.

The dose of an RNAi agent administered to a subject is, for example, a desired level of inhibition of TTR gene expression (e.g., TTR mRNA expression, TTR protein expression, or a reduction in amyloid deposits as defined above). as) or adjusted to balance the risks and benefits of a particular dose to achieve the desired therapeutic effect and at the same time avoid undesirable side effects.

In one embodiment, administration of an iRNA agent of the invention to a subject as a “fixed dose” (eg, a dose in mg) means that a single dose of the iRNA agent is dependent on any particular subject-related factor, such as body weight. This means that it is used for all objects regardless of

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

In certain embodiments, the double-stranded RNAi agent is administered to a human subject from about once per quarter to about once per year. In certain embodiments, the double-stranded RNAi agent is administered to a human subject about once per quarter, about once every 6 months, or about once per year.

In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 300 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg to about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg to about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 25 mg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 50 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 75 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 100 mg/kg. In certain embodiments, the double-stranded RNAi agent is administered to a human subject at a fixed dose of about 200 mg/kg. In certain embodiments, the double-stranded RNAi agent is about 25 mg to about 300 mg; about 25 mg to about 200 mg; about 75 mg to about 200 mg; about 25 mg; about 50 mg; about 100 mg; about 200 mg; or a fixed dose of about 300 mg is administered to a human subject once quarterly, ie, about once every 3 months.

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

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

In certain embodiments, the administration is subcutaneous administration, eg, self-administration via a pre-filled syringe or autoinjector syringe. In some embodiments, a dose of an RNAi agent for subcutaneous administration is contained in a volume of eg, 1 ml or less of a pharmaceutically acceptable carrier.

Any of these schedules may optionally be repeated in one or more iterations. The number of repetitions varies depending on the achievement of a desired effect, eg, inhibition of the TTR gene, retinol binding protein levels, vitamin A levels, or achievement of a therapeutic effect, eg, reduction of amyloid deposits or reduction of symptoms of a TTR-related disease. can do. In certain embodiments, the RNAi agent is administered chronically for an indefinite period of time, eg, for the lifetime of the patient.

In some embodiments, the RNAi agent is administered in conjunction with other therapeutic agents or other therapeutic regimens. For example, other agents or other therapeutic regimens suitable for treating TTR-related disorders include liver transplantation, heart transplantation, pacemaker implantation, agents capable of reducing monomeric TTR levels in the body; tafamidis (Vyndaqel® or Vyndamax® or AG10), which kinetically stabilizes TTR tetramers, preventing tetramer dissociation required for TTR amyloidogenesis; may be used, for example, to reduce edema in TTR amyloidosis with cardiac involvement nonsteroidal anti-inflammatory drugs (NSAIDS) such as diflunisal and diuretics.

In one embodiment, the subject receives an initial dose and one or more maintenance doses of the RNAi agent. The maintenance dose or doses may be less than or equal to the initial dose, eg half the initial dose. After treatment, the patient may be monitored for changes in the patient's condition.

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

As used herein, the term "inhibiting" is used interchangeably with "reducing", "silencing", "downregulating", "suppressing", and other similar terms, and any inhibition include the level In some embodiments, inhibition comprises statistically significant or clinically significant inhibition.

The phrase “inhibiting expression of TTR” is intended to refer to inhibition of expression of any TTR gene, including variants or mutants of the TTR gene. Thus, the TTR gene can be a wild-type TTR gene, a mutant TTR gene (eg, a mutant TTR gene that causes amyloid deposits).

Inhibition can be assessed by a decrease in absolute or relative levels of one or more variables associated with TTR expression compared to control levels. A control level is any type of control level used in the art, eg, a pre-administration level, a similar subject, cell or sample that is untreated or treated with a control (eg, a buffer only control or an inactive agent control). It may be a level determined from As used herein, inhibition of TTR expression is typically achieved by determining the level of TTR in an appropriate sample (e.g., a historical control sample, a normal sample, or a level determined in a clinical trial) or as described herein or in PCT publications WO2010048228, WO2013075035, and Prior to treatment of a subject with those provided in WO2017023660, or other agents that inhibit expression of TTR, such as antisense oligonucleotide preparations, dicer substrate preparations (see, eg WO20118139917 and WO20118139917); and after treatment with an iRNA formulation provided herein. It is understood that the iRNA preparations provided herein are durable but slow acting. Thus, the level of knockdown is determined after a time sufficient to reach a nadir, e.g., at least 3 weeks after the first administration of an iRNA agent in a human subject, or when a steady state of TTR knockdown is achieved, e.g., an iRNA provided herein. determined after multiple administrations with the formulation.

Inhibition of the expression of the TTR gene can be achieved by contacting a cell or cells with an iRNA of the present disclosure or by administering an RNAi agent of the present invention to a subject in which the TTR gene is or has been transcribed and treated (e.g., a cell or cells). Expression of the TTR gene is indicated by a decrease in the amount of mRNA expressed by a first cell or group of cells (which may be present, for example, in a sample derived from a subject) by administration) of the first cell or group of cells; substantially the same as the group of cells, but inhibited compared to a second cell or group of cells that have not been so treated (control cell(s)). In some embodiments, percent inhibition is assessed by expressing mRNA levels in treated cells as a percentage of mRNA levels in control cells using the formula:

Similar calculations can be performed for serum TTR protein concentrations in blood samples obtained, for example, from a subject to determine percent inhibition of expression. If TTR is not detected in the serum or plasma sample after treatment, the amount of TTR present is considered to be at the lower detection limit of the assay used.

In some embodiments, percent inhibition is determined using validated and clinically accepted methods.

Alternatively, inhibition of expression of the TTR gene can be assessed in terms of a reduction in TTR gene expression, eg TTR protein expression, retinol binding protein levels, vitamin A levels or parameters functionally linked to the presence of amyloid deposits comprising TTR. can TTR gene silencing can be determined constitutively in any cell expressing TTR, or by genome engineering and any assay known in the art. The liver is the major site of TTR gene expression. Other significant sites of interest include the retina and choroid plexus.

III. iRNAs of the present invention

Suitable iRNAs for use in the methods of the invention are double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the TTR gene in a cell, such as a cell in a subject, eg, a mammal, eg, a human with a TTR-related disease. includes The dsRNA includes an antisense strand having a region of complementarity that is complementary to at least a portion of the mRNA formed in expression of the TTR gene. The region of complementarity is between about 21 and no more than 30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 21 nucleotides in length). . Upon contact with a cell expressing the TTR gene, the iRNA is capable of expressing the TTR gene (e.g. , human, non-human primate, or non-primate) when Hep3B cells are transfected with 10 nM of the iRNA preparation using the methods provided herein. mammalian TTR gene) by at least about 70% as assessed by real-time PCR using, for example, the method provided in Example 4 of WO2013075035.

A dsRNA comprises two RNA strands that are complementary and hybridize to form a duplex structure under the conditions in which the dsRNA is used. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary to the target sequence and is usually fully complementary. The target sequence may be derived from the sequence of mRNA formed during expression of the TTR gene. The other strand (the antisense strand) contains a region complementary to the antisense strand so that the two strands hybridize to form a duplex structure when combined under suitable conditions. As described elsewhere herein and known in the art, complementary sequences of dsRNAs can also be included as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, duplex structures are 21 to 30 base pairs in length. Similarly, the length of the region of complementarity with the target sequence is 22 and 30 nucleotides.

dsRNA can be synthesized by standard methods known in the art, as discussed further below.

iRNA compounds of the invention can be prepared using a two-step process. First, the individual strands of a double-stranded RNA molecule are prepared separately. The component strands are then annealed. Individual strands of siRNA compounds can be prepared using either solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage of being able to easily prepare oligonucleotide strands comprising unnatural or modified nucleotides. The single-stranded oligonucleotides of the present invention can be prepared using either solution-phase or solid-phase organic synthesis or both.

IV. Modified iRNAs of the present invention

RNAi preparations for use in the methods of the invention include defined chemical modifications in the sense and antisense strands. When the length of either strand is extended to provide an antisense strand of greater than 23 nucleotides and a sense strand of greater than 21 nucleotides, the nucleotides include modifications, including but not limited to sugar modifications, backbone modifications and base modifications Modifications may include, for example, terminal modifications such as 5'-end modifications (phosphorylation, conjugation, inverted ligation) or 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.); base modifications, eg, replacement of stabilizing bases or bases that form base pairs with an expanded repertoire of partners, removal of bases (baseless nucleotides), or conjugated bases; sugar modifications ( eg , at the 2'-position or 4'-position) or replacement of sugars; or backbone modifications including modification or replacement of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones or do not contain any natural internucleotide linkages. RNAs with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For purposes herein and as sometimes referred to in the art, modified RNAs that do not have a phosphorus atom in their internucleotide backbone may also be considered oligonucleotides. In some embodiments, a modified iRNA has a phosphorus atom in its internucleoside backbone.

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

Modified RNA backbones herein that do not contain a phosphorus atom include short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatomic or heterocyclic nucleoside linkages. It has a skeleton formed by side-to-side connections. These include morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbone; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbone; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbone; and others having mixed N, O, S and CH ₂ component parts.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs in which both sugar and internucleoside linkages, i.e., the backbone of nucleotide units are replaced by new groups. Base units are retained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, which is an RNA mimetic that has been shown to have good hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the backbone.

Some embodiments featured in the present disclosure include phosphorothioate backbones, and heteroatom backbones and particularly --CH ₂ --NH--CH ₂ -, --CH ₂ --N(CH of US Pat. No. 5,489,677). ₃ )--O--CH ₂ --[known as methylene (methylimino) or MMI backbone], --CH ₂ --O--N(CH ₃ )--CH ₂ --, --CH ₂ --N(CH ₃ )--N(CH ₃ )--CH ₂ -- and --N(CH ₃ )--CH ₂ --CH ₂ -- and the amide backbone of the aforementioned US Pat. No. 5,602,240 It includes RNA having an oligonucleotide having a. In some embodiments, the RNA featured herein has the morpholino backbone structure of the aforementioned U.S. Patent No. 5,034,506. The native phosphodiester backbone can be represented as OP(O)(OH)-OCH2-.

Modified RNAs may also contain one or more substituted sugar moieties. An iRNA, e.g., a dsRNA featured herein, has an OH at the 2'-position;F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl can be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl. . An exemplary suitable variant is O[(CH ₂ ) _n O] _m CH ₃ , O(CH ₂ ). _n OCH ₃ , O(CH ₂ ) _n NH ₂ , O(CH ₂ ) _n CH ₃ , O(CH ₂ ) _n ONH ₂ , and O(CH ₂ ) _n ON[(CH ₂ ) _n CH ₃ )] ₂ , wherein n and m are from 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH , SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkyl Amino, polyalkylamino, substituted silyl, RNA cleavage groups, reporter groups, intercalants, groups for improving the pharmacokinetic properties of iRNA, or groups for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification is 2'-methoxyethoxy (2'-O--CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Reference: Martin et al . , Helv. Chim. Acta, 1995, 78: 486-504 ), that is, it contains an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, ie, O(CH ₂ ) ₂ ON(CH ₃ ) ₂ groups, and 2 as described in the Examples herein below. '-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH ₂ --O--CH ₂ --N(CH ₃ ) ₂ . Additional exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides, (these three series of both R and S isomers); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

Other modifications include 2'-methoxy (2'-OCH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ) and 2'-fluoro (2'-F). . Similar modifications can also be made at other positions on the RNA of the iRNA, particularly at the 3' position of the sugar on the 3' terminal nucleotide or at the 5' position of the 2'-5' linked dsRNA and 5' terminal nucleotide. An iRNA may also have a sugar mimetic such as a cyclobutyl moiety in place of a pentofuranosyl sugar.

The RNA of an iRNA of the invention may also contain nucleobase (commonly referred to simply as a “base”) modification or substitution. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). include The modified nucleobases are deoxythymidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine, and 6 guanine. -methyl and other alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-haluracil and cytosine, 5-propynyluracil and cytosine, 6 -azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and Guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and other synthetic and natural nucleobases such as 7- and 3-deazaguanine and 3-deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propyluracil and 5-propynylcytosine. include 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2 °C (see Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) exemplarily base substitutions, even more particularly in combination with modifications per 2'-O-methoxyethyl.

RNAi agents of the present disclosure may also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a ring formed by the bridging of two carbons, regardless of adjacent or non-adjacent atoms. A “bicyclic nucleoside” (“BNA”) is an adjacent A nucleoside having a sugar moiety comprising a ring formed by bridging, including a bridge connecting two carbon atoms of the sugar ring, whether contiguous or not, to form a bicyclic ring system. In, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring through a 2'-acyclic oxygen atom. Thus, in some embodiments, an agent of the present disclosure comprises one or more locked nucleic acids ( LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety, wherein said ribose moiety contains an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a 4 A nucleotide comprising a bicyclic sugar moiety comprising a '-CH2-O-2' bridge. This structure effectively locks ribose in the 3'-endo conformational form. Addition of the locked nucleic acid to siRNA is siRNA It has been shown to increase stability and reduce off-target effects (see Elmen, J. et al . , (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al . , (2007 ) Mol Canc Ther 6(3):833-843 Grunweller, A. et al . , (2003) Nucleic Acids Research 31(12):3185-3193 Bicyclics for Use in Polynucleotides of the Disclosure Examples of nucleosides include, but are not limited to, nucleosides comprising bridges between 4' and 2' ribosyl ring atoms. One or more bicyclic nucleosides containing bridges.

A locked nucleoside can be represented by a structure (stereochemistry omitted), where B is a nucleobase or modified nucleobase and L is a linking group connecting the 2'-carbon to the 4'-carbon of the ribose ring.

Examples of the above 4' to 2' bridged bicyclic nucleosides include 4'-(CH2)-O-2'(LNA);4′-(CH2)-S-2′; 4′-(CH2)-O-2′ (ENA); 4'-CH(CH3)-O-2' (also referred to as "bound ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and analogs thereof; see, e.g., the United States See Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof; see, eg, US Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, eg, US Pat. No. 8,278,425); 4′-CH2-ON(CH3)-2′ (see, eg, US Patent Publication No. 2004/0171570); 4'-CH2-N(R)-O-2' where R is H, C1-C12 alkyl or a protecting group (see, eg, US Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ ( see, eg , Chattopadhyaya et al ., J. Org. Chem ., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogues thereof; see eg US Pat. No. 8,278,426). The entire contents of each of the foregoing documents are incorporated herein by reference.

Additional representative US patents and US patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to: US Patent Nos. 6,268,490, each of which is incorporated herein by reference in its entirety; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US Publication No. 2008/0039618; and US Publication No. 2009/0012281.

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

RNAi agents of the present disclosure may also be modified to include one or more constrained ethyl nucleotides. As used herein, "tethered ethyl nucleotide" or "cEt" refers to a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge (i.e., L in the previous structure). It is a locked nucleic acid. In one embodiment, the constrained ethyl nucleotides are in the S form, referred to herein as "S-cEt".

An iRNA of the invention may also contain one or more “conformationally restricted nucleotides” (“CRNs”). CRN is a nucleotide analog with a linker connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRN locks the ribose ring into a stable conformation and increases the hybridization affinity for mRNA. The linker is of sufficient length to reduce ribose ring puckering by positioning the oxygen in an optimal position for stability and affinity.

One or more nucleotides of an iRNA of the invention may also include hydroxymethyl substituted nucleotides. A "hydroxymethyl substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide also referred to as an "unlocked" ("UNA") variant.

Potentially stabilizing modifications to the ends of RNA molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxypyrrolinol (Hyp-C6-NHAc), -C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxy prolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted base dT (idT), etc. The disclosure of such modifications can be found in WO 2011/005861 can

Other modifications of the nucleotides of an iRNA of the invention include a 5' phosphate or 5' phosphate mimetic on the antisense strand of an RNAi agent, eg , a 5'-terminal phosphate or phosphate mimetic. Suitable phosphate mimetics are described, for example, in US Patent Publication No. 2012/0157511, incorporated herein by reference in its entirety.

V. iRNA conjugated to a ligand

Another modification of the RNA of the iRNA of the present invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity of the iRNA, cellular distribution, or cellular uptake of the iRNA. Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA , 1989,86:6553-6556), cholic acid (Manoharan et al . , Biorg. Med. Chem. Let ., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (see Manoharan et al . , Ann. NY Acad. Sci ., 1992 , 660 : 306-309 ; 533-538), aliphatic chains such as dodecanediol or undecyl residues (see Saison-Behmoaras et al . , EMBOJ , 1991, 10:1111-1118; Kabanovet et al . , FEBS Lett ., 1990 Svinarchuk et al . , Biochimie , 1993, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O- Hexadecyl-rac-glycero-3-phosphonate (see Manoharan et al . , Tetrahedron Lett ., 1995, 36:3651-3654; Shea et. al. , Nucl. Acids Res ., 1990, 18: 3777-3783), polyamine or polyethylene glycol chains (see Manoharan et al . , Nucleosides & Nucleotides , 1995, 14:969-973), or adamantane acetic acid (see Manoharan et al . , Tetrahedron Lett ., 1995, 36:3651-3654), palmityl moiety (see Mishra et al . , Biochim.B iophys . Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (see Crooke et al . , J. Pharmacol. Exp. Ther ., 1996, 277:923-937), but is not limited thereto.

In one embodiment, the ligand alters the distribution, targeting or longevity of the iRNA agent into which it is introduced. In some embodiments, a ligand is directed against a selected target, e.g., a molecule, cell or cell type, compartment, e.g., cell or organ compartment, tissue, organ, or region of the body, compared to a species in which the ligand is absent. Provides enhanced affinity. Exemplary ligands do not participate in duplex pairing in duplexed nucleic acids.

A ligand may also include a targeting group, eg, a cell or tissue targeting agent, eg, a lectin, glycoprotein, lipid or protein, eg, an antibody that binds to a specific cell type, such as kidney cells. Targeting groups include thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, monovalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, multivalent fu course, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folates, vitamin B12, vitamin A, biotin, or RGD peptides or RGD peptide mimetics can be In certain embodiments, the ligand comprises monovalent or polyvalent galactose. In certain embodiments, the ligand includes cholesterol.

The ligand-conjugated oligonucleotides of the present invention can be synthesized by the use of oligonucleotides containing pendant reactive functional groups such as those derived from the attachment of a linking molecule onto the oligonucleotide (described below). The reactive oligonucleotides can react directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands with linking moieties attached thereto.

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

A. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate conjugated iRNAs are advantageous for in vivo delivery of nucleic acids as well as compositions suitable for therapeutic use in vivo as described herein. As used herein, “carbohydrate” refers to one or more monosaccharide units (which may be linear, branched or cyclic) having at least 6 carbon atoms with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. A compound that is a carbohydrate itself composed of; or having as part a carbohydrate moiety consisting of one or more monosaccharide units (which may be linear, branched or cyclic) having at least 6 carbon atoms each with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. mention the compound. Representative carbohydrates include sugars (mono-, di-, tri-, and oligosaccharides containing about 4, 5, 6, 7, 8 or 9 monosaccharide units) and polysaccharides such as starch, These include glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 or higher (eg , C5, C6, C7, or C8) sugars; Disaccharides and trisaccharides include sugars having two or three monosaccharide units (eg , C5, C6, C7, or C8).

In one embodiment, carbohydrate conjugates for use in the compositions and methods of the present invention are monosaccharides. In another embodiment, the carbohydrate conjugate for use in the compositions and methods of the present invention is selected from the group consisting of:

화학식 II,

formula II,

화학식 III,

formula III,

화학식 IV,

formula IV,

화학식 V,

formula V,

화학식 VI,

formula VI,

화학식 VII,

formula VII,

화학식 VIII,

formula VIII,

화학식 IX,

formula IX,

화학식 X,

formula X,

화학식 XI,

formula XI,

화학식 XII,

formula XII,

화학식 XIII,

formula XIII,

화학식 XIV,

formula XIV,

화학식 XV,

formula XV,

화학식 XVI,

formula XVI,

화학식 XVII,

formula XVII,

화학식 XVIII,

formula XVIII,

화학식 XIX,

formula XIX,

화학식 XX,

formula XX,

화학식 XXI,

formula XXI,

화학식 XXII.

Formula XXII.

In one embodiment, the monosaccharide is N-acetylgalactosamine, such as

화학식 II이다.

Formula II.

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to:

(화학식 XXIII), X 또는 Y 중 하나가 올리고뉴클레오타이드인 경우, 다른 하나는 수소이다.

(Formula XXIII), when one of X or Y is an oligonucleotide, the other is hydrogen.

In certain embodiments of the invention, GalNAc or a GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, GalNAc or a GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In another embodiment of the present invention, GalNAc or a GalNAc derivative is attached to an iRNA agent of the present invention via a trivalent linker.

In one embodiment, the double-stranded RNAi agent of the present invention comprises one GalNAc or GalNAc derivative attached to the iRNA agent. In another embodiment, the double-stranded RNAi agent of the invention comprises multiple (e.g. , 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently containing multiple monovalent linkers. It is attached to a number of nucleotides of the double-stranded RNAi agent via.

In some embodiments, for example, the two strands of an RNAi agent of the invention are linked by uninterrupted nucleotide chains between the 3'-end of one strand and the 5'-end of each other strand to form multiple pairs. When part of one larger molecule that forms a hairpin loop containing nucleotides that do not form a nucleotide, each unpaired nucleotide within the hairpin loop may independently contain a GalNAc or GalNAc derivative attached via a monovalent linker. can A hairpin loop may also be formed by an extended overhang on one strand of the duplex.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as but not limited to, PK modulators or cell penetrating peptides.

Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, each of which is incorporated herein by reference in its entirety.

B. linker

In some embodiments, a conjugate or ligand described herein may be attached to an iRNA oligonucleotide using a variety of linkers that may or may not be cleaved.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, eg, covalently attaches the two parts of a compound. Linkers typically include direct bonds or atoms, units such as oxygen or sulfur, such as NR8, C(O), C(O)NH, SO, SO ₂ , SO ₂ NH, or chains of atoms, and are now not limited to but for example, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroaryl Alkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, al kenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, Alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylal kenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclyl including alkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylene is O, S, S(O), SO ₂ , N(R8 ), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7 -17, 8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is a group that can be sufficiently stable outside the cell, but upon entry into the target cell is cleaved, releasing the two parts that the linker holds together. In one embodiment, the cleavable linking group is in the target cell or in a first, rather than in the subject's blood or under a second reference condition (eg, which can be selected to mimic or represent a condition found in blood or serum). at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more under reference conditions (which may be selected to mimic or represent intracellular conditions, for example) or at least about 100 times faster.

A cleavable linking group may be sensitive to the presence of a cleavage agent, eg pH, redox potential or degrading molecule. Generally, cleavage agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degradants include: redox agents selected for a particular substrate or having no substrate specificity, which are capable of redox cleavage, for example by oxidizing or reducing enzymes or reducing agents, for example reduction. redox agents including mercaptans present in cells capable of degrading linking groups; esterase; Agents that can create an endosome or an acidic environment, such as those that induce a pH of 5 or less; Enzymes capable of hydrolyzing or cleaving acid cleavable linking groups by acting as common acids, peptidases (which may be substrate specific), and phosphatases.

A cleavable linking group, such as a disulfide bond, may be pH sensitive. The pH of human serum is 7.4, and the average intracellular pH is slightly lower, about 7.1-7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH at about 5.0. Some linkers have cleavable linking groups that are cleaved at a selected pH to release the cationic lipid from the ligand inside the cell or to the desired compartment of the cell.

A linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker comprising an ester group. Liver cells are rich in esterases and therefore linkers are cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, neocortex and testis.

Linkers containing peptide bonds can be used when targeting peptidase-rich cell types such as liver cells and synovial cells.

In general, the suitability of a candidate cleavable linking group can be assessed by testing the ability of a degrading agent (or condition) to cleave the candidate linking group. It may also be desirable to test candidate cleavable linking groups for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, one skilled in the art can determine the relative susceptibility to cleavage between a first condition and a second condition, wherein the first condition is selected to exhibit cleavage in the target cell and the second condition is selected to exhibit cleavage in another tissue or biological fluid, such as For example, it is selected to show cleavage in blood or serum. Assessments can be performed in cell-free systems, cells, cell cultures, organ or tissue cultures, or whole animals. It may be useful to perform initial assessments in cell-free or cultured conditions and to confirm by further assessments in whole animals. In some embodiments, useful candidate compounds have a concentration of at least about 2, 4 in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions). , 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly.

i. Redox cleavable linking group

In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-S-S-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or suitable for use with, for example, a specific iRNA moiety and a specific targeting agent, one skilled in the art will look to the methods described herein. can For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rates observed in cells, eg, target cells. have. Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. In one, the candidate compound is cleaved in the blood by up to about 10%. In another embodiment, a useful candidate compound has a concentration of at least about 2, 4, 10 in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). , 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster. The rate of cleavage of a candidate compound can be determined using standard enzyme kinetic assays by comparing conditions selected to mimic the intracellular medium and conditions selected to mimic the extracellular medium.

ii. Phosphate-based cleavable linking groups

In certain embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. Tablesphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. Examples of agents that cleave phosphate groups in cells are enzymes such as intracellular phosphatases. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O-P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk) )-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O-P(S)(ORk)-S-, -S-P(S)(ORk)- O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)-O- , -S-P(O)(Rk)-S-, -O-P(S)(Rk)-S, wherein each Rk is independently C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7- C12 aralkyl. Exemplary embodiments include -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)- O-, -O-P(O)(OH)-S-, -S-P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O- , -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O, -S-P(S)(H)-O-, -S-P (O)(H)-S-, and -O-P(S)(H)-S-. In one embodiment, the phosphate based linking group is -O-P(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

iii. acid cleavable linking group

In certain embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, the acid cleavable linking group is an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5,75, 5.5, 5.25, 5.0 or less) or an agent such as an enzyme that can act as a common acid. is cut by Certain low pH organelles in cells, such as endosomes and lysosomes, can provide a cleavage environment for acid cleavable linking groups. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. An acid cleavable group can have the formula -C=NN-, C(O)O, or -OC(O). An exemplary embodiment is when the carbon (alkoxy group) attached to the oxygen of the ester is an aryl group, substituted alkyl group or tertiary alkyl group, such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

iv. Ester-based linking groups

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

v. Peptide-based cleavage group

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids, resulting in oligopeptides (eg, dipeptides, tripeptides, etc.) and polypeptides. A peptide-based cleavable group does not contain an amide group (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids, resulting in peptides and proteins. Peptide-based cleavage groups are generally limited to peptide bonds formed between the peptide and the amino acids that make up the protein (i.e., amide bonds), and do not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula NHCHRAC(O)NHCHRBC(O)-, where RA and Rb are R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

In one embodiment, an iRNA of the invention is conjugated to a carbohydrate via a linker. Non-limiting examples of linker and iRNA carbohydrate conjugates of the compositions and methods of the present invention include, but are not limited to:

(화학식 XXIV),

(Formula XXIV),

(화학식 XXV),

(Formula XXV),

(화학식 XXVI),

(formula XXVI),

(화학식 XXVII),

(Formula XXVII),

(화학식 XXVIII),

(Formula XXVIII),

(화학식 XXIX),

(Formula XXIX),

(화학식 XXX), 및

(Formula XXX), and

(화학식 XXXI),

(formula XXXI),

When either X or Y is an oligonucleotide, the other is hydrogen.

In certain embodiments of the compositions and methods of the present invention, the ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a divalent or trivalent side chain linker.

In one embodiment, a dsRNA of the invention is conjugated to a divalent or trivalent side chain linker selected from the group of structures represented by any of Formulas (XXXII) - (XXXV):

Formula XXXII Formula XXXIII

Formula XXXIV Formula XXXV

here:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C each independently represents 0 to 20, wherein the repeating units may be the same or different;

P ^2A , P ^2B , P ^3A , P ^3B , P ^4A , P ^4B , P ^5A , P ^5B , P ^5C , T ^2A , T ^2B , T ^3A , T ^3B , T ^4A , T ^4B , T ^4A , T ^5B , T ^5C are each independently absent or CO, NH, O, S, OC(O), NHC(O), CH ₂ , CH ₂ NH or CH ₂ O;

Q ^2A , Q ^2B , Q ^3A , Q ^3B , Q ^4A , Q ^4B , Q ^5A , Q ^5B , Q ^5C are each independently absent, alkylene, substituted alkylene, wherein at least one methylene is O, S , S(O), SO ₂ , N(R ^N ), C(R′)=C(R″), C≡C or C(O);

,

또는 헤테로사이클릴이고;R ^2A , R ^2B , R ^3A , R ^3B , R ^4A , R ^4B , R ^5A , R ^5B , R ^5C are each independently absent, or NH, O, S, CH ₂ , C(O)O, C( O) NH, NHCH (R ^a ) C (O), -C (O) -CH (R ^a ) -NH-, CO, CH=NO,

,

or heterocyclyl;

L ^2A , L ^2B , L ^3A , L ^3B , L ^4A , L ^4B , L ^5A , L ^5B and L ^5C are ligands, ie, each independently a monosaccharide (eg GalNAc), a disaccharide, a trisaccharide saccharides, tetrasaccharides, oligosaccharides or polysaccharides; R ^a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly useful for use with RNAi agents to inhibit expression of target genes, such as those of formula XXXVI:

Formula XXXVI

Here, L ^5A , L ^5B and L ^5C represent monosaccharides such as GalNAc derivatives.

Examples of suitable divalent and trivalent side chain linker groups for conjugating GalNAc derivatives include, but are not limited to, the structures set forth above as Formulas II, VII, XI, X, and XIII.

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

Not all positions of a given compound need be uniformly modified, and indeed one or more of the above modifications may be introduced into a single compound or even a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

VI. Delivery of the iRNA of the invention

Delivery of an iRNA of the invention to a cell, eg , a cell in a human subject (eg, a subject in need thereof, eg, a subject having a disease, disorder or condition associated with contact activation pathway gene expression) It can be accomplished in a number of different ways. For example, delivery can be performed by contacting a cell with an iRNA of the invention in vitro or in vivo. in vivo Delivery can also be performed directly by administering to a subject a composition comprising an iRNA, eg, a dsRNA. Delivery can be by, for example, intravenous or subcutaneous administration. In certain embodiments, iRNA preparations are delivered by subcutaneous administration. In certain embodiments, the iRNA preparation is administered by self-administration using a pre-filled syringe or auto-injector device.

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

VII. Pharmaceutical composition of the present invention

The invention also includes pharmaceutical compositions and formulations comprising the iRNAs described herein for use in the methods of the invention. In one embodiment, provided herein is a pharmaceutical composition containing an iRNA as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing the iRNA are useful for treating diseases or disorders associated with the expression or activity of the TTR gene. The pharmaceutical composition is formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery, eg, by subcutaneous (SC) or intravenous (IV) delivery. The pharmaceutical composition of the present invention can be administered in a dose sufficient to inhibit expression of the TTR gene. In one embodiment, an iRNA preparation, eg a dsRNA preparation, of the invention is formulated for subcutaneous administration in a pharmaceutically acceptable carrier.

In another embodiment, a single dose of the pharmaceutical composition is sustained over an extended period of time such that subsequent doses are administered at 1-month intervals, 1, 2, 3, or 4-month intervals or quarterly (about every 3-monthly) intervals. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once per month. In another embodiment of the present invention, a single dose of the pharmaceutical composition of the present invention is administered once every other month. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered quarterly, ie once every 3 months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every 4 months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every 5 months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every 6 months. In another embodiment, a single dose of the pharmaceutical composition of the present invention is administered once every 12 months.

VIII. kit

The invention also provides kits for performing any of the methods of the invention. Such kits include one or more double-stranded RNAi agent(s) and a label providing instructions for use of the double-stranded agent(s) for use in any method for the present invention. The kit optionally includes means for contacting cells with the RNAi agent (eg, an injection device or infusion pump), or means for measuring inhibition of TTR (eg, means for measuring inhibition of TTR mRNA or TTR protein). ) may be further included. Such means for determining inhibition of TTR may include means for obtaining a sample from a subject, such as, for example, a plasma sample. Kits of the present invention may optionally further include means for administering the RNAi agent(s) to a subject or means for determining a therapeutically effective amount or a prophylactically effective amount.

The RNAi preparation may be provided in any convenient form, such as sterile water solution or other suitable solution for resuspension and injection, such as PBS, physiological saline, 5 mM phosphate buffer. For example, the RNAi preparation may be provided in a 300 mg, 200 mg, 100 mg, or 50 mg vial with water or other suitable solution in sterile water for injection. In certain embodiments, the RNAi agent is provided as a kit for self-administration comprising a pre-filled syringe or autoinjector containing 300 mg, 200 mg, 100 mg, or 50 mg of the RNAi agent in an appropriate volume of an excipient for administration, optionally further comprising: Instructions for use are included. In certain embodiments, the RNAi agent may be provided in multiple vials or devices for administration of doses by multiple injections given at about the same time, eg, within 1 week, within 1 day, within 1 hour.

IX. TTR amyloidosis polyneuropathy diagnosis and disease burden assessment

TTR amyloidosis is a complex, multifactorial disease. An extended list of criteria was used to monitor the progression of TTR-FAP: Neuropathic Impairment Score (NIS), NIS + 7, and Modified NIS (mNIS) + 7 and mNIS + 7 _Ionis . These diagnostic criteria are well known in the art and highlights of the criteria are provided below. As used herein, meeting the diagnostic criteria for TTR-FAP is understood to be fulfilling the FAP stage 1 criteria regardless of the presence or absence of a mutation associated with hereditary TTR-FAP. Progression of the neuropathic index is considered as an increase of at least 2 points in the modified neuropathic impairment score (mNIS) + 7.

Familial Amyloid Polyneuropathy (FAP) Stage.

Coutinho et al. developed a clinical staging system for the neuropathic symptoms of hATTR (formerly called familial amyloid neuropathy). The scale ranges from 1 to 3 as follows (reference: Ando et al. Orphanet J Rare Dis. 2013;8:31): FAP stage 1: walking without assistance, mild neuropathy of the lower extremities (sensory, autonomic and Exercise).

FAP Stage 2: Moderate impairment of assisted walking, lower extremities, trunk, and upper extremities.

FAP stage 3: wheelchair or bed-bound, severe neuropathy.

Subjects without neuropathy are considered FAP stage 0.

Neuropathic damage scoring method

Methods for assessing neuropathy are known in the art. For example, even in the Mayo Clinic Neurologic Examination Sheet and the Weakness subscore of the Neuropathy Impairment Score (NIS), Weakness (NIS-W) is a 25% decrease from 1 to 4 points. , and separately scored for major muscle groups on each side of the body (see Dyck et al., Quantitating overall neuropathic symptoms, impairments, and outcomes. In: Dyck PJ, Thomas PK, editors. Peripheral neuropathy. 4th ed. Philadelphia: Elsevier; 2005. p. 1031-52). A broad group, particularly the cranial, proximal and distal limb muscles, is rated for a maximum score of 192 on the NIS-W. Decreases in the major five muscle stretch reflexes are commonly assessed by neurologists, and foot and hand touch, vibration, joint motion, and pin prick sensation are 25% on the Mayo Clinic Neurology Examination Sheet. Decrease rate and scored from 1 to 4. To complete the NIS of reflexes (NIS-R) and sensory (NIS-S), Mayo Clinic record scores are converted to NIS score scores (i.e., a Mayo Clinic score of 1 or 2 is assigned to a NIS score score of 1 and a Mayo Clinic score of 3 or 4 is assigned an NIS score of 2). Thus, the maximum NIS score for common reflexes assessed by neurologists (NIS-R) is 5 × 2 × 2 = 20 points, and the four sensory modalities commonly assessed by neurologists (NIS-S) are 8 × 2 × 2 2 = 32 points. Thus, the maximum NIS score is 192 + 20 + 32 = 244 points. NIS has been described in previous publications (Dyck et al. 2005 and Dyck et al., Neurol. 1997;49:229-39).

NIS + 7 has been used as the primary or co-primary outcome measure in trials of diabetic sensorimotor polyneuropathy, TTR FAP, and other generalized sensorimotor polyneuropathy (N. Suanprasert et al. J Neurol Sci). 344 (2014) 121-128). The NIS + 7 adequately assesses the graded severity of muscle weakness and muscle stretch reflex abnormalities with minimal ceiling effects on reflexes only. At NIS + 7, 5 out of 7 tests were characteristic of nerve conduction, expressed as normal deviations (Ζ scores) or scores. The features included in the NIS+7 were chosen because their abnormalities sensitively detect diabetic sensorimotor polyneuropathy (Dyck et al. Muscle Nerve 2003;27(2):202-10). Included characteristics were peroneal nerve complex muscle action potential (CMAP) amplitude, motor nerve conduction velocity (MNCV) and motor nerve distal latency (MNDL), tibial MNDL and sciatic sensory nerve action potential (SNAP) amplitudes. These measured values can be converted to normal deviations in percentage values correcting for the corresponding variables of age, sex, height or weight based on earlier studies of large healthy subject reference cohorts. Additionally, these percentage values can be expressed as NIS scores at the percentage values obtained (i.e., N5th = 0 points, ≤5th-N 1st = 1 point, and ≤1st = 2 points) (if the abnormality is in the upper tail of the normal distribution) similarly, if any).

Weakness and dysreflexia assessment, sensory loss assessment, autonomic dysfunction and neurophysiological examination abnormalities are not adequately assessed by the NIS + 7 for use in the TTR FAP test. Sensory loss in the NIS + 7 is not optimally assessed: 1) body distribution of sensory loss is not adequately considered, 2) large sensory loss is overemphasized compared to small fibers, and 3) test method is improved over clinical evaluation. and comparisons with reference values are preferred. Also, using heart rate deep breathing (HRdb) alone does not adequately assess autonomic dysfunction. The properties of nerve conduction used to assess NIS+7 are not ideal for TTR FAP studies.

An updated version of the Neuropathic Impairment Score +7 (mNIS+7) and NIS+7 are composite scores that measure motor strength, reflexes, sensation, nerve conduction and autonomic function. Two versions of this composite measure were adjusted at NIS+7 to better reflect hATTR amyloidosis with polyneuropathy and were used as primary outcomes in the innotersen and patisiran clinical trial. The main differences between these two versions and other neuropathic scoring systems are summarized in the table below (Adams et al., BMC Neurology, volume 17, Article number: 181 (2017)). A lower score on either scale indicates better nervous system function (eg, an increase in score reflects worsening nervous system damage).

1. CMAP compound muscle action potential; exam; mNIS+7 modified NIS+7; NIS neuropathy

damage score; NIS-LL but NIS based inspection; QST quantitative sensory test;

SNAP sensory nerve action potential

2. ^a Score (0-3.72) expressed as normal deviation based on the healthy-subject parameter

3. ^b Scores graded according to defined categories; Normal (95 percent) = 0 points; slightly decreased (≥95 to <99 percent) = 1 point; and very reduced (≥99 percent) = 2 points

4. ^c may also be referred to as fibula

Diagnosis and disease burden assessment of TTR amyloidotic polyneuropathy (ATTR-CM)

Patients with hATTR amyloidosis and cardiomyopathy typically experience progressive symptoms of heart failure (HF) and cardiac arrhythmias, and death typically occurs 2.5 to 5 years after diagnosis. Cardiac infiltration of the extracellular matrix by TTR amyloid fibrils causes progressive increases in ventricular wall thickness and marked increases in chamber stiffness, resulting in impaired diastolic function. Systolic function is also impaired and is typically reflected by abnormal longitudinal tension despite a normal ejection fraction, which is maintained until the end of the disease. Both longitudinal tension of brain natriuretic peptide (NT-proBNP) and N-terminal prohormone have been shown to be independent predictors of survival in patients with ATTR amyloidosis and light chain (AL) cardiac amyloidosis.

Echocardiography is routinely used to evaluate heart structure and function; Parameters pre-specified in the statistical analysis plan include mean left ventricular (LV) wall thickness, LV mass, longitudinal tension and ejection fraction. Cardiac output, left atrial volume, LV end-diastolic volume (LVEDV) and LV end-systolic volume (LVESV). Echocardiography is routinely used for heart imaging. Myocardial tone can be assessed by speckle tracking using vendor-independent software (TOMTEC, Munich, Germany). Analysis of NT-proBNP and troponin I levels is routinely performed in clinical laboratories using commercially available diagnostic tests, e.g., using chemiluminescence assays (Roche Diagnostic Cobas, Indianapolis, IN, USA for NT -proBNP; Siemens Centaur XP, Camberley, Surrey, UK for troponin I). Similarly, clinical practices routinely include measurements of creatinine levels and estimated glomerular filtration rate (eGFR) based on creatinine levels, for example using a variation of the renal disease study formula.

A review providing screening and diagnostic methods for ATTR-CM was recently published by Witteles et al., 2019 (JACC: Heart Failure, 2019. 7:709-716), which Diagnostic methods including a list of "red flags" suggesting the presence of echocardiography, electrocardiogram, cardiac magnetic resonance, presence of systemic symptoms related to the peripheral or autonomic nervous system and bilateral following an ascending pattern starting in the lower extremities screening methods including cardiac dysfunction including sensorimotor polyneuropathy, autonomic dystonia in the form of orthostatic hypotension, diarrhea/constipation and erectile dysfunction, and ocular involvement such as glaucoma, intravitreal deposition and scallop pupil; Provides information on screening methods for carpal tunnel syndromes, including bilateral carpal tunnel syndrome, lumbar spinal canal stenosis, and biceps tendon rupture. Other diagnostic methods include bone scintigraphy using technetium (Tc) labeled bisphosphonates, which for unknown reasons localize to TTR cardiac amyloid deposits. A biopsy is also used to confirm the presence of TTR amyloidosis in the heart.

Methods for assessment and classification of cardiac function of the parameters provided above are known in the art. As used herein, certain methods of assessment or classification of cardiac function refer to any clinically significant reduction in cardiac function such that standard of care includes medical intervention, e.g., administration of pharmacological agents, surgery. may be an acceptable standard.

Serum biomarkers as indicators of nerve damage and TTR amyloidosis progression

The diagnostic and monitoring methods described above are complex and often subjective. In addition, since TTR amyloidosis is rare and the signs and symptoms can be present in a number of other diseases, clinically validated non-invasive plasma biomarkers may facilitate early diagnosis and facilitate monitoring of disease progression. In a study by Ticau et al. (2019) (ref: www.medrxiv.org/content/10.1101/19011155v2.full.pdf), plasma levels of >1000 proteins were associated with hATTR amyloidosis with polyneuropathy. and in patients receiving placebo or patisiran in the Phase 3 APOLLO study (NCT01960348) and a cohort of healthy individuals. The effect of treatment with patisiran, a lipid formulated RNAi agent that inhibits hepatic expression of TTR, on the temporal profile of each protein was determined by a linear mixed model at 0, 9 and 18 months. Neurofilament light chain (NfL) protein was further evaluated using an orthogonal quantitative approach. Significant changes in the levels of 66 proteins were observed with patissiran compared to placebo, and the change in NfL, an indicator of nerve damage, was the most prominent. Analysis of changes in protein levels demonstrated that the proteome of patisiran-treated patients tended toward healthy individuals at 18 months. Plasma NfL levels in healthy controls were 4-fold lower than in patients with TTR amyloidosis with polyneuropathy (16.3 [SD 12.0] pg/mL vs. 69.4 [SD 42.1] pg/mL, p<10 ^-16 ). 18 months , levels of NfL increased with placebo (99.5 [SD 60.1] pg/mL) and decreased with patisiran treatment (48.8 [SD 29.9] pg/mL). At 18 months, improvement in modified neuropathic impairment score+7 (mNIS+7) in patisiran-treated patients significantly correlated with reduction in NfL level (R=0.43, p<10 ^-7 ) there is The reduction in NfL with patisiran treatment that correlated with improvement in mNIS+7 suggests that it may serve as a biomarker of nerve damage and polyneuropathy in TTR amyloidosis. Such biomarkers may enable early diagnosis of polyneuropathy in patients with hATTR amyloidosis and facilitate monitoring of disease progression.

Decreased levels of other proteins, particularly RSPO3, CCDC80, EDA2R and NT-proBNP, have been found to correlate with improvements in mNIS+7, either alone or in combination with each other or Nfl, indicating that they are associated with neuronal damage and polyneuropathy in ATTR amyloidosis. It suggests that it can act as a biomarker of pathology. Increases in N-CDase levels have been found to correlate with improvements in mNIS+7, which alone or in combination with the other markers listed above may serve as biomarkers of nerve damage and polyneuropathy in ATTR amyloidosis. suggests that there is

Additional potential biomarkers observed to change in response to treatment with patisiran are listed in the table below. If the subject has an increase in protein level with a positive beta coefficient relative to the reference level indicative of progression of ATTR amyloidosis and if the subject has a decrease in protein level with a negative beta coefficient relative to the reference level indicative of progression of ATTR amyloidosis. Changes in the level of one or more of these markers may indicate amelioration, stabilization, or reduction of ongoing nerve damage and polyneuropathy progression in ATTR amyloidosis.

[ Table 1 ]

Biomarkers with altered levels in response to patisiran treatment

Sequences for these biomarkers are provided herein as SEQ ID NOs: 17-34.

The invention is further illustrated by the following examples which should not be construed as limiting. The contents and sequence listings of all references, published patents and patent applications cited throughout this application are hereby incorporated by reference.

Example

Exemplary double-stranded RNAi agents for use in the methods of the present invention are provided in Table 3 below. Table 2 below provides abbreviations for nucleotide monomers and ligands used to provide nucleic acid sequences. When present in an oligonucleotide, these monomers are understood to be interconnected by 5'-3'-phosphodiester linkages unless otherwise indicated.

[Table 2]

[Table 3]

Example 1: TTR protein knockdown by RNAi agents in V30M transgenic mice

The V30M mutation is a common amyloidogenic mutation in human TTR. Transgenic mice lacking mouse TTR and expressing human TTR with the V30M mutation were used in the study. Based on the sequence and chemistry of AD-65492, the RNAi agent previously described in WO2018112320, or AD-65942 where the chemical modification at a single position of the antisense strand was changed to a GNA modification as shown in Table 2 above, mice (n = 3 per group) were injected. A single subcutaneous 1 mg/kg dose of different RNAi agents was administered. Blood samples were obtained on day 0 (pre-dose), 3, 7, 10, 14, 21, 35, and 49 days. Serum was prepared and human TTR levels were determined using an ELISA assay (see, eg , Coelho, et al . (2013) N Engl J Med 369:819). Relative TTR levels compared to day 0 are shown in FIG. 1 .

Incorporation of GNA at position 7 or 8 in the antisense strand was well tolerated (AD-87404 and AD-87405). Similar kinetics and maximal protein knockdown were similar to the parent RNAi agent AD-65492. The durability of knockdown by AD-87404 was similar to that of AD-65492. Incorporation of GNA at positions 3-6 in the antisense strand was less well tolerated.

Example 2: TTR protein knockdown by RNAi agents in non-human primates

The sequences of the iRNA preparations in Table 2 are completely cross-reactive with Cynomolgus monkey TTR. A single subcutaneous dose of AD-65492 (1 mg/kg) or AD-87404 (1 mg/kg or 3 mg/kg) was not effective in cynomolgus monkeys on day 0 (n=3 per group) in three separate studies. administered. Blood samples were variously collected on day -7 (7 days prior to dosing) over a total of 119 days as shown in FIG. 2 . Serum was prepared and Cynomolgus monkey TTR levels were determined using an ELISA assay (see, eg , Coelho, et al . (2013) N Engl J Med 369:819). Relative TTR levels compared to day 0 are shown in FIG. 2 . TTR knockdown was similar in monkeys dosed with 1 mg/kg of AD-65492 and 3 mg/kg of AD-87404.

Example 3: Administration of a Single Dose of AD-87404 in Healthy Human Subjects

In a phase I, randomized, single-blind, placebo-controlled study, AD-87404 (sense: 5'-usgsggauUfuCfAfUfguaaccaaga - 3' (SEQ ID NO: 6), wherein the L96 ligand is conjugated to the 3' strand of the sense strand) ; Antisense: 5′-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7)) is administered to healthy human volunteers at doses of 25 mg, 75 mg, 100 mg, 200 mg, or 400 mg, and the possible dose groups are 600 mg, 700 mg, 900 mg, and 1000 mg. Groups are balanced according to relevant demographic characteristics such as age, gender, and weight.

Demographically matched control subjects will also receive a single dose of placebo.

Plasma samples were collected and TTR protein levels in samples from subjects in the placebo group and subjects in all treatment groups were measured using an ELISA assay (see, e.g., Coelho, et al. (2013) N Engl J Med 369:819 ) at predetermined intervals, e.g., on days 1, 2, 3, 8, 15, 22, 29, 43, 57, 90, and then every 28 days for subjects in the active treatment group, TTR levels reached 80% of pre-treatment levels. % until recovery (up to about 1 year after maximal dose). The level and duration of the knockdown is determined.

Subjects in both the AD-87404 group and the control group are also monitored for side effects. Exemplary side effects monitored in the study include injection site erythema, injection site pain, pruritus, cough, nausea, fatigue and abdominal pain and physical examination, ECG, vital signs or clinical laboratory parameters such as renal function, hematological parameters, and clinically significant changes in liver function (eg, alanine aminotransferase (ALT), aspartate aminotransferase (AST)).

The results of this study demonstrate that a single subcutaneous dose of AD-87404 potently and consistently knocks down TTR protein levels in a dose dependent manner.

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

equivalent

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods of the disclosure described herein. Such equivalents are intended to be included within the scope of the following claims.

SEQUENCE LISTING <110> ALNYLAM PHARMACEUTICALS, INC. <120> COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF TRANSTHYRETIN (TTR) <130> 121301-12320 <140> <141> <150> 62/985,950 <151> 2020-03-06 <160> 34 <170> PatentIn version 3.5 <210> 1 <400> 1 000 <210> 2 <400> 2 000 <210> 3 <400> 3 000 <210> 4 <400> 4 000 <210> 5 <400> 5 000 <210> 6 <211> 21 <212> RNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 6 ugggauuuca uguaaccaag a 21 <210 > 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> source <223> /note= "Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide" <400> 7 ucuuggtuac augaaauccc auc 23 <210> 8 <211> 21 <212> RNA <213> Artificial Sequence <220> <221> source <223> /note= "Description of Artificial Sequence: Synthetic oligonucleotide" <400> 8 ugggauuuca uguaaccaag a 21 <210> 9 <211> 23 <212> RNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 9 ucuugguuac augaaauccc auc 23 <210> 10 <211> 21 <212> RNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 10 u gggauuuca uguaaccaag a 21 <210> 11 <211> 23 <212> RNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 11 ucuugguuac augaaauccc auc 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> source <223> /note="Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide" <400> 12 uctugguuac augaaauccc auc 23 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> source <223> /note="Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide" <400> 13 ucutgguuac augaaauccc auc 23 <210> 14 < 211> 23 <212> RNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 14 ucuugguuac augaaauccc auc 23 <210> 15 <211> 23 <212> RNA <213> Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> 15 ucuugguuac augaaauccc auc 23 <210> 16 <211> 23 <212> DNA <213 >Artificial Sequence <220> <221> source <223> /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> <221> source <223> /note="Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide" <400> 16 ucuuggutac augaaauccc auc 23 <210> 17 <211> 543 <212> PRT <213> Homo sapiens <400> 17 Met Ser Ser Phe Ser Tyr Glu Pro Tyr Tyr Ser Thr Ser Tyr Lys Arg 1 5 10 15 Arg Tyr Val Glu Thr Pro Arg Val His Ile Ser Ser Val Arg Ser Gly 20 25 30 Tyr Ser Thr Ala Arg Ser Ala Tyr Ser Ser Tyr Ser Ala Pro Val Ser 35 40 45 Ser Ser Leu Ser Val Arg Arg Ser Tyr Ser Ser Ser Ser Gly Ser Leu 50 55 60 Met Pro Ser Leu Glu Asn Leu Asp Leu Ser Gln Val Ala Ala Ile Ser 65 70 75 80 Asn Asp Leu Lys Ser Ile Arg Thr Gln Glu Lys Ala Gln Leu Gln Asp 85 90 95 Leu Asn Asp Arg Phe Ala Ser Phe Ile Glu Arg Val His Glu Leu Glu 100 105 110 Gln Gln Asn Lys Val Leu Glu Ala Glu Leu Leu Val Leu Arg Gln Lys 115 120 125 His Ser Glu Pro Ser Arg Phe Arg Ala Leu Tyr Glu Gln Glu Ile Arg 130 135 140 Asp Leu Arg Leu Ala Ala Glu Asp Ala Thr Asn Glu Lys Gln Ala Leu 145 150 155 160 Gln Gly Glu Arg Glu Gly Leu Glu Glu Thr Leu Arg Asn Leu Gln Ala 165 170 175 Arg Tyr Glu Glu Glu Val Leu Ser Arg Glu Asp Ala Glu Gly Arg Leu 180 185 190 Met Glu Ala Arg Lys Gly Ala Asp Glu Ala Ala Leu Ala Arg Ala Glu 195 200 205 Leu Glu Lys Arg Ile Asp Ser Leu Met Asp Glu Ile Ser Phe Leu Lys 210 215 220 Lys Val His Glu Glu Glu Ile Ala Glu Leu Gln Ala Gln Ile Gln Tyr 225 230 235 240 Ala Gln Ile Ser Val Glu Met Asp Val Thr Lys Pro Asp Leu Ser Ala 245 250 255 Ala Leu Lys Asp Ile Arg Ala Gln Tyr Glu Lys Leu Ala Ala Lys Asn 260 265 270 Met Gln Asn Ala Glu Glu Trp Phe Lys Ser Arg Phe Thr Val Leu Thr 275 280 285 Glu Ser Ala Ala Lys Asn Thr Asp Ala Val Arg Ala Ala Lys Asp Glu 290 295 300 Val Ser Glu Ser Arg Arg Leu Leu Lys Ala Lys Thr Leu Glu Ile Glu 305 310 315 320 Ala Cys Arg Gly Met Asn Glu Ala Leu Glu Lys Gln Leu Gln Glu Leu 325 330 335 Glu Asp Lys Gln Asn Ala Asp Ile Ser Ala Met Gln Asp Thr Ile Asn 340 345 350 Lys Leu Glu Asn Glu Leu Arg Thr Thr Lys Ser Glu Met Ala Arg Tyr 355 360 365 Leu Lys Glu Tyr Gln Asp Leu Leu Asn Val Lys Met Ala Leu Asp Ile 370 375 380 Glu Ile Ala Ala Tyr Arg Lys Leu Leu Glu Gly Glu Glu Thr Arg Leu 385 390 395 400 Ser Phe Thr Ser Val Gly Ser Ile Thr Ser Gly Tyr Ser Gln Ser Ser Ser 405 410 415 Gln Val Phe Gly Arg Ser Ala Tyr Gly Gly Leu Gln Thr Ser Ser Tyr 420 425 430 Leu Met Ser Thr Arg Ser Phe Pro Ser Tyr Tyr Thr Ser His Val Gln 435 440 445 Glu Glu Gln Ile Glu Val Glu Glu Thr Ile Glu Ala Ala Lys Ala Glu 450 455 460 Glu Ala Lys Asp Glu Pro Pro Ser Glu Gly Glu Ala Glu Glu Glu Glu 465 470 475 480 Lys Asp Lys Glu Glu Ala Glu Glu Glu Glu Ala Ala Glu Glu Glu Glu Glu 485 490 495 Ala Ala Lys Glu Glu Glu Glu Glu Glu Ala Lys Glu Glu Glu Glu Gly Gly 500 505 510 Glu Gly Glu Glu Gly Glu Glu Thr Lys Glu Ala Glu Glu Glu Glu Lys 515 520 525 Lys Val Glu Gly Ala Gly Glu Glu Gln Ala Ala Lys Lys Lys Asp 530 535 540 <210> 18 <211> 284 <212> PRT <213> Homo sapiens <400> 18 Met Ser Ser Phe Ser Tyr Glu Tyr Ser Thr Ser Tyr Lys Arg 1 5 10 15 Arg Tyr Val Glu Thr Pro Arg Val His Ile Ser Ser Val Arg Ser Gly 20 25 30 Tyr Ser Thr Ala Arg Ser Ala Tyr Ser Ser Tyr Ser Ala Pro Val Ser 35 40 45 Ser Ser Leu Ser Val Arg Arg Ser Tyr Ser Ser Ser Ser Gly Ser Leu 50 55 60 Met Pro Ser Leu Glu Asn Leu Asp Leu Ser Gln Val Ala Ala Ile Ser 65 70 75 80 Asn Asp Leu Lys Ser Ile Arg Thr Gln Glu Lys Ala Gln Leu Gln Asp 85 90 95 Leu Asn Asp Arg Phe Ala Ser Phe Ile Glu Arg Val His Glu Leu Glu 100 105 110 Gln Gln Asn Lys Val Leu Glu Ala Glu Leu Leu Val Leu Arg Gln Lys 115 120 125 His Ser Glu Pro Ser Arg Phe Arg Ala Leu Tyr Glu Gln Glu Ile Arg 130 135 140 Asp Leu Arg Leu Ala Ala Glu Asp Ala Thr Asn Glu Lys Gln Ala Leu 145 150 155 160 Gln Gly Glu Arg Glu Gly Leu Glu Glu Thr Leu Arg Asn Leu Gln Ala 165 170 175 Arg Tyr Glu Glu Glu Val Leu Ser Arg Glu Asp Ala Glu Gly Arg Leu 180 185 190 Met Glu Ala Arg Lys Gly Ala Lys Asn Thr Asp Ala Val Arg Ala Ala 195 200 205 Lys Asp Glu Val Ser Glu Ser Arg Arg Leu Leu Lys Ala Lys Thr Leu 210 215 220 Glu Ile Glu Ala Cys Arg Gly Met Asn Glu Ala Leu Glu Lys Gln Leu 225 230 235 240 Gln Glu Leu Glu Asp Lys Gln Asn Ala Asp Ile Ser Val Met Gln Asp 245 250 255 Thr Ile Asn Lys Leu Glu Asn Glu Leu Arg Thr Thr Lys Ser Glu Met 260 265 270 Ala Arg Tyr Leu Lys Glu Tyr Gln Asp Leu Leu Thr 275 280 <210> 19 <211> 272 <212> PRT <213> Homo sapiens <400> 19 Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Il e Leu Asn Phe Met 1 5 10 15 Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg 20 25 30 Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40 45 Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala 50 55 60 Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys 65 70 75 80 Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95 Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr 100 105 110 Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn 115 120 125 Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser 130 135 140 Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr 145 150 155 160 Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val 165 170 175 Arg Glu Ile Ile Gln His Pro Se r Ala Lys Gly Asn Leu Cys Pro Pro 180 185 190 Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys 195 200 205 Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn 210 215 220 Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser 225 230 235 240 Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys 245 250 255 Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His 260 265 270 <210> 20 <211> 203 <212> PRT <213> Homo sapiens <400> 20 Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys Pro Ser Gly Tyr Tyr 1 5 10 15 Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr Lys Cys Lys Ala Asp 20 25 30 Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr Lys Cys Lys Ser Gly 35 40 45 Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn Cys Pro Glu Gly Leu 50 55 6 0 Glu Ala Asn Asn His Thr Met Glu Cys Val Ser Ile Val His Cys Glu 65 70 75 80 Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr Lys Lys Gly Lys Thr 85 90 95 Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val Arg Glu Ile Ile Gln 100 105 110 His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro Thr Asn Glu Thr Arg 115 120 125 Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys Gly Glu Arg Gly Lys 130 135 140 Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn Lys Gly Glu Ser Lys 145 150 155 160 Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser Ser Lys Glu Ile Pro 165 170 175 Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys Arg Lys Val Gln Asp 180 185 190 Lys Gln Lys Ser Val Ser Val Ser Thr Val His 195 200 <210> 21 <211> 224 <212> PRT <213> Homo sapiens <400> 21 Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met 1 5 10 15 Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg 20 25 30 Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40 45 Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala 50 55 60 Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys 65 70 75 80 Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90 95 Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr 100 105 110 Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn 115 120 125 Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser 130 135 140 Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr 145 150 155 160 Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val 165 170 175 Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro 180 185 190 Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys 195 200 205 Gly Glu Arg Gly Arg Thr Ser Arg Gly Asp Glu Arg Cys Phe Asn His 210 215 220 <210> 22 <211> 553 <212> PRT <213> Homo sapiens <400> 22 Met Thr Trp Arg Met Gly Pro Arg Phe Thr Met Leu Leu Ala Met Trp 1 5 10 15 Leu Val Cys Gly Ser Glu Pro His Pro His Ala Thr Ile Arg Gly Ser 20 25 30 His Gly Gly Arg Lys Val Pro Leu Val Ser Pro Asp Ser Ser Arg Pro 35 40 45 Ala Arg Phe Leu Arg His Thr Gly Arg Ser Arg Gly Ile Glu Arg Ser 50 55 60 Thr Leu Glu Glu Pro Asn Leu Gln Pro Leu Gln Arg Arg Arg Ser Val 65 70 75 80 Pro Val Leu Arg Leu Ala Arg Pro Thr Glu Pro Pro Ala Arg Ser Asp 85 90 95 Ile Asn Gly Ala Ala Val Arg Pro Glu Gln Arg Pro Ala Ala Arg Gly 100 105 110 Ser Pro Arg Glu Met Ile Arg Asp Glu Gly Ser Ser Ala Arg Ser Arg 115 120 125 Met Leu Arg Phe Pro Ser Gly Ser Ser Ser Pro Asn Ile Leu Ala Ser 130 135 140 Phe Ala Gly Lys Asn Arg Val Trp Val Ile Ser Ala Pro His Ala Ser 145 150 155 160 Glu Gly Tyr Tyr Arg Leu Met Met Ser Leu Leu Lys Asp Asp Val Tyr 165 170 175 Cys Glu Leu Ala Glu Arg His Ile Gln Gln Ile Val Leu Phe His Gln 180 185 190 Ala Gly Glu Glu Gly Gly Lys Val Arg Arg Ile Thr Ser Glu Gly Gln 195 200 205 Ile Leu Glu Gln Pro Leu Asp Pro Ser Leu Ile Pro Lys Leu Met Ser 210 215 220 Phe Leu Lys Leu Glu Lys Gly Lys Phe Gly Met Val Leu Leu Lys Lys 225 230 235 240 Thr Leu Gln Val Glu Glu Arg Tyr Pro Tyr Pro Val Arg Leu Glu Ala 245 250 255 Met Tyr Glu Val Ile Asp Gln Gly Pro Ile Arg Arg Ile Glu Lys Ile 260 265 270 Arg Gln Lys Gly Phe Val Gln Lys Cys Lys Ala Ser Gly Val Glu Gly 275 280 285 Gln Val Val Ala Glu Gly Asn Asp Gly Gly Gly Gly Ala Gly Arg Pro 290 295 300 Ser Leu Gly Ser Glu Lys Lys Lys Glu Asp Pro Arg Arg Ala Gln Val 305 310 315 320 Pro Pro Thr Arg Glu Ser Arg Val Lys Val Leu Arg Lys Leu Ala Ala 325 330 335 Thr Ala Pro Ala Leu Pro Gln Pro Pro Ser Thr Pro Arg Ala Thr Thr 340 345 350 Leu Pro Pro Ala Pro Ala Thr Thr Val Thr Arg Ser Thr Ser Arg Ala 355 360 365 Val Thr Val Ala Ala Arg Pro Met Thr Thr Thr Ala Phe Pro Thr Thr Thr 370 375 380 Gln Arg Pro Trp Thr Pro Ser Pro Ser His Arg Pro Pro Thr Thr Thr 385 390 395 400 Glu Val Ile Thr Ala Arg Arg Pro Ser Val Ser Glu Asn Leu Tyr Pro 405 410 415 Pro Ser Arg Lys Asp Gln His Arg Glu Arg Pro Gln Thr Thr Arg Arg 420 425 430 Pro Ser Lys Ala Thr Ser Leu Glu Ser Phe Thr Asn Ala Pro Pro Thr 435 440 445 Thr Ile Ser Glu Pro Ser Thr Arg Ala Ala Gly Pro Gly Arg Phe Arg 450 455 460 Asp Asn Arg Met Asp Arg Arg Glu His Gly His Arg Asp Pro Asn Val 465 470 475 480 Val Pro Gly Pro Pro Lys Pro Ala Lys Glu Lys Pro Pro Lys Lys Lys 485 490 495 Ala Gln Asp Lys Ile Leu Ser Asn Glu Tyr Glu Glu Lys Tyr Asp Leu 500 505 510 Ser Arg Pro Thr Ala Ser Gln Leu Glu Asp Glu Leu Gln Val Gly Asn 515 520 525 Val Pro Leu Lys Lys Ala Lys Glu Ser Lys Lys His Glu Lys Leu Glu 530 535 540 Lys Pro Glu Lys Lys Lys Glu Lys Lys 545 550 <210> 23 <211> 331 <212> PRT <213> Homo sapiens <400> 23 Glu Gly Lys Arg Arg Leu Leu Leu Ile Thr Ala Pro Lys Ala Glu Asn 1 5 10 15 Asn Met Tyr Val Gln Gln Arg Asp Glu Tyr Leu Glu Ser Phe Cys Lys 20 25 30 Met Ala Thr Arg Lys Ile Ser Val Ile Thr Ile Phe Gly Pro Val Asn 35 40 45 Asn Ser Thr Met Lys Ile Asp His Phe Gln Leu Asp Asn Glu Lys Pro 50 55 60 Met Arg Val Val Asp Asp Glu Asp Leu Val Asp Gln Arg Leu Ile Ser 65 70 75 80 Glu Leu Arg Lys Glu Tyr Gly Met Thr Tyr Asn Asp Phe Phe Met Val 85 90 95 Leu Thr Asp Val Asp Leu Arg Val Lys Gln Tyr Tyr Glu Val Pro Ile 100 105 110 Thr Met Lys Ser Val Phe Asp Leu Ile Asp Thr Phe Gln Ser Arg Ile 115 120 125 Lys Asp Met Glu Lys Gln Lys Lys Glu Gly Ile Val Cys Lys Glu Asp 130 135 140 Lys Lys Gln Ser Leu Glu Asn Phe Leu Ser Arg Phe Arg Trp Arg Arg 145 150 155 160 Arg Leu Leu Val Ile Ser Ala Pro Asn Asp Glu Asp Trp Ala Tyr Ser 165 170 175 Gln Gln Leu Ser Ala Leu Ser Gly Gln Ala Cys Asn Phe Gly Leu Arg 180 185 190 His Ile Thr Ile Leu Lys Leu Leu Gly Val Gly Glu Glu Val Gly Gly 195 200 205 Val Leu Glu Leu Phe Pro Ile Asn Gly Gly Ser Val Val Glu Arg Glu 210 215 220 Asp Val Pro Ala His Leu Val Lys Asp Ile Arg Asn Tyr Phe Gln Val 225 230 235 240 Ser Pro Glu Tyr Phe Ser Met Leu Leu Val Gly Lys Asp Gly Asn Val 245 250 255 Lys Ser Trp Tyr Pro Ser Pro Met Trp Ser Met Val Ile Val Tyr Asp 260 265 270 Leu Ile Asp Ser Met Gln Leu Arg Arg Gln Glu Met Ala Ile Gln Gln 275 280 285 Ser Leu Gly Met Arg Cys Pro Glu Asp Glu Tyr Ala Gly Tyr Gly Tyr 290 295 300 His Ser Tyr His Gln Gly Tyr Gln Asp Gly Tyr Gln Asp Asp Tyr Arg 305 310 315 320 His His Glu Ser Tyr His His Gly Tyr Pro Tyr 325 330 <210> 24 <211> 85 <212> PRT <213> Homo sapiens <400> 24 Met Leu Leu Val Gly Lys Asp Gly Asn Val Lys Ser Trp Tyr Pro Ser 1 5 10 15 Pro Met Trp Ser Met Val Ile Val Tyr Asp Leu Ile Asp Ser Met Gln 20 25 30 Leu Arg Arg Gln Glu Met Ala Ile Gln Gln Ser Leu Gly Met Arg Cys 35 40 45 Pro Glu Asp Glu Tyr Ala Gly Tyr Gly Tyr His Ser Tyr His Gln Gly 50 55 60 Tyr Gln Asp Gly Tyr Gln Asp Asp Tyr Arg His His Glu Ser Tyr His 65 70 75 80 His Gly Tyr Pro Tyr 85 <210> 25 <211> 297 <212> PRT <213> Homo sapiens <400> 25 Met Asp Cys Gln Glu Asn Glu Tyr Trp Asp Gln Trp Gly Arg Cys Val 1 5 10 15 Thr Cys Gln Arg Cys Gly Pro Gly Gln Glu Leu Ser Lys Asp Cys Gly 20 25 30 Tyr Gly Glu Gly Gly Asp Ala Tyr Cys Thr Ala Cys Pro Pro Arg Arg 35 40 45 Tyr Lys Ser Ser Trp Gly His Ar g Cys Gln Ser Cys Ile Thr Cys 50 55 60 Ala Val Ile Asn Arg Val Gln Lys Val Asn Cys Thr Ala Thr Ser Asn 65 70 75 80 Ala Val Cys Gly Asp Cys Leu Pro Arg Phe Tyr Arg Lys Thr Arg Ile 85 90 95 Gly Gly Leu Gln Asp Gln Glu Cys Ile Pro Cys Thr Lys Gln Thr Pro 100 105 110 Thr Ser Glu Val Gln Cys Ala Phe Gln Leu Ser Leu Val Glu Ala Asp 115 120 125 Ala Pro Thr Val Pro Pro Gln Glu Ala Thr Leu Val Ala Leu Val Ser 130 135 140 Ser Leu Leu Val Val Phe Thr Leu Ala Phe Leu Gly Leu Phe Phe Leu 145 150 155 160 Tyr Cys Lys Gln Phe Phe Asn Arg His Cys Gln Arg Gly Gly Leu Leu 165 170 175 Gln Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu Ser Leu Phe Pro Val 180 185 190 Pro Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln Val Ser Glu Asn Ile 195 200 205 Phe Gln Thr Gln Pro Leu Asn Pro Ile Leu Glu Asp Asp Cys Ser Ser 210 215 220 Thr Ser Gly Phe Pro Thr Gln Glu Ser Phe Thr Met Ala Ser Cys Thr 225 230 235 240 Ser Glu Ser His Ser His Trp Val His Ser Pro Ile Glu Cys Thr Glu 245 250 255 Leu Asp Leu Gln Lys Phe Ser Ser Ser Ala Ser Tyr Thr Gly Ala Glu 260 265 270 Thr Leu Gly Gly Asn Thr Val Glu Ser Thr Gly Asp Arg Leu Glu Leu 275 280 285 Asn Val Pro Phe Glu Val Pro Ser Pro 290 295 <210 > 26 <211> 318 <212> PRT <213> Homo sapiens <400> 26 Met Asp Cys Gln Glu Asn Glu Tyr Trp Asp Gln Trp Gly Arg Cys Val 1 5 10 15 Thr Cys Gln Arg Cys Gly Pro Gly Gln Glu Leu Ser Lys Asp Cys Gly 20 25 30 Tyr Gly Glu Gly Gly Asp Ala Tyr Cys Thr Ala Cys Pro Pro Arg Arg 35 40 45 Tyr Lys Ser Ser Trp Gly His Arg Cys Gln Ser Cys Ile Thr Cys 50 55 60 Ala Val Ile Asn Arg Val Gln Lys Val Asn Cys Thr Ala Thr Ser Asn 65 70 75 80 Ala Val Cys Gly Asp Cys Leu Pro Arg Phe Tyr Arg Lys Thr Arg Ile 85 90 95 Gly Gly Leu Gln Asp Gln Glu Cys Ile Pro Cys Thr Lys Gln Thr Pro 100 105 110 Thr Ser Glu Val Gln Cys Ala Phe Gln Leu Ser Leu Val Glu Ala Asp 115 120 125 Ala Pro Thr Val Pro Pro Gln Glu Ala Thr Leu Val Ala Leu Val Ser 130 135 140 Ser Leu Leu Val Val Phe Thr Leu Ala Phe Leu Gly Leu Phe Phe Leu 145 150 155 160 Tyr Cys Lys Gln Phe Phe Asn Arg His Cys Gln Arg Glu Lys Leu Ile 165 170 175 Ile Phe Ser Asp Pro Val Pro Ala Ser Leu Asn Leu Ile Pro Glu Phe 180 185 190 Ala Gly Gly Leu Leu Gln Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu 195 200 205 Ser Leu Phe Pro Val Pro Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln 210 215 220 Val Ser Glu Asn Ile Phe Gln Thr Gln Pro Leu Asn Pro Ile Leu Glu 225 230 235 240 Asp Asp Cys Ser Ser Thr Ser Gly Phe Pro Thr Gln Glu Ser Phe Thr 245 250 255 Met Ala Ser Cys Thr Ser Glu Ser His Ser His Trp Val His Ser Pro 260 265 270 Ile Glu Cys Thr Glu Leu Asp Leu Gln Lys Phe Ser Ser Ser Ser Ala Ser 275 280 285 Tyr Thr Gly Ala Glu Thr Leu Gly Gly Asn Thr Val Glu Ser Thr Gly 290 295 300 Asp Arg Leu Glu Leu Asn Val Pro Phe Glu Val Pro Ser Pro 305 310 315 <210> 27 <211> 173 <212> PRT <213> Homo sapiens <400> 27 Met Ser Thr Gly Thr Asn Gly Asp Gly Val Ser Pro Ala Asn Gly Val 1 5 10 15 Val Leu Asp Arg Ser Tyr Pro Arg Cys Leu Pro Val Glu Leu Ser Gly 20 25 30 Gly Arg Tyr Thr His Ser Ala Pro Ser Gly Gly His Thr Cys Cys Thr 35 40 45 Gly Gly Leu Leu Gln Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu Ser 50 5 5 60 Leu Phe Pro Val Pro Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln Val 65 70 75 80 Ser Glu Asn Ile Phe Gln Thr Gln Pro Leu Asn Pro Ile Leu Glu Asp 85 90 95 Asp Cys Ser Ser Ser Thr Ser Gly Phe Pro Thr Gln Glu Ser Phe Thr Met 100 105 110 Ala Ser Cys Thr Ser Glu Ser His Ser His Trp Val His Ser Pro Ile 115 120 125 Glu Cys Thr Glu Leu Asp Leu Gln Lys Phe Ser Ser Ser Ala Ser Tyr 130 135 140 Thr Gly Ala Glu Thr Leu Gly Gly Asn Thr Val Glu Ser Thr Gly Asp 145 150 155 160 Arg Leu Glu Leu Asn Val Pro Phe Glu Val Pro Ser Pro 165 170 <210> 28 <211> 265 <212> PRT <213> Homo sapiens <400> 28 Met Ser Thr Gly Thr Asn Gly Asp Gly Val Ser Pro Ala Asn Gly Val 1 5 10 15 Val Leu Asp Arg Ser Tyr Pro Arg Ile Val Val Met Glu Arg Val Glu 20 25 30 Met Pro Thr Ala Gln Pro Ala Leu Leu Ala Gly Thr Lys Ala Ala Gly 35 40 45 Ala Thr Thr Asp Val Arg Val Ala Ser Pro Val Leu Ser Ser Ile Val 50 55 60 Phe Arg Arg Ser Thr Ala Gln Leu Pro Leu Met Leu Ser Val Gly Thr 65 70 75 80 Val Cys Pro Gly Ala Phe Gln Leu Ser Leu Val Glu Ala Asp Thr Pro 85 90 95 Thr Val Pro Pro Gln Glu Ala Thr Leu Val Ala Leu Val Ser Ser Leu 100 105 110 Leu Val Val Phe Thr Leu Ala Phe Leu Gly Leu Phe Phe Leu Tyr Cys 115 120 125 Lys Gln Phe Phe Asn Arg His Cys Gln Arg Val Ala Gly Gly Leu Leu 130 135 140 Gln Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu Ser Leu Phe Pro Val 145 150 155 160 Pro Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln Val Ser Glu Asn Ile 165 170 175 Phe Gln Thr Gln Pro Leu Asn Pro Ile Leu Glu Asp Asp Cys Ser Ser 180 185 190 Thr Ser Gly Phe Pro Thr Gln Glu Ser Phe Thr Met Ala Ser Cys Thr 195 200 205 Ser Glu Ser His Ser His Trp V al His Ser Pro Ile Glu Cys Thr Glu 210 215 220 Leu Asp Leu Gln Lys Phe Ser Ser Ser Ala Ser Tyr Thr Gly Ala Glu 225 230 235 240 Thr Leu Gly Gly Asn Thr Val Glu Ser Thr Gly Asp Arg Leu Glu Leu 245 250 255 Asn Val Pro Phe Glu Val Pro Ser Pro 260 265 <210> 29 <211> 107 <212> PRT <213> Homo sapiens <400> 29 Met Ser Thr Gly Thr Asn Gly Asp Gly Val Ser Pro Ala Asn Gly Val 1 5 10 15 Val Leu Asp Arg Ser Tyr Pro Arg Cys Leu Pro Val Glu Leu Ser Gly 20 25 30 Gly Arg Tyr Thr His Ser Ala Pro Ser Gly Gly His Thr Cys Cys Thr 35 40 45 Gly Gly Leu Leu Gln Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu Ser 50 55 60 Leu Phe Pro Val Pro Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln Val 65 70 75 80 Ser Trp Ala Pro Gly Ser Leu Ala Gln Leu Phe Ser Leu Asp Ser Val 85 90 95 Pro Ile Pro Gln Gln Gln Gln Gly Pro Glu Met 100 105 <210> 30 <211> 134 <212> PRT <213> Homo sapi ens <400> 30 Met Gly Thr Val Cys His Leu Pro Thr Val Trp Ser Trp Thr Gly Ala 1 5 10 15 Ile Gln Gly Ala Phe Gln Leu Ser Leu Val Glu Ala Asp Thr Pro Thr 20 25 30 Val Pro Pro Gln Glu Ala Thr Leu Val Ala Leu Val Ser Ser Leu Leu 35 40 45 Val Val Phe Thr Leu Ala Phe Leu Gly Leu Phe Phe Leu Tyr Cys Lys 50 55 60 Gln Phe Phe Asn Arg His Cys Gln Arg Val Ala Gly Gly Leu Leu Gln 65 70 75 80 Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu Ser Leu Phe Pro Val Pro 85 90 95 Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln Val Ser Trp Ala Pro Gly 100 105 110 Ser Leu Ala Gln Leu Phe Ser Leu Asp Ser Val Pro Ile Pro Gln Gln 115 120 125 Gln Gln Gly Pro Glu Met 130 <210> 31 <211> 132 <212> PRT <213> Homo sapiens <400> 31 Met Gly Thr Val Cys His Leu Pro Thr Val Trp Ser Trp Thr Gly Ala 1 5 10 15 Ile Gln Gly Ala Phe Gln Leu Ser Leu Val Glu Ala Asp Thr Pro Thr 20 25 30 Val Pro Pro Gln Glu Ala Thr Leu Val Ala Leu Val Ser Ser Leu Leu 35 40 45 Val Val Phe Thr Leu Ala Phe Leu Gly Leu Phe Phe Leu Tyr Cys Lys 50 55 60 Gln Phe Phe Asn Arg His Cys Gln Arg Gly Gly Leu Leu Gln Phe Glu 65 70 75 80 Ala Asp Lys Thr Ala Lys Glu Glu Ser Leu Phe Pro Val Pro Pro Ser 85 90 95 Lys Glu Thr Ser Ala Glu Ser Gln Val Ser Trp Ala Pro Gly Ser Leu 100 105 110 Ala Gln Leu Phe Ser Leu Asp Ser Val Pro Ile Pro Gln Gln Gln Gln 115 120 125 Gly Pro Glu Met 130 <210> 32 <211> 299 <212> PRT <213> Homo sapiens <400> 32 Met Asp Cys Gln Glu Asn Glu Tyr Trp Asp Gln Trp Gly Arg Cys Val 1 5 10 15 Thr Cys Gln Arg Cys Gly Pro Gly Gln Glu Leu Ser Lys Asp Cys Gly 20 25 30 Tyr Gly Glu Gly Gly Asp Ala Tyr Cys Thr Ala Cys Pro Pro Arg Arg 35 40 45 Tyr Lys Ser Ser Trp Gly His Arg Cys Gln Ser Cys Ile Thr Cys 50 55 60 Ala Val Ile Asn Arg Val Gln Lys Val Asn Cys Thr Ala Thr Ser Asn 65 70 75 80 Ala Val Cys Gly Asp Cys Leu Pro Arg Phe Tyr Arg Lys Thr Arg Ile 85 90 95 Gly Gly Leu Gln Asp Gln G lu Cys Ile Pro Cys Thr Lys Gln Thr Pro 100 105 110 Thr Ser Glu Val Gln Cys Ala Phe Gln Leu Ser Leu Val Glu Ala Asp 115 120 125 Thr Pro Thr Val Pro Pro Gln Glu Ala Thr Leu Val Ala Leu Val Ser 130 135 140 Ser Leu Leu Val Val Phe Thr Leu Ala Phe Leu Gly Leu Phe Phe Leu 145 150 155 160 Tyr Cys Lys Gln Phe Phe Asn Arg His Cys Gln Arg Val Ala Gly Gly 165 170 175 Leu Leu Gln Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu Ser Leu Phe 180 185 190 Pro Val Pro Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln Val Ser Glu 195 200 205 Asn Ile Phe Gln Thr Gln Pro Leu Asn Pro Ile Leu Glu Asp Asp Cys 210 215 220 Ser Ser Thr Ser Gly Phe Pro Thr Gln Glu Ser Phe Thr Met Ala Ser 225 230 235 240 Cys Thr Ser G lu Ser His Ser His Trp Val His Ser Pro Ile Glu Cys 245 250 255 Thr Glu Leu Asp Leu Gln Lys Phe Ser Ser Ser Ala Ser Tyr Thr Gly 260 265 270 Ala Glu Thr Leu Gly Gly Asn Thr Val Glu Ser Thr Gly Asp Arg Leu 275 280 285 Glu Leu Asn Val Pro Phe Glu Val Pro Ser Pro 290 295 <210> 33 <211> 297 <212> PRT <213> Homo sapiens <400> 33 Met Asp Cys Gln Glu Asn Glu Tyr Trp Asp Gln Trp Gly Arg Cys Val 1 5 10 15 Thr Cys Gln Arg Cys Gly Pro Gly Gln Glu Leu Ser Lys Asp Cys Gly 20 25 30 Tyr Gly Glu Gly Gly Asp Ala Tyr Cys Thr Ala Cys Pro Pro Arg Arg 35 40 45 Tyr Lys Ser Ser Trp Gly His Arg Cys Gln Ser Cys Ile Thr Cys 50 55 60 Ala Val Ile Asn Arg Val Gln Lys Val Asn Cys Thr Ala Thr Ser Asn 65 70 75 80 Ala Val Cys Gly Asp Cys Leu Pro Arg Phe Tyr Arg Lys Thr Arg Ile 85 90 95 Gly Gly Leu Gln Asp Gln Glu Cys Ile Pro Cys Thr Lys Gln Thr Pro 100 105 110 Thr Ser Glu Val Gln Cys Ala Phe Gln Leu Ser Leu Val Glu Ala Asp 115 120 125 Thr Pro Thr Val Pro Pro Gln Glu Ala Thr Leu Val Ala Leu Val Ser 130 135 140 Ser Leu Leu Val Val Phe Thr Leu Ala Phe Leu Gly Leu Phe Phe Leu 145 150 155 160 Tyr Cys Lys Gln Phe Phe Asn Arg His Cys Gln Arg Gly Gly Leu Leu 165 170 175 Gln Phe Glu Ala Asp Lys Thr Ala Lys Glu Glu Ser Leu Phe Pro Val 180 185 190 Pro Pro Ser Lys Glu Thr Ser Ala Glu Ser Gln Val Ser Glu Asn Ile 195 200 205 Phe Gln Thr Gln Pro Leu Asn Pro Ile Leu Glu Asp Asp Cys Ser Ser Ser 210 215 220 Thr Ser Gly Phe Pro Thr Gln Glu Ser Phe Thr Met Ala Ser Cys Thr 225 230 235 240 Ser Glu Ser His Ser His Trp Val His Ser Pro Ile Glu Cys Thr Glu 245 250 255 Leu Asp Leu Gln Lys Phe Ser Ser Ser Ser Ala Ser Tyr Thr Gly Ala Glu 260 265 270 Thr Leu Gly Gly Asn Thr Val Glu Ser Thr Gly Asp Arg Leu Glu Leu 275 280 285 Asn Val Pro Phe Glu Val Pro Ser Pro 290 295 <210> 34 <211> 134 <212> PRT <213> Homo sapiens <400> 34 Met Asp Pro Gln Thr Ala Pro Ser Arg Ala Leu Leu Leu Leu Leu Leu Phe 1 5 10 15 Leu His Leu Ala Phe Leu Gly Gly Arg Ser His Pro Leu Gly Ser Pro 20 25 30 Gly Ser Ala Ser Asp Leu Glu Thr Ser Gly Leu Gln Glu Gln Arg Asn 35 40 45 His Leu Gln Gly Lys Leu Ser Glu Leu Gln Val Glu Gln Thr Ser Leu 50 55 60 Glu Pro Leu Gln Glu Ser Pro Arg Pro Thr Gly Val Trp Lys Ser Arg 65 70 75 80 Glu Val Ala Thr Glu Gly Ile Arg Gly His Arg Lys Met Val Leu Tyr 85 90 95 Thr Leu Arg Ala Pro Arg Ser Pro Lys Met Val Gln Gly Ser Gly Cys 100 105 110 Phe Gly Arg Lys Met Asp Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys 115 120 125 Lys Val Leu Arg Arg His 130

Claims (51)

A double-stranded RNAi agent comprising a sense strand and an antisense strand, wherein
wherein each of the sense strand and antisense strand is independently 30 nucleotides or less in length;
the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6);
wherein the antisense strand comprises the modified nucleotide sequence 5′-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7);
Here, a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, and 2'-O-methylguanosine-3', respectively. -phosphate, and 2'-O-methyluridine-3'-phosphate;
Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2, respectively. '-fluorouridine-3'-phosphate;
(Tgn) is the thymidine-glycol nucleic acid (GNA) S-isomer;
and s is a phosphorothioate linker. The RNAi agent of claim 1 , wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. 3. The RNAi agent of claim 2, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent side chain linker. 4. The method of claim 3, wherein the ligand

Phosphorus, RNAi agent. The RNAi ligand according to any one of claims 2 to 4, wherein the ligand is attached to the 3' end of the sense strand. 6. The method of claim 5, wherein the double-stranded RNAi agent is conjugated to a ligand as shown in the scheme below,

wherein X is O or S. 7. The RNAi agent according to any one of claims 1 to 6, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. Use of an RNAi agent in a method of treating a human subject suffering from a TTR-related disease, wherein the method comprises administering to the subject a fixed dose of 25 mg to 1000 mg of a double-stranded RNAi agent comprising a sense strand and an antisense strand. include, where
wherein each of the sense strand and antisense strand is independently 30 nucleotides or less in length;
the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6);
wherein the antisense strand comprises the modified nucleotide sequence 5′-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7);
Here, a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, and 2'-O-methylguanosine-3', respectively. -phosphate, and 2'-O-methyluridine-3'-phosphate;
Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2, respectively. '-fluorouridine-3'-phosphate;
(Tgn) is the thymidine-glycol nucleic acid (GNA) S-isomer;
s is a phosphorothioate linker. Use of an RNAi agent in a method of inhibiting the expression of TTR in a human subject who does not meet the criteria for diagnosis of a TTR-related disease, wherein the method comprises fixing 25 mg to 1000 mg of a double-stranded RNAi agent comprising a sense strand and an antisense strand administering the dose to the subject, wherein
wherein each of the sense strand and antisense strand is independently 30 nucleotides or less in length;
the sense strand comprises the modified nucleotide sequence 5'-usgsggauUfuCfAfUfguaaccaaga-3' (SEQ ID NO: 6);
wherein the antisense strand comprises the modified nucleotide sequence 5′-usCfsuugGf(Tgn)uAfcaugAfaAfucccasusc-3′ (SEQ ID NO: 7);
Here, a, c, g, and u are 2'-O-methyladenosine-3'-phosphate, 2'-O-methylcytidine-3'-phosphate, and 2'-O-methylguanosine-3', respectively. -phosphate, and 2'-O-methyluridine-3'-phosphate;
Af, Cf, Gf, and Uf are 2'-fluoroadenosine-3'-phosphate, 2'-fluorocytidine-3'-phosphate, 2'-fluoroguanosine-3'-phosphate, and 2, respectively. '-fluorouridine-3'-phosphate;
(Tgn) is the thymidine-glycol nucleic acid (GNA) S-isomer;
s is a phosphorothioate linker. 10. The use according to claim 8 or 9, wherein the sense strand of the double-stranded RNAi agent is conjugated to at least one ligand. 11. Use according to claim 10, wherein the ligand is one or more GalNAc derivatives attached via a bivalent or trivalent side chain linker. 12. The method of claim 11, wherein the ligand

person, use. 13. The use according to any one of claims 9 to 12, wherein the ligand is attached to the 3' end of the sense strand. 14. The method of claim 13, wherein the double-stranded RNAi agent is conjugated to a ligand as shown in the scheme below,

wherein X is O or S. 15. The use according to any one of claims 8 to 14, wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. 16. The use according to any one of claims 8 to 15, wherein the method comprises improving at least one indicator of nervous system damage, quality of life, ongoing neurological damage, or cardiovascular damage. 17. The use according to claim 16, wherein the indicator is a nervous system damage indicator. The method of claim 17, wherein the neuropathic impairment index is a neuropathic impairment (NIS) score, a modified NIS (mNIS+7) score, a NIS-W score, a composite autonomic symptom score (COMPASS-31), and a median body mass index (mBMI). A change from baseline in an index selected from the group of score, 6-minute walk test (6MWT) score, and 10-meter walk test score. 17. The use according to claim 16, wherein the indicator is a quality of life indicator. 20. The method of claim 19, wherein the quality of life index is SF-36® Health Survey score, Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) score, and Rasch-Constructed Total Disability Scale (R-ODS) score Use, which is a change from baseline in an indicator selected from the group of 17. The use according to claim 16, wherein the indicator is ongoing nerve damage. 22. The method of claim 21, wherein the indicator of ongoing nerve damage is a change from baseline in plasma protein levels of one or more proteins selected from the group of neurofilament light chain (NfL), RSPO3, CCDC80, EDA2R, NT-proBNP, and N-CDase. , purpose. 22. The use according to claim 21, wherein the indicator of ongoing nerve damage is a change in plasma levels of neurofilament light chain (NfL) protein levels. 17. The use according to claim 16, wherein the indicator is an indicator of cardiovascular damage. 25. The method of claim 24, wherein the indices of cardiovascular damage are determined by cardiovascular hospital admissions, change from baseline using the Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS) with an increased score indicating better health status, echocardiographic evaluation. change from baseline in mean left ventricular (LV) wall thickness by echocardiographic assessment, change from baseline in global longitudinal tension by echocardiographic assessment, and change from baseline in the N-terminal prohormone type B natriuretic peptide (NTproBNP), purpose. 26. The use according to any one of claims 8 to 25, wherein the human subject has a TTR gene mutation associated with development of a TTR related disease. 27. The method according to any one of claims 8 to 26, wherein the TTR-related disease is senile systemic amyloidosis (SSA), familial systemic amyloidosis, familial amyloidogenic polyneuropathy (FAP), familial amyloidogenic cardiomyopathy (FAC) ), leptomeningeal/central nervous system (CNS) amyloidosis, hyperthyroxinemia and cardiac amyloidosis. 26. The use of any one of claims 8-25, wherein the human subject has transthyretin-mediated amyloidosis (ATTR amyloidosis) and the use of the RNAi formulation method reduces amyloid TTR deposits in the human subject. . 29. The use according to claim 28, wherein the ATTR is a dielectric ATTR (h-ATTR). 29. The use according to claim 28, wherein the ATTR is a non-heritable ATTR (wt ATTR). 31. The use according to any one of claims 8 to 30, wherein the double-stranded RNAi agent is administered to the human subject by subcutaneous or intravenous administration. 32. The use according to claim 31, wherein the subcutaneous administration is self-administration. 33. The use of claim 32, wherein the self-administration is via a pre-filled syringe or an auto-injecting syringe. 34. The use of any one of claims 8-33, further comprising assessing the level of TTR mRNA expression or TTR protein expression in a sample derived from the human subject. 35. The use according to any one of claims 8 to 34, wherein the double-stranded RNAi agent is administered to the human subject once every 3 months to once a year. 36. The use of any one of claims 8-35, wherein the fixed dose of the double-stranded RNAi agent is administered to the human subject once about every 3 months, once about every 6 months, or once a year . 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 25 mg to 300 mg once every 3 months. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 25 mg to 200 mg once every 3 months. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 75 mg to 200 mg once every 3 months. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 25 mg once every 3 months. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 75 mg once every 3 months. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 100 mg once every 3 months. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 200 mg once every 3 months. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 300 mg once every 3 months. 36. The method of any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg to 600 mg once every 6 months to once every 12 months. to be, use. 36. The method of any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg to 600 mg once every 6 months to once every 12 months. to be, use. 36. The method of any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 400 mg or 600 mg once every 6 months to once every 12 months. to be, use. 36. The use according to any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 700 mg to 1000 mg once every 12 months. 36. The method of any one of claims 8 to 35, wherein the fixed dose double-stranded RNAi agent is administered to the human subject at a fixed dose of 700 mg, 800 mg, 900 mg or 1000 mg once every 12 months. to be, use. 50. The use of any one of claims 8-49, further comprising administering to the human subject an additional therapeutic agent. 51. The use of claim 50, wherein the additional therapeutic agent is a TTR tetramer stabilizer or a non-steroidal anti-inflammatory agent.

Images