# Patent

## Title
RNAi agents, compositions and methods of use thereof for treating  transthyretin (TTR) associated diseases

## Abstract

The present invention provides RNAi agents, e.g., double stranded RNAi agents, that target the transthyretin (TTR) gene and methods of using such RNAi agents for treating or preventing TTR-associated diseases.

## Background
RELATED APPLICATIONS
This application is a 35 U.S.C. §371 national stage filing of International Application No. PCT/US2012/065691, filed Nov. 16, 2012, which claims priority to U.S. Provisional Application No. 61/561,710, filed on Nov. 18, 2011, U.S. Provisional Application No. 61/615,618, filed on Mar. 26, 2012, and U.S. Provisional Application No. 61/680,098, filed on Aug. 6, 2012, the entire contents of each of which are hereby incorporated herein by reference.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 13, 2012, is named 121301WO.txt and is 541,508 bytes in size.
BACKGROUND OF THE INVENTION
Transthyretin (TTR) (also known as prealbumin) is found in serum and cerebrospinal fluid (CSF). TTR transports retinol-binding protein (RBP) and thyroxine (T4) and also acts as a carrier of retinol (vitamin A) through its association with RBP in the blood and the CSF. Transthyretin is named for its transport of thyroxine and retinol. TTR also functions as a protease and can cleave proteins including apoA-I (the major HDL apolipoprotein), amyloid β-peptide, and neuropeptide Y. See Liz, M. A. et al. (2010) IUBMB Life, 62(6):429-435.
TTR is a tetramer of four identical 127-amino acid subunits (monomers) that are rich in beta sheet structure. Each monomer has two 4-stranded beta sheets and the shape of a prolate ellipsoid. Antiparallel beta-sheet interactions link monomers into dimers. A short loop from each monomer forms the main dimer-dimer interaction. These two pairs of loops separate the opposed, convex beta-sheets of the dimers to form an internal channel.
The liver is the major site of TTR expression. Other significant sites of expression include the choroid plexus, retina (particularly the retinal pigment epithelium) and pancreas.
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. (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi:10.1152/ajpheart.00815.2011. Extracellular deposition of amyloid fibrils in organs and tissues is the hallmark of amyloidosis. Amyloid fibrils are composed of misfolded protein aggregates, which may result from either excess production of or specific mutations in precursor proteins. The amyloidogenic potential of TTR may be related to its extensive beta sheet structure; X-ray crystallographic studies indicate that certain amyloidogenic mutations destabilize the tetrameric structure of the protein. See, e.g., Saraiva M. J. M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11.
Amyloidosis is a general term for the group of amyloid diseases that are characterized by amyloid deposits. Amyloid diseases are classified based on their precursor protein; for example, the name starts with “A” for amyloid and is followed by an abbreviation of the precursor protein, e.g., ATTR for amloidogenic transthyretin. Ibid.
There are numerous TTR-associated diseases, most of which are amyloid diseases. Normal-sequence TTR is associated with cardiac amyloidosis in people who are elderly and is termed senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA often is accompanied by microscopic deposits in many other organs. TTR amyloidosis manifests in various forms. When the peripheral nervous system is affected more prominently, the disease is termed familial amyloidotic polyneuropathy (FAP). When the heart is primarily involved but the nervous system is not, the disease is called familial amyloidotic cardiomyopathy (FAC). A third major type of TTR amyloidosis is leptomeningeal amyloidosis, also known as leptomeningeal or meningocerebrovascular amyloidosis, central nervous system (CNS) amyloidosis, or amyloidosis VII form. Mutations in TTR may also cause amyloidotic vitreous opacities, carpal tunnel syndrome, and euthyroid hyperthyroxinemia, which is a non-amyloidotic 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, e.g., Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.
Abnormal amyloidogenic proteins may be either inherited or acquired through somatic mutations. Guan, J. et al. (Nov. 4, 2011) Current perspectives on cardiac amyloidosis, Am J Physiol Heart Circ Physiol, doi:10.1152/ajpheart.00815.2011. Transthyretin associated ATTR is the most frequent form of hereditary systemic amyloidosis. Lobato, L. (2003) J. Nephrol., 16:438-442. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of ATTR. More than 85 amyloidogenic TTR variants are known to cause systemic familial 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 opacities. Ibid.
The V30M mutation is the most prevalent TTR mutation. See, e.g., 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. It is estimated that SSA affects more than 25% of the population over age 80. Westermark, P. et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87 (7): 2843-5.
Accordingly, there is a need in the art for effective treatments for TTR-associated diseases.
SUMMARY OF THE INVENTION
The present invention provides RNAi agents, e.g., double stranded RNAi agents, targeting the Transthyretin (TTR) gene. The present invention also provides methods of inhibiting expression of TTR and methods of treating or preventing a TTR-associated disease in a subject using the RNAi agents, e.g. 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 expression of TTR. Agents including a certain pattern of chemical modifications (e.g., an alternating pattern) and a ligand are shown herein to be effective in silencing the activity of the TTR gene. Furthermore, agents including one or more motifs of three identical modifications on three consecutive nucleotides, including one such motif at or near the cleavage site of the agents, show surprisingly enhanced TTR gene silencing activity. When a single such chemical motif is present in the agent, it is preferred to be at or near the cleavage region for enhancing of the gene silencing activity. Cleavage region is the region surrounding the cleavage site, i.e., the site on the target mRNA at which cleavage occurs.
Accordingly, in one aspect, the present invention features RNAi agents, e.g., double stranded RNAi agents, for inhibiting expression of a transthyretin (TTR). The double stranded RNAi agent includes a sense strand complementary to an antisense strand. The antisense strand includes a region complementary to a part of an mRNA encoding transthyretin. Each strand has 14 to 30 nucleotides, and the double stranded RNAi agent is represented by 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).

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 represents an oligonucleotide sequence including 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence including at least two differently modified nucleotides; each Nb and Nb′ independently represents an oligonucleotide sequence including 0-10 nucleotides which are either modified or unmodified or combinations thereof; each np, np′, nq, and nq′ independently represents an overhang nucleotide; XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represents one motif of three identical modifications on three consecutive nucleotides; modifications on Nb differ from the modification on Y and modifications on Nb′ differ from the modification on Y′. In some embodiments, the sense strand is conjugated to at least one ligand, e.g., at least one ligand, e.g., at least one ligand attached to the 3′ end of the sense strand. In other embodiments, the ligand may be conjugated to the antisense strand.

In some embodiments, i is 1; j is 1; or both i and j are 1.
In some embodiments, k is 1; l is 1; or both k and 1 are 1.
In some embodiments, i is 0; j is 1.
In some embodiments, i is 1, j is 0.
In some embodiments, k is 0; l is 1.
In some embodiments, k is 1; l is 0.
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′.
In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand.
In some embodiments, the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end.
In some embodiments, the Y′ is 2′-O-methyl.
In some embodiments, the Y′ is 2′-fluoro.
In some embodiments, formula (III) is represented as formula (IIIa):

sense: 5′np-Na-YYY—Nb-ZZZ-Na-nq3′

antisense: 3′np′-Na′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′nq′5′   (IIIa).

In formula IIIa, each Nb and Nb′ independently represents an oligonucleotide sequence including 1-5 modified nucleotides.

In some embodiments, formula (III) is represented as formula (IIIb):

sense: 5′np-Na-XXX-Nb-YYY-Na-nq3′

antisense: 3′np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Na′-nq′5′   (IIIb).

In formula IIIb each Nb and Nb′ independently represents an oligonucleotide sequence including 1-5 modified nucleotides.

In some embodiments, formula (III) is represented as formula (IIIc):

sense: 5′np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3′

antisense: 3′np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′-nq′5′   (IIIc).

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.

In many embodiments, the duplex region is 15-30 nucleotide pairs in length. In some embodiments, the duplex region is 17-23 nucleotide pairs in length, 17-25 nucleotide pairs in length, 23-27 nucleotide pairs in length, 19-21 nucleotide pairs in length, or 21-23 nucleotide pairs in length.
In certain embodiments, each strand has 15-30 nucleotides.
In some embodiments, the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof. In some preferred embodiments, the modifications on the nucleotides are 2′-O-methyl or 2′-fluoro.
In some embodiments, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a bivalent or trivalent branched linker. In particular embodiments, the ligand is




In some embodiments, the ligand is attached to the 3′ end of the sense strand.
In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following schematic




wherein X is O or S.
In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following schematic




In some embodiments, the RNAi agent further includes at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand. In some embodiments, the strand is the antisense strand. In other embodiments, the strand is the sense strand.
In certain embodiments, the base pair at the 1 position of the 5′-end of the duplex is an AU base pair.
In some embodiments, the Y nucleotides contain a 2′-fluoro modification.
In some embodiments, the Y′ nucleotides contain a 2′-O-methyl modification.
In some embodiments, 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 embodiments, p′=1 or 2. In some preferred embodiments, p′=2. In some such embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are complementary to the target mRNA. In other such embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides are non-complementary to the target mRNA.
In some embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.
In certain embodiments, linkages between np′ include phosphorothioate linkages. In some such embodiments, the linkages between np′ are phosphorothioate linkages.
In some embodiments, the RNAi agent is selected from the group of agents listed in Table 1.
In preferred embodiments, the RNAi agent is selected from the group consisting of AD-51544, AD-51545, AD-51546, and AD-51547.
In an even more preferred embodiment, the RNAi agent is AD-51547 having the following structure:









sense:



(SEQ ID NO: 2)



5′- UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′



 



antisense:



(SEQ ID NO: 3)



5′- uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′






wherein lowercase nucleotides (a, u, g, c) indicate 2′-O-methyl nucleotides; Nf (e.g., Af) indicates a 2′-fluoro nucleotide; s indicates a phosphothiorate linkage; L96 indicates a GalNAc3 ligand.

In another aspect, the present invention features a cell containing the RNAi agent for inhibiting expression of TTR.
In a further aspect, the present invention features a pharmaceutical composition comprising an RNAi agent for inhibiting expression of TTR. In some embodiments, the pharmaceutical composition is a solution comprising the RNAi agent. In some embodiments, the solution comprising the RNAi agent is an unbuffered solution, e.g., saline solution or water. In other embodiments, the solution is a buffered solution, e.g., a solution of phosphate buffered saline (PBS). In other embodiments, the pharmaceutical composition is a liposome or a lipid formulation. In some embodiments, the lipid formulation comprises a XTC or MC3.
In yet another aspect, the present invention features methods of inhibiting expression of transthyretin (TTR) in a cell. The methods include contacting a cell with an RNAi agent, e.g., a double stranded RNAi agent, in an amount effective to inhibit expression of TTR in the cell, thereby inhibiting expression of TTR in the cell.
In some embodiments, the expression of TTR is inhibited by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.
In other embodiments, the cell is contacted in vitro with the RNAi agent. In other embodiments, the cell is present within a subject. In preferred embodiments, the subject is a human.
In further embodiments, the subject is a subject suffering from a TTR-associated disease and the effective amount is a therapeutically effective amount. In other embodiments, the subject is a subject at risk for developing a TTR-associated disease and the effective amount is a prophylactically effective amount. In some embodiments, a subject at risk for developing a TTR-associated disease is a subject who carries a TTR gene mutation that is associated with the development of a TTR-associated disease.
In certain embodiments, the TTR-associated disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS) amyloidosis, and hyperthyroxinemia.
In some embodiments, the subject has a TTR-associated amyloidosis and the method reduces an amyloid TTR deposit in the subject.
In other embodiments, the RNAi agent is administered to the subject by an administration means selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combinations thereof. In certain embodiments, the RNAi agent is administered to the subject via subcutaneous or intravenous administration. In preferred embodiments, the RNAi agent is administered to the subject via subcutaneous administration. In some such embodiments, the subcutaneous administration includes administration via a subcutaneous pump or subcutaneous depot.
In certain embodiments, the RNAi agent is administered to the subject such that the RNAi agent is delivered to a specific site within the subject. In some embodiments, the site is selected from the group consisting of liver, choroid plexus, retina, and pancreas. In preferred embodiments, the site is the liver. In some embodiments, the delivery of the RNAi agent is mediated by asialoglycoprotein receptor (ASGP-R) present in hepatocytes.
In some embodiments, the RNAi agent is administered at a dose of between about 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg to about 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, between about 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10 mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg to about 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about 15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg, between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg to about 50 mg/kg.
In some embodiments, the RNAi agent is administered at a dose of about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about 31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, about 49 mg/kg or about 50 mg/kg.
In some embodiments, the RNAi agent is administered in two or more doses. In particular embodiments, the RNAi agent is administered at intervals selected from the group consisting of once every about 2 hours, once every about 3 hours, once every about 4 hours, once every about 6 hours, once every about 8 hours, once every about 12 hours, once every about 24 hours, once every about 48 hours, once every about 72 hours, once every about 96 hours, once every about 120 hours, once every about 144 hours, once every about 168 hours, once every about 240 hours, once every about 336 hours, once every about 504 hours, once every about 672 hours and once every about 720 hours.
In other embodiments, the method further includes assessing the level of TTR mRNA expression or TTR protein expression in a sample derived from the subject.
In preferred embodiments, administering the RNAi agent does not result in an inflammatory response in the subject as assessed based on the level of a cytokine or chemokine selected from the group consisting of G-CSF, IFN-γ, IL-10, IL-12 (p70), IL1β, IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, and any combinations thereof, in a sample from the subject.
In some embodiments, the RNAi agent is administered using a pharmaceutical composition
In preferred embodiments, the RNAi agent is administered in a solution. In some such embodiments, the siRNA is administered in an unbuffered solution. In one embodiment, the siRNA is administered in water. In other embodiments, the 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).
In another embodiment, the pharmaceutical composition is a liposome or a lipid formulation comprising SNALP or XTC. In one embodiment, the lipid formulation comprises an MC3.
In another aspect, the invention provides methods of treating or preventing a TTR-associated disease in a subject. The methods include administering to the subject a therapeutically effective amount or prophylactically effective amount of an RNAi agent, e.g., a double stranded RNAi agent, thereby treating or preventing the TTR-associated disease in the subject.
In some embodiments, TTR expression in a sample derived from the subject is inhibited by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60% or at least about 70% at least about 80%, or at least about 90%.
In some embodiments, the subject is a human.
In some embodiments, the subject is a subject suffering from a TTR-associated disease. In other embodiments, the subject is a subject at risk for developing a TTR-associated disease.
In some embodiments, the subject is a subject who carries s a TTR gene mutation that is associated with the development of a TTR-associated disease.
In certain embodiments, the TTR-associated disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS) amyloidosis, and hyperthyroxinemia.
In some embodiments, the subject has a TTR-associated amyloidosis and the method reduces an amyloid TTR deposit in the subject.
In some embodiments, the RNAi agent is administered to the subject by an administration means selected from the group consisting of subcutaneous, intravenous, intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combinations thereof. In certain embodiments, the RNAi agent is administered to the subject via subcutaneous or intravenous administration. In preferred embodiments, the RNAi agent is administered to the subject via subcutaneous administration. In some such embodiments, the subcutaneous administration includes administration via a subcutaneous pump or subcutaneous depot.
In certain embodiments, the RNAi agent is administered to the subject such that the RNAi agent is delivered to a specific site within the subject. In some such embodiments, the site is selected from the group consisting of liver, choroid plexus, retina, and pancreas. In preferred embodiments, the site is the liver. In some embodiments, the delivery of the RNAi agent is mediated by asialoglycoprotein receptor (ASGP-R) present in hepatocytes.
In some embodiments, the RNAi agent is administered at a dose of between about 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg to about 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, between about 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10 mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg to about 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about 15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg, between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg to about 50 mg/kg.
In some embodiments, the RNAi agent is administered at a dose of about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about 31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, about 49 mg/kg or about 50 mg/kg.
In some embodiments, the RNAi agent is administered in two or more doses. In particular embodiments, the RNAi agent is administered at intervals selected from the group consisting of once every about 2 hours, once every about 3 hours, once every about 4 hours, once every about 6 hours, once every about 8 hours, once every about 12 hours, once every about 24 hours, once every about 48 hours, once every about 72 hours, once every about 96 hours, once every about 120 hours, once every about 144 hours, once every about 168 hours, once every about 240 hours, once every about 336 hours, once every about 504 hours, once every about 672 hours and once every about 720 hours.
In other embodiments, the method further includes assessing the level of TTR mRNA expression or TTR protein expression in a sample derived from the subject.
In preferred embodiments, administering the RNAi agent does not result in an inflammatory response in the subject as assessed based on the level of a cytokine or chemokine selected from the group consisting of G-CSF, IFN-γ, IL-10, IL-12 (p70), IL1β, IL-1ra, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, and any combinations thereof, in a sample from the subject.
In some embodiments, the RNAi agent is administered using a pharmaceutical composition, e.g., a liposome.
In some embodiments, the RNAi agent is administered in a solution. In some such embodiments, the siRNA is administered in an unbuffered solution. In one embodiment, the siRNA is administered in saline or water. In other embodiments, the 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).
In another aspect, the present invention provides a method of inhibiting expression of transthyretin (TTR) in a cell, including contacting a cell with an RNAi agent, e.g., a double stranded RNAi agent, in an amount effective to inhibit expression of TTR in the cell. In one aspect, the double stranded RNAi agent is selected from the group of agents listed in Table 1, thereby inhibiting expression of transthyretin (TTR) in the cell.
In another aspect, the present invention provides a method of inhibiting expression of transthyretin (TTR) in a cell, including contacting a cell with an RNAi agent, e.g., a double stranded RNAi agent, in an amount effective to inhibit expression of TTR 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 expression of transthyretin (TTR) in the cell.
In a further aspect, the present invention provides a method of treating or preventing a TTR-associated disease in a subject, including administering to the subject a therapeutically effective amount or a prophylactically effective amount of an RNAi agent, e.g., 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 TTR-associated disease in the subject.
In yet another aspect, the present invention provides a method of treating or preventing a TTR-associated disease in a subject, including administering to the subject a therapeutically effective amount or a prophylactically effective amount of an RNAi agent, e.g., 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 TTR-associated disease in the subject.
In further aspects, the invention provides kits for performing the methods of the invention. In one aspect, the invention provides a kit for performing a method of inhibiting expression of transthyretin (TTR) in a cell comprising contacting a cell with an RNAi agent, e.g., a double stranded RNAi agent, in an amount effective to inhibit 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 subject.
The present invention is further illustrated by the following detailed description and drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph depicting that administering to mice a single subcutaneous dose of a GalNAc-conjugated RNAi agent targeting TTR resulted in dose-dependent suppression of TTR mRNA.
FIG. 2 is a graph depicting that administering to mice a single subcutaneous dose of 7.5 mg/kg or 30 mg/kg of a GalNAc conjugated RNAi agent targeting TTR resulted in long lasting suppression of TTR mRNA.
FIG. 3 depicts the human TTR mRNA sequence.
FIG. 4 is a graph depicting improved silencing activity of RNAi agents modified relative to the parent AD-45163.
FIG. 5 is a graph depicting improved silencing activity of RNAi agents modified relative to the parent AD-45165.
FIG. 6 is a graph depicting improved free uptake silencing following 4 hour incubation with RNAi agents modified relative to the parent AD-45163.
FIG. 7 is a graph depicting improved free uptake silencing following 24 hour incubation with RNAi agents modified relative to the parent AD-45163.
FIG. 8 is a graph depicting improved free uptake silencing following 4 hour incubation with RNAi agents modified relative to the parent AD-45165.
FIG. 9 is a graph depicting improved free uptake silencing following 24 hour incubation with RNAi agents modified relative to the parent AD-45165.
FIG. 10 is a graph depicting silencing of TTR mRNA in transgenic mice that express hTTR V30M following administration of a single subcutaneous dose of RNAi agents AD-51544, AD-51545, AD-45163, AD-51546, AD-51547, or AD-45165.
FIG. 11 is a graph depicting TTR protein suppression in transgenic mice that express hTTR V30M following administration of a single subcutaneous dose of 5 mg/kg or 1 mg/kg of RNAi agents AD-51544, AD-51545, or AD-45163.
FIG. 12 is a graph depicting TTR protein suppression in transgenic mice that express hTTR V30M following administration of a single subcutaneous dose of 5 mg/kg or 1 mg/kg of RNAi agents AD-51546, AD-51547, or AD-45165.
FIG. 13 depicts the protocol for post-dose blood draws in monkeys that received 5×5 mg/kg RNAi agent (top line) or 1×25 mg/kg RNAi agent (bottom line).
FIG. 14 is a graph depicting suppression of TTR protein in non-human primates following subcutaneous administration of five 5 mg/kg doses (top panel) or a single 25 mg/kg dose (bottom panel) of AD-45163, AD-51544, AD-51545, AD-51546, or AD-51547.
FIG. 15 is a graph depicting suppression of TTR protein in non-human primates following subcutaneous administration of AD-51547 at 2.5 mg/kg (white squares), 5 mg/kg (black squares) or 10 mg/kg (patterned squares) per dose, or administration of PBS as a negative control (gray squares).



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides RNAi agents, e.g., double stranded RNAi agents, and compositions targeting the Transthyretin (TTR) gene. The present invention also provides methods of inhibiting expression of TTR and methods of treating or preventing a TTR-associated disease in a subject using the RNAi agents, e.g., 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 expression of TTR. Agents including a certain pattern of chemical modifications (e.g., an alternating pattern) and a ligand are shown herein to be effective in silencing the activity of the TTR gene. Furthermore, agents including one or more motifs of three identical modifications on three consecutive nucleotides, including one such motif at or near the cleavage site of the agents, show surprisingly enhanced TTR gene silencing activity. When a single such chemical motif is present in the agent, it is preferred to be at or near the cleavage region for enhancing of the gene silencing activity. Cleavage region is the region surrounding the cleavage site, i.e., the site on the target mRNA at which cleavage occurs.
I. Definitions
As used herein, each of the following terms has the meaning associated with it in this section.
The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.
The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
As used herein, a “transthyretin” (“TTR”) refers to the well known gene and protein. TTR is also known as prealbumin, HsT2651, PALB, and TBPA. TTR functions as a transporter of retinol-binding protein (RBP), thyroxine (T4) and retinol, and it also acts as a protease. The liver secretes TTR into the blood, and the choroid plexus secretes TTR into the cerebrospinal fluid. TTR is also expressed in the pancreas and the retinal pigment epithelium. The greatest clinical relevance of TTR is that both normal and mutant TTR protein can form amyloid fibrils that aggregate into extracellular deposits, causing amyloidosis. See, e.g., Saraiva M. J. M. (2002) Expert Reviews in Molecular Medicine, 4(12):1-11 for a review. The molecular cloning and nucleotide sequence of rat transthyretin, as well as the distribution of mRNA expression, was described by Dickson, P. W. et al. (1985) J. Biol. Chem. 260(13)8214-8219. The X-ray crystal structure of human TTR was described in Blake, C. C. et al. (1974) J Mol Biol 88, 1-12. The sequence of a human TTR mRNA transcript can be found at National Center for Biotechnology Information (NCBI) RefSeq accession number NM_000371. The sequence of mouse TTR mRNA can be found at RefSeq accession number NM_013697.2, and the sequence of rat TTR mRNA can be found at RefSeq accession number NM_012681.1
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a TTR gene, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. “T” and “dT” are used interchangeably herein and refer to a deoxyribonucleotide wherein the nucleobase is thymine, e.g., 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 further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
A “double stranded RNAi agent,” double-stranded RNA (dsRNA) molecule, also referred to as “dsRNA agent,” “dsRNA”, “siRNA”, “iRNA agent,” as used interchangeably herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined below, nucleic acid strands. In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.
In another embodiment, the RNAi agent may 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 endonuclease Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150: 883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150; 883-894.
The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi agent may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to an RNAi agent as described above.
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 the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The antisense RNA molecule may have about 15-30 nucleotides that are complementary to the target mRNA. For example, the antisense RNA molecule may have a sequence of at least 15, 16, 17, 18, 19, 20 or more contiguous nucleotides from one of the antisense sequences of Table 1.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of an RNAi agent when a 3′-end of one strand of the RNAi agent extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNAi agent, i.e., no nucleotide overhang. A “blunt ended” RNAi agent is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The RNAi agents of the invention include RNAi agents with nucleotide overhangs at one end (i.e., agents with one overhang and one blunt end) or with nucleotide overhangs at both ends.
The term “antisense strand” refers to the strand of a double stranded RNAi agent which includes a region that is substantially complementary to a target sequence (e.g., a human TTR mRNA). As used herein, the term “region complementary to part of an mRNA encoding transthyretin” refers to a region on the antisense strand that is substantially complementary to part of a TTR mRNA sequence. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are generally in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
As used herein, 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 cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.
As used herein, and 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 can, for example, be stringent conditions, where stringent conditions may 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 may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
Sequences can be “fully complementary” with respect to each when there is base-pairing of the nucleotides of the first nucleotide sequence with the nucleotides of the second nucleotide sequence over 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 herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding TTR) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR. For example, a polynucleotide is complementary to at least a part of a TTR mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding TTR.
The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.
The phrase “inhibiting expression of a TTR,” as used herein, includes inhibition of expression of any TTR gene (such as, e.g., a mouse TTR gene, a rat TTR gene, a monkey TTR gene, or a human TTR gene) as well as variants or mutants of a TTR gene. Thus, the TTR gene may be a wild-type TTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise to systemic amyloid deposition), or a transgenic TTR gene in the context of a genetically manipulated cell, group of cells, or organism.
“Inhibiting expression of a TTR gene” includes any level of inhibition of a TTR gene, e.g., at least partial suppression of the expression of a TTR gene, such as an inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
The expression of a TTR gene may be assessed based on the level of any variable associated with TTR gene expression, e.g., TTR mRNA level, TTR protein level, retinol binding protein level, vitamin A level, or the number or extent of amyloid deposits. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).
The phrase “contacting a cell with an RNAi agent,” as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent, e.g., 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. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.
Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., a GalNAc3 ligand, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. In connection with the methods of the invention, a cell might also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.
A “patient” or “subject,” as used herein, is intended to include either a human or non-human animal, preferably a mammal, e.g., a monkey. Most preferably, the subject or patient is a human.
A “TTR-associated disease,” as used herein, is intended to include any disease associated with the TTR gene or protein. Such a disease may be caused, for example, by excess production of the TTR protein, by TTR gene mutations, by abnormal cleavage of the TTR protein, by abnormal interactions between TTR and other proteins or other endogenous or exogenous substances. A “TTR-associated disease” includes any type of TTR amyloidosis (ATTR) wherein TTR plays a role in the formation of abnormal extracellular aggregates or amyloid deposits. TTR-associated diseases include senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS) amyloidosis, amyloidotic vitreous opacities, carpal tunnel syndrome, and hyperthyroxinemia. Symptoms of TTR amyloidosis include sensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and corneal lattice dystrophy.
“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a patient for treating a TTR associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by TTR expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
“Prophylactically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject who does not yet experience or display symptoms of a TTR-associated disease, but who may be predisposed to the disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Symptoms that may be ameliorated include sensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and corneal lattice dystrophy. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. RNAi gents employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes), the retina or parts of the retina (e.g., retinal pigment epithelium), the central nervous system or parts of the central nervous system (e.g., ventricles or choroid plexus), or the pancreas or certain cells or parts of the pancreas. In some embodiments, a “sample derived from a subject” refers to cerebrospinal fluid obtained from the subject. In preferred embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject. In further embodiments, a “sample derived from a subject” refers to liver tissue (or subcomponents thereof) or retinal tissue (or subcomponents thereof) derived from the subject.
II. RNAi Agents
The present invention provides RNAi agents with superior gene silencing activity. It is shown herein and in Provisional Application No. 61/561,710 (to which the present application claims priority) that a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand and/or antisense strand of a RNAi agent, particularly at or near the cleavage site. The sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense and/or antisense strand. The RNAi agent also optionally conjugates with a GalNAc derivative ligand, for instance on the sense strand. The resulting RNAi agents present superior gene silencing activity.
The inventors surprisingly discovered that when the sense strand and antisense strand of the RNAi agent are completely modified, having one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of a RNAi agent superiorly enhanced the gene silencing activity of the RNAi agent.
Accordingly, the invention provides RNAi agents, e.g., 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 strand and an antisense strand. 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, 27-30 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.
The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs 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.
In one embodiment, the RNAi agent may contain one or more overhang regions and/or capping groups of RNAi agent at 3′-end, or 5′-end or both ends of a strand. The overhang can be 1-6 nucleotides in length, for instance 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 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
The RNAi agents provided by the present invention include agents with chemical modifications as disclosed, for example, in U.S. Provisional Application No. 61/561,710, filed on Nov. 18, 2011, International Application No. PCT/US2011/051597, filed on Sep. 15, 2010, and PCT Publication WO 2009/073809, the entire contents of each of which are incorporated herein by reference.
In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, 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 combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.
The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.
The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang is located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While the Applicants are not bound by theory, the theoretical mechanism is that the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.
In one embodiment, the RNAi agent is a double ended bluntmer of 19 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7,8,9 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.
In one embodiment, the RNAi agent is a double ended bluntmer of 20 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8,9,10 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.
In one embodiment, the RNAi agent is a double ended bluntmer of 21 nt in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end.
In one embodiment, the RNAi agent comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9,10,11 from the 5′ end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′ end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nt overhang. Preferably, the 2 nt overhang is at the 3′-end of the antisense. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).
In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce 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 on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.
In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11,12,13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along 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 wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.
In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.
In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand
For RNAi agent having a duplex region of 17-23 nt in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus, the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1st nucleotide from the 5′-end of the antisense strand, or, the count starting from the 1st paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.
The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.
In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif should occur at or near the cleavage site of the strand and the other motifs may be wing modifications. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either 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 are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.
Like the sense strand, the antisense strand of the RNAi agent may contain at least two motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that is present on the sense strand.
In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.
In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.
When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.
When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.
In one embodiment, every nucleotide in the sense strand and antisense strand of the RNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.
As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.
It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.
In one embodiment, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-β-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.
At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.
In one embodiment, the Na and/or Nb comprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.
The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.
In one embodiment, the RNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′-3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 5′-3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.
In one embodiment, the RNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.
The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or antisense strand interrupts the initial modification pattern present in the sense strand and/or antisense strand. This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense and/or antisense strand surprisingly enhances the gene silencing activity to the target gene.
In one embodiment, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, 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 containing the motif is “ . . . NaYYYNb . . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “Na” and “Nb” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where Na and Nb can be the same or different modifications. Alternatively, Na and/or Nb may be present or absent when there is a wing modification present.
The RNAi agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.
In one embodiment, the RNAi comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paried nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.
In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.
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 from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.
In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand 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 from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.
In one embodiment, the sense strand sequence may be represented by formula (I):

5′np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq3′  (I)

wherein:
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 an overhang nucleotide;
wherein Nb and Y do not have the same modification; and
XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.
In one embodiment, the Na and/or Nb comprise modifications of alternating pattern.
In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: 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 duplex region, from the 5′-end.
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 strand can therefore be represented by the following formulas:

5′np-Na-YYY-Nb-ZZZ-Na-nq3′  (Ia);

5′np-Na-XXX-Nb-YYY-Na-nq3′  (Ib); or

5′np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq3′  (Ic).

When the sense strand is represented by formula (Ia), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the sense strand is represented as formula (Ib), 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 can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the sense strand is represented as formula (Ic), each Nb independently represents an oligonucleotide sequence 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.
Each of X, Y and Z may be the same or different from each other.
In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

5′nq′-Na′-(Z′Z′Z′)k-Nb′-Y′Y′Y′-Nb′-(X′X′X′)l-N′a-np′3′  (II)

wherein:
k and l 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 an overhang nucleotide;
wherein Nb′ and Y′ do not have the same modification;
and
X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.
In one embodiment, the Na′ and/or Nb′ comprise modifications of alternating pattern.
The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nt in length, 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 strand, with the count starting from the 1st nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.
In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.
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.
The antisense strand can therefore 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′-Na′-np′3′  (IIb); or

5′nq′-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Nb′-X′X′X′-Na′-np′3′  (IIc).

When the antisense strand is represented by formula (IIa), 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.
When the antisense strand is represented as 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.
When the antisense strand is represented as 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.
Each of X′, Y′ and Z′ may be the same or different from each other.
Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, 2′-deoxy or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.
In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1st nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.
In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1st nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand 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 represents a 2′-OMe modification or 2′-F modification.
The sense strand represented by any one of the above formulas (Ia), (Ib) and (Ic) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb) and (IIc), respectively.
Accordingly, the RNAi agents of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):







(III)


sense: 5′ np -Na-(X X X)i -Nb- Y Y Y -Nb -(Z Z Z)j- 


 


Na-nq 3′


 


antisense: 3′ np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′-Nb′-


 


(Z′Z′Z′)l-Na′-nq′ 5′






wherein:
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 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 an overhang nucleotide; and
XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.
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.
Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:







(IIIa)


5′ np -Na-Y Y Y -Nb -Z Z Z -Na-nq 3′


 


3′ np′-Na′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′nq′ 5′


 


(IIIb)


5′ np-Na- X X X -Nb -Y Y Y - Na-nq 3′


 


3′ np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Na′-nq′ 5′


 


(IIIc)


5′ np-Na-X X X -Nb-Y Y Y -Nb- Z Z Z -Na-nq 3′


 


3′ np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Nb′-Z′Z′Z′-Na-nq′ 5′






When the RNAi agent is represented by formula (IIIa), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the RNAi agent is represented as 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.
When the RNAi agent is represented as 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 alternating pattern.
Each of X, Y and Z in formulas (III), (IIIa), (IIIb) and (IIIc) may be the same or different from each other.
When the RNAi agent is represented by formula (III), (IIIa), (IIIb) or (IIIc), at least one of the Y nucleotides may 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.
When the RNAi agent is represented by formula (IIIa) or (IIIc), at least one of the Z nucleotides may 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.
When the RNAi agent is represented as formula (IIIb) or (IIIc), at least one of the X nucleotides may 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.
In one embodiment, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, and/or the modification on the X nucleotide is different than the modification on the X′ nucleotide.
In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprise a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.
In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb) or (IIIc), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.
In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb) or (IIIc) are linked to each other at the 5′ end, and one or both of the 3′ ends of the are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.
Various publications describe multimeric RNAi agents. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520 the entire contents of which are hereby incorporated herein by reference.
The RNAi agent that contains conjugations of one or more carbohydrate moieties to a RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached 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 with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.
The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier 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, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.
In certain specific embodiments, the RNAi agent of the invention is an 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, D1034, D1035, D1036, D1037, D1038, D1039, D1040, D1041, D1042, D1043, D1044, D1045, D1046, D1047, D1048, D1049, D1050, D1051, D1052, D1053, D1054, D1055, D1056, D1057, D1058, D1059, D1060, D1061, D1062, D1063, D1064, D1065, D1066, D1067, D1068, D1069, D1070, D1071, D1072, D1073, D1074, D1075, D1076, D1077, D1078, D1079, D1080, D1081, D1082, D1083, D1084, D1085, D1086, D1087, D1088, D1089, D1090, D1091, D1092, D1093, D1094, D1095, D1096, D1097, D1098, D1099, D1100, D1101, D1102, D1103, D1104, D1105, D1106, D1107, D1108, D1109, D1110, D1111, D1112, D1113, D1114, D1115, D1116, D1117, D1118, D1119, D1120, D1121, D1122, D1123, D1124, D1125, D1126, D1127, D1128, D1129, D1130, D1131, D1132, D1133, D1134, D1135, D1136, D1137, D1138, D1139, D1140, D1141, D1142, D1143, D1144, D1145, D1146, D1147, D1148, D1149, D1150, D1151, D1152, D1153, D1154, D1155, D1156, D1157, D1158, D1159, D1160, D1161, D1162, D1163, D1164, D1165, D1166, D1167, D1168, D1169, D1170, D1171, D1172, D1173, D1174, D1175, D1176, D1177, D1178, D1179, D1180, D1181, D1182, D1183, D1184, D1185, D1186, D1187, D1188, D1189, D1190, D1191, D1192, D1193, D1194, D1195, D1196, D1197, D1198, D1199, D1200, D1201, D1202, D1203, D1204, D1205, D1206, D1207, D1208, D1209, D1210, D1211, D1212, D1213, D1214, D1215, D1216, D1217, D1218, D1219, D1220, D1221, D1222, D1223, D1224, D1225, D1226, D1227, D1228, D1229, D1230, D1231, D1232, D1233, D1234, D1235, D1236, D1237, D1238, D1239, D1240, D1241, D1242, D1243, D1244, D1245, D1246, D1247, D1248, D1249, D1250, D1251, D1252, D1253, D1254, D1255, D1256, D1257, D1258, D1259, D1260, D1261, D1262, D1263, D1264, D1265, D1266, D1267, D1268, D1269, D1270, D1271, D1272, D1273, D1274, D1275, D1276, D1277, D1278, D1279, D1280, D1281, D1282, D1283, D1284, D1285, D1286, D1287, D1288, D1289, D1290, D1291, D1292, D1293, D1294, D1295, D1296, D1297, D1298, D1299, D1300, D1301, D1302, D1303, D1304, D1305, D1306, D1307, D1308, D1309, D1310, D1311, D1312, D1313, D1314, D1315, D1316, D1317, D1318, D1319, D1320, D1321, D1322, D1323, D1324, D1325, D1326, D1327, D1328, D1329, D1330, D1331, D1332, D1333, D1334, D1335, D1336, D1337, D1338, D1339, D1340, D1341, D1342, D1343, D1344, D1345, D1346, D1347, D1348, D1349, D1350, D1351, D1352, D1353, D1354, D1355, D1356, D1357, D1358, D1359, D1360, D1361, D1362, D1363, D1364, D1365, D1366, D1367, D1368, D1369, D1370, D1371, D1372, D1373, D1374, D1375, D1376, D1377, D1378, D1379, D1380, D1381, D1382, D1383, D1384, D1385, D1386, D1387, D1388, D1389, D1390, D1391, D1392, D1393, D1394, D1395, D1396, D1397, D1398, D1399, D1400, D1401, D1402, D1403, D1404, D1405, D1406, D1407, D1408, D1409, D1410, D1411, D1412, D1413, D1414, D1415, D1416, D1417, D1418, D1419, D1420, D1421, D1422, D1423, D1424, D1425, D1426, D1427, D1428, D1429, D1430, D1431, D1432, D1433, D1434, D1435, D1436, D1437, D1438, D1439, D1440, D1441, D1442, D1443, D1444, D1445, D1446, D1447, D1448, D1449, D1450, D1451, D1452, D1453, D1454, D1455, D1456, D1457, D1458, D1459, D1460, D1461, D1462, D1463, D1464, D1465, D1466, D1467, D1468, D1469, D1470, D1471, D1472, D1473, D1474, D1475, D1476, D1477, D1478, D1479, D1480, D1481, D1482, D1483, D1484, D1485, D1486, D1487, D1488, D1489, D1490, D1491, D1492, D1493, D1494, D1495, D1496, D1497, D1498, D1499, D1500, D1501, D1502, D1503, D1504, D1505, D1506, D1507, D1508, D1509, D1510, D1511, D1512, D1513, D1514, D1515, D1516, D1517, D1518, D1519, D1520, D1521, D1522, D1523, D1524, D1525, D1526, D1527, D1528, D1529, D1530, D1531, D1532, D1533, D1534, D1535, D1536, D1537, D1538, D1539, D1540, D1541, D1542, D1543, D1544, D1545, D1546, D1547, D1548, D1549, D1550, D1551, D1552, D1553, D1554, D1555, D1556, D1557, D1558, D1559, D1560, D1561, D1562, D1563, D1564, D1565, D1566, D1567, D1568, D1569, D1570, D1571, D1572, D1573, D1574, D1575, D1576, D1577, D1578, D1579, D1580, D1581, D1582, D1583, D1584, D1585, D1586, D1587, D1588, D1589, D1590, D1591, D1592, D1593, D1594, D1595, D1596, D1597, D1598, D1599, D1600, D1601, D1602, D1603, D1604, D1605, D1606, D1607, D1608, D1609, D1610, D1611, D1612, D1613, D1614, D1615, D1616, D1617, D1618, D1619, D1620, D1621, D1622, D1623, D1624, D1625, D1626, D1627, D1628, D1629, D1630, D1631, D1632, D1633, D1634, D1635, D1636, D1637, D1638, D1639, D1640, D1641, D1642, D1643, D1644, D1645, D1646, D1647, D1648, D1649, D1650, D1651, D1652, D1653, D1654, D1655, D1656, D1657, D1658, D1659, D1660, D1661, D1662, D1663, D1664, D1665, D1666, D1667, D1668, D1669, D1670, D1671, D1672, D1673, D1674, D1675, D1676, D1677, D1678, D1679, D1680, D1681, D1682, D1683, D1684, D1685, D1686, D1687, D1688, D1689, D1690, D1691, D1692, D1693, D1694, D1695, D1696, D1697, D1698, D1699, D1700, D1701, D1702, D1703, D1704, D1705, D1706, D1707, D1708, D1709, D1710, D1711, D1712, D1713, D1714, D1715, D1716, D1717, D1718, D1719, D1720, D1721, D1722, D1723, D1724, D1725, D1726, D1727, D1728, D1729, D1730, D1731, D1732, D1733, D1734, D1735, D1736, D1737, D1738, D1739, D1740, D1741, D1742, D1743, D1744, D1745, D1746, D1747, D1748, D1749, D1750, D1751, D1752, D1753, D1754, D1755, D1756, D1757, D1758, D1759, D1760, D1761, D1762, D1763, D1764, D1765, D1766, D1767, D1768, D1769, D1770, D1771, D1772, D1773, D1774, D1775, D1776, D1777, D1778, D1779, D1780, D1781, D1782, D1783, D1784, D1785, D1786, D1787, D1788, D1789, D1790, D1791, D1792, D1793, D1794, D1795, D1796, D1797, D1798, D1799, D1800, D1801, D1802, D1803, D1804, D1805, D1806, D1807, D1808, D1809, D1810, D1811, D1812, D1813, D1814, D1815, D1816, D1817, D1818, D1819, D1820, D1821, D1822, D1823, D1824, D1825, D1826, D1827, D1828, D1829, D1830, D1831, D1832, D1833, D1834, D1835, D1836, D1837, D1838, D1839, D1840, D1841, D1842, D1843, D1844, D1845, D1846, D1847, D1848, D1849, D1850, D1851, D1852, D1853, D1854, D1855, D1856, D1857, D1858, D1859, D1860, D1861, D1862, D1863, D1864, D1865, D1866, D1867, D1868, D1869, D1870, D1871, D1872, D1873, D1874, D1875, D1876, D1877, D1878, D1879, D1880, D1881, D1882, D1883, D1884, D1885, D1886, D1887, D1888, D1889, D1890, D1891, D1892, D1893, D1894, D1895, D1896, D1897, D1898, D1899, D1900, D1901, D1902, D1903, D1904, D1905, D1906, D1907, D1908, D1909, D1910, D1911, D1912, D1913, D1914, D1915, D1916, D1917, D1918, D1919, D1920, D1921, D1922, D1923, D1924, D1925, D1926, D1927, D1928, D1929, D1930, D1931, D1932, D1933, D1934, D1935, D1936, D1937, D1938, D1939, D1940, D1941, D1942, D1943, D1944, D1945, D1946, D1947, D1948, D1949, D1950, D1951, D1952, D1953, D1954, D1955, D1956, D1957, D1958, D1959, D1960, D1961, D1962, D1963, D1964, D1965, D1966, D1967, D1968, D1969, D1970, D1971, D1972, D1973, D1974, D1975, D1976, D1977, D1978, D1979, D1980, D1981, D1982, D1983, D1984, D1985, D1986, D1987, D1988, D1989, D1990, D1991, D1992, D1993, D1994, D1995, D1996, D1997, D1998, D1999, D2000, D2001, D2002, D2003, D2004, D2005, D2006, D2007, D2008, D2009, D2010, D2011, D2012, D2013, D2014, D2015, D2016, D2017, D2018, D2019, D2020, D2021, D2022, D2023, D2024, D2025, D2026, D2027, D2028, D2029, D2030, D2031, D2032, D2033, D2034, D2035, D2036, D2037, D2038, D2039, D2040, D2041, D2042, D2043, D2044, D2045, D2046, D2047, D2048, D2049, D2050, D2051, D2052, D2053, D2054, D2055, D2056, D2057, D2058, D2059, D2060, D2061, D2062, D2063, D2064, D2065, D2066, D2067, D2068, D2069, D2070, D2071, D2072, D2073, D2074, D2075, D2076, D2077, D2078, D2079, D2080, D2081, D2082, D2083, D2084, D2085, D2086, D2087, D2088, D2089, D2090 and D2091.
These agents may further comprise a ligand, such as a GalNAc ligand.
Ligands
The RNAi agents of the invention, e.g., double stranded RNAi agents, may optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, antisense strand or both strands, at the 3′-end, 5′-end or both ends. For instance, the ligand may be conjugated to the sense strand. In preferred embodiments, the ligand is conjugated to the 3′-end of the sense strand. In one preferred embodiment, the ligand is a GalNAc ligand. In particularly preferred embodiments, the ligand is GalNAc3:




A wide variety of entities can be coupled to the RNAi agents of the present invention. Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether.
In preferred embodiments, a ligand alters the distribution, targeting or lifetime of the molecule into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Ligands providing enhanced affinity for a selected target are also termed targeting ligands.
Some ligands can have endosomolytic properties. The endosomolytic ligands promote the lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endo some and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands 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 their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.
Ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g., an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, 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.
Other examples of ligands include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or a chelator (e.g., EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
The ligand can increase the uptake of the oligonucleotide into the cell by, for example, activating an inflammatory response. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.
In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include B vitamins, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HAS, low density lipoprotein (LDL) and high-density lipoprotein (HDL).
In another aspect, the ligand 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 peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:4). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP) (SEQ ID NO:5) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) (SEQ ID NO:6) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) (SEQ ID NO:7) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized. An RGD peptide moiety 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., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing αvβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers enriched in proliferating cells can be used. For example, RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type of ligand target PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.
A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., 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 fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
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, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. 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 salt bridges with the anionic residues glutamate or aspartate.
Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
The targeting ligand can be any ligand that is capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, or an apatamer. A cluster is a combination of two or more sugar units. The targeting ligands also include integrin receptor ligands, Chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands. The ligands can also be based on nucleic acid, e.g., an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.
Endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations, masked oligo or poly cations or anions, acetals, polyacetals, ketals/polyketals, orthoesters, polymers with masked or unmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.
PK modulator stands for pharmacokinetic modulator. PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Examplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g., as PK modulating ligands).
In addition, aptamers that bind serum components (e.g., serum proteins) are also amenable to the present invention as PK modulating ligands.
Other ligand conjugates amenable to the invention are described in U.S. patent application Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser. No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filed Aug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser. No. 11/944,227 filed Nov. 21, 2007, which are incorporated by reference in their entireties for all purposes.
When two or more ligands are present, the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties. For example, a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In a preferred embodiment, all the ligands have different properties.
Ligands can be coupled to the oligonucleotides at various places, for example, 3′-end, 5′-end, and/or at an internal position. In preferred embodiments, the ligand is attached to the oligonucleotides via an intervening tether, e.g., a carrier described herein. The ligand or tethered ligand may be present on a monomer when the monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated via coupling to a “precursor” monomer after the “precursor” monomer has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH2)nNH2 may be incorporated into a growing oligonucleotide strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer's tether.
In another example, a monomer having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated, e.g., an azide or alkyne terminated tether/linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having complementary chemical group, e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
For double-stranded oligonucleotides, ligands can be attached to one or both strands. 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.
In some embodiments, ligand can be conjugated to nucleobases, sugar moieties, or internucleosidic linkages of nucleic acid molecules. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a conjugate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2′, 3′, and 5′ carbon atoms. The 1′ position can also be attached to a conjugate moiety, such as in an abasic residue. Internucleosidic linkages can also bear conjugate moieties. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleosidic linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
Any suitable ligand in the field of RNA interference may be used, although the ligand is typically a carbohydrate e.g. monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, polysaccharide.
Linkers that conjugate the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N-acetylglucosamine) derivatives attached through a bivalent or trivalent branched linker.
In one embodiment, the dsRNA of the invention is conjugated to a bivalent and trivalent branched linkers include the structures shown in any of formula (IV)-(VII):




wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently for each occurrence absent, 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 occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R′′), C≡C or C(O);
R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO, CH═N—O,




or heterocyclyl;

L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and
Ra is H or amino acid side chain.
Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (VII):




    
    
        wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the following compounds:

    
    












In other embodiments, the RNAi agent of the invention is an agent selected from the group consisting of AD-45163, AD-45165, AD-51544, AD-51545, AD-51546, and AD-51547.
III. Pharmaceutical Compositions
The RNAi agents of the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals. The pharmaceutical compositions comprising RNAi agents of the invention may be, for example, solutions with or without a buffer, or compositions containing pharmaceutically acceptable carriers. Such compositions include, for example, aqueous or crystalline compositions, liposomal formulations, micellar formulations, emulsions, and gene therapy vectors.
In the methods of the invention, the RNAi agent may be administered in a solution. A free RNAi agent may be administered in an unbuffered solution, e.g., in saline or in water. Alternatively, the free siRNA may also be administered in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In 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 administering to a subject.
In some embodiments, the buffer solution further comprises an agent for controlling the osmolarity of the solution, such that the osmolarity is kept at a desired value, e.g., at the physiologic values of the human plasma. Solutes which can be added to the buffer solution to control the osmolarity include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolarity of the solution is a salt. In certain embodiments, the agent for controlling the osmolarity of the solution is sodium chloride or potassium chloride.
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 herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
In one embodiment, the RNAi agent preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, an RNAi agent composition for the treatment of a TTR-associated disease, e.g., a transthyretin-related hereditary amyloidosis (familial amyloid polyneuropathy, FAP), may include a known drug for the amelioration of FAP, e.g., Tafamidis (INN, or Fx-1006A or Vyndaqel).
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 (e.g., it contains less than 80, 50, 30, 20, or 10% of water). In another example, the RNAi agent is in an aqueous phase, e.g., in a solution that includes water.
The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the RNAi agent composition is formulated in a manner that is compatible with the intended method of administration, as described herein. For example, in particular embodiments 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, freeze-drying, condensation and other self-assembly.
An RNAi agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes RNAi agent, e.g., a protein that complexes with the RNAi agent to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
In one embodiment, the RNAi agent preparation includes another siRNA compound, e.g., a second RNAi agent that can mediate RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different RNAi agent species. Such RNAi agents can mediate RNAi with respect to a similar number of different genes.
The iRNA agents of the invention may 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 preceding embodiments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.
Methods of preparing pharmaceutical compositions of the invention include the step of bringing into association 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 bringing into association an RNAi agent of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
The pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, 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 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 patch 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.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the patient. Some examples of materials which 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 state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, 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) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical compositions.
The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of RNAi agent which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The RNAi agent which can be combined with a carrier material to produce a single dosage form will generally be that amount of the RNAi agent which produces a desired effect, e.g., therapeutic or prophylactic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of RNAi agent, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
In certain embodiments, a composition of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and an RNAi agent of the present invention. In certain embodiments, an aforementioned composition renders orally bioavailable an RNAi agent of the present invention.
In some cases, in order to prolong the effect of an RNAi agent, it is desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the RNAi agent then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered RNAi agent may be accomplished by dissolving or suspending the agent in an oil vehicle.
Liposomes
An RNAi agent of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one 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. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the RNAi agent are delivered into the cell where the RNAi agent can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the RNAi agent to particular cell types.
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 can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.
If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favor condensation.
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, e.g., WO 96/37194, the entire contents of which are incorporated herein 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; U.S. Pat. No. 4,897,355; U.S. 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. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging RNAi agent preparations into liposomes.
Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. 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.
In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.
Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from 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.
A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. LipofectinTM Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.
Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (TransfectamTM, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Choi”) which has been formulated into 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 containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
Liposomal formulations are particularly suited for topical administration, liposomes present 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 at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. 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, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of 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 into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.
Liposomes that include RNAi agent can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.
Other formulations amenable to the present invention are described in U.S. provisional application Ser. Nos. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.
Surfactants
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). RNAi agent (or a precursor, e.g., a larger dsiRNA which can be processed into a siRNA, or a DNA which encodes a siRNA or precursor) compositions can include a surfactant. In one embodiment, the siRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile 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, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their 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. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
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, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
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. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Micelles and Other Membranous Formulations
The RNAi agents of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8 to C22 alkyl sulphate, 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, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.
In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. 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 sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.
The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
Particles
In another embodiment, an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may 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
The present invention also provides methods of inhibiting expression of a transthyretin (TTR) in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of TTR in the cell, thereby inhibiting expression of TTR in the cell.
Contacting of a cell with an RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc3 ligand, or any other ligand that directs the RNAi agent to a site of interest, e.g., the liver of a subject.
The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.
The phrase “inhibiting expression of a TTR” is intended to refer to inhibition of expression of any TTR gene (such as, e.g., a mouse TTR gene, a rat TTR gene, a monkey TTR gene, or a human TTR gene) as well as variants or mutants of a TTR gene. Thus, the TTR gene may be a wild-type TTR gene, a mutant TTR gene (such as a mutant TTR gene giving rise to amyloid deposition), or a transgenic TTR gene in the context of a genetically manipulated cell, group of cells, or organism.
“Inhibiting expression of a TTR gene” includes any level of inhibition of a TTR gene, e.g., at least partial suppression of the expression of a TTR gene. The expression of the TTR gene may be assessed based on the level, or the change in the level, of any variable associated with TTR gene expression, e.g., TTR mRNA level, TTR protein level, or the number or extent of amyloid deposits. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.
Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with TTR expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).
In some embodiments of the methods of the invention, expression of a TTR gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
Inhibition of the expression of a TTR gene may be manifested by a reduction of 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 a subject) in which a TTR gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an RNAi agent of the invention, or by administering an RNAi agent of the invention to a subject in which the cells are or were present) such that the expression of a TTR gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s)). In preferred embodiments, the 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:




  
    
      
        
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Alternatively, inhibition of the expression of a TTR gene may be assessed in terms of a reduction of a parameter that is functionally linked to TTR gene expression, e.g., TTR protein expression, retinol binding protein level, vitamin A level, or presence of amyloid deposits comprising TTR. TTR gene silencing may be determined in any cell expressing TTR, either constitutively or by genomic engineering, and by any assay known in the art. The liver is the major site of TTR expression. Other significant sites of expression include the choroid plexus, retina and pancreas.
Inhibition of the expression of a TTR protein may be manifested by a reduction in the level of the TTR protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.
A control cell or group of cells that may be used to assess the inhibition of the expression of a TTR gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.
The level of TTR mRNA that is expressed by a cell or group of cells, or the level of circulating TTR mRNA, may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of TTR in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the TTR gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in situ hybridization, and microarray analysis. Circulating TTR mRNA may be detected using methods the described in PCT/US2012/043584, the entire contents of which are hereby incorporated herein by reference.
In one embodiment, the level of expression of TTR is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific TTR. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to TTR mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of TTR mRNA.
An alternative method for determining the level of expression of TTR in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (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 nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of TTR is determined by quantitative fluorogenic RT-PCR (i.e., the TaqManTM System).
The expression levels of TTR mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of TTR expression level may also comprise using nucleic acid probes in solution.
In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein.
The level of TTR protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.
In some embodiments, the efficacy of the methods of the invention can be monitored by detecting or monitoring a reduction in an amyloid TTR deposit. Reducing an amyloid TTR deposit, as used herein, includes any decrease in the size, number, or severity of TTR deposits, or to a prevention or reduction in the formation of TTR deposits, within an organ or area of a subject, as may be assessed 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. (Editors) (2010), Amyloidosis: Diagnosis and Treatment, New York: Humana Press. Methods of assessing amyloid deposits may include biochemical analyses, as well as visual or computerized assessment of amyloid deposits, as made visible, e.g., using immunohistochemical staining, fluorescent labeling, light microscopy, electron microscopy, fluorescence microscopy, or other types of microscopy. Invasive or noninvasive imaging modalities, including, e.g., CT, PET, or NMR/MRI imaging may be employed to assess amyloid deposits.
The methods of the invention may 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.
The term “sample” as used herein refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, lymph, urine, cerebrospinal fluid, saliva, ocular fluids, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organics. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes), the retina or parts of the retina (e.g., retinal pigment epithelium), the central nervous system or parts of the central nervous system (e.g., ventricles or choroid plexus), or the pancreas or certain cells or parts of the pancreas. In preferred embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject. In further embodiments, a “sample derived from a subject” refers to liver tissue or retinal tissue derived from the subject.
In some embodiments of the methods of the invention, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of TTR may be assessed using measurements of the level or change in the level of TTR mRNA or TTR protein in a sample derived from fluid or tissue from the specific site within the subject. In preferred embodiments, the site is selected from the group consisting of liver, choroid plexus, retina, and pancreas. The site may also be a subsection or subgroup of cells from any one of the aforementioned sites (e.g., hepatocytes or retinal pigment epithelium). The site may also include cells that express a particular type of receptor (e.g., hepatocytes that express the asialogycloprotein receptor).
V. Methods for Treating or Preventing a TTR-Associated Disease
The present invention also provides methods for treating or preventing a TTR-associated disease in a subject. The methods include administering to the subject a therapeutically effective amount or prophylactically effective amount of an RNAi agent of the invention.
As used herein, a “subject” includes either a human or a non-human animal, preferably a vertebrate, and more preferably a mammal. A subject may include a transgenic organism. Most preferably, the subject is a human, such as a human suffering from or predisposed to developing a TTR-associated disease.
In some embodiments, the subject is suffering from a TTR-associated disease. In other embodiments, the subject is a subject at risk for developing a TTR-associated disease, e.g., a subject with a TTR gene mutation that is associated with the development of a TTR associated disease, a subject with a family history of TTR-associated disease, or a subject who has signs or symptoms suggesting the development of TTR amyloidosis.
A “TTR-associated disease,” as used herein, includes any disease caused by or associated with the formation of amyloid deposits in which the fibril precursors consist of 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 misfolded 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 amyloidotic cardiomyopathy (FAC); and leptomeningeal amyloidosis, also known as leptomeningeal or meningocerebrovascular amyloidosis, central nervous system (CNS) amyloidosis, or amyloidosis VII form.
In some embodiments of the methods of the invention, RNAi agents of the invention are administered to subjects suffering from familial amyloidotic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Normal-sequence TTR causes cardiac amyloidosis in people who are elderly and is termed senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA) or cardiac amyloidosis). SSA often is accompanied by microscopic deposits in many other organs. TTR mutations accelerate the process of TTR amyloid formation and are the most important risk factor for the development of clinically significant TTR amyloidosis (also called ATTR (amyloidosis-transthyretin type)). More than 85 amyloidogenic TTR variants are known to cause systemic familial amyloidosis.
In some embodiments of the methods of the invention, RNAi agents of the invention are administered to subjects suffering from transthyretin (TTR)-related familial amyloidotic polyneuropathy (FAP). Such subjects may suffer from ocular manifestations, such as vitreous opacity and glaucoma. It is known to one of skill in the art that amyloidogenic transthyretin (ATTR) synthesized by retinal pigment epithelium (RPE) plays important roles in the progression of ocular amyloidosis. Previous studies have shown that panretinal laser photocoagulation, which reduced the RPE cells, prevented the progression of amyloid deposition in the vitreous, indicating that the effective suppression of ATTR expression in RPE may become a novel therapy for ocular amyloidosis (see, e.g., Kawaji, T., et al., Ophthalmology. (2010) 117: 552-555). The methods of the invention are useful for treatment of ocular manifestations of TTR related FAP, e.g., ocular amyloidosis. The RNAi agent can be delivered in a manner suitable for targeting a particular tissue, such as the eye. Modes of ocular delivery include retrobulbar, subcutaneous eyelid, subconjunctival, subtenon, anterior chamber or intravitreous injection (or internal injection or infusion). Specific formulations for ocular delivery include eye drops or ointments.
Another TTR-associated disease is hyperthyroxinemia, also known as “dystransthyretinemic hyperthyroxinemia” or “dysprealbuminemic hyperthyroxinemia”. This type of hyperthyroxinemia may be secondary to an increased association of thyroxine with TTR due to a mutant TTR molecule with increased affinity for thyroxine. See, e.g., Moses et al. (1982) J. Clin. Invest., 86, 2025-2033.
The RNAi agents of the invention may be administered to a subject using any mode of administration known in the art, including, but not limited to subcutaneous, intravenous, intramuscular, intraocular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, cerebrospinal, and any combinations thereof. In preferred embodiments, the agents are administered subcutaneously.
In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of TTR, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.
In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the RNAi agent to the liver.
Other modes of administration include epidural, intracerebral, intracerebroventricular, nasal administration, intraarterial, intracardiac, intraosseous infusion, intrathecal, and intravitreal, and pulmonary. The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.
In some embodiments, the RNAi agent is administered to a subject in an amount effective to inhibit TTR expression in a cell within the subject. The amount effective to inhibit TTR expression in a cell within a subject may be assessed using methods discussed above, including methods that involve assessment of the inhibition of TTR mRNA, TTR protein, or related variables, such as amyloid deposits.
In some embodiments, the RNAi agent is administered to a subject in a therapeutically or prophylactically effective amount.
“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a patient for treating a TTR associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, stage of pathological processes mediated by TTR expression, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
“Prophylactically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject who does not yet experience or display symptoms of a TTR-associated disease, but who may be predisposed to the disease, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Symptoms that may be ameliorated include sensory neuropathy (e.g., paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and corneal lattice dystrophy. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. RNAi agents employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
As used herein, the 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 pathological processes mediated by TTR expression. Symptoms of TTR amyloidosis include sensory neuropathy (e.g. paresthesia, hypesthesia in distal limbs), autonomic neuropathy (e.g., gastrointestinal dysfunction, such as gastric ulcer, or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, vitreous opacities, renal insufficiency, nephropathy, substantially reduced mBMI (modified Body Mass Index), cranial nerve dysfunction, and corneal lattice dystrophy.
The dose of an RNAi agent that is administered to a subject may be tailored to balance the risks and benefits of a particular dose, for example, to achieve a desired level of TTR gene suppression (as assessed, e.g., based on TTR mRNA suppression, TTR protein expression, or a reduction in an amyloid deposit, as defined above) or a desired therapeutic or prophylactic effect, while at the same time avoiding undesirable side effects.
In one embodiment, the RNAi agent is administered at a dose of between about 0.25 mg/kg to about 50 mg/kg, e.g., between about 0.25 mg/kg to about 0.5 mg/kg, between about 0.25 mg/kg to about 1 mg/kg, between about 0.25 mg/kg to about 5 mg/kg, between about 0.25 mg/kg to about 10 mg/kg, between about 1 mg/kg to about 10 mg/kg, between about 5 mg/kg to about 15 mg/kg, between about 10 mg/kg to about 20 mg/kg, between about 15 mg/kg to about 25 mg/kg, between about 20 mg/kg to about 30 mg/kg, between about 25 mg/kg to about 35 mg/kg, or between about 40 mg/kg to about 50 mg/kg.
In some embodiments, the RNAi agent is administered at a dose of about 0.25 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, about 20 mg/kg, about 21 mg/kg, about 22 mg/kg, about 23 mg/kg, about 24 mg/kg, about 25 mg/kg, about 26 mg/kg, about 27 mg/kg, about 28 mg/kg, about 29 mg/kg, 30 mg/kg, about 31 mg/kg, about 32 mg/kg, about 33 mg/kg, about 34 mg/kg, about 35 mg/kg, about 36 mg/kg, about 37 mg/kg, about 38 mg/kg, about 39 mg/kg, about 40 mg/kg, about 41 mg/kg, about 42 mg/kg, about 43 mg/kg, about 44 mg/kg, about 45 mg/kg, about 46 mg/kg, about 47 mg/kg, about 48 mg/kg, about 49 mg/kg or about 50 mg/kg.
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, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable. In some embodiments, the number or amount of subsequent doses is dependent on the achievement of a desired effect, e.g., the suppression of a TTR gene, or the achievement of a therapeutic or prophylactic effect, e.g., reducing an amyloid deposit or reducing a symptom of a TTR-associated disease. In some embodiments, the RNAi agent is administered according to a schedule. For example, the RNAi agent may be administered twice per week, three times per week, four times per week, or five times per week. In some embodiments, the schedule involves regularly spaced administrations, e.g., hourly, 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, biweekly, or monthly. In other embodiments, the schedule involves closely 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 (e.g., about every 6 hours, about every 12 hours, about every 24 hours, about every 48 hours, or about every 72 hours) followed by a longer time period (e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, or about 8 weeks) during which the RNAi agent is not administered. In one embodiment, the RNAi agent is initially administered hourly and is later administered at a longer interval (e.g., daily, weekly, biweekly, or monthly). In another embodiment, the RNAi agent is initially administered daily and is later administered at a longer interval (e.g., weekly, biweekly, or monthly). In certain embodiments, the longer 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 during a first week, followed by weekly dosing starting on the eighth day of administration. In another specific embodiment, the RNAi agent is administered every other day during a first week followed by weekly dosing starting on the eighth day of administration.
Any of these schedules may optionally be repeated for one or more iterations. The number of iterations may depend on the achievement of a desired effect, e.g., the suppression of a TTR gene, retinol binding protein level, vitamin A level, and/or the achievement of a therapeutic or prophylactic effect, e.g., reducing an amyloid deposit or reducing a symptom of a TTR-associated disease.
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 TTR-associated disease may include a liver transplant, which can reduce mutant TTR levels in the body; Tafamidis (Vyndaqel), which kinetically stabilizes the TTR tetramer preventing tetramer dissociation required for TTR amyloidogenesis; and diuretics, which may be employed, for example, to reduce edema in TTR amyloidosis with cardiac involvement.
In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an RNAi agent. The maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 15 mg/kg of body weight per day, e.g., 10 mg/kg, 1 mg/kg, 0.1 mg/kg, 0.01 mg/kg, 0.001 mg/kg, or 0.00001 mg/kg of bodyweight per day. The maintenance doses are, for example, 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 every 21 days, or once every 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In certain embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in his/her condition. The dosage of the RNAi agent may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
VI. Kits
The present invention also provides kits for performing any of the methods of the invention. Such kits include one or more RNAi agent(s) and instructions for use, e.g., instructions for inhibiting expression of a TTR in a cell by contacting the cell with the RNAi agent(s) in an amount effective to inhibit expression of the TTR. The kits may optionally further comprise means for contacting the cell with the RNAi agent (e.g., an injection device), or means for measuring the inhibition of TTR (e.g., means for measuring the inhibition of TTR mRNA or TTR protein). Such means for measuring the inhibition of TTR may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the invention may optionally further comprise means for administering the RNAi agent(s) to a subject or means for determining the therapeutically effective or prophylactically effective amount.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references and published patents and patent applications cited throughout the application are hereby incorporated herein by reference.
EXAMPLES
Example 1
Inhibition of TTR with TTR-GalNAc Conjugates
A single dose of the TTR RNAi agent AD-43527 was administered to mice subcutaneously and TTR mRNA levels were determined 72 hours post administration.
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 liver. The sequence of each strand of AD-43527 is shown below.













Duplex #
Strand
Oligo
Sequence 5′ to 3′







AD-43527
s
A-
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfaAfL96 (SEQ ID NO: 8)




89592



 



as
A-
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu (SEQ ID NO: 9)




83989






L96 = GalNAc3; lowercase nts (a, u, g, c) are





2′-O-methyl nucleotides, Nf (i.e., Af) is a





2′-fluoro nucleotide





Strand: s = sense; as = antisense






The ligand used was GalNAc3:





This GalNAc3 ligand was conjugated to the 3′-end of the sense strand using the linker and tether as shown below:





The structure of the resulting GalNAc3 conjugated sense strand is shown in the following schematic:





Additional RNAi agents that target TTR and have the following sequences and modifications were synthesized and assayed.











Mouse/rat cross reactive TTR RNAi agents









Duplex
Sense strand 5′-3′
Antisense strand 5′-3′





AD-
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfaAfQ11L96
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu


43528
(SEQ ID NO: 10)
(SEQ ID NO: 11)









Human/cyno cross reactive TTR RNAi agents; parent duplex is AD-18328 [having a sense strand 5′-3′ sequence of GuAAccAAGAGuAuuccAudTdT (SEQ ID NO: 12) and antisense strand 5′ to 3′ sequence of AUGGAAuACUCUUGGUuACdTdT (SEQ ID NO: 13) with the following modifications: alternating 2′F/2′OMe w/2 PS on AS.













Duplex
Sense strand 5′-3′
Antisense strand 5′-3′







AD-
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfaUfL96
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa


45163
(SEQ ID NO: 14)
(SEQ ID NO: 16)


 


AD-
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfaUfQ11L96
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa


45164
(SEQ ID NO: 15)
(SEQ ID NO: 17)









L96=GalNAc3; lowercase nts (a,u,g,c) are 2′-O-methyl nucleotides, Nf (i.e., Af) is a 2′-fluoro nucleotide; Q11 is cholesterol; s is phosphorothioate.

AD-43527 was administered to female C57BL/6 mice (6-10 weeks, 5 per group) via subcutaneous injection at a dose volume of 10 μl/g at 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 at the same dose volume.
After approximately seventy two hours, mice were anesthetized with 200 μl of ketamine, and then exsanguinated by severing the right caudal artery. Liver tissue was collected, flash-frozen and stored at −80° C. until processing.
Efficacy of treatment was evaluated by measurement of TTR mRNA in the liver at 72 hours post-dose. TTR liver mRNA levels were assayed utilizing the Branched DNA assays—QuantiGene 1.0 (Panomics). Briefly, mouse liver samples were ground and tissue lysates were prepared. Liver lysis mixture (a mixture of 1 volume of lysis mixture, 2 volume of nuclease-free water and 10 μl of Proteinase-K/ml for a final concentration of 20 mg/ml) was incubated at 65° C. for 35 minutes. 5 μl of liver lysate and 95 μl of working probe set (TTR probe for gene target and GAPDH for endogenous control) were added into the Capture Plate. Capture Plates were incubated at 53° C.±1° C. (aprx. 16-20 hrs). The next day, the Capture Plates were washed 3 times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 240 g. 100 μl of Amplifier Probe mix per well was added into the Capture Plate, which was sealed with aluminum foil and incubated for 1 hour at 46° C.±1° C. Following a 1 hour incubation, the wash step was repeated, then 100 μl of Label Probe mix per well was added. Capture plates were incubated at 46° C.±1° C. for 1 hour. The plates were then washed with 1× Wash Buffer, dried and 100 μl substrate per well was added into the Capture Plates. Capture Plates were incubated for 30 minutes at 46° C. followed by incubation for 30 minutes at room temperature. Plates were read using the SpectraMax Luminometer following incubation. bDNA data were analyzed by subtracting the average background from each duplicate sample, averaging the resultant duplicate GAPDH (control probe) and TTR (experimental probe) values, and then computing the ratio: (experimental probe-background)/(control probe-background). The average TTR mRNA level was calculated for each group and normalized to the PBS group average to give relative TTR mRNA as a % of the PBS control group.
The results are shown in FIG. 1. The GalNAc conjugated RNAi agent targeting TTR had an ED50 of approximately 5 mg/kg for TTR mRNA knockdown. These results demonstrate that GalNAc conjugated RNAi agents that target TTR are effective at inhibiting expression of TTR mRNA.
Example 2
Inhibition of TTR with TTR-GalNAc Conjugates is Durable
Mice were administered a subcutaneous dose (either 7.5 or 30.0 mg/kg) of AD-43527, a GalNAc conjugated RNAi agent that targets TTR. The TTR mRNA levels in the liver were evaluated at 1, 3, 5, 7, 10, 13, 15, 19, 26, 33, and 41 days post treatment using the method described in Example 1.
The results are shown in FIG. 2. At day 19, administration of 30.0 mg/kg GalNAc conjugated RNAi agents still showed about 50% silencing. Full recovery of expression occurred at day 41.
These results demonstrated that the inhibition provided by GalNAc conjugated siRNA targeting TTR is durable, lasting up to 3, 5, 7, 10, 13, 15, 19, 26 or 33 days post treatment.
Example 3
RNA Synthesis and Duplex Annealing
1. Oligonucleotide Synthesis
Oligonucleotides were synthesized on an AKTAoligopilot synthesizer or an ABI 394 synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500 Å, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis unless otherwise specified. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite were purchased from (Promega). All phosphoramidites were used at a concentration of 0.2M in acetonitrile (CH3CN) except for guanosine which was used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes was used. The activator was 5-ethyl thiotetrazole (0.75M, American International Chemicals), for the PO-oxidation Iodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) was used.
Ligand conjugated strands were synthesized using a solid support containing the corresponding ligand. For example, the introduction of a carbohydrate moiety/ligand (for e.g., GalNAc) at the 3′-end of a sequence was achieved by starting the synthesis with the corresponding carbohydrate solid support. Similarly a cholesterol moiety at the 3′-end was introduced by starting the synthesis on the cholesterol support. In general, the ligand moiety was tethered to trans-4-hydroxyprolinol via a tether of choice as described in the previous examples to obtain a hydroxyprolinol-ligand moiety. The hydroxyprolinol-ligand moiety was then coupled to a solid support via a succinate linker or was converted to phosphoramidite via standard phosphitylation conditions to obtain the desired carbohydrate conjugate building blocks. Fluorophore labeled siRNAs were synthesized from the corresponding phosphoramidite or solid support, purchased from Biosearch Technologies. The oleyl lithocholic (GalNAc)3 polymer support made in house at a loading of 38.6 μmol/gram. The Mannose (Man)3 polymer support was also made in house at a loading of 42.0 μmol/gram.
Conjugation of the ligand of choice at the desired position, for example at the 5′-end of the sequence, was achieved by coupling of the corresponding phosphoramidite to the growing chain under standard phosphoramidite coupling conditions unless otherwise specified. An extended 15 minute coupling of 0.1M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate was carried out 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, K. G., Egli, M. and Manoharan, M. (2006) Gene Silencing Activity of siRNAs with a Ribo-difluorotoluoyl Nucleotide. ACS Chem. Biol., 1, 176-183 or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with a 10 minute oxidation wait time conjugated oligonucleotide. Phosphorothioate was introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent The cholesterol phosphoramidite was synthesized in house, and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite was 16 minutes.
2. Deprotection-I (Nucleobase Deprotection)
After completion of 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 cooled briefly on ice and then the ethanolic ammonia mixture was filtered into a new 250 ml bottle. The CPG was washed with 2×40 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 on a speed vac.
3. Deprotection-II (Removal of 2′ TBDMS group)
The dried 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 the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction was then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to 6.5, and stored in freezer until purification.

4. Analysis

The oligonucleotides were analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.

5. HPLC Purification

The ligand conjugated oligonucleotides were purified by reverse phase preparative HPLC. The unconjugated oligonucleotides were purified by anion-exchange HPLC on a TSK gel column packed in house. 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 full-length oligonucleotides were pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess were diluted in water to 150 μl and then pipetted in special vials for CGE and LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

6. RNAi Agent Preparation

For the preparation of an RNAi agent, equimolar amounts of sense and antisense strand were heated in 1×PBS 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 which target human or rodent TTR mRNA.








TABLE 1







RNAi Agents and Results of In Vitro Screening






















% of mRNA remained





SEQ


SEQ

conc. of siRNA


















Duplex

ID


ID


0.1
0.01
IC50


ID
S ID
NO:
Sense strand (S)
AS ID
NO:
Antisense strand (AS)
1 nM
nM
nM
(nM)




















D1000
S1000
18
AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasu
AS1000
1110
AfUfgGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.03
0.1
0.47
0.006


 


D1001
S1001
19
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1001
1111
aUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.03
0.10
0.49
0.0065


 


D1002
S1002
20
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1002
1112
aUfgGfAfAfuAfcUfcuuGfgsUfuAfcAfusGfsa
0.04
0.10
0.46
0.0068


 


D1003
S1003
21
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1003
1113
aUfgGfAfAfuAfcUfcuuGfgUfsuAfcAfusGfsa
0.05
0.12
0.56
0.0073


 


D1004
S1004
22
aUGuaACccAGagUAuuCCasu
AS1004
1114
AUggAAuaCUcuUGguUAcaUsGsa
0.07
0.13
0.44
0.008


 


D1005
S1005
23
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1005
1115
aUfgGfAfAfuAfcUfcuuGfgsUfsuAfcAfusGfsa
0.06
0.11
0.53
0.0093


 


D1006
S1006
24
AfuGfuAfAfccAfAfGfaGfuAfuUfcCfasUf
AS1006
1116
aUfgGfaAfuAfcUfcuuGfGfuuAfcAfusGfsa
0.05
0.16
0.55
0.0095


 


D1007
S1007
25
AfuGfuAfAfCfcAfAfGfaGfuAfuUfcCfasUf
AS1007
1117 
aUfgGfaAfuAfcUfcuuGfguuAfcAfusGfsa
0.05
0.14
0.48
0.0098


 


D1008
S1008
26
auguaaccaadGadGudAudAcdGasu
AS1008
1118
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.07
0.11
0.33
0.010


 


D1009
S1009
27
UfgGfGfAfuUfuCfAfUfgUfaAfcCfAfAfgsAf
AS1009
1119
uCfuugGfuUfaCfaugAfaAfuccCfasUfsc
0.03
0.14
0.56
0.0101


 


D1010
S1010
28
UfgGfgauUfuCfAfUfgUfaAfcCfaAfgsAf
AS1010
1120
uCfuUfgGfuUfaCfaugAfaAfUfCfcCfasUfsc
0.03
0.14
0.65
0.0101


 


D1011
S1011
29
aUfGfuAfAfccAfAfGfaGfuAfuUfcCfasUf
AS1011
1121
aUfgGfaAfuAfcUfcuuGfGfuuAfcaUfsgsa
0.06
0.10
0.55
0.011


 


D1012
S1012
30
UfgGfgAfuUfuCfAfUfgUfaacCfaAfgsAf
AS1012
1122
uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc
0.04
0.13
0.54
0.0114


 


D1013
S1013
31
auguaaccaadGadGudAudAcdGasu
AS1013
1123
aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa
0.11
0.19
0.49
0.011


 


D1014
S1014
32
AfuGfuaaCfcAfAfGfaGfuAfuUfcCfasUf
AS1014
1124
aUfgGfaAfuAfcUfcuuGfgUfUfAfcAfusGfsa
0.04
0.16
0.59
0.013


 


D1015
S1015
33
AfuguAfaccAfaGfdAGfdTAdTudCcdAsu
AS1015
1125
dAUdGgdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa
0.07
0.15
0.51
0.013


 


D1016
S1016
34
auGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf
AS1016
1126
aUfgGfaAfuAfcUfcuuGfgUfuAfcAfUfsGfsa
0.05
0.14
0.64
0.013


 


D1017
S1017
35
UfGfggAfuUfuCfAfUfgUfAfAfcCfaAfgsAf
AS1017
1127
uCfuUfgGfuuaCfaugAfaAfuCfCfcasUfsc
0.09
0.41
0.74
0.0133


 


D1018
S1018
36
AfuguAfaCfcAfAfGfaGfuAfuUfcCfasUf
AS1018
1128
aUfgGfaAfuAfcUfcuuGfgUfuAfCfAfusGfsa
0.03
0.14
0.61
0.014


 


D1019
S1019
37
AfuGfuAfaccAfAfGfaGfuAfuUfcCfasUf
AS1019
1129
aUfgGfaAfuAfcUfcuuGfGfUfuAfcAfusGfsa
0.02
0.2
0.7
0.014


 


D1020
S1020
38
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1020
1130
asUfsgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.04
0.16
0.67
0.0156


 


D1021
S1021
39
aUfguAfAfccAfAfgagUfaUfuCfcasUf
AS1021
1131
aUfGfgAfaUfaCfUfCfuuGfGfuuAfCfaUfsgsa
0.11
0.24
0.64
0.016


 


D1022
S1022
40
dTdGggdAdTuudCdAugdTdAacdCdAagsdA
AS1022
1132
udCdTugdGdTuadCdAugdAdAaudCdCcasdTsc
0.08
0.27
0.64
0.0161


 


D1023
S1023
41
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1023
1133
aUfgsGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.03
0.19
0.63
0.0163


 


D1024
S1024
42
UfgGfgAfuUfuCfAfUfguaAfcCfaAfgsAf
AS1024
1134
uCfuUfgGfuUfAfCfaugAfaAfuCfcCfasUfsc
0.05
0.25
0.69
0.0164


 


D1025
S1025
43
UfgGfgAfuUfuCfAfUfgUfAfAfcCfaAfgsAf
AS1025
1135
uCfuUfgGfuuaCfaugAfaAfuCfcCfasUfsc
0.04
0.18
0.75
0.0166


 


D1026
S1026
44
UfgGfgAfuUfuCfAfUfgUfaAfcCfaAfgsAf
AS1026
1136
uCfuUfgGfuUfaCfaugAfaAfuCfcCfasUfsc
0.04
0.19
0.66
0.0178


 


D1027
S1027
45
UfgGfgAfuUfuCfAfUfgUfaAfccaAfgsAf
AS1027
1137
uCfuUfGfGfuUfaCfaugAfaAfuCfcCfasUfsc
0.04
0.19
0.69
0.018


 


D1028
S1028
46
dAdTgudAdAccdAdAgadGdTaudTdCcasdT
AS1028
1138
adTdGgadAdTacdTdCuudGdGuudAdCausdGsa
0.15
0.29
0.72
0.018


 


D1029
S1029
47
AdTGdTAdACdCAdAGdAGdTAdTUdCCdAsU
AS1029
1139
dAUdGGdAAdTAdCUdCUdTGdGUdTAdCAdTsGsdA
0.1
0.27
0.61
0.018


 


D1030
S1030
48
UfgGfGfAfuuuCfAfUfgUfaAfcCfaAfgsAf
AS1030
1140
uCfuUfgGfuUfaCfaugAfAfAfuccCfasUfsc
0.04
0.21
0.64
0.0187


 


D1031
S1031
49
AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu
AS1031
1141
AfUfGfGfAfAfuAfCfUfCfUfuGfGfuuAfcAfusGfsa
0.06
0.15
0.62
0.019


 


D1032
S1032
50
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1032
1142
asUfgGfAfAfuAfcUfcuuGfgUfsuAfcAfusGfsa
0.09
0.34
0.78
0.021


 


D1033
S1033
51
UfgGfgAfuUfuCfaUfGfUfaacCfaAfgsAf
AS1033
1143
uCfuUfgGfUfUfacaUfgAfaAfuCfcCfasUfsc
0.06
0.26
0.57
0.0212


 


D1034
S1034
52
AfuGfuAfAfccAfaGfaGfuAfuUfcCfasUf
AS1034
1144
aUfgGfaAfuAfcUfcUfuGfGfuuAfcAfusGfsa
0.11
0.39
0.82
0.0216


 


D1035
S1035
53
UfgGfgAfuuuCfAfUfgUfaAfcCfaAfgsAf
AS1035
1145
uCfuUfgGfuUfaCfaugAfAfAfuCfcCfasUfsc
0.04
0.16
0.56
0.0222


 


D1036
S1036
54
UfgGfGfAfuUfuCfaUfgUfaAfcCfAfAfgsAf
AS1036
1146
uCfuugGfuUfaCfaUfgAfaAfuccCfasUfsc
0.06
0.31
0.78
0.0234


 


D1037
S1037
55
UfgGfGfAfuUfuCfAfUfgUfaAfcCfaAfgsAf
AS1037
1147
uCfuUfgGfuUfaCfaugAfaAfuccCfasUfsc
0.03
0.14
0.62
0.0235


 


D1038
S1038
56
UfGfggAfUfuuCfAfugUfAfacCfAfagsAf
AS1038
1148
uCfUfugGfUfuaCfAfugAfAfauCfCfcasUfsc
0.09
0.39
0.78
0.0239


 


D1039
S1039
57
AfuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1039
1149
aUfgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.03
0.14
0.59
0.025


 


D1040
S1040
58
AfuGfuAfaCfcAfAfGfaGfuAfuUfccasUf
AS1040
1150
aUfGfGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.03
0.13
0.56
0.025


 


D1041
S1041
59
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1041
1151
asUfgGfAfAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.06
0.27
0.79
0.0252


 


D1042
S1042
60
UfgGfgAfuuuCfAfUfgUfAfAfcCfaAfgsAf
AS1042
1152
uCfuUfgGfuuaCfaugAfAfAfuCfcCfasUfsc
0.05
0.27
0.67
0.0259


 


D1043
S1043
61
AfuGfuAfaCfcAfAfGfaGfuauUfcCfasUf
AS1043
1153
aUfgGfaAfUfAfcUfcuuGfgUfuAfcAfusGfsa
0.02
0.16
0.63
0.027


 


D1044
S1044
62
AfsuGfuAfaCfcAfAfGfaGfuAfuucCfasUf
AS1044
1154
asUfgGfAfAfuAfcUfcuuGfgsUfsuAfcAfusGfsa
0.06
0.30
0.81
0.0271


 


D1045
S1045
63
aUfguAfAfccAfAfgaGfGfauUfCfcasUf
AS1045
1155
aUfGfgaAfUfacUfCfuuGfGfuuAfCfaUfsgsa
0.12
0.29
0.8
0.028


 


D1046
S1046
64
AfuGfuAfaCfcAfAfGfaguAfuUfcCfasUf
AS1046
1156
aUfgGfaAfuAfCfUfcuuGfgUfuAfcAfusGfsa
0.03
0.15
0.59
0.030


 


D1047
S1047
65
UfgGfGfAfuUfuCfaUfgUfAfAfcCfaAfgsAf
AS1047
1157
uCfuUfgGfuuaCfaUfgAfaAfuccCfasUfsc
0.08
0.44
0.83
0.0324


 


D1048
S1048
66
AfuGfuAfaCfcAfAfGfaGfuAfuUfcCfasUf
AS1048
1158
aUfgGfaAfuAfcUfcuuGfgUfuAfcAfusGfsa
0.07
0.23
0.67
0.036


 


D1049
S1049
67
AfuGfuAfAfccAfAfGfAfGfuAfuuccAfsu
AS1049
1159
AfUfGfGfAfAfuAfCfUfCfUfUfGfGfUfuAfCfAfusGfsa
0.08
0.23
0.73
0.037


 


D1050
S1050
68
UfgGfgAfuuuCfaUfgUfaAfcCfAfAfgsAf
AS1050
1160
uCfuugGfuUfaCfaUfgAfAfAfuCfcCfasUfsc
0.06
0.29
0.78
0.0372


 


D1051
S1051
69
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1051
1161
aUfgGfaAfudAcdTcdTudGgdTuAfcAfusgsa
0.12
0.41
0.86
0.040


 


D1052
S1052
70
AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu
AS1052
1162
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.1
0.22
0.72
0.042


 


D1053
S1053
71
AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu
AS1053
1163
dAUdGGdAAfuAfcUfcUfuGfGfUfuAfCfAfusGfsa
0.09
0.31
0.69
0.044


 


D1054
S1054
72
AfuGfuAfaCfcAfaGfadGdTAfuUfcdCdAsUf
AS1054
1164
adTdGGfaAfudAdCUfcUfuGfgUfuAfcAfusGfsa
0.1
0.45
0.75
0.047


 


D1055
S1055
73
AfuguAfaccAfaGfaGfdTAdTUdCcdAsu
AS1055
1165
dAUdGGdAadTAfcUfcUfuGfgUfuAfcAfusGfsa
0.12
0.26
0.7
0.049


 


D1056
S1056
74
AuGuAaCcAaGaGuAuUcCasU
AS1056
1166
aUgGaAuAcUcUuGgUuAcAusGsa
0.08
0.24
0.65
0.050


 


D1057
S1057
75
AfuguAfaccAfagaGfuauUfccasUf
AS1057
1167
aUfGfGfaAfUfAfcUfCfUfuGfGfUfuAfCfAfusGfsa
0.14
0.42
0.62
0.051


 


D1058
S1058
76
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1058
1168
aUfgGfaAfudAcdTcdTudGgdTuAfcAfusGfsa
0.12
0.36
0.86
0.053


 


D1059
S1059
77
AfuguAfaccAfaGfdAGfdTAdTUdCcdAsu
AS1059
1169
dAUdGGdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa
0.09
0.27
0.7
0.054


 


D1060
S1060
78
adTgudAdAccdAdAgagdTadTudCcasdT
AS1060
1170
adTdGgdAadTadCdTdCuudGdGuudAdCadTsgsa
0.11
0.37
0.66
0.056


 


D1061
S1061
79
AfuGfuAfaCfcAfaGfdAdGuAfuUfcdCdAsUf
AS1061
1171
adTdGGfaAfuAfdCdTcUfuGfgUfuAfcAfusGfsa
0.1
0.31
0.77
0.059


 


D1062
S1062
80
AfuguAfaccAfaGfdAGfdTAdTudCcdAsu
AS1062
1172
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.1
0.27
0.65
0.059


 


D1063
S1063
81
adTdGuadAdCccdAdGagdTdAuudCdCasu
AS1063
1173
dAdTggdAdAuadCdTcudTdGgudTdAcadTsdGsa
0.12
0.44
0.82
0.064


 


D1064
S1064
82
AfuGfuAfaCfcAfaGfaGfdTdAuUfcdCdAsUf
AS1064
1174
adTdGGfaAfdTdAcUfcUfuGfgUfuAfcAfusGfsa
0.12
0.32
0.83
0.064


 


D1065
S1065
83
AfuguAfaccAfaGfaGfdTAdTudCcdAsu
AS1065
1175
dAUdGgdAadTAfcUfcUfuGfgUfuAfcAfusGfsa
0.13
0.34
0.72
0.066


 


D1066
S1066
84
AfuGfuAfaCfcAfaGfaGfudAdTUfcdCdAsUf
AS1066
1176
adTdGGfadAdTAfcUfcUfuGfgUfuAfcAfusGfsa
0.11
0.33
0.72
0.067


 


D1067
S1067
85
AfuguAfaccAfaGfaGfdTAdTUdCcdAsu
AS1067
1177
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.11
0.37
0.62
0.070


 


D1068
S1068
86
AfuguAfaccAfaGfaGfdTAdTUdCcdAsu
AS1068
1178
dAUdGGdAAuAfcUfcUfuGfGfUfuAfCfAfusGfsa
0.16
0.33
0.64
0.072


 


D1069
S1069
87
aUfGfuaAfCfccAfGfagUfAfuuCfCfasu
AS1069
1179
AfUfggAfAfuaCfUfcuUfGfguUfAfcaUfsGfsa
0.14
0.43
0.73
0.074


 


D1070
S1070
88
AfuGfuAfaCfCfAfaGfaguAfuUfcCfasUf
AS1070
1180
aUfgGfaAfuAfCfUfcUfuggUfuAfcAfusGfsa
0.08
0.42
0.94
0.075


 


D1071
S1071
89
UfgGfgAfuuuCfaUfgUfaAfcCfaAfgsAf
AS1071
1181
uCfuUfgGfuUfaCfaUfgAfAfAfuCfcCfasUfsc
0.14
0.28
0.83
0.0797


 


D1072
S1072
90
AfuGfuAfaCfcAfaGfAfGfuauUfcCfasUf
AS1072
1182
aUfgGfaAfUfAfcucUfuGfgUfuAfcAfusGfsa
0.05
0.26
0.8
0.082


 


D1073
S1073
91
AfuGfuAfaCfcAfaGfadGdTdAdTUfcCfasUf
AS1073
1183
aUfgGfadAdTdAdCUfcUfuGfgUfuAfcAfusGfsa
0.12
0.41
0.73
0.083


 


D1074
S1074
92
AfUfguAfAfccAfAfgaGfUfauUfCfcasUf
AS1074
1184
aUfGfgaAfUfacUfCfuuGfGfuuAfCfausGfsa
0.14
0.44
0.75
0.086


 


D1075
S1075
93
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1075
1185
aUfgGfdAdAdTdAcUfcUfuGfgUfuAfcAfusGfsa
0.1
0.41
0.72
0.088


 


D1076
S1076
94
AfuGfuAfaCfcAfaGfaGfudAdTdTdCCfasUf
AS1076
1186
aUfgdGdAdAdTAfcUfcUfuGfgUfuAfcAfusGfsa
0.15
0.45
0.86
0.088


 


D1077
S1077
95
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasu
AS1077
1187
AfUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.08
0.46
0.95
0.092


 


D1078
S1078
96
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1078
1188
dAUdGGdAadTAfcUfcUfuGfgUfuAfcAfusGfsa
0.09
0.32
0.76
0.093


 


D1079
S1079
97
AfuguAfaccAfaGfaGfdTadTudCcdAsu
AS1079
1189
dAudGgdAadTAfcUfcUfuGfgUfuAfcAfusGfsa
0.14
0.38
0.76
0.095


 


D1080
S1080
98
AfuGfuAfaCfcAfaGfAfGfuAfuucCfasUf
AS1080
1190
aUfgGfAfAfuAfcucUfuGfgUfuAfcAfusGfsa
0.05
0.42
0.86
0.099


 


D1081
S1081
99
AfuGfuAfaCfcAfaGfaGfuAfuUfdCdCdAsdT
AS1081
1191
dAdTdGdGaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.17
0.47
0.9
0.105


 


D1082
S1082
100
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1082
1192
aUfgGfaAfudACfudCUfudGGfudTAfcAfusgsa
0.12
0.44
0.83
0.106


 


D1083
S1083
101
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1083
1193
adTdGGfaAfdTdAcUfcUfuGfgUfuAfcAfusGfsa
0.11
0.34
0.74
0.109


 


D1084
S1084
102
AfuGfuAfAfCfcAfaGfaGfuauUfcCfasUf
AS1084
1194
aUfgGfaAfUfAfcUfcUfuGfguuAfcAfusGfsa
0.1
0.45
0.93
0.117


 


D1085
S1085
103
AfuGfUfAfaCfcAfaGfaGfuauUfcCfasUf
AS1085
1195
aUfgGfaAfUfAfcUfcUfuGfgUfuacAfusGfsa
0.07
0.42
0.78
0.120


 


D1086
S1086
104
aUfguAfAfccAfAfgaGfuAfuUfcCfasUf
AS1086
1196
aUfgGfaAfuAfcUfCfuuGfGfuuAfCfaUfsgsa
0.17
0.45
0.83
0.1197


 


D1087
S1087
105
AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasu
AS1087
1197
AfUfgGfaAfuacUfcUfuGfgUfuAfcAfusGfsa
0.05
0.3
0.7
0.120


 


D1088
S1088
106
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1088
1198
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusgsa
0.11
0.46
0.8
0.120


 


D1089
S1089
107
AfuGfuAfaCfcAfaGfaGfUfAfuUfcCfasUf
AS1089
1199
aUfgGfaAfuacUfcUfuGfgUfuAfcAfusGfsa
0.14
0.49
0.85
0.122


 


D1090
S1090
108
AfuGfuAfaCfcAfaGfaGfuauUfcCfasUf
AS1090
1200
aUfgGfaAfUfAfcUfcUfuGfgUfuAfcAfusGfsa
0.1
0.41
0.85
0.125


 


D1091
S1091
109
AfuguAfaccAfaGfaGfdTAdTudCcdAsu
AS1091
1201
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.16
0.38
0.77
0.125


 


D1092
S1092
110
AfuGfuAfaCfcAfaGfAfGfuAfuUfcCfasu
AS1092
1202
AfUfgGfaAfuAfcucUfuGfgUfuAfcAfusGfsa
0.05
0.31
0.93
0.126


 


D1093
S1093
111
auGfuAfaCfcAfaGfAfGfuAfuUfcCfasUf
AS1093
1203
aUfgGfaAfuAfcucUfuGfgUfuAfcAfUfsGfsa
0.06
0.33
0.9
0.135


 


D1094
S1094
112
AfuGfuAfaCfcAfaGfaGfUfAfuUfccasUf
AS1094
1204
aUfGfGfaAfuacUfcUfuGfgUfuAfcAfusGfsa
0.07
0.39
0.85
0.142


 


D1095
S1095
113
AfuGfuAfaCfcAfaGfAfGfuAfuUfcCfasUf
AS1095
1205
aUfgGfaAfuAfcucUfuGfgUfuAfcAfusGfsa
0.09
0.39
0.76
0.146


 


D1096
S1096
114
AfuGfuAfaCfcAfaGfaGfUfAfuucCfasUf
AS1096
1206
aUfgGfAfAfuacUfcUfuGfgUfuAfcAfusGfsa
0.06
0.38
0.85
0.147


 


D1097
S1097
115
AfuGfUfAfaCfcAfaGfaGfuAfuucCfasUf
AS1097
1207
aUfgGfAfAfuAfcUfcUfuGfgUfuacAfusGfsa
0.12
0.47
0.87
0.147


 


D1098
S1098
116
AfuGfuAfaCfcAfaGfaGfuAfUfUfccasUf
AS1098
1208
aUfGfGfaauAfcUfcUfuGfgUfuAfcAfusGfsa
0.06
0.42
0.85
0.151


 


D1099
S1099
117
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1099
1209
dAUdGGdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa
0.16
0.41
0.85
0.152


 


D1100
S1100
118
AfuguAfaccAfaGfaGfuAfuUfcCfasUf
AS1100
1210
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.15
0.48
0.72
0.152


 


D1101
S1101
119
AfuGfuAfaCfcAfaGfAfGfuAfuUfccasUf
AS1101
1211
aUfGfGfaAfuAfcucUfuGfgUfuAfcAfusGfsa
0.06
0.38
0.94
0.158


 


D1102
S1102
120
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1102
1212
aUfgGfaAfuAfdCuCfdTuGfdG uUfacAfusGfsa
0.21
0.45
0.89
0.162


 


D1103
S1103
121
AfuGfuaaCfCfAfaGfaGfuAfuUfcCfasUf
AS1103
1213
aUfgGfaAfuAfcUfcUfuggUfUfAfcAfusGfsa
0.14
0.49
0.95
0.163


 


D1104
S1104
122
AfuGfuAfaccAfaGfaGfUfAfuUfcCfasUf
AS1104
1214
aUfgGfaAfuacUfcUfuGfGfUfuAfcAfusGfsa
0.06
0.36
0.92
0.163


 


D1105
S1105
123
AfuGfuAfaCfcAfaGfaGfuAfuucCfasUf
AS1105
1215
aUfgGfAfAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.1
0.45
0.84
0.167


 


D1106
S1106
124
AfuGfuaaCfcAfaGfAfGfuAfuUfcCfasUf
AS1106
1216
aUfgGfaAfuAfcucUfuGfgUfUfAfcAfusGfsa
0.09
0.43
0.91
0.170


 


D1107
S1107
125
AfuGfuAfaccAfaGfAfGfuAfuUfcCfasUf
AS1107
1217
aUfgGfaAfuAfcucUfuGfGfUfuAfcAfusGfsa
0.09
0.46
1
0.171


 


D1108
S1108
126
AfuguAfaccAfaGfaGfdTadTudCcdAsu
AS1108
1218
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.11
0.39
0.71
0.176


 


D1109
S1109
127
AfuGfUfAfaCfcAfaGfaGfuAfuUfccasUf
AS1109
1219
aUfGfGfaAfuAfcUfcUfuGfgUfuacAfusGfsa
0.1
0.43
0.9
0.180


 


D1110
S1110
128
AfuGfuAfaCfcAfaGfaguAfUfUfcCfasUf
AS1110
1220
aUfgGfaauAfCfUfcUfuGfgUfuAfcAfusGfsa
0.06
0.42
0.88
0.182


 


D1111
S1111
129
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1111
1221
dAUdGGdAAuAfcUfcUfuGfGfUfuAfCfAfusGfsa
0.18
0.49
0.79
0.183


 


D1112
S1112
130
AfuGfUfAfaccAfaGfaGfuAfuUfcCfasUf
AS1112
1222
aUfgGfaAfuAfcUfcUfuGfGfUfuacAfusGfsa
0.14
0.48
0.85
0.195


 


D1113
S1113
131
AfuGfuAfaCfcAfaGfaguAfuUfcCfasUf
AS1113
1223
aUfgGfaAfuAfCfUfcUfuGfgUfuAfcAfusGfsa
0.09
0.41
0.85
0.201


 


D1114
S1114
132
auGfuAfaCfcAfaGfaGfUfAfuUfcCfasUf
AS1114
1224
aUfgGfaAfuacUfcUfuGfgUfuAfcAfUfsGfsa
0.05
0.44
0.94
0.201


 


D1115
S1115
133
AfuguAfaCfcAfaGfaGfUfAfuUfcCfasUf
AS1115
1225
aUfgGfaAfuacUfcUfuGfgUfuAfCfAfusGfsa
0.08
0.41
0.96
0.204


 


D1116
S1116
134
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1116
1226
adTdGGfadAdTAfcUfcUfuGfgUfuAfcAfusGfsa
0.15
0.47
0.79
0.208


 


D1117
S1117
135
AfuGfuaaCfcAfaGfaGfUfAfuUfcCfasUf
AS1117
1227
aUfgGfaAfuacUfcUfuGfgUfUfAfcAfusGfsa
0.08
0.42
0.92
0.224


 


D1118
S1118
136
auguaaccaagaguauuccasu
AS1118
1228
AfUfGfGfAfAfUfAfCfUfCfUfUfGfGfUfUfAfCfAfUfsgsa
0.19
0.5
0.87
0.303


 


D1119
S1119
137
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1119
1229
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.14
0.55
0.89



 


D1120
S1120
138
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1120
1230
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.19
0.63
0.72



 


D1121
S1121
139
AfuGfuAfaccAfaGfaGfuAfuUfcCfasUf
AS1121
1231
aUfgGfaAfuAfcUfcUfuGfGfUfuAfcAfusGfsa
0.14
0.61
0.91



 


D1122
S1122
140
AfUfGfuAfaCfcAfaGfaGfuAfuUfccasUf
AS1122
1232
aUfGfGfaAfuAfcUfcUfuGfgUfuAfcausGfsa
0.14
0.54
0.95



 


D1123
S1123
141
auGfuAfAfCfcAfaGfaGfuAfuUfcCfasUf
AS1123
1233
aUfgGfaAfuAfcUfcUfuGfguuAfcAfUfsGfsa
0.13
0.61
0.97



 


D1124
S1124
142
AfuGfuAfaCfcAfaGfaGfuAfUfUfcCfasUf
AS1124
1234
aUfgGfaauAfcUfcUfuGfgUfuAfcAfusGfsa
0.14
0.56
0.94



 


D1125
S1125
143
AfuGfuAfaCfcaaGfaGfuAfuUfcCfasUf
AS1125
1235
aUfgGfaAfuAfcUfcUfUfGfgUfuAfcAfusGfsa
0.21
0.74
0.95



 


D1126
S1126
144
AfUfGfuAfaCfcAfaGfaGfuAfuucCfasUf
AS1126
1236
aUfgGfAfAfuAfcUfcUfuGfgUfuAfcausGfsa
0.2
0.69
0.91



 


D1127
S1127
145
AfuguAfAfCfcAfaGfaGfuAfuUfcCfasUf
AS1127
1237
aUfgGfaAfuAfcUfcUfuGfguuAfCfAfusGfsa
0.17
0.7
0.96



 


D1128
S1128
146
AfUfGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1128
1238
aUfgGfaAfuAfcUfcUfuGfgUfuAfcausGfsa
0.19
0.62
0.85



 


D1129
S1129
147
AfuGfuAfaCfcAfaGfaGfuAfuUfCfCfasUf
AS1129
1239
aUfggaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.23
0.76
0.98



 


D1130
S1130
148
AfuGfuAfaCfcAfagaGfuAfuUfcCfasUf
AS1130
1240
aUfgGfaAfuAfcUfCfUfuGfgUfuAfcAfusGfsa
0.21
0.64
0.9



 


D1131
S1131
149
AfuGfuAfAfCfcaaGfaGfuAfuUfcCfasUf
AS1131
1241
aUfgGfaAfuAfcUfcUfUfGfguuAfcAfusGfsa
0.17
0.7
1.01



 


D1132
S1132
150
AfuGfUfAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1132
1242
aUfgGfaAfuAfcUfcUfuGfgUfuacAfusGfsa
0.17
0.58
0.87



 


D1133
S1133
151
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1133
1243
augGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.33
0.89
1.05



 


D1134
S1134
152
AfUfGfuAfaCfcAfaGfaguAfuUfcCfasUf
AS1134
1244
aUfgGfaAfuAfCfUfcUfuGfgUfuAfcausGfsa
0.16
0.64
0.96



 


D1135
S1135
153
AfuGfUfAfaCfcAfaGfaguAfuUfcCfasUf
AS1135
1245
aUfgGfaAfuAfCfUfcUfuGfgUfuacAfusGfsa
0.12
0.53
0.96



 


D1136
S1136
154
AfuGfuAfAfCfcAfagaGfuAfuUfcCfasUf
AS1136
1246
aUfgGfaAfuAfcUfCfUfuGfguuAfcAfusGfsa
0.16
0.58
0.98



 


D1137
S1137
155
AfuGfuAfAfCfcAfaGfaGfuAfuUfcCfasUf
AS1137
1247
aUfgGfaAfuAfcUfcUfuGfguuAfcAfusGfsa
0.16
0.6
0.91



 


D1138
S1138
156
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1138
1248
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsAf
0.1
0.54
0.91



 


D1139
S1139
157
AfUfGfuAfaCfcAfagaGfuAfuUfcCfasUf
AS1139
1249
aUfgGfaAfuAfcUfCfUfuGfgUfuAfcausGfsa
0.24
0.68
0.98



 


D1140
S1140
158
AfuGfUfAfaCfcAfagaGfuAfuUfcCfasUf
AS1140
1250
aUfgGfaAfuAfcUfCfUfuGfgUfuacAfusGfsa
0.13
0.75
0.9



 


D1141
S1141
159
AfuGfuAfAfCfcAfaGfaguAfuUfcCfasUf
AS1141
1251
aUfgGfaAfuAfCfUfcUfuGfguuAfcAfusGfsa
0.15
0.52
1.05



 


D1142
S1142
160
AfuGfuAfaCfCfAfaGfaGfuAfuUfcCfasUf
AS1142
1252
aUfgGfaAfuAfcUfcUfuggUfuAfcAfusGfsa
0.16
0.66
0.89



 


D1143
S1143
161
auGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1143
1253
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfUfsGfsa
0.12
0.51
0.89



 


D1144
S1144
162
AfUfGfuAfaCfcaaGfaGfuAfuUfcCfasUf
AS1144
1254
aUfgGfaAfuAfcUfcUfUfGfgUfuAfcausGfsa
0.25
0.71
0.95



 


D1145
S1145
163
AfuGfUfAfaCfcaaGfaGfuAfuUfcCfasUf
AS1145
1255
aUfgGfaAfuAfcUfcUfUfGfgUfuacAfusGfsa
0.17
0.74
0.98



 


D1146
S1146
164
AfuguAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1146
1256
aUfgGfaAfuAfcUfcUfuGfgUfuAfCfAfusGfsa
0.11
0.51
0.86



 


D1147
S1147
165
AfuGfuAfaCfcAfaGfaGfuAfuUfccasUf
AS1147
1257
aUfGfGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.1
0.52
0.83



 


D1148
S1148
166
AfUfGfuAfaccAfaGfaGfuAfuUfcCfasUf
AS1148
1258
aUfgGfaAfuAfcUfcUfuGfGfUfuAfcausGfsa
0.14
0.63
0.98



 


D1149
S1149
167
AfuGfuAfAfCfcAfaGfaGfuAfuucCfasUf
AS1149
1259
aUfgGfAfAfuAfcUfcUfuGfguuAfcAfusGfsa
0.13
0.58
0.88



 


D1150
S1150
168
AfuGfuaaCfcAfaGfaGfuAfuUfcCfasUf
AS1150
1260
aUfgGfaAfuAfcUfcUfuGfgUfUfAfcAfusGfsa
0.15
0.62
0.94



 


D1151
S1151
169
AfUfGfuaaCfcAfaGfaGfuAfuUfcCfasUf
AS1151
1261
aUfgGfaAfuAfcUfcUfuGfgUfUfAfcausGfsa
0.18
0.73
0.94



 


D1152
S1152
170
auGfUfAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1152
1262
aUfgGfaAfuAfcUfcUfuGfgUfuacAfUfsGfsa
0.13
0.53
0.97



 


D1153
S1153
171
AfuGfuAfAfCfcAfaGfaGfuAfuUfccasUf
AS1153
1263
aUfGfGfaAfuAfcUfcUfuGfguuAfcAfusGfsa
0.13
0.53
0.98



 


D1154
S1154
172
UfgGfgAfuUfuCfaUfgUfaAfcCfaAfgsAf
AS1154
1264
uCfuUfgGfuUfaCfaUfgAfaAfuCfcCfasUfsc
0.09
0.5
0.78



 


D1155
S1155
173
UfgGfGfAfuuuCfaUfgUfAfAfcCfaAfgsAf
AS1155
1265
uCfuUfgGfuuaCfaUfgAfAfAfuccCfasUfsc
0.13
0.62
0.89



 


D1156
S1156
174
UfgGfgAfuuuCfaUfGfUfaAfcCfaAfgsAf
AS1156
1266
uCfuUfgGfuUfacaUfgAfAfAfuCfcCfasUfsc
0.12
0.65
0.85



 


D1157
S1157
175
UfgGfgAfuUfuCfaUfgUfAfAfcCfaAfgsAf
AS1157
1267
uCfuUfgGfuuaCfaUfgAfaAfuCfcCfasUfsc
0.11
0.54
0.85



 


D1158
S1158
176
UfgGfgAfuuuCfaUfgUfAfAfcCfaAfgsAf
AS1158
1268
uCfuUfgGfuuaCfaUfgAfAfAfuCfcCfasUfsc
0.13
0.53
0.8



 


D1159
S1159
177
UfGfggAfUfuUfcAfuGfuAfAfccAfAfgsAf
AS1159
1269
uCfuuGfGfuuAfcAfuGaAfauCfCfcasUfsc
0.59
0.89
0.81



 


D1160
S1160
178
UfGfggAfUfuuCfaUfgUfAfAfcCfaAfgsAf
AS1160
1270
uCfuUfgGfuuaCfaUfgAfAfauCfCfcasUfsc
0.16
0.72
0.9



 


D1161
S1161
179
UfgGfgAfuUfucaUfGfUfaAfcCfaAfgsAf
AS1161
1271
uCfuUfgGfuUfacaUfGfAfaAfuCfcCfasUfsc
0.27
0.69
0.86



 


D1162
S1162
180
AfuGfuAfaCfcaaGfaGfUfAfuUfcCfasUf
AS1162
1272
aUfgGfaAfuacUfcUfUfGfgUfuAfcAfusGfsa
0.12
0.6
0.95



 


D1163
S1163
181
AfuGfuAfaccAfaGfaGfuAfUfUfcCfasUf
AS1163
1273
aUfgGfaauAfcUfcUfuGfGfUfuAfcAfusGfsa
0.05
0.56
1.02



 


D1164
S1164
182
AfuGfuAfaCfcAfagaGfUfAfuUfcCfasUf
AS1164
1274
aUfgGfaAfuacUfCfUfuGfgUfuAfcAfusGfsa
0.13
0.55
1



 


D1165
S1165
183
AfuGfuAfaCfcaaGfaGfuAfUfUfcCfasUf
AS1165
1275
aUfgGfaauAfcUfcUfUfGfgUfuAfcAfusGfsa
0.09
0.6
0.97



 


D1166
S1166
184
AfuguAfaCfCfAfaGfaGfuAfuUfcCfasUf
AS1166
1276
aUfgGfaAfuAfcUfcUfuggUfuAfCfAfusGfsa
0.15
0.59
0.91



 


D1167
S1167
185
AfuGfuAfaCfcAfagaGfuAfUfUfcCfasUf
AS1167
1277
aUfgGfaauAfcUfCfUfuGfgUfuAfcAfusGfsa
0.11
0.59
1



 


D1168
S1168
186
AfuGfuAfaCfCfAfagaGfuAfuUfcCfasUf
AS1168
1278
aUfgGfaAfuAfcUfCfUfuggUfuAfcAfusGfsa
0.13
0.57
0.94



 


D1169
S1169
187
auGfuAfaCfcAfaGfaGfuAfUfUfcCfasUf
AS1169
1279
aUfgGfaauAfcUfcUfuGfgUfuAfcAfUfsGfsa
0.08
0.5
0.9



 


D1170
S1170
188
AfuguAfaCfcAfaGfaGfuAfUfUfcCfasUf
AS1170
1280
aUfgGfaauAfcUfcUfuGfgUfuAfCfAfusGfsa
0.06
0.53
0.91



 


D1171
S1171
189
auGfuAfaCfcAfaGfaGfuAfuUfCfCfasUf
AS1171
1281
aUfggaAfuAfcUfcUfuGfgUfuAfcAfUfsGfsa
0.07
0.56
0.89



 


D1172
S1172
190
AfuGfuAfaCfCfAfaGfaGfuAfuucCfasUf
AS1172
1282
aUfgGfAfAfuAfcUfcUfuggUfuAfcAfusGfsa
0.13
0.59
0.98



 


D1173
S1173
191
AfuGfuAfaCfcaaGfAfGfuAfuUfcCfasUf
AS1173
1283
aUfgGfaAfuAfcucUfUfGfgUfuAfcAfusGfsa
0.2
0.65
1.03



 


D1174
S1174
192
AfuGfuaaCfcAfaGfaGfuAfUfUfcCfasUf
AS1174
1284
aUfgGfaauAfcUfcUfuGfgUfUfAfcAfusGfsa
0.07
0.51
0.95



 


D1175
S1175
193
AfuguAfaCfcAfaGfaGfuAfuUfCfCfasUf
AS1175
1285
aUfggaAfuAfcUfcUfuGfgUfuAfCfAfusGfsa
0.2
0.53
0.76



 


D1176
S1176
194
auGfuAfaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1176
1286
augGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.74
0.98
0.81



 


D1177
S1177
195
AfuGfuAfaCfcAfaGfaGfuAfuucCfAfsUf
AS1177
1287
augGfAfAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.43
0.64
0.88



 


D1178
S1178
196
auguaaccAfaGfaGfuAfuUfcCfasUf
AS1178
1288
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.17
0.49
0.81



 


D1179
S1179
197
AfuGfuaaCfcAfaGfaGfuAfuUfCfCfasUf
AS1179
1289
aUfggaAfuAfcUfcUfuGfgUfUfAfcAfusGfsa
0.22
0.65
0.73



 


D1180
S1180
198
AfuguAfaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1180
1290
augGfaAfuAfcUfcUfuGfgUfuAfcAfUfsGfsa
0.6
1.09
0.8



 


D1181
S1181
199
auGfuAfaCfcAfaGfaGfuAfuUfccasu
AS1181
1291
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.3
0.78
0.78



 


D1182
S1182
200
auguaaccaaGfaGfuAfuUfcCfasUf
AS1182
1292
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.35
0.73
0.84



 


D1183
S1183
201
AfuGfuAfaccAfaGfaGfuAfuUfCfCfasUf
AS1183
1293
aUfggaAfuAfcUfcUfuGfGfUfuAfcAfusGfsa
0.19
0.6
0.94



 


D1184
S1184
202
AfuGfuaaCfcAfaGfaGfuAfuUfcCfAfsUf
AS1184
1294
augGfaAfuAfcUfcUfuGfgUfuAfCfAfusGfsa
0.61
1.08
0.8



 


D1185
S1185
203
auGfuAfaCfcAfaGfaGfuAfuuccasu
AS1185
1295
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.16
0.52
0.72



 


D1186
S1186
204
auguaaccaagaGfuAfuUfcCfasUf
AS1186
1296
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.2
0.53
0.74



 


D1187
S1187
205
AfuGfuAfaCfcaaGfaGfuAfuUfCfCfasUf
AS1187
1297
aUfggaAfuAfcUfcUfUfGfgUfuAfcAfusGfsa
0.34
0.66
0.85



 


D1188
S1188
206
AfuGfuAfaccAfaGfaGfuAfuUfcCfAfsUf
AS1188
1298
augGfaAfuAfcUfcUfuGfgUfUfAfcAfusGfsa
0.61
0.98
1.02



 


D1189
S1189
207
AfuGfuAfaCfcAfaGfaGfuAfuuccasu
AS1189
1299
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.3
0.73
0.85



 


D1190
S1190
208
auguaaccaagaguauuccasu
AS1190
1300
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.28
0.69
0.78



 


D1191
S1191
209
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1191
1301
aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa
0.33
0.88
0.64



 


D1192
S1192
210
AfuGfuAfaCfcAfagaGfuAfuUfCfCfasUf
AS1192
1302
aUfggaAfuAfcUfCfUfuGfgUfuAfcAfusGfsa
0.31
0.64
0.83



 


D1193
S1193
211
AfuGfuAfaCfcaaGfaGfuAfuUfcCfAfsUf
AS1193
1303
augGfaAfuAfcUfcUfuGfGfUfuAfcAfusGfsa
0.64
0.82
0.92



 


D1194
S1194
212
AfuGfuAfaCfcAfaGfaGfuauuccasu
AS1194
1304
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.21
0.62
0.77



 


D1195
S1195
213
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1195
1305
aUfgGfaAfuAfcUfcUfuGfGfUfuAfCfAfusGfsa
0.17
0.7
0.95



 


D1196
S1196
214
AfuGfuAfaCfcAfaGfaguAfuUfCfCfasUf
AS1196
1306
aUfggaAfuAfCfUfcUfuGfgUfuAfcAfusGfsa
0.19
0.71
0.65



 


D1197
S1197
215
AfuGfuAfaCfcAfagaGfuAfuUfcCfAfsUf
AS1197
1307
augGfaAfuAfcUfcUfUfGfgUfuAfcAfusGfsa
0.64
0.82
0.93



 


D1198
S1198
216
auguAfaCfcAfaGfaGfuAfuUfccasu
AS1198
1308
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.19
0.65
0.72



 


D1199
S1199
217
AfuGfuAfaCfcAfaGfaGfuauUfCfCfasUf
AS1199
1309
aUfggaAfUfAfcUfcUfuGfgUfuAfcAfusGfsa
0.15
0.52
0.64



 


D1200
S1200
218
AfuGfuAfaCfcAfaGfaguAfuUfcCfAfsUf
AS1200
1310
augGfaAfuAfcUfCfUfuGfgUfuAfcAfusGfsa
0.48
0.74
0.92



 


D1201
S1201
219
auguAfaCfcAfaGfaGfuAfuUfcCfasu
AS1201
1311
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.17
0.71
0.77



 


D1202
S1202
220
AfuGfuAfaCfcAfaGfaGfuauUfcCfAfsUf
AS1202
1312
augGfaAfuAfCfUfcUfuGfgUfuAfcAfusGfsa
0.43
0.69
0.85



 


D1203
S1203
221
auguaaCfcAfaGfaGfuAfuUfcCfasUf
AS1203
1313
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.14
0.61
0.76



 


D1204
S1204
222
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1204
1314
adTdGGfaAfudAdCUfcUfuGfgUfuAfcAfusGfsa
0.16
0.56
0.89



 


D1205
S1205
223
AfuGfuAfaCfcAfaGfaGfdTdAdTdTcCfasUf
AS1205
1315
aUfgGfdAdAdTdAcUfcUfuGfgUfuAfcAfusGfsa
0.13
0.57
0.9



 


D1206
S1206
224
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1206
1316
adTdGdGdAAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.29
0.73
0.89



 


D1207
S1207
225
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1207
1317
adTdGGfaAfuAfdCdTcUfuGfgUfuAfcAfusGfsa
0.16
0.56
0.78



 


D1208
S1208
226
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1208
1318
aUfdGdGdAdAuAfcUfcUfuGfgUfuAfcAfusGfsa
0.22
0.67
0.89



 


D1209
S1209
227
AfuguAfaccAfaGfaGfuAfuUfcCfasUf
AS1209
1319
aUfgGfaAfuAfcUfcUfuGfGfUfuAfCfAfusGfsa
0.14
0.55
0.78



 


D1210
S1210
228
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1210
1320
aUfgdGdAdAdTAfcUfcUfuGfgUfuAfcAfusGfsa
0.14
0.5
0.84



 


D1211
S1211
229
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1211
1321
aUfgGfadAdTdAdCUfcUfuGfgUfuAfcAfusGfsa
0.14
0.59
0.72



 


D1212
S1212
230
auguaaccaaGfaGfuAfuUfcCfasUf
AS1212
1322
aUfgGfaAfuAfcUfcUfugdGudTadCadTsgsa
0.21
0.74
0.77



 


D1213
S1213
231
AfuGfuAfaCfcAfaGfaGfuAfudTdCdCdAsUf
AS1213
1323
adTdGdGdAAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.15
0.53
0.91



 


D1214
S1214
232
aUfgUfaAfcCfaAfgAfgUfaUfuCfcAfsu
AS1214
1324
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.12
0.71
0.87



 


D1215
S1215
233
AfuGfuAfaCfcAfaGfaGfuAfdTdTdCdCasUf
AS1215
1325
aUfdGdGdAdAuAfcUfcUfuGfgUfuAfcAfusGfsa
0.18
0.67
0.97



 


D1216
S1216
234
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1216
1326
aUfgGfaAfuacucuuggUfuAfcAfusgsa
0.36
0.87
1.07



 


D1217
S1217
235
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1217
1327
aUfgGfaAfuAfCfUfCfUfuGfGfuuAfcAfusgsa
0.37
0.73
1.03



 


D1218
S1218
236
AfUfguAfAfccAfAfgaGfUfauUfCfcasUf
AS1218
1328
aUfGfgaAfUfacUfCfuuGfGfuuAfCfausGfsa
0.23
0.42
0.84



 


D1219
S1219
237
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1219
1329
aUfgGfaAfuaCfUfcUfUfgGfuuAfcAfusgsa
0.43
0.71
1.03



 


D1220
S1220
238
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1220
1330
aUfgGfaAfuAfcUfCfUfuGfGfuuAfcAfusgsa
0.37
0.63
0.99



 


D1221
S1221
239
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1221
1331
aUfgGfaAfuAfcUfCfUfuGfGfuUfacAfusgsa
0.29
0.84
0.88



 


D1222
S1222
240
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1222
1332
aUfgGfaAfuaCfuCfuUfgGfuuAfcAfusgsa
0.31
0.8
0.99



 


D1223
S1223
241
auGfuAfAfccAfaGfagUfaUfUfcCfasUf
AS1223
1333
aUfgGfaaUfaCfUfcUfuGfGfuuAfcAfAfsgsa
0.09
0.52
0.82



 


D1224
S1224
242
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1224
1334
aUfgGfaAfuadCudCudTgdGuuAfcAfusgsa
0.22
0.79
1



 


D1225
S1225
243
auGfuaAfccAfagAfguAfuuCfcasUf
AS1225
1335
aUfGfgAfAfuAfCfuCfUfuGfGfuUfAfcAfUfsGfsa
0.31
0.76
0.84



 


D1226
S1226
244
AfuGfuAfaccaagaguAfuUfcCfasUf
AS1226
1336
aUfgGfaAfuadCUfcdTUfgdGuuAfcAfusgsa
0.26
0.64
0.87



 


D1227
S1227
245
augUfaacCfaagAfguaUfuccAfsu
AS1227
1337
aUfgGfAfaUfAfCfuCfUfUfgGfUfUfaCfAfUfsGfsa
0.33
0.79
0.81



 


D1228
S1228
246
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1228
1338
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.464
0.932
0.978



 


D1229
S1229
247
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1229
1339
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.453
1.047
1.178



 


D1230
S1230
248
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1230
1340
aUfgGfaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.831
0.967
1.151



 


D1231
S1231
249
auGfuAfAfCfcAfaGfaGfuAfuUfcCfasu
AS1231
1341
AfUfgGfaAfuAfcUfcUfuGfguuAfcAfUfsGfsa
0.09
0.5
1.07



 


D1232
S1232
250
AfuGfuAfaCfCfAfaGfaGfuAfuUfcCfasu
AS1232
1342
AfUfgGfaAfuAfcUfcUfuggUfuAfcAfusGfsa
0.11
0.54
1.1



 


D1233
S1233
251
AfuGfuAfaCfcAfaGfaGfuAfuUfCfCfasu
AS1233
1343
AfUfggaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.19
0.61
0.74



 


D1234
S1234
252
aUfgUfaAfcCfaAfgAfgUfaUfuCfcAfsu
AS1234
1344
AfuGfgAfaUfaCfuCfuUfgGfuUfaCfaUfsgsAf
0.22
0.61
0.98



 


D1235
S1235
253
aUfgUfaAfcCfaAfgAfgUfaUfuCfcAfsu
AS1235
1345
AfuGfgAfaUfaCfuCfuUfgGfuUfaCfaUfsgsAf
0.27
0.69
0.92



 


D1236
S1236
254
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1236
1346
AfuGfgAfaUfaCfuCfuUfgGfuUfaCfaUfsgsAf
0.54
1.08
0.8



 


D1237
S1237
255
augUfaAfccaAfgAfguaUfuCfcasu
AS1237
1347
AfUfGfgAfaUfAfCfuCfuUfGfGfuUfaCfAfUfsgsa
0.29
0.61
0.79



 


D1238
S1238
256
AfugUfaAfccaAfgAfguaUfuCfcasu
AS1238
1348
AfUfGfgAfaUfAfCfuCfuUfGfGfuUfaCfAfusgsa
0.31
0.6
0.88



 


D1239
S1239
257
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1239
1349
dAUdGGdAauAfcUfcUfuGfgUfuAfcAfusGfsa
0.2
0.67
0.85



 


D1240
S1240
258
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1240
1350
dAUdGgdAauAfcUfcUfuGfgUfuAfcAfusGfsa
0.23
0.58
0.68



 


D1241
S1241
259
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1241
1351
dAudGgdAauAfcUfcUfuGfgUfuAfcAfusGfsa
0.25
0.65
0.78



 


D1242
S1242
260
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1242
1352
dAUdGgdAadTAfcUfcUfuGfgUfuAfcAfusGfsa
0.18
0.64
0.84



 


D1243
S1243
261
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1243
1353
dAUdGGdAAfuAfcUfcUfuGfGfUfuAfCfAfusGfsa
0.19
0.72
0.87



 


D1244
S1244
262
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1244
1354
dAUdGgdAadTAfdCUfcUfuGfgUfuAfcAfusGfsa
0.16
0.55
0.8



 


D1245
S1245
263
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1245
1355
dAUdGGdAAuAfcUfcUfuGfgUfuAfcAfusGfsa
0.22
0.51
0.9



 


D1246
S1246
264
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1246
1356
dAudGgdAadTAfcUfcUfuGfgUfuAfcAfusGfsa
0.27
0.78
0.66



 


D1247
S1247
265
AfuGfuAfaCfcAfaGfaGfuAfuUfcCfasUf
AS1247
1357
dAdTdGdGaAfuAfcUfcUfuGfgUfuAfcAfusGfsa
0.16
0.57
0.97



 


D1248
S1248
266
AfacaAfuguUfcUfuGfdCUdCudAudAsa
AS1248
1358
dTUdAudAgdAGfcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.09
0.36
0.0047


 


D1249
S1249
267
AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasa
AS1249
1359
UfUfaUfaGfagcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.10
0.47
0.005


 


D1250
S1250
268
AfaCfaGfuGfuUfcUfugcUfcUfAfUfasAf
AS1250
1360
uUfauaGfaGfCfAfaGfaAfcAfcUfgUfusUfsu
0.07
0.14
0.55
0.005


 


D1251
S1251
269
AfaCfaGfuGfuUfcUfuGfcucUfAfUfasAf
AS1251
1361
uUfauaGfAfGfcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.14
0.49
0.006


 


D1252
S1252
270
cAGuGuucuuGcucuAuAAdTdT
AS1252
1362
UuAuAGAGcAAGAAcACUGdTdT



0.006


 


D1253
S1253
271
AfaCfaGfuGfuUfcUfugcUfCfUfaUfasAf
AS1253
1363
uUfaUfagaGfCfAfaGfaAfcAfcUfgUfusUfsu
0.05
0.12
0.43
0.006


 


D1254
S1254
272
AfaCfaGfuGfuUfCfUfuGfcUfcUfaUfasa
AS1254
1364
UfUfaUfaGfaGfcAfagaAfcAfcUfgUfusUfsu
0.06
0.13
0.39
0.006


 


D1255
S1255
273
AfaCfaGfuGfuUfcUfuGfcUfCfUfaUfasa
AS1255
1365
UfUfaUfagaGfcAfaGfaAfcAfcUfgUfusUfsu
0.08
0.17
0.48
0.007


 


D1256
S1256
274
AfaCfaGfuGfuUfcUfUfGfcUfcUfaUfasa
AS1256
1366
UfUfaUfaGfaGfcaaGfaAfcAfcUfgUfusUfsu
0.08
0.14
0.40
0.007


 


D1257
S1257
275
AfaCfaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1257
1367
uUfaUfagaGfcAfaGfaAfcAfcUfgUfusUfsUf
0.07
0.12
0.40
0.007


 


D1258
S1258
276
AfaCfaguGfuUfCfUfuGfcUfcUfaUfasAf
AS1258
1368
uUfaUfaGfaGfcAfagaAfcAfCfUfgUfusUfsu
0.08
0.13
0.41
0.007


 


D1259
S1259
277
AfaCfAfGfuGfuUfcUfuGfcucUfaUfasAf
AS1259
1369
uUfaUfaGfAfGfcAfaGfaAfcAfcugUfusUfsu
0.05
0.11
0.35
0.008


 


D1260
S1260
278
AfacaGfuGfuUfCfUfuGfcUfcUfaUfasAf
AS1260
1370
uUfaUfaGfaGfcAfagaAfcAfcUfGfUfusUfsu
0.06
0.12
0.40
0.008


 


D1261
S1261
279
AfacaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1261
1371
uUfaUfagaGfcAfaGfaAfcAfcUfGfUfusUfsu
0.06
0.13
0.42
0.008


 


D1262
S1262
280
AfaCfaGfuGfuUfcUfuGfcucUfaUfasAf
AS1262
1372
uUfaUfaGfAfGfcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.13
0.37
0.008


 


D1263
S1263
281
cAGuGuucuuGcucuAuAAdTdT
AS1263
1373
UuAuAGAGcAAGAAcACUGdTdT



0.008


 


D1264
S1264
282
AfaCfaGfuGfuUfcUfuGfCfUfcUfauasAf
AS1264
1374
uUfAfUfaGfagcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.12
0.50
0.008


 


D1265
S1265
283
AfaCfaGfuguUfCfUfuGfcUfcUfaUfasAf
AS1265
1375
uUfaUfaGfaGfcAfagaAfCfAfcUfgUfusUfsu
0.12
0.13
0.48
0.009


 


D1266
S1266
284
AfacaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1266
1376
uUfauaGfaGfcAfaGfaAfcAfcUfGfUfusUfsu
0.07
0.15
0.51
0.009


 


D1267
S1267
285
AfacaAfuguUfcUfuGfdCudCudAudAsa
AS1267
1377
dTudAudAgdAGfcAfaGfaAfcAfcAfgUfusUfsu
0.06
0.14
0.48
0.0088


 


D1268
S1268
286
AfaCfaGfuGfuUfCfUfuGfcucUfaUfasAf
AS1268
1378
uUfaUfaGfAfGfcAfagaAfcAfcUfgUfusUfsu
0.05
0.09
0.35
0.009


 


D1269
S1269
287
cAGuGuucuuGcucuAuAAdTdT
AS1269
1379
UuAuAGAGcAAGAAcACUGdTdT



0.009


 


D1270
S1270
288
aaCfaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1270
1380
uUfaUfagaGfcAfaGfaAfcAfcUfgUfUfsUfsu
0.07
0.14
0.49
0.009


 


D1271
S1271
289
AfaCfaGfUfGfuUfcUfuGfcucUfaUfasAf
AS1271
1381
uUfaUfaGfAfGfcAfaGfaAfcacUfgUfusUfsu
0.06
0.10
0.36
0.009


 


D1272
S1272
290
cAGuGuucuuGcucuAuAAdTdT
AS1272
1382
UuAuAGAGcAAGAAcACUGdTdT



0.009


 


D1273
S1273
291
AfaCfaGfUfGfuUfcUfuGfcUfcUfaUfasAf
AS1273
1383
uUfaUfaGfaGfcAfaGfaAfcacUfgUfusUfsUf
0.06
0.13
0.51
0.009


 


D1274
S1274
292
AfaCfaGfuGfuUfCfUfuGfcUfcuaUfasAf
AS1274
1384
uUfaUfAfGfaGfcAfagaAfcAfcUfgUfusUfsu
0.06
0.12
0.46
0.010


 


D1275
S1275
293
cAGuGuucuuGcucuAuAAdTdT
AS1275
1385
UuAuAGAGcAAGAAcACUGdTdT



0.010


 


D1276
S1276
294
AfaCfaGfuGfuUfCfUfuGfcUfcUfauasAf
AS1276
1386
uUfAfUfaGfaGfcAfagaAfcAfcUfgUfusUfsu
0.06
0.14
0.47
0.010


 


D1277
S1277
295
AfaCfaguGfuUfcUfuGfcUfCfUfaUfasAf
AS1277
1387
uUfaUfagaGfcAfaGfaAfcAfCfUfgUfusUfsu
0.07
0.15
0.50
0.010


 


D1278
S1278
296
AfaCfaGfuGfuUfCfUfugcUfcUfaUfasAf
AS1278
1388
uUfaUfaGfaGfCfAfagaAfcAfcUfgUfusUfsu
0.06
0.13
0.43
0.010


 


D1279
S1279
297
cAGuGuucuuGcucuAuAAdTdT
AS1279
1389
UuAuAGAGcAAGAAcACUGdTdT



0.010


 


D1280
S1280
298
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasa
AS1280
1390
UfUfaUfaGfaGfcAfaGfaAfcAfcUfgUfususu
0.06
0.14
0.45
0.010


 


D1281
S1281
299
AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasa
AS1281
1391
UfUfaUfaGfaGfcAfaGfaAfcAfcugUfusUfsu
0.07
0.18
0.46
0.011


 


D1282
S1282
300
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1282
1392
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.15
0.55
0.011


 


D1283
S1283
301
AfaCfaGfuGfuUfcUfuGfcucUfaUfasAf
AS1283
1393
uUfaUfaGfAfGfcAfaGfaAfcAfcUfgUfususu
0.07
0.12
0.45
0.011


 


D1284
S1284
302
AfacaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1284
1394
uUfaUfaGfaGfcAfaGfaAfcAfcUfGfUfusUfsu
0.06
0.13
0.48
0.011


 


D1285
S1285
303
AfAfCfaGfuGfuUfcUfuGfcucUfaUfasAf
AS1285
1395
uUfaUfaGfAfGfcAfaGfaAfcAfcUfguusUfsu
0.06
0.11
0.40
0.011


 


D1286
S1286
304
AfaCfAfGfuGfuUfcUfuGfcUfcUfauasAf
AS1286
1396
uUfAfUfaGfaGfcAfaGfaAfcAfcugUfusUfsu
0.06
0.16
0.47
0.011


 


D1287
S1287
305
AfaCfaGfuGfuUfcUfugcUfcUfaUfasAf
AS1287
1397
uUfaUfaGfaGfCfAfaGfaAfcAfcUfgUfususu
0.07
0.19
0.46
0.012


 


D1288
S1288
306
AfaCfaGfuGfuUfcUfugcUfcUfaUfasAf
AS1288
1398
uUfaUfaGfaGfCfAfaGfaAfcAfcUfgUfusUfsu
0.06
0.17
0.46
0.012


 


D1289
S1289
307
AfaCfaGfuGfuUfcUfUfGfcucUfaUfasAf
AS1289
1399
uUfaUfaGfAfGfcaaGfaAfcAfcUfgUfusUfsu
0.05
0.09
0.31
0.012


 


D1290
S1290
308
AfAfCfaGfuGfuUfcUfuGfcUfcUfaUfasa
AS1290
1400
UfUfaUfaGfaGfcAfaGfaAfcAfcUfguusUfsu
0.06
0.16
0.49
0.013


 


D1291
S1291
309
AfaCfaGfuGfuUfCfUfuGfcUfcUfaUfasAf
AS1291
1401
uUfaUfaGfaGfcAfagaAfcAfcUfgUfusUfsUf
0.06
0.11
0.32
0.013


 


D1292
S1292
310
AfaCfAfGfuGfuUfcUfugcUfcUfaUfasAf
AS1292
1402
uUfaUfaGfaGfCfAfaGfaAfcAfcugUfusUfsu
0.06
0.14
0.44
0.013


 


D1293
S1293
311
AfaCfaGfUfGfuUfcUfuGfcUfcUfaUfasa
AS1293
1403
UfUfaUfaGfaGfcAfaGfaAfcacUfgUfusUfsu
0.07
0.16
0.39
0.013


 


D1294
S1294
312
AfaCfAfGfuGfuUfcUfuGfcUfcuaUfasAf
AS1294
1404
uUfaUfAfGfaGfcAfaGfaAfcAfcugUfusUfsu
0.07
0.18
0.41
0.014


 


D1295
S1295
313
AfaCfaGfUfGfuUfcUfuGfcUfcuaUfasAf
AS1295
1405
uUfaUfAfGfaGfcAfaGfaAfcacUfgUfusUfsu
0.07
0.18
0.47
0.014


 


D1296
S1296
314
adAdCagdTdGuudCdTugdCdTcudAdTasa
AS1296
1406
dTdTaudAdGagdCdAagdAdAcadCdTgudTsdTsu
0.12
0.21
0.68
0.0146


 


D1297
S1297
315
AfacaGfUfGfuUfcUfuGfcUfcUfaUfasAf
AS1297
1407
uUfaUfaGfaGfcAfaGfaAfcacUfGfUfusUfsu
0.06
0.15
0.50
0.016


 


D1298
S1298
316
AfaCfaGfUfGfuUfcUfuGfcUfcUfauasAf
AS1298
1408
uUfAfUfaGfaGfcAfaGfaAfcacUfgUfusUfsu
0.08
0.17
0.50
0.016


 


D1299
S1299
317
AfaCfaguGfuUfcUfuGfcUfcUfaUfasAf
AS1299
1409
uUfaUfaGfaGfcAfaGfaAfcAfCfUfgUfususu
0.07
0.16
0.50
0.018


 


D1300
S1300
318
AfaCfaGfuGfuUfcUfUfGfcUfcUfauasAf
AS1300
1410
uUfAfUfaGfaGfcaaGfaAfcAfcUfgUfusUfsu
0.06
0.12
0.43
0.020


 


D1301
S1301
319
AfaCfaGfUfGfuUfcUfugcUfcUfaUfasAf
AS1301
1411
uUfaUfaGfaGfCfAfaGfaAfcacUfgUfusUfsu
0.07
0.17
0.45
0.021


 


D1302
S1302
320
AfaCfaGfuguUfcUfUfGfcUfcUfaUfasAf
AS1302
1412
uUfaUfaGfaGfcaaGfaAfCfAfcUfgUfusUfsu
0.06
0.14
0.49
0.021


 


D1303
S1303
321
AfAfCfaguGfuUfcUfuGfcUfcUfaUfasAf
AS1303
1413
uUfaUfaGfaGfcAfaGfaAfcAfCfUfguusUfsu
0.07
0.24
0.51
0.022


 


D1304
S1304
322
AfaCfaGfuGfuucUfuGfcUfcUfaUfasAf
AS1304
1414
uUfaUfaGfaGfcAfaGfAfAfcAfcUfgUfususu
0.09
0.27
0.47
0.033


 


D1305
S1305
323
aadCdAgudGdTucdTdTgcdTdCuadTdAsa
AS1305
1415
udTadTdAgadGdCaadGdAacdAdCugdTdTsusu
0.19
0.36
0.86
0.045


 


D1306
S1306
324
AfacaGfuguUfcUfuGfdCUdCUdAudAsa
AS1306
1416
dTUdAUdAGfaGfcAfaGfaAfCfAfcUfGfUfusUfsu
0.08
0.22
0.61



 


D1307
S1307
325
AfacaGfuguUfcUfdTGfdCUdCUdAudAsa
AS1307
1417
dTUdAUdAGfaGfcAfaGfaAfCfAfcUfGfUfusUfsu
0.13
0.39
0.84



 


D1308
S1308
326
AfacaGfuguUfcUfuGfdCUdCUdAudAsa
AS1308
1418
dTUdAUdAgdAGfcAfaGfaAfcAfcUfgUfusUfsu
0.09
0.13
0.48



 


D1309
S1309
327
AfacaGfuguUfcUfdTGfdCUdCUdAudAsa
AS1309
1419
dTUdAUdAgdAGfdCAfaGfaAfcAfcUfgUfusUfsu
0.07
0.13
0.58



 


D1310
S1310
328
AfacaAfuguUfcUfdTGfdCUdCudAudAsa
AS1310
1420
dTUdAudAgdAGfdCAfaGfaAfcAfcAfgUfusUfsu
0.07
0.14
0.55



 


D1311
S1311
329
AfaCfaAfuGfuUfcUfuGfcUfcUfdAdTdAsdA
AS1311
1421
dTdTdAdTaGfaGfcAfaGfaAfcAfcAfgUfusUfsu
0.10
0.30
0.66



 


D1312
S1312
330
AfacaGfuguUfcUfuGfdCUdCUdAudAsa
AS1312
1422
dTUdAUdAgdAGfcAfaGfaAfcAfcUfgUfusUfsu
0.09
0.13
0.48



 


D1313
S1313
331
AfAfCfaGfuGfuucUfuGfcUfcUfaUfasAf
AS1313
1423
uUfaUfaGfaGfcAfaGfAfAfcAfcUfguusUfsu
0.14
0.38
0.74



 


D1314
S1314
332
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1314
1424
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.19
0.54



 


D1315
S1315
333
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1315
1425
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.15
0.55



 


D1316
S1316
334
AfaCfaGfuGfuUfcUfuGfcUfcUfauasAf
AS1316
1426
uUfAfUfaGfaGfcAfaGfaAfcAfcUfgUfususu
0.07
0.16
0.53



 


D1317
S1317
335
AfacaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1317
1427
uUfaUfaGfaGfcAfaGfaAfcAfcUfGfUfususu
0.07
0.16
0.55



 


D1318
S1318
336
AfAfCfaGfuguUfcUfuGfcUfcUfaUfasAf
AS1318
1428
uUfaUfaGfaGfcAfaGfaAfCfAfcUfguusUfsu
0.10
0.32
0.61



 


D1319
S1319
337
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1319
1429
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfususu
0.08
0.16
0.53



 


D1320
S1320
338
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1320
1430
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfususu
0.08
0.16
0.61



 


D1321
S1321
339
AfaCfaGfuGfuUfcUfuGfcUfCfUfaUfasAf
AS1321
1431
uUfaUfagaGfcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.14
0.58



 


D1322
S1322
340
AfaCfaGfuGfuUfcuuGfcUfcUfaUfasAf
AS1322
1432
uUfaUfaGfaGfcAfAfGfaAfcAfcUfgUfusUfsu
0.15
0.49
0.84



 


D1323
S1323
341
AfaCfaGfuGfuUfcUfuGfcUfcuaUfasAf
AS1323
1433
uUfaUfAfGfaGfcAfaGfaAfcAfcUfgUfususu
0.07
0.20
0.62



 


D1324
S1324
342
AfAfCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1324
1434
uUfaUfaGfaGfcAfaGfaAfcAfcUfguusUfsu
0.08
0.25
0.78



 


D1325
S1325
343
AfAfCfaGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1325
1435
uUfaUfaGfaGfcAfaGfaAfcAfcUfguusUfsu
0.08
0.18
0.80



 


D1326
S1326
344
AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1326
1436
uUfauaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.21
0.66



 


D1327
S1327
345
AfaCfaGfuGfuucUfuGfcUfcUfaUfasAf
AS1327
1437
uUfaUfaGfaGfcAfaGfAfAfcAfcUfgUfusUfsu
0.10
0.31
0.70



 


D1328
S1328
346
AfAfCfaGfuGfuUfcUfuGfcUfcUfauasAf
AS1328
1438
uUfAfUfaGfaGfcAfaGfaAfcAfcUfguusUfsu
0.07
0.15
0.55



 


D1329
S1329
347
AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1329
1439
uUfaUfaGfaGfcAfaGfaAfcAfcugUfusUfsu
0.08
0.19
0.71



 


D1330
S1330
348
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1330
1440
uuaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.09
0.27
0.76



 


D1331
S1331
349
AfaCfaGfuguUfcUfuGfcUfcUfaUfasAf
AS1331
1441
uUfaUfaGfaGfcAfaGfaAfCfAfcUfgUfusUfsu
0.07
0.21
0.65



 


D1332
S1332
350
AfAfCfaGfuGfuUfcUfuGfcUfcuaUfasAf
AS1332
1442
uUfaUfAfGfaGfcAfaGfaAfcAfcUfguusUfsu
0.07
0.17
0.53



 


D1333
S1333
351
AfaCfaGfUfGfuUfcUfuGfcUfcUfaUfasAf
AS1333
1443
uUfaUfaGfaGfcAfaGfaAfcacUfgUfusUfsu
0.08
0.25
0.73



 


D1334
S1334
352
AfaCfaguGfuUfcUfuGfcUfcUfaUfasAf
AS1334
1444
uUfaUfaGfaGfcAfaGfaAfcAfCfUfgUfusUfsu
0.07
0.18
0.54



 


D1335
S1335
353
AfaCfaGfuGfuUfcuuGfcUfcUfaUfasAf
AS1335
1445
uUfaUfaGfaGfcAfAfGfaAfcAfcUfgUfususu
0.14
0.38
0.57



 


D1336
S1336
354
AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasAf
AS1336
1446
uUfaUfaGfaGfcAfaGfaacAfcUfgUfusUfsu
0.16
0.50
0.96



 


D1337
S1337
355
AfaCfaGfuGfuUfcUfuGfcUfcUfauasAf
AS1337
1447
uUfAfUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.08
0.19
0.54



 


D1338
S1338
356
AfAfCfaGfuGfuUfcUfugcUfcUfaUfasAf
AS1338
1448
uUfaUfaGfaGfCfAfaGfaAfcAfcUfguusUfsu
0.08
0.20
0.69



 


D1339
S1339
357
AfaCfaGfuGfuUfCfUfuGfcUfcUfaUfasAf
AS1339
1449
uUfaUfaGfaGfcAfagaAfcAfcUfgUfusUfsu
0.07
0.16
0.55



 


D1340
S1340
358
AfaCfaGfuGfuUfcUfuGfcUfcuaUfasAf
AS1340
1450
uUfaUfAfGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.08
0.17
0.57



 


D1341
S1341
359
AfaCfaGfuguUfcUfuGfcUfcUfaUfasAf
AS1341
1451
uUfaUfaGfaGfcAfaGfaAfCfAfcUfgUfususu
0.08
0.22
0.63



 


D1342
S1342
360
AfAfCfaGfuGfuUfcuuGfcUfcUfaUfasAf
AS1342
1452
uUfaUfaGfaGfcAfAfGfaAfcAfcUfguusUfsu
0.21
0.56
0.86



 


D1343
S1343
361
AfacaGfuGfUfUfcUfuGfcUfcUfaUfasAf
AS1343
1453
uUfaUfaGfaGfcAfaGfaacAfcUfGfUfusUfsu
0.14
0.37
0.73



 


D1344
S1344
362
AfaCfaGfuGfuucUfUfGfcUfcUfaUfasAf
AS1344
1454
uUfaUfaGfaGfcaaGfAfAfcAfcUfgUfusUfsu
0.08
0.20
0.66



 


D1345
S1345
363
AfaCfAfGfuGfuUfcuuGfcUfcUfaUfasAf
AS1345
1455
uUfaUfaGfaGfcAfAfGfaAfcAfcugUfusUfsu
0.12
0.34
0.73



 


D1346
S1346
364
AfaCfaGfuGfUfUfcUfuGfcUfcUfauasAf
AS1346
1456
uUfAfUfaGfaGfcAfaGfaacAfcUfgUfusUfsu
0.16
0.42
0.90



 


D1347
S1347
365
AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasAf
AS1347
1457
uUfaUfaGfaGfcAfaGfaacAfcUfgUfusUfsUf
0.17
0.43
0.85



 


D1348
S1348
366
AfaCfAfGfuGfuucUfuGfcUfcUfaUfasAf
AS1348
1458
uUfaUfaGfaGfcAfaGfAfAfcAfcugUfusUfsu
0.08
0.21
0.58



 


D1349
S1349
367
AfaCfaGfuGfUfUfcUfuGfcUfcuaUfasAf
AS1349
1459
uUfaUfAfGfaGfcAfaGfaacAfcUfgUfusUfsu
0.21
0.39
0.88



 


D1350
S1350
368
AfaCfaguGfuUfcUfUfGfcUfcUfaUfasAf
AS1350
1460
uUfaUfaGfaGfcaaGfaAfcAfCfUfgUfusUfsu
0.06
0.13
0.52



 


D1351
S1351
369
AfaCfAfGfuguUfcUfuGfcUfcUfaUfasAf
AS1351
1461
uUfaUfaGfaGfcAfaGfaAfCfAfcugUfusUfsu
0.08
0.21
0.58



 


D1352
S1352
370
AfaCfaGfUfGfuUfcuuGfcUfcUfaUfasAf
AS1352
1462
uUfaUfaGfaGfcAfAfGfaAfcacUfgUfusUfsu
0.18
0.49
0.84



 


D1353
S1353
371
AfaCfaGfuGfUfUfcUfuGfcucUfaUfasAf
AS1353
1463
uUfaUfaGfAfGfcAfaGfaacAfcUfgUfusUfsu
0.11
0.25
0.68



 


D1354
S1354
372
AfacaGfuGfuUfcUfUfGfcUfcUfaUfasAf
AS1354
1464
uUfaUfaGfaGfcaaGfaAfcAfcUfGfUfusUfsu
0.07
0.15
0.52



 


D1355
S1355
373
AfaCfaGfUfGfuucUfuGfcUfcUfaUfasAf
AS1355
1465
uUfaUfaGfaGfcAfaGfAfAfcacUfgUfusUfsu
0.10
0.26
0.63



 


D1356
S1356
374
AfaCfaGfuGfUfUfcUfugcUfcUfaUfasAf
AS1356
1466
uUfaUfaGfaGfCfAfaGfaacAfcUfgUfusUfsu
0.16
0.33
0.79



 


D1357
S1357
375
AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1357
1467
uUfaUfaGfaGfcAfaGfaAfcAfcugUfusUfsUf
0.09
0.19
0.51



 


D1358
S1358
376
AfaCfaGfuGfUfUfcuuGfcUfcUfaUfasAf
AS1358
1468
uUfaUfaGfaGfcAfAfGfaacAfcUfgUfusUfsu
0.22
0.48
0.71



 


D1359
S1359
377
AfaCfaGfuGfuUfcUfUfGfcUfcUfaUfasAf
AS1359
1469
uUfaUfaGfaGfcaaGfaAfcAfcUfgUfusUfsUf
0.10
0.17
0.61



 


D1360
S1360
378
AfaCfaguGfUfUfcUfuGfcUfcUfaUfasAf
AS1360
1470
uUfaUfaGfaGfcAfaGfaacAfCfUfgUfusUfsu
0.14
0.40
0.87



 


D1361
S1361
379
AfaCfaGfuGfuUfcUfUfGfcUfcuaUfasAf
AS1361
1471
uUfaUfAfGfaGfcaaGfaAfcAfcUfgUfusUfsu
0.07
0.14
0.52



 


D1362
S1362
380
aaCfaGfuGfuUfcUfuGfCfUfcUfaUfasAf
AS1362
1472
uUfaUfaGfagcAfaGfaAfcAfcUfgUfUfsUfsu
0.10
0.28
0.81



 


D1363
S1363
381
AfaCfaGfuGfuucUfuGfcUfcUfAfUfasAf
AS1363
1473
uUfauaGfaGfcAfaGfAfAfcAfcUfgUfusUfsu
0.06
0.16
0.68



 


D1364
S1364
382
AfaCfaGfuGfuUfcUfugcUfcUfaUfAfsAf
AS1364
1474
uuaUfaGfaGfCfAfaGfaAfcAfcUfgUfusUfsu
0.09
0.26
0.67



 


D1365
S1365
383
aacaguguucuugcucuauasa
AS1365
1475
uUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.20
0.59
0.95



 


D1366
S1366
384
AfaCfaGfuGfuUfcUfuGfCfUfcUfauasAf
AS1366
1476
uUfAfUfaGfagcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.13
0.53



 


D1367
S1367
385
AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasAf
AS1367
1477
uUfaUfaGfagcAfaGfaAfcAfcUfgUfusUfsUf
0.08
0.16
0.53



 


D1368
S1368
386
AfaCfaGfuguUfcUfuGfcUfcUfAfUfasAf
AS1368
1478
uUfauaGfaGfcAfaGfaAfCfAfcUfgUfusUfsu
0.07
0.15
0.54



 


D1369
S1369
387
AfaCfaGfuGfuUfcuuGfcUfcUfaUfAfsAf
AS1369
1479
uuaUfaGfaGfcAfAfGfaAfcAfcUfgUfusUfsu
0.23
0.56
0.89



 


D1370
S1370
388
AfaCfaGfuGfuUfcUfuGfCfUfcuaUfasAf
AS1370
1480
uUfaUfAfGfagcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.12
0.55



 


D1371
S1371
389
AfaCfaGfuGfuUfcUfuGfCfUfcuaUfasAf
AS1371
1481
uUfaUfAfGfagcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.18
0.58



 


D1372
S1372
390
AfaCfaguGfuUfcUfuGfcUfcUfAfUfasAf
AS1372
1482
uUfauaGfaGfcAfaGfaAfcAfCfUfgUfusUfsu
0.06
0.15
0.56



 


D1373
S1373
391
AfaCfaGfuGfuucUfuGfcUfcUfaUfAfsAf
AS1373
1483
uuaUfaGfaGfcAfaGfAfAfcAfcUfgUfusUfsu
0.21
0.51
0.89



 


D1374
S1374
392
AfacaGfuguUfcUfuGfcUfcUfaUfasAf
AS1374
1484
uUfaUfaGfaGfcAfaGfaAfCfAfcUfGfUfusUfsu
0.08
0.21
0.64



 


D1375
S1375
393
AfaCfaGfuGfuUfcuuGfCfUfcUfaUfasAf
AS1375
1485
uUfaUfaGfagcAfAfGfaAfcAfcUfgUfusUfsu
0.15
0.40
0.94



 


D1376
S1376
394
AfaCfaGfuGfuUfcuuGfCfUfcUfaUfasAf
AS1376
1486
uUfaUfaGfagcAfAfGfaAfcAfcUfgUfusUfsu
0.13
0.40
0.96



 


D1377
S1377
395
AfaCfaGfuGfuUfcUfuGfcUfCfUfauasAf
AS1377
1487
uUfAfUfagaGfcAfaGfaAfcAfcUfgUfusUfsu
0.08
0.17
0.64



 


D1378
S1378
396
AfaCfaGfuguUfcUfuGfcUfcUfaUfAfsAf
AS1378
1488
uuaUfaGfaGfcAfaGfaAfCfAfcUfgUfusUfsu
0.18
0.50
0.97



 


D1379
S1379
397
AfaCfaGfuGfuucUfuGfCfUfcUfaUfasAf
AS1379
1489
uUfaUfaGfagcAfaGfAfAfcAfcUfgUfusUfsu
0.08
0.24
0.79



 


D1380
S1380
398
aaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1380
1490
uUfauaGfaGfcAfaGfaAfcAfcUfgUfUfsUfsu
0.07
0.14
0.58



 


D1381
S1381
399
AfaCfaguGfuUfcUfuGfcUfcUfaUfAfsAf
AS1381
1491
uuaUfaGfaGfcAfaGfaAfcAfCfUfgUfusUfsu
0.11
0.34
0.96



 


D1382
S1382
400
AfaCfaGfuguUfcUfuGfCfUfcUfaUfasAf
AS1382
1492
uUfaUfaGfagcAfaGfaAfCfAfcUfgUfusUfsu
0.08
0.18
0.69



 


D1383
S1383
401
AfaCfaGfuGfuUfcuuGfcUfCfUfaUfasAf
AS1383
1493
uUfaUfagaGfcAfAfGfaAfcAfcUfgUfusUfsu
0.14
0.38
0.85



 


D1384
S1384
402
AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasAf
AS1384
1494
uUfauaGfaGfcAfaGfaAfcAfcUfgUfusUfsUf
0.07
0.16
0.54



 


D1385
S1385
403
AfacaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1385
1495
uuaUfaGfaGfcAfaGfaAfcAfcUfGfUfusUfsu
0.08
0.20
0.75



 


D1386
S1386
404
aacaguguucUfuGfcUcUaudAsa
AS1386
1496
uUfdAUdAGfaGfcAfaGfaadCadCudGdTdTsusu
0.25
0.56
0.90



 


D1387
S1387
405
AfaCfaguGfuUfcUfuGfCfUfcUfaUfasAf
AS1387
1497
uUfaUfaGfagcAfaGfaAfcAfCfUfgUfusUfsu
0.08
0.19
0.70



 


D1388
S1388
406
AfaCfaGfuGfuucUfuGfcUfCfUfaUfasAf
AS1388
1498
uUfaUfagaGfcAfaGfAfAfcAfcUfgUfusUfsu
0.08
0.14
0.60



 


D1389
S1389
407
AfaCfaGfuGfuUfcUfuGfcUfcuaUfAfsAf
AS1389
1499
uuaUfAfGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.08
0.19
0.62



 


D1390
S1390
408
aaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1390
1500
uuaUfaGfaGfcAfaGfaAfcAfcUfgUfUfsUfsu
0.08
0.27
0.76



 


D1391
S1391
409
aacaguguucdTudGcdTcdTadTasa
AS1391
1501
uUfdAUdAGfaGfcAfaGfaadCadCudGudTsusu
0.18
0.36
0.81



 


D1392
S1392
410
AfacaGfuGfuUfcUfuGfCfUfcUfaUfasAf
AS1392
1502
uUfaUfaGfagcAfaGfaAfcAfcUfGfUfusUfsu
0.07
0.17
0.55



 


D1393
S1393
411
AfaCfaGfuguUfcUfuGfcUfCfUfaUfasAf
AS1393
1503
uUfaUfagaGfcAfaGfaAfCfAfcUfgUfusUfsu
0.07
0.15
0.57



 


D1394
S1394
412
AfaCfaGfuGfuUfcuuGfcUfcUfAfUfasAf
AS1394
1504
uUfauaGfaGfcAfAfGfaAfcAfcUfgUfusUfsu
0.26
0.68
1.06



 


D1395
S1395
413
AfaCfaGfuGfuUfcUfuGfcucUfaUfAfsAf
AS1395
1505
uuaUfaGfAfGfcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.18
0.58



 


D1396
S1396
414
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfAfsAf
AS1396
1506
uuaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsUf
0.09
0.27
0.73



 


D1397
S1397
415
AfaCfaAfuGfuUfcUfuGfcdAdCdTdAUfasAf
AS1397
1507
uUfadTdAdGdAGfcAfaGfaGfcAfcAfgUfusUfsu
0.20
0.51
0.73



 


D1398
S1398
416
AfacaGfuguUfcuuGfcucUfauasAf
AS1398
1508
uUfAfUfaGfAfGfcAfAfGfaAfCfAfcUfGfUfusUfsu
0.13
0.34
0.86



 


D1399
S1399
417
dAacadGugudTcuudGcucdTauasdA
AS1399
1509
udTdAdTadGdAdGcdAdAdGadAdCdAcdTdGdTusdTsu
0.24
0.42
0.82



 


D1400
S1400
418
AfaCfaAfuGfuUfcUfuGfdCdAdCdTaUfasAf
AS1400
1510
uUfaUfdAdGdAdGcAfaGfaGfcAfcAfgUfusUfsu
0.49
0.85
0.78



 


D1401
S1401
419
AfaCfaAfuGfuUfcUfudGdCdAdCUfaUfasAf
AS1401
1511
uUfaUfadGdAdGdCAfaGfaGfcAfcAfgUfusUfsu
0.67
0.83
0.85



 


D1402
S1402
420
aaCfAfguGfUfucUfUfgcUfCfuaUfAfsa
AS1402
1512
uUfaUfAfgaGfCfaaGfAfacAfCfugUfUfsusu
0.18
0.47
0.80



 


D1403
S1403
421
AfaCfaAfuGfuUfcUfuGfcdAdCUfadTdAsAf
AS1403
1513
udTdAUfadGdAGfcAfaGfaGfcAfcAfgUfusUfsu
0.73
0.89
0.77



 


D1404
S1404
422
aacAgugUucuUgcuCuauAsa
AS1404
1514
uUaUAgAGCaAGAaCACuGUUsusu
0.12
0.39
0.79



 


D1405
S1405
423
AacaGuguUcuuGcucUauasA
AS1405
1515
uUAUaGAGcAAGaACAcUGUusUsu
0.12
0.37
0.77



 


D1406
S1406
424
AfaCfaAfuGfuUfcUfudGdCAfcUfadTdAsAf
AS1406
1516
udTdAUfaGfadGdCAfaGfaGfcAfcAfgUfusUfsu
0.59
0.93
0.89



 


D1407
S1407
425
aACagUGuuCUugCUcuAUasa
AS1407
1517
UUauAGagCAagAAcaCUguUsUsu
0.09
0.16
0.55



 


D1408
S1408
426
AfaCfaAfuGfuUfcUfuGfcAfcdTdAdTdAsAf
AS1408
1518
udTdAdTdAGfaGfcAfaGfaAfcAfcAfgUfusUfsu
0.22
0.64
0.86



 


D1409
S1409
427
aaCAguGUucUUgcUCuaUAsa
AS1409
1519
uUaUAgaGCaaGAacACugUUsusu
0.13
0.31
0.76



 


D1410
S1410
428
AfaCfaAfuGfuUfcUfuGfcAfdCdTdAdTdAsAf
AS1410
1520
udTdAdTdAdGaGfcAfaGfaGfcAfcAfgUfusUfsu
0.77
0.94
0.93



 


D1411
S1411
429
aacAfgugUfucuUfgcuCfuauAfsa
AS1411
1521
uUfaUfAfgAfGfCfaAfGfAfaCfAfCfuGfUfUfsusu
0.23
0.53
1.04



 


D1412
S1412
430
aacdAgugdTucudTgcudCuaudAsa
AS1412
1522
udTadTdAgdAdGdCadAdGdAadCdAdCudGdTdTsusu
0.30
0.64
0.90



 


D1413
S1413
431
AfaCfaGfuGfuUfcUfuGfcUfcUfaUfasa
AS1413
1523
UfUfaUfaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.09
0.19
0.63



 


D1414
S1414
432
AfaCfaGfuGfUfUfcUfuGfcUfcUfaUfasa
AS1414
1524
UfUfaUfaGfaGfcAfaGfaacAfcUfgUfusUfsu
0.11
0.28
0.66



 


D1415
S1415
433
AfaCfaGfuGfuUfcUfuGfCfUfcUfaUfasa
AS1415
1525
UfUfaUfaGfagcAfaGfaAfcAfcUfgUfusUfsu
0.06
0.13
0.53



 


D1416
S1416
434
aacaguguucuugcucuauasa
AS1416
1526
UfUfAfUfAfGfAfGfCfAfAfGfAfAfCfAfCfUfGfUfUfsusu
0.20
0.53
0.99



 


D1417
S1417
435
AfaCfaGfuGfuUfcUfuGfcUfcUfAfUfasa
AS1417
1527
UfUfauaGfaGfcAfaGfaAfcAfcUfgUfusUfsu
0.07
0.17
0.53



 


D1418
S1418
436
aAfCfagUfGfuuCfUfugCfUfcuAfUfasa
AS1418
1528
UfUfauAfGfagCfAfagAfAfcaCfUfguUfsUfsu
0.08
0.20
0.70



 


D1419
S1419
437
AfaCfAfGfuGfuUfcUfuGfcUfcUfaUfasAf
AS1419
1529
uUfaUfaGfaGfcAfaGfaAfcAfcugUfusUfsUf
0.08
0.20
0.70



 


D1420
S1420
438
GfaCfuUfcUfcCfUfCfcAfgugGfaCfcUfL96
AS1420
1530
aGfgUfcCfAfCfuGfgagGfaGfaAfgUfcsCfsc






 


D1421
S1421
439
GfaCfuUfcUfcCfUfCfcAfgUfGfGfaCfcUfL96
AS1421
1531
aGfgUfccaCfuGfgagGfaGfaAfgUfcsCfsc






 


D1422
S1422
440
AfcUfuCfuCfcUfCfCfaGfuggAfcCfuGfL96
AS1422
1532
cAfgGfuCfCfAfcUfggaGfgAfgAfaGfusCfsc






 


D1423
S1423
441
AfcUfuCfuCfcUfCfCfaGfuGfGfAfcCfuGfL96
AS1423
1533
cAfgGfuccAfcUfggaGfgAfgAfaGfusCfsc






 


D1424
S1424
442
CfuUfcUfcCfuCfCfAfgUfggaCfcUfgAfL96
AS1424
1534
uCfaGfgUfCfCfaCfuggAfgGfaGfaAfgsUfsc






 


D1425
S1425
443
CfuUfcUfcCfuCfCfAfgUfgGfAfCfcUfgAfL96
AS1425
1535
uCfaGfgucCfaCfuggAfgGfaGfaAfgsUfsc






 


D1426
S1426
444
UfuCfuCfcUfcCfAfGfuGfgacCfuGfaAfL96
AS1426
1536
uUfcAfgGfUfCfcAfcugGfaGfgAfgAfasGfsu






 


D1427
S1427
445
UfuCfuCfcUfcCfAfGfuGfgAfCfCfuGfaAfL96
AS1427
1537
uUfcAfgguCfcAfcugGfaGfgAfgAfasGfsu






 


D1428
S1428
446
UfcUfcCfuCfcAfGfUfgGfaccUfgAfaGfL96
AS1428
1538
cUfuCfaGfGfUfcCfacuGfgAfgGfaGfasAfsg






 


D1429
S1429
447
UfcUfcCfuCfcAfGfUfgGfaCfCfUfgAfaGfL96
AS1429
1539
cUfuCfaggUfcCfacuGfgAfgGfaGfasAfsg






 


D1430
S1430
448
CfuCfcUfcCfaGfUfGfgAfccuGfaAfgGfL96
AS1430
1540
cCfuUfcAfGfGfuCfcacUfgGfaGfgAfgsAfsa






 


D1431
S1431
449
CfuCfcUfcCfaGfUfGfgAfcCfUfGfaAfgGfL96
AS1431
1541
cCfuUfcagGfuCfcacUfgGfaGfgAfgsAfsa






 


D1432
S1432
450
UfcCfuCfcAfgUfGfGfaCfcugAfaGfgAfL96
AS1432
1542
uCfcUfuCfAfGfgUfccaCfuGfgAfgGfasGfsa






 


D1433
S1433
451
UfcCfuCfcAfgUfGfGfaCfcUfGfAfaGfgAfL96
AS1433
1543
uCfcUfucaGfgUfccaCfuGfgAfgGfasGfsa






 


D1434
S1434
452
CfcUfcCfaGfuGfGfAfcCfugaAfgGfaCfL96
AS1434
1544
gUfcCfuUfCfAfgGfuccAfcUfgGfaGfgsAfsg






 


D1435
S1435
453
CfcUfcCfaGfuGfGfAfcCfuGfAfAfgGfaCfL96
AS1435
1545
gUfcCfuucAfgGfuccAfcUfgGfaGfgsAfsg






 


D1436
S1436
454
CfuCfcAfgUfgGfAfCfcUfgaaGfgAfcGfL96
AS1436
1546
cGfuCfcUfUfCfaGfgucCfaCfuGfgAfgsGfsa






 


D1437
S1437
455
CfuCfcAfgUfgGfAfCfcUfgAfAfGfgAfcGfL96
AS1437
1547
cGfuCfcuuCfaGfgucCfaCfuGfgAfgsGfsa






 


D1438
S1438
456
UfcCfaGfuGfgAfCfCfuGfaagGfaCfgAfL96
AS1438
1548
uCfgUfcCfUfUfcAfgguCfcAfcUfgGfasGfsg






 


D1439
S1439
457
UfcCfaGfuGfgAfCfCfuGfaAfGfGfaCfgAfL96
AS1439
1549
uCfgUfccuUfcAfgguCfcAfcUfgGfasGfsg






 


D1440
S1440
458
CfcAfgUfgGfaCfCfUfgAfaggAfcGfaGfL96
AS1440
1550
cUfcGfuCfCfUfuCfaggUfcCfaCfuGfgsAfsg






 


D1441
S1441
459
CfcAfgUfgGfaCfCfUfgAfaGfGfAfcGfaGfL96
AS1441
1551
cUfcGfuccUfuCfaggUfcCfaCfuGfgsAfsg






 


D1442
S1442
460
CfaGfuGfgAfcCfUfGfaAfggaCfgAfgGfL96
AS1442
1552
cCfuCfgUfCfCfuUfcagGfuCfcAfcUfgsGfsa






 


D1443
S1443
461
CfaGfuGfgAfcCfUfGfaAfgGfAfCfgAfgGfL96
AS1443
1553
cCfuCfgucCfuUfcagGfuCfcAfcUfgsGfsa






 


D1444
S1444
462
AfgUfgGfaCfcUfGfAfaGfgacGfaGfgGfL96
AS1444
1554
cCfcUfcGfUfCfcUfucaGfgUfcCfaCfusGfsg






 


D1445
S1445
463
AfgUfgGfaCfcUfGfAfaGfgAfCfGfaGfgGfL96
AS1445
1555
cCfcUfcguCfcUfucaGfgUfcCfaCfusGfsg






 


D1446
S1446
464
GfuGfgAfcCfuGfAfAfgGfacgAfgGfgAfL96
AS1446
1556
uCfcCfuCfGfUfcCfuucAfgGfuCfcAfcsUfsg






 


D1447
S1447
465
GfuGfgAfcCfuGfAfAfgGfaCfGfAfgGfgAfL96
AS1447
1557
uCfcCfucgUfcCfuucAfgGfuCfcAfcsUfsg






 


D1448
S1448
466
UfgGfaCfcUfgAfAfGfgAfcgaGfgGfaUfL96
AS1448
1558
aUfcCfcUfCfGfuCfcuuCfaGfgUfcCfasCfsu






 


D1449
S1449
467
UfgGfaCfcUfgAfAfGfgAfcGfAfGfgGfaUfL96
AS1449
1559
aUfcCfcucGfuCfcuuCfaGfgUfcCfasCfsu






 


D1450
S1450
468
GfgAfcCfuGfaAfGfGfaCfgagGfgAfuGfL96
AS1450
1560
cAfuCfcCfUfCfgUfccuUfcAfgGfuCfcsAfsc






 


D1451
S1451
469
GfgAfcCfuGfaAfGfGfaCfgAfGfGfgAfuGfL96
AS1451
1561
cAfuCfccuCfgUfccuUfcAfgGfuCfcsAfsc






 


D1452
S1452
470
GfaCfcUfgAfaGfGfAfcGfaggGfaUfgGfL96
AS1452
1562
cCfaUfcCfCfUfcGfuccUfuCfaGfgUfcsCfsa






 


D1453
S1453
471
GfaCfcUfgAfaGfGfAfcGfaGfGfGfaUfgGfL96
AS1453
1563
cCfaUfcccUfcGfuccUfuCfaGfgUfcsCfsa






 


D1454
S1454
472
AfcCfuGfaAfgGfAfCfgAfgggAfuGfgGfL96
AS1454
1564
cCfcAfuCfCfCfuCfgucCfuUfcAfgGfusCfsc






 


D1455
S1455
473
AfcCfuGfaAfgGfAfCfgAfgGfGfAfuGfgGfL96
AS1455
1565
cCfcAfuccCfuCfgucCfuUfcAfgGfusCfsc






 


D1456
S1456
474
CfcUfgAfaGfgAfCfGfaGfggaUfgGfgAfL96
AS1456
1566
uCfcCfaUfCfCfcUfcguCfcUfuCfaGfgsUfsc






 


D1457
S1457
475
CfcUfgAfaGfgAfCfGfaGfgGfAfUfgGfgAfL96
AS1457
1567
uCfcCfaucCfcUfcguCfcUfuCfaGfgsUfsc






 


D1458
S1458
476
CfuGfaAfgGfaCfGfAfgGfgauGfgGfaUfL96
AS1458
1568
aUfcCfcAfUfCfcCfucgUfcCfuUfcAfgsGfsu






 


D1459
S1459
477
CfuGfaAfgGfaCfGfAfgGfgAfUfGfgGfaUfL96
AS1459
1569
aUfcCfcauCfcCfucgUfcCfuUfcAfgsGfsu






 


D1460
S1460
478
UfgAfaGfgAfcGfAfGfgGfaugGfgAfuUfL96
AS1460
1570
aAfuCfcCfAfUfcCfcucGfuCfcUfuCfasGfsg






 


D1461
S1461
479
UfgAfaGfgAfcGfAfGfgGfaUfGfGfgAfuUfL96
AS1461
1571
aAfuCfccaUfcCfcucGfuCfcUfuCfasGfsg






 


D1462
S1462
480
GfaAfgGfaCfgAfGfGfgAfuggGfaUfuUfL96
AS1462
1572
aAfaUfcCfCfAfuCfccuCfgUfcCfuUfcsAfsg






 


D1463
S1463
481
GfaAfgGfaCfgAfGfGfgAfuGfGfGfaUfuUfL96
AS1463
1573
aAfaUfcccAfuCfccuCfgUfcCfuUfcsAfsg






 


D1464
S1464
482
AfaGfgAfcGfaGfGfGfaUfgggAfuUfuCfL96
AS1464
1574
gAfaAfuCfCfCfaUfcccUfcGfuCfcUfusCfsa






 


D1465
S1465
483
AfaGfgAfcGfaGfGfGfaUfgGfGfAfuUfuCfL96
AS1465
1575
gAfaAfuccCfaUfcccUfcGfuCfcUfusCfsa






 


D1466
S1466
484
AfgGfaCfgAfgGfGfAfuGfggaUfuUfcAfL96
AS1466
1576
uGfaAfaUfCfCfcAfuccCfuCfgUfcCfus Ufsc






 


D1467
S1467
485
AfgGfaCfgAfgGfGfAfuGfgGfAfUfuUfcAfL96
AS1467
1577
uGfaAfaucCfcAfuccCfuCfgUfcCfusUfsc






 


D1468
S1468
486
GfgAfcGfaGfgGfAfUfgGfgauUfuCfaUfL96
AS1468
1578
aUfgAfaAfUfCfcCfaucCfcUfcGfuCfcsUfsu






 


D1469
S1469
487
GfgAfcGfaGfgGfAfUfgGfgAfUfUfuCfaUfL96
AS1469
1579
aUfgAfaauCfcCfaucCfcUfcGfuCfcsUfsu






 


D1470
S1470
488
GfaCfgAfgGfgAfUfGfgGfauuUfcAfuGfL96
AS1470
1580
cAfuGfaAfAfUfcCfcauCfcCfuCfgUfcsCfsu






 


D1471
S1471
489
GfaCfgAfgGfgAfUfGfgGfaUfUfUfcAfuGfL96
AS1471
1581
cAfuGfaaaUfcCfcauCfcCfuCfgUfcsCfsu






 


D1472
S1472
490
AfcGfaGfgGfaUfGfGfgAfuuuCfaUfgUfL96
AS1472
1582
aCfaUfgAfAfAfuCfccaUfcCfcUfcGfusCfsc






 


D1473
S1473
491
AfcGfaGfgGfaUfGfGfgAfuUfUfCfaUfgUfL96
AS1473
1583
aCfaUfgaaAfuCfccaUfcCfcUfcGfusCfsc






 


D1474
S1474
492
CfgAfgGfgAfuGfGfGfaUfuucAfuGfuAfL96
AS1474
1584
uAfcAfuGfAfAfaUfcccAfuCfcCfuCfgsUfsc






 


D1475
S1475
493
CfgAfgGfgAfuGfGfGfaUfuUfCfAfuGfuAfL96
AS1475
1585
uAfcAfugaAfaUfcccAfuCfcCfuCfgsUfsc






 


D1476
S1476
494
GfaGfgGfaUfgGfGfAfuUfucaUfgUfaAfL96
AS1476
1586
uUfaCfaUfGfAfaAfuccCfaUfcCfcUfcsGfsu






 


D1477
S1477
495
GfaGfgGfaUfgGfGfAfuUfuCfAfUfgUfaAfL96
AS1477
1587
uUfaCfaugAfaAfuccCfaUfcCfcUfcsGfsu






 


D1478
S1478
496
AfgGfgAfuGfgGfAfUfuUfcauGfuAfaCfL96
AS1478
1588
gUfuAfcAfUfGfaAfaucCfcAfuCfcCfusCfsg






 


D1479
S1479
497
AfgGfgAfuGfgGfAfUfuUfcAfUfGfuAfaCfL96
AS1479
1589
gUfuAfcauGfaAfaucCfcAfuCfcCfusCfsg






 


D1480
S1480
498
GfgGfaUfgGfgAfUfUfuCfaugUfaAfcCfL96
AS1480
1590
gGfuUfaCfAfUfgAfaauCfcCfaUfcCfcsUfsc






 


D1481
S1481
499
GfgGfaUfgGfgAfUfUfuCfaUfGfUfaAfcCfL96
AS1481
1591
gGfuUfacaUfgAfaauCfcCfaUfcCfcsUfsc






 


D1482
S1482
500
GfgAfuGfgGfaUfUfUfcAfuguAfaCfcAfL96
AS1482
1592
uGfgUfuAfCfAfuGfaaaUfcCfcAfuCfcsCfsu






 


D1483
S1483
501
GfgAfuGfgGfaUfUfUfcAfuGfUfAfaCfcAfL96
AS1483
1593
uGfgUfuacAfuGfaaaUfcCfcAfuCfcsCfsu






 


D1484
S1484
502
GfaUfgGfgAfuUfUfCfaUfguaAfcCfaAfL96
AS1484
1594
uUfgGfuUfAfCfaUfgaaAfuCfcCfaUfcsCfsc






 


D1485
S1485
503
GfaUfgGfgAfuUfUfCfaUfgUfAfAfcCfaAfL96
AS1485
1595
uUfgGfuuaCfaUfgaaAfuCfcCfaUfcsCfsc






 


D1486
S1486
504
AfuGfgGfaUfuUfCfAfuGfuaaCfcAfaGfL96
AS1486
1596
cUfuGfgUfUfAfcAfugaAfaUfcCfcAfusCfsc






 


D1487
S1487
505
AfuGfgGfaUfuUfCfAfuGfuAfAfCfcAfaGfL96
AS1487
1597
cUfuGfguuAfcAfugaAfaUfcCfcAfusCfsc






 


D1488
S1488
506
UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96
AS1488
1598
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D1592
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D1594
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612
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1704
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D1595
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613
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1705
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D1596
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614
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1706
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D1597
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615
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1707
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D1598
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616
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1708
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D1599
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617
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1709
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D1600
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618
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1710
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D1601
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619
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1711
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D1602
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620
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1712
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D1603
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621
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1713
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D1604
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622
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1714
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D1605
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623
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1715
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D1606
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624
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1716
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D1607
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625
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1717
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D1608
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626
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1718
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D1609
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627
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1710
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D1610
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628
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1720
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D1611
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629
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1721
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D1612
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630
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1722
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D1613
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631
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1723
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D1614
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632
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1724
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D1615
S1615
633
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1725
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D1616
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634
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1726
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D1617
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635
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1727
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D1618
S1618
636
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1728
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D1619
S1619
637
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1729
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D1620
S1620
638
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1730
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D1621
S1621
639
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1731
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D1622
S1622
640
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1732
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D1623
S1623
641
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1733
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D1624
S1624
642
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AS1624
1734
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D1625
S1625
643
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1735
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D1626
S1626
644
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1736
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D1627
S1627
645
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1737
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D1628
S1628
646
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1738
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D1629
S1629
647
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1739
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D1630
S1630
648
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AS1630
1740
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D1631
S1631
649
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1741
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D1632
S1632
650
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1742
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D1633
S1633
651
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1743
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D1634
S1634
652
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1744
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D1635
S1635
653
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1745
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D1636
S1636
654
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1746
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D1637
S1637
655
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1747
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D1638
S1638
656
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1748
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D1639
S1639
657
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1749
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D1640
S1640
658
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1750
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D1641
S1641
659
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1751
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D1642
S1642
660
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1752
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D1643
S1643
661
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1753
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D1644
S1644
662
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1754
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D1645
S1645
663
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1755
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D1646
S1646
664
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1756
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D1647
S1647
665
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1757
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D1648
S1648
666
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1758
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D1649
S1649
667
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1759
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D1650
S1650
668
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1760
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D1651
S1651
669
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1761
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D1652
S1652
670
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1762
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D1653
S1653
671
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1763
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D1654
S1654
672
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1764
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D1655
S1655
673
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1765
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D1656
S1656
674
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1766
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D1657
S1657
675
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1767
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D1658
S1658
676
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1768
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D1659
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677
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1769
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D1660
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678
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1770
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D1661
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679
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1771
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D1662
S1662
680
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1772
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D1663
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681
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1773
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D1664
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682
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1774
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D1665
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683
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1775
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D1666
S1666
684
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1776
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D1667
S1667
685
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1777
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D1668
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686
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1778
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D1669
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687
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1779
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D1670
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688
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1780
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D1671
S1671
689
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1781
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D1672
S1672
690
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1782
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D1673
S1673
691
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1783
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D1674
S1674
692
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1784
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D1675
S1675
693
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1785
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D1676
S1676
694
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1786
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D1677
S1677
695
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AS1677
1787
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D1678
S1678
696
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AS1678
1788
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D1679
S1679
697
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AS1679
1789
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D1680
S1680
698
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AS1680
1790
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D1681
S1681
699
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AS1681
1791
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D1682
S1682
700
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AS1682
1792
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D1683
S1683
701
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AS1683
1793
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D1684
S1684
702
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AS1684
1794
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D1685
S1685
703
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AS1685
1795
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D1686
S1686
704
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AS1686
1796
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D1687
S1687
705
UfgUfgAfaAfgGfCfAfcUfuUfUfCfaUfuCfL96
AS1687
1797
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D1688
S1688
706
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AS1688
1798
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D1689
S1689
707
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AS1689
1799
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D1690
S1690
708
UfgAfaAfgGfcAfCfUfuUfucaUfuCfcAfL96
AS1690
1800
uGfgAfaUfGfAfaAfaguGfcCfuUfuCfasCfsa






 


D1691
S1691
709
UfgAfaAfgGfcAfCfUfuUfuCfAfUfuCfcAfL96
AS1691
1801
uGfgAfaugAfaAfaguGfcCfuUfuCfasCfsa






 


D1692
S1692
710
GfaAfaGfgCfaCfUfUfuUfcauUfcCfaCfL96
AS1692
1802
gUfgGfaAfUfGfaAfaagUfgCfcUfuUfcsAfsc






 


D1693
S1693
711
GfaAfaGfgCfaCfUfUfuUfcAfUfUfcCfaCfL96
AS1693
1803
gUfgGfaauGfaAfaagUfgCfcUfuUfcsAfsc






 


D1694
S1694
712
AfaAfgGfcAfcUfUfUfuCfauuCfcAfcUfL96
AS1694
1804
aGfuGfgAfAfUfgAfaaaGfuGfcCfuUfusCfsa






 


D1695
S1695
713
AfaAfgGfcAfcUfUfUfuCfaUfUfCfcAfcUfL96
AS1695
1805
aGfuGfgaaUfgAfaaaGfuGfcCfuUfusCfsa






 


D1696
S1696
714
AfaGfgCfaCfuUfUfUfcAfuucCfaCfuUfL96
AS1696
1806
aAfgUfgGfAfAfuGfaaaAfgUfgCfcUfusUfsc






 


D1697
S1697
715
AfaGfgCfaCfuUfUfUfcAfuUfCfCfaCfuUfL96
AS1697
1807
aAfgUfggaAfuGfaaaAfgUfgCfcUfusUfsc






 


D1698
S1698
716
AfgGfcAfcUfuUfUfCfaUfuccAfcUfuUfL96
AS1698
1808
aAfaGfuGfGfAfaUfgaaAfaGfuGfcCfusUfsu






 


D1699
S1699
717
AfgGfcAfcUfuUfUfCfaUfuCfCfAfcUfuUfL96
AS1699
1809
aAfaGfuggAfaUfgaaAfaGfuGfcCfusUfsu






 


D1700
S1700
718
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1810
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D1701
S1701
719
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1811
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D1702
S1702
720
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AS1702
1812
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D1703
S1703
721
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1813
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D1704
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722
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1814
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D1705
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723
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1815
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D1706
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724
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1816
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D1707
S1707
725
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1817
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D1708
S1708
726
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1818
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D1709
S1709
727
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1819
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D1710
S1710
728
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1820
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D1711
S1711
729
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1821
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D1712
S1712
730
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1822
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D1713
S1713
731
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1823
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D1714
S1714
732
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1824
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D1715
S1715
733
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1825
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D1716
S1716
734
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1826
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D1717
S1717
735
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1827
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D1718
S1718
736
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1828
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D1719
S1719
737
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1829
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D1720
S1720
738
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1830
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D1721
S1721
739
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1831
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D1722
S1722
740
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1832
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D1723
S1723
741
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1833
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D1724
S1724
742
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1834
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D1725
S1725
743
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1835
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D1726
S1726
744
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1836
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D1727
S1727
745
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1837
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D1728
S1728
746
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1838
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D1729
S1729
747
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1839
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D1730
S1730
748
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1840
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D1731
S1731
749
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1841
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D1732
S1732
750
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AS1732
1842
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D1733
S1733
751
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1843
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D1734
S1734
752
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1844
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D1735
S1735
753
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1845
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D1736
S1736
754
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1846
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D1737
S1737
755
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1847
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D1738
S1738
756
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1848
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D1739
S1739
757
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1849
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D1740
S1740
758
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1850
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D1741
S1741
759
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1851
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D1742
S1742
760
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1852
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D1743
S1743
761
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1853
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D1744
S1744
762
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1854
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D1745
S1745
763
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1855
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D1746
S1746
764
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1856
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D1747
S1747
765
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1857
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D1748
S1748
766
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1858
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D1749
S1749
767
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1859
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D1750
S1750
768
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1860
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D1751
S1751
769
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1861
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D1752
S1752
770
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1862
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D1753
S1753
771
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1863
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D1754
S1754
772
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1864
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D1755
S1755
773
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1865
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D1756
S1756
774
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1866
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D1757
S1757
775
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1867
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D1758
S1758
776
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1868
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D1759
S1759
777
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1869
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D1760
S1760
778
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1870
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D1761
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779
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1871
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D1762
S1762
780
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1872
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D1763
S1763
781
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1873
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D1764
S1764
782
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1874
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D1765
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783
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1875
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D1766
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784
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1876
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D1767
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785
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1877
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D1768
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786
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1878
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D1769
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787
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1879
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D1770
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788
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1880
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D1771
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1881
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D1772
S1772
790
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1882
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D1773
S1773
791
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1883
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D1774
S1774
792
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1884
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D1775
S1775
793
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1885
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D1776
S1776
794
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1886
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D1777
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795
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1887
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D1778
S1778
796
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1888
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D1779
S1779
797
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1889
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D1780
S1780
798
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1890
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D1781
S1781
799
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1891
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D1782
S1782
800
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1892
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D1783
S1783
801
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1893
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D1784
S1784
802
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1894
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D1785
S1785
803
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1895
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D1786
S1786
804
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1896
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D1787
S1787
805
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1897
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D1788
S1788
806
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1898
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D1789
S1789
807
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1899
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D1790
S1790
808
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AS1790
1900
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D1791
S1791
809
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AS1791
1901
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D1792
S1792
810
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1902
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D1793
S1793
811
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AS1793
1903
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D1794
S1794
812
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AS1794
1904
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D1795
S1795
813
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AS1795
1905
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D1796
S1796
814
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AS1796
1906
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D1797
S1797
815
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AS1797
1907
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D1798
S1798
816
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AS1798
1908
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D1799
S1799
817
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AS1799
1909
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D1800
S1800
818
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AS1800
1910
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D1801
S1801
819
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AS1801
1911
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D1802
S1802
820
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AS1802
1912
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D1803
S1803
821
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AS1803
1913
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D1804
S1804
822
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AS1804
1914
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D1805
S1805
823
UfcCfaAfaAfaAfAfAfgAfgAfAfUfcAfaAfL96
AS1805
1915
uUfuGfauuCfuCfuuuUfuUfuUfgGfasAfsg






 


D1806
S1806
824
CfcAfaAfaAfaAfAfGfaGfaauCfaAfaAfL96
AS1806
1916
uUfuUfgAfUfUfcUfcuuUfuUfuUfuGfgsAfsa






 


D1807
S1807
825
CfcAfaAfaAfaAfAfGfaGfaAfUfCfaAfaAfL96
AS1807
1917
uUfuUfgauUfcUfcuuUfuUfuUfuGfgsAfsa






 


D1808
S1808
826
CfaAfaAfaAfaAfGfAfgAfaucAfaAfaUfL96
AS1808
1918
aUfuUfuGfAfUfuCfucuUfuUfuUfuUfgsGfsa






 


D1809
S1809
827
CfaAfaAfaAfaAfGfAfgAfaUfCfAfaAfaUfL96
AS1809
1919
aUfuUfugaUfuCfucuUfuUfuUfuUfgsGfsa






 


D1810
S1810
828
AfaAfaAfaAfaGfAfGfaAfucaAfaAfuUfL96
AS1810
1920
aAfuUfuUfGfAfuUfcucUfuUfuUfuUfusGfsg






 


D1811
S1811
829
AfaAfaAfaAfaGfAfGfaAfuCfAfAfaAfuUfL96
AS1811
1921
aAfuUfuugAfuUfcucUfuUfuUfuUfusGfsg






 


D1812
S1812
830
AfaAfaAfaAfgAfGfAfaUfcaaAfaUfuUfL96
AS1812
1922
aAfaUfuUfUfGfaUfucuCfuUfuUfuUfusUfsg






 


D1813
S1813
831
AfaAfaAfaAfgAfGfAfaUfcAfAfAfaUfuUfL96
AS1813
1923
aAfaUfuuuGfaUfucuCfuUfuUfuUfusUfsg






 


D1814
S1814
832
AfaAfaAfaGfaGfAfAfuCfaaaAfuUfuUfL96
AS1814
1924
aAfaAfuUfUfUfgAfuucUfcUfuUfuUfusUfsu






 


D1815
S1815
833
AfaAfaAfaGfaGfAfAfuCfaAfAfAfuUfuUfL96
AS1815
1925
aAfaAfuuuUfgAfuucUfcUfuUfuUfusUfsu






 


D1816
S1816
834
AfaAfaAfgAfgAfAfUfcAfaaaUfuUfuAfL96
AS1816
1926
uAfaAfaUfUfUfuGfauuCfuCfuUfuUfusUfsu






 


D1817
S1817
835
AfaAfaAfgAfgAfAfUfcAfaAfAfUfuUfuAfL96
AS1817
1927
uAfaAfauuUfuGfauuCfuCfuUfuUfusUfsu






 


D1818
S1818
836
AfaAfaGfaGfaAfUfCfaAfaauUfuUfaCfL96
AS1818
1928
gUfaAfaAfUfUfuUfgauUfcUfcUfuUfusUfsu






 


D1819
S1819
837
AfaAfaGfaGfaAfUfCfaAfaAfUfUfuUfaCfL96
AS1819
1929
gUfaAfaauUfuUfgauUfcUfcUfuUfusUfsu






 


D1820
S1820
838
AfaAfgAfgAfaUfCfAfaAfauuUfuAfcAfL96
AS1820
1930
uGfuAfaAfAfUfuUfugaUfuCfuCfuUfusUfsu






 


D1821
S1821
839
AfaAfgAfgAfaUfCfAfaAfaUfUfUfuAfcAfL96
AS1821
1931
uGfuAfaaaUfuUfugaUfuCfuCfuUfusUfsu






 


D1822
S1822
840
AfaGfaGfaAfuCfAfAfaAfuuuUfaCfaAfL96
AS1822
1932
uUfgUfaAfAfAfuUfuugAfuUfcUfcUfusUfsu






 


D1823
S1823
841
AfaGfaGfaAfuCfAfAfaAfuUfUfUfaCfaAfL96
AS1823
1933
uUfgUfaaaAfuUfuugAfuUfcUfcUfusUfsu






 


D1824
S1824
842
AfgAfgAfaUfcAfAfAfaUfuuuAfcAfaAfL96
AS1824
1934
uUfuGfuAfAfAfaUfuuuGfaUfuCfuCfusUfsu






 


D1825
S1825
843
AfgAfgAfaUfcAfAfAfaUfuUfUfAfcAfaAfL96
AS1825
1935
uUfuGfuaaAfaUfuuuGfaUfuCfuCfusUfsu






 


D1826
S1826
844
GfaGfaAfuCfaAfAfAfuUfuuaCfaAfaGfL96
AS1826
1936
cUfuUfgUfAfAfaAfuuuUfgAfuUfcUfcsUfsu






 


D1827
S1827
845
GfaGfaAfuCfaAfAfAfuUfuUfAfCfaAfaGfL96
AS1827
1937
cUfuUfguaAfaAfuuuUfgAfuUfcUfcsUfsu






 


D1828
S1828
846
AfgAfaUfcAfaAfAfUfuUfuacAfaAfgAfL96
AS1828
1938
uCfuUfuGfUfAfaAfauuUfuGfaUfuCfusCfsu






 


D1829
S1829
847
AfgAfaUfcAfaAfAfUfuUfuAfCfAfaAfgAfL96
AS1829
1939
uCfuUfuguAfaAfauuUfuGfaUfuCfusCfsu






 


D1830
S1830
848
GfaAfuCfaAfaAfUfUfuUfacaAfaGfaAfL96
AS1830
1940
uUfcUfuUfGfUfaAfaauUfuUfgAfuUfcsUfsc






 


D1831
S1831
849
GfaAfuCfaAfaAfUfUfuUfaCfAfAfaGfaAfL96
AS1831
1941
uUfcUfuugUfaAfaauUfuUfgAfuUfcsUfsc






 


D1832
S1832
850
AfaUfcAfaAfaUfUfUfuAfcaaAfgAfaUfL96
AS1832
1942
aUfuCfuUfUfGfuAfaaaUfuUfuGfaUfusCfsu






 


D1833
S1833
851
AfaUfcAfaAfaUfUfUfuAfcAfAfAfgAfaUfL96
AS1833
1943
aUfuCfuuuGfuAfaaaUfuUfuGfaUfusCfsu






 


D1834
S1834
852
AfuCfaAfaAfuUfUfUfaCfaaaGfaAfuCfL96
AS1834
1944
gAfuUfcUfUfUfgUfaaaAfuUfuUfgAfusUfsc






 


D1835
S1835
853
AfuCfaAfaAfuUfUfUfaCfaAfAfGfaAfuCfL96
AS1835
1945
gAfuUfcuuUfgUfaaaAfuUfuUfgAfusUfsc






 


D1836
S1836
854
UfcAfaAfaUfuUfUfAfcAfaagAfaUfcAfL96
AS1836
1946
uGfaUfuCfUfUfuGfuaaAfaUfuUfuGfasUfsu






 


D1837
S1837
855
UfcAfaAfaUfuUfUfAfcAfaAfGfAfaUfcAfL96
AS1837
1947
uGfaUfucuUfuGfuaaAfaUfuUfuGfasUfsu






 


D1838
S1838
856
CfaAfaAfuUfuUfAfCfaAfagaAfuCfaAfL96
AS1838
1948
uUfgAfuUfCfUfuUfguaAfaAfuUfuUfgsAfsu






 


D1839
S1839
857
CfaAfaAfuUfuUfAfCfaAfaGfAfAfuCfaAfL96
AS1839
1949
uUfgAfuucUfuUfguaAfaAfuUfuUfgsAfsu






 


D1840
S1840
858
AfaAfaUfuUfuAfCfAfaAfgaaUfcAfaAfL96
AS1840
1950
uUfuGfaUfUfCfuUfuguAfaAfaUfuUfusGfsa






 


D1841
S1841
859
AfaAfaUfuUfuAfCfAfaAfgAfAfUfcAfaAfL96
AS1841
1951
uUfuGfauuCfuUfuguAfaAfaUfuUfusGfsa






 


D1842
S1842
860
AfaAfuUfuUfaCfAfAfaGfaauCfaAfaGfL96
AS1842
1952
cUfuUfgAfUfUfcUfuugUfaAfaAfuUfusUfsg






 


D1843
S1843
861
AfaAfuUfuUfaCfAfAfaGfaAfUfCfaAfaGfL96
AS1843
1953
cUfuUfgauUfcUfuugUfaAfaAfuUfusUfsg






 


D1844
S1844
862
AfaUfuUfuAfcAfAfAfgAfaucAfaAfgGfL96
AS1844
1954
cCfuUfuGfAfUfuCfuuuGfuAfaAfaUfusUfsu






 


D1845
S1845
863
AfaUfuUfuAfcAfAfAfgAfaUfCfAfaAfgGfL96
AS1845
1955
cCfuUfugaUfuCfuuuGfuAfaAfaUfusUfsu






 


D1846
S1846
864
AfuUfuUfaCfaAfAfGfaAfucaAfaGfgAfL96
AS1846
1956
uCfcUfuUfGfAfuUfcuuUfgUfaAfaAfusUfsu






 


D1847
S1847
865
AfuUfuUfaCfaAfAfGfaAfuCfAfAfaGfgAfL96
AS1847
1957
uCfcUfuugAfuUfcuuUfgUfaAfaAfusUfsu






 


D1848
S1848
866
UfuUfuAfcAfaAfGfAfaUfcaaAfgGfaAfL96
AS1848
1958
uUfcCfuUfUfGfaUfucuUfuGfuAfaAfasUfsu






 


D1849
S1849
867
UfuUfuAfcAfaAfGfAfaUfcAfAfAfgGfaAfL96
AS1849
1959
uUfcCfuuuGfaUfucuUfuGfuAfaAfasUfsu






 


D1850
S1850
868
UfuUfaCfaAfaGfAfAfuCfaaaGfgAfaUfL96
AS1850
1960
aUfuCfcUfUfUfgAfuucUfuUfgUfaAfasAfsu






 


D1851
S1851
869
UfuUfaCfaAfaGfAfAfuCfaAfAfGfgAfaUfL96
AS1851
1961
aUfuCfcuuUfgAfuucUfuUfgUfaAfasAfsu






 


D1852
S1852
870
UfuAfcAfaAfgAfAfUfcAfaagGfaAfuUfL96
AS1852
1962
aAfuUfcCfUfUfuGfauuCfuUfuGfuAfasAfsa






 


D1853
S1853
871
UfuAfcAfaAfgAfAfUfcAfaAfGfGfaAfuUfL96
AS1853
1963
aAfuUfccuUfuGfauuCfuUfuGfuAfasAfsa






 


D1854
S1854
872
UfaCfaAfaGfaAfUfCfaAfaggAfaUfuCfL96
AS1854
1964
gAfaUfuCfCfUfuUfgauUfcUfuUfgUfasAfsa






 


D1855
S1855
873
UfaCfaAfaGfaAfUfCfaAfaGfGfAfaUfuCfL96
AS1855
1965
gAfaUfuccUfuUfgauUfcUfuUfgUfasAfsa






 


D1856
S1856
874
AfcAfaAfgAfaUfCfAfaAfggaAfuUfcUfL96
AS1856
1966
aGfaAfuUfCfCfuUfugaUfuCfuUfuGfusAfsa






 


D1857
S1857
875
AfcAfaAfgAfaUfCfAfaAfgGfAfAfuUfcUfL96
AS1857
1967
aGfaAfuucCfuUfugaUfuCfuUfuGfusAfsa






 


D1858
S1858
876
CfaAfaGfaAfuCfAfAfaGfgaaUfuCfuAfL96
AS1858
1968
uAfgAfaUfUfCfcUfuugAfuUfcUfuUfgsUfsa






 


D1859
S1859
877
CfaAfaGfaAfuCfAfAfaGfgAfAfUfuCfuAfL96
AS1859
1969
uAfgAfauuCfcUfuugAfuUfcUfuUfgsUfsa






 


D1860
S1860
878
AfaAfgAfaUfcAfAfAfgGfaauUfcUfaGfL96
AS1860
1970
cUfaGfaAfUfUfcCfuuuGfaUfuCfuUfusGfsu






 


D1861
S1861
879
AfaAfgAfaUfcAfAfAfgGfaAfUfUfcUfaGfL96
AS1861
1971
cUfaGfaauUfcCfuuuGfaUfuCfuUfusGfsu






 


D1862
S1862
880
AfaGfaAfuCfaAfAfGfgAfauuCfuAfgAfL96
AS1862
1972
uCfuAfgAfAfUfuCfcuuUfgAfuUfcUfusUfsg






 


D1863
S1863
881
AfaGfaAfuCfaAfAfGfgAfaUfUfCfuAfgAfL96
AS1863
1973
uCfuAfgaaUfuCfcuuUfgAfuUfcUfusUfsg






 


D1864
S1864
882
AfgAfaUfcAfaAfGfGfaAfuucUfaGfaAfL96
AS1864
1974
uUfcUfaGfAfAfuUfccuUfuGfaUfuCfusUfsu






 


D1865
S1865
883
AfgAfaUfcAfaAfGfGfaAfuUfCfUfaGfaAfL96
AS1865
1975
uUfcUfagaAfuUfccuUfuGfaUfuCfusUfsu






 


D1866
S1866
884
GfaAfuCfaAfaGfGfAfaUfucuAfgAfaAfL96
AS1866
1976
uUfuCfuAfGfAfaUfuccUfuUfgAfuUfcsUfsu






 


D1867
S1867
885
GfaAfuCfaAfaGfGfAfaUfuCfUfAfgAfaAfL96
AS1867
1977
uUfuCfuagAfaUfuccUfuUfgAfuUfcsUfsu






 


D1868
S1868
886
AfaUfcAfaAfgGfAfAfuUfcuaGfaAfaGfL96
AS1868
1978
cUfuUfcUfAfGfaAfuucCfuUfuGfaUfusCfsu






 


D1869
S1869
887
AfaUfcAfaAfgGfAfAfuUfcUfAfGfaAfaGfL96
AS1869
1979
cUfuUfcuaGfaAfuucCfuUfuGfaUfusCfsu






 


D1870
S1870
888
AfuCfaAfaGfgAfAfUfuCfuagAfaAfgUfL96
AS1870
1980
aCfuUfuCfUfAfgAfauuCfcUfuUfgAfusUfsc






 


D1871
S1871
889
AfuCfaAfaGfgAfAfUfuCfuAfGfAfaAfgUfL96
AS1871
1981
aCfuUfucuAfgAfauuCfcUfuUfgAfusUfsc






 


D1872
S1872
890
UfcAfaAfgGfaAfUfUfcUfagaAfaGfuAfL96
AS1872
1982
uAfcUfuUfCfUfaGfaauUfcCfuUfuGfasUfsu






 


D1873
S1873
891
UfcAfaAfgGfaAfUfUfcUfaGfAfAfaGfuAfL96
AS1873
1983
uAfcUfuucUfaGfaauUfcCfuUfuGfasUfsu






 


D1874
S1874
892
CfaAfaGfgAfaUfUfCfuAfgaaAfgUfaUfL96
AS1874
1984
aUfaCfuUfUfCfuAfgaaUfuCfcUfuUfgsAfsu






 


D1875
S1875
893
CfaAfaGfgAfaUfUfCfuAfgAfAfAfgUfaUfL96
AS1875
1985
aUfaCfuuuCfuAfgaaUfuCfcUfuUfgsAfsu






 


D1876
S1876
894
AfaAfgGfaAfuUfCfUfaGfaaaGfuAfuCfL96
AS1876
1986
gAfuAfcUfUfUfcUfagaAfuUfcCfuUfusGfsa






 


D1877
S1877
895
AfaAfgGfaAfuUfCfUfaGfaAfAfGfuAfuCfL96
AS1877
1987
gAfuAfcuuUfcUfagaAfuUfcCfuUfusGfsa






 


D1878
S1878
896
AfaGfgAfaUfuCfUfAfgAfaagUfaUfcUfL96
AS1878
1988
aGfaUfaCfUfUfuCfuagAfaUfuCfcUfusUfsg






 


D1879
S1879
897
AfaGfgAfaUfuCfUfAfgAfaAfGfUfaUfcUfL96
AS1879
1989
aGfaUfacuUfuCfuagAfaUfuCfcUfusUfsg






 


D1880
S1880
898
AfgGfaAfuUfcUfAfGfaAfaguAfuCfuGfL96
AS1880
1990
cAfgAfuAfCfUfuUfcuaGfaAfuUfcCfusUfsu






 


D1881
S1881
899
AfgGfaAfuUfcUfAfGfaAfaGfUfAfuCfuGfL96
AS1881
1991
cAfgAfuacUfuUfcuaGfaAfuUfcCfusUfsu






 


D1882
S1882
900
GfgAfaUfuCfuAfGfAfaAfguaUfcUfgGfL96
AS1882
1992
cCfaGfaUfAfCfuUfucuAfgAfaUfuCfcsUfsu






 


D1883
S1883
901
GfgAfaUfuCfuAfGfAfaAfgUfAfUfcUfgGfL96
AS1883
1993
cCfaGfauaCfuUfucuAfgAfaUfuCfcsUfsu






 


D1884
S1884
902
GfaAfuUfcUfaGfAfAfaGfuauCfuGfgGfL96
AS1884
1994
cCfcAfgAfUfAfcUfuucUfaGfaAfuUfcsCfsu






 


D1885
S1885
903
GfaAfuUfcUfaGfAfAfaGfuAfUfCfuGfgGfL96
AS1885
1995
cCfcAfgauAfcUfuucUfaGfaAfuUfcsCfsu






 


D1886
S1886
904
AfaUfuCfuAfgAfAfAfgUfaucUfgGfgCfL96
AS1886
1996
gCfcCfaGfAfUfaCfuuuCfuAfgAfaUfusCfsc






 


D1887
S1887
905
AfaUfuCfuAfgAfAfAfgUfaUfCfUfgGfgCfL96
AS1887
1997
gCfcCfagaUfaCfuuuCfuAfgAfaUfusCfsc






 


D1888
S1888
906
AfuUfcUfaGfaAfAfGfuAfucuGfgGfcAfL96
AS1888
1998
uGfcCfcAfGfAfuAfcuuUfcUfaGfaAfusUfsc






 


D1889
S1889
907
AfuUfcUfaGfaAfAfGfuAfuCfUfGfgGfcAfL96
AS1889
1999
uGfcCfcagAfuAfcuuUfcUfaGfaAfusUfsc






 


D1890
S1890
908
UfuCfuAfgAfaAfGfUfaUfcugGfgCfaGfL96
AS1890
2000
cUfgCfcCfAfGfaUfacuUfuCfuAfgAfasUfsu






 


D1891
S1891
909
UfuCfuAfgAfaAfGfUfaUfcUfGfGfgCfaGfL96
AS1891
2001
cUfgCfccaGfaUfacuUfuCfuAfgAfasUfsu






 


D1892
S1892
910
UfcUfaGfaAfaGfUfAfuCfuggGfcAfgAfL96
AS1892
2002
uCfuGfcCfCfAfgAfuacUfuUfcUfaGfasAfsu






 


D1893
S1893
911
UfcUfaGfaAfaGfUfAfuCfuGfGfGfcAfgAfL96
AS1893
2003
uCfuGfcccAfgAfuacUfuUfcUfaGfasAfsu






 


D1894
S1894
912
CfuAfgAfaAfgUfAfUfcUfgggCfaGfaAfL96
AS1894
2004
uUfcUfgCfCfCfaGfauaCfuUfuCfuAfgsAfsa






 


D1895
S1895
913
CfuAfgAfaAfgUfAfUfcUfgGfGfCfaGfaAfL96
AS1895
2005
uUfcUfgccCfaGfauaCfuUfuCfuAfgsAfsa






 


D1896
S1896
914
UfaGfaAfaGfuAfUfCfuGfggcAfgAfaCfL96
AS1896
2006
gUfuCfuGfCfCfcAfgauAfcUfuUfcUfasGfsa






 


D1897
S1897
915
UfaGfaAfaGfuAfUfCfuGfgGfCfAfgAfaCfL96
AS1897
2007
gUfuCfugcCfcAfgauAfcUfuUfcUfasGfsa






 


D1898
S1898
916
AfgAfaAfgUfaUfCfUfgGfgcaGfaAfcGfL96
AS1898
2008
cGfuUfcUfGfCfcCfagaUfaCfuUfuCfusAfsg






 


D1899
S1899
917
AfgAfaAfgUfaUfCfUfgGfgCfAfGfaAfcGfL96
AS1899
2009
cGfuUfcugCfcCfagaUfaCfuUfuCfusAfsg






 


D1900
S1900
918
GfaAfaGfuAfuCfUfGfgGfcagAfaCfgCfL96
AS1900
2010
gCfgUfuCfUfGfcCfcagAfuAfcUfuUfcsUfsa






 


D1901
S1901
919
GfaAfaGfuAfuCfUfGfgGfcAfGfAfaCfgCfL96
AS1901
2011
gCfgUfucuGfcCfcagAfuAfcUfuUfcsUfsa






 


D1902
S1902
920
AfaAfgUfaUfcUfGfGfgCfagaAfcGfcUfL96
AS1902
2012
aGfcGfuUfCfUfgCfccaGfaUfaCfuUfusCfsu






 


D1903
S1903
921
AfaAfgUfaUfcUfGfGfgCfaGfAfAfcGfcUfL96
AS1903
2013
aGfcGfuucUfgCfccaGfaUfaCfuUfusCfsu






 


D1904
S1904
922
AfaGfuAfuCfuGfGfGfcAfgaaCfgCfuAfL96
AS1904
2014
uAfgCfgUfUfCfuGfcccAfgAfuAfcUfusUfsc






 


D1905
S1905
923
AfaGfuAfuCfuGfGfGfcAfgAfAfCfgCfuAfL96
AS1905
2015
uAfgCfguuCfuGfcccAfgAfuAfcUfusUfsc






 


D1906
S1906
924
AfgUfaUfcUfgGfGfCfaGfaacGfcUfaGfL96
AS1906
2016
cUfaGfcGfUfUfcUfgccCfaGfaUfaCfusUfsu






 


D1907
S1907
925
AfgUfaUfcUfgGfGfCfaGfaAfCfGfcUfaGfL96
AS1907
2017
cUfaGfcguUfcUfgccCfaGfaUfaCfusUfsu






 


D1908
S1908
926
GfuAfuCfuGfgGfCfAfgAfacgCfuAfgGfL96
AS1908
2018
cCfuAfgCfGfUfuCfugcCfcAfgAfuAfcsUfsu






 


D1909
S1909
927
GfuAfuCfuGfgGfCfAfgAfaCfGfCfuAfgGfL96
AS1909
2019
cCfuAfgcgUfuCfugcCfcAfgAfuAfcsUfsu






 


D1910
S1910
928
UfaUfcUfgGfgCfAfGfaAfcgcUfaGfgAfL96
AS1910
2020
uCfcUfaGfCfGfuUfcugCfcCfaGfaUfasCfsu






 


D1911
S1911
929
UfaUfcUfgGfgCfAfGfaAfcGfCfUfaGfgAfL96
AS1911
2021
uCfcUfagcGfuUfcugCfcCfaGfaUfasCfsu






 


D1912
S1912
930
AfuCfuGfgGfcAfGfAfaCfgcuAfgGfaGfL96
AS1912
2022
cUfcCfuAfGfCfgUfucuGfcCfcAfgAfusAfsc






 


D1913
S1913
931
AfuCfuGfgGfcAfGfAfaCfgCfUfAfgGfaGfL96
AS1913
2023
cUfcCfuagCfgUfucuGfcCfcAfgAfusAfsc






 


D1914
S1914
932
UfcUfgGfgCfaGfAfAfcGfcuaGfgAfgAfL96
AS1914
2024
uCfuCfcUfAfGfcGfuucUfgCfcCfaGfasUfsa






 


D1915
S1915
933
UfcUfgGfgCfaGfAfAfcGfcUfAfGfgAfgAfL96
AS1915
2025
uCfuCfcuaGfcGfuucUfgCfcCfaGfasUfsa






 


D1916
S1916
934
CfuGfgGfcAfgAfAfCfgCfuagGfaGfaGfL96
AS1916
2026
cUfcUfcCfUfAfgCfguuCfuGfcCfcAfgsAfsu






 


D1917
S1917
935
CfuGfgGfcAfgAfAfCfgCfuAfGfGfaGfaGfL96
AS1917
2027
cUfcUfccuAfgCfguuCfuGfcCfcAfgsAfsu






 


D1918
S1918
936
UfgGfgCfaGfaAfCfGfcUfaggAfgAfgAfL96
AS1918
2028
uCfuCfuCfCfUfaGfcguUfcUfgCfcCfasGfsa






 


D1919
S1919
937
UfgGfgCfaGfaAfCfGfcUfaGfGfAfgAfgAfL96
AS1919
2029
uCfuCfuccUfaGfcguUfcUfgCfcCfasGfsa






 


D1920
S1920
938
GfgGfcAfgAfaCfGfCfuAfggaGfaGfaUfL96
AS1920
2030
aUfcUfcUfCfCfuAfgcgUfuCfuGfcCfcsAfsg






 


D1921
S1921
939
GfgGfcAfgAfaCfGfCfuAfgGfAfGfaGfaUfL96
AS1921
2031
aUfcUfcucCfuAfgcgUfuCfuGfcCfcsAfsg






 


D1922
S1922
940
GfgCfaGfaAfcGfCfUfaGfgagAfgAfuCfL96
AS1922
2032
gAfuCfuCfUfCfcUfagcGfuUfcUfgCfcsCfsa






 


D1923
S1923
941
GfgCfaGfaAfcGfCfUfaGfgAfGfAfgAfuCfL96
AS1923
2033
gAfuCfucuCfcUfagcGfuUfcUfgCfcsCfsa






 


D1924
S1924
942
GfcAfgAfaCfgCfUfAfgGfagaGfaUfcCfL96
AS1924
2034
gGfaUfcUfCfUfcCfuagCfgUfuCfuGfcsCfsc






 


D1925
S1925
943
GfcAfgAfaCfgCfUfAfgGfaGfAfGfaUfcCfL96
AS1925
2035
gGfaUfcucUfcCfuagCfgUfuCfuGfcsCfsc






 


D1926
S1926
944
CfaGfaAfcGfcUfAfGfgAfgagAfuCfcAfL96
AS1926
2036
uGfgAfuCfUfCfuCfcuaGfcGfuUfcUfgsCfsc






 


D1927
S1927
945
CfaGfaAfcGfcUfAfGfgAfgAfGfAfuCfcAfL96
AS1927
2037
uGfgAfucuCfuCfcuaGfcGfuUfcUfgsCfsc






 


D1928
S1928
946
AfgAfaCfgCfuAfGfGfaGfagaUfcCfaAfL96
AS1928
2038
uUfgGfaUfCfUfcUfccuAfgCfgUfuCfusGfsc






 


D1929
S1929
947
AfgAfaCfgCfuAfGfGfaGfaGfAfUfcCfaAfL96
AS1929
2039
uUfgGfaucUfcUfccuAfgCfgUfuCfusGfsc






 


D1930
S1930
948
GfaAfcGfcUfaGfGfAfgAfgauCfcAfaAfL96
AS1930
2040
uUfuGfgAfUfCfuCfuccUfaGfcGfuUfcsUfsg






 


D1931
S1931
949
GfaAfcGfcUfaGfGfAfgAfgAfUfCfcAfaAfL96
AS1931
2041
uUfuGfgauCfuCfuccUfaGfcGfuUfcsUfsg






 


D1932
S1932
950
AfaCfgCfuAfgGfAfGfaGfaucCfaAfaUfL96
AS1932
2042
aUfuUfgGfAfUfcUfcucCfuAfgCfgUfusCfsu






 


D1933
S1933
951
AfaCfgCfuAfgGfAfGfaGfaUfCfCfaAfaUfL96
AS1933
2043
aUfuUfggaUfcUfcucCfuAfgCfgUfusCfsu






 


D1934
S1934
952
AfcGfcUfaGfgAfGfAfgAfuccAfaAfuUfL96
AS1934
2044
aAfuUfuGfGfAfuCfucuCfcUfaGfcGfusUfsc






 


D1935
S1935
953
AfcGfcUfaGfgAfGfAfgAfuCfCfAfaAfuUfL96
AS1935
2045
aAfuUfuggAfuCfucuCfcUfaGfcGfusUfsc






 


D1936
S1936
954
CfgCfuAfgGfaGfAfGfaUfccaAfaUfuUfL96
AS1936
2046
aAfaUfuUfGfGfaUfcucUfcCfuAfgCfgsUfsu






 


D1937
S1937
955
CfgCfuAfgGfaGfAfGfaUfcCfAfAfaUfuUfL96
AS1937
2047
aAfaUfuugGfaUfcucUfcCfuAfgCfgsUfsu






 


D1938
S1938
956
GfcUfaGfgAfgAfGfAfuCfcaaAfuUfuCfL96
AS1938
2048
gAfaAfuUfUfGfgAfucuCfuCfcUfaGfcsGfsu






 


D1939
S1939
957
GfcUfaGfgAfgAfGfAfuCfcAfAfAfuUfuCfL96
AS1939
2049
gAfaAfuuuGfgAfucuCfuCfcUfaGfcsGfsu






 


D1940
S1940
958
CfuAfgGfaGfaGfAfUfcCfaaaUfuUfcCfL96
AS1940
2050
gGfaAfaUfUfUfgGfaucUfcUfcCfuAfgsCfsg






 


D1941
S1941
959
CfuAfgGfaGfaGfAfUfcCfaAfAfUfuUfcCfL96
AS1941
2051
gGfaAfauuUfgGfaucUfcUfcCfuAfgsCfsg






 


D1942
S1942
960
UfaGfgAfgAfgAfUfCfcAfaauUfuCfcAfL96
AS1942
2052
uGfgAfaAfUfUfuGfgauCfuCfuCfcUfasGfsc






 


D1943
S1943
961
UfaGfgAfgAfgAfUfCfcAfaAfUfUfuCfcAfL96
AS1943
2053
uGfgAfaauUfuGfgauCfuCfuCfcUfasGfsc






 


D1944
S1944
962
AfgGfaGfaGfaUfCfCfaAfauuUfcCfaUfL96
AS1944
2054
aUfgGfaAfAfUfuUfggaUfcUfcUfcCfusAfsg






 


D1945
S1945
963
AfgGfaGfaGfaUfCfCfaAfaUfUfUfcCfaUfL96
AS1945
2055
aUfgGfaaaUfuUfggaUfcUfcUfcCfusAfsg






 


D1946
S1946
964
GfgAfgAfgAfuCfCfAfaAfuuuCfcAfuUfL96
AS1946
2056
aAfuGfgAfAfAfuUfuggAfuCfuCfuCfcsUfsa






 


D1947
S1947
965
GfgAfgAfgAfuCfCfAfaAfuUfUfCfcAfuUfL96
AS1947
2057
aAfuGfgaaAfuUfuggAfuCfuCfuCfcsUfsa






 


D1948
S1948
966
GfaGfaGfaUfcCfAfAfaUfuucCfaUfuGfL96
AS1948
2058
cAfaUfgGfAfAfaUfuugGfaUfcUfcUfcsCfsu






 


D1949
S1949
967
GfaGfaGfaUfcCfAfAfaUfuUfCfCfaUfuGfL96
AS1949
2059
cAfaUfggaAfaUfuugGfaUfcUfcUfcsCfsu






 


D1950
S1950
968
AfgAfgAfuCfcAfAfAfuUfuccAfuUfgUfL96
AS1950
2060
aCfaAfuGfGfAfaAfuuuGfgAfuCfuCfusCfsc






 


D1951
S1951
969
AfgAfgAfuCfcAfAfAfuUfuCfCfAfuUfgUfL96
AS1951
2061
aCfaAfuggAfaAfuuuGfgAfuCfuCfusCfsc






 


D1952
S1952
970
GfaGfaUfcCfaAfAfUfuUfccaUfuGfuCfL96
AS1952
2062
gAfcAfaUfGfGfaAfauuUfgGfaUfcUfcsUfsc






 


D1953
S1953
971
GfaGfaUfcCfaAfAfUfuUfcCfAfUfuGfuCfL96
AS1953
2063
gAfcAfaugGfaAfauuUfgGfaUfcUfcsUfsc






 


D1954
S1954
972
AfgAfuCfcAfaAfUfUfuCfcauUfgUfcUfL96
AS1954
2064
aGfaCfaAfUfGfgAfaauUfuGfgAfuCfusCfsu






 


D1955
S1955
973
AfgAfuCfcAfaAfUfUfuCfcAfUfUfgUfcUfL96
AS1955
2065
aGfaCfaauGfgAfaauUfuGfgAfuCfusCfsu






 


D1956
S1956
974
GfaUfcCfaAfaUfUfUfcCfauuGfuCfuUfL96
AS1956
2066
aAfgAfcAfAfUfgGfaaaUfuUfgGfaUfcsUfsc






 


D1957
S1957
975
GfaUfcCfaAfaUfUfUfcCfaUfUfGfuCfuUfL96
AS1957
2067
aAfgAfcaaUfgGfaaaUfuUfgGfaUfcsUfsc






 


D1958
S1958
976
AfuCfcAfaAfuUfUfCfcAfuugUfcUfuGfL96
AS1958
2068
cAfaGfaCfAfAfuGfgaaAfuUfuGfgAfusCfsu






 


D1959
S1959
977
AfuCfcAfaAfuUfUfCfcAfuUfGfUfcUfuGfL96
AS1959
2069
cAfaGfacaAfuGfgaaAfuUfuGfgAfusCfsu






 


D1960
S1960
978
UfcCfaAfaUfuUfCfCfaUfuguCfuUfgCfL96
AS1960
2070
gCfaAfgAfCfAfaUfggaAfaUfuUfgGfasUfsc






 


D1961
S1961
979
UfcCfaAfaUfuUfCfCfaUfuGfUfCfuUfgCfL96
AS1961
2071
gCfaAfgacAfaUfggaAfaUfuUfgGfasUfsc






 


D1962
S1962
980
CfcAfaAfuUfuCfCfAfuUfgucUfuGfcAfL96
AS1962
2072
uGfcAfaGfAfCfaAfuggAfaAfuUfuGfgsAfsu






 


D1963
S1963
981
CfcAfaAfuUfuCfCfAfuUfgUfCfUfuGfcAfL96
AS1963
2073
uGfcAfagaCfaAfuggAfaAfuUfuGfgsAfsu






 


D1964
S1964
982
CfaAfaUfuUfcCfAfUfuGfucuUfgCfaAfL96
AS1964
2074
uUfgCfaAfGfAfcAfaugGfaAfaUfuUfgsGfsa






 


D1965
S1965
983
CfaAfaUfuUfcCfAfUfuGfuCfUfUfgCfaAfL96
AS1965
2075
uUfgCfaagAfcAfaugGfaAfaUfuUfgsGfsa






 


D1966
S1966
984
AfaAfuUfuCfcAfUfUfgUfcuuGfcAfaGfL96
AS1966
2076
cUfuGfcAfAfGfaCfaauGfgAfaAfuUfusGfsg






 


D1967
S1967
985
AfaAfuUfuCfcAfUfUfgUfcUfUfGfcAfaGfL96
AS1967
2077
cUfuGfcaaGfaCfaauGfgAfaAfuUfusGfsg






 


D1968
S1968
986
AfaUfuUfcCfaUfUfGfuCfuugCfaAfgCfL96
AS1968
2078
gCfuUfgCfAfAfgAfcaaUfgGfaAfaUfusUfsg






 


D1969
S1969
987
AfaUfuUfcCfaUfUfGfuCfuUfGfCfaAfgCfL96
AS1969
2079
gCfuUfgcaAfgAfcaaUfgGfaAfaUfusUfsg






 


D1970
S1970
988
AfuUfuCfcAfuUfGfUfcUfugcAfaGfcAfL96
AS1970
2080
uGfcUfuGfCfAfaGfacaAfuGfgAfaAfusUfsu






 


D1971
S1971
989
AfuUfuCfcAfuUfGfUfcUfuGfCfAfaGfcAfL96
AS1971
2081
uGfcUfugcAfaGfacaAfuGfgAfaAfusUfsu






 


D1972
S1972
990
UfuUfcCfaUfuGfUfCfuUfgcaAfgCfaAfL96
AS1972
2082
uUfgCfuUfGfCfaAfgacAfaUfgGfaAfasUfsu






 


D1973
S1973
991
UfuUfcCfaUfuGfUfCfuUfgCfAfAfgCfaAfL96
AS1973
2083
uUfgCfuugCfaAfgacAfaUfgGfaAfasUfsu






 


D1974
S1974
992
UfuCfcAfuUfgUfCfUfuGfcaaGfcAfaAfL96
AS1974
2084
uUfuGfcUfUfGfcAfagaCfaAfuGfgAfasAfsu






 


D1975
S1975
993
UfuCfcAfuUfgUfCfUfuGfcAfAfGfcAfaAfL96
AS1975
2085
uUfuGfcuuGfcAfagaCfaAfuGfgAfasAfsu






 


D1976
S1976
994
UfcCfaUfuGfuCfUfUfgCfaagCfaAfaGfL96
AS1976
2086
cUfuUfgCfUfUfgCfaagAfcAfaUfgGfasAfsa






 


D1977
S1977
995
UfcCfaUfuGfuCfUfUfgCfaAfGfCfaAfaGfL96
AS1977
2087
cUfuUfgcuUfgCfaagAfcAfaUfgGfasAfsa






 


D1978
S1978
996
CfcAfuUfgUfcUfUfGfcAfagcAfaAfgCfL96
AS1978
2088
gCfuUfuGfCfUfuGfcaaGfaCfaAfuGfgsAfsa






 


D1979
S1979
997
CfcAfuUfgUfcUfUfGfcAfaGfCfAfaAfgCfL96
AS1979
2089
gCfuUfugcUfuGfcaaGfaCfaAfuGfgsAfsa






 


D1980
S1980
998
CfaUfuGfuCfuUfGfCfaAfgcaAfaGfcAfL96
AS1980
2090
uGfcUfuUfGfCfuUfgcaAfgAfcAfaUfgsGfsa






 


D1981
S1981
999
CfaUfuGfuCfuUfGfCfaAfgCfAfAfaGfcAfL96
AS1981
2091
uGfcUfuugCfuUfgcaAfgAfcAfaUfgsGfsa






 


D1982
S1982
1000
AfuUfgUfcUfuGfCfAfaGfcaaAfgCfaCfL96
AS1982
2092
gUfgCfuUfUfGfcUfugcAfaGfaCfaAfusGfsg






 


D1983
S1983
1001
AfuUfgUfcUfuGfCfAfaGfcAfAfAfgCfaCfL96
AS1983
2093
gUfgCfuuuGfcUfugcAfaGfaCfaAfusGfsg






 


D1984
S1984
1002
UfuGfuCfuUfgCfAfAfgCfaaaGfcAfcGfL96
AS1984
2094
cGfuGfcUfUfUfgCfuugCfaAfgAfcAfasUfsg






 


D1985
S1985
1003
UfuGfuCfuUfgCfAfAfgCfaAfAfGfcAfcGfL96
AS1985
2095
cGfuGfcuuUfgCfuugCfaAfgAfcAfasUfsg






 


D1986
S1986
1004
UfgUfcUfuGfcAfAfGfcAfaagCfaCfgUfL96
AS1986
2096
aCfgUfgCfUfUfuGfcuuGfcAfaGfaCfasAfsu






 


D1987
S1987
1005
UfgUfcUfuGfcAfAfGfcAfaAfGfCfaCfgUfL96
AS1987
2097
aCfgUfgcuUfuGfcuuGfcAfaGfaCfasAfsu






 


D1988
S1988
1006
GfuCfuUfgCfaAfGfCfaAfagcAfcGfuAfL96
AS1988
2098
uAfcGfuGfCfUfuUfgcuUfgCfaAfgAfcsAfsa






 


D1989
S1989
1007
GfuCfuUfgCfaAfGfCfaAfaGfCfAfcGfuAfL96
AS1989
2099
uAfcGfugcUfuUfgcuUfgCfaAfgAfcsAfsa






 


D1990
S1990
1008
UfcUfuGfcAfaGfCfAfaAfgcaCfgUfaUfL96
AS1990
2100
aUfaCfgUfGfCfuUfugcUfuGfcAfaGfasCfsa






 


D1991
S1991
1009
UfcUfuGfcAfaGfCfAfaAfgCfAfCfgUfaUfL96
AS1991
2101
aUfaCfgugCfuUfugcUfuGfcAfaGfasCfsa






 


D1992
S1992
1010
CfuUfgCfaAfgCfAfAfaGfcacGfuAfuUfL96
AS1992
2102
aAfuAfcGfUfGfcUfuugCfuUfgCfaAfgsAfsc






 


D1993
S1993
1011
CfuUfgCfaAfgCfAfAfaGfcAfCfGfuAfuUfL96
AS1993
2103
aAfuAfcguGfcUfuugCfuUfgCfaAfgsAfsc






 


D1994
S1994
1012
UfuGfcAfaGfcAfAfAfgCfacgUfaUfuAfL96
AS1994
2104
uAfaUfaCfGfUfgCfuuuGfcUfuGfcAfasGfsa






 


D1995
S1995
1013
UfuGfcAfaGfcAfAfAfgCfaCfGfUfaUfuAfL96
AS1995
2105
uAfaUfacgUfgCfuuuGfcUfuGfcAfasGfsa






 


D1996
S1996
1014
UfgCfaAfgCfaAfAfGfcAfcguAfuUfaAfL96
AS1996
2106
uUfaAfuAfCfGfuGfcuuUfgCfuUfgCfasAfsg






 


D1997
S1997
1015
UfgCfaAfgCfaAfAfGfcAfcGfUfAfuUfaAfL96
AS1997
2107
uUfaAfuacGfuGfcuuUfgCfuUfgCfasAfsg






 


D1998
S1998
1016
GfcAfaGfcAfaAfGfCfaCfguaUfuAfaAfL96
AS1998
2108
uUfuAfaUfAfCfgUfgcuUfuGfcUfuGfcsAfsa






 


D1999
S1999
1017
GfcAfaGfcAfaAfGfCfaCfgUfAfUfuAfaAfL96
AS1999
2109
uUfuAfauaCfgUfgcuUfuGfcUfuGfcsAfsa






 


D2000
S2000
1018
CfaAfgCfaAfaGfCfAfcGfuauUfaAfaUfL96
AS2000
2110
aUfuUfaAfUfAfcGfugcUfuUfgCfuUfgsCfsa






 


D2001
S2001
1019
CfaAfgCfaAfaGfCfAfcGfuAfUfUfaAfaUfL96
AS2001
2111
aUfuUfaauAfcGfugcUfuUfgCfuUfgsCfsa






 


D2002
S2002
1020
AfaGfcAfaAfgCfAfCfgUfauuAfaAfuAfL96
AS2002
2112
uAfuUfuAfAfUfaCfgugCfuUfuGfcUfusGfsc






 


D2003
S2003
1021
AfaGfcAfaAfgCfAfCfgUfaUfUfAfaAfuAfL96
AS2003
2113
uAfuUfuaaUfaCfgugCfuUfuGfcUfusGfsc






 


D2004
S2004
1022
AfgCfaAfaGfcAfCfGfuAfuuaAfaUfaUfL96
AS2004
2114
aUfaUfuUfAfAfuAfcguGfcUfuUfgCfusUfsg






 


D2005
S2005
1023
AfgCfaAfaGfcAfCfGfuAfuUfAfAfaUfaUfL96
AS2005
2115
aUfaUfuuaAfuAfcguGfcUfuUfgCfusUfsg






 


D2006
S2006
1024
GfcAfaAfgCfaCfGfUfaUfuaaAfuAfuGfL96
AS2006
2116
cAfuAfuUfUfAfaUfacgUfgCfuUfuGfcsUfsu






 


D2007
S2007
1025
GfcAfaAfgCfaCfGfUfaUfuAfAfAfuAfuGfL96
AS2007
2117
cAfuAfuuuAfaUfacgUfgCfuUfuGfcsUfsu






 


D2008
S2008
1026
CfaAfaGfcAfcGfUfAfuUfaaaUfaUfgAfL96
AS2008
2118
uCfaUfaUfUfUfaAfuacGfuGfcUfuUfgsCfsu






 


D2009
S2009
1027
CfaAfaGfcAfcGfUfAfuUfaAfAfUfaUfgAfL96
AS2009
2119
uCfaUfauuUfaAfuacGfuGfcUfuUfgsCfsu






 


D2010
S2010
1028
AfaAfgCfaCfgUfAfUfuAfaauAfuGfaUfL96
AS2010
2120
aUfcAfuAfUfUfuAfauaCfgUfgCfuUfusGfsc






 


D2011
S2011
1029
AfaAfgCfaCfgUfAfUfuAfaAfUfAfuGfaUfL96
AS2011
2121
aUfcAfuauUfuAfauaCfgUfgCfuUfusGfsc






 


D2012
S2012
1030
AfaGfcAfcGfuAfUfUfaAfauaUfgAfuCfL96
AS2012
2122
gAfuCfaUfAfUfuUfaauAfcGfuGfcUfusUfsg






 


D2013
S2013
1031
AfaGfcAfcGfuAfUfUfaAfaUfAfUfgAfuCfL96
AS2013
2123
gAfuCfauaUfuUfaauAfcGfuGfcUfusUfsg






 


D2014
S2014
1032
AfgCfaCfgUfaUfUfAfaAfuauGfaUfcUfL96
AS2014
2124
aGfaUfcAfUfAfuUfuaaUfaCfgUfgCfusUfsu






 


D2015
S2015
1033
AfgCfaCfgUfaUfUfAfaAfuAfUfGfaUfcUfL96
AS2015
2125
aGfaUfcauAfuUfuaaUfaCfgUfgCfusUfsu






 


D2016
S2016
1034
GfcAfcGfuAfuUfAfAfaUfaugAfuCfuGfL96
AS2016
2126
cAfgAfuCfAfUfaUfuuaAfuAfcGfuGfcsUfsu






 


D2017
S2017
1035
GfcAfcGfuAfuUfAfAfaUfaUfGfAfuCfuGfL96
AS2017
2127
cAfgAfucaUfaUfuuaAfuAfcGfuGfcsUfsu






 


D2018
S2018
1036
CfaCfgUfaUfuAfAfAfuAfugaUfcUfgCfL96
AS2018
2128
gCfaGfaUfCfAfuAfuuuAfaUfaCfgUfgsCfsu






 


D2019
S2019
1037
CfaCfgUfaUfuAfAfAfuAfuGfAfUfcUfgCfL96
AS2019
2129
gCfaGfaucAfuAfuuuAfaUfaCfgUfgsCfsu






 


D2020
S2020
1038
AfcGfuAfuUfaAfAfUfaUfgauCfuGfcAfL96
AS2020
2130
uGfcAfgAfUfCfaUfauuUfaAfuAfcGfusGfsc






 


D2021
S2021
1039
AfcGfuAfuUfaAfAfUfaUfgAfUfCfuGfcAfL96
AS2021
2131
uGfcAfgauCfaUfauuUfaAfuAfcGfusGfsc






 


D2022
S2022
1040
CfgUfaUfuAfaAfUfAfuGfaucUfgCfaGfL96
AS2022
2132
cUfgCfaGfAfUfcAfuauUfuAfaUfaCfgsUfsg






 


D2023
S2023
1041
CfgUfaUfuAfaAfUfAfuGfaUfCfUfgCfaGfL96
AS2023
2133
cUfgCfagaUfcAfuauUfuAfaUfaCfgsUfsg






 


D2024
S2024
1042
GfuAfuUfaAfaUfAfUfgAfucuGfcAfgCfL96
AS2024
2134
gCfuGfcAfGfAfuCfauaUfuUfaAfuAfcsGfsu






 


D2025
S2025
1043
GfuAfuUfaAfaUfAfUfgAfuCfUfGfcAfgCfL96
AS2025
2135
gCfuGfcagAfuCfauaUfuUfaAfuAfcsGfsu






 


D2026
S2026
1044
UfaUfuAfaAfuAfUfGfaUfcugCfaGfcCfL96
AS2026
2136
gGfcUfgCfAfGfaUfcauAfuUfuAfaUfasCfsg






 


D2027
S2027
1045
UfaUfuAfaAfuAfUfGfaUfcUfGfCfaGfcCfL96
AS2027
2137
gGfcUfgcaGfaUfcauAfuUfuAfaUfasCfsg






 


D2028
S2028
1046
AfuUfaAfaUfaUfGfAfuCfugcAfgCfcAfL96
AS2028
2138
uGfgCfuGfCfAfgAfucaUfaUfuUfaAfusAfsc






 


D2029
S2029
1047
AfuUfaAfaUfaUfGfAfuCfuGfCfAfgCfcAfL96
AS2029
2139
uGfgCfugcAfgAfucaUfaUfuUfaAfusAfsc






 


D2030
S2030
1048
UfuAfaAfuAfuGfAfUfcUfgcaGfcCfaUfL96
AS2030
2140
aUfgGfcUfGfCfaGfaucAfuAfuUfuAfasUfsa






 


D2031
S2031
1049
UfuAfaAfuAfuGfAfUfcUfgCfAfGfcCfaUfL96
AS2031
2141
aUfgGfcugCfaGfaucAfuAfuUfuAfasUfsa






 


D2032
S2032
1050
UfaAfaUfaUfgAfUfCfuGfcagCfcAfuUfL96
AS2032
2142
aAfuGfgCfUfGfcAfgauCfaUfaUfuUfasAfsu






 


D2033
S2033
1051
UfaAfaUfaUfgAfUfCfuGfcAfGfCfcAfuUfL96
AS2033
2143
aAfuGfgcuGfcAfgauCfaUfaUfuUfasAfsu






 


D2034
S2034
1052
AfaAfuAfuGfaUfCfUfgCfagcCfaUfuAfL96
AS2034
2144
uAfaUfgGfCfUfgCfagaUfcAfuAfuUfusAfsa






 


D2035
S2035
1053
AfaAfuAfuGfaUfCfUfgCfaGfCfCfaUfuAfL96
AS2035
2145
uAfaUfggcUfgCfagaUfcAfuAfuUfusAfsa






 


D2036
S2036
1054
AfaUfaUfgAfuCfUfGfcAfgccAfuUfaAfL96
AS2036
2146
uUfaAfuGfGfCfuGfcagAfuCfaUfaUfusUfsa






 


D2037
S2037
1055
AfaUfaUfgAfuCfUfGfcAfgCfCfAfuUfaAfL96
AS2037
2147
uUfaAfuggCfuGfcagAfuCfaUfaUfusUfsa






 


D2038
S2038
1056
AfuAfuGfaUfcUfGfCfaGfccaUfuAfaAfL96
AS2038
2148
uUfuAfaUfGfGfcUfgcaGfaUfcAfuAfusUfsu






 


D2039
S2039
1057
AfuAfuGfaUfcUfGfCfaGfcCfAfUfuAfaAfL96
AS2039
2149
uUfuAfaugGfcUfgcaGfaUfcAfuAfusUfsu






 


D2040
S2040
1058
UfaUfgAfuCfuGfCfAfgCfcauUfaAfaAfL96
AS2040
2150
uUfuUfaAfUfGfgCfugcAfgAfuCfaUfasUfsu






 


D2041
S2041
1059
UfaUfgAfuCfuGfCfAfgCfcAfUfUfaAfaAfL96
AS2041
2151
uUfuUfaauGfgCfugcAfgAfuCfaUfasUfsu






 


D2042
S2042
1060
AfuGfaUfcUfgCfAfGfcCfauuAfaAfaAfL96
AS2042
2152
uUfuUfuAfAfUfgGfcugCfaGfaUfcAfusAfsu






 


D2043
S2043
1061
AfuGfaUfcUfgCfAfGfcCfaUfUfAfaAfaAfL96
AS2043
2153
uUfuUfuaaUfgGfcugCfaGfaUfcAfusAfsu






 


D2044
S2044
1062
UfgAfuCfuGfcAfGfCfcAfuuaAfaAfaGfL96
AS2044
2154
cUfuUfuUfAfAfuGfgcuGfcAfgAfuCfasUfsa






 


D2045
S2045
1063
UfgAfuCfuGfcAfGfCfcAfuUfAfAfaAfaGfL96
AS2045
2155
cUfuUfuuaAfuGfgcuGfcAfgAfuCfasUfsa






 


D2046
S2046
1064
GfaUfcUfgCfaGfCfCfaUfuaaAfaAfgAfL96
AS2046
2156
uCfuUfuUfUfAfaUfggcUfgCfaGfaUfcsAfsu






 


D2047
S2047
1065
GfaUfcUfgCfaGfCfCfaUfuAfAfAfaAfgAfL96
AS2047
2157
uCfuUfuuuAfaUfggcUfgCfaGfaUfcsAfsu






 


D2048
S2048
1066
AfuCfuGfcAfgCfCfAfuUfaaaAfaGfaCfL96
AS2048
2158
gUfcUfuUfUfUfaAfuggCfuGfcAfgAfusCfsa






 


D2049
S2049
1067
AfuCfuGfcAfgCfCfAfuUfaAfAfAfaGfaCfL96
AS2049
2159
gUfcUfuuuUfaAfuggCfuGfcAfgAfusCfsa






 


D2050
S2050
1068
UfcUfgCfaGfcCfAfUfuAfaaaAfgAfcAfL96
AS2050
2160
uGfuCfuUfUfUfuAfaugGfcUfgCfaGfasUfsc






 


D2051
S2051
1069
UfcUfgCfaGfcCfAfUfuAfaAfAfAfgAfcAfL96
AS2051
2161
uGfuCfuuuUfuAfaugGfcUfgCfaGfasUfsc






 


D2052
S2052
1070
CfuGfcAfgCfcAfUfUfaAfaaaGfaCfaCfL96
AS2052
2162
gUfgUfcUfUfUfuUfaauGfgCfuGfcAfgsAfsu






 


D2053
S2053
1071
CfuGfcAfgCfcAfUfUfaAfaAfAfGfaCfaCfL96
AS2053
2163
gUfgUfcuuUfuUfaauGfgCfuGfcAfgsAfsu






 


D2054
S2054
1072
UfgCfaGfcCfaUfUfAfaAfaagAfcAfcAfL96
AS2054
2164
uGfuGfuCfUfUfuUfuaaUfgGfcUfgCfasGfsa






 


D2055
S2055
1073
UfgCfaGfcCfaUfUfAfaAfaAfGfAfcAfcAfL96
AS2055
2165
uGfuGfucuUfuUfuaaUfgGfcUfgCfasGfsa






 


D2056
S2056
1074
GfcAfgCfcAfuUfAfAfaAfagaCfaCfaUfL96
AS2056
2166
aUfgUfgUfCfUfuUfuuaAfuGfgCfuGfcsAfsg






 


D2057
S2057
1075
GfcAfgCfcAfuUfAfAfaAfaGfAfCfaCfaUfL96
AS2057
2167
aUfgUfgucUfuUfuuaAfuGfgCfuGfcsAfsg






 


D2058
S2058
1076
CfaGfcCfaUfuAfAfAfaAfgacAfcAfuUfL96
AS2058
2168
aAfuGfuGfUfCfuUfuuuAfaUfgGfcUfgsCfsa






 


D2059
S2059
1077
CfaGfcCfaUfuAfAfAfaAfgAfCfAfcAfuUfL96
AS2059
2169
aAfuGfuguCfuUfuuuAfaUfgGfcUfgsCfsa






 


D2060
S2060
1078
AfgCfcAfuUfaAfAfAfaGfacaCfaUfuCfL96
AS2060
2170
gAfaUfgUfGfUfcUfuuuUfaAfuGfgCfusGfsc






 


D2061
S2061
1079
AfgCfcAfuUfaAfAfAfaGfaCfAfCfaUfuCfL96
AS2061
2171
gAfaUfgugUfcUfuuuUfaAfuGfgCfusGfsc






 


D2062
S2062
1080
GfcCfaUfuAfaAfAfAfgAfcacAfuUfcUfL96
AS2062
2172
aGfaAfuGfUfGfuCfuuuUfuAfaUfgGfcsUfsg






 


D2063
S2063
1081
GfcCfaUfuAfaAfAfAfgAfcAfCfAfuUfcUfL96
AS2063
2173
aGfaAfuguGfuCfuuuUfuAfaUfgGfcsUfsg






 


D2064
S2064
1082
CfcAfuUfaAfaAfAfGfaCfacaUfuCfuGfL96
AS2064
2174
cAfgAfaUfGfUfgUfcuuUfuUfaAfuGfgsCfsu






 


D2065
S2065
1083
CfcAfuUfaAfaAfAfGfaCfaCfAfUfuCfuGfL96
AS2065
2175
cAfgAfaugUfgUfcuuUfuUfaAfuGfgsCfsu






 


D2066
S2066
1084
CfaUfuAfaAfaAfGfAfcAfcauUfcUfgUfL96
AS2066
2176
aCfaGfaAfUfGfuGfucuUfuUfuAfaUfgsGfsc






 


D2067
S2067
1085
CfaUfuAfaAfaAfGfAfcAfcAfUfUfcUfgUfL96
AS2067
2177
aCfaGfaauGfuGfucuUfuUfuAfaUfgsGfsc






 


D2068
S2068
1086
AfuUfaAfaAfaGfAfCfaCfauuCfuGfuAfL96
AS2068
2178
uAfcAfgAfAfUfgUfgucUfuUfuUfaAfusGfsg






 


D2069
S2069
1087
AfuUfaAfaAfaGfAfCfaCfaUfUfCfuGfuAfL96
AS2069
2179
uAfcAfgaaUfgUfgucUfuUfuUfaAfusGfsg






 


D2070
S2070
1088
UfuAfaAfaAfgAfCfAfcAfuucUfgUfaAfL96
AS2070
2180
uUfaCfaGfAfAfuGfuguCfuUfuUfuAfasUfsg






 


D2071
S2071
1089
UfuAfaAfaAfgAfCfAfcAfuUfCfUfgUfaAfL96
AS2071
2181
uUfaCfagaAfuGfuguCfuUfuUfuAfasUfsg






 


D2072
S2072
1090
UfaAfaAfaGfaCfAfCfaUfucuGfuAfaAfL96
AS2072
2182
uUfuAfcAfGfAfaUfgugUfcUfuUfuUfasAfsu






 


D2073
S2073
1091
UfaAfaAfaGfaCfAfCfaUfuCfUfGfuAfaAfL96
AS2073
2183
uUfuAfcagAfaUfgugUfcUfuUfuUfasAfsu






 


D2074
S2074
1092
AfaAfaAfgAfcAfCfAfuUfcugUfaAfaAfL96
AS2074
2184
uUfuUfaCfAfGfaAfuguGfuCfuUfuUfusAfsa






 


D2075
S2075
1093
AfaAfaAfgAfcAfCfAfuUfcUfGfUfaAfaAfL96
AS2075
2185
uUfuUfacaGfaAfuguGfuCfuUfuUfusAfsa






 


D2076
S2076
1094
AfaAfaGfaCfaCfAfUfuCfuguAfaAfaAfL96
AS2076
2186
uUfuUfuAfCfAfgAfaugUfgUfcUfuUfusUfsa






 


D2077
S2077
1095
AfaAfaGfaCfaCfAfUfuCfuGfUfAfaAfaAfL96
AS2077
2187
uUfuUfuacAfgAfaugUfgUfcUfuUfusUfsa






 


D2078
S2078
1096
AfaAfgAfcAfcAfUfUfcUfguaAfaAfaAfL96
AS2078
2188
uUfuUfuUfAfCfaGfaauGfuGfuCfuUfusUfsu






 


D2079
S2079
1097
AfaAfgAfcAfcAfUfUfcUfgUfAfAfaAfaAfL96
AS2079
2189
uUfuUfuuaCfaGfaauGfuGfuCfuUfusUfsu






 


D2080
S2080
1098
AfaGfaCfaCfaUfUfCfuGfuaaAfaAfaAfL96
AS2080
2190
uUfuUfuUfUfAfcAfgaaUfgUfgUfcUfusUfsu






 


D2081
S2081
1099
AfaGfaCfaCfaUfUfCfuGfuAfAfAfaAfaAfL96
AS2081
2191
uUfuUfuuuAfcAfgaaUfgUfgUfcUfusUfsu






 


D2082
S2082
1100
AfgAfcAfcAfuUfCfUfgUfaaaAfaAfaAfL96
AS2082
2192
uUfuUfuUfUfUfaCfagaAfuGfuGfuCfusUfsu






 


D2083
S2083
1101
AfgAfcAfcAfuUfCfUfgUfaAfAfAfaAfaAfL96
AS2083
2193
uUfuUfuuuUfaCfagaAfuGfuGfuCfusUfsu






 


D2084
S2084
1102
GfaCfaCfaUfuCfUfGfuAfaaaAfaAfaAfL96
AS2084
2194
uUfuUfuUfUfUfuAfcagAfaUfgUfgUfcsUfsu






 


D2085
S2085
1103
GfaCfaCfaUfuCfUfGfuAfaAfAfAfaAfaAfL96
AS2085
2195
uUfuUfuuuUfuAfcagAfaUfgUfgUfcsUfsu






 


D2086
S2086
1104
AfcAfcAfuUfcUfGfUfaAfaaaAfaAfaAfL96
AS2086
2196
uUfuUfuUfUfUfuUfacaGfaAfuGfuGfusCfsu






 


D2087
S2087
1105
AfcAfcAfuUfcUfGfUfaAfaAfAfAfaAfaAfL96
AS2087
2197
uUfuUfuuuUfuUfacaGfaAfuGfuGfusCfsu






 


D2088
S2088
1106
CfaCfaUfuCfuGfUfAfaAfaaaAfaAfaAfL96
AS2088
2198
uUfuUfuUfUfUfuUfuacAfgAfaUfgUfgsUfsc






 


D2089
S2089
1107
CfaCfaUfuCfuGfUfAfaAfaAfAfAfaAfaAfL96
AS2089
2199
uUfuUfuuuUfuUfuacAfgAfaUfgUfgsUfsc






 


D2090
S2090
1108
AfcAfuUfcUfgUfAfAfaAfaaaAfaAfaAfL96
AS2090
2200
uUfuUfuUfUfUfuUfuuaCfaGfaAfuGfusGfsu






 


D2091
S2091
1109
AfcAfuUfcUfgUfAfAfaAfaAfAfAfaAfaAfL96
AS2091
2201
uUfuUfuuuUfuUfuuaCfaGfaAfuGfusGfsu





Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides; Nf(e.g., Af) is a 2′-fluoro nucleotide; s is aphosphothiorate linkage; L96 indicates a GalNAc3 ligand.






Example 4
In Vitro Screening of RNAi Agents
Cell Culture and Transfections
Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, Va.) were grown to near 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. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing  ̃2×104 Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done using 8, 4 fold serial dilutions with a maximum dose of 10 nM final duplex concentration.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand 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, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)
A master mix of 1 μl 10× Buffer, 0.4 μl 25×dNTPs, 1 μl Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitor and 1.6 μl of H2O per reaction were added into 5 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR
2 μl of cDNA were added to a master mix containing 0.50 GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E (human) Cat #4308313 (rodent)), 0.50 TTR TaqMan probe (Applied Biosystems cat #HS00174914_m1 (human) cat #Rn00562124_m1 (rat)) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plate (Roche cat #04887301001). Real time PCR was done in a Roche LC 480 Real Time PCR machine (Roche). Each duplex was tested in at least two independent transfections and each transfection was assayed in duplicate, unless otherwise noted.
To calculate relative fold change, real time data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 (sense sequence: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 2202); antisense sequence: UCGAAGuCUcAGCGuAAGdTsdT (SEQ ID NO: 2203)) or naïve cells over the same dose range, or to its own lowest dose. IC50s were calculated for each individual transfection as well as in combination, where a single IC50 was fit to the data from both transfections.
The results of gene silencing of the exemplary siRNA duplex 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
The following experiments demonstrated the beneficial effects of chemical modifications, including the introduction of triplet repeat motifs, together with a GalNAc3 ligand, on the silencing activity of RNAi agents that target TTR. The sequences of the agents investigated are provided in Table 2 below. The regions of complementarity to the TTR mRNA are as follows: the region of complementarity of RNAi agents AD-45165, AD-51546 and AD-51547 is GGATGGGATTTCATGTAACCAAGA (SEQ ID NO: 2204) and the region or complemetarity of RNAi agents AD-45163, AD-51544, and AD-51545 is TTCATGTAACCAAGAGTATTCCAT (SEQ ID NO: 2205).
Protocol for Assessment of IC50 in Hep3B Cells
The IC50 for each modified siRNA was determined in Hep3B cells (a human hepatoma cell line) by standard reverse transfection using Lipofectamine RNAiMAX. In brief, reverse transfection was carried out by adding 5 μL of Opti-MEM to 5 μL of siRNA duplex per well into a 96-well plate along with 10 μL of Opti-MEM plus 0.5 μL of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubating at room temperature for 15-20 minutes. Following incubation, 100 μL of complete growth media without antibiotic containing 12,000-15,000 Hep3B cells was then added to each well. Cells were incubated for 24 hours at 37° C. in an atmosphere of 5% CO2 prior to lysis and analysis of TTR and GAPDH mRNA by bDNA (Quantigene). Seven different siRNA concentrations ranging from 10 nM to 0.6 μM were assessed for IC50 determination and TTR/GAPDH for siRNA transfected cells was normalized to cells transfected with 10 nM Luc siRNA. The results are shown in Table 2.
Protocol for Assessment of Free-Uptake IC50 
Free uptake silencing in primary cynomolgus hepatocytes was assessed following incubation with TTR siRNA for either 4 hours or 24 hours. Silencing was measured at 24 hours from the initial exposure. In brief, 96-well culture plates were coated with 0.05%-0.1% collagen (Sigma C3867-1VL) at room temperature, 24 hours prior to the start of the experiment. On the day of assay, siRNAs were diluted in pre-warmed Plating Media consisting of DMEM supplemented with GIBCO's Maintenance Media Kit (Serum-Free, Life Technologies CM4000), and added to the collagen-coated 96-well culture plates. Cryopreserved primary cynomolgus hepatocytes were rapidly thawed in a 37° C. water bath, and immediately diluted in Plating Media to a concentration of 360,000 cells/mL. A volume of cell suspension was gently pipetted on top of the pre-plated siRNAs such that the final cell count was 18,000 cells/well. The plate was lightly swirled to mix and spread cells evenly across the wells and placed in a 37° C., 5% CO2 incubator for 24 hours prior to lysis and analysis of TTR and GAPDH mRNA by bDNA (Quantigene, Affymetrix). In the case of the 4 h incubation with siRNA, the media was decanted after 4 hours of exposure to the cells, and replaced with fresh Plating Media for the remaining 20 hours of incubation. Downstream analysis for TTR and GAPDH mRNA was the same as described above. For a typical dose response curve, siRNAs were titrated from 1 uM to 0.24 nM by 4 fold serial dilution.







TABLE 2







In vitro Activity Summary for Alternating TTR-GalNAc and Variants with Triplet Motifs














Free-Uptake






IC50 (μM)
Hep3B IC50












Duplex ID
S (5′-3′)
AS (5′-3′′)
4 h
24 h
(nM)





AD-45163
AfuGfuAfaCfcAfaGfaGfuAfuU
aUfgGfaAfuAfcUfcUfuGfgUfu
0.04101
0.00820
0.0115



fcCfaUfL96
AfcAfusGfsa






(SEQ ID NO: 2206)
(SEQ ID NO: 2212)





 


AD-51544
AfuGfuAfaCfcAfAfGfaGfuA
aUfgGfAfAfuAfcUfcuuGfgUfuA
0.00346
0.00374
0.0014



fuucCfaUfL96
fcAfusGfsa






(SEQ ID NO: 2207)
(SEQ ID NO: 2213)





 


AD-51545
AfuGfuAfAfCfcAfAfGfaGfuAf
aUfgGfaAfuAfcUfcuuGfguuA
0.00395
0.00389
0.0018



uUfcCfaUfL96
fcAfusGfsa






(SEQ ID NO: 2208)
(SEQ ID NO: 2214)





 


AD-45165
UfgGfgAfuUfuCfaUfgUfaAf
uCfuUfgGfuUfaCfaUfgAfaAfuC
0.02407
0.00869
0.0112



cCfaAfgAfL96
fcCfasUfsc






(SEQ ID NO: 2209)
(SEQ ID NO: 2215)





 


AD-51546
UfgGfGfAfuUfuCfAfUfgUfaA
uCfuugGfuUfaCfaugAfaAf
0.00317
0.00263
0.0017



fcCfAfAfgAfL96 
uccCfasUfsc






(SEQ ID NO: 2210)
(SEQ ID NO: 2216)





 


AD-51547
UfgGfgAfuUfuCfAfUfgUfaa
uCfuUfgGfUfUfaCfaugAfaAf
0.00460
0.00374
0.0028



cCfaAfgAfL96
uCfcCfasUfsc






(SEQ ID NO: 2211)
(SEQ ID NO: 2217)





Lowercase nucleotides (a, u, g, c) indicate 2′-O-methyl nucleotides; Nf (e.g., Af) indicates a 2′-fluoro nucleotide; s indicates a phosphothiorate linkage; L96 indicates a GalNAc3 ligand; bold nucleotides indicate changes relative to the corresponding parent agent. Each bold nucleotide is at the center of a triplet motif.






The results are provided in Table 2 and demonstrate that modified RNAi agents that target TTR provide enhanced silencing activity.
Results: Improved Activity of Modified RNAi Agents
Parent RNAi agents with alternating chemical modifications and a GalNAc3 ligand provided an IC50 in Hep3B cells of about 0.01 nM. As shown in FIGS. 4-5 and in Table 2, agents modified relative to the parent agents, for example, by the addition of one or more repeating triplets of 2′-fluoro and 2′-O-methyl modifications, showed unexpectedly enhanced silencing activity, achieving IC50 values in Hep3B cells that were 5-8 fold better than the corresponding parent agent.
Results: Free Uptake IC50s in Hep3B Cells
As shown in Table 2 and FIGS. 6-7, RNAi agents modified relative to the parent AD-45163 also showed enhanced free uptake silencing. The modified agents showed more than double the silencing activity of the parent after a 24 hour incubation period and nearly 10 times the silencing activity of the parent after a 4 hour incubation period.
As shown in Table 2 and FIGS. 8-9, RNAi agents modified relative to the parent AD-45165 also showed enhanced free uptake silencing. The modified agents showed 2-3 times the silencing activity of the parent after a 24 hour incubation period and 5-8 times the silencing activity of the parent after a 4 hour incubation period.
Taken collectively, these results demonstrate that the modified RNAi agents presented herein, e.g., AD-51544, AD-51545, AD-51546, and AD-51547, all showed unexpectedly good inhibition of TTR mRNA in in vitro silencing experiments.
Example 6
TTR mRNA Silencing and TTR Protein Suppression in Transgenic Mice
To assess the efficacy of the RNAi agents AD-45163, AD-51544, AD-51545, AD45165, AD-51546, and AD-51547, these agents were administered to transgenic mice that express human transthyretin with the V30M mutation (see Santos, S D., Fernaandes, R., and Saraiva, M J. (2010) Neurobiology of Aging, 31, 280-289). The V30M mutation is known to cause familial amyloid polyneuropathy type I in humans. See, e.g., Lobato, L. (2003) J Nephrol., 16(3):438-42.
The RNAi agents (in PBS buffer) or PBS control were administered to mice (2 male and 2 female) of 18-24 months of age in a single subcutaneous dose of 5 mg/kg or 1 mg/kg. After approximately 48 hours, mice were anesthetized with 200 μl of ketamine, and then exsanguinated by severing the right caudal artery. Whole blood was isolated and plasma was isolated and stored at −80° C. until assaying. Liver tissue was collected, flash-frozen and stored at −80° C. until processing.
Efficacy of treatment was evaluated by (i) measurement of TTR mRNA in liver at 48 hours post-dose, and (ii) measurement of TTR protein in plasma at pre-bleed and at 48 hours post-dose. TTR liver mRNA levels were assayed utilizing the Branched DNA assays—QuantiGene 2.0 (Panomics cat #: QS0011). Briefly, mouse liver samples were ground and tissue lysates were prepared. Liver lysis mixture (a mixture of 1 volume of lysis mixture, 2 volume of nuclease-free water and 10 ul of Proteinase-K/ml for a final concentration of 20 mg/ml) was incubated at 65° C. for 35 minutes. 20 μl of Working Probe Set (TTR probe for gene target and GAPDH for endogenous control) and 80 ul of tissue-lysate were then added into the Capture Plate. Capture Plates were incubated at 55° C.±1° C. (aprx. 16-20 hrs). The next day, the Capture Plates were washed 3 times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 240 g. 100 μl of pre-Amplifier Working Reagent was added into the Capture Plate, which was sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C. Following 1 hour incubation, the wash step was repeated, then 100 μl of Amplifier Working Reagent was added. After 1 hour, the wash and dry steps were repeated, and 100 μl of Label Probe was added. Capture plates were incubated 50° C.±1° C. for 1 hour. The plate was then washed with 1× Wash Buffer, dried and 100 μl Substrate was added into the Capture Plate. Capture Plates were read using the SpectraMax Luminometer following a 5 to 15 minute incubation. bDNA data were analyzed by subtracting the average background from each triplicate sample, averaging the resultant triplicate GAPDH (control probe) and TTR (experimental probe) values, and then computing the ratio: (experimental probe-background)/(control probe-background).
Plasma TTR levels were assayed utilizing the commercially available kit “AssayMax Human Prealbumin ELISA Kit” (AssayPro, St. Charles, Mo., Catalog #EP3010-1) according to manufacturer's guidelines. Briefly, mouse plasma was diluted 1:10,000 in 1× mix diluents and added to pre-coated plates along with kit standards, and incubated for 2 hours at room temperature followed by 5× washes with kit wash buffer. Fifty microliters of biotinylated prealbumin antibody was added to each well and incubated for 1 hr at room temperature, followed by 5× washes with wash buffer. Fifty microliters of streptavidin-peroxidase conjugate was added to each well and plates were incubated for 30 minutes at room temperature followed by washing as previously described. The reaction was developed by the addition of 50 μl/well of chromogen substrate and incubation for 10 minutes at room temperature with stopping of reaction by the addition of 50 μl/well of stop solution. Absorbance at 450 nm was read on a Versamax microplate reader (Molecular Devices, Sunnyvale, Calif.) and data were analyzed utilizing the Softmax 4.6 software package (Molecular Devices).
The results are shown in FIGS. 10-12. FIG. 10 shows that the RNAi agents modified relative to the parent agents AD-45163 and AD-45165 showed RNA silencing activity that was similar or more potent compared with that of the parent agents. FIG. 11 shows that the agents AD-51544 and AD-51545 showed dose dependent silencing activity and that the silencing activity of these agents at a dose of 5 mg/kg was similar to that of the corresponding parent AD-45163. FIG. 12 shows that the agents AD-51546 and AD-51547 also showed dose-dependent silencing activity. Furthermore, the silencing activity of AD-51546 and AD-51547 at a dose of 5 mg/kg was superior to that of the corresponding parent AD-45165.
Example 7
Serum and Liver Pharmacokinetic Profiles of RNAi Agents that Target TTR in Mice
To assess 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 a single IV bolus or subcutaneous (SC) administration. The plasma concentrations and liver concentrations of the agents were assessed at various timepoints after the administration.
The plasma pharmacokinetic parameters are presented in Tables 3 and 4 below. The mean resident time (MRT) in plasma was about 0.2 hours after IV dosing and about 1 hour after SC dosing. At a dose of 25 mg/kg, the agents AD-51544, AD-51545, AD-51546, and AD-51547 showed similar plasma pharmacokinetic properties. Each of these agents had more than 75% bioavailability from the subcutaneous space. Their bioavailability was superior to that of the parent agent AD-45163 that was administered at a higher dose of 30 mg/kg. The subcutaneous bioavailability of AD-51544 and AD-51547 was about 100%, whereas that of AD-51545 was 90% and that of and AD-51546 was 76%.







TABLE 3







Summary of Plasma PK Parameter Estimates After


SC Administration of TTR-GalNAc siRNAs in Mice













30 mpk
25 mpk
25 mpk
25 mpk
25 mpk



AD-45163
AD-51544
AD-51545
AD-51546
AD-51547



(h/c
(h/c
(h/c
(h/c
(h/c



TTR-
TTR-
TTR-
TTR-
TTR-


Parameter
GalNAc)
GalNAc)
GalNAc)
GalNAc)
GalNAc)















Plasma
0.25
1
0.5
1
0.5


Tmax (h)


Plasma
9.6
11.7
10.9
11.7
12.1


Cmax


(μg/mL)


Plasma
12.4
21.9
19.9
20.9
25.3


AUC


(h*μg/mL)


Fsc(%)
79
100
90.1
76.0
99.2
















TABLE 4







Plasma siRNA PK Parameters 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


siRNA Dose (mg/kg)
25
25
25
25















Route of Administration
IV
SC
IV
SC
IV
SC
IV
SC





tmax (h)
 0.083
 1
 0.083
 0.5
 0.083
 1
 0.083
 0.5


Cmax (μg/mL)
96.5a
 11.7
108a
10.9
128a
10.9
123a
12.1


AUC0-last (h · μg/mL)
21.6
 21.9
 22.1
19.9
 27.5
20.9
 25.5
25.3


MRT0-last (h)
 0.17
 1.2
 0.16
 1.1
 0.22
 1.4
 0.19
 1.3


Apparent t1/2β (h)b
ND
ND
ND
 0.49
ND
 1.2
ND
 0.56


Fsc (%)c
—
102
—
90.1
—
76.0
—
99.2





aConcentration at the 1st sampling time (5 min) after IV dosing


bApparent elimination half-life (t1/2β) could not be determined (ND) for all 4 test articles after IV dosing as the terminal phase of the concentration-time profiles was not well defined, as a result, the t1/2β-associated PK parameters (eg, AUC0-∞, CL and Vss) were not reported.


cSC bioavailability, calculated as percentage ratio of AUC0-last after SC and IV dosing at 25 mg/kg






The results also indicated that the RNAi agents AD-45163, AD-51544, AD-51545, AD-51546, and AD-51547 achieved similar or higher concentrations in the liver when administered subcutaneously than when administered by IV bolus. The liver pharmacokinetic parameters are presented in Tables 5 and 6 below. The peak concentration (Cmax) and area under the curve (AUC0-last) in the liver were two to three times higher after subcutaneous administration as compared with IV administration of the same agent at the same dose. Liver exposures were highest for AD-51547 and lowest for AD-51545. The mean resident time (MRT) and elimination half-life were longer for AD-51546 and AD-51547 compared with AD-51544 and AD-51545. Following subcutaneous administration, the approximate MRTs were 40 hours for AD-51546 and 25 hours for AD-51547, whereas the MRTs for AD-51544 and AD-51545 were lower (about 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 Liver PK Parameter Estimates After


SC Administration of TTR-GalNAc siRNAs in Mice













30 mpk
25 mpk
25 mpk
25 mpk
25 mpk



AD-45163
AD-51544
AD-51545
AD-51546
AD-51547



(h/c
(h/c
(h/c
(h/c
(h/c



TTR-
TTR-
TTR-
TTR-
TTR-


Parameter
GalNAc)
GalNAc)
GalNAc)
GalNAc)
GalNAc)















Liver Tmax
8
4
4
2
8


(h)


Liver Cmax
313
126
80
117
174


(μg/g)


Liver AUC
4519
1092
763
2131
4583


(h*μg/g)
















TABLE 6







Liver siRNA PK Parameters 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


siRNA Dose (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


Cmax (μg/g)
67.9
126
37.0
80.5
35.3
117
73.8
174


AUC0-last 
632
1092
324
763
984
2131
1429
4583


(h · μg/g)










MRT0-last (h)
8.7
6.5
5.9
8.5
45.7
40.2
29.4
25.3


Apparent t1/2β (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
The serum stability of RNAi agents AD-51544, AD-51545, AD-51546, and AD-51547 was also assessed in monkeys. The results demonstrated that the antisense and sense strands of AD-51544, AD-51545, and AD-51547 showed serum stability over a period of about 24 hours (data not shown).
Example 9
RNAi Agents Produce Lasting Suppression of TTR Protein in Non-Human Primates
The RNA silencing activity of RNAi agents AD-45163, AD-51544, AD-51545, AD-51546, and AD-51547 was assessed by measuring suppression of TTR protein in serum of cynomologous monkeys following subcutaneous administration of five 5 mg/kg doses (one dose each day for 5 days) or a single 25 mg/kg dose. Pre-dose TTR protein levels in serum were assessed by averaging the levels at 11 days prior to the first dose, 7 days prior to the first dose, and 1 day prior to the first dose. Post-dose serum levels of TTR protein were assessed by determining the level in serum beginning at 1 day after the final dose (i.e., study day 5 in the 5×5 mg/kg group and study day 1 in the 1×25 mg/kg group) until 49 days after the last dose (i.e., study day 53 in the 5×5 mg/kg group and study day 49 in the 1×25 mg/kg group). See FIG. 13.
TTR protein levels were assessed as described in Example 6. The results are shown in FIG. 14 and in Tables 7 and 8.
A maximal suppression of TTR protein of up to about 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 maximal suppression of TTR protein of about 70% was achieved in the groups that received 5×5 mg/kg of AD-45163, AD-51544, AD-51546, and AD-51547 (see Table 7). The agent AD-51545 produced a lesser degree of suppression in both administration protocols. Significant suppression of about 20% or more persisted for up to 49 days after the last dose of AD-51546 and AD-51547 in both the 1×25 mg/kg and 5×5 mg/kg protocols. Generally, better suppression was achieved in the 5×5 mg/kg protocol than in the 1×25 mg/kg protocol.







TABLE 7







Fraction Serum Transthyretin Relative to Pre-dose in Cynomolgus Monkeys (5 mg/kg daily for 5 days)























D-11
D-7
D-1
D5
D7
D9
D11
D14
D18
D22
D26
D32
D39
D46
D53





AD-
0.98
0.99
1.03
0.71
0.52
0.40
0.34
0.27
0.31
0.39
0.48
0.64
0.68
0.81
0.88


45163

















AD-
1.02
0.99
0.99
0.60
0.47
0.37
0.35
0.39
0.48
0.58
0.66
0.74
0.83
0.91
0.92


51544

















AD-
1.03
0.97
1.00
0.73
0.65
0.63
0.69
0.68
0.78
0.87
0.97
1.00
1.03
1.06
1.09


51545

















AD-
1.01
0.97
1.02
0.59
0.42
0.35
0.30
0.32
0.43
0.58
0.66
0.77
0.92
0.93
0.97


51546

















AD-
0.99
0.99
1.02
0.74
0.54
0.41
0.34
0.34
0.39
0.49
0.51
0.53
0.65
0.70
0.77


51547
















TABLE 8







Fraction Serum Transthyretin Relative to Pre-dose in Cynomolgus Monkeys (25 mg/kg)























D-11
D-7
D-1
D1
D3
D5
D7
D10
D14
D18
D22
D28
D35
D42
D49





AD-
1.04
1.01
0.95
0.99
0.84
0.67
0.57
0.44
0.45
0.51
0.58
0.66
0.72
0.78
0.85


45163

















AD-
1.01
1.04
0.95
0.92
0.69
0.57
0.49
0.48
0.56
0.65
0.69
0.77
0.83
0.87
0.94


51544

















AD-
0.98
1.02
0.99
0.87
0.77
0.69
0.71
0.72
0.84
0.90
0.92
0.99
1.00
1.00
1.00


51545

















AD-
1.04
1.03
0.93
0.89
0.71
0.62
0.53
0.50
0.55
0.70
0.70
0.69
0.72
0.79
0.84


51546

















AD-
0.96
1.03
1.01
1.19
0.90
0.70
0.54
0.48
0.50
0.50
0.52
0.58
0.62
0.70
0.72


51547









Example 10
Tolerability of RNAi Agents that Target TTR
In Cytokine Evaluation in Whole Blood Assay
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 either 300 nM DOTAP transfected or 1 μM without transfection reagent (free siRNA). There was less than a two fold change for the following cytokines/chemokines: G-CSF, IFN-γ, 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
To assess in vivo tolerability, RNAi agents were injected subcutaneously in 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 observed at 6 or 24 hours after subcucutaneous injection of AD-51544, AD-51545, AD-51546, or AD-51547.
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 (cynomologous monkeys) with dose volumes between 1-2 ml per site. No erythema or edema was observed at injection sites.
Single SC Dose Rat Tolerability Study
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); gross and microscopic evaluation (kidney, liver, lung, lymph node, spleen, testes, thymus, aorta, heart, intestine (small and large).







TABLE 9







Single SC Dose Rat Tolerability Study: 100, 250, 500


& 750 mg/kg of AD-45163 in Sprague Dawley Rats













Dose
Dose

No. Male




Level
Volume
Route &
Sprague
Day of


Group
(mg/kg)
(ml/kg)
Regimen
Dawley Rats
Necropsy















PBS
0
10
SC
7/group
Day 4


AD-45163
100

Injection
(5 Tox


Parent
250

Day 1
animals,



500

(2 sites)
2 TK



750


animals)









The results showed no test article-related clinical signs of toxicity, effects on body weight, organ weights, or clinical chemistry. No histopathology was observed in heart, kidneys, testes, spleen, liver, and thymus. There was a non-adverse, slight test article-related increase in WBC (↑68%, primarily attributed to increase in NEUT and MONO) at 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
To assess the tolerability of repeated subcutaneous administrations of AD-45163, daily subcutaneous injections of 300 mg/kg were given for 5 days, and a necropsy was performed on day 6. The study design is shown in Table 10.







TABLE 10







Five Day Repeat Dose Tolerability Study in Rat














Dose







Level
Conc
No of Tox



Group
(kmg/kg
(mg/mL)
Animals
Nx Day 6

















PBS
0
0
2M, 2F
2M, 2F



AD-45163
300
150
2M, 2F
2M, 2F










The following outcome variables were assessed: clinical signs, body weights, hematology, clinical chemistry and coagulation, organ weights, gross and microscopic evaluation (liver, spleen, kidney, heart, GI tract and first and last injection site). The results showed no test article-related clinical signs, body weight or organ weight effects, and also no test article-related findings in clinical hematology or chemistry. There was a possible slight prolongation of activated partial thromboplastin time (APTT) on day 6 (20.4 vs. 17.4 sec). Histopathology revealed no test article-related findings 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 multifocal mononuclear infiltration, not adverse. These results indicate that five daily 300 mg/kg doses of the parent RNAi agent AD-45163 are well tolerated in rats.
Example 11
RNAi Agents Produce Lasting Suppression of TTR Protein in Non-Human Primates
The RNA silencing activity of RNAi agent AD-51547 was assessed by measuring suppression of TTR protein in the serum of cynomologous monkeys following subcutaneous administration of a “loading phase” of the RNAi agent: five daily doses of either 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 either 2.5 mg/kg, 5 mg/kg or 10 mg/kg for 4 weeks. Pre-dose TTR protein levels in serum were assessed by averaging the levels at 11 days prior to the first dose, 7 days prior to the first dose, and 1 day prior to the first dose. Post-dose serum levels of TTR protein were assessed by determining the level in serum relative to pre-dose beginning at 1 day after the loading phase was completed until 40 days after the last dose of the maintenance phase (i.e., study day 70).
TTR protein levels were assessed as described in Example 6. The results are shown in FIG. 15.
A maximal suppression of TTR protein of up to about 80% was achieved in all of the groups that received either 2.5 mg/kg, 5 mg/kg or 10 mg/kg of AD-51547. Nadir knockdown was achieved in all of the groups by about day 14, the suppression sustained at nadir knockdown levels with a weekly maintenance dose of either 2.5 mg/kg, 5 mg/kg or 10 mg/kg of AD-51547. The levels of TTR had not returned to baseline more than 40 days after the day of administration of the last maintenance dose for the 5 and 2.5 mg/kg dose levels.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

## Claims

1. A double stranded RNAi agent comprising a sense strand complementary to an antisense strand, wherein said antisense strand comprises a sequence that is complementary to nucleotides 504 to 526 of the transthyretin (TTR) gene (SEQ ID NO:1), wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein said double stranded RNAi agent is represented by formula (III):







(III)


sense: 5′ np -Na -(X X X)i-Nb -Y Y Y -Nb -(Z Z Z)j- 


 


Na-nq 3′


 


antisense: 3′ np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′-Nb′-


 


(Z′Z′Z′)l-Na′- nq′ 5′





wherein:
j=1; and i, k, and l are 0;
p′ is 2; p, q, and q′ are 0;
each Na and Na′ independently represents an oligonucleotide sequence comprising 2-10 nucleotides which are modified nucleotides;
each Nb and Nb′ independently represents an oligonucleotide sequence comprising 0-7 nucleotides which are modified nucleotides;
np′ represents an overhang nucleotide;
YYY, ZZZ, and Y′Y′Y′, each independently represent one motif of three identical modifications on three consecutive nucleotides, wherein the Y nucleotides contain a 2′-fluoro modification, the Y′ nucleotides contain a 2′-O-methyl modification, and the Z nucleotides contain a 2′-O-methyl modification;
and
wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

2. The RNAi agent of claim 1, wherein the YYY motif occurs at or near the cleavage site of the sense strand; or wherein the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end.

3. The RNAi agent of claim 1, wherein the modifications on the Na, Na′, Nb and Nb′ nucleotides are each independently selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and combinations thereof.

4. The RNAi agent of claim 3, wherein the modifications on the Na, Na′, Nb and Nb′ nucleotides are 2′-O-methyl, 2′-fluoro or both.

5. The RNAi agent of claim 1, wherein the ligand is

6. The RNAi agent of claim 1, wherein the ligand is attached to the 3′ end of the sense strand.

7. The RNAi agent of claim 6, wherein the RNAi agent is conjugated to the ligand as shown in the following schematic




wherein X is O or S.

8. The RNAi agent of claim 7, wherein the RNAi agent is conjugated to the ligand as shown in the following schematic

9. The RNAi agent of claim 1 further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

10. The RNAi agent of claim 9, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand.

11. The RNAi agent of claim 10, wherein said strand is the antisense strand or the sense strand.

12. The RNAi agent of claim 1, wherein the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

13. The RNAi agent of claim 1, wherein the p′ overhang nucleotides are complementary to the target mRNA or wherein the p′ overhang nucleotides are non-complementary to the target mRNA.

14. The RNAi agent of claim 1, wherein at least one np′ is linked to a neighboring nucleotide via a phosphorothioate linkage.

15. The RNAi agent of claim 14, wherein all np′ are linked to neighboring nucleotides via phosphorothioate linkages.

16. The RNAi agent of claim 1, wherein said RNAi agent is selected from the group consisting of AD-51546 and AD-51547.

17. The RNAi agent of claim 1, wherein the RNAi agent is AD-51547.

18. The double stranded RNAi agent of claim 1, wherein the sense strand comprises the nucleotide sequence 5′-UGGGAUUUCAUGUAACCAAGA-3′ (SEQ ID NO:2211).

19. The double stranded RNAi agent of claim 1, wherein the sense strand comprises the nucleotide sequence 5′-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′ (SEQ ID NO:2) and the antisense strand comprises the nucleotide sequence 5′-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′ (SEQ ID NO:3),
wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; and L96 is a GalNAc3 ligand.

20. A double stranded RNAi agent, comprising a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence 5′-UfgGfgAfuUfuCfAfUfgUfaacCfaAfgAfL96-3′ (SEQ ID NO:2) and the antisense strand comprises the nucleotide sequence 5′-uCfuUfgGfUfUfaCfaugAfaAfuCfcCfasUfsc-3′ (SEQ ID NO:3),
wherein a, g, c, and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf, and Uf are 2′-fluoro A, G, C, and U; s is a phosphorothioate linkage; and L96 is a GalNAc3 ligand.

21. An isolated cell containing an RNAi agent of any one of claims 1, 17, or 20.

22. A pharmaceutical composition comprising an RNAi agent of any one of claims 1, 17, or 20.

23. The pharmaceutical composition of claim 22, wherein the RNAi agent is administered in an unbuffered solution.

24. The pharmaceutical composition of claim 23, wherein said unbuffered solution is saline or water.

25. The pharmaceutical composition of claim 22, wherein said RNAi agent is administered with a buffer solution.

26. The pharmaceutical composition of claim 25, wherein said buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

27. The pharmaceutical composition of claim 26, wherein said buffer solution is phosphate buffered saline (PBS).

28. A method of inhibiting expression of a transthyretin (TTR) in a cell comprising contacting said cell with an RNAi agent of any one of claims 1, 17, or 20 in an amount effective to inhibit expression of said TTR in said cell, thereby inhibiting expression of said transthyretin (TTR) in said cell.

29. A kit for performing the method of claim 28, comprising
a) said RNAi agent, and
b) instructions for use.

30. A method of treating a TTR-associated disease in a subject, comprising administering to said subject a therapeutically effective amount of an RNAi agent of any one of claims 1, 17, or 20, thereby treating said TTR-associated disease in said subject.

31. The method of claim 30, wherein said subject is a human.

32. The method of claim 30, wherein said subject carries a TTR gene mutation that is associated with the development of a TTR-associated disease.

33. The method of claim 30, wherein said TTR-associated disease is selected from the group consisting of senile systemic amyloidosis (SSA), systemic familial amyloidosis, familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/Central Nervous System (CNS) amyloidosis, and hyperthyroxinemia.

34. The method of claim 30, wherein said subject has a TTR-associated amyloidosis and said method reduces an amyloid TTR deposit in said subject.

35. The method of claim 30, wherein said RNAi agent is administered to the subject subcutaneously.

36. A kit for performing the method of claim 30, comprising
a) said RNAi agent,
b) instructions for use, and
c) optionally, means for administering said RNAi agent to said subject.