# Patent

## Title
Lipid formulated compositions and methods for inhibiting expression of transthyretin (TTR)

## Abstract

The invention relates to lipid formulated double-stranded ribonucleic acid (dsRNA) targeting a transthyretin (TTR) gene, and methods of using the dsRNA to inhibit expression of TTR.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a National Stage of International Application No. PCT/US2010/055311 filed Nov. 3, 2010, which claims the benefit of U.S. Provisional Application Ser. No. 61/257,763, filed Nov. 3, 2009, and claims the benefit of U.S. Provisional Application Ser. No. 61/285,606, filed Dec. 11, 2009; and claims the benefit of U.S. Provisional Application Ser. No. 61/288,224, filed Dec. 18, 2009 and claims the benefit of U.S. Provisional Application Ser. No. 61/300,299, filed Feb. 1, 2010, all of which are incorporated herein by reference, in their entirety, for all purposes.


REFERENCE TO SEQUENCE LISTING
This application includes a Sequence Listing submitted electronically as a text file named 17628PCT_CRF_sequencelisting.txt, created on Nov. 3, 2010, with a size of 466,944 bytes. The sequence listing is incorporated by reference.
FIELD OF THE INVENTION
The invention relates to lipid formulated double-stranded ribonucleic acid (dsRNA) targeting a transthyretin (TTR) gene, and methods of using the dsRNA to inhibit expression of TTR.
BACKGROUND OF THE INVENTION
Transthyretin (TTR) is a secreted thyroid hormone-binding protein. TTR binds and transports retinol binding protein (RBP)/Vitamin A, and serum thyroxine (T4) in plasma and cerebrospinal fluid.
Both normal-sequence TTR and variant-sequence TTR cause amyloidosis. Normal-sequence TTR causes cardiac amyloidosis in people who are elderly and is termed senile systemic amyloidosis (SSA) (also called senile cardiac amyloidosis (SCA)). 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. The liver is the major site of TTR expression. Other significant sites of expression include the choroid plexus, retina and pancreas.
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 called leptomeningeal/CNS (Central Nervous System) amyloidosis.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) disclosed the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.).
U.S. Publication No. 20070207974 (U.S. patent application Ser. No. 11/095,383, filed Mar. 30, 2005) discloses functional and hyperfunctional siRNAs. U.S. Publication No. 20090082300 (U.S. patent application Ser. No. 12/273,731, filed Nov. 19, 2008) discloses antisense molecules targeting transthyretin (TTR). U.S. Pat. No. 7,250,496 (U.S. patent application Ser. No. 10/310,914, filed Dec. 6, 2002) discloses microRNAs targeting transthyretin (TTR). WO 2010/048228 (International application no. PCT/US2009/061381, filed Oct. 20, 2009) and U.S. publication no. 20100120893 (U.S. patent application Ser. No. 12/582,669, filed Oct. 20, 2009) disclose compositions (e.g., siRNA) and methods for inhibiting expression of transthyretin (TTR).
SUMMARY OF THE INVENTION
Disclosed is a method for reducing a TTR amyloid deposit in a subject by administering an effective amount of TTR dsRNA for inhibiting expression of TTR in the subject, wherein the TTR dsRNA having a sense strand and an antisense strand and a duplex structure between 15 and 30 basepairs in length, the antisense strand having 15 or more contiguous nucleotides of SEQ ID NO:170. In some embodiments, the antisense strand comprises SEQ ID NO:170 and the sense strand comprises SEQ ID NO:169; in other embodiments, the antisense strand consists of SEQ ID NO:730 and the sense strand consists of SEQ ID NO:729.
The dsRNA can include at least one modified nucleotide, e.g., a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
In one embodiment, antisense strand consists of SEQ ID NO:1010 and the sense strand consists of SEQ ID NO:1009.
The dsRNA composition can be in a nucleic acid lipid formulation comprising a cationic lipid, e.g., DLinDMA, MC3, or TechG1 (C12-200).
In some embodiments, TTR amyloid deposit comprises V30M mutant TTR. The subject can be a human subject. The effective amount can be, e.g., a dose of 0.01-30.0 mg/kg.
In some embodiments the method includes determining TTR deposition in the subject.
Also disclosed in a composition consisting of the drug product ALN-TTR01 of Table 17 comprising a pH in the range of 6.8 to 7.8; a osmolality in the range of 250-350 mOsm/kg; a lipid: siRNA Ratio in the range of 5.6-8.4 mg/mg, and a particle size in the range of 60-120 nm≦0.15, and methods of using this composition for reducing a TTR amyloid deposit in a subject.
Additionally disclosed is a method for reducing TTR mRNA expression in a primate for at least 3 days by administering to the primate a single dose of 0.01-30.0 mg/kg of a TTR dsRNA consisting of AD-18328. In some embodiments, the TTR mRNA expression is reduced for 30 days. The primate can be a human. In some embodiments, the single dose is 1.0 or 3.0 or 10.0 mg/kg. The TTR dsRNA can be lipid formulated, e.g., SNALP formulated (ALN-TTR01). In some embodiments, the method includes determining TTR mRNA expression in the subject.
Also disclosed is a method for reducing TTR mRNA expression in a subject comprising administering to the subject a composition at a dose of 0.03 mg/kg, the composition comprising a TTR dsRNA AD-18328 and a lipid formulation. In some embodiments, the subject is a human. The lipid formulation can include a cationic lipid MC3 or Tech G1 (C12-200) and can be LNP-12 (Tech G1/DSPC/Cholesterol/PEG200-DMG in a mol ratio of 50/10/38.5/1.5). The method can include determining TTR mRNA expression in the subject.
The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.



DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of TNFalpha and IFNalpha levels in cultured human PBMCs following transfection with TTR siRNAs.
FIGS. 2A and 2B are dose response curves for AD-18324 and AD-18328, respectively, in HepG2 cells.
FIG. 3 is a dose response curve for AD-18246 in HepG2 cells.
FIG. 4A and FIG. 4B show inhibition of liver mRNA and plasma protein levels, respectively, in transgenic H129-mTTR-KO/iNOS-KO/hTTR mice by an intravenous bolus administration of TTR-dsRNA (AD-18324, AD-18328 and AD-18246) formulated in LNP01.
FIG. 5 is a graph summarizing the measurements of TTR mRNA levels in livers of non-human primates following 15-minute intravenous infusion of TTR-dsRNA (AD-18324 and AD-18328) formulated in SNALP.
FIG. 6A and FIG. 6B show inhibition of human V30M TTR liver mRNA and serum protein levels, respectively, in transgenic mice by an intravenous bolus administration of SNALP-18328. Group means were determined, normalized to the PBS control group, and then plotted. Error bars represent standard deviations. The percentage reduction of the group mean, relative to PBS, is indicated for the SNALP-1955 and SNALP-18328 groups. (*** p<0.001, One-way ANOVA, with Dunn's post-hoc test).
FIG. 7A and FIG. 7B show the durability of reduction of human V30M TTR liver mRNA and serum protein levels, respectively, in transgenic mice over 22 days following a single intravenous bolus administration of SNALP-18328. Group means were determined. TTR/GAPDH mRNA levels were normalized to day 0 levels and plotted. The percent reduction of normalized TTR mRNA levels relative to SNALP-1955 for each time point were calculated and are indicated for the SNALP-18328 groups. (*** p<0.001, One-way ANOVA, with Dunn's post-hoc test).
FIG. 8 shows the timecourse of TTR serum protein levels in non-human primates over 14 days following a single 15-minute intravenous infusion of SNALP-18328.
FIG. 9 shows reduction of TTR-immunoreactivity in various tissues of human V30M TTR/HSF-1 knock-out mice following intravenous bolus administration of SNALP-18328. E, esophagus; S, stomach; I1, intestine/duodenum; I4, intestine/colon; N, nerve; D, dorsal root ganglia.
FIG. 10 shows the measurements of TTR mRNA levels in livers of non-human primates following 15-minute intravenous infusion of XTC-SNALP-18328.
FIGS. 11A and 11B show the measurements of TTR mRNA and serum protein levels, respectively, in livers of non-human primates following 15-minute intravenous infusion of LNP09-18328 or LNP11-18328. FIG. 11C shows the timecourse of TTR serum protein levels over 28 days following a 15-minute intravenous infusion of 0.3 mg/kg LNP09-18328, as compared to the PBS control group.
FIG. 12 shows the sequence of human TTR mRNA (Ref. Seq. NM—000371.3, SEQ ID NO:1331).
FIGS. 13A and 13B are the sequences of human and rat TTR mRNA, respectively. FIG. 13A is the sequence of human TTR mRNA (Ref. Seq. NM—000371.2, SEQ ID NO:1329). FIG. 13B is the sequence of rat TTR mRNA (Ref. Seq. NM—012681.1, SEQ ID NO:1330).
FIG. 14 shows the nucleotide alignment of NM—000371.3, NM—000371.2, and AD-18328.
FIG. 15 illustrates symptoms and mutations in TTR associated with familial amyloidotic neuropathy, familial amyloidotic cardiomyopathy and CNS amyloidosis.
FIG. 16 shows reduction of TTR mRNA levels in the liver with SNALP-18534 with different infusion durations. Groups of animals (n=4/group) were administered 1 mg/kg SNALP-18534 via a 15-minute, or 1, 2, or 3 hour infusion. Forty-eight hours later, rats were euthanized and livers harvested. TTR and GAPDH mRNA levels were measured from liver lysates using the Quantigene bDNA assay. The ratio of TTR to GAPDH mRNA levels was calculated for each animal. Group means were determined and normalized to a PBS control group, and then plotted. Error bars represent standard deviations. (*** p<0.001, One-way ANOVA with Bonferroni post-hoc test, relative to PBS).
FIG. 17 shows the measurements of TTR mRNA levels in livers of rats following 15-minute intravenous infusion of LNP07-18534 or LNP08-18534.
FIG. 18 shows in vivo inhibition of endogenous TTR mRNA levels in livers of Sprague-Dawley Rats following a 15-min IV infusion of LNP09-18534 or LNP11-18534. Groups of animals (n=4/group) were intravenously administered 0.01, 0.03, 0.1, or 0.3 mg/kg LNP09-18534, LNP-11-18534; or PBS via a 15-minute infusion. Forty-eight hours later, animals were euthanized and livers harvested. TTR and GAPDH mRNA levels were measured from liver biopsy lysates using the Quantigene bDNA assay. The ratio of TTR to GAPDH mRNA levels was calculated for each animal. Group means were determined, normalized to the PBS control group, and then plotted. Error bars represent standard deviations.
FIG. 19 shows the efficacy of LNP12 formulated siRNA targeting TTR in non-human primates. Data points represent group mean±s.d.
FIG. 20 is a graph with results from a sGLP study in NHP illustrating the durability of mRNA suppression by ALN-TTR01.
FIG. 21 is a graph illustrating regression of TTR deposits in various tissues of mature animals (hV30M TTR/HSF-1 knock-out mice) following intravenous bolus administration of SNALP-18328 (ALN-TTR01).



DETAILED DESCRIPTION OF THE INVENTION
The invention provides dsRNAs and methods of using the dsRNAs for inhibiting the expression of a TTR gene in a cell or a mammal where the dsRNA targets a TTR gene. The invention also provides compositions and methods for treating pathological conditions and diseases, such as a TTR amyloidosis, in a mammal caused by the expression of a TTR gene. dsRNA directs the sequence-specific degradation of mRNA.
1. DEFINITIONS
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“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. 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.
As used herein, “transthyretin” (“TTR”) refers to a gene in a cell. TTR is also known as ATTR, HsT2651, PALB, prealbumin, TBPA, and transthyretin (prealbumin, amyloidosis type I). The sequence of a human TTR mRNA transcript can be found at NM—000371. The sequence of mouse TTR mRNA can be found at NM—013697.2, and the sequence of rat TTR mRNA can be found at 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.
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.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. 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 “double-stranded RNA” or “dsRNA,” as used herein, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, 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 at least one non-ribonucleotide, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, as used in this specification, “dsRNA” may include chemical modifications to ribonucleotides, including substantial modifications at multiple nucleotides and including all types of modifications disclosed herein or known in the art. Any such modifications, as used in an siRNA type molecule, are encompassed by “dsRNA” for the purposes of this specification and claims.
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, a dsRNA may comprise one or more nucleotide overhangs. The term “siRNA” is also used herein to refer to a dsRNA as described above.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA 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 dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. 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.
Exemplary dsRNA targeting TTR include the duplex AD-18328 (SEQ ID NOS: 1009 and 1010) and the like:














location

SEQ



on

ID


strand
NM_000371.2
Sequence 5′ to 3′
NO:







sense
518
GuAAccAAGAGuAuuccAudTdT
1009


 


antisense
536
AUGGAAuACUCUUGGUuACdTdT
1010


 


sense
518
AUGGAAUACUCUUGGUUAC
 170


 


antisense
536
AUGGAAUACUCUUGGUUACNN
 450


 


sense
518
AUGGAAUACUCUUGGUUACdTdT
 730


 


antisense
536
AUGGAAuACUCUUGGUuACdTdT
1010









As used herein, the term“nucleoc acid lipid particle” includes the term “SNALP” and refers to a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as a dsRNA or a plasmid from which a dsRNA is transcribed. Nucleic acid lipid particles, e.g., SNALP are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed on Apr. 15, 2008. These applications are hereby incorporated by reference.
“Introducing into a cell,” when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein or known in the art.
The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of” and the like in as far as they refer to a TTR gene, herein refer to the at least partial suppression of the expression of a TTR gene, as manifested by a reduction of the amount of mRNA which may be isolated from a first cell or group of cells in which a TTR gene is transcribed and which has or have been treated such that the expression of a TTR gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of




  
    
      
        
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to TTR gene expression, e.g., the amount of protein encoded by a TTR gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g., apoptosis. In principle, TTR gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given dsRNA inhibits the expression of a TTR gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of a TTR gene is suppressed by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a TTR gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide featured in the invention. In some embodiments, a TTR gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide featured in the invention.
As used herein in the context of TTR expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by TTR expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by TTR expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition; or to slow or reverse the progression of such condition, such as the slowing the progression of a TTR amyloidosis, such as FAP. 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.
As used herein, the phrases “effective amount” refers to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by TTR expression or an overt symptom of pathological processes mediated by TTR expression, e.g., reduction of TTR amyloid deposit. The specific amount that is effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by TTR expression, the patient's history and age, the stage of pathological processes mediated by TTR expression, and the administration of other anti-pathological processes mediated by TTR expression agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter. For example, a therapeutically effective amount of a dsRNA targeting TTR can reduce TTR serum levels by at least 25%. In another example, a therapeutically effective amount of a dsRNA targeting TTR can improve liver function or renal function by at least 25%.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
II. Double-Stranded Ribonucleic Acid (dsRNA)
As described in more detail herein, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a TTR gene in a cell or mammal, e.g., in a human having a TTR amyloidosis, where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of aTTR gene, and where the region of complementarity is less than 30 nucleotides in length, generally 19-24 nucleotides in length, and where said dsRNA, upon contact with a cell expressing said TTR gene, inhibits the expression of said TTR gene by at least 30% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. Expression of a TTR gene can be reduced by at least 30% when measured by an assay as described in the Examples below. For example, expression of a TTR gene in cell culture, such as in Hep3B cells, can be assayed by measuring TTR mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by ELISA assay. The dsRNA of the invention can further include one or more single-stranded nucleotide overhangs.
The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. The dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of a TTR gene, the other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 or between 25 and 30, or between 18 and 25, or between 19 and 24, or between 19 and 21, or 19, 20, or 21 base pairs in length. In one embodiment the duplex is 19 base pairs in length. In another embodiment the duplex is 21 base pairs in length. When two different siRNAs are used in combination, the duplex lengths can be identical or can differ.
Each strand of the dsRNA of invention is generally between 15 and 30, or between 18 and 25, or 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, each is strand is 25-30 nucleotides in length. Each strand of the duplex can be the same length or of different lengths. When two different siRNAs are used in combination, the lengths of each strand of each siRNA can be identical or can differ.
The dsRNA of the invention can include one or more single-stranded overhang(s) of one or more nucleotides. In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. In another embodiment, the antisense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the sense strand. In further embodiments, the sense strand of the dsRNA has 1-10 nucleotides overhangs each at the 3′ end and the 5′ end over the antisense strand.
A dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties than the blunt-ended counterpart. In some embodiments the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. A dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Generally, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA can also have a blunt end, generally located at the 5′-end of the antisense strand. Such dsRNAs can have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In one embodiment, a TTR gene is a human TTR gene. In specific embodiments, the sense strand of the dsRNA is one of the sense sequences from Tables 3A, 3B, 4, 6A, 6B, or 7, and the antisense strand is one of the antisense sequences of Tables 3A, 3B, 4, 6A, 6B, or 7. Alternative antisense agents that target elsewhere in the target sequence provided in Tables 3A, 3B, 4, 6A, 6B, or 7 can readily be determined using the target sequence and the flanking TTR sequence.
The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 3A, 3B, 4, 6A, 6B, and 7, the dsRNAs featured in the invention can include at least one strand of a length described herein. It can be reasonably expected that shorter dsRNAs having one of the sequences of Tables 3A, 3B, 4, 6A, 6B, or 7 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 3, 4, 6 or 7, and differing in their ability to inhibit the expression of a TTR gene in an assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention. Further, dsRNAs that cleave within a desired TTR target sequence can readily be made using the corresponding TTR antisense sequence and a complementary sense sequence.
In addition, the dsRNAs provided in Tables 3A, 3B, 4, 6A, 6B, or 7 identify a site in a TTR that is susceptible to RNAi based cleavage. As such, the present invention further features dsRNAs that target within the sequence targeted by one of the agents of the present invention. As used herein, a second dsRNA is said to target within the sequence of a first dsRNA if the second dsRNA cleaves the message anywhere within the mRNA that is complementary to the antisense strand of the first dsRNA. Such a second dsRNA will generally consist of at least 15 contiguous nucleotides from one of the sequences provided in Tables 3A, 3B, 4, 6A, 6B, or 7 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a TTR gene.
Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. The cleavage site on the target mRNA of a dsRNA can be determined using methods generally known to one of ordinary skill in the art, e.g., the 5′-RACE method described in Soutschek et al., Nature; 2004, Vol. 432, pp. 173-178 (which is herein incorporated by reference for all purposes). In an embodiment, using the 5′-RACE method described by Soutschek et al., ALN-18328 was determined to cleave a TTR mRNA between the guanine nucleotide at position 636 of SEQ ID NO:1331 (NM—000371.3) and the adenine nucleotide at position 637 of SEQ ID NO:1331. In an embodiment, it was determined that ALN-18328 does not cleave a TTR mRNA between the adenine nucleotide at position 637 of SEQ ID NO:1331 and the guanine nucleotide at position 638 of SEQ ID NO:1331.
The dsRNA featured in the invention can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA featured in the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of a TTR gene, the dsRNA generally does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a TTR gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of a TTR gene is important, especially if the particular region of complementarity in a TTR gene is known to have polymorphic sequence variation within the population.
Modifications
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Specific examples of dsRNA compounds useful in this invention include dsRNAs containing modified backbones or no natural internucleoside linkages. As defined in this specification, dsRNAs having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified dsRNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
Modified dsRNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference
Modified dsRNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or ore or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other suitable dsRNA mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, a dsRNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of a dsRNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
Other embodiments of the invention are dsRNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. Also preferred are dsRNAs having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified dsRNAs may also contain one or more substituted sugar moieties. Preferred dsRNAs comprise one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred dsRNAs comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an dsRNA, or a group for improving the pharmacodynamic properties of an dsRNA, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxy-alkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
Other preferred modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the dsRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
dsRNAs may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
Conjugates
Another modification of the dsRNAs of the invention involves chemically linking to the dsRNA one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the dsRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
Representative U.S. patents that teach the preparation of such dsRNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within a dsRNA. The present invention also includes dsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compounds or “chimeras,” in the context of this invention, are dsRNA compounds, particularly dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These dsRNAs typically contain at least one region wherein the dsRNA is modified so as to confer upon the dsRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid: An additional region of the dsRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of dsRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region.
In certain instances, the dsRNA may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to dsRNAs in order to enhance the activity, cellular distribution or cellular uptake of the dsRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such dsRNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of dsRNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the dsRNA still bound to the solid support or following cleavage of the dsRNA in solution phase. Purification of the dsRNA conjugate by HPLC typically affords the pure conjugate.
Vector Encoded dsRNAs
In another aspect, TTR dsRNA molecules are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are generally DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
Any viral vector capable of accepting the coding sequences for the dsRNA molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate.
For example, lentiviral vectors featured in the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors featured in the invention can be made to target different cells by engineering the vectors to express different capsid protein serotypes. For example, an AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Selection of recombinant viral vectors suitable for use in the invention, methods for inserting nucleic acid sequences for expressing the dsRNA into the vector, and methods of delivering the viral vector to the cells of interest are within the skill in the art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire disclosures of which are herein incorporated by reference.
Viral vectors can be derived from AV and AAV. In one embodiment, the dsRNA featured in the invention is expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
A suitable AV vector for expressing the dsRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector featured in the invention may be a eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA polymerase II (e.g., CMV early promoter or actin promoter or U1 snRNA promoter) or generally RNA polymerase III promoter (e.g., U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g., the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Generally, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
dsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKOTM). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single TTR gene or multiple TTR genes over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
TTR specific dsRNA molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
III. Pharmaceutical Compositions Containing dsRNA
In one embodiment, the invention provides pharmaceutical compositions containing a dsRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the dsRNA is useful for treating a disease or disorder associated with the expression or activity of a TTR gene, such as pathological processes mediated by TTR expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.
The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of TTR genes.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.0059 mg/kg, 0.01 mg/kg, 0.0295 mg/kg, 0.05 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.628 mg/kg, 2 mg/kg, 3 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose.
In one embodiment, the dosage is between 0.01 and 0.2 mg/kg. For example, the dsRNA can be administered at a dose of 0.01 mg/kg, 0.02 mg/kg, 0.03 mg/kg, 0.04 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.07 mg/kg 0.08 mg/kg 0.09 mg/kg, 0.10 mg/kg, 0.11 mg/kg, 0.12 mg/kg, 0.13 mg/kg, 0.14 mg/kg, 0.15 mg/kg, 0.16 mg/kg, 0.17 mg/kg, 0.18 mg/kg, 0.19 mg/kg, or 0.20 mg/kg.
In one embodiment, the dosage is between 0.005 mg/kg and 1.628 mg/kg. For example, the dsRNA can be administered at a dose of 0.0059 mg/kg, 0.0295 mg/kg, 0.0590 mg/kg, 0.163 mg/kg, 0.543 mg/kg, 0.5900 mg/kg, or 1.628 mg/kg.
In one embodiment, the dosage is between 0.2 mg/kg and 1.5 mg/kg. For example, the dsRNA can be administered at a dose of 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, or 1.5 mg/kg.
The dsRNA can be administered at a dose of 0.03 mg/kg, or 0.03, 0.1, 0.2, or 0.4 mg/kg.
The pharmaceutical composition may be administered once daily or the dsRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on TTR levels is long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals, or at not more than 5, 6, 7, 8, 9, or 10 week intervals.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by TTR expression. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a plasmid expressing human TTR. Another suitable mouse model is a transgenic mouse carrying a transgene that expresses human TTR.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
The dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by target gene expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Administration
The present invention also includes pharmaceutical compositions and formulations which include the dsRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical, pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intraparenchymal, intrathecal or intraventricular, administration.
The dsRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
The present invention includes pharmaceutical compositions that can be delivered by injection directly into the brain. The injection can be by stereotactic injection into a particular region of the brain (e.g., the substantia nigra, cortex, hippocampus, striatum, or globus pallidus), or the dsRNA can be delivered into multiple regions of the central nervous system (e.g., into multiple regions of the brain, and/or into the spinal cord). The dsRNA can also be delivered into diffuse regions of the brain (e.g., diffuse delivery to the cortex of the brain).
In one embodiment, a dsRNA targeting TTR can be delivered by way of a cannula or other delivery device having one end implanted in a tissue, e.g., the brain, e.g., the substantia nigra, cortex, hippocampus, striatum, corpus callosum or globus pallidus of the brain. The cannula can be connected to a reservoir of the dsRNA composition. The flow or delivery can be mediated by a pump, e.g., an osmotic pump or minipump, such as an Alzet pump (Durect, Cupertino, Calif.). In one embodiment, a pump and reservoir are implanted in an area distant from the tissue, e.g., in the abdomen, and delivery is effected by a conduit leading from the pump or reservoir to the site of release. Infusion of the dsRNA composition into the brain can be over several hours or for several days, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for delivery to the brain are described, for example, in U.S. Pat. Nos. 6,093,180, and 5,814,014.
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the dsRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearoylphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, dsRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
Liposomal Formulations
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
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. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
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 drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., 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.
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 liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that 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 a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is 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, 1992, 19, 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.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
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 NovasomeTM I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NovasomeTM II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. 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 (also known as the “head”) 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 and cosmetic 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).
Nucleic Acid Lipid Particles
In one embodiment, a TTR dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. In some embodiments the lipid to dsRNA ratio can be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 11:1.
In general, the lipid-nucleic acid particle is suspended in a buffer, e.g., PBS, for administration. In one embodiment, the pH of the lipid formulated siRNA is between 6.8 and 7.8, e.g., 7.3 or 7.4. The osmolality can be, e.g., between 250 and 350 mOsm/kg, e.g., around 300, e.g., 298, 299, 300, 301, 302, 303, 304, or 305.
The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200 or Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Cl6), or a PEG-distearyloxypropyl (C18). Other examples of PEG conjugates include PEG-cDMA (N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine), mPEG2000-DMG (mPEG-dimyrystylglycerol (with an average molecular weight of 2,000) and PEG-C-DOMG (R-3-[(w-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxlpropyl-3-amine). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 1.0, 1.1., 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
In one embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
For example, the lipid-siRNA particle can include 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
In still another embodiment, the compound 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1) can be used to prepare lipid-siRNA particles. For example, the dsRNA can be formulated in a lipid formulation comprising Tech-G1, distearoyl phosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG at a molar ratio of 50:10:38.5:1.5 at a total lipid to siRNA ratio of 7:1 (wt:wt).
LNP01
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula 1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared, as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-siRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.




LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference. Additional exemplary formulations are described in Table A.










TABLE A








cationic lipid/non-cationic lipid/




cholesterol/PEG-lipid conjugate



Cationic
Mol % ratios



Lipid
Lipid:siRNA ratio


















SNALP
DLinDMA
DLinDMA/DPPC/Cholesterol/PEG-cDMA




(57.1/7.1/34.4/1.4)




lipid:siRNA ~7:1


S-XTC
XTC
XTC/DPPC/Cholesterol/PEG-cDMA




57.1/7.1/34.4/1.4




lipid:siRNA ~7:1


LNP05
XTC
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA ~6:1


LNP06
XTC
XTC/DSPC/Cholesterol/PEG-DMG




57.5/7.5/31.5/3.5




lipid:siRNA ~11:1


LNP07
XTC
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA ~6:1


LNP08
XTC
XTC/DSPC/Cholesterol/PEG-DMG




60/7.5/31/1.5,




lipid:siRNA ~11:1


LNP09
XTC
XTC/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP10
ALN100
ALN100/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid: siRNA 10:1


LNP11
MC3
MC-3/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP12
C12-200
C12-200/DSPC/Cholesterol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA 10:1


LNP13
XTC
XTC/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 33:1


LNP14
MC3
MC3/DSPC/Chol/PEG-DMG




40/15/40/5




Lipid:siRNA: 11:1


LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG




50/10/35/4.5/0.5




Lipid:siRNA: 11:1


LNP16
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP17
MC3
MC3/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP18
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 12:1


LNP19
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/35/5




Lipid:siRNA: 8:1


LNP20
MC3
MC3/DSPC/Chol/PEG-DPG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP22
XTC
XTC/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1









SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International patent application no. PCT/US10/22614, filed Jan. 29, 2010, which are hereby incorporated by reference.
MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, and U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, which are hereby incorporated by reference.
ALN100, i.e., ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
C12-200, i.e., Tech G1 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009, which is hereby incorporated by reference.
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total siRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated siRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” siRNA content (as measured by the signal in the absence of surfactant) from the total siRNA content. Percent entrapped siRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Emulsions
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of dsRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or dsRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of dsRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of dsRNAs and nucleic acids.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the dsRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly dsRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of dsRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of dsRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
Non-chelating non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more dsRNA compounds and (b) one or more anti-cytokine biologic agents which function by a non-RNAi mechanism. Examples of such biologics include, biologics that target IL1β (e.g., anakinra), IL6 (tocilizumab), or TNF (etanercept, infliximab, adlimumab, or certolizumab).
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the dsRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by TTR expression. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Treating Diseases Caused by Expression of a TTR Gene
The invention relates in particular to the use of a dsRNA targeting TTR and compositions containing at least one such dsRNA for the treatment of a TTR-mediated disorder or disease. For example, a dsRNA targeting a TTR gene can be useful for the treatment of a TTR amyloidosis, such as familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy (FAC), leptomeningeal/CNS amyloidosis, amyloidosis VII form (also known as leptomeningeal or meningocerebrovascular amyloidosis), hyperthyroxinemia, and cardiac amyloidosis (also called senile systemic amyloidosis (SSA) and senile cardiac amyloidosis (SCA)). The treatment can be therapeutic and/or prophylactic.
FIG. 15 illustrates symptoms and mutations in TTR associated with familial amyloidotic neuropathy, familial amyloidotic cardiomyopathy and CNS amyloidosis. The invention includes compositions and methods for treatment of these diseases and symptoms, and directed to these mutant versions of TTR.
A dsRNA targeting a TTR gene is also used for treatment of symptoms and disorders, such as TTR amyloidosis. Symptoms associated with such amyloidosis include, e.g., seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic insufficiency, cardiomyopathy, gastrointestinal dysfunction (e.g., gastric ulcers, diarrhea, constipation, malabsorption), weight loss, hepatomegaly, lymphadenopathy, goiter, vitreous opacities, renal insufficiency (including proteinuria and kidney failure), nephropathy, cranial nerve dysfunction, corneal lattice dystrophy, and congestive heart failure with generalized weakness and difficulties breathing from fluid retention.
In some embodiments, the TTR siRNA is used for treatment of a TTR related disorder that results in deposition of TTR amyloid plaques, e.g., deposition of TTR amyloid plaques in the peripheral nervous system. Examples include Transthyretin amyloidosis (ATTR) and familial amyloidotic polyneuropathy (FAP). The treatment can be prophylactic, e.g., treatment results in prevention of deposition. Alternatively, or in addition, treatment can be therapeutic, e.g., treatment results in regression of plaques already present. Treatment can reduce or prevent TTR deposits in any number of tissues including but not limited to esophagus, stomach, intestine (duodenum and colon), sciatic nerve, and/or dorsal root ganglion.
For example, the invention includes methods of treatment of TTR deposition in a human subject in need of such treatment, e.g., a FAP patient. The methods include administering a TTR siRNA, e.g., ALN-TTR01, wherein treatment results in a reduction in the amount TTR amyloid deposition following treatment and/or results in a reduction in the pre-existing TTR amyloid deposition.
Owing to the inhibitory effects on TTR expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
The invention further relates to the use of a dsRNA or a pharmaceutical composition thereof, e.g., for treating a TTR amyloidosis, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. In one example, a dsRNA targeting TTR can be administered in combination with a liver transplant. In other examples, a dsRNA targeting TTR can be administered in combination with a pharmaceutical or therapeutic method for treating a symptom of a TTR disease, such as diuretics, ACE (angiotensin converting enzyme) inhibitors, angiotensin receptor blockers (ARBs), or dialysis, e.g., for management of renal function.
The dsRNA and an additional therapeutic agent can be administered in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
The invention features a method of administering a dsRNA targeting TTR to a patient having a disease or disorder mediated by TTR expression, such as a TTR amyloidosis, e.g., FAP. Administration of the dsRNA can stabilize and improve peripheral neurological function, for example, in a patient with FAP. Patients can be administered a therapeutic amount of dsRNA, such as 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dsRNA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, 25 minute, 60 minute, 120 minute or 180 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the dsRNA can reduce TTR levels in the blood or urine of the patient by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more.
Before administration of a full dose of the dsRNA, patients can be administered a smaller dose, such as a dose that is 5% of the full dose, and monitored for adverse effects, such as an allergic reaction or a change in liver function. For example, in patients monitored for changes in liver function, a low incidence of LFT (Liver Function Test) change (e.g., a 10-20% incidence of LFT) is acceptable (e.g., a reversible, 3-fold increase in ALT (alanine aminotransferase) and/or AST (aspartate aminotransferase) levels).
Many TTR-associated diseases and disorders are hereditary. Therefore, a patient in need of a TTR dsRNA can be identified by taking a family history. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering a TTR dsRNA. A DNA test may also be performed on the patient to identify a mutation in the TTR gene, before a TTR dsRNA is administered to the patient.
The patient may have a biopsy performed before receiving a TTR dsRNA. The biopsy can be, for example, on a tissue, such as the gastric mucosa, peripheral nerve, skin, abdominal fat, liver, or kidney, and the biopsy may reveal amyloid plaques, which are indicative of a TTR-mediated disorder. Upon the identification of amyloid plaques, the patient is administered a TTR dsRNA.
Methods for Inhibiting Expression of a TTR Gene
In yet another aspect, the invention provides a method for inhibiting the expression of a TTR gene in a mammal. The method includes administering a composition featured in the invention to the mammal such that expression of the target TTR gene is silenced.
When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the dsRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
dsRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
siRNA Synthesis
Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.
For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol-3′), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA




A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB




Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC




3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride, salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD




The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE




Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF




Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG




Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
Succinic acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH




Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.




Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1.







TABLE 1







Abbreviations of nucleotide monomers used


in nucleic acid sequence representation.










Abbreviation
Nucleotide(s)







A
adenosine-3′-phosphate



C
cytidine-3′-phosphate



G
guanosine-3′-phosphate



U
uridine-3′-phosphate



N
any nucleotide (G, A, C, or T)



a
2′-O-methyladenosine-3′-phosphate



c
2′-O-methylcytidine-3′-phosphate



g
2′-O-methylguanosine-3′-phosphate



u
2′-O-methyluridine-3′-phosphate



T, dT
2′-deoxythymidine-3v-phosphate



sT; sdT
2′-deoxy-thymidine-5′phosphate-phosphorothioate







It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds.






Example 2A
TTR siRNA Design
Transcripts
siRNA design was carried out to identify siRNAs targeting the gene transthyretin from human (symbol TTR) and rat (symbol Ttr). The design used the TTR transcripts NM—000371.2 (SEQ ID NO:1329) (human) and NM—012681.1 (SEQ ID NO:1330) (rat) from the NCBI Refseq collection. The siRNA duplexes were designed with 100% identity to their respective TTR genes.
siRNA Design and Specificity Prediction
The predicted specificity of all possible 19mers was determined for each sequence. The TTR siRNAs were used in a comprehensive search against the human and rat transcriptomes (defined as the set of NM_ and XM_ records within the NCBI Refseq set) using the FASTA algorithm. The Python script ‘offtargetFasta.py’ was then used to parse the alignments and generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8; mismatches in the putative cleavage sites 10 and 11 are given a penalty of 1.2, and mismatches in positions 12-19 a penalty of 1. Mismatches in position 1 are not considered. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNA strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific, and between 2.2 and 2.8 as moderately specific. In picking which oligos to synthesize, off-target scores of the antisense strand were sorted from high to low, and the 144 best (lowest off-target score) oligo pairs from human, and the best 26 pairs from rat were selected.
siRNA Sequence Selection
A total of 140 sense and 140 antisense human TTR derived siRNA oligos were synthesized and formed into duplexes. A total of 26 sense and 26 antisense rat TTR derived siRNA oligos were synthesized and formed into duplexes. Duplexes included The oligos are presented in Tables 2-4 (human TTR) and Tables 5-7 (rat TTR).







TABLE 2







Identification numbers for human TTR dsRNAs











Duplex #
Sense Oligo #
Antisense Oligo #







AD-18243
A-32153
A-32154



AD-18244
A-32155
A-32156



AD-18245
A-32157
A-32158



AD-18246
A-32159
A-32160



AD-18247
A-32163
A-32164



AD-18248
A-32165
A-32166



AD-18249
A-32167
A-32168



AD-18250
A-32169
A-32170



AD-18251
A-32171
A-32172



AD-18252
A-32175
A-32176



AD-18253
A-32177
A-32178



AD-18254
A-32179
A-32180



AD-18255
A-32181
A-32182



AD-18256
A-32183
A-32184



AD-18257
A-32187
A-32188



AD-18258
A-32189
A-32190



AD-18259
A-32191
A-32192



AD-18260
A-32193
A-32194



AD-18261
A-32195
A-32196



AD-18262
A-32199
A-32200



AD-18263
A-32201
A-32202



AD-18264
A-32203
A-32204



AD-18265
A-32205
A-32206



AD-18266
A-32207
A-32208



AD-18267
A-32211
A-32212



AD-18268
A-32213
A-32214



AD-18269
A-32215
A-32216



AD-18270
A-32217
A-32218



AD-18271
A-32219
A-32220



AD-18272
A-32221
A-32222



AD-18273
A-32223
A-32224



AD-18274
A-32225
A-32226



AD-18275
A-32227
A-32228



AD-18276
A-32229
A-32230



AD-18277
A-32231
A-32232



AD-18278
A-32233
A-32234



AD-18279
A-32235
A-32236



AD-18280
A-32237
A-32238



AD-18281
A-32239
A-32240



AD-18282
A-32241
A-32242



AD-18283
A-32243
A-32244



AD-18284
A-32247
A-32248



AD-18285
A-32249
A-32250



AD-18286
A-32251
A-32252



AD-18287
A-32253
A-32254



AD-18288
A-32255
A-32256



AD-18289
A-32259
A-32260



AD-18290
A-32261
A-32262



AD-18291
A-32263
A-32264



AD-18292
A-32265
A-32266



AD-18293
A-32267
A-32268



AD-18294
A-32269
A-32270



AD-18295
A-32271
A-32272



AD-18296
A-32273
A-32274



AD-18297
A-32275
A-32276



AD-18298
A-32277
A-32278



AD-18299
A-32279
A-32280



AD-18300
A-32281
A-32282



AD-18301
A-32283
A-32284



AD-18302
A-32285
A-32286



AD-18303
A-32287
A-32288



AD-18304
A-32289
A-32290



AD-18305
A-32291
A-32292



AD-18306
A-32295
A-32296



AD-18307
A-32297
A-32298



AD-18308
A-32299
A-32300



AD-18309
A-32301
A-32302



AD-18310
A-32303
A-32304



AD-18311
A-32307
A-32308



AD-18312
A-32309
A-32310



AD-18313
A-32311
A-32312



AD-18314
A-32313
A-32314



AD-18315
A-32315
A-32316



AD-18316
A-32319
A-32320



AD-18317
A-32321
A-32322



AD-18318
A-32323
A-32324



AD-18319
A-32325
A-32326



AD-18320
A-32327
A-32328



AD-18321
A-32331
A-32332



AD-18322
A-32333
A-32334



AD-18323
A-32335
A-32336



AD-18324
A-32337
A-32338



AD-18325
A-32339
A-32340



AD-18326
A-32341
A-32342



AD-18327
A-32343
A-32344



AD-18328
A-32345
A-32346



AD-18329
A-32347
A-32348



AD-18330
A-32349
A-32350



AD-18331
A-32351
A-32352



AD-18332
A-32353
A-32354



AD-18333
A-32355
A-32356



AD-18334
A-32357
A-32358



AD-18335
A-32359
A-32360



AD-18336
A-32363
A-32364



AD-18337
A-32367
A-32368



AD-18338
A-32369
A-32370



AD-18339
A-32371
A-32372



AD-18340
A-32373
A-32374



AD-18341
A-32375
A-32376



AD-18342
A-32379
A-32380



AD-18343
A-32381
A-32382



AD-18344
A-32383
A-32384



AD-18345
A-32385
A-32386



AD-18346
A-32387
A-32388



AD-18347
A-32391
A-32392



AD-18348
A-32393
A-32394



AD-18349
A-32395
A-32396



AD-18350
A-32397
A-32398



AD-18351
A-32399
A-32400



AD-18352
A-32401
A-32402



AD-18353
A-32403
A-32404



AD-18354
A-32405
A-32406



AD-18355
A-32407
A-32408



AD-18356
A-32409
A-32410



AD-18357
A-32411
A-32412



AD-18358
A-32415
A-32416



AD-18359
A-32417
A-32418



AD-18360
A-32419
A-32420



AD-18361
A-32421
A-32422



AD-18362
A-32423
A-32424



AD-18363
A-32427
A-32428



AD-18364
A-32429
A-32430



AD-18446
A-32161
A-32162



AD-18447
A-32173
A-32174



AD-18448
A-32185
A-32186



AD-18449
A-32197
A-32198



AD-18450
A-32209
A-32210



AD-18451
A-32245
A-32246



AD-18452
A-32257
A-32258



AD-18453
A-32293
A-32294



AD-18454
A-32305
A-32306



AD-18455
A-32317
A-32318



AD-18456
A-32329
A-32330



AD-18457
A-32361
A-32362



AD-18458
A-32365
A-32366



AD-18459
A-32377
A-32378



AD-18460
A-32389
A-32390



AD-18461
A-32401
A-32402



AD-18462
A-32413
A-32414



AD-18463
A-32425
A-32426







See Table 4 for sequences and modifications of oligos.













TABLE 3A







Sense and antisense strand sequences of human TTR dsRNAs


Strand: s = sense; as = antisense; Position: position of


5′ base on transcript (NM_000371.2, SEQ ID NO: 1329)















SEQ
Sequence with 3′ dinucleotide
SEQ




Sequence
ID
overhang
ID


Strand
Position
(5′ to 3′)
NO:
(5′ to 3′)
NO:





S
100
CCGGUGAAUCCAAGUGUCC
  1
CCGGUGAAUCCAAGUGUCCNN
281


 


as
118
GGACACUUGGAUUCACCGG
  2
GGACACUUGGAUUCACCGGNN
282


 


S
 11
ACUCAUUCUUGGCAGGAUG
  3
ACUCAUUCUUGGCAGGAUGNN
283


 


as
 29
CAUCCUGCCAAGAAUGAGU
  4
CAUCCUGCCAAGAAUGAGUNN
284


 


S
111
AAGUGUCCUCUGAUGGUCA
  5
AAGUGUCCUCUGAUGGUCANN
285


 


as
129
UGACCAUCAGAGGACACUU
  6
UGACCAUCAGAGGACACUUNN
286


 


S
 13
UCAUUCUUGGCAGGAUGGC
  7
UCAUUCUUGGCAGGAUGGCNN
287


 


as
 31
GCCAUCCUGCCAAGAAUGA
  8
GCCAUCCUGCCAAGAAUGANN
288


 


s
130
AAGUUCUAGAUGCUGUCCG
  9
AAGUUCUAGAUGCUGUCCGNN
289


 


as
148
CGGACAGCAUCUAGAACUU
 10
CGGACAGCAUCUAGAACUUNN
290


 


s
132
GUUCUAGAUGCUGUCCGAG
 11
GUUCUAGAUGCUGUCCGAGNN
291


 


as
150
CUCGGACAGCAUCUAGAAC
 12
CUCGGACAGCAUCUAGAACNN
292


 


s
135
CUAGAUGCUGUCCGAGGCA
 13
CUAGAUGCUGUCCGAGGCANN
293


 


as
153
UGCCUCGGACAGCAUCUAG
 14
UGCCUCGGACAGCAUCUAGNN
294


 


s
138
GAUGCUGUCCGAGGCAGUC
 15
GAUGCUGUCCGAGGCAGUCNN
295


 


as
156
GACUGCCUCGGACAGCAUC
 16
GACUGCCUCGGACAGCAUCNN
296


 


s
 14
CAUUCUUGGCAGGAUGGCU
 17
CAUUCUUGGCAGGAUGGCUNN
297


 


as
 32
AGCCAUCCUGCCAAGAAUG
 18
AGCCAUCCUGCCAAGAAUGNN
298


 


s
140
UGCUGUCCGAGGCAGUCCU
 19
UGCUGUCCGAGGCAGUCCUNN
299


 


as
158
AGGACUGCCUCGGACAGCA
 20
AGGACUGCCUCGGACAGCANN
300


 


s
146
CCGAGGCAGUCCUGCCAUC
 21
CCGAGGCAGUCCUGCCAUCNN
301


 


as
164
GAUGGCAGGACUGCCUCGG
 22
GAUGGCAGGACUGCCUCGGNN
302


 


s
152
CAGUCCUGCCAUCAAUGUG
 23
CAGUCCUGCCAUCAAUGUGNN
303


 


as
170
CACAUUGAUGGCAGGACUG
 24
CACAUUGAUGGCAGGACUGNN
304


 


s
164
CAAUGUGGCCGUGCAUGUG
 25
CAAUGUGGCCGUGCAUGUGNN
305


 


as
182
CACAUGCACGGCCACAUUG
 26
CACAUGCACGGCCACAUUGNN
306


 


s
178
AUGUGUUCAGAAAGGCUGC
 27
AUGUGUUCAGAAAGGCUGCNN
307


 


as
196
GCAGCCUUUCUGAACACAU
 28
GCAGCCUUUCUGAACACAUNN
308


 


s
  2
CAGAAGUCCACUCAUUCUU
 29
CAGAAGUCCACUCAUUCUUNN
309


 


as
 20
AAGAAUGAGUGGACUUCUG
 30
AAGAAUGAGUGGACUUCUGNN
310


 


s
 21
GGCAGGAUGGCUUCUCAUC
 31
GGCAGGAUGGCUUCUCAUCNN
311


 


as
 39
GAUGAGAAGCCAUCCUGCC
 32
GAUGAGAAGCCAUCCUGCCNN
312


 


s
210
GAGCCAUUUGCCUCUGGGA
 33
GAGCCAUUUGCCUCUGGGANN
313


 


as
228
UCCCAGAGGCAAAUGGCUC
 34
UCCCAGAGGCAAAUGGCUCNN
314


 


s
 23
CAGGAUGGCUUCUCAUCGU
 35
CAGGAUGGCUUCUCAUCGUNN
315


 


as
 41
ACGAUGAGAAGCCAUCCUG
 36
ACGAUGAGAAGCCAUCCUGNN
316


 


s
 24
AGGAUGGCUUCUCAUCGUC
 37
AGGAUGGCUUCUCAUCGUCNN
317


 


as
 42
GACGAUGAGAAGCCAUCCU
 38
GACGAUGAGAAGCCAUCCUNN
318


 


s
245
AGAGCUGCAUGGGCUCACA
 39
AGAGCUGCAUGGGCUCACANN
319


 


as
263
UGUGAGCCCAUGCAGCUCU
 40
UGUGAGCCCAUGCAGCUCUNN
320


 


s
248
GCUGCAUGGGCUCACAACU
 41
GCUGCAUGGGCUCACAACUNN
321


 


as
266
AGUUGUGAGCCCAUGCAGC
 42
AGUUGUGAGCCCAUGCAGCNN
322


 


s
 25
GGAUGGCUUCUCAUCGUCU
 43
GGAUGGCUUCUCAUCGUCUNN
323


 


as
 43
AGACGAUGAGAAGCCAUCC
 44
AGACGAUGAGAAGCCAUCCNN
324


 


s
251
GCAUGGGCUCACAACUGAG
 45
GCAUGGGCUCACAACUGAGNN
325


 


as
269
CUCAGUUGUGAGCCCAUGC
 46
CUCAGUUGUGAGCCCAUGCNN
326


 


s
253
AUGGGCUCACAACUGAGGA
 47
AUGGGCUCACAACUGAGGANN
327


 


as
271
UCCUCAGUUGUGAGCCCAU
 48
UCCUCAGUUGUGAGCCCAUNN
328


 


s
254
UGGGCUCACAACUGAGGAG
 49
UGGGCUCACAACUGAGGAGNN
329


 


as
272
CUCCUCAGUUGUGAGCCCA
 50
CUCCUCAGUUGUGAGCCCANN
330


 


s
270
GAGGAAUUUGUAGAAGGGA
 51
GAGGAAUUUGUAGAAGGGANN
331


 


as
288
UCCCUUCUACAAAUUCCUC
 52
UCCCUUCUACAAAUUCCUCNN
332


 


s
276
UUUGUAGAAGGGAUAUACA
 53
UUUGUAGAAGGGAUAUACANN
333


 


as
294
UGUAUAUCCCUUCUACAAA
 54
UGUAUAUCCCUUCUACAAANN
334


 


s
277
UUGUAGAAGGGAUAUACAA
 55
UUGUAGAAGGGAUAUACAANN
335


 


as
295
UUGUAUAUCCCUUCUACAA
 56
UUGUAUAUCCCUUCUACAANN
336


 


s
278
UGUAGAAGGGAUAUACAAA
 57
UGUAGAAGGGAUAUACAAANN
337


 


as
296
UUUGUAUAUCCCUUCUACA
 58
UUUGUAUAUCCCUUCUACANN
338


 


s
281
AGAAGGGAUAUACAAAGUG
 59
AGAAGGGAUAUACAAAGUGNN
339


 


as
299
CACUUUGUAUAUCCCUUCU
 60
CACUUUGUAUAUCCCUUCUNN
340


 


s
295
AAGUGGAAAUAGACACCAA
 61
AAGUGGAAAUAGACACCAANN
341


 


as
313
UUGGUGUCUAUUUCCACUU
 62
UUGGUGUCUAUUUCCACUUNN
342


 


s
299
GGAAAUAGACACCAAAUCU
 63
GGAAAUAGACACCAAAUCUNN
343


 


as
317
AGAUUUGGUGUCUAUUUCC
 64
AGAUUUGGUGUCUAUUUCCNN
344


 


s
300
GAAAUAGACACCAAAUCUU
 65
GAAAUAGACACCAAAUCUUNN
345


 


as
318
AAGAUUUGGUGUCUAUUUC
 66
AAGAUUUGGUGUCUAUUUCNN
346


 


s
303
AUAGACACCAAAUCUUACU
 67
AUAGACACCAAAUCUUACUNN
347


 


as
321
AGUAAGAUUUGGUGUCUAU
 68
AGUAAGAUUUGGUGUCUAUNN
348


 


s
304
UAGACACCAAAUCUUACUG
 69
UAGACACCAAAUCUUACUGNN
349


 


as
322
CAGUAAGAUUUGGUGUCUA
 70
CAGUAAGAUUUGGUGUCUANN
350


 


s
305
AGACACCAAAUCUUACUGG
 71
AGACACCAAAUCUUACUGGNN
351


 


as
323
CCAGUAAGAUUUGGUGUCU
 72
CCAGUAAGAUUUGGUGUCUNN
352


 


s
317
UUACUGGAAGGCACUUGGC
 73
UUACUGGAAGGCACUUGGCNN
353


 


as
335
GCCAAGUGCCUUCCAGUAA
 74
GCCAAGUGCCUUCCAGUAANN
354


 


s
 32
UUCUCAUCGUCUGCUCCUC
 75
UUCUCAUCGUCUGCUCCUCNN
355


 


as
 50
GAGGAGCAGACGAUGAGAA
 76
GAGGAGCAGACGAUGAGAANN
356


 


s
322
GGAAGGCACUUGGCAUCUC
 77
GGAAGGCACUUGGCAUCUCNN
357


 


as
340
GAGAUGCCAAGUGCCUUCC
 78
GAGAUGCCAAGUGCCUUCCNN
358


 


s
326
GGCACUUGGCAUCUCCCCA
 79
GGCACUUGGCAUCUCCCCANN
359


 


as
344
UGGGGAGAUGCCAAGUGCC
 80
UGGGGAGAUGCCAAGUGCCNN
360


 


s
333
GGCAUCUCCCCAUUCCAUG
 81
GGCAUCUCCCCAUUCCAUGNN
361


 


as
351
AUGGAAUGGGGAGAUGCCTT
 82
AUGGAAUGGGGAGAUGCCTTNN
362


 


s
334
GCAUCUCCCCAUUCCAUGA
 83
GCAUCUCCCCAUUCCAUGANN
363


 


as
352
UCAUGGAAUGGGGAGAUGC
 84
UCAUGGAAUGGGGAGAUGCNN
364


 


s
335
CAUCUCCCCAUUCCAUGAG
 85
CAUCUCCCCAUUCCAUGAGNN
365


 


as
353
CUCAUGGAAUGGGGAGAUG
 86
CUCAUGGAAUGGGGAGAUGNN
366


 


s
336
AUCUCCCCAUUCCAUGAGC
 87
AUCUCCCCAUUCCAUGAGCNN
367


 


as
354
GCUCAUGGAAUGGGGAGAU
 88
GCUCAUGGAAUGGGGAGAUNN
368


 


s
338
CUCCCCAUUCCAUGAGCAU
 89
CUCCCCAUUCCAUGAGCAUNN
369


 


as
356
AUGCUCAUGGAAUGGGGAG
 90
AUGCUCAUGGAAUGGGGAGNN
370


 


s
341
CCCAUUCCAUGAGCAUGCA
 91
CCCAUUCCAUGAGCAUGCANN
371


 


as
359
UGCAUGCUCAUGGAAUGGG
 92
UGCAUGCUCAUGGAAUGGGNN
372


 


s
347
CCAUGAGCAUGCAGAGGUG
 93
CCAUGAGCAUGCAGAGGUGNN
373


 


as
365
CACCUCUGCAUGCUCAUGG
 94
CACCUCUGCAUGCUCAUGGNN
374


 


s
352
AGCAUGCAGAGGUGGUAUU
 95
AGCAUGCAGAGGUGGUAUUNN
375


 


as
370
AAUACCACCUCUGCAUGCU
 96
AAUACCACCUCUGCAUGCUNN
376


 


s
354
CAUGCAGAGGUGGUAUUCA
 97
CAUGCAGAGGUGGUAUUCANN
377


 


as
372
UGAAUACCACCUCUGCAUG
 98
UGAAUACCACCUCUGCAUGNN
378


 


s
355
AUGCAGAGGUGGUAUUCAC
 99
AUGCAGAGGUGGUAUUCACNN
379


 


as
373
GUGAAUACCACCUCUGCAU
100
GUGAAUACCACCUCUGCAUNN
380


 


s
362
GGUGGUAUUCACAGCCAAC
101
GGUGGUAUUCACAGCCAACNN
381


 


as
380
GUUGGCUGUGAAUACCACC
102
GUUGGCUGUGAAUACCACCNN
382


 


s
363
GUGGUAUUCACAGCCAACG
103
GUGGUAUUCACAGCCAACGNN
383


 


as
381
CGUUGGCUGUGAAUACCAC
104
CGUUGGCUGUGAAUACCACNN
384


 


s
364
UGGUAUUCACAGCCAACGA
105
UGGUAUUCACAGCCAACGANN
385


 


as
382
UCGUUGGCUGUGAAUACCA
106
UCGUUGGCUGUGAAUACCANN
386


 


s
365
GGUAUUCACAGCCAACGAC
107
GGUAUUCACAGCCAACGACNN
387


 


as
383
GUCGUUGGCUGUGAAUACC
108
GUCGUUGGCUGUGAAUACCNN
388


 


s
366
GUAUUCACAGCCAACGACU
109
GUAUUCACAGCCAACGACUNN
389


 


as
384
AGUCGUUGGCUGUGAAUAC
110
AGUCGUUGGCUGUGAAUACNN
390


 


s
367
UAUUCACAGCCAACGACUC
111
UAUUCACAGCCAACGACUCNN
391


 


as
385
GAGUCGUUGGCUGUGAAUA
112
GAGUCGUUGGCUGUGAAUANN
392


 


s
370
UCACAGCCAACGACUCCGG
113
UCACAGCCAACGACUCCGGNN
393


 


as
388
CCGGAGUCGUUGGCUGUGA
114
CCGGAGUCGUUGGCUGUGANN
394


 


s
390
CCCCGCCGCUACACCAUUG
115
CCCCGCCGCUACACCAUUGNN
395


 


as
408
CAAUGGUGUAGCGGCGGGG
116
CAAUGGUGUAGCGGCGGGGNN
396


 


s
  4
GAAGUCCACUCAUUCUUGG
117
GAAGUCCACUCAUUCUUGGNN
397


 


as
 22
CCAAGAAUGAGUGGACUUC
118
CCAAGAAUGAGUGGACUUCNN
398


 


s
412
CCCUGCUGAGCCCCUACUC
119
CCCUGCUGAGCCCCUACUCNN
399


 


as
430
GAGUAGGGGCUCAGCAGGG
120
GAGUAGGGGCUCAGCAGGGNN
400


 


s
417
CUGAGCCCCUACUCCUAUU
121
CUGAGCCCCUACUCCUAUUNN
401


 


as
435
AAUAGGAGUAGGGGCUCAG
122
AAUAGGAGUAGGGGCUCAGNN
402


 


s
418
UGAGCCCCUACUCCUAUUC
123
UGAGCCCCUACUCCUAUUCNN
403


 


as
436
GAAUAGGAGUAGGGGCUCA
124
GAAUAGGAGUAGGGGCUCANN
404


 


s
422
CCCCUACUCCUAUUCCACC
125
CCCCUACUCCUAUUCCACCNN
405


 


as
440
GGUGGAAUAGGAGUAGGGG
126
GGUGGAAUAGGAGUAGGGGNN
406


 


s
425
CUACUCCUAUUCCACCACG
127
CUACUCCUAUUCCACCACGNN
407


 


as
443
CGUGGUGGAAUAGGAGUAG
128
CGUGGUGGAAUAGGAGUAGNN
408


 


s
426
UACUCCUAUUCCACCACGG
129
UACUCCUAUUCCACCACGGNN
409


 


as
444
CCGUGGUGGAAUAGGAGUA
130
CCGUGGUGGAAUAGGAGUANN
410


 


s
427
ACUCCUAUUCCACCACGGC
131
ACUCCUAUUCCACCACGGCNN
411


 


as
445
GCCGUGGUGGAAUAGGAGU
132
GCCGUGGUGGAAUAGGAGUNN
412


 


s
429
UCCUAUUCCACCACGGCUG
133
UCCUAUUCCACCACGGCUGNN
413


 


as
447
CAGCCGUGGUGGAAUAGGA
134
CAGCCGUGGUGGAAUAGGANN
414


 


s
432
UAUUCCACCACGGCUGUCG
135
UAUUCCACCACGGCUGUCGNN
415


 


as
450
CGACAGCCGUGGUGGAAUA
136
CGACAGCCGUGGUGGAAUANN
416


 


s
433
AUUCCACCACGGCUGUCGU
137
AUUCCACCACGGCUGUCGUNN
417


 


as
451
ACGACAGCCGUGGUGGAAU
138
ACGACAGCCGUGGUGGAAUNN
418


 


s
437
CACCACGGCUGUCGUCACC
139
CACCACGGCUGUCGUCACCNN
419


 


as
455
GGUGACGACAGCCGUGGUG
140
GGUGACGACAGCCGUGGUGNN
420


 


s
438
ACCACGGCUGUCGUCACCA
141
ACCACGGCUGUCGUCACCANN
421


 


as
456
UGGUGACGACAGCCGUGGU
142
UGGUGACGACAGCCGUGGUNN
422


 


s
439
CCACGGCUGUCGUCACCAA
143
CCACGGCUGUCGUCACCAANN
423


 


as
457
UUGGUGACGACAGCCGUGG
144
UUGGUGACGACAGCCGUGGNN
424


 


s
441
ACGGCUGUCGUCACCAAUC
145
ACGGCUGUCGUCACCAAUCNN
425


 


as
459
GAUUGGUGACGACAGCCGU
146
GAUUGGUGACGACAGCCGUNN
426


 


s
442
CGGCUGUCGUCACCAAUCC
147
CGGCUGUCGUCACCAAUCCNN
427


 


as
460
GGAUUGGUGACGACAGCCG
148
GGAUUGGUGACGACAGCCGNN
428


 


s
449
CGUCACCAAUCCCAAGGAA
149
CGUCACCAAUCCCAAGGAANN
429


 


as
467
UUCCUUGGGAUUGGUGACG
150
UUCCUUGGGAUUGGUGACGNN
430


 


s
455
CAAUCCCAAGGAAUGAGGG
151
CAAUCCCAAGGAAUGAGGGNN
431


 


as
473
CCCUCAUUCCUUGGGAUUG
152
CCCUCAUUCCUUGGGAUUGNN
432


 


s
491
CCUGAAGGACGAGGGAUGG
153
CCUGAAGGACGAGGGAUGGNN
433


 


as
509
CCAUCCCUCGUCCUUCAGG
154
CCAUCCCUCGUCCUUCAGGNN
434


 


s
497
GGACGAGGGAUGGGAUUUC
155
GGACGAGGGAUGGGAUUUCNN
435


 


as
515
GAAAUCCCAUCCCUCGUCC
156
GAAAUCCCAUCCCUCGUCCNN
436


 


s
  5
AAGUCCACUCAUUCUUGGC
157
AAGUCCACUCAUUCUUGGCNN
437


 


as
 23
GCCAAGAAUGAGUGGACUU
158
GCCAAGAAUGAGUGGACUUNN
438


 


s
508
GGGAUUUCAUGUAACCAAG
159
GGGAUUUCAUGUAACCAAGNN
439


 


as
526
CUUGGUUACAUGAAAUCCC
160
CUUGGUUACAUGAAAUCCCNN
440


 


s
509
GGAUUUCAUGUAACCAAGA
161
GGAUUUCAUGUAACCAAGANN
441


 


as
527
UCUUGGUUACAUGAAAUCC
162
UCUUGGUUACAUGAAAUCCNN
442


 


s
514
UCAUGUAACCAAGAGUAUU
163
UCAUGUAACCAAGAGUAUUNN
443


 


as
532
AAUACUCUUGGUUACAUGA
164
AAUACUCUUGGUUACAUGANN
444


 


s
516
AUGUAACCAAGAGUAUUCC
165
AUGUAACCAAGAGUAUUCCNN
445


 


as
534
GGAAUACUCUUGGUUACAU
166
GGAAUACUCUUGGUUACAUNN
446


 


s
517
UGUAACCAAGAGUAUUCCA
167
UGUAACCAAGAGUAUUCCANN
447


 


as
535
UGGAAUACUCUUGGUUACA
168
UGGAAUACUCUUGGUUACANN
448


 


s
518
GUAACCAAGAGUAUUCCAU
169
GUAACCAAGAGUAUUCCAUNN
449


 


as
536
AUGGAAUACUCUUGGUUAC
170
AUGGAAUACUCUUGGUUACNN
450


 


s
 54
UGCCUUGCUGGACUGGUAU
171
UGCCUUGCUGGACUGGUAUNN
451


 


as
 72
AUACCAGUCCAGCAAGGCA
172
AUACCAGUCCAGCAAGGCANN
452


 


s
543
UAAAGCAGUGUUUUCACCU
173
UAAAGCAGUGUUUUCACCUNN
453


 


as
561
AGGUGAAAACACUGCUUUA
174
AGGUGAAAACACUGCUUUANN
454


 


s
 55
GCCUUGCUGGACUGGUAUU
175
GCCUUGCUGGACUGGUAUUNN
455


 


as
 73
AAUACCAGUCCAGCAAGGC
176
AAUACCAGUCCAGCAAGGCNN
456


 


s
551
UGUUUUCACCUCAUAUGCU
177
UGUUUUCACCUCAUAUGCUNN
457


 


as
569
AGCAUAUGAGGUGAAAACA
178
AGCAUAUGAGGUGAAAACANN
458


 


s
552
GUUUUCACCUCAUAUGCUA
179
GUUUUCACCUCAUAUGCUANN
459


 


as
570
UAGCAUAUGAGGUGAAAAC
180
UAGCAUAUGAGGUGAAAACNN
460


 


s
553
UUUUCACCUCAUAUGCUAU
181
UUUUCACCUCAUAUGCUAUNN
461


 


as
571
AUAGCAUAUGAGGUGAAAA
182
AUAGCAUAUGAGGUGAAAANN
462


 


s
555
UUCACCUCAUAUGCUAUGU
183
UUCACCUCAUAUGCUAUGUNN
463


 


as
573
ACAUAGCAUAUGAGGUGAA
184
ACAUAGCAUAUGAGGUGAANN
464


 


s
557
CACCUCAUAUGCUAUGUUA
185
CACCUCAUAUGCUAUGUUANN
465


 


as
575
UAACAUAGCAUAUGAGGUG
186
UAACAUAGCAUAUGAGGUGNN
466


 


s
 56
CCUUGCUGGACUGGUAUUU
187
CCUUGCUGGACUGGUAUUUNN
467


 


as
 74
AAAUACCAGUCCAGCAAGG
188
AAAUACCAGUCCAGCAAGGNN
468


 


s
563
AUAUGCUAUGUUAGAAGUC
189
AUAUGCUAUGUUAGAAGUCNN
469


 


as
581
GACUUCUAACAUAGCAUAU
190
GACUUCUAACAUAGCAUAUNN
470


 


s
564
UAUGCUAUGUUAGAAGUCC
191
UAUGCUAUGUUAGAAGUCCNN
471


 


as
582
GGACUUCUAACAUAGCAUA
192
GGACUUCUAACAUAGCAUANN
472


 


s
566
UGCUAUGUUAGAAGUCCAG
193
UGCUAUGUUAGAAGUCCAGNN
473


 


as
584
CUGGACUUCUAACAUAGCA
194
CUGGACUUCUAACAUAGCANN
474


 


s
 57
CUUGCUGGACUGGUAUUUG
195
CUUGCUGGACUGGUAUUUGNN
475


 


as
 75
CAAAUACCAGUCCAGCAAG
196
CAAAUACCAGUCCAGCAAGNN
476


 


s
578
AGUCCAGGCAGAGACAAUA
197
AGUCCAGGCAGAGACAAUANN
477


 


as
596
AUUGUCUCUGCCUGGACUTT
198
AUUGUCUCUGCCUGGACUTTNN
478


 


s
580
UCCAGGCAGAGACAAUAAA
199
UCCAGGCAGAGACAAUAAANN
479


 


as
598
UUUAUUGUCUCUGCCUGGA
200
UUUAUUGUCUCUGCCUGGANN
480


 


s
607
GUGAAAGGCACUUUUCAUU
201
GUGAAAGGCACUUUUCAUUNN
481


 


as
625
AAUGAAAAGUGCCUUUCAC
202
AAUGAAAAGUGCCUUUCACNN
482


 


s
 62
UGGACUGGUAUUUGUGUCU
203
UGGACUGGUAUUUGUGUCUNN
483


 


as
 80
AGACACAAAUACCAGUCCA
204
AGACACAAAUACCAGUCCANN
484


 


s
 77
GUCUGAGGCUGGCCCUACG
205
GUCUGAGGCUGGCCCUACGNN
485


 


as
 95
CGUAGGGCCAGCCUCAGAC
206
CGUAGGGCCAGCCUCAGACNN
486


 


s
 79
CUGAGGCUGGCCCUACGGG
207
CUGAGGCUGGCCCUACGGGNN
487


 


as
 97
CCCGUAGGGCCAGCCUCAG
208
CCCGUAGGGCCAGCCUCAGNN
488


 


s
 81
GAGGCUGGCCCUACGGGCA
209
GAGGCUGGCCCUACGGGCANN
489


 


as
 99
UGCCCGUAGGGCCAGCCUC
210
UGCCCGUAGGGCCAGCCUCNN
490


 


s
 82
AGGCUGGCCCUACGGGCAC
211
AGGCUGGCCCUACGGGCACNN
491


 


as
100
GUGCCCGUAGGGCCAGCCU
212
GUGCCCGUAGGGCCAGCCUNN
492


 


s
 84
GCUGGCCCUACGGGCACCG
213
GCUGGCCCUACGGGCACCGNN
493


 


as
102
CGGUGCCCGUAGGGCCAGC
214
CGGUGCCCGUAGGGCCAGCNN
494


 


s
 85
CUGGCCCUACGGGCACCGG
215
CUGGCCCUACGGGCACCGGNN
495


 


as
103
CCGGUGCCCGUAGGGCCAG
216
CCGGUGCCCGUAGGGCCAGNN
496


 


s
 87
GGCCCUACGGGCACCGGUG
217
GGCCCUACGGGCACCGGUGNN
497


 


as
105
CACCGGUGCCCGUAGGGCC
218
CACCGGUGCCCGUAGGGCCNN
498


 


s
  9
CCACUCAUUCUUGGCAGGA
219
CCACUCAUUCUUGGCAGGANN
499


 


as
 27
UCCUGCCAAGAAUGAGUGG
220
UCCUGCCAAGAAUGAGUGGNN
500


 


s
 90
CCUACGGGCACCGGUGAAU
221
CCUACGGGCACCGGUGAAUNN
501


 


as
108
AUUCACCGGUGCCCGUAGG
222
AUUCACCGGUGCCCGUAGGNN
502


 


s
 91
CUACGGGCACCGGUGAAUC
223
CUACGGGCACCGGUGAAUCNN
503


 


as
109
GAUUCACCGGUGCCCGUAG
224
GAUUCACCGGUGCCCGUAGNN
504


 


s
 92
UACGGGCACCGGUGAAUCC
225
UACGGGCACCGGUGAAUCCNN
505


 


as
110
GGAUUCACCGGUGCCCGUA
226
GGAUUCACCGGUGCCCGUANN
506


 


s
 93
ACGGGCACCGGUGAAUCCA
227
ACGGGCACCGGUGAAUCCANN
507


 


as
111
UGGAUUCACCGGUGCCCGU
228
UGGAUUCACCGGUGCCCGUNN
508


 


s
 97
GCACCGGUGAAUCCAAGUG
229
GCACCGGUGAAUCCAAGUGNN
509


 


as
115
CACUUGGAUUCACCGGUGC
230
CACUUGGAUUCACCGGUGCNN
510


 


s
 98
CACCGGUGAAUCCAAGUGU
231
CACCGGUGAAUCCAAGUGUNN
511


 


as
116
ACACUUGGAUUCACCGGUG
232
ACACUUGGAUUCACCGGUGNN
512


 


s
167
UGUGGCCAUGCAUGUGUUC
233
UGUGGCCAUGCAUGUGUUCNN
513


 


as
185
GAACACAUGCAUGGCCACA
234
GAACACAUGCAUGGCCACANN
514


 


s
168
GUGGCCAUGCAUGUGUUCA
235
GUGGCCAUGCAUGUGUUCANN
515


 


as
186
UGAACACAUGCAUGGCCAC
236
UGAACACAUGCAUGGCCACNN
516


 


s
171
GCCAUGCAUGUGUUCAGAA
237
GCCAUGCAUGUGUUCAGAANN
517


 


as
189
UUCUGAACACAUGCAUGGC
238
UUCUGAACACAUGCAUGGCNN
518


 


s
432
UAUUCCACCACGGCUGUCA
239
UAUUCCACCACGGCUGUCANN
519


 


as
449
UGACAGCCGUGGUGGAAUA
240
UGACAGCCGUGGUGGAAUANN
520


 


s
447
GUCAUCACCAAUCCCAAGG
241
GUCAUCACCAAUCCCAAGGNN
521


 


as
465
CCUUGGGAUUGGUGAUGAC
242
CCUUGGGAUUGGUGAUGACNN
522


 


s
115
GUCCUCUGAUGGUCAAAGU
243
GUCCUCUGAUGGUCAAAGUNN
523


 


as
133
ACUUUGACCAUCAGAGGAC
244
ACUUUGACCAUCAGAGGACNN
524


 


s
122
GAUGGUCAAAGUUCUAGAU
245
GAUGGUCAAAGUUCUAGAUNN
525


 


as
140
AUCUAGAACUUUGACCAUC
246
AUCUAGAACUUUGACCAUCNN
526


 


s
139
AUGCUGUCCGAGGCAGUCC
247
AUGCUGUCCGAGGCAGUCCNN
527


 


as
157
GGACUGCCUCGGACAGCAU
248
GGACUGCCUCGGACAGCAUNN
528


 


s
172
CCGUGCAUGUGUUCAGAAA
249
CCGUGCAUGUGUUCAGAAANN
529


 


as
190
UUUCUGAACACAUGCACGG
250
UUUCUGAACACAUGCACGGNN
530


 


s
238
AGUCUGGAGAGCUGCAUGG
251
AGUCUGGAGAGCUGCAUGGNN
531


 


as
256
CCAUGCAGCUCUCCAGACU
252
CCAUGCAGCUCUCCAGACUNN
532


 


s
252
CAUGGGCUCACAACUGAGG
253
CAUGGGCUCACAACUGAGGNN
533


 


as
270
CCUCAGUUGUGAGCCCAUG
254
CCUCAGUUGUGAGCCCAUGNN
534


 


s
 33
UCUCAUCGUCUGCUCCUCC
255
UCUCAUCGUCUGCUCCUCCNN
535


 


as
 51
GGAGGAGCAGACGAUGAGA
256
GGAGGAGCAGACGAUGAGANN
536


 


s
340
CCCCAUUCCAUGAGCAUGC
257
CCCCAUUCCAUGAGCAUGCNN
537


 


as
358
GCAUGCUCAUGGAAUGGGG
258
GCAUGCUCAUGGAAUGGGGNN
538


 


s
421
GCCCCUACUCCUAUUCCAC
259
GCCCCUACUCCUAUUCCACNN
539


 


as
439
GUGGAAUAGGAGUAGGGGC
260
GUGGAAUAGGAGUAGGGGCNN
540


 


s
431
CUAUUCCACCACGGCUGUC
261
CUAUUCCACCACGGCUGUCNN
541


 


as
449
GACAGCCGUGGUGGAAUAG
262
GACAGCCGUGGUGGAAUAGNN
542


 


s
440
CACGGCUGUCGUCACCAAU
263
CACGGCUGUCGUCACCAAUNN
543


 


as
458
AUUGGUGACGACAGCCGUG
264
AUUGGUGACGACAGCCGUGNN
544


 


s
496
AGGACGAGGGAUGGGAUUU
265
AGGACGAGGGAUGGGAUUUNN
545


 


as
514
AAAUCCCAUCCCUCGUCCU
266
AAAUCCCAUCCCUCGUCCUNN
546


 


s
556
UCACCUCAUAUGCUAUGUU
267
UCACCUCAUAUGCUAUGUUNN
547


 


as
574
AACAUAGCAUAUGAGGUGA
268
AACAUAGCAUAUGAGGUGANN
548


 


s
559
CCUCAUAUGCUAUGUUAGA
269
CCUCAUAUGCUAUGUUAGANN
549


 


as
577
UCUAACAUAGCAUAUGAGG
270
UCUAACAUAGCAUAUGAGGNN
550


 


s
570
AUGUUAGAAGUCCAGGCAG
271
AUGUUAGAAGUCCAGGCAGNN
551


 


as
588
CUGCCUGGACUUCUAACAU
272
CUGCCUGGACUUCUAACAUNN
552


 


s
 78
UCUGAGGCUGGCCCUACGG
273
UCUGAGGCUGGCCCUACGGNN
553


 


as
 96
CCGUAGGGCCAGCCUCAGA
274
CCGUAGGGCCAGCCUCAGANN
554


 


s
 87
GGCCCUACGGGCACCGGUG
275
GGCCCUACGGGCACCGGUGNN
555


 


as
105
CACCGGUGCCCGUAGGGCC
276
CACCGGUGCCCGUAGGGCCNN
556


 


s
 95
GGGCACCGGUGAAUCCAAG
277
GGGCACCGGUGAAUCCAAGNN
557


 


as
113
CUUGGAUUCACCGGUGCCC
278
CUUGGAUUCACCGGUGCCCNN
558


 


s
167
CCAUGCAUGUGUUCAGAAA
279
CCAUGCAUGUGUUCAGAAANN
559


 


as
185
UUUCUGAACACAUGCAUGG
280
UUUCUGAACACAUGCAUGGNN
560
















TABLE 3B







Sense and antisense strand sequences of human TTR


dsRNAs (NM_000371.2, SEQ ID NO: 1329)













SEQ




Sequence with 3′deoxythimidine
ID


Strand
Position
overhang (5′ to 3′)
NO:













s
100
CCGGUGAAUCCAAGUGUCCdTdT
561


 


as
118
GGACACUUGGAUUCACCGGdTdT
562


 


s
11
ACUCAUUCUUGGCAGGAUGdTdT
563


 


as
29
CAUCCUGCCAAGAAUGAGUdTdT
564


 


s
111
AAGUGUCCUCUGAUGGUCAdTdT
565


 


as
129
UGACCAUCAGAGGACACUUdTdT
566


 


s
13
UCAUUCUUGGCAGGAUGGCdTdT
567


 


as
31
GCCAUCCUGCCAAGAAUGAdTdT
568


 


s
130
AAGUUCUAGAUGCUGUCCGdTdT
569


 


as
148
CGGACAGCAUCUAGAACUUdTdT
570


 


s
132
GUUCUAGAUGCUGUCCGAGdTdT
571


 


as
150
CUCGGACAGCAUCUAGAACdTdT
572


 


s
135
CUAGAUGCUGUCCGAGGCAdTdT
573


 


as
153
UGCCUCGGACAGCAUCUAGdTdT
574


 


s
138
GAUGCUGUCCGAGGCAGUCdTdT
575


 


as
156
GACUGCCUCGGACAGCAUCdTdT
576


 


s
14
CAUUCUUGGCAGGAUGGCUdTdT
577


 


as
32
AGCCAUCCUGCCAAGAAUGdTdT
578


 


s
140
UGCUGUCCGAGGCAGUCCUdTdT
579


 


as
158
AGGACUGCCUCGGACAGCAdTdT
580


 


s
146
CCGAGGCAGUCCUGCCAUCdTdT
581


 


as
164
GAUGGCAGGACUGCCUCGGdTdT
582


 


s
152
CAGUCCUGCCAUCAAUGUGdTdT
583


 


as
170
CACAUUGAUGGCAGGACUGdTdT
584


 


s
164
CAAUGUGGCCGUGCAUGUGdTdT
585


 


as
182
CACAUGCACGGCCACAUUGdTdT
586


 


s
178
AUGUGUUCAGAAAGGCUGCdTdT
587


 


as
196
GCAGCCUUUCUGAACACAUdTdT
588


 


s
2
CAGAAGUCCACUCAUUCUUdTdT
589


 


as
20
AAGAAUGAGUGGACUUCUGdTdT
590


 


s
21
GGCAGGAUGGCUUCUCAUCdTdT
591


 


as
39
GAUGAGAAGCCAUCCUGCCdTdT
592


 


s
210
GAGCCAUUUGCCUCUGGGAdTdT
593


 


as
228
UCCCAGAGGCAAAUGGCUCdTdT
594


 


s
23
CAGGAUGGCUUCUCAUCGUdTdT
595


 


as
41
ACGAUGAGAAGCCAUCCUGdTdT
596


 


s
24
AGGAUGGCUUCUCAUCGUCdTdT
597


 


as
42
GACGAUGAGAAGCCAUCCUdTdT
598


 


s
245
AGAGCUGCAUGGGCUCACAdTdT
599


 


as
263
UGUGAGCCCAUGCAGCUCUdTdT
600


 


s
248
GCUGCAUGGGCUCACAACUdTdT
601


 


as
266
AGUUGUGAGCCCAUGCAGCdTdT
602


 


s
25
GGAUGGCUUCUCAUCGUCUdTdT
603


 


as
43
AGACGAUGAGAAGCCAUCCdTdT
604


 


s
251
GCAUGGGCUCACAACUGAGdTdT
605


 


as
269
CUCAGUUGUGAGCCCAUGCdTdT
606


 


s
253
AUGGGCUCACAACUGAGGAdTdT
607


 


as
271
UCCUCAGUUGUGAGCCCAUdTdT
608


 


s
254
UGGGCUCACAACUGAGGAGdTdT
609


 


as
272
CUCCUCAGUUGUGAGCCCAdTdT
610


 


s
270
GAGGAAUUUGUAGAAGGGAdTdT
611


 


as
288
UCCCUUCUACAAAUUCCUCdTdT
612


 


s
276
UUUGUAGAAGGGAUAUACAdTdT
613


 


as
294
UGUAUAUCCCUUCUACAAAdTdT
614


 


s
277
UUGUAGAAGGGAUAUACAAdTdT
615


 


as
295
UUGUAUAUCCCUUCUACAAdTdT
616


 


s
278
UGUAGAAGGGAUAUACAAAdTdT
617


 


as
296
UUUGUAUAUCCCUUCUACAdTdT
618


 


s
281
AGAAGGGAUAUACAAAGUGdTdT
619


 


as
299
CACUUUGUAUAUCCCUUCUdTdT
620


 


s
295
AAGUGGAAAUAGACACCAAdTdT
621


 


as
313
UUGGUGUCUAUUUCCACUUdTdT
622


 


s
299
GGAAAUAGACACCAAAUCUdTdT
623


 


as
317
AGAUUUGGUGUCUAUUUCCdTdT
624


 


s
300
GAAAUAGACACCAAAUCUUdTdT
625


 


as
318
AAGAUUUGGUGUCUAUUUCdTdT
626


 


s
303
AUAGACACCAAAUCUUACUdTdT
627


 


as
321
AGUAAGAUUUGGUGUCUAUdTdT
628


 


s
304
UAGACACCAAAUCUUACUGdTdT
629


 


as
322
CAGUAAGAUUUGGUGUCUAdTdT
630


 


s
305
AGACACCAAAUCUUACUGGdTdT
631


 


as
323
CCAGUAAGAUUUGGUGUCUdTdT
632


 


s
317
UUACUGGAAGGCACUUGGCdTdT
633


 


as
335
GCCAAGUGCCUUCCAGUAAdTdT
634


 


s
32
UUCUCAUCGUCUGCUCCUCdTdT
635


 


as
50
GAGGAGCAGACGAUGAGAAdTdT
636


 


s
322
GGAAGGCACUUGGCAUCUCdTdT
637


 


as
340
GAGAUGCCAAGUGCCUUCCdTdT
638


 


s
326
GGCACUUGGCAUCUCCCCAdTdT
639


 


as
344
UGGGGAGAUGCCAAGUGCCdTdT
640


 


s
333
GGCAUCUCCCCAUUCCAUGdTdT
641


 


as
351
AUGGAAUGGGGAGAUGCCTTdTdT
642


 


s
334
GCAUCUCCCCAUUCCAUGAdTdT
643


 


as
352
UCAUGGAAUGGGGAGAUGCdTdT
644


 


s
335
CAUCUCCCCAUUCCAUGAGdTdT
645


 


as
353
CUCAUGGAAUGGGGAGAUGdTdT
646


 


s
336
AUCUCCCCAUUCCAUGAGCdTdT
647


 


as
354
GCUCAUGGAAUGGGGAGAUdTdT
648


 


s
338
CUCCCCAUUCCAUGAGCAUdTdT
649


 


as
356
AUGCUCAUGGAAUGGGGAGdTdT
650


 


s
341
CCCAUUCCAUGAGCAUGCAdTdT
651


 


as
359
UGCAUGCUCAUGGAAUGGGdTdT
652


 


s
347
CCAUGAGCAUGCAGAGGUGdTdT
653


 


as
365
CACCUCUGCAUGCUCAUGGdTdT
654


 


s
352
AGCAUGCAGAGGUGGUAUUdTdT
655


 


as
370
AAUACCACCUCUGCAUGCUdTdT
656


 


s
354
CAUGCAGAGGUGGUAUUCAdTdT
657


 


as
372
UGAAUACCACCUCUGCAUGdTdT
658


 


s
355
AUGCAGAGGUGGUAUUCACdTdT
659


 


as
373
GUGAAUACCACCUCUGCAUdTdT
660


 


s
362
GGUGGUAUUCACAGCCAACdTdT
661


 


as
380
GUUGGCUGUGAAUACCACCdTdT
662


 


s
363
GUGGUAUUCACAGCCAACGdTdT
663


 


as
381
CGUUGGCUGUGAAUACCACdTdT
664


 


s
364
UGGUAUUCACAGCCAACGAdTdT
665


 


as
382
UCGUUGGCUGUGAAUACCAdTdT
666


 


s
365
GGUAUUCACAGCCAACGACdTdT
667


 


as
383
GUCGUUGGCUGUGAAUACCdTdT
668


 


s
366
GUAUUCACAGCCAACGACUdTdT
669


 


as
384
AGUCGUUGGCUGUGAAUACdTdT
670


 


s
367
UAUUCACAGCCAACGACUCdTdT
671


 


as
385
GAGUCGUUGGCUGUGAAUAdTdT
672


 


s
370
UCACAGCCAACGACUCCGGdTdT
673


 


as
388
CCGGAGUCGUUGGCUGUGAdTdT
674


 


s
390
CCCCGCCGCUACACCAUUGdTdT
675


 


as
408
CAAUGGUGUAGCGGCGGGGdTdT
676


 


s
4
GAAGUCCACUCAUUCUUGGdTdT
677


 


as
22
CCAAGAAUGAGUGGACUUCdTdT
678


 


s
412
CCCUGCUGAGCCCCUACUCdTdT
679


 


as
430
GAGUAGGGGCUCAGCAGGGdTdT
680


 


s
417
CUGAGCCCCUACUCCUAUUdTdT
681


 


as
435
AAUAGGAGUAGGGGCUCAGdTdT
682


 


s
418
UGAGCCCCUACUCCUAUUCdTdT
683


 


as
436
GAAUAGGAGUAGGGGCUCAdTdT
684


 


s
422
CCCCUACUCCUAUUCCACCdTdT
685


 


as
440
GGUGGAAUAGGAGUAGGGGdTdT
686


 


s
425
CUACUCCUAUUCCACCACGdTdT
687


 


as
443
CGUGGUGGAAUAGGAGUAGdTdT
688


 


s
426
UACUCCUAUUCCACCACGGdTdT
689


 


as
444
CCGUGGUGGAAUAGGAGUAdTdT
690


 


s
427
ACUCCUAUUCCACCACGGCdTdT
691


 


as
445
GCCGUGGUGGAAUAGGAGUdTdT
692


 


s
429
UCCUAUUCCACCACGGCUGdTdT
693


 


as
447
CAGCCGUGGUGGAAUAGGAdTdT
694


 


s
432
UAUUCCACCACGGCUGUCGdTdT
695


 


as
450
CGACAGCCGUGGUGGAAUAdTdT
696


 


s
433
AUUCCACCACGGCUGUCGUdTdT
697


 


as
451
ACGACAGCCGUGGUGGAAUdTdT
698


 


s
437
CACCACGGCUGUCGUCACCdTdT
699


 


as
455
GGUGACGACAGCCGUGGUGdTdT
700


 


s
438
ACCACGGCUGUCGUCACCAdTdT
701


 


as
456
UGGUGACGACAGCCGUGGUdTdT
702


 


s
439
CCACGGCUGUCGUCACCAAdTdT
703


 


as
457
UUGGUGACGACAGCCGUGGdTdT
704


 


s
441
ACGGCUGUCGUCACCAAUCdTdT
705


 


as
459
GAUUGGUGACGACAGCCGUdTdT
706


 


s
442
CGGCUGUCGUCACCAAUCCdTdT
707


 


as
460
GGAUUGGUGACGACAGCCGdTdT
708


 


s
449
CGUCACCAAUCCCAAGGAAdTdT
709


 


as
467
UUCCUUGGGAUUGGUGACGdTdT
710


 


s
455
CAAUCCCAAGGAAUGAGGGdTdT
711


 


as
473
CCCUCAUUCCUUGGGAUUGdTdT
712


 


s
491
CCUGAAGGACGAGGGAUGGdTdT
713


 


as
509
CCAUCCCUCGUCCUUCAGGdTdT
714


 


s
497
GGACGAGGGAUGGGAUUUCdTdT
715


 


as
515
GAAAUCCCAUCCCUCGUCCdTdT
716


 


s
5
AAGUCCACUCAUUCUUGGCdTdT
717


 


as
23
GCCAAGAAUGAGUGGACUUdTdT
718


 


s
508
GGGAUUUCAUGUAACCAAGdTdT
719


 


as
526
CUUGGUUACAUGAAAUCCCdTdT
720


 


s
509
GGAUUUCAUGUAACCAAGAdTdT
721


 


as
527
UCUUGGUUACAUGAAAUCCdTdT
722


 


s
514
UCAUGUAACCAAGAGUAUUdTdT
723


 


as
532
AAUACUCUUGGUUACAUGAdTdT
724


 


s
516
AUGUAACCAAGAGUAUUCCdTdT
725


 


as
534
GGAAUACUCUUGGUUACAUdTdT
726


 


s
517
UGUAACCAAGAGUAUUCCAdTdT
727


 


as
535
UGGAAUACUCUUGGUUACAdTdT
728


 


s
518
GUAACCAAGAGUAUUCCAUdTdT
729


 


as
536
AUGGAAUACUCUUGGUUACdTdT
730


 


s
54
UGCCUUGCUGGACUGGUAUdTdT
731


 


as
72
AUACCAGUCCAGCAAGGCAdTdT
732


 


s
543
UAAAGCAGUGUUUUCACCUdTdT
733


 


as
561
AGGUGAAAACACUGCUUUAdTdT
734


 


s
55
GCCUUGCUGGACUGGUAUUdTdT
735


 


as
73
AAUACCAGUCCAGCAAGGCdTdT
736


 


s
551
UGUUUUCACCUCAUAUGCUdTdT
737


 


as
569
AGCAUAUGAGGUGAAAACAdTdT
738


 


s
552
GUUUUCACCUCAUAUGCUAdTdT
739


 


as
570
UAGCAUAUGAGGUGAAAACdTdT
740


 


s
553
UUUUCACCUCAUAUGCUAUdTdT
741


 


as
571
AUAGCAUAUGAGGUGAAAAdTdT
742


 


s
555
UUCACCUCAUAUGCUAUGUdTdT
743


 


as
573
ACAUAGCAUAUGAGGUGAAdTdT
744


 


s
557
CACCUCAUAUGCUAUGUUAdTdT
745


 


as
575
UAACAUAGCAUAUGAGGUGdTdT
746


 


s
56
CCUUGCUGGACUGGUAUUUdTdT
747


 


as
74
AAAUACCAGUCCAGCAAGGdTdT
748


 


s
563
AUAUGCUAUGUUAGAAGUCdTdT
749


 


as
581
GACUUCUAACAUAGCAUAUdTdT
750


 


s
564
UAUGCUAUGUUAGAAGUCCdTdT
751


 


as
582
GGACUUCUAACAUAGCAUAdTdT
752


 


s
566
UGCUAUGUUAGAAGUCCAGdTdT
753


 


as
584
CUGGACUUCUAACAUAGCAdTdT
754


 


s
57
CUUGCUGGACUGGUAUUUGdTdT
755


 


as
75
CAAAUACCAGUCCAGCAAGdTdT
756


 


s
578
AGUCCAGGCAGAGACAAUAdTdT
757


 


as
596
AUUGUCUCUGCCUGGACUTTdTdT
758


 


s
580
UCCAGGCAGAGACAAUAAAdTdT
759


 


as
598
UUUAUUGUCUCUGCCUGGAdTdT
760


 


s
607
GUGAAAGGCACUUUUCAUUdTdT
761


 


as
625
AAUGAAAAGUGCCUUUCACdTdT
762


 


s
62
UGGACUGGUAUUUGUGUCUdTdT
763


 


as
80
AGACACAAAUACCAGUCCAdTdT
764


 


s
77
GUCUGAGGCUGGCCCUACGdTdT
765


 


as
95
CGUAGGGCCAGCCUCAGACdTdT
766


 


s
79
CUGAGGCUGGCCCUACGGGdTdT
767


 


as
97
CCCGUAGGGCCAGCCUCAGdTdT
768


 


s
81
GAGGCUGGCCCUACGGGCAdTdT
769


 


as
99
UGCCCGUAGGGCCAGCCUCdTdT
770


 


s
82
AGGCUGGCCCUACGGGCACdTdT
771


 


as
100
GUGCCCGUAGGGCCAGCCUdTdT
772


 


s
84
GCUGGCCCUACGGGCACCGdTdT
773


 


as
102
CGGUGCCCGUAGGGCCAGCdTdT
774


 


s
85
CUGGCCCUACGGGCACCGGdTdT
775


 


as
103
CCGGUGCCCGUAGGGCCAGdTdT
776


 


s
87
GGCCCUACGGGCACCGGUGdTdT
777


 


as
105
CACCGGUGCCCGUAGGGCCdTdT
778


 


s
9
CCACUCAUUCUUGGCAGGAdTdT
779


 


as
27
UCCUGCCAAGAAUGAGUGGdTdT
780


 


s
90
CCUACGGGCACCGGUGAAUdTdT
781


 


as
108
AUUCACCGGUGCCCGUAGGdTdT
782


 


s
91
CUACGGGCACCGGUGAAUCdTdT
783


 


as
109
GAUUCACCGGUGCCCGUAGdTdT
784


 


s
92
UACGGGCACCGGUGAAUCCdTdT
785


 


as
110
GGAUUCACCGGUGCCCGUAdTdT
786


 


s
93
ACGGGCACCGGUGAAUCCAdTdT
787


 


as
111
UGGAUUCACCGGUGCCCGUdTdT
788


 


s
97
GCACCGGUGAAUCCAAGUGdTdT
789


 


as
115
CACUUGGAUUCACCGGUGCdTdT
790


 


s
98
CACCGGUGAAUCCAAGUGUdTdT
791


 


as
116
ACACUUGGAUUCACCGGUGdTdT
792


 


s
167
UGUGGCCAUGCAUGUGUUCdTdT
793


 


as
185
GAACACAUGCAUGGCCACAdTdT
794


 


s
168
GUGGCCAUGCAUGUGUUCAdTdT
795


 


as
186
UGAACACAUGCAUGGCCACdTdT
796


 


s
171
GCCAUGCAUGUGUUCAGAAdTdT
797


 


as
189
UUCUGAACACAUGCAUGGCdTdT
798


 


s
432
UAUUCCACCACGGCUGUCAdTdT
799


 


as
449
UGACAGCCGUGGUGGAAUAdTdT
800


 


s
447
GUCAUCACCAAUCCCAAGGdTdT
801


 


as
465
CCUUGGGAUUGGUGAUGACdTdT
802


 


s
115
GUCCUCUGAUGGUCAAAGUdTdT
803


 


as
133
ACUUUGACCAUCAGAGGACdTdT
804


 


s
122
GAUGGUCAAAGUUCUAGAUdTdT
805


 


as
140
AUCUAGAACUUUGACCAUCdTdT
806


 


s
139
AUGCUGUCCGAGGCAGUCCdTdT
807


 


as
157
GGACUGCCUCGGACAGCAUdTdT
808


 


s
172
CCGUGCAUGUGUUCAGAAAdTdT
809


 


as
190
UUUCUGAACACAUGCACGGdTdT
810


 


s
238
AGUCUGGAGAGCUGCAUGGdTdT
811


 


as
256
CCAUGCAGCUCUCCAGACUdTdT
812


 


s
252
CAUGGGCUCACAACUGAGGdTdT
813


 


as
270
CCUCAGUUGUGAGCCCAUGdTdT
814


 


s
33
UCUCAUCGUCUGCUCCUCCdTdT
815


 


as
51
GGAGGAGCAGACGAUGAGAdTdT
816


 


s
340
CCCCAUUCCAUGAGCAUGCdTdT
817


 


as
358
GCAUGCUCAUGGAAUGGGGdTdT
818


 


s
421
GCCCCUACUCCUAUUCCACdTdT
819


 


as
439
GUGGAAUAGGAGUAGGGGCdTdT
820


 


s
431
CUAUUCCACCACGGCUGUCdTdT
821


 


as
449
GACAGCCGUGGUGGAAUAGdTdT
822


 


s
440
CACGGCUGUCGUCACCAAUdTdT
823


 


as
458
AUUGGUGACGACAGCCGUGdTdT
824


 


s
496
AGGACGAGGGAUGGGAUUUdTdT
825


 


as
514
AAAUCCCAUCCCUCGUCCUdTdT
826


 


s
556
UCACCUCAUAUGCUAUGUUdTdT
827


 


as
574
AACAUAGCAUAUGAGGUGAdTdT
828


 


s
559
CCUCAUAUGCUAUGUUAGAdTdT
829


 


as
577
UCUAACAUAGCAUAUGAGGdTdT
830


 


s
570
AUGUUAGAAGUCCAGGCAGdTdT
831


 


as
588
CUGCCUGGACUUCUAACAUdTdT
832


 


s
78
UCUGAGGCUGGCCCUACGGdTdT
833


 


as
96
CCGUAGGGCCAGCCUCAGAdTdT
834


 


s
87
GGCCCUACGGGCACCGGUGdTdT
835


 


as
105
CACCGGUGCCCGUAGGGCCdTdT
836


 


s
95
GGGCACCGGUGAAUCCAAGdTdT
837


 


as
113
CUUGGAUUCACCGGUGCCCdTdT
838


 


s
167
CCAUGCAUGUGUUCAGAAAdTdT
839


 


as
185
UUUCUGAACACAUGCAUGGdTdT
840





Strand: s = sense; as = antisense; Position: position of 5′ base on transcript













TABLE 4







Chemically modified sense and antisense strand


sequences of human TTR dsRNAs (NM_000371.2,


SEQ ID NO: 1329)















SEQ




Posi-

ID


Strand
Oligo #
tion
Sequence(5′ to 3′)
NO:














s
A-32153
100
ccGGuGAAuccAAGuGuccdTdT
841


 


as
A-32154
118
GGAcACUUGGAUUcACCGGdTdT
842


 


s
A-32155
11
AcucAuucuuGGcAGGAuGdTdT
843


 


as
A-32156
29
cAUCCUGCcAAGAAUGAGUdTdT
844


 


s
A-32157
III
AAGuGuccucuGAuGGucAdTdT
845


 


as
A-32158
129
UGACcAUcAGAGGAcACUUdTdT
846


 


s
A-32163
13
ucAuucuuGGcAGGAuGGcdTdT
847


 


as
A-32164
31
GCcAUCCUGCcAAGAAUGAdTdT
848


 


s
A-32165
130
AAGuucuAGAuGcuGuccGdTdT
849


 


as
A-32166
148
CGGAcAGcAUCuAGAACUUdTdT
850


 


s
A-32167
132
GuucuAGAuGcuGuccGAGdTdT
851


 


as
A-32168
150
CUCGGAcAGcAUCuAGAACdTdT
852


 


s
A-32169
135
cuAGAuGcuGuccGAGGcAdTdT
853


 


as
A-32170
153
UGCCUCGGAcAGcAUCuAGdTdT
854


 


s
A-32171
138
GAuGcuGuccGAGGcAGucdTdT
855


 


as
A-32172
156
GACUGCCUCGGAcAGcAUCdTdT
856


 


s
A-32175
14
cAuucuuGGcAGGAuGGcudTdT
857


 


as
A-32176
32
AGCcAUCCUGCcAAGAAUGdTdT
858


 


s
A-32177
140
uGcuGuccGAGGcAGuccudTdT
859


 


as
A-32178
158
AGGACUGCCUCGGAcAGcAdTdT
860


 


s
A-32179
146
ccGAGGcAGuccuGccAucdTdT
861


 


as
A-32180
164
GAUGGcAGGACUGCCUCGGdTdT
862


 


s
A-32181
152
cAGuccuGccAucAAuGuGdTdT
863


 


as
A-32182
170
cAcAUUGAUGGcAGGACUGdTdT
864


 


s
A-32183
164
cAAuGuGGccGuGcAuGuGdTdT
865


 


as
A-32184
182
cAcAUGcACGGCcAcAUUGdTdT
866


 


s
A-32187
178
AuGuGuucAGAAAGGcuGcdTdT
867


 


as
A-32188
196
GcAGCCUUUCUGAAcAcAUdTdT
868


 


s
A-32189
2
cAGAAGuccAcucAuucuudTdT
869


 


as
A-32190
20
AAGAAUGAGUGGACUUCUGdTdT
870


 


s
A-32191
21
GGcAGGAuGGcuucucAucdTdT
871


 


as
A-32192
39
GAUGAGAAGCcAUCCUGCCdTdT
872


 


s
A-32193
210
GAGccAuuuGccucuGGGAdTdT
873


 


as
A-32194
228
UCCcAGAGGcAAAUGGCUCdTdT
874


 


s
A-32195
23
cAGGAuGGcuucucAucGudTdT
875


 


as
A-32196
41
ACGAUGAGAAGCcAUCCUGdTdT
876


 


s
A-32199
24
AGGAuGGcuucucAOcGucdTdT
877


 


as
A-32200
42
GACGAUGAGAAGCcAUCCUdTdT
878


 


s
A-32201
245
AGAGcuGcAuGGGcucAcAdTdT
879


 


as
A-32202
263
UGUGAGCCcAUGcAGCUCUdTdT
880


 


s
A-32203
248
GcuGcAuGGGcucAcAAcudTdT
881


 


as
A-32204
266
AGUUGUGAGCCcAUGcAGCdTdT
882


 


s
A-32205
25
GGAuGGcuucucAucGucudTdT
883


 


as
A-32206
43
AGACGAUGAGAAGCcAUCCdTdT
884


 


s
A-32207
251
GcAuGGGcucAcAAcuGAGdTdT
885


 


as
A-32208
269
CUcAGUUGUGAGCCcAUGCdTdT
886


 


s
A-32211
253
AuGGGcucAcAAcuGAGGAdTdT
887


 


as
A-32212
271
UCCUcAGUUGUGAGCCcAUdTdT
888


 


s
A-32213
254
uGGGcucAcAAcuGAGGAGdTdT
889


 


as
A-32214
272
CUCCUcAGUUGUGAGCCcAdTdT
890


 


s
A-32215
270
GAGGAAuuuGuAGAAGGGAdTdT
891


 


as
A-32216
288
UCCCUUCuAcAAAUUCCUCdTdT
892


 


s
A-32217
276
uuuGuAGAAGGGAuAuAcAdTdT
893


 


as
A-32218
294
UGuAuAUCCCUUCuAcAAAdTdT
894


 


s
A-32219
277
uuGuAGAAGGGAuAuAcAAdTdT
895


 


as
A-32220
295
UUGuAuAUCCCUUCuAcAAdTdT
896


 


s
A-32221
278
uGuAGAAGGGAuAuAcAAAdTdT
897


 


as
A-32222
296
UUUGuAuAUCCCUUCuAcAdTdT
898


 


s
A-32223
281
AGAAGGGAuAuAcAAAGuGdTdT
899


 


as
A-32224
299
cACUUUGuAuAUCCCUUCUdTdT
900


 


s
A-32225
295
AAGuGGAAAuAGAcAccAAdTdT
901


 


as
A-32226
313
UUGGUGUCuAUUUCcACUUdTdT
902


 


s
A-32227
299
GGAAAuAGAcAccAAAucudTdT
903


 


as
A-32228
317
AGAUUUGGUGUCuAUUUCCdTdT
904


 


s
A-32229
300
GAAAuAGAcAccAAAucuudTdT
905


 


as
A-32230
318
AAGAUUUGGUGUCuAUUUCdTdT
906


 


s
A-32231
303
AuAGAcAccAAAucuuAcudTdT
907


 


as
A-32232
321
AGuAAGAUUUGGUGUCuAUdTdT
908


 


s
A-32233
304
uAGAcAccAAAucuuAcuGdTdT
909


 


as
A-32234
322
cAGuAAGAUUUGGUGUCuAdTdT
910


 


s
A-32235
305
AGAcAccAAAucuuAcuGGdTdT
911


 


as
A-32236
323
CcAGuAAGAUUUGGUGUCUdTdT
912


 


s
A-32237
317
uuAcuGGAAGGcAcuuGGcdTdT
913


 


as
A-32238
335
GCcAAGUGCCUUCcAGuAAdTdT
914


 


s
A-32239
32
uucucAucGucuGcuccucdTdT
915


 


as
A-32240
50
GAGGAGcAGACGAUGAGAAdTdT
916


 


s
A-32241
322
GGAAGGcAcuuGGcAucucdTdT
917


 


as
A-32242
340
GAGAUGCcAAGUGCCUUCCdTdT
918


 


s
A-32243
326
GGcAcuuGGcAucuccccAdTdT
919


 


as
A-32244
344
UGGGGAGAUGCcAAGUGCCdTdT
920


 


s
A-32247
333
GGcAucuccccAuuccAuGdTdT
921


 


as
A-32248
351
cAUGGAAUGGGGAGAUGCCdTdT
922


 


s
A-32249
334
GcAucuccccAuuccAuGAdTdT
923


 


as
A-32250
352
UcAUGGAAUGGGGAGAUGCdTdT
924


 


s
A-32251
335
cAucuccccAuuccAuGAGdTdT
925


 


as
A-32252
353
CUcAUGGAAUGGGGAGAUGdTdT
926


 


s
A-32253
336
AucuccccAuuccAuGAGcdTdT
927


 


as
A-32254
354
GCUcAUGGAAUGGGGAGAUdTdT
928


 


s
A-32255
338
cuccccAuuccAuGAGcAudTdT
929


 


as
A-32256
356
AUGCUcAUGGAAUGGGGAGdTdT
930


 


s
A-32259
341
cccAuuccAuGAGcAuGcAdTdT
931


 


as
A-32260
359
UGcAUGCUcAUGGAAUGGGdTdT
932


 


s
A-32261
347
ccAuGAGcAuGcAGAGGuGdTdT
933


 


as
A-32262
365
cACCUCUGcAUGCUcAUGGdTdT
934


 


s
A-32263
352
AGcAuGcAGAGGuGGuAuudTdT
935


 


as
A-32264
370
AAuACcACCUCUGcAUGCUdTdT
936


 


s
A-32265
354
cAuGcAGAGGuGGuAuucAdTdT
937


 


as
A-32266
372
UGAAuACcACCUCUGcAUGdTdT
938


 


s
A-32267
355
AuGcAGAGGuGGuAuucAcdTdT
939


 


as
A-32268
373
GUGAAuACcACCUCUGcAUdTdT
940


 


s
A-32269
362
GGuGGuAuucAcAGccAAcdTdT
941


 


as
A-32270
380
GUUGGCUGUGAAuACcACCdTdT
942


 


s
A-32271
363
GuGGuAuucAcAGccAAcGdTdT
943


 


as
A-32272
381
CGUUGGCUGUGAAuACcACdTdT
944


 


s
A-32273
364
uGGuAuucAcAGccAAcGAdTdT
945


 


as
A-32274
382
UCGUUGGCUGUGAAuACcAdTdT
946


 


s
A-32275
365
GGuAuucAcAGccAAcGAcdTdT
947


 


as
A-32276
383
GUCGUUGGCUGUGAAuACCdTdT
948


 


s
A-32277
366
GuAuucAcAGccAAcGAcudTdT
949


 


as
A-32278
384
AGUCGUUGGCUGUGAAuACdTdT
950


 


s
A-32279
367
uAuucAcAGccAAcGAcucdTdT
951


 


as
A-32280
385
GAGUCGUUGGCUGUGAAuAdTdT
952


 


s
A-32281
370
ucAcAGccAAcGAcuccGGdTdT
953


 


as
A-32282
388
CCGGAGUCGUUGGCUGUGAdTdT
954


 


s
A-32283
390
ccccGccGcuAcAccAuuGdTdT
955


 


as
A-32284
408
cAAUGGUGuAGCGGCGGGGdTdT
956


 


s
A-32285
4
GAAGuccAcucAuucuuGGdTdT
957


 


as
A-32286
22
CcAAGAAUGAGUGGACUUCdTdT
958


 


s
A-32287
412
cccuGcuGAGccccuAcucdTdT
959


 


as
A-32288
430
GAGuAGGGGCUcAGcAGGGdTdT
960


 


s
A-32289
417
cuGAGccccuAcuccuAuudTdT
961


 


as
A-32290
435
AAuAGGAGuAGGGGCUcAGdTdT
962


 


s
A-32291
418
uGAGccccuAcuccuAuucdTdT
963


 


as
A-32292
436
GAAuAGGAGuAGGGGCUcAdTdT
964


 


s
A-32295
422
ccccuAcuccuAuuccAccdTdT
965


 


as
A-32296
440
GGUGGAAuAGGAGuAGGGGdTdT
966


 


s
A-32297
425
cuAcuccuAuuccAccAcGdldT
967


 


as
A-32298
443
CGUGGUGGAAuAGGAGuAGdTdT
968


 


s
A-32299
426
uAcuccuAuuccAccAcGGdTdT
969


 


as
A-32300
444
CCGUGGUGGAAuAGGAGuAdTdT
970


 


s
A-32301
427
AcuccuAuuccAccAcGGcdTdT
971


 


as
A-32302
445
GCCGUGGUGGAAuAGGAGUdTdT
972


 


s
A-32303
429
uccuAuuccAccAcGGcuGdTdT
973


 


as
A-32304
447
cAGCCGUGGUGGAAuAGGAdTdT
974


 


s
A-32307
432
uAuuccAccAcGGcuGucGdTdT
975


 


as
A-32308
450
CGAcAGCCGUGGUGGAAuAdTdT
976


 


s
A-32309
433
AuuccAccAcGGcuGucGudTdT
977


 


as
A-32310
451
ACGAcAGCCGUGGUGGAAUdTdT
978


 


s
A-32311
437
cAccAcGGcuGucGucAccdTdT
979


 


as
A-32312
455
GGUGACGAcAGCCGUGGUGdTdT
980


 


s
A-32313
438
AccAcGGcuGucGucAccAdTdT
981


 


as
A-32314
456
UGGUGACGAcAGCCGUGGUdTdT
982


 


s
A-32315
439
ccAcGGcuGucGucAccAAdTdT
983


 


as
A-32316
457
UUGGUGACGAcAGCCGUGGdTdT
984


 


s
A-32319
441
AcGGcuGucGucAccAAucdTdT
985


 


as
A-32320
459
GAUUGGUGACGAcAGCCGUdTdT
986


 


s
A-32321
442
cGGcuGucGucAccAAuccdTdT
987


 


as
A-32322
460
GGAUUGGUGACGAcAGCCGdTdT
988


 


s
A-32323
449
cGucAccAAucccAAGGAAdTdT
989


 


as
A-32324
467
UUCCUUGGGAUUGGUGACGdTdT
990


 


s
A-32325
455
cAAucccAAGGAAuGAGGGdTdT
991


 


as
A-32326
473
CCCUcAUUCCUUGGGAUUGdTdT
992


 


s
A-32327
491
ccuGAAGGAcGAGGGAuGGdTdT
993


 


as
A-32328
509
CcAUCCCUCGUCCUUcAGGdTdT
994


 


s
A-32331
497
GGAcGAGGGAuGGGAuuucdTdT
995


 


as
A-32332
515
GAAAUCCcAUCCCUCGUCCdTdT
996


 


s
A-32333
5
AAGuccAcucAuucuuGGcdTdT
997


 


as
A-32334
23
GCcAAGAAUGAGUGGACUUdTdT
998


 


s
A-32335
508
GGGAuuucAuGuAAccAAGdTdT
999


 


as
A-32336
526
CUUGGUuAcAUGAAAUCCCdTdT
1000


 


s
A-32337
509
GGAuuucAuGuAAccAAGAdTdT
1001


 


as
A-32338
527
UCUUGGUuAcAUGAAAUCCdTdT
1002


 


s
A-32339
514
ucAuGuAAccAAGAGuAuudTdT
1003


 


as
A-32340
532
AAuACUCUUGGUuAcAUGAdTdT
1004


 


s
A-32341
516
AuGuAAccAAGAGuAuuccdTdT
1005


 


as
A-32342
534
GGAAuACUCUUGGUuAcAUdTdT
1006


 


s
A-32343
517
uGuAAccAAGAGuAuuccAdTdT
1007


 


as
A-32344
535
UGGAAuACUCUUGGUuAcAdTdT
1008


 


s
A-32345
518
GuAAccAAGAGuAuuccAudTdT
1009


 


as
A-32346
536
AUGGAAuACUCUUGGUuACdTdT
1010


 


s
A-32347
54
uGccuuGcuGGAcuGGuAudTdT
1011


 


as
A-32348
72
AuACcAGUCcAGcAAGGcAdTdT
1012


 


s
A-32349
543
uAAAGcAGuGuuuucAccudTdT
1013


 


as
A-32350
561
AGGUGAAAAcACUGCUUuAdTdT
1014


 


s
A-32351
55
GccuuGcuGGAcuGGuAuudTdT
1015


 


as
A-32352
73
AAuACcAGUCcAGcAAGGCdTdT
1016


 


s
A-32353
551
uGuuuucAccucAuAuGcudTdT
1017


 


as
A-32354
569
AGcAuAUGAGGUGAAAAcAdTdT
1018


 


s
A-32355
552
GuuuucAccucAuAuGcuAdTdT
1019


 


as
A-32356
570
uAGcAuAUGAGGUGAAAACdTdT
1020


 


s
A-32357
553
uuuucAccucAuAuGcuAudTdT
1021


 


as
A-32358
571
AuAGcAuAUGAGGUGAAAAdTdT
1022


 


s
A-32359
555
uucAccucAuAuGcuAuGudTdT
1023


 


as
A-32360
573
AcAuAGcAuAUGAGGUGAAdTdT
1024


 


s
A-32363
557
cAccucAuAuGcuAuGuuAdTdT
1025


 


as
A-32364
575
uAAcAuAGcAuAUGAGGUGdTdT
1026


 


s
A-32367
56
ccuuGcuGGAcuGGuAuuudTdT
1027


 


as
A-32368
74
AAAuACcAGUCcAGcAAGGdTdT
1028


 


s
A-32369
563
AuAuGcuAuGuuAGAAGucdTdT
1029


 


as
A-32370
581
GACUUCuAAcAuAGcAuAUdTdT
1030


 


s
A-32371
564
uAuGcuAuGuuAGAAGuccdTdT
1031


 


as
A-32372
582
GGACUUCuAAcAuAGcAuAdTdT
1032


 


s
A-32373
566
uGcuAuGuuAGAAGuccAGdTdT
1033


 


as
A-32374
584
CUGGACUUCuAAcAuAGcAdTdT
1034


 


s
A-32375
57
cuuGcuGGAcuGGuAuuuGdTdT
1035


 


as
A-32376
75
cAAAuACcAGUCcAGcAAGdTdT
1036


 


s
A-32379
578
AGuccAGGcAGAGAcAAuAdTdT
1037


 


as
A-32380
596
uAUUGUCUCUGCCUGGACUdTdT
1038


 


s
A-32381
580
uccAGGcAGAGAcAAuAAAdTdT
1039


 


as
A-32382
598
UUuAUUGUCUCUGCCUGGAdTdT
1040


 


s
A-32383
607
GuGAAAGGcAcuuuucAuudTdT
1041


 


as
A-32384
625
AAUGAAAAGUGCCUUUcACdTdT
1042


 


s
A-32385
62
uGGAcuGGuAuuuGuGucudTdT
1043


 


as
A-32386
80
AGAcAcAAAuACcAGUCcAdTdT
1044


 


s
A-32387
77
GucuGAGGcuGGcccuAcGdTdT
1045


 


as
A-32388
95
CGuAGGGCcAGCCUcAGACdTdT
1046


 


s
A-32391
79
cuGAGGcuGGcccuAcGGGdTdT
1047


 


as
A-32392
97
CCCGuAGGGCcAGCCUcAGdTdT
1048


 


s
A-32393
81
GAGGcuGGcccuAcGGGcAdTdT
1049


 


as
A-32394
99
UGCCCGuAGGGCcAGCCUCdTdT
1050


 


s
A-32395
82
AGGcuGGcccuAcGGGcAcdTdT
1051


 


as
A-32396
100
GUGCCCGuAGGGCcAGCCUdTdT
1052


 


s
A-32397
84
GcuGGcccuAcGGGcAccGdTdT
1053


 


as
A-32398
102
CGGUGCCCGuAGGGCcAGCdTdT
1054


 


s
A-32399
85
cuGGcccuAcGGGcAccGGdTdT
1055


 


as
A-32400
103
CCGGUGCCCGuAGGGCcAGdTdT
1056


 


s
A-32401
87
GGcccuAcGGGcAccGGuGdTdT
1057


 


as
A-32402
105
cACCGGUGCCCGuAGGGCCdTdT
1058


 


s
A-32403
9
ccAcucAuucuuGGcAGGAdTdT
1059


 


as
A-32404
27
UCCUGCcAAGAAUGAGUGGdTdT
1060


 


s
A-32405
90
ccuAcGGGcAccGGuGAAudTdT
1061


 


as
A-32406
108
AUUcACCGGUGCCCGuAGGdTdT
1062


 


s
A-32407
91
cuAcGGGcAccGGuGAAucdTdT
1063


 


as
A-32408
109
GAUUcACCGGUGCCCGuAGdTdT
1064


 


s
A-32409
92
uAcGGGcAccGGuGAAuccdTdT
1065


 


as
A-32410
110
GGAUUcACCGGUGCCCGuAdTdT
1066


 


s
A-32411
93
AcGGGcAccGGuGAAuccAdTdT
1067


 


as
A-324I2
111
UGGAUUcACCGGUGCCCGUdTdT
1068


 


s
A-32415
97
GcAccGGuGAAuccAAGuGdTdT
1069


 


as
A-32416
115
cACUUGGAUUcACCGGUGCdTdT
1070


 


s
A-32417
98
cAccGGuGAAuccAAGuGudTdT
1071


 


as
A-32418
116
AcACUUGGAUUcACCGGUGdTdT
1072


 


s
A-32419
167
uGuGGccAuGcAuGuGuucdTdT
1073


 


as
A-32420
185
GAAcAcAUGcAUGGCcAcAdTdT
1074


 


s
A-32421
168
GuGGccAuGcAuGuGuucAdTdT
1075


 


as
A-32422
186
UGAAcAcAUGcAUGGCcACdTdT
1076


 


s
A-32423
171
GccAuGcAuGuGuucAGAAdTdT
1077


 


as
A-32424
189
UUCUGAAcAcAUGcAUGGCdTdT
1078


 


s
A-32427
432
uAuuccAccAcGGcuGucAdTdT
1079


 


as
A-32428
449
UGAcAGCCGUGGUGGAAuAdTdT
1080


 


s
A-32429
447
GucAucAccAAucccAAGGdTdT
1081


 


as
A-32430
465
CCUUGGGAUUGGUGAUGACdTdT
1082


 


s
A-32159
115
GuccucuGAuGGucAAAGudTdT
1083


 


as
A-32160
133
ACUUUGACcAUcAGAGGACdTdT
1084


 


s
A-32161
122
GAuGGucAAAGuucuAGAudTdT
1085


 


as
A-32162
140
AUCuAGAACUUUGACcAUCdTdT
1086


 


s
A-32173
139
AuGcuGuccGAGGcAGuccdTdT
1087


 


as
A-32174
157
GGACUGCCUCGGAcAGcAUdTdT
1088


 


s
A-32185
172
ccGuGcAuGuGuucAGAAAdTdT
1089


 


as
A-32186
190
UUUCUGAAcAcAUGcACGGdTdT
1090


 


s
A-32197
238
AGucuGGAGAGcuGcAuGGdTdT
1091


 


as
A-32198
256
CcAUGcAGCUCUCcAGACUdTdT
1092


 


s
A-32209
252
cAuGGGcucAcAAcuGAGGdTdT
1093


 


as
A-32210
270
CCUcAGUUGUGAGCCcAUGdTdT
1094


 


s
A-32245
33
ucucAucGucuGcuccuccdTdT
1095


 


as
A-32246
51
GGAGGAGcAGACGAUGAGAdTdT
1096


 


s
A-32257
340
ccccAuuccAuGAGcAuGcdTdT
1097


 


as
A-32258
358
GcAUGCUcAUGGAAUGGGGdTdT
1098


 


s
A-32293
421
GccccuAcuccuAuuccAcdTdT
1099


 


as
A-32294
439
GUGGAAuAGGAGuAGGGGCdTdT
1100


 


s
A-32305
431
cuAuuccAccAcGGcuGucdTdT
1101


 


as
A-32306
449
GAcAGCCGUGGUGGAAuAGdTdT
1102


 


s
A-32317
440
cAcGGcuGucGucAccAAudTdT
1103


 


as
A-32318
458
AUUGGUGACGAcAGCCGUGdTdT
1104


 


s
A-32329
496
AGGAcGAGGGAuGGGAuuudTdT
1105


 


as
A-32330
514
AAAUCCcAUCCCUCGUCCUdTdT
1106


 


s
A-32361
556
ucAccucAuAuGcuAuGuudTdT
1107


 


as
A-32362
574
AAcAuAGcAuAUGAGGUGAdTdT
1108


 


s
A-32365
559
ccucAuAuGcuAuGuuAGAdTdT
1109


 


as
A-32366
577
UCuAAcAuAGcAuAUGAGGdTdT
1110


 


s
A-32377
570
AuGuuAGAAGuccAGGcAGdTdT
1111


 


as
A-32378
588
CUGCCUGGACUUCuAAcAUdTdT
1112


 


s
A-32389
78
ucuGAGGcuGGcccuAcGGdTdT
1113


 


as
A-32390
96
CCGuAGGGCcAGCCUcAGAdTdT
1114


 


s
A-32401
87
GGcccuAcGGGcAccGGuGdTdT
1115


 


as
A-32402
105
cACCGGUGCCCGuAGGGCCdTdT
1116


 


s
A-32413
95
GGGcAccGGuGAAuccAAGdTdT
1117


 


as
A-32414
113
CUUGGAUUcACCGGUGCCCdTdT
1118


 


s
A-32425
167
ccAuGcAuGuGuucAGAAAdTdT
1119


 


as
A-32426
185
UUUCUGAAcAcAUGcAUGGdTdT
1120





See Table 2 for duplex #. Strand: s = sense; as = antisense; Position: position of 5′ base on transcript













TABLE 5







Identification numbers for rat TTR dsRNAs











Duplex #
Sense Oligo #
Antisense Oligo #







AD-18529
A-32745
A-32746



AD-18530
A-32747
A-32748



AD-18531
A-32749
A-32750



AD-18532
A-32751
A-32752



AD-18533
A-32753
A-32754



AD-18534
A-32755
A-32756



AD-18535
A-32757
A-32758



AD-18536
A-32759
A-32760



AD-18537
A-32761
A-32762



AD-18538
A-32763
A-32764



AD-18539
A-32159
A-32160



AD-18540
A-32765
A-32766



AD-18541
A-32767
A-32768



AD-18542
A-32769
A-32770



AD-18543
A-32771
A-32772



AD-18544
A-32773
A-32774



AD-18545
A-32775
A-32776



AD-18546
A-32777
A-32778



AD-18547
A-32779
A-32780



AD-18548
A-32781
A-32782



AD-18549
A-32783
A-32784



AD-18550
A-32785
A-32786



AD-18551
A-32787
A-32788



AD-18552
A-32791
A-32792



AD-18553
A-32793
A-32794



AD-18554
A-32795
A-32796







See Table 7 for sequences.













TABLE 6A







Sense and antisense strand sequences for rat TTR dsRNAs


(NM_012681.1, SEQ ID NO: 1330)















SEQ

SEQ





ID
Sequence with 3′ dinucleotide
ID


Strand
Position
Sequence (5′ to 3′)
NO:
overhang (5′ to 3′)
NO:





s
115
GUCCUCUGAUGGUCAAAGU
1121
GUCCUCUGAUGGUCAAAGUNN
1173


 


as
133
ACUUUGACCAUCAGAGGAC
1122
ACUUUGACCAUCAGAGGACNN
1174


 


s
537
UUCUUGCUCUAUAAACCGU
1123
UUCUUGCUCUAUAAACCGUNN
1175


 


as
555
ACGGUUUAUAGAGCAAGAA
1124
ACGGUUUAUAGAGCAAGAANN
1176


 


s
543
CUCUAUAAACCGUGUUAGC
1125
CUCUAUAAACCGUGUUAGCNN
1177


 


as
561
GCUAACACGGUUUAUAGAG
1126
GCUAACACGGUUUAUAGAGNN
1178


 


s
392
UCGCCACUACACCAUCGCA
1127
UCGCCACUACACCAUCGCANN
1179


 


as
410
UGCGAUGGUGUAGUGGCGA
1128
UGCGAUGGUGUAGUGGCGANN
1180


 


s
538
UCUUGCUCUAUAAACCGUG
1129
UCUUGCUCUAUAAACCGUGNN
1181


 


as
556
CACGGUUUAUAGAGCAAGA
1130
CACGGUUUAUAGAGCAAGANN
1182


 


s
541
UGCUCUAUAAACCGUGUUA
1131
UGCUCUAUAAACCGUGUUANN
1183


 


as
559
UAACACGGUUUAUAGAGCA
1132
UAACACGGUUUAUAGAGCANN
1184


 


s
532
CAGUGUUCUUGCUCUAUAA
1133
CAGUGUUCUUGCUCUAUAANN
1185


 


as
550
UUAUAGAGCAAGAACACUG
1134
UUAUAGAGCAAGAACACUGNN
1186


 


s
542
GCUCUAUAAACCGUGUUAG
1135
GCUCUAUAAACCGUGUUAGNN
1187


 


as
560
CUAACACGGUUUAUAGAGC
1136
CUAACACGGUUUAUAGAGCNN
1188


 


s
134
CCUGGAUGCUGUCCGAGGC
1137
CCUGGAUGCUGUCCGAGGCNN
1189


 


as
152
GCCUCGGACAGCAUCCAGG
1138
GCCUCGGACAGCAUCCAGGNN
1190


 


s
119
UCUGAUGGUCAAAGUCCUG
1139
UCUGAUGGUCAAAGUCCUGNN
1191


 


as
137
CAGGACUUUGACCAUCAGA
1140
CAGGACUUUGACCAUCAGANN
1192


 


s
241
CUGGAGAGCUGCACGGGCU
1141
CUGGAGAGCUGCACGGGCUNN
1193


 


as
259
AGCCCGUGCAGCUCUCCAG
1142
AGCCCGUGCAGCUCUCCAGNN
1194


 


s
544
UCUAUAAACCGUGUUAGCA
1143
UCUAUAAACCGUGUUAGCANN
1195


 


as
562
UGCUAACACGGUUUAUAGA
1144
UGCUAACACGGUUUAUAGANN
1196


 


s
530
AACAGUGUUCUUGCUCUAU
1145
AACAGUGUUCUUGCUCUAUNN
1197


 


as
548
AUAGAGCAAGAACACUGUU
1146
AUAGAGCAAGAACACUGUUNN
1198


 


s
118
CUCUGAUGGUCAAAGUCCU
1147
CUCUGAUGGUCAAAGUCCUNN
1199


 


as
136
AGGACUUUGACCAUCAGAG
1148
AGGACUUUGACCAUCAGAGNN
1200


 


s
140
UGCUGUCCGAGGCAGCCCU
1149
UGCUGUCCGAGGCAGCCCUNN
1201


 


as
158
AGGGCUGCCUCGGACAGCA
1150
AGGGCUGCCUCGGACAGCANN
1202


 


s
239
GUCUGGAGAGCUGCACGGG
1151
GUCUGGAGAGCUGCACGGGNN
1203


 


as
257
CCCGUGCAGCUCUCCAGAC
1152
CCCGUGCAGCUCUCCAGACNN
1204


 


s
531
ACAGUGUUCUUGCUCUAUA
1153
ACAGUGUUCUUGCUCUAUANN
1205


 


as
549
UAUAGAGCAAGAACACUGU
1154
UAUAGAGCAAGAACACUGUNN
1206


 


s
117
CCUCUGAUGGUCAAAGUCC
1155
CCUCUGAUGGUCAAAGUCCNN
1207


 


as
135
GGACUUUGACCAUCAGAGG
1156
GGACUUUGACCAUCAGAGGNN
1208


 


s
131
AGUCCUGGAUGCUGUCCGA
1157
AGUCCUGGAUGCUGUCCGANN
1209


 


as
149
UCGGACAGCAUCCAGGACU
1158
UCGGACAGCAUCCAGGACUNN
1210


 


s
217
UUGCCUCUGGGAAGACCGC
1159
UUGCCUCUGGGAAGACCGCNN
1211


 


as
235
GCGGUCUUCCCAGAGGCAA
1160
GCGGUCUUCCCAGAGGCAANN
1212


 


s
242
UGGAGAGCUGCACGGGCUC
1161
UGGAGAGCUGCACGGGCUCNN
1213


 


as
260
GAGCCCGUGCAGCUCUCCA
1162
GAGCCCGUGCAGCUCUCCANN
1214


 


s
244
GAGAGCUGCACGGGCUCAC
1163
GAGAGCUGCACGGGCUCACNN
1215


 


as
262
GUGAGCCCGUGCAGCUCUC
1164
GUGAGCCCGUGCAGCUCUCNN
1216


 


s
246
GAGCUGCACGGGCUCACCA
1165
GAGCUGCACGGGCUCACCANN
1217


 


as
264
UGGUGAGCCCGUGCAGCUC
1166
UGGUGAGCCCGUGCAGCUCNN
1218


 


s
399
UACACCAUCGCAGCCCUGC
1167
UACACCAUCGCAGCCCUGCNN
1219


 


as
417
GCAGGGCUGCGAUGGUGUA
1168
GCAGGGCUGCGAUGGUGUANN
1220


 


s
132
GUCCUGGAUGCUG UCCGAG
1169
GUCCUGGAUGCUGUCCGAGNN
1221


 


as
150
CUCGGACAGCAUCCAGGAC
1170
CUCGGACAGCAUCCAGGACNN
1222


 


s
245
AGAGCUGCACGGGCUCACC
1171
AGAGCUGCACGGGCUCACCNN
1223


 


as
263
GGUGAGCCCGUGCAGCUCU
1172
GGUGAGCCCGUGCAGCUCUNN
1224





Strand: s = sense; as = antisense; Position: position of 5′ base on transcript













TABLE 6B







Sense and antisense strand sequences for


rat TTR dsRNAs (NM_012681.1, SEQ ID NO: 1330)












Sequence with 3′





deoxythimidine overhang



Strand
Position
(5′ to 3′)
SEQ. ID NO:





s
115
GUCCUCUGAUGGUCAAAGUdTdT
1225


 


as
133
ACUUUGACCAUCAGAGGACdTdT
1226


 


s
537
UUCUUGCUCUAUAAACCGUdTdT
1227


 


as
555
ACGGUUUAUAGAGCAAGAAdTdT
1228


 


s
543
CUCUAUAAACCGUGUUAGCdTdT
1229


 


as
561
GCUAACACGGUUUAUAGAGdTdT
1230


 


s
392
UCGCCACUACACCAUCGCAdTdT
1231


 


as
410
UGCGAUGGUGUAGUGGCGAdTdT
1232


 


s
538
UCUUGCUCUAUAAACCGUGdTdT
1233


 


as
556
CACGGUUUAUAGAGCAAGAdTdT
1234


 


s
541
UGCUCUAUAAACCGUGUUAdTdT
1235


 


as
559
UAACACGGUUUAUAGAGCAdTdT
1236


 


s
532
CAGUGUUCUUGCUCUAUAAdTdT
1237


 


as
550
UUAUAGAGCAAGAACACUGdTdT
1238


 


s
542
GCUCUAUAAACCGUGUUAGdTdT
1239


 


as
560
CUAACACGGUUUAUAGAGCdTdT
1240


 


s
134
CCUGGAUGCUGUCCGAGGCdTdT
1241


 


as
152
GCCUCGGACAGCAUCCAGGdTdT
1242


 


s
119
UCUGAUGGUCAAAGUCCUGdTdT
1243


 


as
137
CAGGACUUUGACCAUCAGAdTdT
1244


 


s
241
CUGGAGAGCUGCACGGGCUdTdT
1245


 


as
259
AGCCCGUGCAGCUCUCCAGdTdT
1246


 


s
544
UCUAUAAACCGUGUUAGCAdTdT
1247


 


as
562
UGCUAACACGGUUUAUAGAdTdT
1248


 


s
530
AACAGUGUUCUUGCUCUAUdTdT
1249


 


as
548
AUAGAGCAAGAACACUGUUdTdT
1250


 


s
118
CUCUGAUGGUCAAAGUCCUdTdT
1251


 


as
136
AGGACUUUGACCAUCAGAGdTdT
1252


 


s
140
UGCUGUCCGAGGCAGCCCUdTdT
1253


 


as
158
AGGGCUGCCUCGGACAGCAdTdT
1254


 


s
239
GUCUGGAGAGCUGCACGGGdTdT
1255


 


as
257
CCCGUGCAGCUCUCCAGACdTdT
1256


 


s
531
ACAGUGUUCUUGCUCUAUAdTdT
1257


 


as
549
UAUAGAGCAAGAACACUGUdTdT
1258


 


s
117
CCUCUGAUGGUCAAAGUCCdTdT
1259


 


as
135
GGACUUUGACCAUCAGAGGdTdT
1260


 


s
131
AGUCCUGGAUGCUGUCCGAdTdT
1261


 


as
149
UCGGACAGCAUCCAGGACUdTdT
1262


 


s
217
UUGCCUCUGGGAAGACCGCdTdT
1263


 


as
235
GCGGUCUUCCCAGAGGCAAdTdT
1264


 


s
242
UGGAGAGCUGCACGGGCUCdTdT
1265


 


as
260
GAGCCCGUGCAGCUCUCCAdTdT
1266


 


s
244
GAGAGCUGCACGGGCUCACdTdT
1267


 


as
262
GUGAGCCCGUGCAGCUCUCdTdT
1268


 


s
246
GAGCUGCACGGGCUCACCAdTdT
1269


 


as
264
UGGUGAGCCCGUGCAGCUCdTdT
1270


 


s
399
UACACCAUCGCAGCCCUGCdTdT
1271


 


as
417
GCAGGGCUGCGAUGGUGUAdTdT
1272


 


s
132
GUCCUGGAUGCUGUCCGAGdTdT
1273


 


as
150
CUCGGACAGCAUCCAGGACdTdT
1274


 


s
245
AGAGCUGCACGGGCUCACCdTdT
1275


 


as
263
GGUGAGCCCGUGCAGCUCUdTdT
1276





Strand: s = sense; as = antisense; Position: position of 5′ base on transcript













TABLE 7







Chemically modified sense and antisense strand


sequences for rat TTR dsRNAs (NM_012681.1,


SEQ ID NO: 1330)















SEQ




Posi-

ID


Strand
Oligo #
tion
Sequence (5′ to 3′)
NO:





s
A-32159
115
GuccucuGAuGGucAAAGudTdT
1277


 


as
A-32160
133
ACUUUGACcAUcAGAGGACdTdT
1278


 


s
A-32745
537
uucuuGcucuAuAAAccGudTdT
1279


 


as
A-32746
555
ACGGUUuAuAGAGcAAGAAdTdT
1280


 


s
A-32747
543
cucuAuAAAccGuGuuAGcdTdT
1281


 


as
A-32748
561
GCuAAcACGGUUuAuAGAGdTdT
1282


 


s
A-32749
392
ucGccAcuAcAccAucGcAdTdT
1283


 


as
A-32750
410
UGCGAUGGUGuAGUGGCGAdTdT
1284


 


s
A-32751
538
ucuuGcucuAuAAAccGuGdTdT
1285


 


as
A-32752
556
cACGGUUuAuAGAGcAAGAdTdT
1286


 


s
A-32753
541
uGcucuAuAAAccGuGuuAdTdT
1287


 


as
A-32754
559
uAAcACGGUUuAuAGAGcAdTdT
1288


 


s
A-32755
532
cAGuGuucuuGcucuAuAAdTdT
1289


 


as
A-32756
550
UuAuAGAGcAAGAAcACUGdTdT
1290


 


s
A-32757
542
GcucuAuAAAccGuGuuAGdTdT
1291


 


as
A-32758
560
CuAAcACGGUUuAuAGAGCdTdT
1292


 


s
A-32759
134
ccuGGAuGcuGuccGAGGcdTdT
1293


 


as
A-32760
152
GCCUCGGAcAGcAUCcAGGdTdT
1294


 


s
A-32761
119
ucuGAuGGucAAAGuccuGdTdT
1295


 


as
A-32762
137
cAGGACUUUGACcAUcAGAdTdT
1296


 


s
A-32763
241
cuGGAGAGcuGcAcGGGcudTdT
1297


 


as
A-32764
259
AGCCCGUGcAGCUCUCcAGdTdT
1298


 


s
A-32765
544
ucuAuAAAccGuGuuAGcAdTdT
1299


 


as
A-32766
562
UGCuAAcACGGUUuAuAGAdTdT
1300


 


s
A-32767
530
AAcAGuGuucuuGcucuAudTdT
1301


 


as
A-32768
548
AuAGAGcAAGAAcACUGUUdTdT
1302


 


s
A-32769
118
cucuGAuGGucAAAGuccudTdT
1303


 


as
A-32770
136
AGGACUUUGACcAUcAGAGdTdT
1304


 


s
A-32771
140
uGcuGuccGAGGcAGcccudTdT
1305


 


as
A-32772
158
AGGGCUGCCUCGGAcAGcAdTdT
1306


 


s
A-32773
239
GucuGGAGAGcuGcAcGGGdTdT
1307


 


as
A-32774
257
CCCGUGcAGCUCUCcAGACdTdT
1308


 


s
A-32775
531
AcAGuGuucuuGcucuAuAdTdT
1309


 


as
A-32776
549
uAuAGAGcAAGAAcACUGUdTdT
1310


 


s
A-32777
117
ccucuGAuGGucAAAGuccdTdT
1311


 


as
A-32778
135
GGACUUUGACcAUcAGAGGdTdT
1312


 


s
A-32779
131
AGuccuGGAuGcuGuccGAdTdT
1313


 


as
A-32780
149
UCGGAcAGcAUCcAGGACUdTdT
1314


 


s
A-32781
217
uuGccucuGGGAAGAccGcdTdT
1315


 


as
A-32782
235
GCGGUCUUCCcAGAGGcAAdTdT
1316


 


s
A-32783
242
uGGAGAGcuGcAcGGGcucdTdT
1317


 


as
A-32784
260
GAGCCCGUGcAGCUCUCcAdTdT
1318


 


s
A-32785
244
GAGAGcuGcAcGGGcucAcdTdT
1319


 


as
A-32786
262
GUGAGCCCGUGcAGCUCUCdTdT
1320


 


s
A-32787
246
GAGcuGcAcGGGcucAccAdTdT
1321


 


as
A-32788
264
UGGUGAGCCCGUGcAGCUCdTdT
1322


 


s
A-32791
399
uAcAccAucGcAGcccuGcdTdT
1323


 


as
A-32792
417
GcAGGGCUGCGAUGGUGuAdTdT
1324


 


s
A-32793
132
GuccuGGAuGcuGuccGAGdTdT
1325


 


as
A-32794
150
CUCGGAcAGcAUCcAGGACdTdT
1326


 


s
A-32795
245
AGAGcuGcAcGGGcucAccdTdT
1327


 


as
A-32796
263
GGUGAGCCCGUGcAGCUCUdTdT
1328





See Table 5 for duplex # (dsRNA name). Strand: s = sense; as = antisense; Position: position of 5′ base on transcript






Synthesis of TTR Sequences
TTR sequences were synthesized on MerMade 192 synthesizer at 1 μmol scale. For all the sequences in the list, ‘endolight’ chemistry was applied as detailed below.

    
    
        All pyrimidines (cytosine and uridine) in the sense strand were replaced with corresponding 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)
        In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides
        A two base dTdT extension at 3′ end of both sense and antisense sequences was introduced
        The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software
    
    


The synthesis of TTR sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. The synthesis of the above sequences was performed at 1 um scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.
The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and triethylamine.3HF in the second step. The crude sequences thus obtained were precipitated using acetone: ethanol mix and the pellet were re-suspended in 0.5M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS and the resulting mass data confirmed the identity of the sequences. A selected set of samples were also analyzed by IEX chromatography.
The next step in the process was purification. All sequences were purified on an AKTA explorer purification system using Source 15Q column. A single peak corresponding to the full length sequence was collected in the eluent and was subsequently analyzed for purity by ion exchange chromatography.
The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted TTR sequences were analyzed for concentration and purity. The single strands were then annealed to form TTR-dsRNA.
Example 2B
In Vitro Screening of TTR siRNAs for mRNA Suppression
Human TTR targeting dsRNAs (Table 2) were assayed for inhibition of endogenous TTR expression in HepG2 and Hep3B cells, using qPCR (real time PCR) and bDNA (branched DNA) assays to quantify TTR mRNA. Rodent TTR targeting dsRNA (Table 5) were synthesized and assayed for inhibition of endogenous TTR expression using bDNA assays in H.4.II.E cells. Results from single dose assays were used to select a subset of TTR dsRNA duplexes for dose response experiments to calculate IC50's. IC50 results were used to select TTR dsRNAs for further testing.
Cell Culture and Transfections:
The hepatocyte cell lines HepG2, Hep3B and H.4.II.E cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. H.4.II.E cells were also grown in Earle's minimal essential medium. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotics containing 4×104 (HepG2), 2×104 (Hep3B) or 2×104 (H.4.II.E) cells were then added. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 10 nM final duplex concentration and dose response experiments were done with 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, 0.00001 nM.
Total RNA Isolation Using MagMAX-96 Total RNA Isolation Kit (Applied Biosystems, Foster City Calif., part #: AM1830):
Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 μl of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA it was eluted with 50 μl of water.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):
A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.41 of H2O per reaction were added into 10 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 was added to a master mix of 1 μl 18S TaqMan Probe (Applied Biosystems Cat #4319413E), 1 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914 M1) and 10 μl TaqMan Universal PCR Master Mix (Applied Biosystems Cat #4324018) per well in a MicroAmp Optical 96 well plate (Applied Biosystems cat #4326659). Real time PCR was done in an ABI 7000 Prism or an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔ Ct(RQ) assay. All reactions were done in triplicate.
Real time data were analyzed using the ΔΔ Ct method and normalized to assays performed from cells transfected with 10 nM BlockIT fluorescent Oligo (Invitrogen Cat #2013) or 10 nM AD-1955 (a control duplex that targets the non-mammalian luciferase gene) to calculate fold change.
Branched DNA Assays—QuantiGene 1.0 (Panomics, Fremont, Calif. cat #: OG0004)—Used to Screen Rodent Specific Duplexes
H.4.II.E cells (ATCC) were transfected with 10 nM siRNA. After removing media, H.4.II.E were lysed in 100 ul of Diluted Lysis Mixture (a mixture of 1 volume of Lysis mixture, 2 volume of nuclease-free water and 10 ul of Proteinase-K per ml for the final concentration of 20 mg/ml) then incubated at 65° C. for 35 minutes. Then, 80 μl of Working Probe Set (a mixture of TTR or GAPDH probe) and 20 ul of cell-lysate were added into the Capture Plate. Capture Plates were incubated at 53° C.±1° C. overnight (approximately 16-20 hrs). Capture Plates were washed 3 times with 1× Wash Buffer (a mixture of nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 1000 rpm. 100 μl of Amplifier Working Reagent was added into the Capture Plate, which was then sealed and incubated for 1 hour at 46° C.±1° C. Wash and dry steps were repeated after 1 hour of incubation and 100 μl of Label Solution Reagent was added. The plate was then washed, dried and 100 μl Substrate (a mixture of Lithium Lauryl Sulfate and Substrate solution) was added. Capture Plates were placed in the incubator for 30 minutes at 46° C.±1° C. Capture Plates were then removed from the incubator and incubated at room temperature for 30 minutes. Finally, the Capture Plates were read using the Victor Luminometer (Perkin Elmer, Waltham, Mass.).
Branched DNA Assays—QuantiGene 2.0 (Panomics cat #: QS0011): Used to Screen All Other Duplexes
After a 24 hour incubation at the dose or doses stated, media was removed and cells were lysed in 100 ul Lysis Mixture (1 volume lysis mixture, 2 volumes nuclease-free water and 10 μl of Proteinase-K/ml for a final concentration of 20 mg/ml) then incubated at 65° C. for 35 minutes. 20 μl Working Probe Set (TTR probe for gene target and GAPDH for endogenous control) and 80 μl of cell-lysate were then added to the Capture Plates. Capture Plates were incubated at 55° C.±1° C. (approx. 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 to the Capture Plates, which were sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C. Following a 1 hour incubation, the wash step was repeated, then 100 μl Amplifier Working Reagent was added. After 1 hour, the wash and dry steps were repeated, and 100 μl Label Probe was added. Capture plates were incubated 50° C.±1° C. for 1 hour. The plates were then washed with 1× Wash Buffer and dried, and then 100 μl Substrate was added to the Capture Plates. Capture Plates were read using the SpectraMax Luminometer (Molecular Devices, Sunnyvale, Calif.) following 5 to 15 minutes incubation.
bDNA Data Analysis:
bDNA data were analyzed by (i) subtracting the average background from each triplicate sample, (ii) averaging the resultant triplicate GAPDH (control probe) and TTR (experimental probe) values, and then (iii) taking the ratio: (experimental probe-background)/(control probe-background).
Results
A summary of the single dose and 1050 results for TTR-dsRNAs (TTR siRNAs) are presented below in Table 8. Single dose results are expressed as % TTR mRNA relative to control, assayed in HepG2 cells. IC50s were determined in HepG2 and/or Hep3B cells, as indicated.







TABLE 8







Single dose and IC50 results of in vitro screens of TTR siRNAs










Single Dose at 10 nM




% relative to control
IC50 (nM)











HepG2
HepG2
Hep3B













Duplex #
qPCR
bDNA
qPCR
bDNA
qPCR
bDNA
















AD-18243
50.35
141.53
ND
ND
ND
ND


AD-18244
64.26
158.55
ND
ND
ND
ND


AD-18245
56.89
107.22
ND
ND
ND
ND


AD-18246
10.53
32.51*
0.265
0.086
ND
ND


AD-18247
125.56
69.57
ND
ND
ND
ND


AD-18248
127.78
66.97
ND
ND
ND
ND


AD-18249
48.77
48.76
ND
ND
ND
ND


AD-18250
96.94
86.42
ND
ND
ND
ND


AD-18251
170.41
129.15
ND
ND
ND
ND


AD-18252
73.52
81.90
ND
ND
ND
ND


AD-18253
25.25
61.25
ND
ND
ND
ND


AD-18254
95.13
103.96
ND
ND
ND
ND


AD-18255
119.46
ND
ND
ND
ND
ND


AD-18256
42.64
95.67
ND
ND
ND
ND


AD-18257
146.25
141.75
ND
ND
ND
ND


AD-18258
10.20
13.41*
0.007
0.005
0.004
0.005


AD-18259
9.30
20.91*
0.102
0.005
ND
ND


AD-18260
125.37
81.36
ND
ND
ND
ND


AD-18261
14.27
19.40*
0.210
ND
ND
ND


AD-18262
84.95
104.05
ND
ND
ND
ND


AD-18263
16.32
23.25*
0.110
ND
ND
ND


AD-18264
104.18
83.69
ND
ND
ND
ND


AD-18265
41.62
64.87
ND
ND
ND
ND


AD-18266
39.98
110.53
ND
ND
ND
ND


AD-18267
149.64
ND
ND
ND
ND
ND


AD-18268
152.93
174.04
ND
ND
ND
ND


AD-18269
37.27
92.28
ND
ND
ND
ND


AD-18270
99.44
164.75
ND
ND
ND
ND


AD-18271
18.89
28.33*
0.503
0.004
ND
ND


AD-18272
128.32
132.58
ND
ND
ND
ND


AD-18273
115.78
201.95
ND
ND
ND
ND


AD-18274
8.97
20.04*
0.009
0.176
0.036
0.012


AD-18275
4.09
22.25*
0.026
0.118
ND
ND


AD-18276
19.73
45.22*
0.198
0.677
ND
ND


AD-18277
10.55
26.31*
0.121
0.426
ND
ND


AD-18278
108.86
116.26
ND
ND
ND
ND


AD-18279
66.59
ND
ND
ND
ND
ND


AD-18280
103.26
170.52
ND
ND
ND
ND


AD-18281
87.98
123.88
ND
ND
ND
ND


AD-18282
82.47
140.32
ND
ND
ND
ND


AD-18283
106.54
182.78
ND
ND
ND
ND


AD-18284
106.93
151.78
ND
ND
ND
ND


AD-18285
26.58
60.05*
ND
0.089
ND
ND


AD-18286
109.95
173.66
ND
ND
ND
ND


AD-18287
54.23
155.45
ND
ND
ND
ND


AD-18288
73.52
174.09
ND
ND
ND
ND


AD-18289
103.36
174.76
ND
ND
ND
ND


AD-18290
17.06
52.04*
1.253
0.181
ND
ND


AD-18291
7.71
169.29*
1.304
0.019
ND
ND


AD-18292
7.51
210.03*
0.604
0.005
ND
ND


AD-18293
3.61
62.53*
0.078
0.003
ND
ND


AD-18294
111.53
107.56
ND
ND
ND
ND


AD-18295
115.88
105.37
ND
ND
ND
ND


AD-18296
57.03
38.03
ND
ND
ND
ND


AD-18297
87.69
73.87
ND
ND
ND
ND


AD-18298
10.39
7.25*
0.455
0.008
ND
ND


AD-18299
18.79
18.06*
0.895
0.014
ND
ND


AD-18300
108.70
ND
ND
ND
ND
ND


AD-18301
114.22
70.50
ND
ND
ND
ND


AD-18302
116.19
122.40
ND
ND
ND
ND


AD-18303
124.89
ND
ND
ND
ND
ND


AD-18304
132.99
89.54
ND
ND
ND
ND


AD-18305
153.10
ND
ND
ND
ND
ND


AD-18306
159.22
ND
ND
ND
ND
ND


AD-18307
116.83
84.57
ND
ND
ND
ND


AD-18308
156.72
87.80
ND
ND
ND
ND


AD-18309
113.22
101.97
ND
ND
ND
ND


AD-18310
132.33
ND
ND
ND
ND
ND


AD-18311
161.68
92.92
ND
ND
ND
ND


AD-18312
103.01
71.17
ND
ND
ND
ND


AD-18313
120.65
53.26
ND
ND
ND
ND


AD-18314
116.33
ND
ND
ND
ND
ND


AD-18315
115.13
ND
ND
ND
ND
ND


AD-18316
118.73
122.34
ND
ND
ND
ND


AD-18317
114.03
121.10
ND
ND
ND
ND


AD-18318
80.85
122.57
ND
ND
ND
ND


AD-18319
119.14
148.87
ND
ND
ND
ND


AD-18320
22.86
55.43*
ND
0.023
0.403
ND


AD-18321
6.44
31.56*
0.001
0.033
ND
ND


AD-18322
54.21
100.46
ND
ND
ND
ND


AD-18323
6.37
28.71*
0.005
0.023
ND
ND


AD-18324
2.53
15.98*
0.002
0.006
0.005
0.014


AD-18325
2.52
11.96*
0.001
0.016
ND
ND


AD-18326
18.34
43.16*
0.025
0.186
ND
ND


AD-18327
18.28
13.90*
0.044
0.215
ND
ND


AD-18328
4.53
26.04*
0.003
0.004
0.006
0.006


AD-18329
96.93
131.54
ND
ND
ND
ND


AD-18330
11.80
45.18*
 0.0004
0.010
0.020
ND


AD-18331
117.77
163.07
ND
ND
ND
ND


AD-18332
11.53
35.09*
0.001
0.076
0.065
ND


AD-18333
12.24
46.94*
0.001
0.115
0.075
ND


AD-18334
16.27
55.28*
 0.0004
0.181
1.071
ND


AD-18335
53.52
112.80
ND
ND
ND
ND


AD-18336
6.39
33.00*
0.001
0.112
0.081
ND


AD-18337
51.77
105.33
ND
ND
ND
ND


AD-18338
48.21
102.86
ND
ND
ND
ND


AD-18339
6.48
26.56*
0.004
0.002
0.018
0.029


AD-18340
4.53
30.76*
0.002
0.002
ND
ND


AD-18341
31.27
100.41
ND
ND
ND
ND


AD-18342
7.60
42.89*
ND
0.016
0.076
ND


AD-18343
3.42
17.45*
ND
0.001
ND
ND


AD-18344
75.08
134.31
ND
ND
ND
ND


AD-18345
13.62
42.75*
0.002
0.013
ND
ND


AD-18346
59.25
121.10
ND
ND
ND
ND


AD-18347
91.23
139.54
ND
ND
ND
ND


AD-18348
89.95
159.29
ND
ND
ND
ND


AD-18349
108.01
144.96
ND
ND
ND
ND


AD-18350
123.65
125.87
ND
ND
ND
ND


AD-18351
108.36
104.02
ND
ND
ND
ND


AD-18352
87.82
128.72
ND
ND
ND
ND


AD-18353
14.40
65.77
0.012
0.027
ND
ND


AD-18354
99.27
123.53
ND
ND
ND
ND


AD-18355
135.04
150.88
ND
ND
ND
ND


AD-18356
100.76
178.96
ND
ND
ND
ND


AD-18357
125.30
162.85
ND
ND
ND
ND


AD-18358
103.15
136.01
ND
ND
ND
ND


AD-18359
34.74
140.48
ND
ND
ND
ND


AD-18360
103.86
146.86
ND
ND
ND
ND


AD-18361
105.74
152.74
ND
ND
ND
ND


AD-18362
106.96
188.22
ND
ND
ND
ND


AD-18363
124.22
58.46
ND
ND
ND
ND


AD-18364
113.75
66.87
ND
ND
ND
ND


AD-18446
29.73
13.30
ND
ND
ND
ND


AD-18447
109.74
53.63
ND
ND
ND
ND


AD-18448
22.96
8.81
ND
ND
ND
ND


AD-18449
112.59
50.11
ND
ND
ND
ND


AD-18450
89.41
34.89
ND
ND
ND
ND


AD-18451
74.35
23.88
ND
ND
ND
ND


AD-18452
125.25
54.86
ND
ND
ND
ND


AD-18453
126.98
56.31
ND
ND
ND
ND


AD-18454
113.88
52.48
ND
ND
ND
ND


AD-18455
163.00
48.89
ND
ND
ND
ND


AD-18456
15.70
10.52
ND
ND
ND
ND


AD-18457
12.86
8.22
ND
ND
ND
ND


AD-18458
13.00
7.00
ND
ND
ND
ND


AD-18459
14.41
10.72
ND
ND
ND
ND


AD-18460
121.16
74.87
ND
ND
ND
ND


AD-18461
100.53
71.87
ND
ND
ND
ND


AD-18462
47.75
29.35
ND
ND
ND
ND


AD-18463
58.98
44.79
ND
ND
ND
ND





ND: no data;


*indicates result that represents average of two experiments.






The dose response data used to identify the IC50 for 5 TTR-dsRNAs (AD-18258, AD-18274, AD-18324, AD-18328, and AD-18339), are presented in detail below in Table 9. All 5 siRNAs were determined to have pM IC50s. The IC50 data for dsRNAs in Table 8 is a summary of the data presented in Table 9 below.







TABLE 9







Dose response data for 5 TTR-dsRNAs












% inhibition relative to control AD-1955




Detection
Dose of duplex (nM)
IC50





















Cell type
method
10
1
0.5
0.1
0.05
0.01
0.005
0.001
0.0005
0.0001
0.00005
0.00001
(nM)











Duplex AD-18258






















HepG2
qPCR
14.4
14.1
16.2
23.9
27.26
40.19
68.46
78.1
74.48
104.37
98.28
113.68
0.007


HepG2
bDNA
14.3
14.5
11.1
12.8
18.82
19.77
51.21
56.03
63.63
58.35
43.64
51.05
0.005


Hep3B
qPCR
11.9
8.62
12.4
16.4
28.35
30.49
58.36
54.57
81.26
89.43
81.85
101.87
0.004


Hep3B
bDNA
7.65
7.5
11.3
12.6
28.85
27.89
64.57
73.48
72.03
91.44
86.71
89.31
0.005








Duplex AD-18274






















HepG2
qPCR
6.68
8.45
11.7
24.2
42.08
49.89
56.95
62.99
64.47
54.92
67.39
72.67
0.009


HepG2
bDNA
27.5
69
25.2
34.2
73.03
103.4
121.57
97.31
154.93
156.7
Nd
152.25
0.176


Hep3B
qPCR
7.58
17
15.6
43.9
42.22
60.55
78.8
77.81
79.97
85.84
86.13
83.99
0.036


Hep3B
bDNA
3.77
4.92
7.51
15
35.21
51.66
72.45
70.12
78.31
77.52
90.72
83.01
0.012








Duplex AD-18324






















HepG2
qPCR
2.07
2.27
2.74
6.36
8.18
15.23
28.82
52.79
90.86
94.72
116.07
98.97
0.002


HepG2
bDNA
14.5
7.88
11.8
15.9
17.2
46.44
40.4
91.86
0
95.57
0
52.15
0.006


Hep3B
qPCR
2.07
3.48
5.76
16.2
18.73
44.54
49.77
68.88
63.48
76.61
74.7
77.83
0.005


Hep3B
bDNA
3.48
3.8
5.15
15.2
30.84
55.36
74.75
99.39
88.89
110.83
96.55
110.26
0.014








Duplex AD-18328






















HepG2
qPCR
5.85
3.97
3.32
5.62
8
16.75
55.01
39.76
122.41
102.37
114.02
124.09
0.003


HepG2
bDNA
12.3
10.7
10.7
11.9
20.06
25
69.52
57.29
112.28
98.14
142.26
148.92
0.004


Hep3B
qPCR
3.17
5.52
11.7
13.8
27.68
39.58
61.21
61.87
90.51
87.56
106.03
108.72
0.006


Hep3B
bDNA
3.08
3.66
4.19
7.25
21.05
22.1
73.74
63.19
105.55
96.27
105.97
96.46
0.006








Duplex AD-18339






















HepG2
qPCR
6.27
7.28
Nd
11
15.25
38.69
38.78
71.7
84.09
62.2
75.61
85.46
0.004


HepG2
bDNA
15.1
8.14
5.13
6.89
12.17
32.14
42.98
64.01
60.76
79.95
81.97
95.43
0.002


Hep3B
qPCR
8.3
9.47
13.2
34.5
44.54
77.38
81.04
81.41
93.95
81.04
75.61
78.28
0.018


Hep3B
bDNA
10.5
9.43
11.7
27.1
44.88
72.32
79.88
79.6
87.46
96.53
95.13
89.88
0.029









A summary of the single dose results for rodent specific TTR-dsRNAs (TTR siRNAs) are presented below in Table 10. Single dose results are expressed as % TTR mRNA relative to control, assayed in rat H.4.II.E cells, after transfection of rodent specific TTR siRNAs at 10 nM. These results show that some rodent specific TTR siRNAs are effective in suppressing endogenous rat TTR mRNA in vitro.







TABLE 10







Single dose results of in vitro screen of


rodent specific TTR-dsRNAs (TTR siRNAs)











% Relative to



Duplex #
control at 10 nM














AD-18529
19.83



AD-18530
44.49



AD-18531
6.01



AD-18532
24.06



AD-18533
37.78



AD-18534
8.19



AD-18535
10.18



AD-18536
16.13



AD-18537
15.88



AD-18538
19.93



AD-18539
49.24



AD-18540
2.99



AD-18541
1.32



AD-18542
6.3



AD-18543
16.46



AD-18544
17.55



AD-18545
3.53



AD-18546
2.75



AD-18547
7.01



AD-18548
5.02



AD-18549
1.61



AD-18550
9.58



AD-18551
7.74



AD-18552
3.74



AD-18553
50.39



AD-18554
111.06










Example 3
In Vitro Assay of TTR siRNAs for Induction of TNF-α and IFN-α Secretion
To evaluate potential for immunostimulation, TTR siRNAs were assayed in vitro for induction of TNF-α and IFN-α secretion.
Human PBMC were isolated from freshly collected buffy coats obtained from healthy donors (Research Blood Components, Inc., Boston, Mass.) by a standard Ficoll-Hypaque density centrifugation. Freshly isolated cells (1×105/well/100 μl) were seeded in 96-well plates and cultured in RPMI 1640 GlutaMax medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic (Invitrogen).
siRNAs were transfected into PBMC using DOTAP transfection reagent (Roche Applied Science). The DOTAP was first diluted in Opti-MEM (Invitrogen) for 5 minutes before mixing with an equal volume of Opti-MEM containing the siRNA. siRNA/DOTAP complexes were incubated as specified by the manufacturer's instructions and subsequently added to PBMC (50 μl/well) which were then cultured for 24 hours. Positive and negative control siRNAs were included in all assays. AD-5048 was used as a positive control siRNA. AD-5048 corresponds to a sequence that targets human Apolipoprotein B (Soutschek et al., 2004) and elicits secretion of both IFN-α and TNF-α in this assay. AD-1955, which does not elicit IFN-α and TNF-α secretion in this assay, was used as a negative control siRNA. All siRNAs were used at a final concentration of 133 nM. The ratio of RNA to transfection reagent was 16.5 pmoles per μg of DOTAP.
Cytokines were detected and quantified in culture supernatants with a commercially available ELISA kit for IFN-α (BMS2161NST) and TNF-α (BMS2231NST), both from Bender MedSystems (Vienna, Austria). TTR siRNA cytokine induction is expressed as percent IFN-α or TNF-α produced relative to the positive control siRNA AD-5048.
IFN-α and TNF-α stimulation results for a number of TTR siRNAs are presented in FIG. 1 (mean of quadruplicate wells±SD) and below in Table 11 (percentage compared with AD-5048). None of the TTR siRNAs evaluated induced significant TNF-α or IFN-α secretion by cultured human PBMCs.







TABLE 11







IFN-α and TNF-α stimulation results for TTR siRNAs












IFN-α (% of
TNF-α (% of



Duplex #
AD-5048)
AD-5048)







AD-18246
0
4



AD-18258
0
0



AD-18259
0
0



AD-18261
0
0



AD-18263
0
0



AD-18271
0
0



AD-18274
2
1



AD-18275
0
0



AD-18276
0
0



AD-18277
0
0



AD-18285
0
0



AD-18290
0
0



AD-18291
0
0



AD-18292
0
0



AD-18293
0
0



AD-18298
0
0



AD-18299
0
0



AD-18320
0
0



AD-18321
0
0



AD-18323
0
0



AD-18324
0
0



AD-18325
0
0



AD-18326
0
0



AD-18327
0
0



AD-18328
0
0



AD-18330
0
0



AD-18332
1
0



AD-18333
0
1



AD-18334
0
1



AD-18336
1
0



AD-18339
0
0



AD-18340
0
0



AD-18342
0
0



AD-18343
0
0



AD-18345
0
0



AD-18353
0
0



AD-18448
0
0



AD-18456
0
0



AD-18457
0
0



AD-18458
0
0



AD-18459
0
0










The five lead TTR targeting dsRNAs (TTR siRNAs) were selected based on IC50s in the pM range in the human hepatocyte cell lines HepG2 and Hep3B, and the absence of immunostimulatory activity. Duplexes without any mismatches are more likely to achieve significant knockdown of the target transcript than duplexes with mismatches between the oligo and the mRNA. To better enable interpretation of cross-species toxicology data and to have the broadest applicability to human patients, duplexes that have 100% identity in orthologous genes from rat, cynomolgus monkey and human, and that do not target regions with known polymorphisms are generally preferred. The five lead compounds were selected based on IC50 in hepatocyte cell lines in the pM range, the absence of immunostimulatory activity, specificity to the human TTR transcripts, and absence of known polymorphisms (mutations) in the region of the mRNA targeted by the duplex. In the case of TTR, no 19 base oligos were found with complete identity in human, rat and cynomolgus monkey. A summary of these data are presented in Table 12, which also includes information on known TTR mutations in the region targeted by the duplex and cross-species reactivity.







TABLE 12







Summary of data for five most potent TTR dsRNAs.













IC50
IC50






(qPCR):
(bDNA):

Mutations not


Duplex #
nM HepG2
nM HepG2
IFNa/TNFa
covered
Cross-species reactivity





AD-18258
0.007
0.005
Negative
None
Cyno: 1 mismatch @ position






(non-coding
14 A to G






region)
Rat: no homology at any position


AD-18274
0.009
0.176
Negative
Lys70Asn;
Cyno: no mismatch






Val71Ala;
Rat: no homology at any position






Ile73Val;






Asp74His


AD-18324
0.002
0.006
Negative
None
Cyno: no mismatch






(non-coding
Rat: no homology at any position






region)


AD-18328
0.003
0.004
Negative
None
Cyno: no mismatch






(non-coding
Rat: 7 mismatches






region)


AD-18339
0.004
0.002
Negative
None
None






(non-coding






region)









Example 4
In Vivo Reduction of Liver TTR mRNA and Plasma TTR Protein by LNP01-18324, LNP01-18328 and LNP01-18246 in Transgenic Mice
Two TTR siRNAs, AD-18324 and AD-18328, were chosen for in vivo evaluation. These duplexes exhibited potent dose-dependent silencing in vitro in hepatocyte cell lines (e.g. HepG2). FIG. 2A and FIG. 2B show the dose responses in HepG2 cells after transfection with AD-18324 (FIG. 2A) or AD-18328 (FIG. 2B) where the doses are expressed in nM on the x-axis and the responses are expressed as fraction TTR mRNA remaining relative to control, on the y-axis. In HepG2 cells, the IC50s of AD-18324 and AD-18328 were determined to be 2 pM and 3 pM, respectively. The TTR target sites for both lead dsRNA candidates are in the 3′ untranslated region of the TTR mRNA, in a region where there are no reported mutations in the literature.
The sequences of each strand of the two lead candidates are reproduced below from the Tables. Strand: s=sense; as=antisense; Position: position of 5′ base on transcript NM—000371.2.




















SEQ ID


Duplex #
Strand
Oligo #
Position*
Sequence 5′ to 3′
NO:







AD-18324
s
A-32337
509
GGAuuucAuGuAAccAAGAdTdT
1001


 


AD-18324
as
A-32338
527
UCUUGGUuAcAUGAAAUCCdTdT
1002


 


AD-18328
s
A-32345
518
GuAAccAAGAGuAuuccAudTdT
1009


 


AD-18328
as
A-32346
536
AUGGAAuACUCUUGGUuACdTdT
1010









In addition, a rodent cross-reactive TTR dsRNA, AD-18246, was chosen for further evaluation in vivo. AD-18246 targets a sequence beginning at position 88 of the open reading frame, where there are three mutations reported in the literature. A dose response curve for AD-18246 in HepG2 cells is shown in FIG. 3. AD-18246 is substantially less potent than AD-18324 and AD-18328; the IC50 of AD-18246 was determined to be 265 pM.
AD-18324, AD-18328, and AD-18246 were administered to transgenic mice after formulation in LNP01. 3-5 month old H129-mTTR-KO/iNOS-KO/hTTR transgenic mice (mouse transthyretin knock-out/inducible nitric oxide synthase knock-out/human transthyretin transgenic) were intravenously (IV) administered 200 μl of LNP01-formulated transthyretin-specific siRNA (AD-18324, AD-18328, or AD-18246), LNP01-formulated control siRNA targeting the non-mammalian luciferase gene (AD-1955) or PBS via the tail vein at concentrations of 1.0 mg/kg, 3.0 mg/kg, or 6.0 mg/kg for siRNAs AD-18324 and AD-18328, 3.0 mg/kg for siRNA AD-18246, and 6.0 mg/kg for siRNA AD-1955. LNP01 is a lipidoid formulation comprised of ND98, Cholesterol, and PEG-Ceramide C16.
After approximately forty-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 prebleed 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 Plate 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 ul 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).
TTR plasma 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).
LNP01-18324 and LNP01-18328 were found to reduce liver TTR mRNA (FIG. 4A) and plasma TTR protein (FIG. 4B) levels in a dose-dependent manner with IV bolus administration. The mRNA ED50 of LNP01-18328 was determined to be  ̃1 mg/kg whereas the ED50 of LNP01-18324 was determined to be  ̃2 mg/kg. The effects of LNP01-18324 and LNP01-18328 were specific, because the control, LNP01-1955 at 6 mg/kg, did not significantly affect liver TTR mRNA levels, as compared with the PBS group. LNP01-18324 and LNP01-18328 reduced plasma TTR protein levels relative to the PBS group, with potencies that were similar to those on TTR mRNA levels. At 3 mg/kg, LNP01-18246 reduced liver TTR mRNA levels to a lessor extent than 3 mg/kg LNP01-18324 or LNP01-18328.
These results demonstrate that LNP01-18324 and LNP01-18328, administered by IV bolus, substantially reduce human TTR mRNA expressed by the transgenic mouse liver, which results in reduction of human TTR protein in the circulation.
Example 5
In Vivo Reduction of Wild-Type TTR mRNA in the Non-Human Primate Liver by SNALP-18324 and SNALP-18328
To evaluate the efficacy of TTR siRNAs AD-18324 and AD-18328 in non-human primates on liver TTR mRNA levels, the siRNAs were formulated in SNALP and administered by 15-minute IV infusion. Cynomolgus monkeys (Macaca fascicularis) (2 to 5 kg, 3 animals per group) were administered 15-minute IV infusions of SNALP-18324 (0.3, 1.0 or 3.0 mg/kg), SNALP-18328 (0.3, 1 or 3 mg/kg), or SNALP-1955 (3 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase). At forty-eight hours post-dosing, monkeys were anesthetized with sodium pentobarbital and exsanguinated. Liver tissue for TTR mRNA determination was collected, flash-frozen, and stored at −80° C. until processing.
TTR mRNA levels in the liver were assayed utilizing a custom designed Branched DNA assay, utilizing the QuantiGene1.0 technology. Briefly, monkey liver samples were ground and tissue lysates were prepared. Liver lysis mixture (1 volume lysis mixture, 2 volume 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. 20 μl Working Probe Set (TTR probe for gene target and GAPDH for endogenous control) and 80 μl tissue-lysate were then added into the Capture Plate. Capture Plates were incubated at 55° C.±1° C. (approx. 16-20 hrs). The next day, the Capture Plates were washed three 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 a 1-hour incubation, the wash step was repeated, and then 100 μl Amplifier Working Reagent was added. After 1 hour, the wash and dry steps were repeated, and 100 μl Label Probe was added. Capture plates were incubated 50° C.±1° C. for 1 hour. The plates were then washed with 1× Wash Buffer and dried, and then 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 (i) subtracting the average background from each triplicate sample, (ii) averaging the resultant GAPDH (control probe) and TTR (experimental probe) values, and then (iii) taking the ratio: (experimental probe-background)/(control probe-background).
The results are shown in FIG. 5. SNALP-18324 and SNALP-18328 reduced TTR mRNA levels in the liver in a dose-dependent manner, compared to the negative control SNALP-1955. The mRNA ED50s of SNALP-18328 and SNALP-18324 were determined to be  ̃0.3 and  ̃1 mg/kg, respectively.
These results demonstrate that SNALP-18324 and SNALP-18328 are effective in suppressing wild-type TTR mRNA in non-human primate liver when administered by IV infusion.
Example 6
In Vivo Reduction of Mutant (V30M) TTR mRNA and Protein by SNALP-18328 in the Transgenic Mouse
To evaluate the efficacy of TTR siRNA AD-18328 on mutant (V30M) TTR mRNA in the liver and mutant (V30M) TTR protein in the serum, AD-18328 was formulated in SNALP and administered by IV bolus to V30M hTTR transgenic mice. 8 to 12-week old V30M hTTR transgenic mice (5 animals/group) were intravenously (IV) administered 200 μl SNALP-18328 (0.03, 0.3 or 3 mg/kg), SNALP-1955 (3 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase), or PBS. Mice used were the Mus musculus strain H129-hTTR KO from Institute of Molecular and Cellular Biology, Porto, Portugal. Briefly, hTTR H129 transgenic mice were crossed with a H129 endogenous TTR KO mice (null mice to generate the H129-hTTR transgenic mice, in a null mouse TTR background (Maeda, S., (2003), Use of genetically altered mice to study the role of serum amyloid P component in amyloid deposition. Amyloid Suppl. 1, 17-20.).
At 48 hrs post-injection, animals in all five treatment groups were given a lethal dose of ketamine/xylazine. Serum samples were collected and stored at −80° C. until analysis. Liver tissue was collected, flash-frozen and stored at −80° C. until processing.
For TTR mRNA quantitation, frozen liver tissue was ground into powder, and lysates were prepared. TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group means of the normalized values were then further normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression.
For TTR protein quantitation, serum was assayed using the AssayPro (St. Charles, Mo.) Assaymax PreAlbumin ELISA Kit according to the manufacturer's protocol.
The results are shown in FIG. 6A and FIG. 6B for liver mRNA and serum protein, respectively. SNALP-18328 treated V30M hTTR transgenic mice had a dose-dependent and significant decrease in liver TTR mRNA levels relative to the PBS control group, reaching a maximum reduction of 97% (p<0.001) at 3 mg/kg SNALP-18328, and a 50% reduction (ED50) at  ̃0.15 mg/kg SNALP-18328. Serum TTR protein was also suppressed in a dose-dependent manner, with a maximum reduction of serum TTR protein of 99% (p<0.01) (relative to pre-dose levels) at 3 mg/kg SNALP-18328, consistent with the reduction in TTR mRNA levels. SNALP-1955 at 3 mg/kg did not have a statistically significant effect on either TTR mRNA or protein levels, compared to PBS.
These results demonstrate that SNALP-18328, when administered IV, is active in suppressing mutant V30M TTR mRNA in the transgenic mouse liver, which results in reduction of mutant V30M TTR protein in the circulation.
Example 7
Durability of TTR mRNA and Protein Suppression by SNALP-18328 in the Transgenic Mouse
To evaluate the durability of TTR mRNA and protein suppression by SNALP-18328, AD-18328 was formulated in SNALP and administered by IV bolus to V30M hTTR transgenic mice. At various timepoints post-dose, liver TTR mRNA levels and serum TTR protein levels were quantified. 8- to 12-week old V30M hTTR transgenic mice (4 animals/group) were intravenously (IV) administered 200 μl SNALP-18328 (1 mg/kg) or SNALP-1955 (1 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase). Mice used were Mus musculus strain H129-hTTR KO from Institute of Molecular and Cellular Biology, Porto, Portugal. Briefly, hTTR H129 transgenic mice were crossed with a H129 endogenous TTR KO mice (null mice to generate the H129-hTTR transgenic mice, in a null mouse TTR background (Maeda, S., (2003), Use of genetically altered mice to study the role of serum amyloid P component in amyloid deposition. Amyloid Suppl. 1, 17-20). Days 3, 8, 15, or 22 post-dose, animals in both treatment groups were given a lethal dose of ketamine/xylazine. Serum samples were collected and stored at −80° C. until analysis. Liver tissue was collected, flash-frozen and stored at −80° C. until processing.
For TTR mRNA quantitation, frozen liver tissue was ground into powder, and lysates were prepared. TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group means of the normalized values were then further normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression.
For TTR protein quantitation, serum was assayed using the AssayPro (St. Charles, Mo.) Assaymax PreAlbumin ELISA Kit according to the manufacturer's protocol.
The results are shown in FIG. 7A and FIG. 7B for liver mRNA and serum protein, respectively. A single IV bolus administration of SNALP-18328 in the hTTR V30M transgenic mouse resulted in durable inhibition of TTR mRNA levels in the liver and TTR protein levels in the serum. Compared to the control group (1 mg/ml SNALP-1955), a single IV administration of SNALP-18328 at 1 mg/kg significantly reduced relative TTR mRNA levels on Days 3, 8, 15 and 22 post-dose by 96% (p<0.001), 90% (p<0.001), 82% (p<0.001) and 73% (p<0.001), respectively, and did not return to baseline levels at termination of the study (Day 22 post-dose). Protein levels also decreased with a maximum reduction of serum TTR of 97% (p<0.001) (relative to SNALP-1955) at Day 3 post-dose. At Days 8, 15, and 22 post-dose, TTR protein levels were suppressed by 72% (p<0.05), 32% (p<0.05), and 40% (p<0.001), respectively, relative to SNALP-1955.
These results demonstrate that a single IV administration of SNALP-18328 produces durable suppression of target liver mRNA and serum protein levels in the V30M hTTR transgenic mouse, with significant reductions of both liver TTR mRNA and serum TTR protein at 22 days post-dose.
Example 8
Durability of Serum TTR Protein and Liver mRNA Suppression by SNALP-18328 in the Non-Human Primate
To evaluate the durability of serum TTR protein suppression by SNALP-18328, AD-18328 was formulated in SNALP and administered by IV infusion to non-human primates. At various timepoints post-dose, serum TTR protein levels were quantified.
Cynomolgus monkeys (Macaca fascicularis) (n=5 animals/group for SNALP-18328 groups and n=3 animals/group for SNALP-1955 and PBS groups) were administered a 15-minute IV infusion of SNALP-18328 (0.3, 1 or 3 mg/kg), SNALP-1955 (3 mg/kg) with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase), or PBS. At Days 0, 1, 2, 3, 4, 5, 7, 10, and 14 of the dosing phase, serum samples were collected and stored at −80° C. until analysis.
Western blot analysis was used to evaluate TTR protein levels in serum samples. Serum samples from each group were pooled and diluted 1:1 with Laemmli sample buffer (β-mercaptoethanol was added at a 1:20 dilution). The samples were heated at 95° C. for 10 minutes. 12.5 μl of each sample was loaded in each lane of a 10-20% Criterion (Biorad, Hercules, Calif.) prep gel and separated by SDS-PAGE at 120V for 1.5 hrs, then transferred to a nitrocellulose membrane using a semi-dry system at 15V for 1 hour. The membrane was blocked overnight at 4° C. in LiCOR (Lincoln, Nebr.) blocking buffer diluted 1:1 with 1×PBS. The blot was probed first with primary antibodies (goat anti-TTR from Santa Cruz (Santa Cruz, Calif.) at a dilution of 1:1000 diluted in LiCOR blocking buffer/PBS on a rocker for 1 hr at room temperature. Blots were washed 4× with PBS+0.2% Tween 20 (10 minutes per wash). The fluorescent labeled secondary antibodies (anti-goat 680 nm from Invitrogen (Carlsbad, Calif.) were added at a dilution of 1:10,000 in LiCOR blocking buffer/PBS and the blot was incubated for 1 hour at room temperature. After incubation, blots were washed 4× with PBS+0.2% Tween 20 followed by one wash with 1×PBS. The Li-COR's Odyssey Infrared Imaging System was used to detect the protein bands. TTR monomer migrates at 15 kDa.
The results are shown in FIG. 8. Serum TTR protein levels showed a dose-dependent reduction with 1 or 3 mg/kg SNALP-18328, as compared to pre-dose (Day 0) levels. The duration of suppression, following a single IV administration of SNALP-18328 is at least 14 days after 1 or 3 mg/kg SNALP-18328 treatment.
These results demonstrate that a single IV administration of SNALP-18328 produces durable suppression of TTR protein in the circulation in the non-human primate (Macaca fascicularis), with significant reduction of TTR protein at 14 days post-dose.
To evaluate the durability of liver TTR mRNA suppression by SNALP-18328, AD-18328 was formulated in SNALP (ALN-TTR01) and administered by single IV infusion to non-human primates. Liver mRNA levels wer measured as described herein at day 3 or day 30 post-administration.
The results are shown in FIG. 20 and demonstrate that ALN-TTR01 suppression of wild type TTR mRNA is durable in non-human primates after 3 days for dosages 1.0 and 3.0 mg/kg and for 30 days at a dose of 10 mg/kg.
Example 9
In Vivo Reduction of Mutant (V30M) TTR in Peripheral Tissues by SNALP-18328 in the Transgenic Mouse
Propylactic Efficacy
To evaluate the efficacy of SNALP-18328 (ALN-TTR01) in reducing TTR in peripheral tissues, hTTR V30M/HSF-1 knock-out mice were evaluated with immunohistochemical staining for TTR. Two-month old hTTR V30M/HSF-1 knock-out mice (Maeda, S., (2003), Use of genetically altered mice to study the role of serum amyloid P component in amyloid deposition. Amyloid Suppl. 1, 17-20) were administered an IV bolus of 3 mg/kg SNALP-18328 (12 animals), 3 mg/kg SNALP-1955 (with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase, 4 animals), or PBS (4 animals) once every two weeks for a total of four doses on days 0, 14, 28, and 42. TTR liver mRNA levels and TTR-immunoreactivity in multiple peripheral tissues were evaluated at 8 weeks post-first dose on day 56.
Mice were anesthetised with 1 mg/kg medetomidine, and given a lethal dose of ketamine. Tissues and organs of interest were collected. For immunohistochemistry, esophagus (E), stomach (S), intestine (duodenum (I1) and colon (I4)), nerve (N) and dorsal root ganglia (D) were fixed in neutral buffered formalin and embedded in paraffin. For TTR detection, rabbit anti-human TTR primary antibody (1:1000, DAKO, Denmark), and anti-rabbit biotin-conjugated secondary antibody (1:20 Sigma, USA) were followed by extravidin labelling (1:20, Sigma, USA) in order to stain for the TTR protein. The reaction was developed with 3-amino-9-ethyl carbaxole, AEC (Sigma, USA). Semi-quantitative analysis of immunohistochemical slides was performed using Scion image quant program that measures the area occupied by the substrate reaction color and normalizes this value to the total image area. Mean values of % occupied area are displayed with the corresponding standard deviation. Each animal tissue was evaluated in four different areas. The presence of human TTR in parasympathetic ganglia of the stomach and intestine was studied by double immunofluorescent staining with rabbit anti-human TTR (1:1000, DAKO, Denmark) and mouse anti-PGP9.5 (1:40, Serotec, USA) as the primary antibodies; secondary antibodies were, respectively: anti-rabbit Alexa Fluor 488 (Molecular probes, UK) and goat anti-mouse Alexa Fluor 568 (Molecular probes, UK). Slides were mounted with vectashield (Vector) and visualized in a Zeiss Cell Observer System microscope (Carl Zeiss, Germany) equipped with filters for FITC and rhodamine.
The results are graphed in FIG. 9. In contrast with PBS and SNALP-1955 treated animals, SNALP-18328 treated animals had a significant reduction of TTR-immunoreactivity in all tissues examined (esophagus (E), stomach (S), intestine (duodenum (I1) and colon (I4)), nerve (N) and dorsal root ganglia (D).
These results demonstrate that SNALP-18328 administration to hTTR V30M/HSF-1 knock-out mice causes a significant reduction of TTR protein deposition in peripheral tissues and organs, including esophagus, stomach, intestine (duodenum and colon), nerve, and dorsal root ganglion.
Therapeutic Efficacy
ALN-TTR01 was administered to mature hTTR V30M/HSF-1 knock-out mice to determine the effects of TTR siRNA treatment on regression of mutant human TTR deposits.
Groups of 21 month old animals (hTTR V30M/HSF-1 knock-out mice) were intravenously administered an IV bolus of ALN-TTR01 or control siRNA at a dose of 3 mg/kg on days 0, 14, 28, 14, 56, and 70. On day 77, the mice were euthanized, tissue was harvested, and TTR deposition was assayed via semi-quantitative analysis of immunohistochemical stained slides using Scion image quant program as described herein. Esophagus, colon; stomach, sciatic nerve; and dorsal root ganglia tissue were examined and results were compared to historical data in this animal model demonstrating both TTR deposition and TTR fibrils present in tissues at this age.
The results are shown in the graph in FIG. 21. The results demonstrate that treatment with TTR siRNA resulted in >90% regression of existing V30M hTTR tissue deposits.
Example 10
In Vivo Reduction of Wild-Type TTR mRNA in the Non-Human Primate Liver by XTC-SNALP-18328
To evaluate the efficacy of the novel lipid nanoparticle formulation XTC-SNALP for delivery of siRNA in non-human primate, TTR siRNA AD-18328 was formulated in XTC-SNALP (XTC-SNALP-18328) and administered by 15-minute IV infusion, and liver TTR mRNA was quantified. Cynomolgus monkeys (Macaca fascicularis) were administered 15-minute IV infusions of XTC-SNALP-18328 (0.03, 0.1, 0.3 or 1 mg/kg) or XTC-SNALP-1955 (1 mg/kg, with negative control siRNA AD-1955 which targets the non-mammalian gene luciferase). At forty-eight hours post-dosing, monkeys were anesthetized with sodium pentobarbital and exsanguinated. Liver tissue for TTR mRNA determination was collected, flash-frozen, and stored at −80° C. until processing. Methods used for TTR mRNA quantitation in liver tissue were similar to those described in Example 5 above.
The results are shown in FIG. 10. XTC-SNALP-18328 reduced TTR mRNA levels in the liver in a dose-dependent manner, compared to the negative control XTC-SNALP-1955. The mRNA ED50 was determined to be  ̃0.1 mg/kg XTC-SNALP-18328. These results demonstrate that XTC-SNALP-18328 is effective in suppressing wild-type TTR mRNA in non-human primate liver when administered by IV infusion.
Example 11
In Vivo Reduction of Wild-Type TTR mRNA in the Non-Human Primate Liver by LNP09-18328 and LNP11-18328
To evaluate the efficacy of two novel lipid nanoparticle formulations, LNP09 and LNP11, for delivery of siRNA in non-human primate, TTR siRNA AD-18328 was formulated in LNP09 (LNP09-18328) or LNP11 (LNP11-18328), and administered by 15-minute IV infusion, and liver TTR mRNA and serum TTR protein levels were assayed. Cynomolgus monkeys (Macaca fascicularis) were administered 15-minute IV infusions of LNP09-18328 (0.03, 0.1, or 0.3 mg/kg), LNP11-18328 (0.03, 0.1, or 0.3 mg/kg), or PBS. Liver biopsy samples were collected at 48 hrs post-dosing, flash-frozen, and stored at −80° C. until processing. Serum was collected before dosing (pre-bleed), and on Days 1, 2, 4, 7, 14, 21 and 28 post-dosing and stored at −80° C. until processing. Methods used for TTR mRNA quantitation in liver tissue and serum TTR protein evaluation were similar to those described in Examples 5 and 8 above.
The results are shown in FIG. 11A for mRNA, and in FIG. 11B and FIG. 11C for protein. LNP09-18328 and LNP11-18328 treated animals showed a dose-dependent decrease in TTR mRNA levels in the liver, reaching a maximum reduction at 0.3 mg/kg of  ̃85% (LNP09-18328) and  ̃90% (LNP11-18328) mRNA relative to the PBS control. The mRNA ED50 was determined to be  ̃0.02 mg/kg for both LNP09-18328 and LNP11-18328. At Day 7 post-dosing, serum samples also exhibited a dose-dependent reduction of TTR protein for 0.1 and 0.3 mg/kg LNP09-18328 and LNP11-18328, compared to PBS control levels. FIG. 11C shows a decrease in TTR protein levels with a 0.3 mg/kg dose of LNP09-18328 that persisted over at least 28 days post-dosing, as compared to the PBS control group and as compared with the pre-bleed samples.
These results demonstrate that LNP09-18328 and LNP11-18328 are effective in suppressing wild-type TTR mRNA in non-human primate liver and wild-type TTR protein in the circulation, when administered by IV infusion. Furthermore, the suppression with LN09-18328 is durable, persisting for at least 28 days following the IV infusion.
Example 12
In Vivo Reduction of Wild-Type TTR mRNA in the Non-Human Primate Liver by LNP12-18328
LNP12 formulated AD-18328 was administered to non-human primates to evaluate the efficacy of this formulation.
LNP12-AD-18328 formulations were prepared using a method adapted from Jeffs et al. (Jeffs L B, et al. (2004) A Scalable, Extrusion-Free Method for Efficient Liposomal Encapsulation of Plasmid DNA. Pharm Res 22:362-372) Briefly, Tech-G1 (described above), distearoyl phosphatidylcholine (DSPC), cholesterol and mPEG2000-DMG were solubilized in 90% ethanol at a molar ratio of 50:10:38.5:1.5. The siRNA was solubilized in 10 mM citrate, pH 3 buffer at a concentration of 0.4 mg/mL. The ethanolic lipid solution and the aqueous siRNA solution were pumped by means of a peristaltic pump fitted with dual pump heads at equivalent volumetric flow rates and mixed in a “T”-junction. Lipids were combined with siRNA at a total lipid to siRNA ratio of 7:1 (wt:wt). The spontaneously formed LNP12-AD18323 formulations were dialyzed against PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) to remove ethanol and exchange buffer. This formulation yields a mean particle diameter of 80 nm with approximately 90 percent siRNA entrapment efficiency.
Cynomolgus monkeys (n=3 per group) received either PBS or 0.03, 0.1, or 0.3 mg/kg LNP12-AD-18328 as 15 minute intravenous infusions (5 mL/kg) via the cephalic vein. Liver biopsies were collected from animals at 48 hours post-administration. TTR mRNA levels relative to GAPDH mRNA levels were determined in liver samples as described herein.
As shown in FIG. 19, high levels of specific knockdown of the wild-type transthyretin (TTR) gene was observed at doses as low as 0.03 mg/kg. This demonstrated that the LNP12 formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously-described siRNA liver delivery system.
Example 13
Synthesis of TTR Tiled Sequences
A set of TTR duplexes (“tiled duplexes”) were designed that targeted the TTR gene near the target region of AD-18328, which targets the human TTR gene starting at nucleotide 628 of NM—000371.3.
In the examples below, the numbering representing the position of the 5′ base of an siRNA on the transcript is based on NM—000371.3 (FIG. 12; SEQ ID NO:1331). In the examples shown above, the numbering for siRNA targeting human siRNA was based on NM—000371.2 (FIG. 13A). NM—000371.3 extends the sequence of the 5′ UTR by 110 bases compared to NM—000371.2, as shown in FIG. 14. Thus, as an example, the starting position of AD-18328 is 628 on NM—000371.3 and 518 on NM—000371.2 (FIG. 14).
TTR tiled sequences were synthesized on MerMade 192 synthesizer at 1 umol scale. For all the sequences in the list, ‘endolight’ chemistry was applied as detailed below.

    
    
        All pyrimidines (cytosine and uridine) in the sense strand contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)
        In the antisense strand, pyrimidines adjacent to (towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides
        A two base dTdT extension at 3′ end of both sense and anti sense sequences was introduced
        The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software
    
    


Synthesis, Cleavage and Deprotection:
The synthesis of TTR sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. The synthesis of the sequences was performed at 1 um scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator. The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. The crude sequences were precipitated using acetone:ethanol (80:20) mix and the pellet were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.
Purification and Desalting:
TTR tiled sequences were purified on AKTA explorer purification system using Source 15Q column. A column temperature of 65C was maintained during purification. Sample injection and collection was performed in 96 well (1.8 mL—deep well) plates. A single peak corresponding to the full length sequence was collected in the eluent. The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted TTR sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The single strands were then submitted for annealing.
TTR Single Strands and Duplexes:
A detailed list of TTR tiled duplexes and corresponding single strands (sense and antisense) are shown in the table below (Table 13).







TABLE 13







TTR tiled duplexes and corresponding single strands


(NM_000371.3, SEQ ID NO: 1331)












Duplex #
Position
Oligo #
Strand
Sequence (5′ to 3′′)
SEQ ID NO:





AD-18323
618
A-32335
S
GGGAuuucAuGuAAccAAGdTdT
1332




A-32336
AS
CUUGGUuAcAUGAAAUCCCdTdT
1333


 


AD-18324
619
A-32337
S
GGAuuucAuGuAAccAAGAdTdT
1334




A-32338
AS
UCUUGGUuAcAUGAAAUCCdTdT
1335


 


AD-23000
620
A-42927
S
GAuuucAuGuAAccAAGAGdTdT
1336




A-42928
AS
CUCUUGGUuAcAUGAAAUCdTdT
1337


 


AD-23001
621
A-42929
S
AuuucAuGuAAccAAGAGudTdT
1338




A-42930
AS
ACUCUUGGUuAcAUGAAAUdTdT
1339


 


AD-23002
622
A-42931
S
uuucAuGuAAccAAGAGuAdTdT
1340




A-42932
AS
uACUCUUGGUuAcAUGAAAdTdT
1341


 


AD-23003
623
A-42933
S
uucAuGuAAccAAGAGuAudTdT
1342




A-42934
AS
AuACUCUUGGUuAcAUGAAdTdT
1343


 


AD-18325
624
A-32339
S
ucAuGuAAccAAGAGuAuudTdT
1344




A-32340
AS
AAuACUCUUGGUuAcAUGAdTdT
1345


 


AD-23004
625
A-42935
S
cAuGuAAccAAGAGuAuucdTdT
1346




A-42936
AS
GAAuACUCUUGGUuAcAUGdTdT
1347


 


AD-18326
626
A-32341
S
AuGuAAccAAGAGuAuuccdTdT
1348




A-32342
AS
GGAAuACUCUUGGUuAcAUdTdT
1349


 


AD-18327
627
A-32343
S
uGuAAccAAGAGuAuuccAdTdT
1350




A-32344
AS
UGGAAuACUCUUGGUuAcAdTdT
1351


 


AD-23005
628
A-42937
S
uAAccAAGAGuAuuccAuudTdT
1352




A-42938
AS
AAUGGAAuACUCUUGGUuAdTdT
1353


 


AD-23006
629
A-42939
S
AAccAAGAGuAuuccAuuudTdT
1354




A-42940
AS
AAAUGGAAuACUCUUGGUUdTdT
1355


 


AD-23007
631
A-42941
S
AccAAGAGuAuuccAuuuudTdT
1356




A-42942
AS
AAAAUGGAAuACUCUUGGUdTdT
1357


 


AD-23008
632
A-42943
S
ccAAGAGuAuuccAuuuuudTdT
1358




A-42944
AS
AAAAAUGGAAuACUCUUGGdTdT
1359


 


AD-23009
633
A-42945
S
cAAGAGuAuuccAuuuuuAdTdT
1360




A-42946
AS
uAAAAAUGGAAuACUCUUGdTdT
1361


 


AD-23010
634
A-42947
S
AAGAGuAuuccAuuuuuAcdTdT
1362




A-42948
AS
GuAAAAAUGGAAuACUCUUdTdT
1363


 


AD-23011
635
A-42949
S
AGAGuAuuccAuuuuuAcudTdT
1364




A-42950
AS
AGuAAAAAUGGAAuACUCUdTdT
1365


 


AD-23012
636
A-42951
S
GAGuAuuccAuuuuuAcuAdTdT
1366




A-42952
AS
uAGuAAAAAUGGAAuACUCdTdT
1367


 


AD-23013
637
A-42953
S
AGuAuuccAuuuuuAcuAAdTdT
1368




A-42954
AS
UuAGuAAAAAUGGAAuACUdTdT
1369


 


AD-23014
638
A-42955
S
GuAuuccAuuuuuAcuAAAdTdT
1370




A-42956
AS
UUuAGuAAAAAUGGAAuACdTdT
1371


 


AD-23015
639
A-42957
S
uAuuccAuuuuuAcuAAAGdTdT
1372




A-42958
AS
CUUuAGuAAAAAUGGAAuAdTdT
1373


 


AD-23016
640
A-42959
S
AuuccAuuuuuAcuAAAGcdTdT
1374




A-42960
AS
GCUUuAGuAAAAAUGGAAUdTdT
1375


 


AD-23017
641
A-42961
S
uuccAuuuuuAcuAAAGcAdTdT
1376




A-42962
AS
UGCUUuAGuAAAAAUGGAAdTdT
1377


 


AD-23018
642
A-42963
S
uccAuuuuuAcuAAAGcAGdTdT
1378




A-42964
AS
CUGCUUuAGuAAAAAUGGAdTdT
1379


 


AD-23019
643
A-42965
S
ccAuuuuuAcuAAAGcAGudTdT
1380




A-42966
AS
ACUGCUUuAGuAAAAAUGGdTdT
1381


 


AD-23020
644
A-42967
S
cAuuuuuAcuAAAGcAGuGdTdT
1382




A-42968
AS
cACUGCUUuAGuAAAAAUGdTdT
1383


 


AD-23021
645
A-42969
S
AuuuuuAcuAAAGcAGuGudTdT
1384




A-42970
AS
AcACUGCUUuAGuAAAAAUdTdT
1385


 


AD-23022
646
A-42971
S
uuuuuAcuAAAGcAGuGuudTdT
1386




A-42972
AS
AAcACUGCUUuAGuAAAAAdTdT
1387


 


AD-23023
647
A-42973
S
uuuuAcuAAAGcAGuGuuudTdT
1388




A-42974
AS
AAAcACUGCUUuAGuAAAAdTdT
1389


 


AD-23024
648
A-42975
S
uuuAcuAAAGcAGuGuuuudTdT
1390




A-42976
AS
AAAAcACUGCUUuAGuAAAdTdT
1391


 


AD-23025
649
A-42977
S
uuAcuAAAGcAGuGuuuucdTdT
1392




A-42978
AS
GAAAAcACUGCUUuAGuAAdTdT
1393


 


AD-23026
650
A-42979
S
uAcuAAAGcAGuGuuuucAdTdT
1394




A-42980
AS
UGAAAAcACUGCUUuAGuAdTdT
1395


 


AD-23027
651
A-42981
S
AcuAAAGcAGuGuuuucAcdTdT
1396




A-42982
AS
GUGAAAAcACUGCUUuAGUdTdT
1397


 


AD-23028
652
A-42983
S
cuAAAGcAGuGuuuucAccdTdT
1398




A-42984
AS
GGUGAAAAcACUGCUUuAGdTdT
1399


 


AD-18330
653
A-32349
S
uAAAGcAGuGuuuucAccudTdT
1400




A-32350
AS
AGGUGAAAAcACUGCUUuAdTdT
1401


 


AD-23029
654
A-42985
S
AAAGcAGuGuuuucAccucdTdT
1402




A-42986
AS
GAGGUGAAAAcACUGCUUUdTdT
1403


 


AD-23030
655
A-42987
S
AAGcAGuGuuuucAccucAdTdT
1404




A-42988
AS
UGAGGUGAAAAcACUGCUUdTdT
1405


 


AD-23031
656
A-42989
S
AGcAGuGuuuucAccucAudTdT
1406




A-42990
AS
AUGAGGUGAAAAcACUGCUdTdT
1407


 


AD-18328
628
A-32345
S
GuAAccAAGAGuAuuccAudTdT
1408




A-32346
AS
AUGGAAuACUCUUGGUuACdTdT
1409





Strand: s = sense; as = antisense; Position: position of 5′ base on transcript






Example 14
In Vitro Screening of TTR Tiled siRNAs
Tiled TTR duplexes were assayed in Hep3B cells for inhibition of endogenous TTR expression using real time PCR assays.
Cell culture and transfection: Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in Eagle's Minimum Essential Medium (EMEM, ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of each siRNA in individual wells of a 96-well plate. To this 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax was added per well (Invitrogen, Carlsbad Calif. cat #13778-150) and the mixture was incubated at room temperature for 15 minutes. 80 μl of complete growth media described above, but without antibiotic containing 2.0×104 Hep3B cells were then added. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 0.1 or 10 nM final duplex concentration.
Total RNA isolation using MagMAX-96 Total RNA Isolation Kit (Applied Biosystems, Foster City Calif., part #: AM1830): Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 μl of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 μl of water.
cDNA synthesis using ABI High capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, Calif., Cat #4368813): A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 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 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl TTR TaqMan probe (Applied Biosystems cat #HS00174914 M1) and 10 μl Roche Probes Master Mix (Roche Cat #04887301001) per well in a LightCycler 480 384 well plate (Roche cat #0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex was tested in two independent transfections and each transfection was assayed in duplicate.
Real time data were analyzed using the ΔΔCt method. Each sample was normalized to GAPDH expression and knockdown was assessed relative to cells transfected with the non-targeting duplex AD-1955. Table 14 shows the knockdown of TTR using the siRNAs. Data are expressed as the percent of message remaining relative to cells targeted with AD-1955.
Many but not all tiled TTR-dsRNAs, targeting TTR near the target of AD-18328, reduced TTR mRNA by at least 70% when transfected into Hep3B cells at 0.1 nM.







TABLE 14







Inhibition of TTR by tiled dsRNA targeting


TTR near target of AD-18328.












% message

% message




remaining
% SD
remaining
% SD


Duplex #
0.1 nM
0.1 nM
10 nM
10 nM














AD-18323
6.7
1.90
1.7
0.02


AD-18324
1.8
0.58
0.9
0.10


AD-23000
5.5
0.93
2.1
0.87


AD-23001
15.2
4.89
4.9
1.74


AD-23002
3.1
1.12
1.4
0.55


AD-23003
17.3
3.13
1.7
0.06


AD-18325
1.5
0.27
1.4
0.66


AD-23004
9.0
0.15
10.5
0.96


AD-18326
22.0
1.85
7.6
0.78


AD-18327
11.6
2.64
9.6
1.67


AD-18328
1.1
0.70
0.6
0.16


AD-23005
0.8
0.31
0.6
0.21


AD-23006
1.5
0.46
1.2
0.43


AD-23007
2.4
0.91
1.9
0.46


AD-23008
0.6
0.10
0.8
0.26


AD-23009
1.0
0.13
0.9
0.22


AD-23010
60.1
15.66
66.2
22.71


AD-23011
56.5
16.99
53.6
4.70


AD-23012
7.7
2.36
7.7
3.25


AD-23013
7.0
0.64
8.0
1.06


AD-23014
0.7
0.01
0.6
0.10


AD-23015
15.4
0.25
16.5
7.07


AD-23016
27.1
0.37
6.7
1.80


AD-23017
4.5
1.26
1.4
0.40


AD-23018
44.6
9.45
7.5
1.09


AD-23019
2.2
0.68
0.8
0.10


AD-23020
52.7
6.45
29.7
1.17


AD-23021
95.4
16.16
45.0
3.00


AD-23022
70.1
3.01
60.8
12.11


AD-23023
2.7
1.12
1.8
0.07


AD-23024
1.7
0.30
1.8
0.33


AD-23025
64.2
13.21
10.5
1.34


AD-23026
1.9
0.15
1.9
0.78


AD-23027
2.5
0.21
1.6
0.49


AD-23028
6.7
4.41
1.2
0.50


AD-18330
6.0
0.56
5.7
1.15


AD-23029
4.5
0.47
1.6
0.10


AD-23030
3.9
0.25
3.3
0.84


AD-23031
3.4
0.78
1.7
0.02









Example 15
Evaluation of Infusion Duration on Efficacy of a Single Intravenous Administration of SNALP-18534 in Sprague-Dawley Rats
Objectives
To determine the effect of infusion duration on efficacy of a single IV infusion of SNALP-18534 on liver TTR mRNA levels in Sprague-Dawley rats.







TABLE 15





Abbreviations and definitions used
















SNALP-18534
Rodent transthyretin specific siRNA formulated in



SNALP


SNALP-1955
Non-mammalian luciferase specific siRNA formulated



in SNALP









The sequences of the sense and antisense strands of AD-18534 are reproduced below from the tables above:


















SEQ




Posi-

ID


Strand
Oligo #
tion
Sequence 5′ to 3′
NO:







s
A-32755
532
cAGuGuucuuGcucuAuAAdTdT
1289


 


as
A-32756
550
UuAuAGAGcAAGAAcACUGdTdT
1290









Study Materials
Test Article(s)
SNALP-18534 is comprised of an siRNA targeting rodent TTR mRNA (AD-18534), formulated in stable nucleic acid lipid particles (SNALP) for delivery to target tissues. The SNALP formulation (lipid particle) consists of a novel aminolipid (DLinDMA), a PEGylated lipid (mPEG2000-C-DMA), a neutral lipid (DPPC) and cholesterol. The ratio of lipid:nucleic acid in the SNALP formulation is approximately 5.8:1 (w:w). SNALP-1955 contains an siRNA targeting the non-mammalian luciferase mRNA, is formulated with the identical lipid particle as SNALP-18534, and serves as a non-pharmacologically active control. Dose levels are expressed as mg/kg based on the weight of siRNA content.
Study Design & Procedures
Animals and Test Article Administration:
The study was comprised of 9 groups of Sprague-Dawley rats (4.males/group). The animals were allowed to have at least a 2 day acclimation period before the study and all animals were 7 weeks old at the initiation of dosing. The dose administered was calculated based upon body weight data collected prior to dosing on Day 1. The test and control articles were administered as a single 15-minute, 1-hour, 2-hour, or 3-hour IV infusion via the tail vein using a 24G 3⁄4′′ cannula sealed with a Baxter Injection Site septum connected via 27G Terumo butterfly needle to a Baxter AS40A Syringe Pump. The dose volume was 3 ml/kg, the infusion rate was 12 ml/kg/hr, and animals were freely moving in the cages during dosing. Rats were divided into nine treatment groups and administered a single IV infusion of SNALP-18534, SNALP-1955, or PBS as shown in Table 16:







TABLE 16







Test Animal Dosage Groups











Group
N
Test Article
Infusion Duration
Dose















A
4
PBS
15
minute
—


B
4
PBS
3
hour
—


C
4
SNALP-1955
1
hour
1 mg/kg


D
4
SNALP-1955
2
hour
1 mg/kg


E
4
SNALP-1955
3
hour
1 mg/kg


F
4
SNALP-18534
15
minute
1 mg/kg


G
4
SNALP-18534
1
hour
1 mg/kg


H
4
SNALP-18534
2
hour
1 mg/kg


I
4
SNALP-18534
3
hour
1 mg/kg









Tissue Collection and RNA Isolation:
On Day 0, animals were anesthetized by isofluorane inhalation and pre-dosing blood samples were collected into serum separator tubes by retro-orbital bleed. The blood samples were allowed to clot at room temperature for approximately 30 minutes prior to centrifugation at 4° C. Serum samples were then stored at −80° C. until analysis was performed. On Day 3, animals in all nine treatment groups were given a lethal dose of ketamine/xylazine. Blood was collected via caudal vena cava into serum separation tubes, and then allowed to clot at room temperature for approximately 30 minutes prior to centrifugation at 4° C. Serum samples were stored at −80° C. until analysis was performed. Liver tissue was harvested and snap frozen on dry ice. Frozen liver tissue was ground and tissue lysates were prepared for liver mRNA quantitation.
TTR mRNA Quantitation:
TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Pommies, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample.
To obtain the relative level of TTR mRNA expression, group mean values for SNALP-1955 and SNALP-18534 treated groups with 15-minute, 1 hour and 2 hour infusion durations were then normalized to the mean value for the PBS treated group with 15-minute infusion whereas group mean values for SNALP-1955 and SNALP-18534 treated groups with 3 hour infusion duration were then normalized to the mean value for the PBS treated group with 3 hour infusion duration.
Results
As shown in FIG. 16, a single IV infusion of 1 mg/kg SNALP-18534 with different infusion durations of 15 minutes to 3 hours results in comparable inhibition of liver TTR mRNA levels measured two days after dosing. A single IV infusion of 1 mg/kg SNALP-18534 also showed durable TTR downregulation over 29 days following a single 15 minute IV infusion, as compared to SNALP-1955 control (data not shown). Compared to the PBS-treated group, a single 15-minute, 1-hour, 2-hour, or 3-hour IV infusion of SNALP-18534 at 1 mg/kg significantly reduced relative TTR mRNA expression levels by 94% (p<0.001), 94% (p<0.001), 92% (p<0.001) and 93% (p<0.001), respectively. Specificity of SNALP-18534 activity is demonstrated by lack of significant target inhibition by SNALP-1955 administration via 1-hour, 2-hour, or 3-hour IV infusion at the same dose level.
Conclusions
This study demonstrates that varying the infusion duration from 15 minutes to up to 3 hours does not affect the efficacy of a single IV administration of 1 mg/kg SNALP-18534 in rats, as assessed by reduction of TTR mRNA levels in the liver.
Example 16
In Vivo Reduction of Wild-Type TTR mRNA in the Rat Liver by LNP07-18534 and LNP08-18534
To evaluate the efficacy of 2 novel lipid nanoparticle formulations, LNP07 and LNP08, for delivery of siRNA in the rat, the rodent-specific TTR siRNA, AD-18534, was formulated in LNP07 (LNP07-18534) or LNP08 (LNP08-18534), and administered by 15-minute IV infusion, and liver TTR mRNA was quantified. Sprague-Dawley rats (4 animals per group) were administered 15-minute IV infusions of LNP07-18534 (0.03, 0.1, 0.3 or 1 mg/kg), LNP08-18534 (0.01, 0.03 or 0.1 mg/kg), or LNP07-1955 (1 mg/kg) or LNP08-1955 (0.1 mg/kg) containing the negative control siRNA AD-1955 which targets the non-mammalian gene luciferase. Forty-eight hours later, animals were euthanized and liver tissue was collected, flash-frozen and stored at −80° C. until processing.
For TTR mRNA quantitation, frozen liver tissue was ground into powder, and lysates were prepared. TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group means of the normalized values were then further normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression.
The results are shown in FIG. 17. LNP07-18534 reduced TTR mRNA levels in the liver in a dose-dependent manner, with 94% suppression of TTR mRNA at 1 mg/kg. The effect was specific, since the negative control LNP07-1955 at 1 mg/kg did not significantly affect TTR mRNA levels compared to the PBS control. The mRNA ED50 was determined to be  ̃0.05 mg/kg LNP07-18534. LNP08-18534 reduced TTR mRNA levels in the liver in a dose-dependent manner, with 86% suppression of TTR mRNA at 0.1 mg/kg. The effect was specific, since the negative control LNP08-1955 at 0.1 mg/kg did not significantly affect TTR mRNA levels compared to the PBS control. The mRNA ED50 was determined to be  ̃0.02 mg/kg LNP08-18534.
These results demonstrate that LNP07-18534 and LNP08-18534 are effective in suppressing wild-type TTR mRNA in the rat liver when administered by IV infusion, and that LNP07 and LNP08 are effective formulations for delivering siRNA to the liver.
Example 17
Reduction of TTR Liver mRNA by a Single Intravenous Administration of LNP09-18534 or LNP11-18534 in Sprague-Dawley Rats
Objective
To evaluate the efficacy of two novel lipid nanoparticle (LNP) formulations for delivery of the rodent TTR-specific siRNA, AD-18534 in the Sprague-Dawley rat for reducing endogenous (wild type) liver TTR mRNA levels. Rats were intravenously dosed via a 15 minute infusion with either 0.01, 0.03, 0.1, or 0.3 mg/kg LNP09-18534, LNP11-18534, or phosphate buffered saline (PBS) and TTR liver mRNA levels were assayed at 48 hrs post-treatment.
Material and Methods:
LNP09 formulation: (XTC/DSPC/Chol/PEG2000-C14)=50/10/38.5/1.5 mol %; Lipid:siRNA ̃11:1. LNP11 formulation: (MC3/DSPC/Chol/PEG2000-C14)=50/10/38.5/1.5 mol %; Lipid:siRNA ̃11.1
Tissue collection and RNA isolation: On Day 3, animals in all treatment groups were given a lethal dose of ketamine/xylazine. Blood was collected via caudal vena cava into serum separation tubes, and then allowed to clot at room temperature for approximately 30 minutes prior to centrifugation at 4° C. Serum samples were stored at −80° C. until for future analysis. Liver tissues were harvested and snap frozen on dry ice. Frozen liver tissue was ground and tissue lysates were prepared for liver mRNA quantitation.
TTR mRNA Quantitation: TTR mRNA levels relative to those of GAPDH mRNA were determined in the lysates by using a branched DNA assay (QuantiGene Reagent System, Panomics, Fremont, Calif.). Briefly, the QuantiGene assay (Genospectra) was used to quantify mRNA levels in tissue sample lysates according to the manufacturer's instructions. The mean level of TTR mRNA was normalized to the mean level of GAPDH mRNA for each sample. Group mean values were then normalized to the mean value for the PBS treated group, to obtain the relative level of TTR mRNA expression.
Results:
As shown in FIG. 18, in contrast with PBS treated animals, LNP09-18534 and LNP11-18534 treated animals had a significant dose-dependent decrease in TTR mRNA levels in the liver, reaching maximum reduction of 90% mRNA reduction for both LNP09 and LNP11 formulated groups, relative to PBC control group at 0.3 mg/kg, and a dose achieving 50% reduction (ED50) of <0.03 mg/kg for LNP11-18534 and <0.1 mg/kg for LNP09-18534.
Conclusions
This study demonstrates that a single 15 minute IV infusion of LNP09-18534 or LNP11-18534 in Sprague-Dawley rats results in a dose-dependent reduction of liver TTR mRNA. These data demonstrate the efficacy of LNP09-18328 and LNP11-18328 in reducing endogenously expressed (wild type) TTR mRNA with ED50 levels of <0.03 and <0.1 mg/kg for LNP11-18534 and LNP09-18534, respectively.
Example 18
Assaying for Toxicity in Animals
ALN-TTR01 was assayed for safety and toxicology under non-GLP and GLP conditions. ALN-TTR01 is the siRNA AD-18328 in a SNALP formulation (DLinDMA/DPPC/Cholesterol/PEG2000-cDMA (57.1/7.1/34.4/1.4) lipid:siRNA 7). Assays were performed in Cynomolgus monkey (1, 3, 6, 10, 30, 100 mg/kg) and Sprague-Dawley Rat (0.3, 1, 3, 6, 10 mg/kg). No toxicity was found at <1 mg/kg in rats and <10 mg/kg in NI-IP. (data not shown).
Example 19
Drug Product ALN-TTR01
The drug product, ALN TTR01 Injection, is a white to off-white, homogeneous sterile liquid suspension of the siRNA ALN 18328 with lipid excipients (referred to as stable nucleic acid lipid particles [SNALP]) in isotonic phosphate buffered saline. The composition of ALN TTR01 is shown in the table below.







TABLE 17







Composition of drug product ALN-TTR01











Concentra-
Per




tion
vial


Component, grade
(mg/mL)
(mg)
Function













ALN-18328, cGMP
2.0
11.0
Active ingredient


DLinDMA
7.3
40.2
Novel excipient;


(1,2-Dilinoleyloxy-N,N-


titratable aminolipid


dimethyl-3-aminopropane),


for interaction with


cGMP


the active ingredient


PEG2000-C-DMA
0.8
4.4
Novel excipient;


(3-N-[(α-Methoxy


stability of drug


poly(ethylene glycol) 2000)


product and desired


carbamoyl]-1,2-


biodistribution


dimyristyloxy-


propylamine), cGMP


DPPC (R-1,2-Dipalmitoyl-sn-
1.1
6.1
Structural integrity


glycero-3-phosphocholine),


of SNALP particles


cGMP


Cholesterol, synthetic, cGMP
2.8
15.4
Structural integrity





of SNALP particles


Phosphate buffered saline,
q.s.
to 5.5
Buffer


cGMP

mL









The lipid excipients have the molecular weights and structures shown in the table below.







TABLE 18







Lipid excipients










Molecular



Lipid
Weight
Chemical Name and Structure





DLinDMA
 616
1,2-Dilinoleyloxy-N,N-dimethyl-3-aminopropane   





 


PEG2000-CDMAa
2824 Polydispersity index 1.01
3-N-[(ω-Methoxy poly(ethylene glycol)2000)carbamoyl]-1,2- dimyristyloxy-propylamine   





 


DPPC
 734
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine   





 


Cholesterol
 387
Cholest-5-en-3β-ol   








aalternate name: mPEG2000-C-DMA






The ALN TTR01 drug product is packaged in 10 mL glass vials with a fill volume of 5.5 mL (11 mg ALN-18328 per vial). The container closure system consists of a USP/EP Type I borosilicate glass vial, a teflon faced butyl rubber stopper and an aluminum flip off cap. The drug product will be stored at 5±3° C.
Stability of the drug product is assayed for up to 24 months and determined using the following criteria:
Appearance: White to off-white, homogeneous opalescent liquid, no foreign particles
pH: 6.8-7.8
Osmolality: 250-350 mOsm/kg
Lipid: siRNA Ratio:5.6-8.4 mg/mg
Particle Size (Z-Average): 60-120 nm
Example 20
Inhibition of TTR in Humans
A human subject is treated with a dsRNA targeted to a TTR gene to inhibit expression of the TTR gene to treat a condition.
A subject in need of treatment is selected or identified. The subject can have a liver disorder, transthyretin amyloidosis, and/or a transplanted liver. For example, patients of any mutant TTR genotype with a biopsy-proven diagnosis of ATTR (FAP or FAC) who have not undergone liver transplantation can be selected. In some embodiments the patients have an adequate performance status, adequate hepatic and renal function, no active infection or inflammatory disorder, and stable cardiac status.
The identification of the subject can occur in a clinical setting, or elsewhere, e.g., in the subject's home through the subject's own use of a self-testing kit.
At time zero, a suitable first dose of an anti-TTR siRNA is administered to the subject. The dsRNA is formulated as described herein. After a period of time following the first dose, e.g., 7 days, 14 days, and 21 days, the subject's condition is evaluated, e.g., by measuring liver function. This measurement can be accompanied by a measurement of TTR expression in said subject, and/or the products of the successful siRNA-targeting of TTR mRNA. Other relevant criteria can also be measured. The number and strength of doses are adjusted according to the subject's needs.
For example, a single dose of ALN-TTR01 or normal saline placebo can be administered by 15 minute IV infusion. A starting dose level can be 0.03 mg/kg, with dose escalation over 4 dose levels: 0.03, 0.1, 0.2 and 0.4 mg/kg. A pharmacodynamic effect in ATTR patients may be seen starting at a dose of 0.1-0.3 mg/kg. The top dose will be, e.g., the lower of 0.4 mg/kg or the ED70 dose (the dose of ALN-TTR01 that results in 70% or greater suppression from baseline of total TTR in at least 2 patients within a cohort).
Patients can be premedicated prior to dosing to reduce the risk of infusion reaction: 1) dexamethasone 8 mg PO the evening before dosing, 2) dexamethasone 20 mg IV, diphenhydramine 50 mg IV, ranitidine 50 mg or famotidine 20 mg IV, and acetaminophen 650 mg PO 30 minutes prior to dosing. An infusion may be performed at a slower rate (maximum infusion time of 3 hours).
Vitamin A and RBP levels and thyroid function tests can be followed on a weekly basis following dosing with ALN-TTR01 (TTR plays a role in vitamin A transport in conjunction with retinol binding protein (RBP), and a minor role in the binding of circulating thyroxine relative to thyroglobulin). Testicular function will be closely monitored in males through serial measurements of testosterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels.
Serial measurements of serum TTR levels will allow for determination of the nadir following dosing as well as the timing of the nadir post-dose and the duration of TTR suppression.
After treatment, the subject's tumor growth rate is lowered relative to the rate existing prior to the treatment, or relative to the rate measured in a similarly afflicted but untreated subject.

## Claims

1. A method for reducing a TTR amyloid deposit by >90% in a subject having the TTR amyloid deposit, comprising administering to the subject an effective amount of a composition comprising a TTR dsRNA and determining TTR amyloid deposition in the subject, wherein: the TTR dsRNA comprises a sense strand and an antisense strand, the antisense strand consisting of SEQ ID NO:1010 and the sense strand consisting of SEQ ID NO:1009; the effective amount is 0.01-30 mg/kg TTR dsRNA; and the composition comprises a lipid formulation comprising a cationic lipid.

2. The method of claim 1, wherein the TTR amyloid deposit comprises V30M mutant TTR.

3. The method of claim 1, wherein the subject is a human subject.

4. The method of claim 1, wherein the TTR amyloid deposit comprises V30M mutant TTR and the subject is a human subject.