RNAI AGENTS, COMPOSITIONS, METHOD OF INHIBITING EXPRESSION OF A TRANSTIRRETIN (TTR), USE OF THESE AG

专利摘要:
RNAI AGENTS, COMPOSITIONS, CELL, METHOD OF INHIBITING EXPRESSION OF A TRANSTIRRETIN (TTR), USE OF THESE AGENTS, AND KITS. The present invention provides RNAi agents, for example, double-stranded RNAi agents, which target the transthyretin (TTR) gene and methods of using such RNAi agents for the treatment or prevention of diseases associated with TTR.
公开号:BR112014011896B1
申请号:R112014011896-5
申请日:2012-11-16
公开日:2021-01-19
发明作者:Klaus Charisse；Rajev G. Kallanthottathil；Tracy Zimmermann；Muthiah Manoharan；Martin Maier；Satyanarayana KUCHIMANCHI
申请人:Alnylam Pharmaceuticals, Inc.；
IPC主号:

专利说明:

Related patent applications
[0001] This patent application claims priority over US Provisional Patent Application No. 61 / 561,710, filed on November 18, 2011, US Provisional Patent Application No. 61 / 615,618, filed on March 26, 2012 , and US Provisional Patent Application No. 61 / 680,098, filed on August 6, 2012, the entire contents of each of which are hereby incorporated into this application by reference. Sequence listing
[0002] The immediate patent application contains a Sequence Listing that was submitted in ASCII format through EFS-web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on November 13, 2012, is called 121301WO.txt and is 541,508 bytes in size. Background of the Invention
[0003] Transthyretin (TTR) (also known as pre-albumin) is found in serum and cerebrospinal fluid (CSF). TTR carries the retinol-binding protein (RBP) and thyroxine (T4) and also acts as a retinol (vitamin A) vehicle for its association with RBP in the blood and CSF. Transthyretin is called its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apoA-I (the main apolipoprotein of HDL), β-amyloid peptide, and neuropeptide Y. See Liz, M.A. et al. (2010) IUBMB Life, 62 (6): 429-435.
[0004] TTR is a tetramer of four identical 127 amino acid subunits (monomers) that are rich in the beta leaf structure. Each monomer has two 4-filament beta sheets and is shaped like an elongated ellipsoid. Antiparallel beta-leaf interactions bind monomers to dimers. A short loop of each monomer forms the main dimer-dimer interaction. These two pairs of handles separate the opposing, convex beta sheets from the dimers to form an internal channel.
[0005] The liver is the main site of TTR expression. Other significant sites of expression include the choroid plexus, retina (particularly the retinal pigment epithelium) and pancreas.
[0006] Transthyretin is one of at least 27 distinct types of proteins that is a precursor protein in the formation of amyloid fibrils. See Guan, J. et al. (November 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi: 10.1152 / ajpheart.00815.2011. The extracellular deposition of amyloid fibrils in organs and tissues is the hallmark of amyloidosis. Amyloid fibrils are composed of protein aggregates with folding errors, which can result from excessive production or specific mutations in precursor proteins. The amyloidogenic potential of TTR may be related to its extensive beta-leaf structure; X-ray crystallographic studies indicate that certain amyloidogenic mutations destabilize the tetrameric structure of the protein. See, for example, Saraiva M.J.M. (2002) Expert Reviews in Molecular Medicine, 4 (12): 1-11.
[0007] Amyloidosis is a general term in the group of amyloid diseases that are characterized by amyloid deposits. Amyloid diseases are classified based on their precursor protein; for example, the name starts with "A" for amyloid and is followed by an abbreviation for the precursor protein, for example, ATTR for amyloidogenic transthyretin. Ibid.
[0008] There are numerous diseases associated with TTRs, most of which are amyloid diseases. Normal-sequence TTR is associated with cardiac amyloidosis in people who are 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 manifests itself in several forms. When the peripheral nervous system is most prominently affected, the disease is called familial amyloidotic polyneuropathy (FAP). When the heart is mainly involved but the nervous system is not, the disease is called familial amyloid cardiomyopathy (FAC). The third main type of TTR amyloidosis is leptomeningeal amyloidosis, also known as leptomeningeal or meningocerebrovascular amyloidosis, central nervous system (CNS) amyloidosis or form VII amyloidosis. TTR mutations can also cause amyloidtic vitreous opacities, carpal tunnel syndrome and euthyroid hyperthyroxinemia, which is a non-amyloid disease thought to be secondary to an increased association of thyroxine with TTR due to a mutant TTR molecule with increased affinity for thyroxine. See, for example, Moisés et al. (1982) J. Clin. Invest., 86, 2025-2033.
[0009] Abnormal amyloidogenic proteins can be inherited or acquired by somatic mutations. Guan, J. et al. (November 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi: 10.1152 / ajpheart.00815.2011. ATTR associated with transthyretin is the most frequent form of hereditary systemic amyloidosis. Lobato, L. (2003) J. Nephrol., 16: 438-442. TTR mutations accelerate the process of amyloid formation by TTR and are the most important risk factor for the development of ATTR. More than 85 amyloidogenic TTR variants are known to cause familial systemic amyloidosis. TTR mutations usually give rise to systemic amyloid deposition, with particular involvement of the peripheral nervous system, although some mutations are associated with cardiomyopathy or vitreous opacity. Ibid.
[00010] The V30M mutation is the most prevalent TTR mutation. See, for example, Lobato, L. (2003) J Nephrol, 16: 438-442. The V122I mutation is carried by 3.9% of the African American population and is the most common cause of FAC. Jacobson, D.R. et al. (1997) N. Engl. J. Med. 336 (7): 466-73. SSA is estimated to affect more than 25% of the population over 80 years of age. Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87 (7): 2843-5.
[00011] Consequently, there is a need in the art for effective treatments of diseases associated with TTRs. Summary of the Invention
[00012] The present invention provides RNAi agents, for example, double-stranded RNAi agents, targeting the Transtirretin (TTR) gene. The present invention also provides methods of inhibiting TTR expression and methods of treating or preventing a disease associated with TTR in an individual using RNAi agents, for example, double-stranded RNAi agents, of the invention. The present invention is based, at least in part, on the discovery that RNAi agents that comprise particular chemical modifications show a superior ability to inhibit TTR expression. Agents including a certain model of chemical modifications (for example, an alternating model) and a linker in this application are effective in silencing the activity of the TTR gene. In addition, agents including one or more motifs of three identical modifications in three consecutive nucleotides, including one such motif within or near the agent's cleavage site, show surprisingly increased TTR gene silencing activity. When a single such chemical motif is present in the agent, it is preferable to be within or close to the cleavage region to increase gene silencing activity. The cleavage region is the region that surrounds the cleavage site, that is, the site on the target mRNA into which the cleavage occurs.
[00013] Consequently, in one aspect, the present invention features RNAi agents, for example, double-stranded RNAi agents, to inhibit the expression of a transthyretin (TTR). The double-stranded RNAi agent includes a complementary sense strand in an antisense strand. The antisense tape includes a complementary region in a part of an mRNA that encodes transthyretin. Each strand has 14 to 30 nucleotides and the double stranded RNAi agent is represented by the formula (III): sense: 5'np-Na- (XXX) i-Nb-YYY-Nb- (ZZZ) j-Na-nq3 'antisense: 3'np'-Na' - (X'X'X ') k-Nb'-Y'Y'Y'-Nb' - (Z'Z'Z ') l-Na'-nq'5 '(III).
[00014] In Formula III, i, j, k, and l are each independently 0 or 1; p, p ', q, and q' are each independently 0-6; each Na and Na 'independently represent a sequence of oligonucleotides including 0-25 nucleotides that are modified or unmodified or combinations thereof, each sequence including at least two differently modified nucleotides; each Na and Na 'independently represents a sequence of oligonucleotides including 0-10 nucleotides that are modified or unmodified or combinations thereof; each np, np ', nq, and nq' independently represents a lump nucleotide; XXX, YYY, ZZZ, X'X'X ', Y’Y'Y', and Z'Z'Z 'each independently represent a motif of three identical modifications in three consecutive nucleotides; changes in Na differ from changes in Y and changes in Na 'differ from changes in Y'. In some embodiments, the sense strand is conjugated to at least one linker, for example, at least one linker, for example, at least one linker attached to the 3 'end of the sense tape. In other embodiments, the ligand can be attached to the antisense tape.
[00015] In some embodiments, i is 1; j is 1; or both i and j are 1.
[00016] In some embodiments, k is 1; l is 1; or both k and l are 1.
[00017] In some embodiments, i is 0; j is 1.
[00018] In some embodiments, i is 1, j is 0.
[00019] In some embodiments, k is 0; l is 1.
[00020] In some embodiments, k is 1; l is 0.
[00021] In some embodiments, XXX is complementary to X'X'X ', YYY is complementary to Y’Y’Y', and ZZZ is complementary to Z’Z’Z '.
[00022] In some embodiments, the YYY motif occurs within or near the cleavage site of the sense tape.
[00023] In some embodiments, the Y'Y'Y 'motif occurs at positions 11, 12 and 13 of the 5' end antisense tape.
[00024] In some embodiments, Y 'is 2'-O-methyl.
[00025] In some embodiments, Y 'is 2'-fluoro.
[00026] In some modalities, the formula (III) is represented according to the formula (IIIa): sense: 5 'np-Na-YYY-Nb-ZZZ-Na-nq 3' antisense: 3 'np'-Na'- Y'Y'Y'-Nb'-Z'Z'Z'-Na'nq'5 '(IIIa).
[00027] In formula IIIa, each Na and Na 'independently represents a sequence of oligonucleotides including 1-5 modified nucleotides.
[00028] In some modalities, the formula (III) is represented according to the formula (IIIb): sense: 5 'np-Na-X XX - Nb-Y Y Y-Na-nq 3' antisense: 3 'np'- Na'-X'X'X'-Nb'-Y'Y'Y'-Na '-nq' 5 '(IIIb).
[00029] In formula IIIb each Na and Na 'independently represents a sequence of oligonucleotides including 1-5 modified nucleotides.
[00030] In some modalities, the formula (III) is represented according to the formula (IIIc): sense: 5 'np-Na-X XX - Nb-Y YY - Nb-Z Z Z-Na-nq 3' antisense: 3 'np'-Na'-X'X'X'-Nb'-Y'Y'Y'-Nb'-Z'Z'Z'-Na'-nq' 5 '(IIIc).
[00031] In formula IIIc, each Nb and -Nb 'independently represents an oligonucleotide sequence including 1-5 modified nucleotides and each Na and Na' independently represents an oligonucleotide sequence including 2-10 modified nucleotides.
[00032] In many embodiments, the duplex region is 15-30 pairs of nucleotides in length. In some embodiments, the duplex region is 17-23 pairs of nucleotides in length, 1725 pairs of nucleotides in length, 23-27 pairs of nucleotides in length, 19-21 pairs of nucleotides in length or 21-23 pairs of nucleotides in length length.
[00033] In certain embodiments, each strand has 15-30 nucleotides.
[00034] In some embodiments, nucleotide modifications are selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-ally, 2'-C-ally , 2'-fluoro, 2'-deoxy, 2'-hydroxyl and combinations thereof. In some preferred embodiments, the changes in the nucleotides are 2'-O-methyl or 2'-fluoro.
[00035] In some embodiments, the linker is one or more derivatives of N-acetylgalactosamine (GalNAc) linked by a divalent or trivalent branched linker. In particular modalities, the ligand is

[00036] In some embodiments, the ligand is attached to the 3 'end of the sense tape.
[00037] In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following scheme
where X is O or S.
[00038] In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following scheme

[00039] In some embodiments, the RNAi agent still includes at least one phosphorothioate or methylphosphonate internucleotide bond. In some embodiments, the phosphorothioate or methylphosphonate internucleotide bond is at the 3 'end of a ribbon. In some embodiments, the tape is the antisense tape. In other modalities, the tape is the sense tape.
[00040] In certain embodiments, the base pair at position 1 of the 5 'end of the duplex is an AU base pair.
[00041] In some embodiments, the Y nucleotides contain a 2'-fluoro modification.
[00042] In some embodiments, the Y 'nucleotides contain a 2'-O-methyl modification.
[00043] In some modalities, p ’> 0. In some such embodiments, each n is complementary to the target mRNA. In other such embodiments, each n is non-complementary to the target mRNA. In some embodiments, p, p ', q and q' are 1-6. In some preferred modalities, p '= 1 or 2. In some preferred modalities, p' = 2. In some such modalities, q '= 0, p = 0, q = 0, and p' lump nucleotides are complementary to the mRNA target. In other such embodiments, q '= 0, p = 0, q = 0, and p' lump nucleotides are non-complementary to the target mRNA.
[00044] In some embodiments, the sense tape has a total of 21 nucleotides and the antisense tape has a total of 23 nucleotides.
[00045] In certain embodiments, the bonds between np 'include phosphorothioate bonds. In some such embodiments, the bonds between np 'are phosphorothioate bonds.
[00046] In some embodiments, the RNAi agent is selected from the group of agents listed in Table 1.
[00047] In preferred embodiments, the RNAi agent is selected from the group consisting of AD-51544, AD-51545, AD-51546 and AD-51547.
[00048] In an even more preferred embodiment, the RNAi agent is AD-51547 having the following structure: sense: 5'-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3 '(SEQ ID NO: 2) antisense: 5'-uCfuUfgGfUfUasCaA ID NO: 3)
[00049] wherein the lowercase nucleotides (a, u, g, c) indicate 2'-O-methyl nucleotides; Nf (for example, Af) indicates a 2'-fluoro nucleotide; s indicates a phosphotiorate bond; L96 indicates a GalNAc3 ligand.
[00050] In another aspect, the present invention features a cell containing the RNAi agent to inhibit TTR expression.
[00051] In a further aspect, the present invention features a pharmaceutical composition comprising an RNAi agent to inhibit TTR expression. In some embodiments, the pharmaceutical composition is a solution that comprises the RNAi agent. In some embodiments, the solution comprising the RNAi agent is an un-buffered solution, for example, saline or water. In other embodiments, the solution is a buffered solution, for example, a phosphate buffered saline solution (PBS). In other embodiments, the pharmaceutical composition is a liposome or a lipid formulation. In some embodiments, the lipid formulation comprises an XTC or MC3.
[00052] In yet another aspect, the present invention features methods of inhibiting transthyretin (TTR) expression in a cell. Methods include contacting a cell with an RNAi agent, for example, a double-stranded RNAi agent, in an amount effective to inhibit TTR expression in the cell, thereby inhibiting TTR expression in the cell.
[00053] In some embodiments, the expression of TTR is inhibited by at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80% or at least approximately 90%.
[00054] In other embodiments, the cell is placed in contact with the RNAi agent in vitro. In other embodiments, the cell is present within an individual. In preferential modalities, the individual is the human being.
[00055] In additional embodiments, the individual is an individual suffering from a disease associated with TTR and the effective amount is a therapeutically effective amount. In other modalities, the individual is an individual at risk of falling ill from a disease associated with TTR and the effective amount is a prophylactically effective amount. In some modalities, an individual at risk of developing a disease associated with TTR is an individual who carries a TTR gene mutation that is associated with the development of a disease associated with TTR.
[00056] In certain modalities, the disease associated with TTR is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / system amyloidosis / Central nervous system (CNS) and hyperthyroxinemia.
[00057] In some embodiments, the individual has an amyloidosis associated with TTR and the method reduces an amyloid deposit by TTR in the individual.
[00058] In other embodiments, the RNAi agent is administered to the individual by a means of administration selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination of these. In certain embodiments, the RNAi agent is administered to the individual via subcutaneous or intravenous administration. In preferred embodiments, the RNAi agent is administered to the individual through subcutaneous administration. In some such embodiments, subcutaneous administration includes administration via a subcutaneous pump or subcutaneous storage.
[00059] In certain embodiments, the RNAi agent is administered to the individual such that the RNAi agent is delivered to a specific site within the individual. In some embodiments, the site is selected from the group consisting of the liver, choroid plexus, retina and pancreas. In preferred modalities, the site is the liver. In some embodiments, the delivery of the RNAi agent is mediated by the asialoglycoprotein receptor (ASGP-R) present in hepatocytes.
[00060] In some embodiments, the RNAi agent is administered at a dose between approximately 0.25 mg / kg to approximately 50 mg / kg, for example, between approximately 0.25 mg / kg to approximately 0.5 mg / kg, between approximately 0.25 mg / kg to approximately 1 mg / kg, between approximately 0.25 mg / kg to approximately 5 mg / kg, between approximately 0.25 mg / kg to approximately 10 mg / kg, between approximately 1 mg / kg to approximately 10 mg / kg, between approximately 5 mg / kg to approximately 15 mg / kg, between approximately 10 mg / kg to approximately 20 mg / kg, between approximately 15 mg / kg to approximately 25 mg / kg, between approximately 20 mg / kg to approximately 30 mg / kg, between approximately 25 mg / kg to approximately 35 mg / kg, or between approximately 40 mg / kg to approximately 50 mg / kg.
[00061] In some embodiments, the RNAi agent is administered at a dose of approximately 0.25 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg , approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, approximately 20 mg / kg, approximately 21 mg / kg, approximately 22 mg / kg, approximately 23 mg / kg, approximately 24 mg / kg, approximately 25 mg / kg, approximately 26 mg / kg, approximately 27 mg / kg, approximately 28 mg / kg , approximately 29 mg / kg, 30 mg / kg, approximately 31 mg / kg, approximately 32 mg / kg, approximately 33 mg / kg, approximately 34 mg / kg, approximately approximately 35 mg / kg, approximately 36 mg / kg, approximately 37 mg / kg, approximately 38 mg / kg, approximately 39 mg / kg, approximately 40 mg / kg, approximately 41 mg / kg, approximately 42 mg / kg, approximately 43 mg / kg, approximately 44 mg / kg, approximately 45 mg / kg, approximately 46 mg / kg, approximately 47 mg / kg, approximately 48 mg / kg, approximately 49 mg / kg or approximately 50 mg / kg.
[00062] In some embodiments, the RNAi agent is administered in two or more doses. In particular embodiments, the RNAi agent is administered at selected intervals from the group consisting of once every approximately 2 hours, once every approximately 3 hours, once every approximately 4 hours, once every approximately 6 hours. , once every approximately 8 hours, once every approximately 12 hours, once every approximately 24 hours, once every approximately 48 hours, once every approximately 72 hours, once every approximately 96 hours, once once every approximately 120 hours, once every approximately 144 hours, once every approximately 168 hours, once every approximately 240 hours, once every approximately 336 hours, once every approximately 504 hours, once every approximately 672 hours and once approximately approximately 720 hours.
[00063] In other modalities, the method also includes the evaluation of the level of expression of TTR mRNA or protein expression of TTR in a sample derived from the individual.
[00064] In preferred embodiments, administration of the RNAi agent does not result in an inflammatory response in the individual as assessed based on the level of a cytokine or chemokine selected from the group consisting of G-CSF, IFN-Y IL-10, IL -12 (p70), IL1 β, IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα and any combinations of these, in a sample of the individual.
[00065] In some embodiments, the RNAi agent is administered using a pharmaceutical composition
[00066] In preferred embodiments, the RNAi agent is administered in a solution. In some such modalities, siRNA is administered in an un-buffered solution. In one embodiment, siRNA is administered in water. In other embodiments, siRNA is administered with a buffer solution, such as an acetate buffer, a citrate buffer, a prolamine buffer, a carbonate buffer, or a phosphate buffer or any combination thereof. In some embodiments, the buffer solution is phosphate buffered saline (PBS).
[00067] In another embodiment, the pharmaceutical composition is a liposome or lipid formulation comprising SNALP or XTC. In one embodiment, the lipid formulation comprises an MC3.
[00068] In another aspect, the invention provides methods of treating or preventing a disease associated with TTR in an individual. The methods include administering to the individual a therapeutically effective amount or prophylactically effective amount of an RNAi agent, for example, a double-stranded RNAi agent, thereby treating or preventing TTR-associated disease in the individual.
[00069] In some embodiments, the expression of TTR in a sample derived from the individual is inhibited by at least approximately 10%, at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 70% or at least approximately 60% at least approximately 80% or at least approximately 90%.
[00070] In some modalities, the individual is the human being.
[00071] In some modalities, the individual is an individual suffering from a disease associated with TTR. In other modalities, the individual is an individual at risk of falling ill from a disease associated with TTR.
[00072] In some embodiments, the individual is an individual who carries a TTR gene mutation that is associated with the development of a disease associated with TTR.
[00073] In certain modalities, the disease associated with TTR is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloid polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / system amyloidosis / Central Nervous System (CNS) and hyperthyroxinemia.
[00074] In some embodiments, the individual has an amyloidosis associated with TTR and the method reduces an amyloid deposit by TTR in the individual.
[00075] In some embodiments, the RNAi agent is administered to the individual by a means of administration selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination of these. In certain embodiments, the RNAi agent is administered to the individual via subcutaneous or intravenous administration. In preferred embodiments, the RNAi agent is administered to the individual through subcutaneous administration. In some such embodiments, subcutaneous administration includes administration via a subcutaneous pump or subcutaneous storage.
[00076] In certain embodiments, the RNAi agent is administered to the individual such that the RNAi agent is delivered to a specific site within the individual. In some such modalities, the site is selected from the group consisting of the liver, choroid plexus, retina and pancreas. In preferred modalities, the site is the liver. In some embodiments, the delivery of the RNAi agent is mediated by the asialoglycoprotein receptor (ASGP-R) present in hepatocytes.
[00077] In some embodiments, the RNAi agent is administered at a dose between approximately 0.25 mg / kg to approximately 50 mg / kg, for example, between approximately 0.25 mg / kg to approximately 0.5 mg / kg , between approximately 0.25 mg / kg to approximately 1 mg / kg, between approximately 0.25 mg / kg to approximately 5 mg / kg, between approximately 0.25 mg / kg to approximately 10 mg / kg, between approximately 1 mg / kg to approximately 10 mg / kg, between approximately 5 mg / kg to approximately 15 mg / kg, between approximately 10 mg / kg to approximately 20 mg / kg, between approximately 15 mg / kg to approximately 25 mg / kg, between approximately 20 mg / kg to approximately 30 mg / kg, between approximately 25 mg / kg to approximately 35 mg / kg, or between approximately 40 mg / kg to approximately 50 mg / kg.
[00078] In some embodiments, the RNAi agent is administered at a dose of approximately 0.25 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg , approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, approximately 20 mg / kg, approximately 21 mg / kg, approximately 22 mg / kg, approximately 23 mg / kg, approximately 24 mg / kg, approximately 25 mg / kg, approximately 26 mg / kg, approximately 27 mg / kg, approximately 28 mg / kg, approximately 29 mg / kg, 30 mg / kg, approximately 31 mg / kg, approximately 32 mg / kg, approximately 33 mg / kg, approximately 34 mg / kg, approximately approximately 35 mg / kg, approximately 36 mg / kg, approximately 37 mg / kg, approximately 38 mg / kg, approximately 39 mg / kg, approximately 40 mg / kg, approximately 41 mg / kg, approximately 42 mg / kg, approximately 43 mg / kg, approximately 44 mg / kg, approximately 45 mg / kg, approximately 46 mg / kg, approximately 47 mg / kg, approximately 48 mg / kg, approximately 49 mg / kg or approximately 50 mg / kg.
[00079] In some embodiments, the RNAi agent is administered in two or more doses. In particular embodiments, the RNAi agent is administered at selected intervals from the group consisting of once every approximately 2 hours, once every approximately 3 hours, once every approximately 4 hours, once every approximately 6 hours. , once every approximately 8 hours, once every approximately 12 hours, once every approximately 24 hours, once every approximately 48 hours, once every approximately 72 hours, once every approximately 96 hours, once once every approximately 120 hours, once every approximately 144 hours, once every approximately 168 hours, once every approximately 240 hours, once every approximately 336 hours, once every approximately 504 hours, once every approximately 672 hours and once approximately approximately 720 hours.
[00080] In other modalities, the method also includes the evaluation of the level of expression of TTR mRNA or protein expression of TTR in a sample derived from the individual.
[00081] In preferred embodiments, administration of the RNAi agent does not result in an inflammatory response in the individual as assessed based on the level of a cytokine or chemokine selected from the group consisting of G-CSF, IFN-Y, IL-10, IL -12 (p70), IL1 β, IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα and any combinations of these, in a sample of the individual.
[00082] In some embodiments, the RNAi agent is administered using a pharmaceutical composition, for example, a liposome.
[00083] In some embodiments, the RNAi agent is administered in a solution. In some such modalities, siRNA is administered in an un-buffered solution. In one embodiment, siRNA is administered in saline or water. In other embodiments, siRNA is administered with a buffer solution, such as an acetate buffer, a citrate buffer, a prolamine buffer, a carbonate buffer, or a phosphate buffer or any combination thereof. In some embodiments, the buffer solution is phosphate buffered saline (PBS).
[00084] In another aspect, the present invention provides a method of inhibiting transthyretin (TTR) expression in a cell, including contacting a cell with an RNAi agent, for example, a double-stranded RNAi agent, in an amount effective to inhibit TTR expression in the cell. In one aspect, the double-stranded RNAi agent is selected from the group of agents listed in Table 1, thereby inhibiting the expression of transthyretin (TTR) in the cell.
[00085] In another aspect, the present invention provides a method of inhibiting transthyretin (TTR) expression in a cell, including contacting a cell with an RNAi agent, for example, a double-stranded RNAi agent, in an amount effective to inhibit TTR expression in the cell. In one aspect, the double-stranded RNAi agent is selected from the group consisting of AD-51544, AD-51545, AD-51546 and AD-51547, thereby inhibiting the expression of transthyretin (TTR) in the cell.
[00086] In a further aspect, the present invention provides a method of treating or preventing a disease associated with TTR in an individual, including administering to the individual a therapeutically effective amount or a prophylactically effective amount of an RNAi agent. , for example, a double-stranded RNAi agent. In one aspect, the double-stranded RNAi agent is selected from the group of agents listed in Table 1, thereby treating or preventing a disease associated with TTR in the individual.
[00087] In yet another aspect, the present invention provides a method of treating or preventing a disease associated with TTR in an individual, including administering to the individual a therapeutically effective amount or a prophylactically effective amount of an RNAi agent. , for example, a double-stranded RNAi agent. In one aspect, the double-stranded RNAi agent is selected from the group consisting of AD-51544, AD-51545, AD-51546 and AD-51547, thereby treating or preventing a disease associated with TTR in the individual.
[00088] In additional aspects, the invention provides kits for carrying out the methods of the invention. In one aspect, the invention provides a kit for carrying out a method of inhibiting transthyretin expression (TTR) in a cell comprising contacting a cell with an RNAi agent, for example, a double-stranded RNAi agent, in an amount effective to inhibit the expression of said TTR in said cell, thereby inhibiting the expression of TTR in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to the individual.
[00089] The present invention is further illustrated by the following detailed description and drawings. Brief Description of Drawings
[00090] Figure 1 is a graph depicting that administration to mice of a single subcutaneous dose of a GalNAc-conjugated RNAi agent targeting TTR resulted in dose-dependent suppression of the TTR mRNA.
[00091] Figure 2 is a graph depicting that administration to mice of a single subcutaneous dose of 7.5 mg / kg or 30 mg / kg of the RNAi agent conjugated to GalNAc targeting TTR resulted in long-term suppression of the TTR mRNA. .
[00092] Figure 3 represents the human TTR mRNA sequence.
[00093] Figure 4 is a graph representing improved silencing of the activity of modified RNAi agents relative to the parental AD-45163.
[00094] Figure 5 is a graph representing improved silencing of the activity of modified RNAi agents relative to the parental AD-45165.
[00095] Figure 6 is a graph depicting improved free absorption silencing following the 4-hour incubation with modified RNAi agents relative to the parental AD-45163.
[00096] Figure 7 is a graph depicting improved free absorption silencing following the 24-hour incubation with modified RNAi agents relative to the parental AD-45163.
[00097] Figure 8 is a graph depicting improved free absorption silencing following the 4-hour incubation with modified RNAi agents relative to the parental AD-45165.
[00098] Figure 9 is a graph depicting improved free absorption silencing following the 24-hour incubation with modified RNAi agents relative to the parental AD-45165.
[00099] Figure 10 is a graph depicting silencing of TTR mRNA in transgenic mice expressing V30M hTTR after administration of a single subcutaneous dose of RNAi agents AD-51544, AD-51545, AD-45163, AD-51546, AD -51547 or AD-45165.
[000100] Figure 11 is a graph depicting TTR protein suppression in transgenic mice expressing V30M hTTR after administration of a single subcutaneous dose of 5 mg / kg or 1 mg / kg of RNAi agents AD-51544, AD-51545 or AD-45163.
[000101] Figure 12 is a graph depicting TTR protein suppression in transgenic mice expressing V30M hTTR after administration of a single subcutaneous dose of 5 mg / kg or 1 mg / kg of RNAi agents AD-51546, AD-51547 or AD-45165
[000102] Figure 13 represents the protocol for post-dose blood tests in monkeys that received 5x5mg / kg of RNAi agent (upper line) or 1x25mg / kg of RNAi agent (lower line).
[000103] Figure 14 is a graph depicting the suppression of TTR protein in non-human primates after subcutaneous administration of five doses of 5 mg / kg (upper panel) or a single 25 mg / kg dose (lower panel) of AD- 45163, AD-51544, AD-51545, AD-51546 or AD-51547.
[000104] Figure 15 is a graph depicting the suppression of TTR protein in non-human primates after subcutaneous administration of AD-51547 at 2.5 mg / kg (white squares), 5 mg / kg (black squares) or 10 mg / kg (standardized squares) per dose or administration of PBS as a negative control (gray squares). Detailed Description of the Invention
[000105] The present invention provides RNAi agents, for example, double-stranded RNAi agents and compositions that target the Transtirretin (TTR) gene. The present invention also provides methods of inhibiting TTR expression and methods of treating or preventing a disease associated with TTR in an individual using the RNAi agents, for example, double-stranded RNAi agents, of the invention. The present invention is based, at least in part, on the discovery that RNAi agents comprising particular chemical modifications show a superior ability to inhibit TTR expression. Agents including a certain model of chemical modifications (for example, an alternating model) and a linker in this application are shown to be effective in silencing the activity of the TTR gene. In addition, agents including one or more motifs of three identical modifications in three consecutive nucleotides, including one such motif within or near the agent's cleavage site, show surprisingly increased TTR gene silencing activity. When a single such chemical motif is present in the agent, it is preferable to be within or close to the cleavage region to increase gene silencing activity. The cleavage region is the region that surrounds the cleavage site, that is, the site on the target mRNA into which the cleavage occurs. I. Definitions
[000106] As used in this application, each of the following terms has the meaning associated with it in this section.
[000107] The term "including" is used in this application to mean, and is used interchangeably with the phrase "including but not limited to".
[000108] The term "or" is used in this application to mean and is used interchangeably with the term "and / or", unless the context clearly indicates otherwise.
[000109] As used in this application, a "transthyretin" ("TTR") refers to the well-known gene and protein. TTR is also known as pre-albumin, HsT2651, PALB and TBPA. TTR functions as a transporter of the retinol-binding protein (RBP), thyroxine (T4) and retinol, and also acts as a protease. The liver secretes TTR into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR is also expressed in the pancreas and in the retinal pigment epithelium. The greatest clinical relevance of TTR is that both the normal and the mutant TTR protein can form amyloid fibrils that aggregate in extracellular deposits, causing amyloidosis. See, for example, Saraiva M.J.M. (2002) Expert Reviews in Molecular Medicine, 4 (12): 1-11 for a review. The nucleotide sequence and molecular cloning of rat transthyretin, as well as distribution of mRNA expression, has been described by Dickson, P.W. et al. (1985) J. Biol. Chem. 260 (13) 8214-8219. The X-ray crystalline structure of human TTR has been described in Blake, C.C. et al. (1974) J Mol Biol 88, 112. The sequence of a human TTR mRNA transcript can be found at the National Center for Biotechnological Information (NCBI) accession number RefSeq NM_000371. The mouse TTR mRNA sequence can be found at RefSeq accession number NM_013697.2 and the mouse TTR mRNA sequence can be found at RefSeq accession number NM_012681.1
[000110] As used in this application, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TTR gene, including the mRNA that is a product of a product's RNA processing primary transcription.
[000111] As used in this application, the term "ribbon comprising a sequence" refers to an oligonucleotide comprising a nucleotide chain that is described by the referred sequence using standard nucleotide nomenclature.
[000112] "G", "C", "A" and "U" each generally represent a nucleotide containing guanine, cytosine, adenine and uracil as a base, respectively. "T" and "dT" are used interchangeably in this application and refer to a deoxyribonucleotide in which the nucleobase is thymine, for example, deoxyribothymine, 2'-deoxythymidine or thymidine. However, it will be understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to a modified nucleotide, as more detailed below, or a replacement replacement portion. The skilled person is well aware that guanine, cytosine, adenine and uracil can be substituted for other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide that carries such a substitution moiety. For example, without limitation, a nucleotide that comprises inosine as its base can pair bases with nucleotides containing adenine, cytosine or uracil. Therefore, nucleotides containing uracil, guanine or adenine can be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. The sequences comprising such substitution portions are the embodiments of the invention.
[000113] A "double-stranded RNAi agent", double-stranded RNA (dsRNA) molecule, also referred to as "dsRNA agent", "dsRNA", "siRNA", "iRNA agent", as used interchangeably in this This application relates to a complex of ribonucleic acid molecules, having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands, as defined below. In general, the majority of the nucleotides in each strand are ribonucleotides, but as described in detail in this application, each or both phytases may also include one or more non-ribonucleotides, for example, a deoxyribonucleotide and / or a modified nucleotide. In addition, as used in this specification, an "RNAi agent" can include ribonucleotides with chemical modifications; an RNAi agent can include substantial modifications to multiple nucleotides. Such modifications may include all types of modifications described in this application or known in the art. Any such modification, as used in a siRNA-like molecule, is encompassed by an "RNAi agent" for the purposes of this specification and claims.
[000114] In another embodiment, the RNAi agent can be a single-stranded siRNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC Argonaute 2 endonuclease, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in US Patent No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of which are hereby incorporated in this order by reference. Any of the antisense nucleotide sequences described in this application can be used as a single-stranded siRNA as described in this application or as chemically modified by the methods described in Lima et al., (2012) Cell 150;: 883-894.
[000115] The two strands that form the duplex structure can be different portions of a larger RNA molecule, or they can be separate RNA molecules. Where the two strands are part of a larger molecule, and therefore are connected by an unbroken chain of nucleotides between the 3 'end of a strand and the 5' end of another respective strand forming the duplex structure, the connector RNA strand is referred to as a “staple handle”. Where the two strands are covalently linked by means in addition to an unbroken chain of nucleotides between the 3 'end of a strand and the 5' end of another respective strand forming the duplex structure, the connector structure is referred to as a "linker". RNA phytases can have the same or different nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any protrusion that is present in the duplex. In addition to the duplex structure, an RNAi agent can comprise one or more nucleotide protrusions. The term "siRNA" is also used in this application to refer to an RNAi agent as described above.
[000116] In another aspect, the agent is a single-stranded antisense RNA molecule. An antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation in a stoichiometric manner by base pairing to mRNA and physically obstructs the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1: 347-355. The antisense RNA molecule can have approximately 15-30 nucleotides that are complementary to the target mRNA. For example, the antisense RNA molecule can have a sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one of the antisense sequences in Table 1.
[000117] As used in this application, a "nucleotide bulge" refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of an RNAi agent when a 3 'end of an RNAi agent strand extends beyond the 5 'end of another tape, or vice versa. "Blind" or "blind end" means that there is no unpaired nucleotide at that end of the double-stranded RNAi agent, that is, no nucleotide protrusion. A blunt-ended RNAi agent is a dsRNA that is double-stranded along its entire length, that is, no nucleotide protuberance at either end of the molecule. The RNAi agents of the invention include RNAi agents with nucleotide protrusions at one end (i.e., agents with a protrusion and a blunt end) or with nucleotide protrusions at both ends.
[000118] The term "antisense strand" refers to the strand of a double stranded RNAi agent that includes a region that is substantially complementary to a target sequence (e.g., a human TTR mRNA). As used in this application, the term "region complementary to the part of an mRNA encoding transthyretin" refers to a region on the antisense strand that is substantially complementary to the part of a TTR mRNA sequence. Where the complementarity region is not completely complementary to the target sequence, incompatibilities are more tolerated in the terminal regions and, if present, are generally in a terminal region or regions, for example, in 6, 5, 4, 3, or 2 nucleotides of the 5 'and / or 3' end.
[000119] The term "sense ribbon", as used in this application, refers to the ribbon of a dsRNA that includes a region that is substantially complementary to a region of the antisense ribbon.
[000120] As used in this application, the term "cleavage region" refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which the cleavage occurs. In some embodiments, the cleavage region comprises three bases at either end, and immediately adjacent, to the cleavage site. In some embodiments, the cleavage region comprises two bases at either end, and immediately adjacent, to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site linked by 10 and 11 nucleotides of the antisense strand, and the cleavage region comprises 11, 12 and 13 nucleotides.
[000121] As used in this application, and unless otherwise indicated, the term "complementary", when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions, for example, can be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 ° C or 70 ° C for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as they can be found within an organism, can be applied. The specialist will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences according to the final application of the hybridized nucleotides.
[000122] The sequences can be "completely complementary" with respect to each other when base nucleotides of the first nucleotide sequence are matched with the nucleotides of the second nucleotide sequence along the entire length of the first and second nucleotide sequences. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence in this application, the two sequences may be fully complementary, or may form one or more, but generally not more than 4, 3 or 2 base pairs incompatible through hybridization, maintaining the ability to hybridize under the most relevant conditions for its final application. However, where two oligonucleotides are designed to form, during hybridization, one or more single-strip protuberances, such protuberances should not be considered as incompatibilities in determining complementarity. For example, a dsRNA comprising an oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, in which the longest oligonucleotide comprises a sequence of 21 nucleotides that is completely complementary to the shortest oligonucleotide, can still be referred to as “totally complementary ”For the purposes described in this application.
[000123] "Complementary" sequences, as used in this application, may also include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed of unnatural and modified nucleotides, as far as the above requirements with respect their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G: U Wobble or Hoogstein base pairing.
[000124] The terms "complementary", "fully complementary" and "substantially complementary" in this application can be used with respect to the base combining the sense tape and the antisense tape of a dsRNA, or between the antisense tape of a dsRNA and a target sequence, as will be understood from the context of its use.
[000125] As used in this application, a polynucleotide that is "substantially complementary to at least the part of" a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (for example, a mRNA encoding TTR) including a 5 'RTU, an open reading region (ORF) or a 3' RTU. For example, a polynucleotide is complementary to at least part of a TTR mRNA if the sequence is substantially complementary to an unbroken portion of an mRNA encoding TTR.
[000126] The term "inhibition", as used in this application, is used interchangeably with "reduction", "silencing", "down regulation", "suppression" and other similar terms, and includes any level of inhibition.
[000127] The phrase "inhibiting the expression of a TTR" as used in this application includes inhibiting the expression of any TTR gene (such as, for example, a mouse TTR gene, a mouse TTR gene, a monkey TTR gene or human TTR gene) as well as variants or mutants of a TTR gene. Thus, the TTR gene can be a wild-type TTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise to systemic amyloid deposition) or transgenic TTR gene in the context of a genetically engineered cell, group of cells or organism.
[000128] "Inhibition of the expression of a TTR gene" includes any level of inhibition of a TTR gene, for example, the at least partial suppression of expression of a TTR gene, such as an inhibition of at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90 %, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%. at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99%.
[000129] TTR gene expression can be assessed based on the level of any variable associated with TTR gene expression, for example, TTR mRNA level, TTR protein level, retinol-binding protein level, vitamin A level, or the number or extent of amyloid deposits. Inhibition can be assessed by a reduction in an absolute or relative level of one or more of these variables compared to a control level. The level of control can be any type of level of control that is used in the art, for example, a pre-dose baseline level or a specific level of a similar individual, cell or sample that is not treated or treated with a control ( such as, for example, control only buffer or inactive agent control).
[000130] The phrase "contacting a cell with an RNAi agent", as used in this application, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent, for example, a double-stranded RNAi agent, includes contacting a cell in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. Putting in contact can be done directly or indirectly. In this way, for example, the RNAi agent can be put in physical contact with the cell by the individual who performs the method, or alternatively, the RNAi agent can be put in a situation that will allow or cause it to come into contact with it later. the cell.
[000131] Contact of a cell in vitro can be done, for example, by incubating the cell with the RNAi agent. Contact of a cell in vivo can be made, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent in another area, for example, in the bloodstream or in the subcutaneous space, such that the agent will later reach the tissue where the cell to be contacted is located. For example, the RNAi agent can contain and / or be coupled to a linker, for example, a GalNAc3 linker, which directs the RNAi agent to a site of interest, for example, the liver. Combinations of in vitro and in vivo methods for contacting are also possible. With respect to the methods of the invention, a cell could also be contacted in vitro with an RNAi agent and subsequently transplanted into an individual.
[000132] A "patient" or "individual", as used in this application, is intended to include a human or non-human animal, preferably a mammal, for example, a monkey. Even more preferably, the individual or the patient is a human being.
[000133] A "disease associated with TTR", as used in this application, is intended to include any disease associated with the TTR gene or protein. Such a disease can be caused, for example, by the excessive production of the TTR protein, by gene TTR mutations, by the abnormal cleavage of the TTR protein, by abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. A "disease associated with TTR" includes any type of TTR amyloidosis (ATTR) in which TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposits. Diseases associated with TTRs include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal / Central Nervous System (CNS) amyloidosis, vitreous amyloid syndrome opacities carpal tunnel and hyperthyroxinemia. Symptoms of TTR amyloidosis include sensory neuropathy (eg paresthesia, hypoaesthesia in distal limbs), autonomic neuropathy (eg gastrointestinal dysfunction, such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, syndrome carpal tunnel, autonomic failure, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction and truss corneal dystrophy.
[000134] "Therapeutically effective amount", as used in this application, is intended to include the amount of an RNAi agent that, when administered to a patient to treat a disease associated with TTR, is sufficient to affect the treatment of the disease (for example, example, decreasing, ameliorating or maintaining existing disease or one or more symptoms of the disease) The “therapeutically effective amount” can vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic construction, stage of pathological processes mediated by TTR expression, types of previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
[000135] "Prophylactically effective amount", as used in this application, is intended to include the amount of an RNAi agent that, when administered to an individual who has not yet experienced or exhibits symptoms of a disease associated with TTR, but which can be predisposed to the disease, it is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Symptoms that can be improved include sensory neuropathy (eg, paresthesia, hypoaesthesia in distal limbs), autonomic neuropathy (eg, gastrointestinal dysfunction, such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction and truss corneal dystrophy. Improving the disease includes slowing the course of the disease or reducing the severity of the disease that develops afterwards. The “prophylactically effective amount” can vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, and the history, age, weight, family history, genetic construction, types of previous or concomitant treatments , if any, and other individual characteristics of the patient to be treated.
[000136] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect in a reasonable benefit / risk ratio applicable to any treatment. RNAi agents employed in the methods of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.
[000137] The term "sample", as used in this application, includes a collection of fluids, cells or similar isolated tissues from an individual, as well as fluids, cells or tissues present within an individual. Examples of biological fluids include blood, serum and serous fluids, plasma, cerebrospinal fluids, eye fluids, lymph, urine, saliva and the like. Tissue samples can include tissue samples, organs or localized regions. For example, samples can be derived from organs, parts of organs, or particular fluids or cells within those organs. In certain embodiments, the samples can be derived from the liver (eg, whole liver or certain liver segments or certain types of cells in the liver, such as, for example, hepatocytes), retina or parts of the retina (eg, epithelium of the liver) retinal pigment), central nervous system or parts of the central nervous system (for example, ventricles or choroid plexus), or pancreas or certain cells or parts of the pancreas. In some embodiments, a “sample derived from an individual” refers to the cerebrospinal fluid obtained from the individual. In preferred embodiments, a “sample derived from an individual” refers to blood or plasma taken from the individual. In additional embodiments, a “sample derived from an individual” refers to liver tissue (or subcomponents thereof) or retinal tissue (or subcomponents thereof) derived from the individual. II. RNAi agents
[000138] The present invention provides RNAi agents with superior gene silencing activity. It is shown in this application and in Provisional Patent Application No. 61 / 561,710 (over which the present patent application claims priority) that a superior result can be obtained by introducing one or more motifs of three identical modifications into three consecutive nucleotides on a ribbon sense and / or antisense tape of an RNAi agent, particularly within or near the cleavage site. The sense strand and the antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these reasons interrupts the modification model, if present, of the sense and / or antisense tape. The RNAi agent also optionally conjugates with a linker derived from GalNAc, for example, in the sense strand. The resulting RNAi agents have superior gene silencing activity.
[000139] The inventors surprisingly found that when the sense strand and the antisense strand of the RNAi agent are completely modified, having one or more motifs of three identical modifications in three consecutive nucleotides within or near the cleavage site of at least one strand of a superior RNAi agent increased the gene silencing activity of the RNAi agent.
[000140] Consequently, the invention provides RNAi agents, for example, double-stranded RNAi agents, capable of inhibiting the expression of a target gene (i.e., a TTR gene) in vivo. The RNAi agent comprises a sense tape and an antisense tape. Each strand of the RNAi agent can range from 12-30 nucleotides in length. For example, each strand can be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 2730 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17- 19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length or 21-23 nucleotides in length.
[000141] Sense tape and antisense tape typically form duplex double-stranded RNA ("dsRNA"), also referred to in this application as an "RNAi agent". The duplex region of an RNAi agent can be 12-30 pairs of nucleotides in length. For example, the duplex region can be between 14-30 pairs of nucleotides in length, 17-30 pairs of nucleotides in length, 27-30 pairs of nucleotides in length, 17-23 pairs of nucleotides in length, 17-21 pairs nucleotides in length, 17-19 pairs of nucleotides in length, 19-25 pairs of nucleotides in length, 19-23 pairs of nucleotides in length, 19-21 pairs of nucleotides in length, 21-25 pairs of nucleotides in length or 21-23 pairs of nucleotides in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27.
[000142] In one embodiment, the RNAi agent can contain one or more hump regions and / or groups ending the RNAi agent at the 3 'end, or at the 5' end, or both ends of a strand. The lump can be 1-6 nucleotides in length, for example, 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1 -3 nucleotides in length, 2-3 nucleotides in length or 12 nucleotides in length. The lumps can be the result of one tape being longer than the other or the result of two tapes of the same length being uncoordinated. The bulge may form an incompatibility with the target mRNA or it may be complementary to the gene sequences that are targeted or it may be another sequence. The first and second tapes can also be joined, for example, by additional bases to form a clamp, or by other non-base binders.
[000143] The RNAi agents provided by the present invention include agents with chemical modifications as described, for example, in US Provisional Patent Application No. 61 / 561,710, filed on November 18, 2011, in International Patent Application No. PCT / US2011 / 051597, filed on September 15, 2010, and PCT Publication WO 2009/073809, the entire contents of which are incorporated into this application by reference.
[000144] In one embodiment, the nucleotides in the hump region of the RNAi agent can each be independently a modified or unmodified nucleotide including, but not limited to, modified 2 'sugar, such as, 2-F, 2'-O -methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination of these . For example, TT can be a sequence of protrusions from either end on any tape. The bulge may form an incompatibility with the target mRNA or it may be complementary to the gene sequences that are targeted or it may be another sequence.
[000145] The 5 'or 3' protuberances in the sense tape, antisense tape or both RNAi agent phytases can be phosphorylated. In some embodiments, the bulge region contains two nucleotides that have a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the protrusion is present at the 3 'end of the sense tape, antisense tape or both strips. In one embodiment, this 3 'protrusion is present in the antisense tape. In one embodiment, this 3 'protuberance is present in the sense tape.
[000146] The RNAi agent can contain only a single protuberance, which can strengthen the RNAi interference activity, without affecting its total stability. For example, the protrusion of single tape is located at the 3 'end of the sense tape or, alternatively, at the 3' end of the antisense tape. The RNAi can also have a blunt end, located at the 5 'end of the antisense tape (or the 3' end of the sense tape) or vice versa. Generally, the RNAi antisense strand has a nucleotide protuberance at the 3 'end, and the 5' end is blunt. Although Applicants are not bound by theory, the theoretical mechanism is that the asymmetric blind end at the 5 'end of the antisense tape and the 3' end protrusion of the antisense tape favors the loading of the guide tape in the RISC process.
[000147] In one embodiment, the RNAi agent is a 19 nt long double ended double ended terminator, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 7, 8, 9 from the 5 'end. The antisense tape contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, 13 of the 5 'end.
[000148] In one embodiment, the RNAi agent is a 20 nt long double ended blunt ended terminator, where the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 8, 9, 10 from the 5 'end. The antisense tape contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, 13 of the 5 'end.
[000149] In one embodiment, the RNAi agent is a 21 nt long double ended blunt terminator, in which the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides at positions 9, 10, 11 of the 5 'end. The antisense tape contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, 13 of the 5 'end.
[000150] In one embodiment, the RNAi agent comprises a 21-nucleotide (nt) sense tape and a 23-nucleotide (nt) antisense tape, wherein the sense tape contains at least one 2'-F modification motif in three consecutive nucleotides at positions 9, 10, 11 of the 5 'end; the antisense tape contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at positions 11, 12, 13 of the 5 'end, where one end of the RNAi agent is blunt, while the other end comprises a 2 nt bulge. Preferably, the 2 nt protrusion is at the 3 'end of the antisense. Optionally, the RNAi agent further comprises a linker (preferably GalNAc3).
[000151] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, in which the sense strand is 25-30 nucleotide residues in length, in which starting from the positions of the 5 'terminal nucleotide (position 1) 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense tape is 36-66 nucleotide residues in length and, starting from the 3 'terminal nucleotide, comprises at least 8 ribonucleotides in positions paired with positions 1-23 of the sense tape to form a duplex; wherein at least the 3 'terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3' terminal nucleotides are not paired with the sense strand, thereby forming a single stranded 3 'protuberance of 1- 6 nucleotides; wherein the 5 'terminal of the antisense tape comprises 10-30 consecutive nucleotides that are not paired with the sense tape, thereby forming a 5' protuberance of 10-30 nucleotides single strip; where at least the 5 'terminal sense strand and 3' terminal nucleotides are paired in bases with nucleotides from the antisense tape when the sense and antisense phytates are aligned with maximum complementarity, thereby forming a substantially duplexed region between the antisense and sense phytases ; and the antisense strand is sufficiently complementary to the target RNA over at least 19 antisense strand length ribonucleotides to reduce the target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2'-F modifications in three consecutive nucleotides, where at least one of the motifs occurs within or near the cleavage site. The antisense tape contains at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides within or near the cleavage site.
[000152] In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length that is at least 25 and a maximum of 29 nucleotides and a second strand having a length that is at least maximum 30 nucleotides with at least one motif of three 2'-O-methyl modifications in three consecutive nucleotides at position 11, 12, 13 of the 5 'end; where the 3 'end of the first strand and the 5' end of the second strand form a blunt end and the second strand has 1-4 more nucleotides at its 3 'end than the first strand, where the duplex region that it is at least 25 nucleotides in length and the second strand is sufficiently complementary to a target mRNA over at least 19 nt of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and in that dicer cleavage of the RNAi agent preferably results in a siRNA comprising the 3 'end of the second strand, thereby reducing the expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a linker.
[000153] In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs occurs at the cleavage site on the sense strand.
[000154] In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications in three consecutive nucleotides, where one of the motifs occurs within or near the cleavage site on the antisense strand.
[000155] For the RNAi agent having a 1723 nt long duplex region, the antisense tape cleavage site is typically around positions 10, 11 and 12 of the 5 'end. In this way, the reasons for three identical changes can occur in positions 9, 10, 11; positions 10, 11, 12; positions 11, 12, 13; positions 12, 13, 14; or positions 13, 14, 15 of the antisense tape, the count starting from the 1st nucleotide of the 5 'end of the antisense tape, or, the count starting of the 1st paired nucleotide within the duplex region of the 5' end of the antisense tape. The cleavage site on the antisense tape can also be modified according to the length of the RNAi duplex region of the 5 'end.
[000156] The RNAi agent sense strand may contain at least one motif of three identical modifications in three consecutive nucleotides at the strand cleavage site; and the antisense tape can have at least one motif of three identical modifications in three consecutive nucleotides within or near the tape's cleavage site. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that a motif of the three nucleotides in the sense strand and a motif of the three nucleotides in the antisense strand have at least one nucleotide overlap. , that is, at least one of the three nucleotides of the motif on the sense tape forms a base pair with at least one of the three nucleotides of the motif on the antisense tape. Alternatively, at least two nucleotides can overlap, or the three nucleotides can overlap.
[000157] In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications in three consecutive nucleotides. The first motif must occur within or near the tape's cleavage site and other motifs may be wing modifications. The term "wing modification" in this application refers to a motif that occurs on another portion of the tape that is separated from the motif within or near the cleavage site of the same tape. The wing modification is adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other than the chemistry of the motifs is distinct from one another and when the motifs are separated by one or more nucleotides than the chemicals can be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification can occur at one end with respect to the first motif that is within or near the cleavage site or on both sides of the leading motif.
[000158] Like the sense strand, the RNAi agent's antisense strand may contain at least two motifs of three identical modifications in three consecutive nucleotides, with at least one motif occurring within or near the strand cleavage site. This antisense tape can also contain one or more wing modifications in an alignment similar to the wing modifications that are present in the sense tape.
[000159] In one embodiment, the wing modification of the sense strand or antisense strand of the RNAi agent typically does not include the first or two terminal nucleotides at the 3 'end, 5' end or both ends of the strand.
[000160] In another embodiment, the wing modification of the sense strand or antisense strand of the RNAi agent typically does not include the first or two paired nucleotides within the duplex region at the 3 'end, 5' end or both ends of the strand.
[000161] When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, wing modifications may fall at the same end of the duplex region and have an overlap of one, two or three nucleotides.
[000162] When the sense tape and the antisense tape of the RNAi agent each contain at least two wing modifications, the sense tape and the antisense tape can be so aligned that two modifications each of a tape falls on one end of the region duplex, having an overlap of one, two or three nucleotides; two modifications each of a strand falls at the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications of a ribbon fall on either side of the leading motif, having an overlap of one, two or three nucleotides in the duplex region.
[000163] In one embodiment, each nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotides that are part of the motifs, can be modified. Each nucleotide can be modified with the same or different modification which can include one or more changes to one or both non-binding oxygen phosphates and / or one or both oxygen binding phosphates; changing a constituent of ribose sugar, for example, 2'-hydroxyl in ribose sugar; massive replacement of the phosphate portion with "defosfo" binders; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.
[000164] Since nucleic acids are polymers of the subunits, many of the modifications occur in a position that is repeated within a nucleic acid, for example, a modification of a base, or a phosphate moiety or a non-binding O of a phosphate moiety . In some cases, the modification will occur at all object positions in the nucleic acid but in many cases they will not occur. By way of example, a modification can only occur in a 3 'or 5' terminal position, it can only occur in a terminal region, for example, in a position in relation to a terminal nucleotide or in the last 2, 3, 4, 5 , or 10 nucleotides from a strand. A modification can occur in a double tape region, a single tape region, or both. A modification can occur only in the double stranded RNA region or can only occur in a single stranded RNA region. For example, a phosphorothioate modification in a non-ligand position can only occur at one or both terminals, it can only occur in a terminal region, for example, in a position relative to a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides from a strand, or it can occur in regions of double strand and single strand, particularly at terminals. The 5 'end or ends can be phosphorylated.
[000165] It may be possible, for example, to increase stability, to include particular bases in protrusions or to include modified nucleotides or nucleotide representatives, in single-stranded protuberances, for example, in a 5 'or 3' protuberance, or both. For example, it may be desirable to include purine nucleotides in lumps. In some embodiments, all or some bases on a 3 'or 5' protuberance can be modified, for example, with a modification described in this application. The modifications may include, for example, the use of modifications in the 2 'position of the ribose sugar with modifications that are known in the art, for example, the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl, modified instead of the ribo-sugar of the nucleobase, and modifications in the phosphate group, for example, phosphorothioate modifications. The protrusions do not have to be homologous to the target sequence.
[000166] In one embodiment, each residue of the sense tape and antisense tape is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-ally, 2'-C-ally , 2'-deoxy, 2'-hydroxyl or 2'-fluoro. Phytasses can contain more than one modification. In one embodiment, each residue of the sense tape and antisense tape is independently modified with 2'-O-methyl or 2'-fluoro.
[000167] At least two different modifications are typically present in the sense tape and antisense tape. Those two modifications can be 2'-O-methyl or 2'-fluoro or others.
[000168] In one embodiment, Na and / or Nb comprise modifications of an alternate model. The term "alternating motif", as used in this application, refers to a motif having one or more modifications, each modification occurring in the alternating nucleotides of a strand. The alternating nucleotide can refer to one for each other nucleotide or one for every three nucleotides or similar model. For example, if A, B and C each represent a type of modification to the nucleotide, the alternating motif can be "ABABABABABABAB ...", "AABBAABBAABB ...", "AABAABAABAAB ...", "AAABAAABAAAB ... ”,“ AAABBBAAABBB. ”, Or“ ABCABCABCABC. ”, Etc.
[000169] The type of modifications contained in the alternating reason can be the same or different. For example, if A, B, C, D each represents a type of modification in the nucleotide, the alternating model, that is, modifications in each other nucleotide, can be the same, but each of the sense tape or antisense tape can be selected from various modification possibilities within the alternating motif such as “ABABAB.”, “ACACAC.” "BDBDBD." or “CDCDCD.”, etc.
[000170] In one embodiment, the RNAi agent of the invention comprises the alternating motif modification model in the sense tape in relation to the alternating motif modification model in the antisense tape is displaced. The displacement may be such that the modified group of nucleotides on the sense tape corresponds to a differently modified group of nucleotides on the antisense tape and vice versa. For example, the sense tape when paired with the antisense tape in the dsRNA duplex, the alternating motif in the sense tape can start with the tape's 5'-3 '“ABABAB” and the alternating motif in the antisense tape can start with “BABABA” 5'-3 'of the tape within the duplex region. As another example, the alternating motif on the sense tape can start with the tape's 'AABBAABB' 5'-3 'and the alternating motif on the antisense tape can start with the tape's' BBAABBAA '5'-3' within the region of the tape. duplex, so that there is a complete or partial displacement of the modification models between the sense tape and the antisense tape.
[000171] In one embodiment, the RNAi agent comprises the alternating motif model of 2'-O-methyl modification and 2'-F modification in the sense strand initially has an offset from the alternating motif model of 2'- O-methyl and 2'-F modification on the antisense tape initially, that is, the 2'-O-methyl modified nucleotide in the sense tape base pairs with a 2'-F modified nucleotide on the antisense tape and vice versa . Position 1 of the sense tape can start with the 2'-F modification, and position 1 of the antisense tape can start with the 2'-O-methyl modification.
[000172] The introduction of one or more motifs of three identical modifications in three consecutive nucleotides to the sense tape and / or antisense tape interrupts the initial modification model present in the sense tape and / or antisense tape. This interruption of the sense and / or antisense strand modification model by introducing one or more motifs of three identical modifications in three consecutive nucleotides to the sense and / or antisense strand surprisingly increases the gene silencing activity for the target gene.
[000173] In one embodiment, when the motif of three identical modifications in three consecutive nucleotides is introduced in any of the phytases, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence that contains the motif is "... NaYYYNb ...", where "Y" represents the motif modification of three identical modifications in three consecutive nucleotides and "Na" and "Nb" represents a modification to the nucleotide next to the “YYY” motif which is different than the Y modification, and where Na and Nb can be the same or different modifications. Alternatively, Na and / or Nb may be present or absent when a wing modification is present.
[000174] The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide bond. The modification of the phosphorothioate or methylphosphonate internucleotide bond can occur in any nucleotide of the sense tape or antisense tape or both in any position of the tape. For example, modification of internucleotide binding can occur in each nucleotide in the sense strand or antisense strand; each modification of internucleotide bonding can occur in an alternating pattern on sense tape or antisense tape; either the sense tape or the antisense tape can contain both modifications of internucleotide binding in an alternating model. The alternating model of the internucleotide bond modification on the sense tape can be the same or different from the antisense tape, and the alternate model of the internucleotide bond modification on the sense tape can have a shift from the alternate model of the internucleotide bond modification on the antisense tape .
[000175] In one embodiment, the RNAi comprises modification of the internucleotide bond phosphorothioate or methylphosphonate in the region of the protuberance. For example, the hump region can contain two nucleotides that have an internucleotide phosphorothioate or methylphosphonate bond between the two nucleotides. Modifications of internucleotide binding can also be done to link the nucleotides of the protuberance to nucleotides with paired terminals within the duplex region. For example, at least 2, 3, 4, or all of the lump nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide bonding, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide bonds that link the lump nucleotide with a paired nucleotide that is next to the lump nucleotide. For example, there can be at least two phosphorothioate internucleotide bonds between the three terminal nucleotides, in which two of the three nucleotides are lump nucleotides, and the third is a nucleotide paired next to the lump nucleotide. Preferably, these three terminal nucleotides can be at the 3 'end of the antisense strip.
[000176] In one embodiment, the RNAi agent comprises incompatibility (s) with the target, within the duplex, or combinations thereof. The mismatch can occur in the hump region or in the duplex region. The base pair can be placed based on its propensity to promote dissociation or fusion (for example, in the free association or dissociation energy of a particular match, the simplest approach is to examine the pairs on an individual pair basis, although later nearby neighborhood or similar analysis can also be used). As for the promotion of decoupling: A: U is preferred over G: C; G: U is preferred over G: C; and I: C is preferred over G: C (I = inosine). Incompatibilities, for example, non-canonical or except canonical pairings (as described elsewhere in this order) are preferred over canonical pairings (A: T, A: U, G: C); and pairings that include a universal base are preferred over canonical pairings.
[000177] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions of the 5 'end of the antisense tape can be chosen independently from the group of: A: U, G: U, I: C, and incompatible pairs, for example, non-canonical pairings or except canonical pairings that include a universal base, to promote the dissociation of the antisense tape at the 5 'end of the duplex.
[000178] In one embodiment, the nucleotide at 1 position within the 5 'end duplex region on the antisense tape is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region of the 5 'end of the antisense tape is an AU base pair. For example, the first base pair within the duplex region of the 5 'end of the antisense tape is an AU base pair.
[000179] In one embodiment, the sense tape sequence can be represented by the formula (I): 5 'np-Na- (XXX) i-Nb-YYY-Nb (ZZZ) j-Na-nq 3' (I) where: i and j are each independently 0 or 1; p and q are each independently 0-6; each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each Nb independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; each np and nq independently represent a lump nucleotide; where Na and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent a motif of three identical modifications on three consecutive nucleotides. Preferably YYY are all 2'-F modified nucleotides.
[000180] In one embodiment, Na and / or Nb comprise modifications of the alternating model.
[000181] In one embodiment, the YYY motif occurs within or near the cleavage site of the sense tape. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur within or in the vicinity of the cleavage site (for example: it can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11,12 or 11, 12, 13) of the sense strand, the count starting from the 1st nucleotide, from the 5 'end; or optionally, the count starting at the 1st paired nucleotide within the 5 'end duplex region.
[000182] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense tape, therefore, can be represented by the following formulas: 5 'np-Na- YYY-Nb-ZZZ-Na-nq 3 '(Ia); 5 'np-Na-XXX-Nb-YYY-Na-nq-3' (Ib); or 5 'np-Na-XXX-Nb-YYY-Na-ZZZ-Nb-nq 3' (Ic).
[000183] When the sense strand is represented by the formula (Ia), Nb represents a sequence of oligonucleotides comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
[000184] When the sense ribbon is represented according to formula (Ib), Nb represents a sequence of oligonucleotides comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
[000185] When the sense stripe is represented according to formula (Ic), each Nb independently represents a sequence of oligonucleotides comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
[000186] Each of X, Y and Z can be the same or different from each other.
[000187] In one embodiment, the RNAi antisense tape sequence can be represented by the formula (II): 5 'nq'-Na' - (Z'Z'Z ') k-Nb'-Y'Y'Y' Nb '- (X'X'X') l-N'a-np '3' (II) where: k and k are each independently 0 or 1; p 'and q' are each independently 0-6;
[000188] each Na 'independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each Nb 'independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; each np 'and nq' independently represents a lump nucleotide; where Nb ’and Y’ do not have the same modification; and X'X'X ', Y’Y’Y' and Z’Z'Z 'each independently represent a motif of three identical modifications on three consecutive nucleotides.
[000189] In one embodiment, Na 'and / or Nb' comprises modifications of the alternating model.
[000190] The Y'Y'Y 'motif occurs within or near the cleavage site of the antisense tape. For example, when the RNAi agent has a 17-23 nt long duplex region, the Y'Y'Y 'motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense tape, with the count starting from the 1st nucleotide, from the 5 'end; or optionally, the count starting at the 1st paired nucleotide within the 5 'end duplex region. Preferably, the Y'Y'Y 'motif occurs at positions 11, 12, 13.
[000191] In one embodiment, motif Y'Y'Y 'are all modified nucleotides of 2'-OMe.
[000192] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.
[000193] The antisense tape, therefore, can be represented by the following formulas: 5 'nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y' -Na '-np' 3 '( IIa); 5 ’nq’-Na’-Y’Y’Y’-Nb‘ -X’X’X ‘-np’ 3 ’(IIb); or 5 ’nq’-Na’-Z’Z’Z’-Nb’ ‘- Y’Y’Y‘ -Nb ‘-X’X’X‘ -Na ’-np’ 3 ’(IIc).
[000194] When the antisense tape is represented by the formula (IIa), Nb 'represents an oligonucleotide sequence comprising 010, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na 'independently represents a sequence of oligonucleotides comprising 2-20, 2-15, or 2-10 modified nucleotides.
[000195] When the antisense tape is represented according to formula (IIb), Nb 'represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0- 2 or 0 modified nucleotides. Each Na 'independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
[000196] When the antisense tape is represented according to formula (IIc), each Nb 'independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na 'independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6.
[000197] Each of X ’, Y’ and Z ’can be the same or different from each other.
[000198] Each nucleotide of the sense tape and antisense tape can be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-ally, 2'-C-ally, 2 ' -hydroxyl, 2'-deoxy or 2'-fluoro. For example, each nucleotide of the sense tape and antisense tape is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X ', Y' and Z ', in particular, can represent a 2'-O-methyl modification or a 2'-fluoro modification.
[000199] In one embodiment, the sense strand of the RNAi agent may contain the YYY motif that occurs at positions 9, 10 and 11 of the strand when the duplex region is 21 nt, the count starting from the 1st nucleotide of the 5 'end , or optionally, counting starting at the 1st paired nucleotide within the 5 'end duplex region; and Y represents 2'-F modification. The sense tape can additionally contain motif XXX or motifs ZZZ as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or 2'-F modification.
[000200] In one embodiment, the antisense tape may contain motif Y'Y'Y 'that occurs at positions 11, 12, 13 of the tape, the count starting from the 1st nucleotide of the 5' end, or optionally, the count starting at 1 ° paired nucleotide within the 5 'end duplex region; and Y 'represents 2'-O-methyl modification. The antisense tape may additionally contain X'X'X 'motif or Z'Z'Z' motifs as wing modifications at the opposite end of the duplex region; and X'X'X 'and Z'Z'Z' each independently represent a 2'-OMe modification or 2'-F modification.
[000201] The sense tape represented by any of the formulas above (Ia), (Ib) and (Ic) forms a duplex with an antisense tape that is represented by any of the formulas (IIa), (IIb) and (IIc) , respectively.
[000202] Consequently, the RNAi agents of the invention can comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III): sense: 5 'np-Na- (XXX ) i- Nb YYY -Na- (ZZZ) j-Na-nq 3 'antisense: 3' np'-Na '- (X'X'X') k-Nb'-Y'Y'Y'-Nb ' - (Z'Z'Z ') l-Na'- nq'5' (III) where: i, j, k, el are each independently 0 or 1; p, p ', q, and q' are each independently 0-6; each Na and Na 'independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each Nb and Nb 'independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; wherein each np ', np, nq', and nq independently represents a lump nucleotide; and XXX, YYY, ZZZ, X'X'X ', Y’Y'Y', and Z'Z'Z 'each independently represent a motif of three identical modifications on three consecutive nucleotides.
[000203] In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 1. In another embodiment, k is 1 and l is 0; k is 0 and l is 1; or both k and l are 1.
[000204] Exemplary combinations of sense tape and antisense tape forming an RNAi duplex include the formulas below: 5 'np- Na-Y YY -Na-Z Z Z-Na-nq 3' 3 'np'-Na'- Y'Y'Y '-Nb' -Z'Z'Z '-Na'nq'5' (IIIa) 5 'np-Na-X XX -Na-Y YY - Na-nq 3' 3 'np'- Na'-X'X'X '-Nb' -Y'Y'Y '-Na'-nq'5' (IIIb) 5 'np- Na-X XX - Nb-Y YY - Nb ZZ Z-Na- nq 3 '' 'X'X'X' 'Y'Y'Y' 'Z'Z'Z' '3 np -Na - -Nb - -Nb- -Na-nq 5 (IIIc)
[000205] When the RNAi agent is represented by the formula (IIIa), each Nb independently represents a sequence of modified oligonucleotides comprising 1-10, 1-7, 1-5 or 1-4 nucleotides. Each Na independently represents a sequence of oligonucleotides comprising 2-20, 2-15, or 2-10 modified nucleotides.
[000206] When the RNAi agent is represented according to formula (IIIb), each Nb, Nb 'independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
[000207] When the RNAi agent is represented according to formula (IIIc), each Nb, Nb 'independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0 -4, 0-2 or 0 modified nucleotides. Each Na, Na 'independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na ', Nb and Nb' independently comprises modifications of the alternating model.
[000208] Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and (IIIc) can be the same or different from each other.
[000209] When the RNAi agent is represented by the formula (III), (IIIa), (IIIb) or (IIIc), at least one of the Y nucleotides can form a base pair with one of the Y 'nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y 'nucleotides; or all three of the Y nucleotides, all form base pairs with the corresponding Y 'nucleotides.
[000210] When the RNAi agent is represented by the formula (IIIa) or (IIIc), at least one of the Z nucleotides can form a base pair with one of the Z 'nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z 'nucleotides; or all three of the Z nucleotides, all form base pairs with the corresponding Z 'nucleotides.
[000211] When the RNAi agent is represented according to formula (IIIb) or (IIIc), at least one of the X nucleotides can form a base pair with one of the X 'nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X 'nucleotides; or all three of the X nucleotides, all form base pairs with the corresponding X 'nucleotides.
[000212] In one embodiment, the change in nucleotide Y is different than the change in nucleotide Y ', the change in nucleotide Z is different than the change in nucleotide Z', and / or the change in nucleotide X is different from than the change in nucleotide X '.
[000213] In one embodiment, the RNAi agent is a multimer that contains at least two duplexes represented by the formula (III), (IIIa), (IIIb) or (IIIc), in which the duplexes are connected by a ligand. The binder can be cleavable or non-cleavable. Optionally, the multimer further comprises a binder. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.
[000214] In one embodiment, the RNAi agent is a multimer that contains three, four, five, six or more duplexes represented by the formula (III), (IIIa), (IIIb) or (IIIc), in which the duplexes are connected by a linker. The binder can be cleavable or non-cleavable. Optionally, the multimer further comprises a binder. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.
[000215] In one embodiment, two RNAi agents represented by the formula (III), (IIIa), (IIIb) or (IIIc) are linked together at the 5 'end, and one or both 3' ends of being optionally conjugated to a binder. Each of the agents can target the same gene or two different genes; or each of the agents can target the same gene at two different target sites.
[000216] Several publications describe multimedia RNAi agents. Such publications include WO2007 / 091269, US Patent No. 7858769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 the entire contents of which are hereby incorporated into this application by reference.
[000217] The RNAi agent that contains conjugations of one or more carbohydrate moieties to an RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be linked to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced by another portion, for example, a non-carbohydrate (preferably cyclic) vehicle to which a carbohydrate linker is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has thus been substituted is referred to in this application as a ribose substitution modification subunit (RRMS). A cyclic vehicle can be a carbocyclic ring system, that is, all ring atoms are carbon atoms or a heterocyclic ring system, i.e., one or more ring atoms can be a hetero atom, for example, nitrogen, oxygen, sulfur. The cyclic vehicle may be a monocyclic ring system or may contain two or more rings, for example, fused rings. The cyclic vehicle can be a fully saturated ring system, or it can contain one or more double bonds.
[000218] The linker can be connected to the polynucleotide via a vehicle. Vehicles include (i) at least one “skeleton connection point”, preferably two “skeleton connection points” and (ii) at least one “mooring connection point”. A "skeleton attachment point", as used in this application, refers to a functional group, for example, a hydroxyl group, or generally, a bond available to, and is suitable for incorporating the vehicle into the skeleton, for example , phosphate, or modified phosphate, for example, sulfur-containing skeleton of a ribonucleic acid. A “mooring connection point” (TAP), in some embodiments, refers to a ring atom that is part of the cyclic vehicle, for example, a carbon atom or a heteroatom (distinct from an atom that provides a connection point to the skeleton), which links a selected portion. The portion can be, for example, a carbohydrate, for example, monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected portion is connected by an intervening mooring to the cyclic vehicle. Thus, the cyclic vehicle will often include a functional group, for example, an amino group, or generally provide a bond, which is suitable for the incorporation or binding of another chemical entity, for example, a linker to the constituent ring.
[000219] RNAi agents can be conjugated to a linker via a vehicle, wherein the vehicle can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, pyridine; preferably, the acyclic group is selected from the serinol backbone or diethanolamine backbone.
[000220] In certain specific embodiments, the RNAi agent of the invention is the agent selected from the group of agents listed in Table 1 and consisting of D1000, D1001, D1002, D1003, D1004, D1005, D1006, D1007, D1008, D1009 , D1010, D1011, D1012, D1013, D1014, D1015, D1016, D1017, D1018, D1019, D1020, D1021, D1022, D1023, D1024, D1025, D1026, D1027, D1028, D1029, D1030, D1031, D1032, D1033, D1032, D1033 , D1035, D1036, D1037, D1038, D1039, D1040, D1041, D1042, D1043, D1044, D1045, D1046, D1047, D1048, D1049, D1050, D1051, D1052, D1053, D1054, D1055, D1056, D1057, D1058 , D1060, D1061, D1062, D1063, D1064, D1065, D1066, D1067, D1068, D1069, D1070, D1071, D1072, D1073, D1074, D1075, D1076, D1077, D1078, D1079, D1080, D1081, D1082, D1083, D1082, D1083 , D1085, D1086, D1087, D1088, D1089, D1090, D1091, D1092, D1093, D1094, D1095, D1096, D1097, D1098, D1099, D1100, D1101, D1102, D1103, D1104, D1105, D1010, D1010, D1010, D1010, D1010, D1010, D1010, D1010, D1010, D1010 , D1110, D1111, D1112, D1113, D1114, D1115, D1116, D1117, D1118, D111 9, D1120, D1121, D1122, D1123, D1124, D1125, D1126, D1127, D1128, D1129, D1130, D1131, D1132, D1133, D1134, D1135, D1136, D1137, D1138, D1139, D1140, D11 D1150, D1151, D1158, D1159, D1160, D1167, D1168, D1169, D1176, D1177, D1178, D1185, D1186, D1187, D1194, D1195, D1196, D1203, D1204, D1205, D1212, D1213, D1214, D1214, D1214, D1214, D1214, D1214, D1214, D1214, D1214, D1214, D1214, D1214 D1223, D1230, D1231, D1232, D1239, D1240, D1241, D1248, D1249, D1250, D1257, D1258, D1259, D1266, D1267, D1268, D1275, D1276, D1277, D1284, D1285, D1286, D1293, D1294, D1294, D1294, D1294 D1302, D1303, D1304, D1311, D1312, D1313, D1320, D1321, D1322, D1329, D1330, D1331, D1338, D1339, D1340, D1347, D1348, D1349, D1356, D1357, D1358, D1365, D1366, D1366, D1366, D1366, D1366, D1366, D1366, D1366, D1366 D1375, D1376, D1383, D1384, D1385, D1392, D1393, D1394, D1401, D1402, D1403, D1143, D1144, D1145, D1152, D1153, D1154, D1161, D1162, D1163, D1170, D1171, D1172, D1172, D1172, D1172 D1181, D1188, D1189, D1190, D1197, D1198, D1199, D1206, D1207, D1208, D1215, D1216, D1217, D1224, D1225, D1226, D1233, D1234, D12 35, D1242, D1243, D1244, D1251, D1252, D1253, D1260, D1261, D1262, D1269, D1270, D1271, D1278, D1279, D1280, D1287, D1288, D1289, D1296, D1297, D1298, D1305, D1306, D1306, D1306, D1306, D1306, D1306 D1314, D1315, D1316, D1323, D1324, D1325, D1332, D1333, D1334, D1341, D1342, D1343, D1350, D1351, D1352, D1359, D1360, D1361, D1368, D1369, D1370, D1377, D1378, D1379, D1379, D1379, D1379 D1387, D1388, D1395, D1396, D1397, D1404, D1405, D1406, D1146, D1147, D1148, D1155, D1156, D1157, D1164, D1165, D1166, D1173, D1174, D1175, D1182, D1183, D1184, D92 D1193, D1200, D1201, D1202, D1209, D1210, D1211, D1218, D1219, D1220, D1227, D1228, D1229, D1236, D1237, D1238, D1245, D1246, D1247, D1254, D1255, D1256, D1263, D1264, D1263, D1264, D1263 D1272, D1273, D1274, D1281, D1282, D1283, D1290, D1291, D1292, D1299, D1300, D1301, D1308, D1309, D1310, D1317, D1318, D1319, D1326, D1327, D1328, D1335, D1336, D1336, D1336 D1345, D1346, D1353, D1354, D1355, D1362, D1363, D1364, D1371, D1372, D1373, D1380, D1381, D1382, D1389, D1390, D1391, D1398, D1 399, D1400, D1407, D1408, D1409, D1410, D1411, D1412, D1419, D1420, D1421, D1428, D1429, D1430, D1437, D1438, D1439, D1446, D1447, D1448, D1455, D1456, D1457, D1445 D1466, D1473, D1474, D1475, D1482, D1483, D1484, D1491, D1492, D1493, D1500, D1501, D1502, D1509, D1510, D1511, D1518, D1519, D1520, D1527, D1528, D1529, D1538, D1529 D1545, D1546, D1547, D1554, D1555, D1556, D1563, D1564, D1565, D1572, D1573, D1574, D1581, D1582, D1583, D1590, D1591, D1592, D1599, D1600, D1601, D1608, D1609, D1060 D1618, D1619, D1626, D1627, D1628, D1635, D1636, D1637, D1644, D1645, D1646, D1653, D1654, D1655, D1662, D1663, D1664, D1671, D1672, D1673, D1413, D1414, D1415, D1422, D1422, D1422 D1424, D1431, D1432, D1433, D1440, D1441, D1442, D1449, D1450, D1451, D1458, D1459, D1460, D1467, D1468, D1469, D1476, D1477, D1478, D1485, D1486, D1487, D1494, D1494, D1494, D1494, D1494 D1503, D1504, D1505, D1512, D1513, D1514, D1521, D1522, D1523, D1530, D1531, D1532, D1539, D1540, D1541, D1548, D1549, D1550, D 1557, D1558, D1559, D1566, D1567, D1568, D1575, D1576, D1577, D1584, D1585, D1586, D1593, D1594, D1595, D1602, D1603, D1604, D1611, D1612, D1613, D1620, D1621, D1621, D1621, D1621, D1621, D1621 D1630, D1631, D1638, D1639, D1640, D1647, D1648, D1649, D1656, D1657, D1658, D1665, D1666, D1667, D1674, D1675, D1676, D1416, D1417, D1418, D1425, D1426, D1427, D1434, D1434, D1434, D1434, D1434 D1436, D1443, D1444, D1445, D1452, D1453, D1454, D1461, D1462, D1463, D1470, D1471, D1472, D1479, D1480, D1481, D1488, D1489, D1490, D1497, D1498, D1499. D1506, D1507, D1508, D1515, D1516, D1517, D1524, D1525, D1526, D1533, D1534, D1535, D1542, D1543, D1544, D1551, D1552, D1553, D1560, D1561, D1562, D1569, D1570, D1569 D1579, D1580, D1587, D1588, D1589, D1596, D1597, D1598, D1605, D1606, D1607, D1614, D1615, D1616, D1623, D1624, D1625, D1632, D1633, D1634, D1641, D1642, D1643, D1642, D1643 D1652, D1659, D1660, D1661, D1668, D1669, D1670, D1677, D1678, D1679, D1680, D1681, D1682, D1689, D1690, D1691, D1698, D1699, D1700, D1707, D1708, D1709, D1716, 1717 D1725, D1726, D1727, D1734, D1735, D1736, D1743, D1744, D1745, D1752, D1753, D1754, D1761, D1762, D1763, D1770, D1771, D1772, D1779, D1780, D1781, D1788, D1789, D1790, D1790, D1790, D1790, D1790 D1798, D1799, D1806, D1807, D1808, D1815, D1816, D1817, D1824, D1825, D1826, D1833, D1834, D1835, D1842, D1843, D1844, D1851, D1852, D1853, D1860, D1861, D1862, D1861, D1862 D1871, D1878, D1879, D1880, D1887, D1888, D1889, D1896, D1897, D1898, D1905, D1906, D1907, D1914, D1915, D1916, D1923, D1924, D1925, D1932, D1933, D1934, D1941, D1942, D1943, D1683, D1684, D1685, D1692, D1693, D1694, D1701, D1702, D1703, D1710, D1711, D1712, D1719, D1720, D1721, D1728, D1730 D1737, D1738, D1739, D1746, D1747, D1748, D1755, D1756, D1757, D1764, D1765, D1766, D1773, D1774, D1775, D1782, D1783, D1784, D1791, D1792, D1793, D1800, D1801, D1801, D1801, D1801 D1810, D1811, D1818, D1819, D1820, D1827, D1828, D1829, D1836, D1837, D1838, D1845, D1846, D1847, D1854, D1855, D1856, D1863, D1864, D1865, D1872, D1873, D1874, D1881 D1883, D1890, D1891, D1892, D1899, D1900, D1901, D1908, D1909, D1910, D1917, D1918, D1919, D1926, D1927, D1928, D1935, D1936, D1937, D1944, D1945, D1946, D1686, D1687, D1695, D1696, D1697, D1704, D1705, D1706, D1713, D1714, D1715, D1722, D1723, D1724, D1731, D1732, D1733, D1740, D1741, D1742, D1749, D1750, D1751, D1758, D1759, D1759, D1759, D1768, D1769, D1776, D1777, D1778, D1785, D1786, D1787, D1794, D1795, D1796, D1803, D1804, D1805, D1812, D1813, D1814, D1821 , D1822, D1823, D1830, D1831, D1832, D1839, D1840, D1841, D1848, D1849, D1850, D1857, D1858, D1859, D1866, D1867, D1868, D1875, D1876, D1877, D1884, D1885, D1886, D1893, D1886, D1893 , D1895, D1902, D1903, D1904, D1911, D1912, D1913, D1920, D1921, D1922, D1929, D1930, D1931, D1938, D1939, D1940, D1947, D1948, D1949, D1950, D1951, D1952, D1953, D1954, D1953 , D1956, D1957, D1958, D1959, D1960, D1961, D1962, D1963, D1964, D1965, D1966, D1967, D1968, D1969, D1970, D1971, D1972, D1973, D1974, D1975, D1976, D1977, D1978, D1979 , D1981, D1982, D1983, D1984, D1985, D1986, D1987, D1988, D1989, D1990, D1991, D1992, D1993, D1994, D1995, D1996, D1997, D1998, D1999, D2000, D2001, D2002, D2003, D2004, D2004, D2004, D2004 , D2006, D2007, D2008, D2009, D2010, D2011, D2012, D2013, D2014, D2015, D2016, D2017, D2018, D2019, D2020, D2021, D2022, D2023, D2024, D2025, D2026, D2027, D2028, D2029, D2028, D2029, D2028, D2029, D2028 , D2031, D2032, D2033, D2034, D2035, D2036, D2037, D2038, D2039, D2040, D2041, D2042, D2043, D2044, D2045, D2046, D2047, D204 8, D2049, D2050, D2051, D2052, D2053, D2054, D2055, D2056, D2057, D2058, D2059, D2060, D2061, D2062, D2063, D2064, D2065, D2066, D2067, D2068, D2069, D2070, D2071, D2072, D2071, D2070 D2073, D2074, D2075, D2076, D2077, D2078, D2079, D2080, D2081, D2082, D2083, D2084, D2085, D2086, D2087, D2088, D2089, D2090 and D2091.
[000221] These agents can further comprise a linker, such as a GalNAc linker. Binders
[000222] The RNAi agents of the invention, for example, double-stranded RNAi agents, can optionally be conjugated to one or more ligands. The binder can be attached to sense tape, antisense tape or both strips, at the 3 'end, 5' end or both ends. For example, the binder can be attached to the sense tape. In preferred embodiments, the binder is attached to the 3 'end of the sense tape. In a preferred embodiment, the ligand is a GalNAc ligand. In particularly preferred embodiments, the ligand is GalNAc3:

[000223] A wide variety of entities can be coupled to the RNAi agents of the present invention. Preferred portions are binders, which are coupled, preferably covalently, directly or indirectly through an intervening bond.
[000224] In preferred modalities, a ligand alters the distribution, targeting or life span of the molecule in which it is incorporated. In preferred embodiments, a ligand provides increased affinity for a selected target, for example, molecule, cell or cell type, compartment, receptor, for example, a cell compartment or compartment of organs, tissue, organ or region of the body, such as, for example, compared to a species that such a linker is absent. Binders that provide increased affinity for a selected target are also called targeting ligands.
[000225] Some binders may have endosolytic properties. Endosomal ligands promote lysis of the endosome and / or transport of the composition of the invention or its components, from the endosome to the cell's cytoplasm. The endossomolytic ligand can be a polyanionic or peptideomimetic peptide that shows pH-dependent membrane activity and fusogenicity. In one embodiment, the endosolytic ligand assumes its active conformation at the endosomal pH. The "active" conformation is that the conformation in which the endosomal ligand promotes lysis of the endosome and / or transport of the composition of the invention or its components, from the endosome to the cell cytoplasm. Exemplary endosolytic linkers include the GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586) , and its derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endosolytic component may contain a chemical group (for example, an amino acid) that will undergo a modification in charge or protonation in response to a change in pH. The endosolytic component can be linear or branched.
[000226] The linkers can improve transport, hybridization and specificity properties and can also improve the nuclease resistance of the resulting natural or modified oligoribonucleotide or polymeric molecule comprising any combination of monomers described in this application and / or natural or modified ribonucleotides.
[000227] Binders, in general, may include therapeutic modifiers, for example, to increase absorption; diagnostic compounds or reporter groups, for example, to monitor distribution; interconnecting agents; and conference portions of nuclease resistance. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimetics.
[000228] The binders may include a naturally occurring substance, such as a protein (for example, human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL) or globulin); a carbohydrate (for example, a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The linker can also be a recombinant or synthetic molecule, such as a synthetic polymer, for example, a synthetic polyamino acid, an oligonucleotide (for example, an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, maleic acid styrene anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, ether-divinyl copolymer maleic anhydride, copolymer of N-2- (hydroxypropyl) methacrylamide (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethylacrylic acid), polymers of N-isopropylacrylamide or polyphosphazine. Examples of polyamines include: polyethyleneimine, polylysine (PLL), sperm, spermidine, polyamine, pseudopeptide-polyamine, peptideomimetic-polyamine, polyamine dendrimer, arginine, amidine, protamine, cationic lipid, cationic porphyrin, salt of a polyamine or an alpha helical peptide.
[000229] The ligands can also include targeting groups, for example, a cell or tissue targeting agent, for example, a lectin, glycoprotein, lipid or protein, for example, an antibody, which binds to a specified cell type like a kidney cell. A targeting group can be a thyrotropin, melano-tropin, lectin, glycoprotein, protein A surfactant, carbohydrate Mucine, multivalent lactose, multivalent galactose, N-acetyl-galactose-mine, N-acetyl-glucosamine, multivalent mannose, multivalent glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.
[000230] Other examples of binders include dyes, intercalating agents (for example, acridines), interlinkers (for example, psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (for example, phenazine, dihydrophenazine) , artificial endonucleases or a chelator (eg EDTA), lipophilic molecules, eg cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O- (hexadecyl) glycerol, group geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, hepta-decyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) collenic acid, dimethoxytrityl, or phenoxazine) and conjugates peptides (for example, antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (for example, PEG-40K), MPEG, [MPEG] 2, polyamine, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (for example, Biotin), transport / absorption facilitators (eg, aspirin, vitamin E, folic acid), synthetic ribonucleases (eg, imidazole, bisimidazole, histamine, imidazole, acridine-imidazole conjugates, Eu3 + complexes of tetra-azamacrocycles ), dinitrophenyl, HRP or AP.
[000231] The ligands can be proteins, for example, glycoproteins or peptides, for example, molecules having a specific affinity for a ligand, or antibodies, for example, an antibody, which binds to a cell type specified as a cancer cell , endothelial cell or bone cell. Binders can also include hormones and hormone receptors. They may also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactose-mine, N-acetyl-glucosamine, multivalent mannose, multivalent fucose or aptamers. The linker can be, for example, lipopolysaccharide, an activator of MAP kinase p38 or an activator of NF-KB.
[000232] The ligand can be a substance, for example, a drug, which can increase the absorption of the iRNA agent in the cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latruncinin A, phalloidin, swinholide A, indanocin, or myoservin.
[000233] The ligand can increase the absorption of the oligonucleotide in the cell, for example, by activating an inflammatory response. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin 1 beta or interferon gamma.
[000234] In one aspect, the linker is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds to a whey protein, for example, human serum albumin (HSA). An HSA-binding ligand allows delivery of the conjugate to a target tissue, for example, a non-renal target tissue in the body. For example, the target tissue can be the liver, including liver parenchyma cells. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase the resistance to degradation of the conjugate, (b) increase targeting or transport in a target cell or cell membrane and / or (c) can be used to adjust binding to a whey protein, for example, HSA.
[000235] A lipid-based ligand can be used to modulate, for example, control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that bind HSA more strongly will be less likely to target the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that bind HSA less strongly can be used to target the conjugate to the kidney.
[000236] In a preferred embodiment, the lipid-based binder binds to HSA. Preferably, it binds to HSA with sufficient affinity such that the conjugate will preferably be delivered to non-renal tissue. However, it is preferable that the affinity is not so strong that the HSA-ligand bond cannot be reversed.
[000237] In another preferred embodiment, the lipid-based ligand binds to the HSA weakly or in no way, such that the conjugate will preferably be distributed to the kidney. Other portions that target kidney cells can also be used in place of or in addition to the lipid-based ligand.
[000238] In another aspect, the ligand is a portion, for example, a vitamin, which is absorbed by a target cell, for example, a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, for example, malignant or non-malignant, for example, cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamins, for example, folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients absorbed by cancer cells. Also included are SAH, low density lipoprotein (LDL) and high density lipoprotein (HDL).
[000239] In another aspect, the linker is a cell permeation agent, preferably a helical cell permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmethyl, invertomers, non-peptide or pseudopeptide bonds and the use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
[000240] The linker can be a peptide or peptideomimetic. A peptideomimetic (also referred to in this application as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptideomimetic portion can be approximately 5-50 amino acids in length, for example, approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. A peptide or peptideomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide or hydrophobic peptide (for example, consisting mainly of Tyr, Trp or Phe). The peptide moiety can be a peptide dendrimer, restricted peptide or interconnected peptide. In another alternative, the peptide moiety may include a hydrophobic membrane displacement (MTS) sequence. An exemplary hydrophobic peptide containing MTS is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 4). An RFGF analogue (e.g., AALLPVLLAAP amino acid sequence) (SEQ ID NO: 5) containing a hydrophobic MTS can also be a targeting moiety. The peptide portion can be a "delivery" peptide, which can transport large polar molecules including peptides, oligonucleotides and protein across cell membranes. For example, the sequences of the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO: 6) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO: 7) have been found to be able to function as delivery peptides. A peptide or peptideomimetic can be encoded by a random DNA sequence, such as a peptide identified from a phage display library or one-count-a-compound combinatorial library (OBOC) (Lam et al., Nature, 354: 82- 84, 1991). Preferably, the peptide or peptideomimetic bound to an iRNA agent via an included monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD) or RGD mimetic peptide. A peptide portion can vary in length from approximately 5 amino acids to approximately 40 amino acids. The peptide moieties may have a structural modification, for example, to increase stability or direct conformational properties. Some of the structural modifications described below can be used. A portion of RGD peptide can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62: 513943, 2002). An RGD peptide can facilitate the targeting of an iRNA agent to tumors in a variety of other tissues, including lung, kidney, spleen or liver (Aoki et al., Cancer Gene Therapy 8: 783-787, 2001). Preferably, the RGD peptide will facilitate targeting an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, for example, glycosylated or methylated to facilitate targeting specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell that expresses αVβ3 (Haubner et al., Jour. Nucl. Med., 42: 326-336, 2001). Peptides that target enriched markers in proliferative cells can be used. For example, RGD-containing peptides and peptideomimetics can target cancer cells, in particular cells that exhibit an integrin. In this way, one can use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimetics. In addition to RGD, other portions that target the integrin ligand can be used. Generally, such ligands can be used to control proliferative cells and angiogenesis. Preferred conjugates of this type of linker target PECAM-1, VEGF, or another cancer gene, for example, a cancer gene described in this application.
[000241] A "cell permeation peptide" is capable of permeating a cell, for example, a microbial cell, such as a bacterial or fungal cell or mammalian cell, such as a human cell. A microbial peptide that permeates the cell can be, for example, an α-helical linear peptide (for example, LL-37 or Ceropine P1), a disulfide-containing peptide (for example, α-defensin, β-defensin or bacterenecin) , or peptide containing only one or two dominant amino acids (for example, PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the HIV 1 gp41 fusion peptide domain and the NLS of the large SV40 T antigen (Simeoni et al., Nucl. Acids Res. 31: 2717-2724, 2003).
[000242] In one embodiment, a targeting peptide can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradoxines, buforin, CPF, bombinin-like peptide (BLP), catelicidins, keratotoxins, S. clava peptides, intestinal myxin antimicrobial peptides (HFIAPs), magainins, brevinines-2, dermaseptins, melitins, pleurocidin, H2A peptides, Xenopus peptides, esculetins-1, and caerines. Preferably, it will be considered that the various factors will maintain the integrity of the propeller stability. For example, a maximum number of helix stabilization residues will be used (eg, leu, ala, or lys), and minimum number helix destabilization residues will be used (eg, proline or cyclic monomeric units). The terminator residue will be considered (for example, Gly is an exemplary N-terminator residue and / or C-terminal amidation can be used to provide an extra H bond to stabilize the helix. The formation of salt bridges between residues with opposite charges, separated by i ± 3 or i ± 4 positions can provide stability, for example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form saline bridges with anionic glutamate or aspartate residues.
[000243] Peptide and peptideomimetic linkers include those peptides having naturally occurring or modified, for example, D or L peptides; α, β, or Y peptides; N-methyl peptides; azapeptides; peptides having one or more amides, i.e., peptide, bonds substituted by one or more of urea, thiourea, carbamates or sulfonyl urea bonds; or cyclic peptides.
[000244] The targeting linker can be any linker that is capable of targeting a specific receptor. Examples are folate, GalNAc, galactose, mannose, mannose-6P, sugar groups such as GalNAc group, mannose group, galactose group or an aptamer. A group is a combination of two or more units of sugar. Targeting ligands also include integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. The linkers can also be based on nucleic acid, for example, an aptamer. The aptamer may be unmodified or have any combination of the modifications described in this application.
[000245] Endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polyaboxylates, polycations, masked or poly-cations or - anions, acetals, polyacetals, cetas / polyetals, orthoesters, polymers with cationic charges or masked or non-masked anionic, dendrimers with masked or non-masked cationic or anionic charges.
[000246] The PK modulator means pharmacokinetic modulator. PK modulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binding agents, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. It is also known that oligonucleotides comprising several phosphorothioate bonds bind to whey protein, thus short oligonucleotides, for example, approximately 5 base, 10 base, 15 base or 20 base oligonucleotides, comprising multiple phosphorothioate bonds in the skeleton they are also accessible to the present invention as binders (for example, as PK modulation of binders).
[000247] In addition, aptamers that bind whey components (e.g., whey proteins) are also accessible to the present invention as PK modulation of ligands.
[000248] Other conjugates of binders accessible to the invention are described in U.S. Patent Applications USSN: 10 / 916,185, filed August 10, 2004; USSN: 10 / 946,873, deposited on September 21, 2004; USSN: 10 / 833,934, deposited on August 3, 2007; USSN: 11 / 115,989 filed on April 27, 2005 and USSN: 11 / 944,227 filed on November 21, 2007, which are incorporated by reference in their entirety for all purposes.
[000249] When two or more ligands are present, the ligands can all have the same properties, all have different properties or some ligands have the same properties while the others have different properties. For example, a ligand may have targeting properties, endosolytic activity or PK modulating properties. In a preferred embodiment, all binders have different properties.
[000250] The linkers can be coupled to the oligonucleotides in various places, for example, 3 'end, 5' end, and / or in an internal position. In preferred embodiments, the linker is attached to the oligonucleotides via an intervening mooring, for example, a vehicle described in this application. The ligand or bound ligand may be present in a monomer when the monomer is incorporated into the growing ribbon. In some embodiments, the binder can be incorporated by coupling it to a "precursor" monomer after the "precursor" monomer has been incorporated into the growing ribbon. For example, a monomer having, for example, an amino-terminated bond (i.e., having no associated linker), for example, TAP- (CH2) nNH2 can be incorporated into a growing oligonucleotide strip. In a subsequent operation, that is, after incorporation of the precursor monomer in the strip, a binder having an electrophilic group, for example, a pentafluorophenyl ester or aldehyde group, can later be linked to the precursor monomer by coupling the electrophilic group of the ligand with the group terminal nucleophilic linkage of the precursor monomer.
[000251] In another example, a monomer having a suitable chemical group to take part in the Click Chemistry reaction can be incorporated, for example, an azide or alkaline terminated linker. In a subsequent operation, that is, after incorporation of the precursor monomer into the strip, a binder that has a complementary chemical group, for example, an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and azide together.
[000252] For double-stranded oligonucleotides, the ligands can be attached to one or both phytases. In some embodiments, a double-stranded iRNA agent contains a ligand conjugated to the sense strand. In other embodiments, a double-stranded iRNA agent contains a ligand conjugated to the antisense strand.
[000253] In some embodiments, the ligand may be conjugated to nucleobases, sugar moieties or internucleoside bonds of nucleic acid molecules. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, positions 2, 6, 7, or 8 of a purine nucleobase are attached to a conjugated portion. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, positions 2, 5, and 6 of a pyrimidine nucleobase can be replaced by a conjugated portion. Conjugation to sugar moieties of nucleosides can occur on any carbon atom. Examples of carbon atoms of a sugar moiety that can be attached to a conjugated moiety include carbon atoms 2 ', 3', and 5 '. The 1 'position can also be attached to a conjugated portion, such as an abasic residue. Internucleoside bonds can also carry conjugated portions. For phosphorus-containing bonds (for example, phosphodiester, phosphorothioate, phosphorodithiothate, phosphoramidate, and the like), the conjugated portion may be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. For internucleoside bonds containing amine or amide (e.g., PNA), the conjugated portion can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
[000254] Any suitable ligand in the field of interfering RNA can be used, although the ligand is typically a carbohydrate, for example, monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, polysaccharide.
[000255] The ligands that conjugate the ligand to the nucleic acid include those discussed above. For example, the linker can be one or more derivatives of GalNAc (N-acetylglucosamine) linked by a divalent or trivalent branched linker.
[000256] In one embodiment, the dsRNA of the invention is conjugated to bivalent and trivalent branched ligands that include the structures shown in any of formulas (IV) - (VII):
where: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5Crepresent independently for each occurrence 0-20 and where the repetition unit can be the same or different; P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5Care each independently for each absent occurrence, CO, NH, O, S , OC (O), NHC (O), CH2, CH2NH or CH2O; Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each missing occurrence, alkylene, substituted alkylene in which one or more methylenes can be interrupted or terminated by one or more of O, S, S (O ), SO2, N (RN), C (R ') = C (R''), C = C or C (O); R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5Care each independently for each missing occurrence, NH, O, S, CH2, C (O) O, C (O) NH, NHCH (Ra) C (O), -C (O) -CH (Ra) -NH-, CO, CH = NO, S — S
or heterocyclyl; L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5Crepresent the ligand; this is one independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide or polysaccharide; and Raé H or amino acid side chain.
[000257] Derivatives of trivalent conjugation of GalNAc are particularly useful for use with RNAi agents to inhibit expression of a target gene, such as those of formula (VII): Formula (VII)
where L5A, L5B and L5C represent a monosaccharide, as derived from GalNAc.
[000258] Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the following compounds


[000259] In other embodiments, the RNAi agent of the invention is the agent selected from the group consisting of AD-45163, AD-45165, AD-51544, AD-51545, AD-51546 and AD-51547 III. Pharmaceutical Compositions
[000260] The RNAi agents of the invention can be formulated for administration in any way suitable for use in veterinary or human medicine, by analogy with other pharmacists. Pharmaceutical compositions comprising the RNAi agents of the invention can, for example, be solutions with or without a buffer or compositions containing pharmaceutically acceptable carriers. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micelle formulations, emulsions and gene therapy vectors.
[000261] In the methods of the invention, the RNAi agent can be administered in a solution. A free RNAi agent can be administered in an un-buffered solution, for example, in saline or water. Alternatively, free siRNA can also be administered in a suitable buffer solution. The buffer solution can comprise acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. In a preferred embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administration to an individual.
[000262] In some embodiments, the buffer solution further comprises an agent for controlling the osmolarity of the solution, such that the osmolarity is maintained at a desired value, for example, by the physiological values of human plasma. The solutes that can be added to the buffer solution to control osmolarity include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids or salts. In some embodiments, the agent for controlling the osmolarity of the solution is a salt. In certain embodiments, the agent for controlling the osmolarity of the solution is sodium chloride or potassium chloride.
[000263] In other embodiments, the RNAi agent is formulated as a composition that includes one or more RNAi agents and a pharmaceutically acceptable carrier. As used in this application, the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics and absorption retarding agents, and the like, compatible with pharmaceutical administration. The use of such means and agents of pharmaceutically active substances is well known in the art. Except to the extent that any conventional means or agent are incompatible with the active compound, their use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
[000264] In one embodiment, the RNAi agent preparation includes at least one second therapeutic agent (for example, an agent except RNA or DNA). For example, an RNAi agent composition for the treatment of a disease associated with TTR, for example, a hereditary transthyretin-related amyloidosis (familial amyloidotic polyneuropathy, FAP), may include a known FAP-enhancing drug, for example, Tafamidis (INN, or Fx-1006A or Vyndaqel).
[000265] A formulated RNAi agent composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (for example, contains less than 80, 50, 30, 20, or 10% water). In another example, the RNAi agent is in an aqueous phase, for example, in a solution that includes water.
[000266] The aqueous phase or crystalline compositions can be incorporated into a delivery vehicle, for example, a liposome (particularly for the aqueous phase) or a particle (for example, a microparticle as may be suitable for a crystalline composition). Generally, the RNAi agent composition is formulated in a manner that is compatible with the desired method of administration, as described in this application. For example, in particular modes the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying or a combination of these techniques; or sonication with a lipid, vacuum freezing, condensation and other self-aggregation.
[000267] An RNAi agent preparation can be formulated in combination with another agent, for example, another therapeutic agent or an agent that stabilizes the RNAi agent, for example, a protein that complexes with the RNAi agent to form an iRNP . Still other agents include chelators, for example, EDTA (for example, to remove divalent cations such as Mg2 +), salts, RNAse inhibitors (for example, a broadly specific RNAse inhibitor such as RNAsin) and the like.
[000268] In one embodiment, the RNAi agent preparation includes another siRNA compound, for example, a second RNAi agent that can mediate RNAi with respect to a second gene, or with respect to the same gene. Yet another preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different species of RNAi agent. Such RNAi agents can mediate RNAi with respect to a similar number of different genes.
[000269] The iRNA agents of the invention can be formulated for pharmaceutical use. Pharmaceutically acceptable compositions comprise a therapeutically or prophylactically effective amount of one or more of the dsRNA agents in any of the foregoing embodiments, taken alone or formulated in conjunction with one or more pharmaceutically acceptable vehicles, excipients and / or diluents ( additions).
[000270] Methods of preparing pharmaceutical compositions of the invention include the step of combining an RNAi agent of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately associating an RNAi agent of the present invention with liquid vehicles, or finely divided solid vehicles, or both, and then, if necessary, by shaping the product.
[000271] Pharmaceutical compositions can be specially formulated for administration in solid or liquid form, including those adapted to the following: (1) oral administration, for example, potions (aqueous solutions or non-aqueous solutions or suspensions), tablets, for example , those aimed at oral, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection, such as, for example, a sterile solution or suspension or sustained release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release adhesive or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.
[000272] The phrase “pharmaceutically acceptable” is used in this application to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, proportional to a reasonable benefit / risk ratio.
[000273] The phrase "pharmaceutically acceptable vehicle", as used in this application, means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (for example, lubricant, magnesium talc, calcium or zinc stearate or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound object of an organ or body part, to another organ or body part. Each vehicle must be "acceptable" in terms of being compatible with other ingredients of the composition and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cotton oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) water without pyrogens; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) solutions with buffered pH; (21) polyester, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) component of serum, such as serum albumin, HDL and LDL; and (22) other compatible non-toxic substances used in pharmaceutical compositions.
[000274] The compositions can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmacy art. The amount of RNAi agent that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. The RNAi agent that can be combined with a carrier material to produce a single dosage form will generally be that amount of the RNAi agent that produces a desired effect, for example, therapeutic or prophylactic effect. Generally, out of one hundred percent, this amount will vary from approximately 0.1 percent to approximately ninety-nine percent of the RNAi agent, preferably from approximately 5 percent to approximately 70 percent, even more preferably from approximately 10 percent approximately 30 percent.
[000275] In certain embodiments, a composition of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, for example, bile acids, and polymeric vehicles, for example, polyester and polyanhydrides ; and an RNAi agent of the present invention. In certain embodiments, a composition mentioned above makes an RNAi agent of the present invention orally bioavailable.
[000276] In some cases, in order to prolong the effect of an RNAi agent, it is desirable to reduce the absorption of the subcutaneous or intramuscular injection agent. This can be accomplished by using a liquid suspension of the crystalline or amorphous material having low water solubility. The rate of absorption of the RNAi agent then depends on its rate of dissolution which, in turn, may depend on the crystalline size and the crystalline form. Alternatively, delayed absorption of a parenterally administered RNAi agent can be accomplished by dissolving or suspending the agent in an oily vehicle. Liposomes
[000277] An RNAi agent of the invention can be formulated for delivery in a membranous molecular aggregation, for example, a liposome or a micelle. As used in this application, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, for example, a bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the RNAi agent composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. As the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer merges with the bilayer of cell membranes. As the liposome and cell fusion progresses, the internal aqueous contents that include the RNAi agent are delivered to the cell where the RNAi agent can specifically bind to the target RNA and can mediate RNAi. In some cases, liposomes are also specifically targeted, for example, to target the RNAi agent to particular cell types.
[000278] A liposome containing an RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent may have a high concentration of critical micelle and may be non-ionic. Exemplary detergents include cholate, CHAPS, octylglycoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. Cationic groups in the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, for example, by dialysis, to produce a liposomal preparation of RNAi agent.
[000279] If necessary, a carrier compound that assists in condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer except a nucleic acid (for example, spermine or spermidine). The pH can also be adjusted to promote condensation.
[000280] Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide / cationic lipid complex as structural components of the delivery vehicle, are further described in, for example, WO 96/37194, the entire contents of which are incorporated into this application by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8: 7413-7417, 1987; Pat. No. 4,897,355; Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23: 238, 1965; Olson, et al. Biochim. Biophys. Acta 557: 9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775: 169, 1984; Kim, et al. Biochim. Biophys. Acta 728: 339, 1983; and Fukunaga, et al. Endocrinol. 115: 757, 1984. Techniques commonly used to prepare lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, for example, Mayer, et al. Biochim. Biophys. Acta 858 : 161, 1986). Microfluidization can be used when small (50 to 200 nm) and relatively uniform aggregates are constantly desired (Mayhew, et al. Biochim. Biophys. Acta 775: 169, 1984). These methods are readily adapted to the packaging of RNAi agent preparations in liposomes.
[000281] Liposomes that are pH sensitive or negatively charged capture nucleic acid molecules instead of complementing them. Since both nucleic acid and lipid molecules are similarly charged, repulsion occurs instead of complex formation. However, some nucleic acid molecules are captured within the aqueous interior of these liposomes. PH-sensitive liposomes were used to deliver DNA encoding the thymidine kinase gene to cultured cell monolayers. The exogenous gene expression was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).
[000282] A major type of liposomal composition includes phospholipids in addition to naturally derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimiristoyl phosphatidylglycerol, while fusogenic anionic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed of phosphatidylcholine (PC) such as, for example, soybean PC and egg PC. Another type is made up of mixtures of phospholipids and / or phosphatidylcholine and / or cholesterol.
[000283] Examples of other methods for introducing liposomes into cells in vitro and in vivo include Pat. No. 5,283,185; Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269: 2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90: 11307, 1993; Nabel, Human Gene Ther. 3: 649, 1992; Gershon, Biochem. 32: 7143, 1993; and Strauss EMBO J. 11: 417, 1992.
[000284] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with the cell membrane. Non-cationic liposomes, although not able to fuse efficiently with the plasma membrane, are absorbed by macrophages in vivo and can be used to deliver RNAi agents to macrophages.
[000285] Additional advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble drugs and lipids; liposomes can protect RNAi agents encapsulated in their internal compartments of metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms”, Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
[000286] A positively charged synthetic cationic lipid, N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium (DOTMA) can be used to form the small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes that are able to fuse with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of the RNAi agent (see, for example, Felgner, PL et al ., Proc. Natl. Acad. Sci., USA 8: 7413-7417, 1987 and US Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
[000287] A DOTMA analog, 1,2-bis (oleoyloxy) -3- (trimethylammonia) propane (DOTAP) can be used in combination with a phospholipid to form complexing DNA vesicles. Lipofectin ™ (Bethesda Research Laboratories, Gaithersburg, Md.) Is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells that comprise positively charged DOTMA liposomes that interact spontaneously with negatively charged polynucleotides to form complexes. When many positively charged liposomes are used, the net charge on the resulting complexes is also positive. The positively charged complexes prepared in this way spontaneously bind to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids, for example, in tissue culture cells. Another commercially available cationic lipid, 1,2-bis (oleoyloxy) -3,3- (trimethylammonium) propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in which the oleyl moieties are bonded by ester instead of ether bonds.
[000288] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxypermin which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyperglycine dioctaoleoylamide ("DOGS") (Transfectam ™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxiespermyl-amide ("DPPES") (see, for example, US Pat. No. 5,171,678).
[000289] Another cationic lipid conjugate includes the derivatization of lipid with cholesterol ("DC-Col") that was formulated in liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179: 280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065: 8, 1991). For certain cell lines, these liposomes that contain conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-CV (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
[000290] Liposomal formulations are particularly suited for topical administration, liposomes have several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug to the desired target and the ability to administer the RNAi agent to the skin. In some implementations, liposomes are used to deliver the RNAi agent to epidermal cells and also to increase the penetration of RNAi agent into dermal tissues, for example, the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, for example, Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259 -265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6: 682-690, 1988; Itani, T. et al. Gene 56: 267-276. 1987; Nicolau, C. et al. Meth. Enz. 149: 157-176, 1987; Straubinger, RM and Papahadjopoulos, D. Meth. Enz. 101: 512-527, 1983; Wang, CY and Huang, L., Proc. Natl. Acad. Sci. USA 84: 7851- 7855, 1987).
[000291] Non-ionic liposomal systems have also been examined to determine their usefulness in delivering drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver a drug to the dermis of the mouse skin. Such formulations with the RNAi agent are useful for treating a dermatological disorder.
[000292] Liposomes that include the RNAi agent can be made highly deformable. Such deformability can allow liposomes to penetrate through pores that are smaller than the average radius of the liposome. For example, transferosomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transferosomes that include the RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver the RNAi agent to keratinocytes in the skin. In order to pass through the intact mammalian skin, the lipid vesicles must pass through a series of fine pores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the properties of the lipids, these transferosomes can be self-optimizing (adaptable to the shape of the pores, for example, in the skin), self-repairing, and can often reach their targets without fragmentation, and often by shuttle.
[000293] Other formulations accessible to the present invention are described in US provisional patent applications nos. serial 61 / 018,616, deposited on January 2, 2008; 61 / 018,611, deposited on January 2, 2008; 61 / 039,748, filed on March 26, 2008; 61 / 047,087, filed on April 22, 2008 and 61 / 051,528, filed on May 8, 2008. PCT patent application no. PCT / US2007 / 080331, filed on October 3, 2007 also describes formulations that are accessible to the present invention. Surfactants
[000294] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). Compositions of RNAi agent (or a precursor, for example, a larger dsiRNA that can be processed into a siRNA, or DNA encoding a siRNA or precursor) can include a surfactant. In one embodiment, siRNA is formulated as an emulsion that includes a surfactant. The most common way to classify and qualify the properties of many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, NY, 1988, p. 285).
[000295] If the surfactant molecule is not ionized, it is classified as a non-ionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general, your HLB values range from 2 to approximately 18 depending on your structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters and ethoxylated esters. Non-ionic alkolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols and ethoxylated / propoxylated block polymers are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
[000296] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, amino acid acyl amides, sulfuric acid esters such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as benzene alkyl sulfonates, acyl isethionates, acyl taurates and sulfosuccinates and phosphates. The most important members of the anionic surfactant class are alkyl sulfate and soaps.
[000297] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most used members of this class.
[000298] If the surfactant molecule has the capacity to carry a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include derivatives of acrylic acids, substituted alkylamides, N-alkylbetaines and phosphatides.
[000299] The use of surfactants in pharmaceutical products, formulations and emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285). Micelles and other Membrane Formulations
[000300] The RNAi agents of the invention can also be supplied as micellar formulations. "Micelles" are defined in this application as a particular type of molecular aggregation in which amphipathic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The opposite arrangement exists if the environment is hydrophobic.
[000301] A varied micellar formulation suitable for delivery by transdermal membranes can be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal sulfate C8 to C22 and a micelle forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, pePEG extract, oleic acid, linoleic acid, linolenic acid, mono-olein, mono-oleates, monolaurates, borage oil, evening primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogs of these, polydocanol alkyl ethers, deoxygenolates - broad, and mixtures of these. Micelle-forming compounds can be added at the same time or after addition of the alkali metal alkyl sulfate. Varied micelles will form with substantially any type of mixing of the ingredients but vigorous mixing to provide smaller micelles.
[000302] In one method, a first micellar composition is prepared containing the composition of siRNA and at least alkali metal sulfate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulfate and at least one of the micelle-forming compounds, followed by the addition of the remaining micelle-forming compounds with vigorous mixing.
[000303] Phenol and / or m-cresol can be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol can be added with the micelle forming ingredients. An isotonic agent such as glycerin can also be added after formation of the mixed micellar composition.
[000304] For the delivery of the micellar formulation as a spray, the formulation can be put in an aerosol dispenser and the dispenser is loaded with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The proportions of the ingredients are adjusted so that the aqueous and propellant phases become one, that is, there is a phase. If there are two phases, it is necessary to shake the dispenser before dispensing a portion of the contents, for example, by a calibrated valve. The dispensed dose of pharmaceutical agent is propelled from the calibrated valve in a fine spray.
[000305] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) can be used.
[000306] The specific concentrations of the essential ingredients can be determined by relatively direct experimentation. For absorption into the oral cavities, it is often desirable to increase, for example, at least double or triple the dosage by injection or administration through the gastrointestinal tract. Particles
[000307] In another embodiment, an RNAi agent of the invention can be incorporated into a particle, for example, a microparticle. Microparticles can be produced by spray drying, but they can also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying or a combination of these techniques. IV. Methods for Inhibiting TTR Expression
[000308] The present invention also provides methods of inhibiting the expression of a transthyretin (TTR) in a cell. Methods include contacting a cell with an RNAi agent, for example, double-stranded RNAi agent, in an amount effective to inhibit TTR expression in the cell, thereby inhibiting TTR expression in the cell.
[000309] Contact of a cell with an RNAi agent, for example, a double-stranded RNAi agent, can be done in vitro or in vivo. Contact of a cell in vivo with the RNAi agent includes contacting a cell or group of cells within an individual, for example, a human, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Cell contact can be direct or indirect, as discussed above. In addition, contact of a cell can be carried out via a targeting linker, including any linker described in this application or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, for example, a GalNAc3 ligand or any other ligand that directs the RNAi agent to a site of interest, for example, an individual's liver.
[000310] The term "inhibition", as used in this application, is used interchangeably with "reduction", "silencing", "down regulation", "suppression" and other similar terms, and includes any level of inhibition.
[000311] The phrase "inhibition of the expression of a TTR" is intended to refer to the inhibition of the expression of any TTR gene (such as, for example, a mouse TTR gene, a mouse TTR gene, a monkey TTR gene or human TTR gene) as well as variants or mutants of a TTR gene. Thus, the TTR gene can be a wild-type TTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise to amyloid deposition) or a transgenic TTR gene in the context of a cell, group of cells or organism genetically manipulated.
[000312] "Inhibition of the expression of a TTR gene" includes any level of inhibition of a TTR gene, for example, the at least partial suppression of the expression of a TTR gene. TTR gene expression can be assessed based on the level or modification in the level, of any variable associated with TTR gene expression, for example, TTR mRNA level, TTR protein level, or the number or extent of deposits amyloid. This level can be assessed in an individual cell or in a group of cells, including, for example, a sample derived from an individual.
[000313] Inhibition can be assessed by a reduction in an absolute or relative level of one or more variables that are associated with the expression of TTR compared to a control level. The level of control can be any type of level of control that is used in the art, for example, a pre-dose base level or a determined level of a similar individual, cell or sample that is untreated or treated with a control ( such as, for example, control buffer only or inactive agent control).
[000314] In some embodiments of the methods of the invention, the expression of a TTR gene is inhibited by at least approximately 5%, at least approximately 10%, at least approximately 15%, at least approximately 20%, at least approximately 25%, at least approximately 30%, at least approximately 35%, at least approximately 40%, at least approximately 45%, at least approximately 50%, at least approximately 55%, at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98% or at least approximately 99%.
[000315] Inhibition of the expression of a TTR gene can be manifested by a reduction in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from an individual) in which a TTR gene is transcribed and which has or has been treated (for example, by contacting the cell or cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to an individual in which the cells are or have been present) such that the expression of a TTR gene is inhibited, compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or has not been treated in this way (control cell (s)). In preferred embodiments, inhibition is assessed by expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula: (mRNA in control cells) - (mRNA in treated cells). 100% (mRNA in control cells)
[000316] Alternatively, the inhibition of the expression of a TTR gene can be assessed for a reduction of a parameter that is functionally linked to TTR gene expression, for example, TTR protein expression, retinol-binding protein level , vitamin A level, or the presence of amyloid deposits comprising TTR. Silencing of the TTR gene can be determined in any cell that expresses TTR, either constitutively or by genomic engineering, and by any assay known in the art. The liver is the main site of TTR expression. Other significant sites of expression include choroid plexus, retina and pancreas.
[000317] Inhibition of the expression of a TTR protein can be manifested by a reduction in the level of the TTR protein that is expressed by a cell or group of cells (for example, the level of the protein expressed in a sample derived from an individual ). As explained above for the evaluation of mRNA suppression, inhibition of protein expression levels in a treated cell or group of cells can similarly be expressed as a percentage of the protein level in a control cell or group of cells.
[000318] A cell or group of control cells that can be used to assess inhibition of expression of a TTR gene includes a cell or group of cells that have not yet been brought into contact with an RNAi agent of the invention. For example, the control cell or group of cells can be derived from an individual (for example, a human or an animal) prior to treating the individual with an RNAi agent.
[000319] The level of TTR mRNA that is expressed by a cell or group of cells or the level of circulating TTR mRNA can be determined using any method known in the art to evaluate mRNA expression. In one embodiment, the level of TTR expression in a sample is determined by detecting a transcribed polynucleotide or portion thereof, for example, the mRNA of the TTR gene. RNA can be extracted from cells using RNA extraction techniques including, for example, using extraction with phenolic acid / guanidine isothiocyanate (RNAzol B; Biogenesis), RNA preparation kits RNeasy (Qiagen) or PAXgene (PreAnalytix, Switzerland) . Typical assay formats using ribonucleic acid hybridization include nuclear accessory assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12: 7035), Northern blotting, in situ hybridization and analysis of microarray. Circulating TTR mRNA can be detected using methods such as that described in PCT / US2012 / 043584, the entire contents of which are hereby incorporated into this application by reference.
[000320] In one embodiment, the level of TTR expression is determined using a nucleic acid probe. The term "probe", as used in this application, refers to any molecule that is capable of selectively binding to a specific TTR. The probes can be synthesized by a person skilled in the art or derived from appropriate biological preparations. The probes can be specifically designed to be marked. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies and organic molecules.
[000321] The isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyzes, polymerase chain reaction (PCR) analyzes and probe arrays. One method of determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize the TTR mRNA. In one embodiment, the mRNA is immobilized on a solid surface and placed in contact with a probe, for example, directing the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe (s) is immobilized on a solid surface and the mRNA is placed in contact with the probe (s), for example, in an Affymetrix gene chip arrangement. A person skilled in the art can readily adapt known mRNA detection methods for use in determining the level of TTR mRNA.
[000322] An alternative method for determining the level of TTR expression in a sample involves the process of nucleic acid amplification and / or reverse transcriptase (to prepare cDNA), for example, of the mRNA in the sample, for example, by RT- PCR (the experimental modality presented in Mullis, 1987, US Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189-193), sequence replication self-sustaining (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173- 1177), Q-Beta Replicase (Lizardi et al. (1988) Bio / Technology 6: 1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other method of acid amplification nucleic acid, followed by the detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of TTR expression is determined by quantitative fluorogenic RT-PCR (i.e., the TaqManTM System).
[000323] TTR mRNA expression levels can be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising linked nucleic acids). See Pat. U.S. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated in this order by reference. Determination of the level of TTR expression can also comprise use of nucleic acid probes in the solution.
[000324] In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) or real-time PCR (qPCR) assays. The use of these methods is described and exemplified with the Examples presented in this application.
[000325] The level of TTR protein expression can be determined using any method known in the art by measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, precipitin reactions in fluid or gel, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescent assays, and the like.
[000326] In some embodiments, the effectiveness of the methods of the invention can be monitored by detecting or monitoring a reduction of an amyloid deposit by TTR. Reducing an amyloid deposit by TTR, as used in this application, includes any reduction in the size, number or severity of deposits by TTR, or a prevention or reduction of the formation of deposits by TTR, within an organ or area of an individual, such as it can be evaluated in vitro or in vivo using any method known in the art. For example, some methods of assessing amyloid deposits are described in Gertz, M.A. & Rajukumar, S.V. (Eds) (2010), Amyloidosis: Diagnosis and Treatment, New York: Humana Press. Methods for assessing amyloid deposits may include biochemical analyzes, as well as visual or computerized assessment of amyloid deposits, as if visible, for example, using immunohistochemical staining, fluorescent staining, optical microscopy, electron microscopy, fluorescence microscopy or other types of microscopy. Invasive or non-invasive imaging modalities, including, for example, CT, PET or NMR / MRI imaging, can be used to assess amyloid deposits.
[000327] The methods of the invention can reduce TTR deposits in any number of tissues or regions of the body including but not limited to the heart, liver, spleen, esophagus, stomach, intestine (ileum, duodenum and colon), brain, sciatic nerve, dorsal root ganglion, kidney and retina.
[000328] The term "sample" as used in this application refers to a collection of fluids, cells or similar tissues isolated from an individual, as well as fluids, cells or tissues present within an individual. Examples of biological fluids include blood, serum and serous fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, eye fluids, and the like. Tissue samples can include tissue samples, organs or localized regions. For example, samples can be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, the samples can be derived from the liver (eg, whole liver or certain segments of the liver or certain types of cells in the liver, such as, for example, hepatocytes), retina or parts of the retina (eg, epithelium of the liver) retinal pigment), central nervous system or parts of the central nervous system (for example, ventricles or choroid plexus), or pancreas or certain cells or parts of the pancreas. In preferred embodiments, a “sample derived from an individual” refers to blood or plasma taken from the individual. In additional embodiments, a “sample derived from an individual” refers to liver tissue or retinal tissue derived from the individual.
[000329] In some embodiments of the methods of the invention, the RNAi agent is administered to an individual such that the RNAi agent is delivered to a specific site within the individual. Inhibition of TTR expression can be assessed using measurements of the level or modification in the level of TTR mRNA or TTR protein in a sample derived from fluid or tissue from the specific site within the individual. In preferred modalities, the site is selected from the group consisting of the liver, choroid plexus, retina and pancreas. The site can also be a subsection or subgroup of cells from any of the sites mentioned above (for example, hepatocytes or retinal pigment epithelium). The site can also include cells that express a particular type of receptor (for example, hepatocytes that express the asialoglycloprotein receptor). V. Methods to treat or prevent a disease associated with TTR
[000330] The present invention also provides methods for treating or preventing a disease associated with TTR in an individual. The methods include administering to the individual a therapeutically effective amount or prophylactically effective amount of an RNAi agent of the invention.
[000331] As used in this application, an "individual" includes a human or non-human animal, preferably a vertebrate, and more preferably a mammal. An individual can include a transgenic organism. Even more preferably, the individual is the human being, as a human being suffering or predisposed to develop a disease associated with TTR.
[000332] In some modalities, the individual is suffering from a disease associated with TTR. In other modalities, the individual is an individual at risk of falling ill from a disease associated with TTR, for example, an individual with a TTR gene mutation that is associated with the development of a disease associated with TTR, an individual with a family history of disease associated with TTR or an individual who has signs or symptoms that suggest the development of amyloidosis by TTR.
[000333] A "disease associated with TTR", as used in this application, includes any disease caused or associated with the formation of amyloid deposits in which the fibril precursors consist of the variant or wild-type TTR protein. Mutant and wild-type TTR give rise to various forms of amyloid deposition (amyloidosis). Amyloidosis involves the formation and aggregation of poorly folded proteins, resulting in extracellular deposits that impair organ function. Clinical syndromes associated with TTR aggregation include, for example, senile systemic amyloidosis (SSA); systemic familial amyloidosis; familial amyloidotic polyneuropathy (FAP); familial amyloid cardiomyopathy (FAC); and leptomenin-geal amyloidosis, also known as leptomeningeal or meningo-cerebrovascular amyloidosis, central nervous system (CNS) amyloidosis or form VII amyloidosis.
[000334] In some embodiments of the methods of the invention, the RNAi agents of the invention are administered to individuals suffering from familial amyloidotic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Normal-sequence TTR causes cardiac amyloidosis in people who are 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 accelerate the process of amyloid formation by TTR and are the most important risk factor for the development of clinically significant TTR amyloidosis (also called ATTR (amyloidosis-transthyretin type)). More than 85 amyloidogenic TTR variants are known to cause familial systemic amyloidosis.
[000335] In some embodiments of the methods of the invention, the RNAi agents of the invention are administered to individuals suffering from familial amyloidotic polyneuropathy (FAP) related to transthyretin (TTR). Such individuals may suffer from ocular manifestations, such as vitreous opacity and glaucoma. It is known by a specialist in the art that amyloidogenic transthyretin (ATTR) synthesized by the retinal pigment epithelium (RPE) plays important roles in the progression of ocular amyloidosis. Previous studies have shown that panretinal laser photocoagulation, which reduced RPE cells, prevented the progression of amyloid deposition in the vitreous, indicating that effective suppression of ATTR expression in RPE may become a new therapy for ocular amyloidosis (see, for example, Kawaji, T., et al., Ophthalmology. (2010) 117: 552-555). The methods of the invention are useful for the treatment of ocular manifestations of FAP related to TTR, for example, ocular amyloidosis. The RNAi agent can be delivered in a manner suitable for targeting a particular tissue, such as the eye. The modes of delivery to the eye include retrobulbar injection, subcutaneous injection into the eyelid, subconjunctival, subetenon, anterior or intravitreal chamber (or internal injection or infusion). Specific formulations for eye delivery include eye drops or ointments.
[000336] Another disease associated with TTR is hyperthyroxinemia, also known as "dystranetirretinemic hyperthyroxinemia" or "dysprealbuminemic hyperthyroxinemia". This type of hyperthyroxinemia may be secondary to an increased association of thyroxine with TTR due to a mutant TTR molecule with increased affinity for thyroxine. See, for example, Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.
[000337] The RNAi agents of the invention can be administered to an individual using any mode of administration known in the art, including, but not limited to, subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combination of these. In preferential modalities, agents are administered subcutaneously.
[000338] In some modalities, the administration is through an injection of storage. A storage injection can release the RNAi agent consistently over an extended period of time. In this way, a storage injection can reduce the dosage frequency necessary to obtain a desired effect, for example, a desired inhibition of TTR or a therapeutic or prophylactic effect. A storage injection can also provide more consistent serum concentrations. Storage injections can include subcutaneous injections or intramuscular injections. In preferred embodiments, the storage injection is a subcutaneous injection.
[000339] In some modalities, the administration is through a pump. The pump can be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump can be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver.
[000340] Other modes of administration include epidural, intracerebral, intracerebroventricular, nasal, intraarterial, intracardiac, intraosseous, intrathecal, and intravitreal, and pulmonary administration. The mode of administration can be chosen based on whether local or systemic treatment is desired and based on the area to be treated. The route and site of administration can be chosen to increase targeting.
[000341] In some embodiments, the RNAi agent is administered to an individual in an amount effective to inhibit TTR expression in a cell within the individual. The amount effective to inhibit TTR expression in a cell within an individual can be assessed using methods discussed above, including methods that involve assessing inhibition of TTR mRNA, TTR protein or related variables, such as amyloid deposits.
[000342] In some embodiments, the RNAi agent is administered to an individual in a therapeutically or prophylactically effective amount.
[000343] "Therapeutically effective amount", as used in this application, is intended to include the amount of an RNAi agent that, when administered to a patient to treat a disease associated with TTR, is sufficient to effect the treatment of the disease (for example, example, decreasing, ameliorating or maintaining the existing disease or one or more symptoms of the disease). The “therapeutically effective amount” can vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic construction, stage of pathological processes mediated by TTR expression, types previous or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
[000344] "Prophylactically effective amount", as used in this application, is intended to include the amount of an RNAi agent that, when administered to an individual who does not yet experience or exhibit symptoms of a disease associated with TTR, but who can be predisposed to the disease, it is sufficient to prevent or improve the disease or one or more symptoms of the disease. Symptoms that can be improved include sensory neuropathy (eg, paresthesia, hypoesthesia in distal limbs), autonomic neuropathy (eg, gastrointestinal dysfunction, such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction and latticed corneal dystrophy. Improving the disease includes slowing the course of the disease or reducing the severity of the disease that develops afterwards. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, and the history, age, weight, family history, genetic construction, types of previous or concomitant treatments, if there are any, and other individual characteristics of the patient to be treated.
[000345] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect in a reasonable benefit / risk ratio applicable to any treatment. The RNAi agents employed in the methods of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.
[000346] As used in this application, the phrases "therapeutically effective amount" and "prophylactically effective amount" also include an amount that provides a benefit in the treatment, prevention, or management of pathological processes or symptom (s) of mediated pathological processes by the expression of TTR. Symptoms of TTR amyloidosis include sensory neuropathy (eg, paresthesia, hypoaesthesia in distal limbs), autonomic neuropathy (eg, gastrointestinal dysfunction, such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, syndrome carpal tunnel, autonomic insufficiency, cardiomyopathy, vitreous opacity, renal failure, nephropathy, substantially reduced mBMI (modified body mass index), cranial nerve dysfunction and latticed corneal dystrophy.
[000347] The dose of an RNAi agent that is administered to an individual can be adapted to balance the risks and benefits of the particular dose, for example, achieving a desired level of TTR gene suppression (as assessed, for example, based on in suppression of TTR mRNA, expression of TTR protein or a reduction of an amyloid deposit, as defined above) or a desired therapeutic or prophylactic effect, while avoiding undesirable side effects.
[000348] In one embodiment, the RNAi agent is administered at a dose between approximately 0.25 mg / kg to approximately 50 mg / kg, for example, between approximately 0.25 mg / kg to approximately 0.5 mg / kg , between approximately 0.25 mg / kg to approximately 1 mg / kg, between approximately 0.25 mg / kg to approximately 5 mg / kg, between approximately 0.25 mg / kg to approximately 10 mg / kg, between approximately 1 mg / kg to approximately 10 mg / kg, between approximately 5 mg / kg to approximately 15 mg / kg, between approximately 10 mg / kg to approximately 20 mg / kg, between approximately 15 mg / kg to approximately 25 mg / kg, between approximately 20 mg / kg to approximately 30 mg / kg, between approximately 25 mg / kg to approximately 35 mg / kg, or between approximately 40 mg / kg to approximately 50 mg / kg.
[000349] In some embodiments, the RNAi agent is administered at a dose of approximately 0.25 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg , approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, approximately 10 mg / kg, approximately 11 mg / kg, approximately 12 mg / kg, approximately 13 mg / kg, approximately 14 mg / kg, approximately 15 mg / kg, approximately 16 mg / kg, approximately 17 mg / kg, approximately 18 mg / kg, approximately 19 mg / kg, approximately 20 mg / kg, approximately 21 mg / kg, approximately 22 mg / kg, approximately 23 mg / kg, approximately 24 mg / kg, approximately 25 mg / kg, approximately 26 mg / kg, approximately 27 mg / kg, approximately 28 mg / kg , approximately 29 mg / kg, 30 mg / kg, approximately 31 mg / kg, approximately 32 mg / kg, approximately 33 mg / kg, approximately 34 mg / kg, approx. approximately 35 mg / kg, approximately 36 mg / kg, approximately 37 mg / kg, approximately 38 mg / kg, approximately 39 mg / kg, approximately 40 mg / kg, approximately 41 mg / kg, approximately 42 mg / kg, approximately 43 mg / kg, approximately 44 mg / kg, approximately 45 mg / kg, approximately 46 mg / kg, approximately 47 mg / kg, approximately 48 mg / kg, approximately 49 mg / kg or approximately 50 mg / kg.
[000350] In some embodiments, the RNAi agent is administered in two or more doses. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, for example, a pump, semi-permanent stent (for example, intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable. In some embodiments, the number or quantity of subsequent doses is dependent on the achievement of a desired effect, for example, the suppression of a TTR gene or the realization of a therapeutic or prophylactic effect, for example, by reducing an amyloid deposit or by reducing a symptom of a disease associated with TTR. In some embodiments, the RNAi agent is administered on a schedule. For example, the RNAi agent can be administered twice a week, three times a week, four times a week, or five times a week. In some modalities, the scheme involves regularly spaced administrations, for example, every hour, every four hours, every six hours, every eight hours, every twelve hours, daily, every 2 days, every 3 days, every 4 days, every 5 days, weekly, every two weeks, or monthly. In other embodiments, the scheme involves narrowly spaced administrations followed by a longer period of time during which the agent is not administered. For example, the schedule may involve an initial set of doses that are administered in a relatively short period of time (for example, approximately every 6 hours, approximately every 12 hours, approximately every 24 hours, approximately every 48 hours, or approximately every 72 hours) hours) followed by a longer period of time (for example, approximately 1 week, approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks or approximately 8 weeks) during which the RNAi agent is not administered. In one embodiment, the RNAi agent is initially administered hourly and is then administered at a longer interval (for example, daily, weekly, every two weeks, or monthly). In another embodiment, the RNAi agent is initially administered daily and is then administered at a longer interval (for example, weekly, every two weeks, or monthly). In certain embodiments, the longest interval increases over time or is determined based on the achievement of a desired effect. In a specific embodiment, the RNAi agent is administered once daily for the first week, followed by weekly dosing that begins on the eighth day of administration. In another specific embodiment, the RNAi agent is administered every other day for a first week followed by the weekly dosage which begins on the eighth day of administration.
[000351] Any of these schemes can be optionally repeated for one or more iterations. The number of iterations may depend on the achievement of a desired effect, for example, the suppression of a TTR gene, protein level of retinol binding, vitamin A level and / or realization of a therapeutic or prophylactic effect, for example, reducing an amyloid deposit or reducing a symptom of a disease associated with TTR.
[000352] In some embodiments, the RNAi agent is administered with other therapeutic agents or other therapeutic regimens. For example, other agents or other therapeutic regimens suitable for treating a disease associated with TTR may include a liver transplant, which can reduce the levels of mutant TTR in the body; Tafamidis (Vyndaqel), which kinetically stabilizes the TTR tetramer that prevents tetramer dissociation required for TTR amyloidogenesis; and diuretics, which can be used, for example, to reduce edema in TTR amyloidosis with cardiac involvement.
[000353] In one embodiment, an individual is administered an initial dose and one or more maintenance doses of an RNAi agent. The dose or maintenance doses can be the same or lower than the starting dose, for example, half the starting dose. A maintenance regimen may include treating the individual with a dose or doses ranging from 0.01 μg to 15 mg / kg of body weight per day, for example, 10 mg / kg, 1 mg / kg, 0.1 mg / kg kg, 0.01 mg / kg, 0.001 mg / kg or 0.00001 mg / kg of body weight per day. Maintenance doses, for example, are administered no more than once every 2 days, once every 5 days, once every 7 days, once every 10 days, once every 14 days, once once every 21 days, or once every 30 days. In addition, the treatment regimen may last for a period of time that will vary depending on the nature of the particular disease, its severity and the total condition of the patient. In certain modalities the dosage can be delivered no more than once a day, for example, not more than once for 24, 36, 48, or more hours, for example, not more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition. The dosage of the RNAi agent can be increased in the event that the patient does not respond significantly to current dosage levels, or the dose can be reduced if a relief of the symptoms of the disease state is observed, if the disease state has been ablated. , or if unwanted side effects are observed. SAW. Kits
[000354] The present invention also provides kits for carrying out any of the methods of the invention. Such kits include one or more RNAi agent (s) and instructions for use, for example, instructions to inhibit the expression of a TTR in a cell by contacting the cell with the RNAi agent (s) in an amount effective to inhibit TTR expression. . The kits may optionally further comprise means for contacting the cell with the RNAi agent (for example, an injection device), or means for measuring TTR inhibition (for example, means for measuring TTR mRNA inhibition or TTR protein). Such means for measuring TTR inhibition may comprise a means for obtaining a sample from an individual, such as, for example, a plasma sample. The kits of the invention may optionally further comprise means for administering the RNAi agent (s) to an individual or means for determining the therapeutically effective or prophylactically effective amount.
[000355] This invention is further illustrated by the following examples that should not be interpreted as a limitation. The contents of all published references and patents and patent applications cited in all parts of the application are hereby incorporated into this application by reference. EXAMPLES Example 1: TTR inhibition with TTR-GalNAc conjugates
[000356] A single dose of the TTR RNAi agent AD-43527 was administered to mice subcutaneously and the levels of TTR mRNA were determined 72 hours post-administration.
[000357] The mouse / rat cross-reactive GalNAc conjugate, AD-43527, was chosen for in vivo evaluation in WT C57BL / 6 mice for silencing of TTR mRNA in the liver. The sequence of each AD-43527 tape is shown below. Duplex # Oligo Tape # Sequence 5 'to 3' AD- 43527 s A-89592 AfaCfaGfuGfuUfcUfuGfcUfcUfaUfaAfL96 (SEQ ID NO: 8) as A-83989 uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUU g, c) are 2'-O-methyl nucleotides, Nf (ie, Af) is a 2'-fluoro nucleotide

[000358] The ligand used was GalNAc3:

[000359] This GalNAc3 ligand was attached to the 3 'end of the sense tape using the ligand and the tie as shown below:

[000360] The structure of the resulting GalNAc3 sense-conjugated tape is shown in the following scheme:

[000361] Additional RNAi agents that target TTR and have the following sequences and modifications have been synthesized and analyzed.
[000362] Mouse / rat cross-reactive TTR RNAi agents Duplex Senso tape 5'-3 '5'-3' antisense tape AD- 43528 AfaCfaGfuGfuUfcUfuGfcUfcUfaU faAfQ11L96 (SEQ ID NO: 10) : 11)

[000363] Cross-reactive TTR RNAi agents from human / cinomolgus; the parent is AD-18328 [having a 5'-3 'sense tape sequence from GuAAccAAGAGuAuuccAudTdT (SEQ ID NO: 12) and 5'-3'AUGGAAuACUCUUGGU- uACdTdT (SEQ ID NO: 13) antisense tape sequence following modifications: alternation 2'F / 2'OMe with 2 PS in AS. Duplex sense strand 5'-3 'antisense 5'-tape 3' AfuGfuAfaCfcAfaGfaGfuAfuUfcCf aUfL96 AD-45163 (SEQ ID NO: 14) aUfgGfaAfuAfcUfcUfuGfgUfuAfcA fusGfsa (SEQ ID NO: 16) AfuGfuAfaCfcAfaGfaGfuAfuUfcCf aUfQ11L96 AD-45164 (SEQ ID NO: 15) aUfgGfaAfuAfcUfcUfuGfgUfuAfcA fusGfsa (SEQ ID NO: 17)

[000364] L96 = GalNAc3; lowercase nts (a, u, g, c) are 2'-O-methyl nucleotides, Nf (ie Af) is a 2'-fluoro nucleotide; Q11 is cholesterol; s is phosphorothioate.
[000365] AD-43527 was administered to female C57BL / 6 mice (6-10 weeks, 5 per group) by subcutaneous injection in a dose volume of 10 μl / g in a dose of 30, 15, 7.5, 3.5, 1.75 or 0.5 mg / kg of AD-43527. Control animals received PBS by subcutaneous injection in the same dose volume.
[000366] After approximately seventy-two hours, the mice were anesthetized with 200 μl of ketamine, and then bled by cutting the right caudal artery. The liver tissue was collected, frozen instantly and stored at -80 ° C until processing.
[000367] The effectiveness of the treatment was assessed by measuring the TTR mRNA in the liver in a 72-hour post-dose. TTR liver mRNA levels were analyzed using the QuantiGene 1.0 Branched DNA assays (Panomics). Briefly, the mouse liver samples were ground and tissue lysates were prepared. The liver lysis mixture (a mixture of 1 volume of lysis mixture, 2 volumes of water without nucleases and 10 μl of Proteinase-K / ml of a final concentration of 20mg / ml) was incubated at 65 ° C for 35 minutes. 5 μl of the liver lysate and 95 μl of the working probe set (TTR probe for the gene target and GAPDH for endogenous control) were added to the Capture Plate. The capture plates were incubated at 53 ° C ± 1 ° C (approx. 16-20hrs). The next day, the Capture Plates were washed 3 times with Wash Buffer 1X (water without nucleases, Component 1 of Buffer and Components 2 of Wash Buffer), then dried by centrifuging for 1 minute in 240g. 100 μl of the Amplifier Probe mixture per well was added to the Capture Plate, which was sealed with aluminum foil and incubated for 1 hour at 46 ° C ± 1 ° C. After a 1 hour incubation, the washing step was repeated, then 100 μl of the Marker Probe mixture per well was added. The capture plates were incubated at 46 ° C ± 1 ° C for 1 hour. The plates were then washed with 1X Wash Buffer, dried and 100 μl of substrate per well was added to the Capture Plates. The Capture Plates were incubated for 30 minutes at 46 ° C followed by incubation for 30 minutes at room temperature. The plates were read using a SpectraMax Luminometer after incubation. The bDNA data were analyzed by subtracting the average background of each duplicate sample, calculating values of the resulting duplicated GAPDH average (control probe) and TTR (experimental probe), and then computing the ratio: (experimental probe background) / ( control probe bottom). The average TTR mRNA level was calculated for each group and normalized to the PBS group average to give the relative TTR mRNA as a% of the PBS control group.
[000368] The results are shown in Figure 1. The RNAi agent conjugated to GalNAc targeting TTR had an ED50 of approximately 5 mg / kg of TTR mRNA silencing. These results demonstrate that GalNAc-conjugated RNAi agents that target TTR are effective in inhibiting TTR mRNA expression. Example 2: TTR inhibition with TTR-GalNAc conjugates is durable
[000369] The mice were administered a subcutaneous dose (7.5 or 30.0 mg / kg) of AD-43527, RNAi agent conjugated to GalNAc that targets TTR. Levels of TTR mRNA in the liver were assessed at 1, 3, 5, 7, 10, 13, 15, 19, 26, 33, and 41 days post-treatment using the method described in Example 1.
[000370] The results are shown in Figure 2. On day 19, the administration of GalNAc-conjugated RNAi agents of 30.0 mg / kg still showed approximately 50% silencing. The complete recovery of expression occurred on day 41.
[000371] These results demonstrated that the inhibition provided by the conjugated siRNA of GalNAc that targets TTR is durable, lasting up to 3, 5, 7, 10, 13, 15, 19, 26 or 33 days after treatment. Example 3. Synthesis of RNA and Duplex Ringing 1. Synthesis of oligonucleotides
[000372] The oligonucleotides were synthesized on an AKTAoligopilot synthesizer or an ABI 394 synthesizer. The commercially available controlled pore glass solid support (dT-CPG, 500Á, Prime Synthesis) and RNA phosphoramidites with standard protection groups, 5 ' -O-dimethoxytrityl N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'- ON, N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine -3'-ON, N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N2 - isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-ON, N'-diisopropyl-2-cyanoethylphosphoramidite, and 5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-ON, N'-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the synthesis of oligonucleotides unless otherwise specified . Phosphoramidites 2'-F, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-O- N, N'-diisopropyl-2-cyanoethyl-phosphoramidite and 5'-O-dimethoxytrityl-2 '-fluro- uridine-3'-ON, N'-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from (Promega). All phosphoramidites were used at a concentration of 0.2 M in acetonitrile (CH3CN) except guanosine, which was used at a concentration of 0.2 M in THF / ANC 10% (v / v). The coupling / recycling time of 16 minutes was used. The activator was 5-ethyl tiotetrazole (0.75M, American International Chemicals), since PO oxidation by Iodine / Water / Pyridine was used and PS oxidation PADS (2%) in 2,6-lutidine / ACN ( 1: 1 v / v) was used.
[000373] The ligand conjugated phytases were synthesized using a solid support containing the corresponding ligand. For example, the introduction of a carbohydrate moiety / linker (e.g., GalNAc) at the 3 'end of a sequence has been achieved by starting synthesis with the corresponding solid carbohydrate support. Similarly, a portion of cholesterol at the 3 'end was introduced starting synthesis on the cholesterol support. In general, the linker portion was tied to trans-4-hydroxyprolinol via a link of choice as described in the previous examples to obtain a hydroxyprolinol-linker portion. The hydroxyprolinol-linker portion was then coupled to a solid support via a succinate linker or was converted to phosphoramidite via standard phosphorylation conditions to obtain desired carbohydrate conjugate building blocks. The fluorophore-labeled siRNAs were synthesized from the corresponding phosphoramidite or solid support, purchased from Biosearch Techno-logies. The support of lithocolic oleyl polymer (GalNAc) 3 is made internally at a load of 38.6 μmol / gram. The polymer support Manose (Man) 3 was also made internally at a load of 42.0 μmol / gram.
[000374] Conjugation of the linker of choice at the desired position, for example, at the 5 'end of the sequence, was achieved by coupling the phosphoramidite corresponding to the growing chain under standard phosphoramidite coupling conditions unless otherwise specified. A 15 minute extended coupling of the 0.1 M phosphoramidite solution in anhydrous CH3CN in the presence of 5- (ethylthio) -1H-tetrazole activator to a solid bound to the oligonucleotide. The oxidation of the phosphite internucleotide to phosphate was performed using standard iodine water as reported in Beaucage, S.L. (2008) Solid-phase synthesis of siRNA oligonucleotides. Curr. Opin. Drug Discov. Devel., 11, 203-216; Mueller, S., Wolf, J. and Ivanov, S.A. (2004) Current Strategies for the Synthesis of RNA. Curr. Org. Synth., 1, 293-307; Xia, J., Noronha, A., Toudjarska, I., Li, F., Akinc, A., Braich, R., Frank-Kamenetsky, M., Rajeev, KG, Egli, M. and Manoharan, M. (2006) Gene Silencing Activity of siRNAs with a Ribodifluorotoluyl Nucleotide. ACS Chem. Biol., 1, 176-183 or by treatment with tert-butylhydroperoxide / acetonitrile / water (10: 87: 3) with an oxidation waiting time of 10 minutes conjugated the oligonucleotide. Phosphorothioate was introduced by oxidizing phosphite to phosphorothioate using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. Cholesterol phosphoramidite was synthesized internally and used at a concentration of 0.1 M in dichloromethane. The coupling time for cholesterol phosphoramidite was 16 minutes. 2. Deprotection-I (Nucleobase Deprotection)
[000375] After carrying out the synthesis, the support was transferred to a 100 ml glass bottle (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3: 1)] for 6.5 h at 55 ° C. The bottle was briefly cooled on ice and then the ethanolic ammonia mixture was filtered into a new 250 ml bottle. The CPG was washed with 2 x 40 ml portions of ethanol / water (1: 1 v / v). The volume of the mixture was then reduced to ~ 30 ml by roto-vap. The mixture was then frozen on dry ice and dried under vacuum in a speed vac. 3. Desprotection-II (Removal of group 2 ’TBDMS)
[000376] The dry residue was resuspended in 26 ml of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3: 4: 6) and heated at 60 ° C for 90 minutes to remove tert-butyldimethylsilyl groups (TBDMS) in position 2 '. The reaction was then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to 6.5, and stored in the refrigerator until purification. 4. Analysis
[000377] Oligonucleotides were analyzed by high performance liquid chromatography (HPLC) before purification and buffer selection and the column depends on the nature of the sequence and or conjugated ligand. 5. HPLC purification
[000378] The ligand conjugated oligonucleotides were purified by preparative reverse phase HPLC. The unconjugated oligonucleotides were purified by anion exchange HPLC on an internally packed TSK gel column. The buffers were 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1M NaBr (buffer B). Fractions containing whole oligonucleotides were grouped, desalted and lyophilized. Approximately 0.15 OD of desalinated oligonucleotides were diluted in water at 150 μl and then pipetted in special flasks for CGE and LC / MS analysis. The compounds were finally analyzed by LC-ESMS and CGE. 6. Preparation of RNAi Agent
[000379] For the preparation of an RNAi agent, the equimolar amounts of the sense and antisense tape were heated in 1xPBS at 95 ° C for 5 minutes and slowly cooled to room temperature. The integrity of the duplex was confirmed by HPLC analysis. Table 1 below reflects the RNAi agents that target human or rodent TTR mRNA.





























































































































































[000380] Lowercase nucleotides (a, u, g, c) are 2'-O-methyl nucleotides; Nf (for example, Af) is a 2'-fluoro nucleotide; s is a phosphotiorate bond; L96 indicates a GalNAc3 ligand. 283/304 Petition 870200118973, of 9/23/2020, p. 290/324 Example 4: In vitro screening of RNAi agents Cell culture and transfections
[000381] Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, VA) were grown close to confluence at 37 ° C in an atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS , streptomycin and glutamine (ATCC) before being released from the plate by trypsinization. The transfection was performed by adding 14.8 μl of Opti-MEM plus 0.2 μl of RNAiMax Lipofectamine per well (Invitrogen, Carlsbad CA. cat # 13778-150) to 5 μl of siRNA duplexes per well in a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete antibiotic-free growth media containing ~ 2 x 10 4 Hep3B cells was then added to the siRNA mixture. The cells were incubated for 24 or 120 hours before RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were performed using 8, 4 times serial dilutions with a maximum dose of 10 nM final duplex concentration. Total RNA isolation using DYNABEADS mRNA Isolation Kit (Invitrogen, part #: 610-12)
[000382] The cells were collected and lysed in 150 μl of the Lysis / Binding Buffer then mixed for 5 minutes at 850 rpm using Eppendorf Thermomixer (mixing speed was the same during the process). Ten microliters of magnetic beads and 80 μl of the Lysis Buffer / Binding mixture were added to a round bottom plate and mixed for 1 minute. The magnetic beads were captured using the magnetic rack and the supernatant was removed without disturbing the beads. After removing the supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing the supernatant, the magnetic beads were washed 2 times with 150 μl of Wash Buffer A and mixed for 1 minute. The beads were captured again and the supernatant removed. The beads were then washed with 150 μl of Wash Buffer B, captured and the supernatant removed. The beads were then washed with 150 μl of Elution Buffer, captured and supernatant removed. The beads were allowed to dry for 2 minutes. After drying, 50 μl of the Elution Buffer was added and mixed for 5 minutes at 70 ° C. The beads were captured on the magnet for 5 minutes. 40 μl of the supernatant was removed and added to another 96-well plate. Synthesis of cDNA using ABI High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, Cat # 4368813)
[000383] A master mix of 1 μl of 10X Buffer, 0.4 μl of 25X dNTPs, 1 μl of Random Primers, 0.5 μl of Reverse Transcriptase, 0.5 μl of RNase inhibitor and 1.6 μl of H2O per reaction was added in 5 μl of total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) by the following steps: 25 ° C 10 min, 37 ° C 120 min, 85 ° C 5 seconds, maintain 4 ° C. Real-time PCR
[000384] 2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan probe (Applied Biosystems Cat # 4326317E Cat (human) # 4308313 (rodent)), 0.5μl of TTR TaqMan probe (Applied Biosystems cat # HS00174914 _m1 (human) cat # Rn00562124_m1 (rat)) and 5 μl of Lightcycler 480 master mix probe (Roche Cat # 04887301001) per well in a 384-well plate (Roche cat # 04887301001). Real-time PCR was performed on the Roche LC 480 (Roche) real-time PCR machine. Each duplex was tested on at least two independent transfections and each transfection was analyzed in duplicate, unless otherwise noted.
[000385] To calculate the change in relative times, the data in real time were analyzed using the ΔΔCt method and normalized to tests performed with cells transfected with 10nM AD-1955, or empty transfected cells. IC50s were calculated using a 4-parameter fit model using XLFit and normalized to cells transfected with AD-1955. (sense sequence: cuuAcGcu- GAGuAcuucGAdTsdT (SEQ ID NO: 2202); antisense sequence: UCGAAGuCUcAGCGuAAGdTsdT (SEQ ID NO: 2203)) or naive cells over the same dose range, or at their own lower dose. IC50s were calculated for each individual transfection as well as in combination, where a single IC50 was fitted to the data from both transfections.
[000386] The results of the gene silencing of the exemplary duplex siRNA with various motif modifications of the invention are shown in Table 1 above. Example 5: In vitro Silencing Activity of Chemically Modified RNAi Agents that target TTR
[000387] The following experiments demonstrated the beneficial effects of chemical modifications, including the introduction of triplet repeat motifs, in conjunction with a GalNAc3 ligand, in the activity that silences RNAi agents that target TTR. The sequences of the investigated agents are provided in Table 2 below. The TTR mRNA complementarity regions are as follows: the RNAi agent complementarity region AD-45165, AD-51546 and AD-51547 are GGATGGGATTTCATGTAACC-AAGA (SEQ ID NO: 2204) and the region or complementarity of agents of RNAi AD-45163, AD-51544 and AD-51545 is TTCATGTAA-CCAAGAGTATTCCAT (SEQ ID NO: 2205). IC50 evaluation protocol in Hep3B cells
[000388] The IC50 of each modified siRNA was determined in Hep3B cells (a human hepatoma cell line) by standard reverse transfection using Lipofectamine RNAiMAX. In a nutshell, reverse transfection was performed by adding 5 μL of Opti-MEM to 5 μL of duplex siRNA per well in a 96-well plate along with 10 μL of Opti-MEM plus 0.5 μL of RNAiMax Lipofectamine per well (Invitrogen , Carlsbad CA. cat # 13778-150) and incubating at room temperature for 15-20 minutes. Following incubation, 100 μL of complete antibiotic-free growth media containing 12,000-15,000 Hep3B cells were then added to each well. The cells were incubated for 24 hours at 37 ° C in a 5% CO2 atmosphere before lysis and analysis of GAPDH TTR and mRNA by bDNA (Quantigene). Seven different siRNA concentrations ranging from 10 nM to 0.6 pM were evaluated for IC50 determination and TTR / GAPDH of transfected siRNA cells was normalized to cells transfected with 10 nM Luc siRNA. The results are shown in Table 2. Protocol for the evaluation of free absorption IC50
[000389] Free absorption silencing in primary cinomolgus hepatocytes was assessed after incubation with the TTR siRNA for 4 hours or for 24 hours. Silence was measured at 24 hours from the initial exposure. In a nutshell, 96-well culture plates were coated with 0.05% -0.1% collagen (Sigma C3867-1VV) at room temperature, 24 hours before the start of the experiment. On the test day, siRNAs were diluted in preheated Plating Media consisting of DMEM supplemented with the GIBCO Maintenance Media Kit (serum free, Life Technologies CM4000), and added to the 96-well culture plates coated with collagen. The primary hepatocytes of cryopreserved cinomolgus were quickly thawed in a 37 ° C water bath, and immediately diluted in the Plating Media at a concentration of 360,000 cells / mL. A volume of the cell suspension was gently pipetted over the pre-plated siRNAs such that the final cell count was 18,000 cells / well. The plate was slightly rotated to mix and disperse cells equally through the wells and placed in a 5% CO2 incubator at 37 ° C for 24 hours before lysis and analysis of GAPDH TTR and mRNA by bDNA (Quantigene, Affymetrix). In the case of the 4th incubation with siRNA, the media were decanted after 4 hours of exposure to the cells and replaced with fresh Plating Media for the remaining 20 hours of incubation. The downstream analysis of GAPDH TTR and mRNA was the same as described above. For a typical dose reset curve, siRNAs were titrated from 1 µM to 0.24 nM by 4 times serial dilution.


[000390] Nucleotides in lower case (a, u, g, c) indicate 2'-O-methyl nucleotides; Nf (for example, Af) indicates a 2'-fluoro nucleotide; s indicates a phosphotiorate bond;
[000391] L96 indicates a GalNAc3 ligand; bold nucleotides indicate changes in the corresponding parenting agent. Each nucleotide in bold is at the center of a triplet motif. Petition 870200118973, of 23/09/2020, p. 297/324
[000392] The results are provided in Table 2 and demonstrate that modified RNAi agents that target TTR provide the increased activity that silences. Results: improved activity of modified RNAi agents
[000393] Parental RNAi agents with alternating chemical modifications and a GalNAc3 ligand provided an IC50 in Hep3B cells of approximately 0.01 nM. As shown in Figures 4-5 and Table 2, agents modified with respect to parental agents, for example, by the addition of one or more repeat triplets of 2'-fluoro and 2'-O-methyl modifications, showed silencing activity unexpectedly increased, reaching IC50 values in Hep3B cells that were 5-8 times better than the corresponding parent agent. Results: Free Absorption IC5os in Hep3B cells
[000394] As shown in Table 2 and Figures 6-7, RNAi agents modified for parental AD-45163 also showed increased free absorption silencing. The modified agents showed more than twice the activity of silencing the parental after an incubation period of 24 hours and almost 10 times the activity that silencing the parental after an incubation period of 4 hours.
[000395] As shown in Table 2 and Figures 8-9, the modified RNAi agents for the parental AD-45165 also showed increased free absorption silencing. The modified agents showed 2-3 times the parental silencing activity after a 24 hour incubation period and 5-8 times the parental silencing activity after a 4 hour incubation period.
[000396] Taken collectively, these results demonstrate that the modified RNAi agents presented in this application, for example, AD-51544, AD-51545, AD-51546 and AD-51547, all showed unexpectedly good inhibition of TTR mRNA in silencing in vitro. Example 6: Silencing of TTR and TTR Protein Suppression mRNA in Transgenic Mice
[000397] To assess the efficacy of RNAi agents AD-45163, AD-51544, AD-51545, AD45165, AD-51546 and AD-51547, these agents were administered to transgenic mice expressing human transthyretin with the V30M mutation ( see Santos, SD., Fernaandes, R., and Saraiva, MJ. (2010) Neurobiology of Aging, 31, 280-289). The V30M mutation is known to cause type of familial amyloidotic polyneuropathy I in humans. See, for example, Lobato, L. (2003) J Nephrol., 16 (3): 438-42.
[000398] RNAi agents (in PBS buffer) or PBS control were administered to mice (2 male and 2 female) 18-24 months of age in a single subcutaneous dose of 5 mg / kg or 1 mg / kg. After approximately 48 hours, the mice were anesthetized with 200 μl of ketamine, and then bled by cutting the right caudal artery. Whole blood was isolated and the plasma was isolated and stored at -80 ° C until analysis. The liver tissue was collected, frozen instantly and stored at - 80 ° C until processing.
[000399] Treatment efficacy was assessed by measuring (i) the TTR mRNA in the liver in a 48-hour post-dose, and (ii) measuring the TTR protein in the plasma in pre-hemorrhage and in a post-dose of 48 hours. TTR liver mRNA levels were analyzed using the QuantiGene 2.0 Branched DNA assays (Panomics cat #: QS0011). Briefly, mouse liver samples were ground and tissue lysates were prepared. The liver lysis mixture (a mixture of 1 volume of lysis mixture, 2 volumes of water without nucleases and 10 µl of Proteinase-K / ml at a final concentration of 20mg / ml) was incubated at 65 ° C for 35 minutes. 20 μl of the Working Probe Set (TTR probe for the gene target and GAPDH of the endogenous control) and 80ul of the tissue lysate were then added to the Capture Plate. The Capture Plates were incubated at 55 ° C ± 1 ° C (approx. 16-20hrs). The next day, the Capture Plates were washed 3 times with 1X Wash Buffer (water without nucleases, Buffer Component 1 and Wash Buffer Components 2), then dried by centrifuging for 1 minute at 240 g. 100 μl of the Pre-Amplifier Working Reagent was added to the Capture Plate, which was sealed with aluminum foil and incubated for 1 hour at 55 ° C ± 1 ° C. Incubation for the next 1 hour, the washing step was repeated, then 100 μl of the Amplifying Work Reagent was added. After 1 hour, the washing and drying steps were repeated, and 100 μl of the Marker Probe was added. The capture plates were incubated at 50 ° C ± 1 ° C for 1 hour. The plate was then washed with Wash Buffer 1X, dried and 100 μl of Substrate was added to the Capture Plate. The Capture Plates were read using a SpectraMax Luminometer after an incubation of 5 to 15 minutes. The bDNA data were analyzed by subtracting the average background of each triple sample, calculating values of the resulting triple GAPDH average (control probe) and TTR (experimental probe), and then computing the ratio: (experimental probe background) / ( bottom of the control probe).
[000400] Plasma TTR levels were analyzed using the commercially available kit “AssayMax Human Prealbumin ELISA Kit” (AssayPro, St. Charles, MO, Catalog # EP3010-1) according to the manufacturer's references. Briefly, the mouse plasma was diluted 1: 10,000 in 1X mix diluents and added to pre-coated plates along with kit standards and incubated for 2 hours at room temperature followed by 5X washes with the wash buffer kit. Fifty microliters of biotinylated pre-albumin antibody were added to each well and incubated for 1 hour at room temperature, followed by washing 5X with wash buffer. Fifty microliters of the streptavidin-peroxidase conjugate was added to each well and the plates were incubated for 30 minutes at room temperature followed by washing as previously described. The reaction was developed by adding 50 μl / well of chromogen substrate and incubating for 10 minutes at room temperature with the reaction stopping by adding 50 μl / well of the stop solution. The absorbance at 450 nm was read on a Versamax microplate reader (Molecular Devices, Sunnyvale, CA) and the data were analyzed using Softmax 4.6 software package (Molecular Devices).
[000401] The results are shown in Figures 10-12. Figure 10 shows that the RNAi agents modified with respect to the parental agents AD-45163 and AD-45165 showed RNA silencing activity that was similar or more potent when compared to that of the parental agents. Figure 11 shows that agents AD-51544 and AD-51545 showed dose-dependent silencing activity and that the silencing activity of these agents at a dose of 5mg / kg was similar to that of the corresponding parental AD-45163. Figure 12 shows that agents AD-51546 and AD-51547 also showed dose-dependent silencing activity. In addition, the silencing activity of AD-51546 and AD-51547 at a dose of 5mg / kg was greater than that of the corresponding parental AD-45165. Example 7: Serum and liver pharmacokinetic profiles of RNAi agents targeting TTR in mice
[000402] To evaluate the pharmacokinetic profiles of the RNAi agents AD-45163, AD-51544, AD-51545, AD-51546 and AD-51547, these agents, in PBS buffer, were administered to C57BL / 6 mice using an IV bolus simple or subcutaneous administration (sc). The plasma concentrations and hepatic concentrations of the agents were assessed at various time points after administration.
[000403] Plasma pharmacokinetic parameters are shown in Tables 3 and 4 below. The mean residence time (MRT) in plasma was approximately 0.2 hours after IV dosing and approximately 1 hour after sc dosing At a dose of 25 mg / kg, agents AD-51544, AD-51545, AD-51546 and AD -51547 showed similar plasma pharmacokinetic properties. Each of these agents had bioavailability of more than 75% of the subcutaneous space. Its bioavailability was superior to that of the parental agent AD-45163, which was administered at a higher dose of 30 mg / kg. The subcutaneous bioavailability of AD-51544 and AD-51547 was approximately 100%, whereas that of AD-51545 was 90% and that of AD-51546 was 76%. Table 3: Summary of Plasma PK Parameter Estimates After Sc Administration of TTR-GalNAc siRNAs in
Table 4: PK parameters of plasma siRNA in mice after an IV bolus or sc dose of AD-51544, 51545, 51546 or 51547 at 25 mg / kg

[000404] The results also indicated that the RNAi agents AD-45163, AD-51544, AD-51545, AD-51546 and AD-51547 achieve similar or higher concentrations in the liver when administered subcutaneously than when administered by bolus IV. The hepatic pharmacokinetic parameters are shown in Tables 5 and 6 below. Peak concentration (Cmax) and area under the curve (AUC0-last) in the liver were two to three times higher after subcutaneous administration compared to IV administration of the same agent at the same dose. Liver exposures were the highest for AD-51547 and the lowest for AD-51545. The average residence time (MRT) and elimination half-life were longer for AD-51546 and AD-51547 compared to AD-51544 and AD-51545. After subcutaneous administration, the approximate MRTs were 40 hours during AD-51546 and 25 hours for AD-51547, while the MRTs of AD-51544 and AD-51545 were lower (approximately 6-9 hours). The elimination half-life of AD-51546 and AD-51547 was also higher (41-53 hours) than was the elimination half-life of AD-51544 and AD-51545 (6-10 hours). Table 5: Summary of Hepatic PK Parameter Estimates After Administration of sc of TTR-GalNAc siRNAs in Mice Parameter 30 mpk AD- 45163 (h / c TTR-GalNAc) 25 mpk AD-51544 (h / c TTR-GalNAc) 25 mpk AD-51545 (h / c TTR- GalNAc) 25 mpk AD-51546 (h / c TTR- GalNAc) 25 mpk AD-51547 (h / c TTR- GalNAc) Tmax in the Liver (h) 8 4 4 2 8 Cmax no Liver (μg / g) 313 126 80 117 174 AUC in the liver (h * μg / g) 4519 1092 763 2131 4583 Table 6: PK parameters of hepatic siRNA in mice after an IV bolus or sc dose of AD-51544, 51545, 51546 or 51547 at 25 mg / kg Test article AD-51544 AD-51545 AD-51546 AD-51547 Dose of siRNA (mg / kg) 25 25 25 25 Route of administration IV SC IV SC IV SC IV SC tmax (h) 1 4 1 4 4 2 2 8 Test article AD-51544 AD-51545 AD-51546 AD-51547 Cmax (μg / g) 67.9 126 37.0 80.5 35.3 117 73.8 174 AUCθ-last (h * μg / g) 632 1092 324 763 984 2131 1429 4583 MRTo-iast (h) 8.7 6.5 5.9 8.5 45.7 40.2 29.4 25.3 Ti / 2β Apparent ( h) 8.1 8.2 5.7 10.0 51.1 45.3 41.1 52.7 Example 8: In vitro stability of RNAi Agents in Monkey Serum



[000405] Serum stability of RNAi agents AD-51544, AD-51545, AD-51546 and AD-51547 has also been evaluated in monkeys. The results demonstrated that the sense and antisense phytases of AD-51544, AD-51545 and AD-51547 showed serum stability over the period of approximately 24 hours (data not shown). Example 9: RNAi Agents Produce Long-Lasting Suppression of TTR Protein in Non-Human Primates
[000406] The RNA silencing activity of RNAi agents AD-45163, AD-51544, AD-51545, AD-51546 and AD-51547 was assessed by measuring the suppression of TTR protein in the serum of cinomolgus monkeys after subcutaneous administration of five doses of 5 mg / kg (one dose each day for 5 days) or a single 25 mg / kg dose. Pre-dose levels of serum TTR protein were assessed by averaging levels at 11 days before the first dose, 7 days before the first dose, and 1 day before the first dose. The post-dose serum levels of the TTR protein were assessed by determining the serum level starting 1 day after the final dose (ie, study day 5 in the 5x5 mg / kg group and study day 1 in the 1x25 mg / group) kg) up to 49 days after the last dose (ie, study day 53 in the 5x5 mg / kg group and study day 49 in the 1x25 mg / kg group). See Figure 13.
[000407] TTR protein levels were assessed as described in Example 6. The results are shown in Figure 14 and Tables 7 and 8.
[000408] A maximum suppression of the TTR protein of up to approximately 50% was achieved in the groups that received 25 mg / kg of AD-45163, AD-51544, AD-51546 and AD-51547 (see Table 8). A greater maximum suppression of the TTR protein of approximately 70% was achieved in the groups that received 5x5 mg / kg of AD-45163, AD-51544, AD-51546 and AD-51547 (see Table 7). The AD-51545 agent produced a lower degree of suppression in both administration protocols. Significant suppression of approximately 20% or more persisting for up to 49 days after the last dose of AD-51546 and AD-51547 in both 1x25 mg / kg and 5x5 mg / kg protocols. Generally, the best suppression was achieved in the 5x5 mg / kg protocol than in the 1x25 mg / kg protocol. Table 7 Fraction of Pre-dose Serum Transtirretin in Cinomolgus Monkeys (5 mg / kg daily for 5 days
Table 8 Fraction of Pre-dose Serum Transtirretin in Cinomolgus Monkeys (25 mg / kg)
Example 10: Tolerability of RNAi Agents Targeting TTR In Cytokine Evaluation in Whole Blood Assay
[000409] To assess the tolerability of RNAi agents that target TTR (including AD-45163, AD-51544, AD-51545, AD-51546 and AD-51547), each agent was tested in a whole blood assay using blood from three human donors. The agents were transfected with DOTAP 300 nM or 1 μM without transfection reagent (free siRNA). There was less than a two-fold modification of the following cytokines / chemokines: G-CSF, IFN-Y, IL-10, IL-12 (p70), IL1β, IL-1ra, IL-6, IL-8, IP-10 , MCP-1, MIP-1α, MIP-1β, TNFα. (Results not shown). In Vivo Evaluation
[000410] To assess in vivo tolerability, RNAi agents were injected subcutaneously into CD1 mice at a dose of 125 mg / kg. No cytokine induction was observed at 2, 4, 6, 24, or 48 hours after subcutaneous injection of AD-45163. No significant cytokine induction was seen at 6 or 24 hours after subcutaneous injection of AD-51544, AD-51545, AD-51546 or AD-51547
[000411] To further assess in vivo tolerability, multiple RNAi agents (including AD-45163, AD-51544, AD-51545, AD-51546 and AD-51547) were tested by subcutaneous injection of 5 and 25 mg in non-human primates (cinomolgus monkeys) with dose volumes between 1-2 ml per site. No erythema or edema was observed at injection sites. Single-dose Rat Tolerability Study
[000412] To assess toxicity, rats were injected with a single subcutaneous dose of 100, 250, 500, or 750 mg / kg of AD-45163 (see Table 9). The following assessments were made: clinical signs of toxicity, body weight, hematology, clinical chemistry and coagulation, organ weights (liver &spleen); naked eye and microscopic evaluation (kidney, liver, lung, lymph node, spleen, testicles, thymus, aorta, heart, intestine (small and large) Table 9: Rat Tolerability Study with Single Dose sc: 100, 250, 500 & 750 mg / kg of AD-45163 in Sprague Rats

[000413] The results showed no clinical signs related to the toxicity test article, effects on body weight, organ weights or clinical chemistry. No histopathology was observed in the heart, kidneys, testicles, spleen, liver and thymus. There was a slight increase related to the non-adverse test article in WBC ($ 68%, mainly attributed to the increase in NEUT and MONO) by 750 mg / kg. These results indicate that a single dose of up to 750 mg / kg is well tolerated in rats. Tolerability of repeated subcutaneous administrations in rats
[000414] To assess the tolerability of repeated subcutaneous administrations of AD-45163, daily subcutaneous injections of 300 mg / kg were given for 5 days, and an autopsy was performed on day 6. The study design is shown in Table 10. Table 10: Five-Day Repeated Dose Tolerability Study in Rats

[000415] The following outcome variables were evaluated: clinical signs, body weights, hematology, clinical chemistry and coagulation, organ weights, naked and microscopic evaluation (liver, spleen, kidney, heart, GI tract and jointly) , the injection site). The results showed no clinical signs related to the test article, body weight or effects on organ weight, and also no findings related to the test article in clinical or chemical hematology. There was a possible slight extension of activated partial thromboplastin time (APTT) on day 6 (20.4 versus 17.4 seconds). Histopathology did not reveal any findings related to the test article in the liver, spleen, heart and GI tract. In the kidney, minimal to slight hypertrophy of the tubular epithelium (not adverse) was observed. At the last injection site, there was minimal, non-adverse, multifocal mononuclear infiltration. These results indicate that five doses of 300 mg / kg daily of the parental RNAi agent AD-45163 are well tolerated in rats. Example 11: RNAi Agents Produce Long-Lasting Suppression of TTR Protein in Non-Human Primates
[000416] The RNA silencing activity of RNAi agent AD-51547 was evaluated by measuring the suppression of TTR protein in the serum of cinomolgus monkeys after subcutaneous administration of a "loading phase" of the RNAi agent: five daily doses of 2 , 5 mg / kg, 5 mg / kg or 10 mg / kg (one dose each day for 5 days) followed by a "maintenance phase" of the RNAi agent: weekly dosing of 2.5 mg / kg, 5 mg / kg or 10 mg / kg for 4 weeks. Pre-dose levels of serum TTR protein were assessed by averaging levels at 11 days before the first dose, 7 days before the first dose, and 1 day before the first dose. The post-dose serum levels of the TTR protein were assessed by determining the serum level in relation to the pre-dose starting 1 day after the loading phase was completed up to 40 days after the last dose of the maintenance phase (ie, study 70).
[000417] TTR protein levels were assessed as described in Example 6. The results are shown in Figure 15.
[000418] A maximum suppression of the TTR protein of up to approximately 80% was achieved in all groups receiving 2.5 mg / kg, 5 mg / kg or 10 mg / kg of AD-51547. Silence at the lowest point was achieved in all groups by day 14, sustained suppression at levels of silencing at the lowest point with a weekly maintenance dose of 2.5 mg / kg, 5 mg / kg or 10 mg / kg of AD-51547. TTR levels had not returned to baseline more than 40 days after the day of administration of the last maintenance dose of dose levels of 5 and 2.5 mg / kg. Equivalents:
[000419] Those skilled in the art will recognize or be able to ascertain the use of experimentation no more than regular, many equivalent to the specific modalities and methods described in this application. Such equivalents are intended to be encompassed by the scope of the following claims.

权利要求:
Claims (28)
[0001]
1. Double-stranded RNAi agent, characterized by the fact that it comprises a sense strand complementary to an antisense strand, in which said strand comprises a region complementary to the nucleotide sequence 5'-GGATGGGATTTCATGTA- ACCAAGA-3 '( SEQ ID NO: 2204), in a total of 21 nucleotides and the antisense tape has a total of 23 nucleotides, in which the said double-stranded RNAi agent is represented by the formula (III): sense: 5 'np -Na - (XXX) i-Nb -YYY -Nb - (ZZZ) j -Na - n. 3 'antisense: 3' np'-Na '- (X'X'X') k-Nb'-Y'Y'Y'-Nb '- (Z'Z'Z') l-Na'- n. ' 5 '(III) where: i, O, k and l are each, independently, 0 or 1, where i is 0, j is 1; p, p ', q and q' are each, independently, 0 to 6; each of Na and Na 'independently represents a modified oligonucleotide sequence, each sequence comprising at least two differently modified nucleotides; each of Nb and Nb 'independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides, wherein the modifications are 2'-O-methyl, 2'-fluoro, or both; each of np, np ', nq and nq' independently represents a protruding nucleotide; XXX, YYY, ZZZ, X'X'X ', Y'Y'Y' and Z'Z'Z 'each represent, independently, a motif of three identical modifications in three consecutive nucleotides; wherein the Y nucleotides contain a 2'-fluoro modification and the Y 'nucleotides contain a 2'-O-methyl modification, wherein said YYY motif occurs at positions 9, 10 and 11 when the duplex region is 21 nucleotides; wherein the sense strand is conjugated to at least one linker, where the linker is one or more GalNAc derivatives linked via a divalent or trivalent branched linker; where "complementary" means no more than 2 incompatible base pairs within 2 nucleotides of the 5 'and / or 3' end; and where a phosphorothioate is present in the last 2 nucleotides at one or both ends.
[0002]
2. RNAi agent according to claim 1, characterized by the fact that V is 0 and l is 1.
[0003]
3. RNAi agent according to claim 1, characterized by the fact that YYY is complementary to Y'Y'Y'e ZZZ is complementary to Z'Z'Z '.
[0004]
4. RNAi agent according to claim 1, characterized in that the Y'Y'Y' motif occurs in positions 11, 12 and 13 of the 5 'end antisense tape.
[0005]
5. RNAi agent according to claim 1, characterized by the fact that the duplex region is 21 pairs of nucleotides in length.
[0006]
6. RNAi agent, according to claim 1, characterized by the fact that the ligand is
[0007]
7. RNAi agent according to claim 1, characterized by the fact that the binder is attached to the 3 'end of the sense tape.
[0008]
8. RNAi agent according to claim 7, characterized in that the RNAi agent is conjugated to the linker as shown in the following schematic representation 3 'where X is O or S.
[0009]
9. RNAi agent according to claim 8, characterized by the fact that the RNAi agent is conjugated to the linker as shown in the schematic representation below
[0010]
10. RNAi agent according to claim 1, characterized by the fact that p '> 0.
[0011]
11. RNAi agent according to claim 1, characterized by the fact that p '= 2.
[0012]
12. RNAi agent according to claim 11, characterized in that the protruding nucleotides q '= 0, p = 0, q = 0 and p' are complementary to the target mRNA.
[0013]
13. RNAi agent according to claim 10, characterized by the fact that at least one np 'is linked to an adjacent nucleotide via a phosphorothioate bond.
[0014]
14. RNAi agent according to claim 13, characterized by the fact that all np 'are linked to adjacent nucleotides via phosphorothioate bonds.
[0015]
15. Pharmaceutical composition, characterized by the fact that it comprises an RNAi agent, as defined in claim 1, and one or more pharmaceutically acceptable vehicles (additives), excipient and / or diluents.
[0016]
16. Pharmaceutical composition according to claim 15, characterized by the fact that the RNAi agent is administered in an un-buffered solution.
[0017]
Pharmaceutical composition according to claim 16, characterized in that said non-buffered solution is saline or water.
[0018]
18. Pharmaceutical composition, according to claim 15, characterized by the fact that said siRNA is administered with a buffer solution.
[0019]
19. Pharmaceutical composition according to claim 18, characterized in that said buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.
[0020]
20. Pharmaceutical composition according to claim 19, characterized by the fact that said buffer solution is phosphate buffered saline (PBS).
[0021]
21. Pharmaceutical composition, according to claim 15, characterized by the fact that it is: (i) a liposome; or (ii) a lipid formulation.
[0022]
22. A method of inhibiting the expression of a transthyretin (TTR) in a cell, characterized by the fact that it comprises putting said cell in contact with an RNAi agent, as defined in claim 1, in an amount effective to inhibit the expression of said TTR in said cell, thereby inhibiting the expression of said transthyretin (TTR) in said cell.
[0023]
23. Use of a therapeutically effective amount or a prophylactically effective amount of an RNAi agent, as defined in any one of claims 1 to 14, characterized by the fact that it is in the preparation of a pharmaceutical composition for the treatment or prevention of a disease associated with TTR in an individual.
[0024]
24. Use, according to claim 23, characterized by the fact that said individual is a human being.
[0025]
25. Use, according to claim 23, characterized by the fact that said individual is an individual suffering from a disease associated with TTR; or (b) at risk of developing a disease associated with TTR.
[0026]
26. Use according to any one of claims 15 to 25, characterized by the fact that said individual has a mutation in the TTR gene that is associated with the development of a disease associated with TTR, in which preferably said disease associated with TTR is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal amyloidosis / central nervous system (CNS) and hyperthyroxinemia.
[0027]
27. Kit for carrying out the method, as defined in king-vindication 22, characterized by the fact that it comprises: (a) said RNAi agent, and (b) instructions for use.
[0028]
28. A kit for carrying out a method of treating or preventing a disease associated with TTR in an individual, comprising administering to that individual a therapeutically effective amount or a prophylactically effective amount of an RNAi agent, as defined in claim 1, of that way, treating or avoiding said disease associated with TTR in said individual, the kit characterized by the fact that it comprises: (a) said RNAi agent, (b) instructions for use, and (c) optionally, means to administer the said RNAi agent to said individual.

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法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-13| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

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2020-02-04| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-06-23| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-11-03| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-01-19| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 16/11/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161561710P| true| 2011-11-18|2011-11-18|

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US61/561,710|2011-11-18|

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US201261615618P| true| 2012-03-26|2012-03-26|

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US61/615,618|2012-03-26|

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US201261680098P| true| 2012-08-06|2012-08-06|

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US61/680,098|2012-08-06|

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PCT/US2012/065691|WO2013075035A1|2011-11-18|2012-11-16|Rnai agents, compositions and methods of use thereof for treating transthyretinassociated diseases|

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