CA2521464A1

CA2521464A1 - Irna conjugates

Info

Publication number: CA2521464A1
Application number: CA002521464A
Authority: CA
Inventors: Muthiah Manoharan; Sayda Elbashir; Jens Harborth
Original assignee: Individual
Current assignee: Alnylam Pharmaceuticals Inc
Priority date: 2003-04-09
Filing date: 2004-04-09
Publication date: 2004-10-28
Anticipated expiration: 2024-04-09
Also published as: AU2004229519B2; CA2521464C; WO2004091515A2; AU2004229519A1; WO2004091515A3

Abstract

Therapeutic iRNA agents and methods of making and using are enclosed.</SDOAB >

Description

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Attorney's Docket No.: 14174-072W01 iRNA CONJUGATES
RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Application No. 60/462,097, filed April 9, 2003; U.S. Provisional Application No.
60/461,915, filed April 10, 2003; U.S. Provisional Application No. 60/463,772, filed April 17, 2003; U.S.
Provisional Application No. 60/465,802, filed April 25, 2003; U.S. Provisional Application No.
60/493,986, filed August 8, 2003; U.S. Provisional Application No. 60/494,597, filed August 11, 2003; U.S. Provisional Application No. 601506,341, filed-September 26, 2003;
U.S. Provisional Application No. 60/518,453, filed November 7, 2003; U.S. Provisional Application No. 60/469,612, filed May 9, 2003; U.S. Provisional Application No.
601510,246, filed October 9, 2003; U.S. Provisional Application No. 601510,318, filed October 10, 2003;
U.S. Provisional Application No. 60/465,665, filed April 25, 2003; U.S. Provisional Application No. 60/462,894, filed April 14, 2003; International Application No. PCT/US04/07070, filed March 8, 2004; and International Application No. [xxxxxx], filed April 5, 2004. The contents of these applications are hereby incorporated by reference in their entirety.
TECHNICAL FIELD
The invention relates to RNAi and related methods, e.g., methods of making and using iRNA agents. It includes methods and compositions for silencing genes expressed in the liver, and methods and compositions for directing iRNA agents to the liver.
2o BACKGROUND
RNA interference or "RNAi" is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al., Nature 391:806-81 l, 1998). Short dsRNA
directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA
degradation.

Attorney's Docket No.: 14174-072W01 Work in this field is typified by comparatively cumbersome approaches to delivery of dsRNA to live mammals. E.g., McCaffrey et al. (Nature 418:38-39, 2002) demonstrated the use of dsRNA to inhibit the expression of a luciferase reporter gene in mice. The dsRNAs were administered by the method of hydrodynamic tail vein injections (in addition, inhibition appeared to depend on the injection of greater than 2 mg/kg dsRNA). The inventors have discovered, inter alia, that the unwieldy methods typical of some reported work are not needed to provide effective amounts of dsRNA to mammals and in particular not needed to provide therapeutic amounts of dsRNA to human subj ects. The advantages of the current invention include practical, uncomplicated methods of administration and therapeutic applications, e.g., at dosages of less than 2 mg/kg.
SUMMARY
Aspects of the invention relate to compositions and methods for silencing genes expressed in the liver, e.g., to treat disorders of or related to the liver.
An iRNA agent composition of the invention can be one which has been modified to alter distribution in favor of the liver. A composition of the invention includes an iRNA agent, e.g., an iRNA agent or sRNA
agent described herein.
In one aspect, the invention features a method for reducing apoB-100 levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets apoB-100. The iRNA agent can be one described here, and can be a dsRNA that is 2o substantially identical to a region of the apoB-100 gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length.
In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.
In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Tables 9 or 10. In a preferred embodiment it targets both sequences of a palindromic pair provided in Tables 9 or 10. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Tables 9 or 10.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Tables 9 or 10. In a preferred embodiment the iRNA
agent will include regions complementary to the palindromic pairs of Tables 9 or 10 as a duplex region.
In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Tables 9 or 10 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than l, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence.
The iRNA agent that targets apoB-100 can be administered in an amount sufficient to reduce expression of apoB-100 mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of apoB-100 protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%). Preferably, the iRNA agent does not reduce expression of apoB-48 mRNA or protein. This can be effected, e.g., by selection of an iRNA agent which specifically targets the nucleotides subject to RNA editing in the apoB-100 transcript.
The iRNA agent that targets apoB-100 can be administered to a subject, wherein the 15 subject is suffering from a disorder characterized by elevated or otherwise unwanted expression of apoB-100, elevated or otherwise unwanted levels of cholesterol, and/or disregulation of lipid metabolism. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder. These disorders include HDL/LDL
cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired 2o hyperlipidemia; hypercholestorolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD) coronary heart disease (CHD) atherosclerosis. In one embodiment, the iRNA that targets apoB-100 is administered to a subject suffering from statin-resistant hypercholesterolemia.
The apoB-100 iRNA agent can be administered in an amount sufficient to reduce levels 2s of serum LDL-C and/or HDL-C and/or total cholesterol in a subj ect. For example, the iRNA is administered in an amount sufficient to decrease total cholesterol by at least 0.5%, 1%, 2.5%, 5%, 10% in the subject. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce the risk of myocardial infarction the subj ect.
In a preferred embodiment the iRNA agent is administered repeatedly.
Administration of 3o an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to Attorney's Docket No.: 14174-072W01 patient, depending on such factors as the severity of a patient's symptoms.
For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.
In one embodiment, the iRNA agent can be targeted to the liver, and apoB
expression level are decreased in the liver following administration of the apoB iRNA
agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.
1 o The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to a lipophilic moiety, e.g., a lipid, cholesterol, oleyl, retinyl, or cholesteryl residue.
Other lipophilic moieties that can be associated, e.g., conjugated with the iRNA agent include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-~5 O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In one embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a low-density lipoprotein (LDL), e.g., a lactosylated LDL. In another embodiment, the iRNA
agent can be 2o targeted to the liver by associating, e.g., conjugating, the iRNA agent to a polymeric Garner complex with sugar residues.
In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a liposome complexed with sugar residues. A
targeting agent that incorporates a sugar, e.g., galactose andlor analogues thereof, is particularly useful. These 25 agents target, in particular, the parenchyma) cells of the liver (see Table 1 ). In a preferred embodiment, the targeting moiety includes more than one galactose moiety, preferably two or three. Preferably, the targeting moiety includes 3 galactose moieties, e.g., spaced about 15 angstroms from each other. The targeting moiety can be lactose. A lactose is a glucose coupled to a galactose. Preferably, the targeting moiety includes three lactoses. The targeting moiety can 3o also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose, or mannose-6-phosphate targeting moiety can be used for macrophage targeting.

Attorney's Docket No.: 14174-072W01 The targeting agent can be linked directly, e.g., covalently or non covalently, to the iRNA
agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.
It is particularly preferred to use an iRNA conjugated to a lipophilic molecule to conjugate to an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase iRNA
targeting agent.
In one embodiment, the iRNA agent has been modified, or is associated with a delivery agent, e.g., a delivery agent described herein, e.g., a liposome, which has been modified to alter distribution in favor of the liver. In one embodiment, the modification mediates association with a serum albumin (SA), e.g., a human serum albumin (HSA), or a fragment thereof.
The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to an SA molecule, e.g., an HSA molecule, or a fragment thereof. In one 15 embodiment, the iRNA agent or composition thereof has an affinity for an SA, e.g., HSA, which is sufficiently high such that its levels in the liver are at least 10, 20, 30, 50, or 100% greater in the presence of SA, e.g., HSA, or is such that addition of exogenous SA will increase delivery to the liver. These criteria can be measured, e.g., by testing distribution in a mouse in the presence or absence of exogenous mouse or human SA.
2o The SA, e.g., HSA, targeting agent can be linked directly, e.g., covalently or non-covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome.
E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.
It is particularly preferred to use an iRNA conjugated to an SA, e.g., an HSA, molecule 25 wherein the iRNA agent is an apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.
In another aspect, the invention features, a method for reducing glucose-6-phosphatase levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets glucose-6-phosphatase. The iRNA agent can be a dsRNA
3o that has a sequence that is substantially identical to a sequence of the glucose-6-phosphatase gene.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment, the subject is treated with an iRNA agent that targets one of the sequences listed in Table 11. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 11. The most preferred targets are listed in descending order of preferability, in other words, the more preferred targets are listed earlier in Table 11.
In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 11. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 11 as a duplex region.
In a preferred embodiment the duplex regionof the iRNA agent will target a sequence listed in Table 11 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence In a preferred embodiment the iRNA agent includes overhangs, e.g., 3' or 5' overhangs, preferably one or more 3' overhangs. Overhangs are discussed in detail elsewhere herein but are . preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
Table 11 refers to sequences from human glucose-6-phosphatase. Table 12 refers to sequences from rat glucose-6-phosphatase. The sequences from table 12 can be used, e.g., in 2o experiments with rats or cultured rat cells.
In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurnng ribonucleotides can be used in the selected iRNA
agent.
The iRNA that targets glucose-6-phosphatase can be administered in an amount sufficient to reduce expression of glucose-6-phosphatase mRNA.
The iRNA that targets glucose-6-phosphatase can be administered to a subject to inhibit hepatic glucose production, for the treatment of glucose-metabolism-related disorders, such as diabetes, e.g., type-2-diabetes mellitus. The iRNA agent can be administered to an individual at 3o risk for the disorder to delay onset of the disorder or a symptom of the disorder.

Attorney's Docket No.: 14174-072W01 In other embodiments, iRNA agents having sequence similarity to the following genes can also be used to inhibit hepatic glucose production. These other genes include "forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase;
glucose-6-s phosphate locator; glucokinase inhibitory regulatory protein; and phosphoenolpyruvate carboxykinase (PEPCK).
In one embodiment, the iRNA agent can be targeted to the liver, and RNA
expression levels of the targeted genes are decreased in the liver following administration of the iRNA
agent.
1o The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of a target gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length.
In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.
15 In another aspect, the invention features a method for reducing beta-catenin levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent that targets beta-catenin. The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the beta-catenin gene.
The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides.
Preferably; the 2o iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.
In a preferred embodiment, the subj ect is treated with an iRNA agent which targets one of the sequences listed in Table 13. In a preferred embodiment it targets both sequences of a 2s palindromic pair provided in Table 13. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 13.
In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 13. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 13 as a duplex region.
3o In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 13 but will not be perfectly complementary with the target sequence, e.g., it will Attorney's Docket No.: 14174-072W01 not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence In a preferred embodiment the iRNA agent includes overhangs, e.g., 3' or 5' overhangs, preferably one or more 3' overhangs. Overhangs are discussed in detail elsewhere herein but are s preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
The iRNA agent that targets beta-catenin can be administered in an amount sufficient to reduce expression of beta-catenin mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of beta-catenin protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%).
The iRNA agent that targets beta-catenin can be administered to a subject, wherein the subject is suffering from a disorder characterized by unwanted cellular proliferation in the liver 15 or of liver tissue, e.g., metastatic tissue originating from the liver.
Examples include , a benign or malignant disorder, e.g., a cancer, e.g., a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma.
The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder 2o In a preferred embodiment the iRNA agent is administered repeatedly.
Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms.
For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite 25 period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.
In one embodiment, the iRNA agent can be targeted to the liver, and beta-catenin expression level are decreased in the liver following administration of the beta-catenin iRNA
3o agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

Attorney's Docket No.: 14174-072W01 In another aspect, the invention provides methods to treat liver disorders, e.g., disorders characterized by unwanted cell proliferation, hematological disorders, disorders characterized by inflammation disorders, and metabolic or viral diseases or disorders of the liver. A proliferation disorder of the liver can be, for example, a benign or malignant disorder, e.g., a cancer, e.g, a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma. A
hepatic hematology or inflammation disorder can be a disorder involving clotting factors, a complement-mediated inflammation or a fibrosis, for example. Metabolic diseases of the liver can include dyslipidemias, and irregularities in glucose regulation. Viral diseases of the liver can include hepatitis C or hepatitis B. In one embodiment, a liver disorder is treated by administering one or more iRNA agents that have a sequence that is substantially identical to a sequence in a gene involved in the liver disorder.
In one embodiment an iRNA agent to treat a liver disorder has a sequence which is substantially identical to a sequence of the beta-catenin or c jun gene. In another embodiment, such as for the treatment of hepatitis C or hepatitis B, the iRNA agent can have a sequence that is 15 substantially identical to a sequence of a gene of the hepatitis C virus or the hepatitis B virus, respectively. For example, the iRNA agent can target the 5' core region of HCV. This region lies just downstream of the ribosomal toe-print straddling the initiator methionine.
Alternatively, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, SA, or SB. For the treatment of hepatitis B, an iRNA agent can target the 2o protein X (HBx) gene, for example.
In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 14. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 14. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 14.
2s In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 14. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 14 as a duplex region.
In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 14, but will not be perfectly complementary with the target sequence, e.g., it will so not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence Attorney's Docket No.: 14174-072W01 In a preferred embodiment the iRNA agent includes overhangs, e.g., 3' or 5' overhangs, preferably one or more 3' overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
In another aspect, an iRNA agent can be administered to modulate blood clotting, e.g., to reduce the tendency to form a blood clot. In a preferred embodiment the iRNA
agent targets Factor V expression, preferably in the liver. One or more iRNA agents can be used to target a wild type allele, a mutant allele, e.g., the Leiden Factor V allele, or both.
Such administration can be used to treat or prevent venous thrombosis, e.g., deep vein thrombosis or pulmonary embolism, or another disorder caused by elevated or otherwise unwanted expression of Factor V, in, e.g., the liver. In one embodiment the iRNA agent can treat a subject, e.g., a human who has Factor V Leiden or other genetic trait associated with an unwanted tendency to form blood clots.
In a preferred embodiment administration of an iRNA agent which targets Factor V is with the administration of a second treatment, e.g, a treatment which reduces the tendency of the blood to clot, e.g., the administration of heparin or of a low molecular weight heparin.
In one embodiment, the iRNA agent that targets Factor V can be used as a prophylaxis in patients, e.g., patients with Factor V Leiden, who are placed at risk for a thrombosis, e.g., those 2o about to undergo surgery, in particular those about to undergo high-risk surgical procedures known to be associated with formation of venous thrombosis, those about to undergo a prolonged period of relative inactivity, e.g., on a motor vehicle, train or airplane flight, e.g., a flight or other trip lasting more than three or five hours. Such a treatment can be an adjunct to the therapeutic use of low molecular weight (LMW) heparin prophylaxis.
In another embodiment, the iRNA agent that targets Factor V can be administered to patients with Factor V Leiden to treat deep vein thrombosis (DVT) or pulmonary embolism (PE).
Such a treatment can be an adjunct to (or can replace) therapeutic uses of heparin or coumadin.
The treatment can be administered by inhalation or generally by pulmonary routes.
In a preferred embodiment, an iRNA agent administered to treat a liver disorder is 3o targeted to the liver. For example, the iRNA agent can be complexed with a targeting moiety, e.g., an antibody or ligand that recognizes a liver-specific receptor.

Attorney's Docket No.: 14174-072W01 The invention also includes preparations, including substantially pure or pharmaceutically acceptable preparations of iRNA agents which silence any of the genes discussed herein and in particular for any of apoB-100, glucose-6-phosphatase, beta-catenin, factor V, or any of the HVC genes discussed herein.
The methods and compositions of the invention, e.g., the methods and compositions to treat diseases and disorders of the liver described herein, can be used with any of the iRNA
agents described. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder described herein, and for the treatment of any subject, e.g., any animal, any mammal, such as any human.
The methods and compositions of the invention, e.g., the methods and iRNA
compositions to treat liver-based diseases described herein, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.
A "substantially identical" sequence includes a region of sufficient homology to the target gene, and is of sufficient length in terms of nucleotides, that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to a target RNA transcript. It is not necessary that there be perfect complementarity between the iRNA
agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the 2o target RNA, e.g., mRNA. Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if 2s present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and/or 3' terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of 3o the invention will be apparent from this description, and from the claims.
This application Attorney's Docket No.: 14174-072W01 incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
s FIG 1 is a structural representation of base pairing in psuedocomplementary siRNA2.
FIG. 2 is a schematic representation of dual targeting siRNAs designed to target the HCV
genome.
FIG 3 is a schematic representation of psuedocomplementary, bifunctional siRNAs designed to target the HCV genome.
FIG 4 is a general synthetic scheme for incorporation of RRMS monomers into an oligonucleotide.
FIG. 5 is a table of representative RRMS carriers. Panel 1 shows pyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel 3 shows piperidine-based RRMSs; panel 4 shows morpholine and piperazine-based RRMSs; and panel 5 shows decalin-15 based RRMSs. Rl is succinate or phosphoramidate and R2 is H or a conjugate ligand.
FIG 6A is a graph depicting blood glucose levels in mice treated with nonspecific Renilla<
RNA or not treated with siRNA. Mice treated with nonspecific Renilla RNA were inj ected on Day 7.
FIG 6B is a graph depicting blood glucose levels in mice treated with siRNA
targeting 2o glucose 6-phosphatase. Mice treated with siRNA targeting glucose 6-phosphatase were injected on Day 7.
FIG 6C is a graph depicting blood glucose levels in mice that were either not injected with siRNA, or were injected but the injection failed. Mice that were injected, were injected on Day 7.
2s FIG 7 is a graph depicting average blood glucose levels in four mice treated with siRNA
targeting glucose 6-phosphatase, and in four mice either treated with nonspecific Renilla RNA or not treated with siRNA (triangles). siRNA or Renilla RNA was administered on day 7 by hydrodynamic tail vein injection.
FIG SA is a graph depicting levels of luciferase mRNA in livers of CMV Luc mice 30 (Xanogen) following intervenous injection (iv) of buffer or siRNA into the tail vein. Each bar represents data from one mouse. RNA levels were quantified by QuantiGene Assay Attorney's Docket No.: 14174-072W01 (Genospectra, Inc.; Fremont, CA)). The Y axis represents chemiluminescence values in counts per second (CPS).
FIG. 8B is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (Xanogen). The values are averaged from the data depicted in FIG. 8A.
s FIG 9 is a graph depicting the pharmacokinetics of cholesterol-conjugated and unconjugated siRNA: The diamonds represent the amount of unconjugated 33P-labeled siRNA
(ALN-3000) in mouse plasma over time; the squares represent the amount of cholesterol-conjugated 33P-labeled siRNA (ALN-3001) in mouse plasma over time. "L1163" is equivalent to ALN3000; "L1163Cho1" is equivalent to ALN-3001.
1o FIG 10 is a graph indicating the amount of cholesterol-conjugated (dark bars) and unconjugated siRNA (light bars) detected in mouse whole liver tissue isolated over a period of time following intravenous tail vein injection. The amount of siRNA is represented as a percentage of the total dose or 33P-labeled siRNA delivered to the mouse.
"L1163" is equivalent to ALN3000 (light bars); "L1163Cho1" is equivalent to ALN-3001 (dark bars).
~5 FIG. 11 is a graph indicating the amount of cholesterol-conjugated siRNA
detected in various tissues of two different CMV-Luc mice ("Mouse 69" (light bars) and "Mouse 63" (dark bars)). Mice were injected with 50 mg/kg AL-3001 siRNA by intravenous tail vein injection, and tissue was harvested 22 hours later. SiRNA was detected by RNAse protection, and phosphorimager scanning was used to quantitate the siRNA. The amount of siRNA
is expressed 2o as ug/g liver tissue.
FIG. 12 is a gel of U/LT siRNA (see Table 19) detected in the liver of Baltic mice at increasing time points following hydrodynamic (hd) tail vein injection. U/LT
siRNA was injected at a concentration of 4 mg/kg. siRNA was detected by RNAse protection assay.
Lanes labeled "stand." were loaded with clean siRNA to serve as size and quality standards.
"non" represents 25 control samples isolated from livers of mice that were not injected with U/U siRNA. The control samples were further used in parallel RNAse protection assays.
FIG. 13 is a gel comparing different siRNA species detected in the livers of Baltic mice at increasing time points following hydrodynamic (hd) or nonhydrodynamic (iv) tail vein inj ection.
U/LT siRNA was injected by hd and by iv injection. 3'C/3'C and 3'C!U (see Table 19) were each 3o injected by iv injection. at a concentration of 4 mg/kg. siRNA was detected by RNAse protection assay. Lanes labeled "stand." were loaded with clean siRNA to serve as size and Attorney's Docket No.: 14174-072W01 quality standards. "non" represents control samples isolated from livers of mice that were not injected with siRNA. The control samples were further used in parallel RNAse protection assays.
FIG 14 is a graph depicting the percentage of luciferase activity in liver extracts of CMV
Luc mice injected with siRNA (ALN-3001). Percentage of luciferase activity was relative to activity in CMV Luc mice injected with PBS, pH 4.7. "Bufferl siRNAl," "Buffer2 siRNA2,"
and "Buffer3 siRNA3" represent the average activity observed in three separate experiments.
DETAILED DESCRIPTION
Double-stranded (dsRNA) directs the sequence-specific silencing of mRNA
through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
It has been demonstrated that 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound ~ 5 by theory, it may be that a molecular signal, which may be merely the specific length of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi.
Described herein are methods for preparing and administering these 21-23 nt fragments, and other iRNAs agents, and their use fox specifically inactivating gene function.
The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides of the same or 2o similar nature) enables the targeting of specific mRNAs for silencing in mammalian cells. In addition, longer dsRNA agent fragments can also be used, e.g., as described below.
Although, in mammalian cells, long dsRNAs can induce the interferon response which is frequently deleterious, sRNAs do not trigger the interferon response, at least not to an extent that is deleterious to the cell and host. In particular, the length of the iRNA
agent strands in an sRNA
2s agent can be less than 31, 30, 28, 25, or 23 nt, e.g., sufficiently short to avoid inducing a deleterious interferon response. Thus, the administration of a composition of sRNA agent (e.g., formulated as described herein) to a mammalian cell can be used to silence expression of a target gene while circumventing the interferon response. Further, use of a discrete species of iRNA

Attorney's Docket No.: 14174-072W01 agent can be used to selectively target one allele of a target gene, e.g., in a subject heterozygous for the allele.
Moreover, in one embodiment, a mammalian cell is treated with an iRNA agent that disrupts a component of the interferon response, e.g., double stranded RNA
(dsRNA)-activated protein kinase PKR. Such a cell can be treated with a second iRNA agent that includes a sequence complementary to a target RNA and that has a length that might otherwise trigger the interferon response.
In a typical embodiment, the subject is a mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject can be a dairy mammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken, turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, the subject is a human, e.g., a normal individual or an individual that has, is diagnosed with, or is predicted to have a disease or disorder.
Further, because iRNA agent mediated silencing persists for several days after administering the iRNA agent composition, in many instances, it is possible to administer the 15 composition with a frequency of less than once per day, or, for some instances, only once for the . entire therapeutic regimen. For example, treatment of some cancer cells may be mediated by a single bolus administration, whereas a chronic viral infection may require regular administration, e.g., bnce per week or once per month.
A number of exemplary routes of delivery are described that can be used to administer an 2o iRNA agent to a subject. In addition, the iRNA agent can be formulated according to an exemplary method described herein.
Liver Diseases Exemplary diseases and disorders that can be treated by the methods and compositions of the invention are liver-based diseases.
25 Disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and cirrhosis, such as cirrhosis, portal hypertension, including ascites, portosystemic shunts, and splenomegaly; infectious disorders, such as viral hepatitis, including hepatitis A-E infection and infection by other Attorney's Docket No.: 14174-072W01 hepatitis viruses, clinicopathologic syndromes, such as the carrier state, asymptomatic infection, acute viral hepatitis, chronic viral hepatitis, and fulminant hepatitis;
autoimmune hepatitis; drug-and toxin-induced liver disease, such as alcoholic liver disease; inborn errors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, a1-antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease, such as secondary biliary cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and anomalies of the biliaxy tree; circulatory disorders, such as impaired blood flow into the liver, including hepatic artery compromise and portal vein obstruction and thrombosis, impaired blood flow through the liver, including passive congestion and,centrilobular necrosis and peliosis hepatis, hepatic vein outflow obstruction, 1o including hepatic vein thrombosis (Budd-Chiari syndrome) and verso-occlusive disease; hepatic disease associated with pregnancy, such as preeclampsia and eclampsia, acute fatty liver of pregnancy, and intrehepatic cholestasis of pregnancy; hepatic complications of organ or bone marrow transplantation, such as drug toxicity after bone marrow transplantation, graft-versus-host disease and liver rejection, and nonimmunologic damage to liver allografts; tumors and ~5 tumorous conditions, such as nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma of the liver and metastatic tumors.
An iRNA agent can also be administered to inhibit Factor V expression in the liver. Two to five percent of the United States population is heterozygous for an allele of the Factor V gene that encodes a single amino acid change at position 1961. These heterozygous individuals have a 20 3-~ fold increased risk of venous thrombosis, a risk that is associated with increased factor V
activity The increased activity leads to increased thrombin generation from the prothrombinase complex. An iRNA agent directed against Factor V can treat or prevent venous thrombosis or treat a human who has Factor V Leiden. The iRNA agent that targets Factor V
can be also be used as a prophylaxis in patients with Factor V Leiden who undergo high-risk surgical 25 procedures, and this prophylaxis can be an adjunct to the therapeutic use of low molecular weight (LMW) heparin prophylaxis.
An iRNA agent that targets Factor V can also be administered to patients with Factor V
Leiden to treat deep vein thrombosis (DVT) or pulmonary embolism (PE), and this treatment can be an adjunct to therapeutic uses of heparin or coumadin. Any other disorder caused by elevated 30 or otherwise unwanted levels of Factor V protein can be treated by administering an iRNA agent against Factor V

Attorney's Docket No.: 14174-072W01 iRNA agents of the invention can be targeted to any gene whose overexpression is associated with the liver diseases.
Tar~etin~ to the Liver The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver through a composition that includes the iRNA agent and a liver-targeting agent. For example, a liver-targeting agent can be a lipophilic moiety.
Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues (see Table 1). Other lipophilic moieties that can function as liver-targeting agents include cholic acid, 1o adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
An iRNA agent can also be targeted to the liver by association with a low-density 15 lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.
A targeting agent that incorporates a sugar, e.g., galactose andlor analogues thereof, is particularly useful. These agents target, in particular, the parenchyma) cells of the liver (see Table 1 ). For example, a targeting moiety can include more than one or preferably two or three 2o galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose.
The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A
mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.
Conjugation of an iRNA agent with a serum albumin (SA), such as human serum 25 albumin, can also be used to target the iRNA agent to the liver.
An iRNA agent can be targeted to a particular cell type in the liver by using specific targeting agents, which recognize particular receptors in the liver. Exemplary targeting moieties and their associated receptors are presented in Table 1.

Attorney's Docket No.: 14174-072W01 Table 1 Tar~etin~ agents (Li~ands) and their associated receptors Liver Cells Li~and Receutor 1) Parenchyma) Cell (PC) Galactose ASGP-R
(Hepatocytes) (Asiologlycoprotein receptor) Gal NAc ASPG-R
(n-acetyl-galactosamine) Gal NAc Receptor Lactose Asialofetuin ASPG-r 2) Sinusoidal Endothelial Hyaluronan Hyaluronan receptor Cell (SEC) Procollagen Procollagen receptor Negatively charged Scavenger receptors molecules Mannose Mannose receptors N-acetyl Glucosamine Scavenger receptors Immunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptor mediated transcytosis Transferrin Receptor mediated transcytosis Albumins Non-specific Sugar-Albumin conjugates Mannose-6-phosphate Mannose-6-phosphate receptor 3) Kupffer Cell (KC) Mannose Mannose receptors Fucose Fucose receptors Albumins Non-specific Mannose-albumin conjugates s iRNA AGENT STRUCTURE
Described herein are isolated iRNA agents, e.g., RNA molecules, (double-stranded;
single-stranded) that mediate RNAi. The iRNA agents preferably mediate RNAi with respect to an endogenous gene of a subject or to a gene of a pathogen.
An "RNA agent" as used herein, is an unmodified RNA, modified RNA, or nucleoside 1 o surrogate, all of which axe defined herein (see, e.g., the section below entitled RNA Agents).
While numerous modified RNAs and nucleoside surrogates are described, preferred examples Attorney's Docket No.: 14174-072W01 include those which have greater resistance to nuclease degradation than do unmodified RNAs.
Preferred examples include those which have a 2' sugar modification, a modification in a single strand overhang, preferably a 3' single strand overhang, or, particularly if single stranded, a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
An "iRNA agent" as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
~5 The iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term "ribonucleotide" or 20 "nucleotide", herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA
agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. Tt is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the 25 iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.
Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but Attorney's Docket No.: 14174-072W01 preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and/or 3' terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule.
As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside surrogates in addition to the RRMS. Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3'- or 5'-terminus of an iRNA
agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also .favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA
synthesis.
iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC
(RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not 2o trigger the interferon response (which molecules can also be cleaved by Dicer andlor enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein.
"sRNA agent or shorter iRNA agent" as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs. The sRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.

Attorney's Docket No.: 14174-072W01 Each strand of an sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA
agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3' overhangs, of 2-3 nucleotides.
In addition to homology to target RNA and the ability to down regulate a taxget gene, an iRNA agent will preferably have one or more of the following properties:
(1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agent section below;
(2) if single stranded it will have a 5' modification which includes one or more 1 o phosphate groups or one or more analogs of a phosphate group;
(3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or~ modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;

(4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have "RNA-like" properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand 2o in which all of the nucleotide sugars contain e.g., 2' fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties.
While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2' position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C3~-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix.
Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA
structures.
(Generally, it is preferred that a modified moiety at the 2' sugar position will be able to enter into Attorney's Docket No.: 14174-072W01 H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C3~-efado pucker in all, or at least 50, 75,80, 85, 90, or 95 % of its sugars; exhibit a C3>-erado pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, orl sugar which is not a C3>-endo pucker structure.
These limitations are particularly preferably in the antisense strand;
(5) regardless of the nature of the modification, and even though the RNA
agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70 % of the nucleotides in the molecule, or more than 50, 60, or 70 % of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2' position, are excluded from the definition of RNA agent.
A "single strand iRNA agent" as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may ~ 5 be, or include, a hairpin or pan-handle structure. Single strand iRNA
agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA
agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate 20 ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5');
5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate;
25 (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygenlsulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (R--alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.
RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkylethezphosphonates Attorney's Docket No.: 14174-072W01 (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-). (These modifications can also be used with the antisense strand of a double stranded iRNA.) A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA
agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or SOnucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.
Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region axe 15-30, 17 to 23, 19 to 23, and 19 1 o to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3', and preferably of the antisense side of the hairpin.
Preferred overhangs are 2-3 nucleotides in length.
A "double stranded (ds) iRNA agent" as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.
The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18,19, 25, 29, 40, or 60 nucleotides in length: It should be equal to or less than 200, 100, or 50, nucleotides in length: Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length.
2o The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length.
The double strand portion of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges axe 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

Attorney's Docket No.: 14174-072W01 In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent formation of an active sRNA/protein or RISC. This can be accomplished by a modification which prevents 5'-phosphorylation of the sense strand, e.g., by modification with a 5'-O-methyl ribonucleotide (see Nykanen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5'-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5'-phosphate turning it into a phosphodiester linkage, though this may be less desirable as ~5 phosphodiesterases can cleave such a linkage and release a functional sRNA
5'-end. Antisense strand modifications include 5' phosphorylation as well as any of the other 5' modifications discussed herein, particularly the 5' modifications discussed above in the section on single stranded iRNA molecules.
It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent 2o includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA
agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3' overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be 2s the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5' ends are preferably phosphorylated.
Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the sRNA agent range discussed above. sRNA
agents can resemble in length and structure the natural Dicer processed products from long Attorney's Docket No.: 14174-072W01 dsRNAs. Embodiments in which the two strands of the sRNA agent are linked, e.g.', covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3' overhang are also within the invention.
The isolated iRNA agents described herein, including ds iRNA agents and sRNA
agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.
1o As used herein, the phrase "mediates RNAi" refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an sRNA
agent of 21~ to 23 nucleotides.
As used herein, "specifically hybridizable" and "complementary" are terms which are 15 used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the 2o case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.
In one embodiment, an iRNA agent is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is "exactly complementary"
(excluding 25 the RRMS containing subunit(s))to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A "sufficiently complementary" target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA.
Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide Attorney's Docket No.: 14174-072W01 difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.
As used herein, the term "oligonucleotide" refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.
RNA agents discussed herein include otherwise unmodified RNA as well as RNA
which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates.
Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, 1o but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary:
the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic ~5 acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified "RNAs," they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the 2o correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.
Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is 25 required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.

Attorney's Docket No.: 14174-072,W01 As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subj ect positions in the nucleic acid but in many, and infact in most cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A
modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a 1 o phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5' end or ends can be phosphorylated.
In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5' or 3' overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3' or 5' overhang will be modified, e.g., with a modification described herein.
Modifications can include, e.g., the use of modifications at the 2' OH group of the ribose sugar, e.g., the use of 2o deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.

Attorney's Docket No.: 14174-072W01 Modifications and nucleotide surrogates are discussed below.
II 5.
I _ H (2' OH) X P Y
BASE
',.
'oH (2' OH) 3' The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid.
The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2' hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide.
Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases.
Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications Attorney's Docket No.: 14174-072W01 to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.
Modified nucleic acids and nucleotide surrogates can include one or more of:
(i) alteration, e.g., replacement, of one or both of the non-linking (X and ~
phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of 1o terminology, except where otherwise noted, the W position at the 5' end of a nucleic acid and the terminal Z position at the 3' end of a nucleic acid, are within the term "linking phosphate oxygens" as used herein.);
(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;
(iii) wholesale replacement of the phosphate moiety (bracket ~ with "dephospho" linkers;
(iv) modification or replacement of a naturally occurnng base;
(v) replacement or modification of the ribose-phosphate backbone (bracket II);
(vi) modification of the 3' end or 5' end of the RNA, e.g., removal, modification or 2o replacement of a terminal phosphate group or conjugation of a moiety, e.g.
a fluorescently labeled moiety, to either the 3' or 5' end of RNA.
The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.

Attorney's Docket No.: 14174-072W01 It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure).
While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures.
Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or "localization" of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X =
O and Y = N
in the above figure.
Specific modifications are discussed in more detail below.
The Phosphate Group 15 The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i. e., X and Y in Formula 1 above).
However, the phosphate group can be modified by replacing one of the oxygens with a different substituent.
One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it 2o can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens 25 replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases. Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding Attorney's Docket No.: 14174-072W01 unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR
(R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.
The phosphate linker can also be modified by replacement of a linking oxygen (i. e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3') or position Z (5'). Replacement of W with carbon or Z with nitrogen is preferred.
Candidate agents can be evaluated fox suitability as described below.
The Sugar Group A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2' hydroxyl group (OH) can be modified or replaced with a number of ~ 5 different "oxy" or "deoxy" substituents. While not being bomid by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2' alkoxide ion. The 2' alkoxide can catalyze degradation by intramolecular riucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2' position is not possible.
2o Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);
polyethyleneglycols (PEG), O(CH2CH20)"CHZCH20R; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; O-AMINE (AMINE =
NHZ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or 25 diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CHz)"AMINE, (e.g., AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH2CH20CH3, a PEG

Attorney's Docket No.: 14174-072W01 derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.
"Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, which are ofparticular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro);
amino (e.g. NHz;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CHzNH)"CH2CHz-AMINE (AMINE = NHz; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano;
mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally 1 o substituted with e.g., an amino functionality. Preferred substitutents are 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, ~'-C- allyl, and 2'-fluoro.
The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.
15 Modified RNAs can also include "abasic" sugars, which lack a nucleobase at C-1'. These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.
To maximize nuclease resistance, the 2' modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called "chimeric"
20 oligonucleotides are those that contain two or more different modifications.
The modificaton can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.

Attorney's Docket No.: 14174-072W01 Candidate modifications can be evaluated as described below.
Replacement of the Phosphate Group The phosphate group can be replaced by non-phosphorus containing connectors (cf.
Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.
Examples of moieties which can replace the phosphate group include siloxane, carbonate, 1o carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.
Candidate modifications can be evaluated as described below.
15 Replacement of Ribophosphate Backbone Oligonucleotide- mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize 2o polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.
Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.
25 Candidate modifications can be evaluated as described below.

Attorney's Docket No.: 14174-072W01 Terminal Modifications The 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end, 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAM12A, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., -(CHZ)n , -(CH2)nN-, -(CHa)"O-, -(CHa)nS-, O(CHZCH20)nCH2CHZOH (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional ~5 molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3' end can be an -OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties.
Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve 2o nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl 25 group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]~,, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption 3o facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, Attorney's Docket No.: 14174-072W01 bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraa.zamacrocycles).
Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogs. E.g., in preferred embodiments iRNA agents, especially antisense strands, are 5' phosphorylated or include a phosphoryl analog at tlae 5' prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate;
(HO)2(S)P-O-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-15 phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.
RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), 2o ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-).
Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 4~5. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-25 linking an RNA agent to another moiety; modifications useful for this include mitomycin C.
Candidate modifications can be evaluated as described below.

Attorney's Docket No.: 14174-072W01 The Bases Adenine, guanine, cytosine and uxacil are the most common bases found in RNA.
These bases can be modified or replaced to provide RNA's having improved properties.
E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural s nucleobases (e.g., inosine, thymine, xanthine, hypoxantlune, nubularine, isoguanisine, or tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases, e.g., "unusual bases" and "universal bases," can be employed.
Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-~ 5 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-'?-thiouracil, 5-methoxycaxbonylmethyl-2-20 thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
Further purines and pyrimidines 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, 2s Kroschwitz, J. L, ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.
Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent.
Modified bases can reduce target specificity. This should be taken into consideration in the 3o design of iRNA agents.

Attorney's Docket No.: 14174-072W01 Candidate modifications can be evaluated as described below.
Evaluation of Candidate RNA's One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu;
which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or CyS. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat 15 inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.
20 A functional assay can also be used to evaluate the candidate agent. A
functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence gene expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA
2s agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914).
For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on Attorney's Docket No.: 14174-072W01 gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.
In an alternative functional assay, a candidate dsRNA agent homologous to an endogenous mouse gene, preferably a maternally expressed gene, such as c-rnos, can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNA agent would cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et al. Nature 370:
65-68, 1994;
Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modified agent on target RNA
levels can be verified by Northern blot to assay for a decrease in the level of taxget mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control. Controls can include cells in which with no agent is added and/or cells in which a non-modified RNA is added.
~5 References General References The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example "Oligonucleotide synthesis, a practical approach", Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and Analogues, A
Practical 2o Approach", Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2'-O--Methyloligoribonucleotide- s: synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside methylphosphonates, and.
Chapter 7, 25 Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv.
Chirn. Acta,1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetralaedf-on,1992, 48, 2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.

Attorney's Docket No.: 14174-072W01 Modification described in WO 00/44895, WO01/75164, or W002/44321 can be used herein.
The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference.
Phosphate Group References The preparation of phosphinate oligoribonucleotides is described in U.S. Pat.
No.
5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat.
No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S.
Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation of phosphotriester oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,17,7,198. The preparation of 3'-Deoxy-3'-amino phosphoramidate oligoribonucleotides is described in U.S. Pat.
No. 5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.
Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References Modifications to the 2' modifications can be found in Verma, S. et al. Annu.
Rev.
Biochem.1998,.67, 99-134 and all references therein. Specific modifications to the ribose can be 2o found in the following references: 2'-fluoro (Kawasaki et. al., J. Med.
Clzezn.,1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chizn. Acta 1996, 79, 1930-1938), "LNA"
(Wengel, J. Acc.
Clzezra. Res. 1999, 32, 301-310).
Replacement of the Phosphate Group References Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and Attorney's Docket No.: 14174-472W01 methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos.
5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/LTS92/04294 and PCT/LTS92104305 (published as WO 92/20822 WO and 92/20823, respectively).
Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos.
5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier,J.F. et al.
Nucleic Acids Res. 1988,16, 4583. Carbonate replacements are described in Tittensor, J.R. J.
Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described in Edge, M.D. et al. J.
Chena. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E.P.
Nucleic Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References Cyclobutyl sugar surrogate compounds can be prepaxed as is described in U.S.
Pat. No.
5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S.
Pat. No.
5,519,134. Morpholino sugar surrogates can be prepared as is described in U.S.
Pat. Nos.
5,142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic &
Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No.
5,539,083.
Terminal Modification References Terminal modifications are described in Manoharan, M. et al. Antisense arad Nucleic Acid Dnug Developrraent 12, 103-128 (2002) and references therein.
Bases References N-2 substitued purine nucleoside amidites can be prepared as is described in U.S. Pat.
No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat.
No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in Attorney's Docket No.: 14174-072.W01 U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications.

Attorney's Docket No.: 14174-072W01 Preferred iRNA Agents Preferred RNA agents have the following structure (see Formula 2 below):
A~
R~

R~

Referring to Formula 2 above, Rt, RZ, and R3 are each, independently, H, (i.e.
abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thyrnine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, S-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-~x i A2 Rd Attorney's Docket No.: 14174-072W01 trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-allcylguanine, 5-alkyl cytosine,7-deazaadenine, 7-s deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, 1 o N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
R4, R5, and R6 are each, independently, ORB, O(CH2CH20)",CH2CH2OR8; O(CH2)nR9;
O(CHz)"OR9, H; halo; NH2; NHRB; N(RB)2; NH(CH2CHZNH)n,CH2CHaNHR9; NHC(O)RB; ;
cyano; mercapto, SRB; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, 15 alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or 2o ureido; or R4, R5, or R6 together combine with R7 to form an [-O-CH2-]
covalently bound bridge between the sugar 2' and 4' carbons.

Attorney's Docket No.: 14174-072W01 A1 is:

X1 ~ Y1 W1 ~ 1 X ~ Y X1 P
1 ~ 1 or Y1 W1 Z1 ~ 1 ~ or X Y X1 P Y1 X1 ( ; H; OH; OCH3; W1; an abasic nucleotide; or absent;
(a preferred A1 , especially with regard to anti-sense strands, is chosen from 5'-monophosphate ((HO)2(O)P-O-5'), 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), 5'-triphosphate ((HO)a(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'), 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), ~s 5'-phosphoramidates ((HO)z(O)P-NH-5', (HO)(NHZ)(O)P-O-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, (OH)z(O)P-5'-CH2-), 5'-Attorney's Docket No.: 14174-072W01 alkyletherphosphonates (R=alkylether=rnethoxymethyl (MeOCH2-), ethoxym.ethyl, etc., e.g.
RP(OH)(O)-O-5'-)).
AZ is:
~ 2 A3 is:
~ 3 X3 ~
Ys and Attorney's Docket No.: 14174-072W01 A4 is:
z1 Xa ~ Y4 ~1 X ~ Y X4 P Y4 or Or X ~ Y ~ X ~ Y
a X4 ~ Y4 a a ~4 Zq Z4 H; Z4; an inverted nucleotide; an abasic nucleotide; or absent.
Wl is OH, (CH2)"Rl°, (CHZ)"NHRI°, (CHZ)" ORl°, (CH2)" SRl°; O(CHZ)nRl°;
O(CHZ)nORI°, O(CH2)nNRI°, O(CH2)nSRI°;
O(CH2)"SS(CH2)"ORl°, O(CHz)"C(O)ORl°, NH(CH2)"Rl°; NH(CH2)"NRl° ;NH(CHZ)"ORl°, NH(CH2)"SRl°; S(CH2)"Rl°, S(CH2)"NRl°, S(CHZ)"ORl°, S(CH2)"SRl° O(CH2CHz0)",CH2CH20R1°;
O(CHZCH20)",CH2CH2NHR1° , NH(CH2CH2NH)mCHzCHZNHRI°; Q-Rl°, O-Q-Rl° N-Q-Rl°, S-Q-Rl° or -O_. W4 is O, CHa, NH, or S.
Xl, X2, X3, and X4 are each, independently, O or S.
Yl, Y2, Y3, and Y4 are each, independently, OH, O-, ORB, S, Se, BH3-, H, NHR9, N(R9)2 alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.
Zl, ZZ, and Z3 are each independently O, CHa, NH, or S. Z4 is OH, (CHZ)~Rlo, (CH2)"NHRI°, (CH2)" ORl°, (CHa)" SRl°; O(CHZ)~Rl°;
O(CH2)"ORl°, O(CHz)"NRl°, Attorney's Docket No.: 14174-072W01 O(CHz)nSRI°, O(CHz)"SS(CHz)"ORl°, O(CHz)"C(O)ORl°;
NH(CHz)"Rl°; NH(CHz)"NRIo ;NH(CHz)nORI°, NH(CHz)"SRl°; S(CHz)nRt°, S(CHz)nNRI°, S(CHz)"ORl°, S(CHz)"SRIo O(CHzCHzO)",CHZCHZOR1°, O(CHZCH20)",CHZCHzNHRIO, NH(CH2CHzNH)mCH2CHzNHRIO; Q-R'o, O-Q-Rlo N-Q-Rio, S-Q-Rlo.
x is 5-100, chosen to comply with a length for an RNA agent described herein.
R' is H; or is together combined with R4, R5, or R6 to form an [-O-CHz-]
covalently bound bridge between the sugar 2' and 4' carbons.
R$ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R9 is NHz, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, 1o diheteroaryl amino, or amino acid; and RI° is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyries (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric ~ 5 acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate; amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]z, polyamino; alkyl, cycloalkyl, aryl, aralkyl, 2o heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, 25 disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.

Attorney's Docket No.: 14174-072W01 Preferred RNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):
'20 Rya 5 Referring to Formula 3, Al°-A4° is L-G-L; Al° andJor A4o may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH2(CHZ)g; N(CH2)g; O(CH2)g; S(CH2)g. G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, 1 o methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

, v i Aso Rso Attorney's Docket No.: 14174-072W01 Rlo, Rao, and R3° are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-annino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including ~-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-~5 methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, S-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
R4o, Rso, and R6° are each, independently, ORg, O(CH2CH20)mCH2CH20Rg;
O(CH2)"R9;
O(CHa)"OR9, H; halo; NH2; NHRg; N(R$)2; NH(CH2CH2NH)mCH2CHZR9; NHC(O)Rg;;
cyano;
2o mercapto, SR'; alkyl-thin-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, 25 arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R4o, Rs°, or R6° together combine with R'° to form an [-O-CH2-] covalently bound bridge between the sugar 2' and 4' carbons.
x is 5-100 or chosen to comply with a length for an RNA agent described herein.

Attorney's Docket No.: 14174-072W01 R'° is H; or is together combined with R4°, RS°, or R6° to form an [-O-CHZ-] covalently bound bridge between the sugar 2' and 4' carbons.
R8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R9 is NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid. m is 0-1,000,000, n is 0-20, and g is 0-2.
Preferred nucleoside surrogates have the following structure (see Formula 4 below):
SLRIOO-(M-SLR2oo)X M-SLR3oo 1 o S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CHz(CH2)g; N(CH2)g; O(CH2)g; S(CH2)g; -C(O)(CH2)n or may be absent. M is an amide bond;
sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.
15 Rloo~ R2oo~ ~d Rsoo ~.e each, independently, H (i. e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-2o halouracil, 5-(2-aminopropyl)uracil, S-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-~, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-25 alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1, 2, 4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-Attorney's Docket No.: 14174-072W01 methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.
Nuclease resistant monomers An RNA, e.g., an iRNA agent, can incorporate a nuclease resistant monomer (NRM), such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/469,612, filed on May 9, 2003, and International Application No. PCT/LTS04/07070, both of which are hereby incorporated by reference.
In addition, the invention includes iRNA agents having an NRM and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA
agent which 15 targets a gene described herein, e.g., a gene active in the liver, an iRNA
agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an NRM.
20 An iRNA agent can include monomers which have been modifed so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRMs, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., 2s serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

Attorney's Docket No.: 14174-072W01 While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase s exonuclease and endonuclease resistance and thus prolong the half life of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3' andlor 5' end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off targeting.
Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex 15 comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein.
The anti sense strand may include modifications at the 3' end andlor the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3' end and/or the 5' end and/or at any one of~the 2o intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3' end andlor the 5' end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular 25 configurations are discussed below.
Modifications that can be usefixl for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical andlor stereochemical modifications of the sugar, base, and/or phosphate backbone:

Attorney's Docket No.: 14174-072W01 (i) chiral (SP) thioates. Thus, preferred NRMs include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr2, or Br3. When X
is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRMs are discussed in more detail below;
(ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRMs include monomers at the terminal position derivatized at a cationic group. As the 5' end of an antisense 1 o sequence should have a terminal -OH or phosphate group this NRM is preferably not used at the 5' end of an anti-sense sequence. The group should be attached at a position on the base which minimizes interference with H bond formation and hybridization, e.g., away form the face which interacts with the complementary base on the other strand, e.g, at the 5' position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;
~ 5 (iii) nonphosphate linkages at the termini. Thus, preferred NRMs include Non-phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3' CH2-NCH3-O-CHZ-5' and 3' CH2-NH-(O=)-CH2-5'.;
(iv) 3'-bridging thiophosphates and 5'-bridging thiophosphates. Thus, preferred NRM's can included these structures;
20 (v) L-RNA, 2'-5' linkages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2'-5' phosphate, non-phosphate and modified phosphate linkages (e.g., thiophosphates, ' phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3'-3' or 5'-5' linkages; monomers having an alpha linkage at the 1' site on the sugar, e.g.;
the structures 25 described herein having an alpha linkage;
(vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar , base, or backbone;

Attorney's Docket No.: 14174-072W01 (vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and (vii) 5'-phosphonates and 5'-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5' position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which 1 o attacks a carbon adj acent to the O of a phosphate and resulting in the production of an unprotected phosphate.
One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number ~5 incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintained.
In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off target silencing.
2o Chiral SP Thioates A modification can include the alteration, e.g., replacement, of one or both of the non-linking (X and 'Y) phosphate oxygens and/or of one or more of the linking (W
and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moieties, SBl and SBZ.

Attorney's Docket No.: 14174-072W01 W
X P Y
Z

FORMULA X
In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR2 (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom 1o in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the "R" configuration (herein RP) or the "S"
configuration (herein SP).
Thus if 60% of a population of stereogenic phosphorus atoms have the RP
configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the SP
configuration.
In some embodiments, iRNA agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the SP configuration. Alternatively, iRNA agents having 2o phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these Attorney's Docket No.: 14174-072W01 atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95%
of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the RP
configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the SP configuration and may be substantially free of stereogenic phosphorus atoms having the RP configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the Rp configuration and may be substantially free of stereogenic phosphorus atoms having the SP configuration. As used herein, the phrase "substantially free of stereogenic phosphorus atoms having the RP configuration" means that moieties containing stereogenic phosphorus atoms having the RP configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase "substantially free of stereogenic phosphorus atoms having the SP
configuration" means that moieties containing stereogenic phosphorus atoms having the Sp configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1H
NMR analysis using chiral shift reagents, etc.).
~5 In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom.
In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the SP configuration and be substantially free of stereogenic phosphorus atoms having the RP configuration., 20 Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X-1 and X-2. The former can be used to introduce phosphorothioate Attorney's Docket No.: 14174-072W01 DMTO DMTO

S P Y S P Y
Z

NC
solid phase reagent p \\~~0~ ~ N i r ( p )2 X_ 1 X_2 at the 3' end of a strand, while the latter can be used to introduce this modification at the 5' end or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, RZ~ can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH3), DMT is dimethoxytrityl, and "BASE" can be a natural, unusual, or a universal base.
X-1 and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms 1o having essentially only the RP configuration (i.e., being substantially free of the SP configuration) or only the SP configuration (i.e., being substantially free of the RP
configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about Attorney's Docket No.: 14174-072W01 50% of the atoms have the RP configuration and about 50% of the atoms have the SP
configuration. Dimers having stereogenic phosphorus atoms with the RP
configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the SP
configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.
Cationic Groups Modifications can also include attachment of one or more cationic groups to the sugar, bases and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A
cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing.
A cationic group can be attached e.g., through the C2' position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE = NHa; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);
aminoalkoxy, 15 e.g., O(CHZ)"AMINE, (e.g., AMINE = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH2CH2NH)nCH2CH2-AMINE (AMINE = NH2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or 2o diheteroaryl amino).
Nonplaosplaate Linkages Modifications can also include the incorporation of nonphosphate linkages at the 5' and/or 3' end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, 2s carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups.

Attorney's Docket No.: 14174-072W01 3'-bridging tlaiophosphates and 5'-bridging thiophosphates; locked-RNA, 2'-5' likages, inverted linkages, a nucleosides; cor jugate groups; abasic linkages; and 5' p7aosphonates afad 5' phosphate prodrugs Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom.
Modifications can also include linking two sugars via a phosphate or modified phosphate 1 o group through the 2' position of a first sugar and the 5' position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are eached linked through the respective3' positions. Modified RNA's can also include "abasic"
sugars, which lack a nucleobase at C-1'. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a ~ 5 modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the a-configuration.
Modifcations can also include L-RNA.
Modifications can also include 5'-phosphonates, e.g., P(O)(O-)2-X-C5~-sugar (X= CH2, CF2, CHF and 5'-phosphate prodrugs, e.g., P(O)[OCH2CH2SC(O)R]2CHZC5'-sugar. In the 20 latter case, the prodrug groups may be decomposed via reaction first with caxboxy esterases. The remaining ethyl thiolate group via intramolecular SN2 displacement can depart as episulfide to afford the underivatized phosphate group.
Modification can also include the addition of conjugating groups described elseqhere herein, which are prefereably attached to an iRNA agent through any amino group available for 25 conjugation.
Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferrable to not use Attorney's Docket No.: 14174-072W01 them at the cleavage site or in the cleavage region of an sequence which targets a subj ect sequence or gene.. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirabable to put the NRM at the cleavage site or in the cleavage region of a s sequence which does not target a subject sequence or gene,as it can minimize off target silencing.
In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.
In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which 15 interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of the first nucleotide which is corriplementary to the guide sequence. As used herein 2o cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucletides of the cleave site, in either direction.) Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence 25 which does not target a sequence in the subject.
An iRNA agent can have a first and a second strand chosen from the following:
a first strand which does not target a sequence and which has an NRM
modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;

Attorney's Docket No.: 14174-072W01 a first strand which does not target a sequence and which has an NRM
modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a first strand which does not target a sequence and which has an NRM
modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM
modification at or s within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a first strand which does not target a sequence and which has an NRM
modification at the cleavage site or in the cleavage region;
a first strand which does not target a sequence and which has an NRM
modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2~ 3, 4, 5 , or 6 positions from both the 3' and the 5' end; and a second strand which targets a sequence and which has an NRM modification at or within l, 2, 3, 4, 5 , or 6 positions from the 3' end;
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM
modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
a second strand which targets a sequence and which has an NRM modification at or 2o within 1, 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM
modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a second strand which targets a sequence and which preferably does not have an an NRM
modification at the cleavage site or in the cleavage region;
a second strand which targets a sequence and which does not have an NRM
modification at the cleavage site or in the cleavage region and one or more of an NRM
modification at or within l, 2, 3, 4, 5 , or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions Attorney's Docket No.: 14174-072W01 from both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position l, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand).
An iRNA agent can also target two sequences and can have a first and second strand chosen from:
s a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end;
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM modifications are preferentially not at the terminus but rather at a position 1, ~, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM
modification at or within l, 2, 3, 4, 5 , or 6 positions from the 5' end;
a first strand which targets a sequence and which preferably does not have an an NRM
modification at the cleavage site or in the cleavage region;
a first strand which targets a sequence and which dose not have an NRM
modification at the cleavage site or in the cleavage region and one or more of an NRM
modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions from 2o both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand) and a second strand which targets a sequence and which has an NRM modification at or within l, 2, 3, 4, 5 , or 6 positions from the 3' end;
a second strand which targets a sequence and which has an NRM modification at or 25 within 1, 2, 3, 4, 5 , or 6 positions from the 5' end (5' end NRM
modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand);

Attorney's Docket No.: 14174-072W01 a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end and which has a NRM
modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end;
a second strand which targets a sequence and which preferably does not have an an NRM
modification at the cleavage site or in the cleavage region;
a second strand which targets a sequence and which dose not have an NRM
modification at the cleavage site or in the cleavage region and one or more of an NRM
modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 3' end, a NRM modification at or within 1, 2, 3, 4, 5 , or 6 positions from the 5' end, or NRM modifications at or within 1, 2, 3, 4, 5 , or 6 positions 1 o from both the 3' and the 5' end(5' end NRM modifications are preferentially not at the terminus but rather at a position l, 2, 3, 4, 5 , or 6 away from the 5' terminus of an antisense strand).
Ribose Mimics An RNA, e.g., an iRNA agent, can incorporate a ribose mimic, such as those described herein and those described in copending co-owned United States Provisional Application Serial ~5 No. 60/454,962, filed on March 13, 2003, and International Application No.
PCT/LTS04/07070, both of which are hereby incorporated by reference.
In addition, the invention includes iRNA agents having a ribose mimic and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA
agent which 2o targets a gene described herein, e.g., a gene active in the liver, an iRNA
agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.
25 . Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent.
Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers Attorney's Docket No.: 14174-072W01 nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3' ribose hydroxyl group, thereby causing it to be less susceptible to degradation.
The secondary hydroxyl group refers to an "OH" radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base.. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal 15 phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.
Preferred iRNA agent delivery modules with five intervening bonds between the terminal 'phosphate group phosphorus and the secondary hydroxyl group have the following structure (see 2o formula Y below):
A~W
Y P X
Z
~CH2 Rs NHT
1'/ ~
R CH~ ~C/
R2 ~ ~ 'R5 Attorney's Docket No.: 14174-072W01 (Y) Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein.
The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to "W"
of the phosphate group. These spacers or linkers can include e.g., -(CH2)n , -(CH~)"N-, -(CHZ)"O-, -(CH2)"S-, O(CHZCH20)nCHZCH20H (e.g., n = 3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.
The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, ~ o and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BHP-, C1-C6 alkyl, C6-Clo aryl, H, O, O-, alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O
and Y is S.
Rl and R3 are each, independently, hydrogen; or C1-Cloo alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.
RZ is hydrogen; Cl-Cloo alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is l, Ra may be taken together with with R4 or R6 to form a ring of 5-12 atoms.
R4 is hydrogen; Cl-Cloo allcyl, optionally substituted with hydroxyl, amino, halo, 2o phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is l, R4 may be taken together with with R2 or RS to form a ring of S-12 atoms.
RS is hydrogen, C1-Cloo alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is l, RS may be taken together with with R4 to form a ring of 5-12 atoms.
25 R6 is hydrogen, C1-Cloo alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R~ may be taken together with with R2 to form a ring of 6-10 atoms;

Attorney's Docket No.: 14174-072W01 R~ is hydrogen, C1-Cloo alkyl, or C(O)(CH2)qC(O)NHR9; T is hydrogen or a functional group; n and q are each independently 1-100; Rg is C1-Clo alkyl or C6-Clo aryl; and R9 is hydrogen, C1-C10 alkyl, C6-C10 aryl or a solid support agent.
Preferred embodiments may include one of more of the following subsets of iRNA
agent delivery modules.
In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3' or 5' ribose sugar carbon of the RNA agent.
In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.
In still yet another subset of RNAi agent delivery modules, n is 1, and R2 and R6 are 1 o taken together to form a ring containing six atoms and R4 and RS are taken together to form a ring containing six atoms. Preferably, the ring system is a tYans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-1):
A
NHT
O\ ~O
S~P~O
The functional group can be, for example, a targeting group (e.g., a steroid or a 15 carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety).
The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, 2o monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.

Attorney's Docket No.: 14174-072W01 As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art.The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization.
Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.GM.
1o Wuts, Protective Groups in Orgaraic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994);
and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis,, John Wiley and Sons (1995), and subsequent editions thereof.
Ribose Reulacement Monomer Subunits iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent. An RNA agent, e.g., an iRNA agent can include a ribose replacement monomer subunit (RRMS), such as those described herein and those described in one or more of United States Provisional Application Serial No. 601493,986, filed on August 8, 2003, which is hereby incorporated by reference; United States Provisional 2o Application Serial No. 60/494,597, filed on August 11, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/506,341, filed on September 26, 2003, which is hereby incorporated by reference; United States Provisional Application Serial No. 60/158,453, filed on November 7, 2003, which is hereby incorporated by reference; and International Application No. PCT/LTS04/07070, filed March 8, 2004, which is hereby 2s incorporated by reference.
In addition, the invention includes iRNA agents having a RRMS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA
agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA
agent having an Attorney's Docket No.: 14174-072W01 architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS.
The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) Garner. A
ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic Garner may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i:e., one or more ring atoms may be~a heteroatom, e.g., nitrogen, oxygen, sulfur.
The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic Garner may be a fully saturated ring system, or it may contain one or more double bonds.
The carriers further include (i) at least two "backbone attachment points" and (ii) at least one "tethering attachment point." A "backbone attachment point" as used herein refers to a ~5 functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A "tethering attachment point" as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety.
2o The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
2s Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA
agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA
molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is Attorney's Docket No.: 14174-072W01 tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).
Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R-1) is incorporated into at least one of said strands.
R~ R6 X

R
(R-1) 1o Referring to formula (R-1), X is N(CO)R~, NR7 or CH2; Y is NRB, O, S, CR9R1°, or absent; and Z is CRllRia or absent.
Each of Rl, R2, R3, R4, R9, and Rl° is, independently, H, OR~, ORb, (CH2)"ORa, or (CH2)"ORb, provided that at least one of Rl, R2, R3, R4, R9, and Rl° is ORa or ORb and that at least one of Rl~ Ra, R3, R4, R9, and RI° is (CH2)"ORa, or (CHZ)"ORb (when the RRMS is terminal, one of RI, R2, R3, R4, R9, and Rl° will include Ra and one will include Rb; when the RRMS is internal, two of Rl, RZ, R3, R4, R9, and Rl° will each include an Rb);
further provided that preferably ORa may only be present with (CHa)nORb and (CH2)nORa may only be present with ORb.
Each of R5, R6, Rll, and RIZ is, independently, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR~; or RS and Rl1 together are C3-C8 cycloalkyl optionally substituted with Rt4.

Attorney's Docket No.: 14174-072W01 R7 is C1-Cao alkyl substituted with NR°Rd; R8 is Cl-C6 alkyl; R13 is hydroxy, C1-C4 alkoxy, or halo; and R14 is NR°R~.
Ra 1S:
A
~P B
C
and Rb is:
A
P O Strand C
Each of A and C is, independently, O or S.
BisOH,O-,or Attorney's Docket No.: 14174-072W01 O O
O P O P OH
O- O-R° is H or C1-C6 alkyl; Ra is H or a ligand; and n is 1-4.
In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is s N(CO)R~ or NR', Y is CR9RI°, and Z is absent.
In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R~ or NR~, Y is CR9Rt°, and Z is CRllRlz In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R~ or NR~, Y is NR8, and Z is CR11R12 In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X
is N(CO)R~ or NR~, Y is O, and Z is CRIIRIZ .
In other preferred embodiments the ribose is replaced with a decalin scaffold, and X
isCHz; Y is CR9R1°; and Z is CRllRlz; and RS and Rl I together are C6 cycloalkyl.
In other preferred embodiments the ribose is replaced with a decalin/indane scafold and , 15 and X is CHz; Y is CR9RI°; and Z is CRllRlz; and RS and RI1 together are CS cycloalkyl.
In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold.
RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the 2o sense strand may include one or more of the RRMSs described herein. An RRMS
can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An Attorney's Docket No.: 14174-072W01 RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.
In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3' end of the sense strand. In an embodiment, an iRNA agent may have an 1 o RRMS at (or within 1, 2, or 3 positions of, the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both ligands are located at the same end of the iRNA agent.
In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.
~ 5 In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end of the antisense strand and an RRMS at the 5' end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a "hairpin ligand."
2o In other embodiments, an iRNA agent may have an RRMS at the 3' end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.
25 In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), Attorney's Docket No.: 14174-072W01 e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.
It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA
agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3' overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result. of one strand being longer than the other, or the result of two strands of the same length being staggered: 5' ends are preferably phosphorylated.
An RNA agent, e.g., an iRNA agent, containing a preferred, but nonlimiting RRMS is presented as formula (R-2) in FIG. 4. The carrier includes two "backbone attachment points"
(hydroxyl groups), a "tethering attachment point," and a ligand, which is connected indirectly to the carrier via an intervening tether. The RRMS may be the 5' or 3' terminal subunit of the RNA
molecule, i.e., one of the two "W" groups may be a hydroxyl group, and the other "W" group may be a chain of two or more unmodified or modified ribonucleotides.
Alternatively, the RRMS may occupy an internal position, and both "W" groups may be one or more unmodified or modified ribonucleotides. More than one RRMS may be present in a RNA
molecule, e.g., an iRNA agent.
2o The modified RNA molecule of formula (R-2) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding RRMS monomer compounds (RRMS monomers, see, e.g., A, B, and C in FIG 4) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies.
The R.RMS monomers generally include two differently functionalized hydroxyl groups (OFGI and OFG2 above), which are linked to the carrier molecule (see A in FIG
4), and a tethering attachment point. As used herein, the term "functionalized hydroxyl group" means that the hydroxyl proton has been replaced by another substituent. As shown in representative structures B and C, one hydroxyl group (OFGI) on the carrier is functionalized with a protecting Attorney's Docket No.: 14174-072W01 group (PG). The other hydroxyl group (OFG2) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in S, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a tether, or a tethered ligand at the time that the monomer is incorporated into the growing sense or antisense strand (see R in Scheme 1).
Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a "precursor" RRMS after a "precursor"
RRMS monomer has been incorporated into the strand.
The (OFGI) protecting group may be selected as desired, e.g., from T.W. Greene and P.G.M. Wuts, Protective Groups ija Organic Synthesis, 2d. Ed., John Wiley and Sons (1991).
The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Hydroxyl groups, -OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl ~ 5 groups in which the hydrogen has been replaced with a protecting group, e.g., a triarylmethyl group or a trialkylsilyl group, are essentially unreactive as nucleophiles in displacement reactions. Thus, the protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. A
preferred protecting group is the dimethoxytrityl group.
20 When the OFG2 in B includes a linker, e.g., a long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed to build up a chain of natural and/or modified ribonucleotides once OFGI is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or 25 oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG2. Subsequent to this operation, OFGI can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group (see FIG. 1). R' can be substituted or unsubstituted alkyl or alkenyl. In preferred embodiments, R' is methyl, allyl or 2-cyanoethyl. R" may a C1-C1o alkyl group, so preferably it is a branched group containing three or more carbons, e.g., isopropyl.

Attorney's Docket No.: 14174-072W01 OFGZ in B can be hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be any support medium that can support the monomers described herein. The monomer can be attached to an insoluble support via a linker, L, which allows the monomer (and the growing s chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent;
unreacted reagents and soluble by-products can be readily washed away from the solid support to which the monomer or monomer-derived products is attached. Alternatively, the monomer can be attached to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be -C(O)(CH2)qC(O)-, or -C(O)(CH2)qS-, preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can not be used in conjunction with fluoride labile 5' silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride-~ 5 stable polystyrene based supports or PEG are preferred.
Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from Rl or RZ; R3 or R4; or R9 and Rl° if Y
is CR9R1° (two positions are chosen to give two backbone attachment points, e.g., R1 and R4, or R4 and R9. Preferred tethering attachment points include R~; RS or R6 when X
is CHZ. The 2o carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., Rl or R2; R3 or R4; or R9 or Rl° (when Y is CR9Rl°), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R
groups can be -25 CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.) Attorney's Docket No.: 14174-072W01 R~ R6 X

R
(R-3) X is N(CO)R~, NR~ or CHa; Y is NRg, O, S, CR9R1°; and Z is CRllRia or absent.
Each of Rl, Ra, R3, R4, R9, and R1° is, independently, H, ORa, or (CHa)nORb, provided that at least two of Rl, Ra, R3, R4, R9, and Rl° are ORa andlor (CH2)nORb.
Each of R5, R6, Rl l, and Rla is, independently, a ligand, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR~; or RS and Rll together are C3-C8 cycloalkyl optionally substituted with Ri4.
R' is H, a ligand, or C1-Ca° allcyl substituted with NR°Rd; R8 is H or C1-C6 alkyl; R13 is hydroxy, Cl-C4 alkoxy, or halo; R14 is NR~R~; Rls is C1-C6 alkyl optionally substituted with cyano, or Ca-C6 alkenyl; R16 is CI-Cl° alkyl; and Rl~ is a liquid or solid phase support reagent.
L is -C(O)(CHa)qC(O)-, or -C(O)(CHa)qS-; Ra is CAr3; Rb is P(O)(O-)H, P(ORIS)N(R16)a or L-Rl~; R° is H or Cl-C6 alkyl; and Ra is H or a ligand.
Each Ar is, independently, C6-C1° aryl optionally substituted with Cl-C4 alkoxy; n is 1-4;
and q is 0-4.
Exemplary Garners include those in which, e.g., X is N(CO)R~ or NR~, Y is CR9R1°, and Z is absent; or X is N(CO)R~ or NR~, Y is CR~RI°, and Z is CR11R12; or X is N(CO)R7 or NR~, Y
is NRB, and Z is CRIIRIa; or X is N(CO)R7 or NR~, Y is O, and Z is CRllRia; or X is CHa; Y is 2o CR9RI°; Z is CRl lRla, and R5 and Rl1 together form C~ cycloalkyl (H, z = 2), or the indane ring Attorney's Docket No.: 14174-072W01 system, e.g., X is CHz; Y is CR9R1°; Z is CRl lRlz, and RS and Rll together form CS cycloallcyl (H, z =1).
In certain embodiments, the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R~ or NR~, Y is CR9R1°, and Z is absent (D). OFGI
is preferably attached to a primary carbon, e.g., an exocyclic alkylene CH~OFG~

N
LIGAND
D
group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (-CHzOFGI in D). OFGz is preferably attached directly to one of the carbons in the five-membered ring (-OFGz in D). For the pyrroline-based carriers, -CHzOFGI may be attached to C-2 and OFGz may be attached to C-3; or -CHZOFGI may be attached to C-3 and OFGz may be attached to C-4. . In certain embodiments, CH20FG1 and OFGz may be geminally substituted to ,.
one of the above-referenced carbons.For the 3-hydroxyproline-based carriers, -CH20FG1 may be attached to C-2 and OFGz may be attached to C-4. The pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is ~s restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
Thus, CH20FG1 and OFGz may be cis or tf ans with respect to one another in any of the pairings delineated above Accordingly, all cisltrans isomers are expressly included.
The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric Attorney's Docket No.: 14174-072W01 forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.
In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R~ or NR~, Y is CR9R1°, and Z is CR11Ri2_ OFGI is preferably C4~
C~(CH2)nOFG~
~C2 N
LIGAND
E
attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [-(CHZ)nOFGI in E]. OFGZ is preferably attached directly to one of the carbons in the six-membered ring (=OFGZ
in E). -(CH2)"OFGI and OFGZ maybe disposed in a geminal manner on the ring, i.e., both 1 o groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4.
Alternatively, -(CH2)"OFGI and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CHa)"OFGl may be attached to C-2 and OFGa may be attached to C-3; -(CH2)nOFGI may be attached to C-3 and OFG2 may be attached to C-2;
-(CHz)"OFGI may be attached to C-3 and OFGa may be attached to C-4; or -(CHz)"OFGI may be attached to C-4 and OFG2 may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)"OFGI
and OFGZ may be cis or trayas with respect to one another in any of the pairings delineated above. Accordingly, all cisltr~aras isomers are expressly included. The monomers may also Attorney's Docket No.: 14174-072W01 contain one or more asyrmnetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.
In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R~ or NR~, Y is NRB, and Z is CR11R12, or the morpholine ring system (G), e.g., X is N(CO)R~ or NR~, Y is O, and Z is CRllRiz. OFGI is preferably R"' N./ O./
wC3 wC3 ~-CH20FG~ ~-CH20FG~
N /CZ N /CZ
LIGAND LIGAND
F G
attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, ' connected to one of the carbons in the six-membered ring (-CHaOFGI in F or G).
OFG2 is preferably attached directly to one of the carbons in the six-membered rings (-OFG2 in F or G).
For both F and G, -CHaOFGI may be attached to C-2 and OFGZ may be attached to C-3; or vice versa. In certain embodiments, CH20FG1 and OFG2 may be geminally substituted to one of the above-referenced carbons.The piperazine- and morpholine-based monomers may therefore 15 contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring.
Thus, CHaOFGI and OFG2 may be cis or tf-aras with respect to one another in any of the pairings delineated above.
Accordingly, all cisltraras isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single Attorney's Docket No.: 14174-072W01 enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R"' can be, e.g., Cl-C6 alkyl, preferably CH3. The tethering attachment point is preferably nitrogen in both F and G.
In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CHz; Y is CR9Rl°; Z is CRllRlz, and Rs and Rl1 together farm C6 cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CHz; Y is CR9R1°; Z is CRl lRlz, and Rs and Rl1 together form Cs cycloalkyl (H, z =1). OFGI is preferably attached to a primary carbon, C7~C~C5~C
z ~ (6 ~--(CH2)nOFG1 C1 ~C~C3 H
e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, 1o C-4, or C-5 [-(CHz)nOFGI in H]. OFGz is preferably attached directly to one of C-2, C-3; C-4, or C-5 (-OFGz in H). -(CHz)"OFGl and OFGz may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5.
Alternatively, -(CHz)"OFGI and OFGz may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CHz)"OFGI may be attached to C-2 15 and OFGz may be attached to C-3; -(CHz)"OFGI may be attached to C-3 and OFGz may be attached to C-2; -(CHz)"OFGI may be attached to C-3 and OFGz may be attached to C-4; or -(CHz)"OFGI may be attached to C-4 and OFGz may be attached to C-3; -(CHz)nOFG' may be attached to C-4 and OFGz may be attached to C-5; or -(CHz)"OFGI may be attached to C-5 and OFGz may be attached to C-4. The decalin or indane-based monomers may therefore contain 20 linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, -(CH2)~OFGI and OFGz may be cis or tans with respect to one another in any of the pairings delineated above. Accordingly, all cisltrafas isomers are expressly included. The monomers may also contain one or more Attorney's Docket No.: 14174-072W01 asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. In a preferred embodiment, the substituents at C-1 and C-6 are traps with respect to one another. The tethering attachment point is preferably C-6 or C-7.
Other Garners may include those based on 3-hydroxyproline (J). Thus, -(CH2)nOFGI and OFG2 may be cis or traps with respect to one another. Accordingly, all cisltrafas isomers are expressly included. The monomers may also contain one or more asymmetric centers OFG~
J
and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included.
The tethering attachment point is preferably nitrogen.
Representative Garners are shown in FIG. 5.
In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the Garner via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any C1-Cloo carbon-containing moiety, (e.g. Cl-C75, Cl-Cso~ C1-C2o~ Cl-Clo~ C1-C6)a preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAP=
CH~"NH~; TAP-C O CH )nN~H-; or TAP-NR" "(CH~),,1~, in which n is 1-6 and R""
is C1-2o CG alkyl. and Ra is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH~, or hydrazino group, -IVHNHz. The tether may optionally Attorney's Docket No.: 14174-072W01 be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP- CH )"NH(LIGAND), TAP-C O CH~,NH(LIGAND), or TAP-NR" "(CH~nNH(LIGAND);
TAP- CH~"ONH(LIGAND), TAP-C O CH ~"ONH(LIGAND~, or TAP-NR"" CH2)nONH(LIGAND); TAP- CH~nNHNH~ LIGAND , TAP-C O CH~"NHNH~ LI( GAND), or TAP-NR" "(CH~"NHNH~ LIGAND .
In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP=
CH~"CHO; TAP-C O CH CHO; or TAP-NR""(CH~"CHO, in which n is 1-6 and R"" is C1-C6 alkyl; or TAP-~CH~~,,C(O)ONHS; TAP-C(O~(CH?~"C(O)ONHS; or TAP-NR" "(CH~"C(O)ONHS, in which n is 1-6 and R"" is C1-C6 alkyl;
~5 TAP CH C O OC6F5; TAP-C O CH C O OC6F5; or TAP-NR""(CHI C O OC6Fs, in __ ~~ n~ _. ~~_z which n is 1-6 and R"" is Cl-C6 alkyl; or -(CH?~nCH~LG; TAP-C O CH~,CH~LG; or TAP-NR""(CH~,CH~LG, in which n is 1-6 and R"" is Cl-C6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carned out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the 2o tether.
Tethered Entities A wide variety of entities can be tethered to an iRNA agent, e.g., to the Garner of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent. Preferred entities are those which 25 target to the liver.

Attorney's Docket No.: 14174-072W01 Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the Garner via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into s the growing strand. In some embodiments, the ligand may be incorporated into a "precursor"
RRMS after a "precursor" RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CHZ)nNH2 may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the .ligand with the terminal nucleophilic group of the precursor RRMS tether.
In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an 15 enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
Preferred ligands can improve transport, hybridization, and specificity properties and may 2o also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake;
diagnostic compounds or reporter groups e.g., for monitoring distribution;
cross-linking agents;
25 and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may Attorney's Docket No.: 14174-072W01 also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-malefic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-malefic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine~
dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a liver cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD
peptide or RGD peptide mimetic.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-2s (oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]a, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, Attorney's Docket No.: 14174-072W01 imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-KB.
1 o The ligand can be a substances e.g, a drug, which can increase the uptake of the iRNA
agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculinA, phalloidin, swinholide A, indanocine, or myoservin.
The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.
In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-liver target tissue of the body Preferably, the target tissue is the liver, preferably parenchymal cells of the liver.
Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (h) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA
more strongly will be less likely to be targeted to the liver and therefore less likely to be cleared from the body Attorney's Docket No.: 14174-072W01 In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA
with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells.
Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B
vitamin, e.g., folic acid, B 12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).
In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is arnphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids.
The helical ~ 5 agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and 2o peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 2, for example).

Attorney's Docket No.: 14174-072W01 Table 2. Exemplary Cell Permeation Peptides Cell Amino acid Sequence Reference Permeation Peptide Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO:6700)Derossi et al., J. Biol.

Chem. 269:10444, Tat fragment GRKKRRQRRRPPQC (SEQ m N0:6701) Vives et al., J. Biol.

(48-6Q) Chem., 272:16010, Signal GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin et al., Sequence- (SEQ ID NO:6702) Biochem. Biophys.

based peptide Res. Commun., 243:601, 1998 PVEC LLIILRd?.R-1RKQAHAHSK (SEQ m N0:6703)Elmquist et al., Exp.

Cell Res., 269:237, .

Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al., FASEB

(SEQ ID N0:6704) J., 12:67, 1998 Attorney's Docket No.: 14174-072W01 Amphiphilic KLALKLALKALKAALKLA (SEQ ID Oehlke et al., Mol.

model peptideN0:6705) Ther., 2:339, Arg9 (SEQ ID N0:6706) Mitchell et al., J.

Pept. Res., 56:318, Bacterial KFFKFFKFFK (SEQ lD N0:6707) cell wall permeating LVPRTES (SEQ ID N0:6708) Cecropin SWLSKTAKKLENSAKKRISEGIAIAIQGGP

R (SEQ ll~ N0:6709) a defensin ACYCRIPACIA.GERRYGTCIYQGRLWAFC

C (SEQ a7 NO:6710) b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYR

GKAKCCK (SEQ ID N0:6711) Bactenecin RKCRIWIRVCR (SEQ ID N0:6712) RFPPRFPGKR-NH2 (SEQ ID N0:6713) Indolicidin ILPWKWPWWPWRR-NH2 (SEQ ID

N0:6714) A peptide or peptidomimetic can be, fox example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. The peptide moiety can be an L-peptide or D-peptide. In another alternative, the Attorney's Docket No.: 14174-072W01 peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID N0:6715). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID N0:6716)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKI~RRQRRRPPQ (SEQ ID N0:6717)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID N0:6718)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD
2o peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. Fox example, a glycosylated RGD
peptide can deliver an iRNA agent to a tumor cell expressing aw133 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).
Peptides that target markers enriched in proliferating cells can be used.
E.g., RGD
containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an a~(33 integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD
peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one 3o can use other moieties that target the a~ (33 integrin ligand. Generally, such ligands can be used Attorney's Docket No.: 14174-072W01 to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA
agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.
A "cell permeation peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A
microbial cell-permeating peptide can be, for example, an a helical linear peptide (e.g., LL-37 or Ceropin Pl), a disulfide bond-containing peptide (e.g., a -defensin, ~3-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from 1 o the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T
antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic a helical peptide. Exemplary amphipathic a helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ~5 ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys),, and a minimum number helix destabilization residues will be utilized (e.g., 2o proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue andlor C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i ~ 3, or i ~ 4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic 25 residues glutamate or aspartate.
Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, (3, or y peptides; N-methyl peptides;
azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

Attorney's Docket No.: 14174-072W01 Methods for making iRNA agents iRNA agents can include modified or non-naturally occuring bases, e.g., bases described in copending and coowned United States Provisional Application Serial No.
601463,772, filed on April 17, 2003, which is hereby incorporated by reference and/or in copending and coowned United States Provisional Application Serial No. 60/465,802, filed on April 25, 2003, which is hereby incorporated by reference. Monomers and iRNA agents which include such bases can be made by the methods found in United States Provisional Application Serial No.
60!463,772, filed on April 17, 2003, and/or in United States Provisional Application Serial No.
60/465,802, filed on April 25, 2003.
In addition, the invention includes iRNA agents having a modified or non-naturally occuring base and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA
agent.having an architecture or structure described herein, an iRNA associated with an 15 amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA
agent formulated as described herein, which also incorporates a modified or non-naturally occuring base.
The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and 2o Manoharan, "Oligonucleotide Conjugates in Antisense Technology," in Antisense Drug Technolo~y, ed. S.T. Crooke, Marcel Dekker, Inc., 2001.
In one embodiment of the invention, a peptidamimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be 25 an azapeptide (Game, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the a carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a "carbonyl donor," e.g., phenylchloroformate.

Attorney's Docket No.: 14174-072W01 In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage.
N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99, 2000; Cell Penetrating Peptides:
Processes and At~plications, Langel, ed., CRC Press, Boca Raton, FL, 2002; Fische et al., Bioconjugate. Chem.
12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide.
1 o In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a (3-peptide. ~i-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions.
Their cyclic derivatives can fold into nanotubes in the solid state. ~i-peptides are resistant to degradation by proteolytic enzymes. ,Q-peptides can be synthesized by methods known in the art. For example, an Ant or 15 Tat peptide can be a (3-peptide.
In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate.
2o In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety.
Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea 25 conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA
agent is directed for passage through a bacterial cell wall, or when an iRNA
agent must traverse Attorney's Docket No.: 14174-072W01 the blood-brain barrier, such as for the treatment of a neurological disorder.
In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).
The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, 1 o such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.
A peptide-carrier complex consists of at least a caxrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system andlor a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the earner complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.
A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A
peptide moiety can be tethered to the carrier molecule by a vaxiety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes.

Attorney's Docket No.: 14174-072W01 Targeting The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipids, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.
A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.
Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to a non-kidney tissue, such as the liver.
An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver.
An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver. For example, the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the cx fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer of the liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of Attorney's Docket No.: 14174-072W01 an HDLILDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis. In another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase;
PPAR-Gamma Coactivator (PGC-1); fructose-1,6-bisphosphatase; glucose-6-phosphatase;
glucose-6-phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes. In another embodiment, an iRNA
agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot. An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). For example, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, SA, or SB. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.
Preferred ligands on RRMSs include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.
Definitions The term "halo" refers to any radical of fluorine, chlorine, bromine or iodine.
The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched 2o chain, containing the indicated number of carbon atoms. For example, C1-Cla alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term "haloalkyl" refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group.
Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "aralkyl" include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

Attorney's Docket No.: 14174-072W01 The term "alkenyl" refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups.
The term "alkynyl" refers to a straight or branched hydrocarbon chain containing 2-8 carbon s atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The sp2 and spa carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.
The term "alkoxy" refers to an -O-alkyl radical. The term "aminoalkyl" refers to an alkyl substituted with an aminoThe term "mercapto" refers to an -SH radical. The term "thioalkoxy"
1 o refers to an -S-alkyl radical.
The term "alkylene" refers to a divalent alkyl (i.e., -R-), e.g., -CHZ-, -CHaCH2-, and -CH2CH2CH2-. The term "alkylenedioxo" refers to a divalent species of the structure -O-R-O-, in which R represents an alkylene.
The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring 15 system, wherein any ring atom capable of substitution can be substituted by a substituent.
Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.
The term "cycloalkyl" as employed herein includes saturated cyclic, bicyclic, tricyclic,or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent. The cycloalkyl groups herein described may also 2o contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.
The term "heterocyclyl" refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms 2s selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond.
Examples of Attorney's Docket No.: 14174-072W01 heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.
The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S
(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.
The term "oxo" refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.
The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.
' The term "substituents" refers to a group "substituted" on an alkyl, cycloalkyl, alkenyl, 15 alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substiW ents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, S03H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)nalkyl (where n is~0-2), S(O)" aryl (where n is 0-2), S(O)" heteroaryl (where n is 0-2), S(O)n 2o heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyh heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are 25 independently any one single, or any subset of the aforementioned substituents.
The terms "adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl" and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.
As used herein, an "unusual" nucleobase can include any one of the following:

Attorney's Docket No.: 14174-072W01 2-methyladeninyl, N6-methyladeninyl, 2-methylthio-N6-methyladeninyl, N6-isopentenyladeninyl, 2-methylthio-N6-isopentenyladeninyl, N6-(cis-hydroxyisopentenyl)adeninyl, 2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl, N6-glycinylcarbamoyladeninyl, N6-threonylcarbamoyladeninyl, 2-methylthio-N6-threonyl carbamoyladeninyl, N6-methyl-N6-threonylcarbamoyladeninyl, N6-hydroxynorvalylcarbamoyladeninyl, 2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl, N6,N6-dimethyladeninyl, 3-methylcytosinyl, 5-methylcytosinyl, , 2-thiocytosinyl, 5-formylcytosinyl, NH
COOH
H2N N N .
H ~ 98 Attorney's Docket No.: 14174-072W01 N4-methylcytosinyl, 5-hydroxymethylcytosinyl, 1-methylguaninyl, N2-methylguaninyl, 7-methylguaninyl, N2,N2-dimethylguaninyl, ~CH3 NHCOOCH3 O O
H3C ~ N~ ) ' ~ ' N N N~N N ~N
CH3 ~ CH3 ~ CH3 "~;~' HO~C OH O H C O

N N HaC~N O H3C~N~N> >
HsC / , ~ y ~ N~N N ~ N~N N
N~N ,N", CH3 ~ CH3 ~

Attorney's Docket No.: 14174-072W01 O O HO HO
N N HO I HO O
HN ~~ H3C~N
~N ,N ~ ~N ,N ~ O 1 NH ~ O NH ' HN \ HN \
H2N~N NN H2N~N N,,, HO HO
beta-galactosyl0 O beta-mannosyl0 O
O NH O NH , O HN NH2 , HN \ ~ HN \ H~ \
H2N~N N H2N~N N H2N ~N
N2,7-dimethylguaninyl, NZ,N2,7-trimethylguaninyl, 1-methylguaninyl, 7-cyano-7-deazaguaninyl, 7-aminomethyl-7-deazaguaninyl, pseudouracilyl, dihydrouracilyl, 5-methyluracilyl, 1-methylpseudouracilyl, 2-thiouracilyl, 4-thiouracilyl, Attorney's Docket No.: 14174-072W01 2-thiothyminyl 5-methyl-2-thiouracilyl, 3-(3-amino-3-carboxypropyl)uracilyl, 5-hydroxyuracilyl, 5-methoxyuracilyl, uracilyl 5-oxyacetic acid, uracilyl 5-oxyacetic acid methyl ester, 5-(carboxyhydroxymethyl)uracilyl, 5-(carboxyhydroxymethyl)uracilyl methyl ester, 5-methoxycarbonylinethyluracilyl, 5-methoxycarbonylmethyl-2-thiouracilyl, 5-aminomethyl-2-thiouracilyl, S-methylaminomethyluracilyl, 5-methylaminomethyl-2-thiouracilyl, 5-methylaminomethyl-2-selenouracilyl, 5-carbamoylmethyluracilyl, 5-carboxymethylaminomethyluracilyl, 5-carboxymethylaminomethyl-2-thiouracilyl, 3-methyluracilyl, 1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl, 1o1 Attorney's Docket No.: 14174-072W01 5-carboxymethyluracilyl, 5-methyldihydrouracilyl, or 3-methylpseudouracilyl.
Palindromes An RNA, e.g., an iRNA agent, can have a palindrome structure as described herein and those described in one or more of United States Provisional Application Serial No. 60/452,682, filed March 7, 2003; United States Provisional Application Serial No.
601462,894, filed April 14,2003; and International Application No. PCT/LJS04/07070, filed March 8, 2004, all of which are hereby incorporated by reference. The iRNA agents of the invention can target more than one RNA region. For example, an iRNA agent can include a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region. The first and second sequences of the iRNA agent can be on different RNA
strands, and the mismatch between the first and second sequences can be less than 50%, 40%, ~5 30%, 20%, 10%, 5%, or 1%. The first and second sequences of the iRNA agent are on the same RNA strand, and in a related embodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent can be in bimolecular form. The first and second sequences of the iRNA agent can be fully complementary to each other.
The first target RNA region can be encoded by a first gene and the second target RNA
2o region can encoded by a second gene, or the first and second target RNA
regions can be different regions of an RNA from a single gene. The first and second sequences can differ by at least 1 nucleotide.
The first and second target RNA regions can be on transcripts encoded by first and second sequence variants, e.g., first and second alleles, of a gene. The sequence variants can be 25 mutations, or polymorphisms, for example. The first target RNA region can include a nucleotide substitution, insertion, or deletion relative to the second target RNA region, or the second target RNA region can a mutant or variant of the first target region.

Attorney's Docket No.: 14174-072W01 The first and second target RNA regions can comprise viral or human RNA
regions. The first and second target RNA regions can also be on variant transcripts of an oncogene or include different mutations of a tumor suppressor gene transcript. In addition, the first and second target RNA regions can correspond to hot-spots for genetic variation.
The compositions of the invention can include mixtures of iRNA agent molecules. For example, one iRNA agent can contain a first sequence and a second sequence sufficiently complementary to each other to hybridize, and in addition the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA
region. The mixture can also include at least one additional iRNA agent variety that includes a third sequence and a fourth sequence sufficiently complementary to each other to hybridize, and where the third sequence is complementary to a third target RNA region and the fourth sequence is complementary to a fourth target RNA region. In addition, the first or second sequence can be sufficiently complementary to the third or fourth sequence to be capable of hybridizing to each other. The first and second sequences can be on the same or different RNA
strands, and the third and fourth sequences can be on the same or different RNA strands.
The target RNA regions can be variant sequences of a viral or human RNA, and in certain embodiments, at least two of the target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. The target RNA regions can correspond to genetic hot-spots.
2o Methods of making an iRNA agent composition can include obtaining or providing information about a region of an RNA of a target gene (e.g., a viral or human gene, or an oncogene or tumor suppressor, e.g., p53), where the region has high variability or mutational frequency (e.g., in humans). In addition, information about a plurality of RNA
targets within the region can be obtained or provided, where each RNA target corresponds to a different variant or 2s mutant of the gene (e.g., a region including the codon encoding p53 24~Q
and/or p53 2495).
The iRNA agent can be constructed such that a first sequence is complementary to a first of the plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second of the plurality of variant RNA targets (e.g., encoding 2495), and the first and second sequences can be sufficiently complementary to hybridize.

Attorney's Docket No.: 14174-072W01 Sequence analysis, e.g., to identify common mutants in the target gene, can be used to identify a region of the target gene that has high variability or mutational frequency. A region of the target gene having high variability or mutational frequency can be identified by obtaining or providing genotype information about the target gene from a population.
s Expression of a target gene can be modulated, e.g., downregulated or silenced, by providing an iRNA agent that has a first sequence and a second sequence sufficiently complementary to each other to hybridize. In addition, the first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region.
An iRNA agent can include a first sequence complementary to a first variant RNA target region and a second sequence complementary to a second variant RNA target region. The first and second variant RNA target regions can correspond to first and second variants or mutants of a target gene, e.g., viral gene, tumor suppressor or oncogene. The first and second variant target RNA regions can include allelic variants, mutations (e.g., point mutations), or polymorphisms of 15 the target gene. The first and second variant RNA target regions can correspond to genetic hot-spots.
A plurality of iRNA agents (e.g., a panel or bank) can be provided.
Other than Canonical Watson-Crick Duplex Structures An RNA, e.g., an iRNA agent can include monomers which can form other than a 2o canonical Watson-Crick pairing with another monomer, e.g., a monomer on another strand, such as those described herein and those described in United States Provisional Application Serial No.
60/465,665, filed April 25, 2003, and International Application No.
PCTlLTS04/07070, filed March ~, 2004, both of which axe hereby incorporated by reference.
The use of "other than canonical Watson-Crick pairing" between monomers of a duplex 2s can be used to control, often to promote, melting of all or part of a duplex. The iRNA agent can include a monomer at a selected or constrained position that results in a first level of stability in the iRNA agent duplex (e.g., between the two separate molecules of a double stranded iRNA
agent) and a second level of stability in a duplex between a sequence of an iRNA agent and Attorney's Docket No.: 14174-072W01 another sequence molecule, e.g., a target or off target sequence in a subject.
In some cases the second duplex has a relatively greater level of stability, e.g., in a duplex between an anti-sense sequence of an iRNA agent and a target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and the target sequence (sometimes referred to herein as the selection or constraint parameters), are selected such that the iRNA agent duplex is has a comparatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting disassociation of the duplex iRNA agent in the context of the RISC) while the duplex formed between an anti-sense targeting sequence and its target sequence, has a relatively higher free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting association of the anti-sense sequence and the target RNA).
In other cases the second duplex has a relatively lower level of stability, e.g.; in a duplex between a sense sequence of an iRNA agent and an off target mRNA. W this case one or more of the monomers, the position of the monomers in the iRNA agent, and an off target sequence, ~5 are selected such that the iRNA agent duplex is has a comparatively higher free energy of association while the duplex formed between a sense targeting sequence and its off target sequence, has a relatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to reduce the level of off target silencing by contribute to efficacy by promoting disassociation of the duplex formed by the sense strand and the off target 2o sequence).
Thus, inherent in the structure of the iRNA agent is the property of having a first stability for the infra-iRNA agent duplex and a second stability for a duplex formed between a sequence from the iRNA agent and another RNA, e.g., a target mRNA. As discussed above, this can be accomplished by judicious selection of one or more of the monomers at a selected or constrained 25 position, the selection of the position in the duplex to place the selected or constrained position, and selection of the sequence of a target sequence (e.g., the particular region of a target gene which is to be targeted). The iRNA agent sequences which satisfy these requirements are sometimes referred herein as constrained sequences. Exercise of the constraint or selection parameters can e, e.g., by inspection, or by computer assisted methods.
Exercise of the Attorney's Docket No.: 14174-072W01 parameters can result in selection of a target sequence and of particular monomers to give a desired result in terms of the stability, or relative stability, of a duplex.
Thus, in another aspect, the invention features, an iRNA agent which includes:
a first sequence which targets a first target region and a second sequence which targets a second target region. The first and second sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the first target region has a first monomer, and the second target region has a second monomer. The first and second monomers occupy complementary or 1 o corresponding positions. One, and preferably both monomers are selected such that the stability of the pairing of the monomers contribute to a duplex between the first and second sequence will differ form the stability of the pairing between the first or second sequence with a target sequence.
Usually, the monomers will be selected (selection of the target sequence may be required 15 as well) such that they form a pairing in the iRNA agent duplex which has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring of the monomer with its complementary monomer in a duplex between the iRNA agent sequence and a target RNA
duplex.
The constraint placed upon the monomers can be applied at a selected site or at more 2o than one selected site. By way of example, the constraint can be applied at more than l, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.
A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3' or 5' of a duplexed sequence. A constrained or selected site can be present in the 2s middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.
In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, Attorney's Docket No.: 14174-072W01 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize.
Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA
agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
The monomers can be selected such that: first and second monomers are naturally occurring ribonuceotides, or modified ribonucleotides having naturally occurring bases, and when occupying complemetary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H
bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a ~ 5 canonical Watson-Crick pairing with the base in the complemetary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-A~.ans, G-A~;S, and GU.
2o The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The 25 monomer is one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value 30 or is less, e.g., than that of a canonical pairing, but when one ( or both) sequences form a duplex Attorney's Docket No.: 14174-072W01 with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick paring.
The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized.
By way of example:
The monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G.
These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
~5 The monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U
or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful 2o wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, 25 A~;S, A~.a"s, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which io8 Attorney's Docket No.: 14174-072W01 will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
A non-naturally occurring or modified monomer or monomers can be chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second ~5 monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with 2o a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA
agent.
An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U
or T.
When placed in complementary positions of the iRNA agent these monomers will pair 25 very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurnng target, or a duplex is between 2-thio U and the A of a naturally occurnng target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. This is shown in the FIG. 1.

Attorney's I?ocket No.: 14174-072W01 The pair shown in FIG. 1 (the 2-amino A and the 2-s U and T) is exemplary. In another embodiment, the monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.
1 o iRNA agents of the invention can include:
A sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other ~5 unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:
The monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H
bonding formed between the monomer at the selected site in the sense strand and its monomer at 2o the corresponding site in the anti-sense strand are less stable than the H
bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner);
The monomer is in the corresponding position in the anti-sense strand is selected such 25 that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;

Attorney's Docket No.: 14174-072W01 Optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off target sequence.
The inclusion of such a monomers will have one or more of the following effects: it will s destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.
The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.
A constrained or selected site can be present at a number of positions in the il~NA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3' or 5' of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a 15 duplexed region.
In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than l, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize.
Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides im 20 length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA
agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
The monomers can be selected such that: first and second monomers are naturally 2s occurring ribonuceotides, or modified ribonucleotides having naturally occurring bases, and when occupying complemetary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H
bonding, which usually have a lower free energy of dissociation than seen in a canonical Attorney's Docket No.: 14174-072W01 Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a canonical Watson-Crick pairing with the base in the complemetary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-A~ns, G-A~;S, and GU.
The monomer in one or both of the sequences is selected such that, it does not pair, or 1 o forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The monomer is one or both strands is also chosen to promote stability in one or both of the duplexes ~5 made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex 2o with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick paring.
The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off target sequences, and duplex 25 interactions between the a sequence and the intended target will not be destabilized.
By way of example:
The monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G.

Attorney's Docket No.: 14174-072W01 These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U
or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful 1 o wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, ~5 A~;S, A~.ans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
2o The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target 25 sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
A non-naturally occurring or modified monomer or monomers can be chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when Attorney's Docket No.: 14174-072W01 one (or both) of them pairs with a naturally occurnng monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA
agent.
An example of such a pairing is 2-amino A and either of a 2-thin pyrimidine analog of U
or T.
When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. , The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is 2o a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.
iRNA agents of the invention can include:
A sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological Attorney's Docket No.: 14174-072W01 conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:
The monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H
bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H
bonds formed~by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner);
The monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;
Optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off target sequence.
The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.
The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than l, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.
A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3' or 5' of a duplexed sequence. A constrained or selected site can be present in the Attorney's Docket No.: 14174-072W01 middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.
The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.
In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.
E.g., the iRNA agent can be less than 30 nucleotides in length,' e.g., 21-23 nucleotides.
Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs.
1 o In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.
In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than l, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize.
15 Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA
agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
2o One or more selection or constraint parameters can be exercised such that:
monomers at the selected site in the sense and anti-sense sequences are both naturally occurnng ribonucleotides, or modified ribonucleotides having naturally occurnng bases, and when occupying complementary sites in the iRNA agent duplex either do not pair and have no substantial level of H-bonding, or form a non-canonical Watson-Crick pairing and thus form a 2s non-canonical pattern of H bonding, which generally have a lower free energy of dissociation than seen in a Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one, usually the anti-sense sequence of the iRNA agent sequences forms a duplex with another Attorney's Docket No.: 14174-072W01 sequence, generally a sequence in the subject, and generally a target sequence, the monomer forms a classic Watson-Crick pairing with the base in the complementary position on the target, or forms a non-canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. Optionally, when the other sequence of the iRNA agent, usually the sense sequences forms a duplex with another sequence, generally a sequence in the subject, and generally an off target sequence, the monomer fails to forms a canonical Watson-Crick pairing with the base in the complementary position on the off target sequence, e.g., it forms or forms a non-canonical Watson-Crick pairing having a lower free energy of dissociation and a lower Tm.
1 o By way of example:
the monomer at the selected site in the anti-sense stand includes an A (or a modified base which pairs with T), the corresponding monomer in the target is a T, and the sense strand is chosen from a base which will not pair or which will form a noncanonical pair, e.g., G;
the monomer at the selected site in the anti-sense stand includes a U (or a modified base ~ 5 which pairs with A), the corresponding monomer in the target is an A, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U
or G;
the monomer at the selected site in the anti-sense stand includes a C (or a modified base which pairs with G), the corresponding monomer in the target is a G, and the sense strand is 2o chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, A~;S, Atrans~ Or U; Or the monomer at the selected site in the anti-sense stand includes a G (or a modified base which pairs with C), the corresponding monomer in the target is a C, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing.
25 In another embodiment a non-naturally occurring or modified monomer or monomers is chosen such that when it occupies complementary a position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that Attorney's Docket No.: 14174-072W01 of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurnng monomer in the target sequence than the pairing it formed in the iRNA agent.
An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U
or T. As is discussed above, when placed in complementary positions of the iRNA agent these 1o monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is formed between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable.
The monomer at the selected position in the sense strand can be a universal pairing 15 moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-vitro pyrrole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. In these cases the monomer at the corresponding position of the anti-sense strand can 2o be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.
In another aspect, the invention features, an iRNA agent which includes:
a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a plurality of target sequences in a subj ect, wherein the targets differ in sequence at only 1 or a small number, e.g., no more than 5, 4, 3 or 2 positions. The 2s sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In the sequence of the anti-sense strand of the iRNA agent is selected such that at one, some, or all of the positions which correspond to positions that differe in sequence between the target sequences, the anti-sense strand will include a monomer Attorney's Docket No.: 14174-072W01 which will form H-bonds with at least two different target sequences. In a preferred example the anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A, 2-thio U or 2-thio T, or other universal base referred to herein.
In a preferred embodiment the iRNA agent targets repeated sequences (which differ at only one or a small number of positions from each other) in a single gene, a plurality of genes, or a viral genome, e.g., the HCV genome.
An embodiment is illustrated in the FIGS. 2 and 3.
In another aspect, the invention features, determining, e.g., by measurement or calculation, the stability of a pairing between monomers at a selected or constrained positoin in the iRNA agent duplex, and preferably determining the stability for the corresponding pairing in a duplex between a sequence form the iRNA agent and another RNA, e.g., a taret sequence. The determinations can be compared. An iRNA agent thus analysed can be used in the devolopement of a further modified iRNA agent or can be administered to a subj ect. This analysis can be ~5 performed successively to refine or desing optimized iRNA agents.
In another aspect, the invention features, a kit which inlcudes one or more of the folowing an iRNA described herein, a sterile container in which the iRNA agent is discolsed, and instructions for use.
In another aspect, the invention features, an iRNA agent containing a constrained 2o sequence made by a method described herein. The iRNA agent can target one or more of the genes referred to herein.
iRNA agents having constrained or selected sites, e.g., as described herein, can be used in any way described herein. Accordingly, they iRNA agents having constrained or selected sites, e.g., as described herein, can be used to silence a target, e.g., in any of the methods 25 described herein and to target any of the genes described herein or to treat any of the disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incorporated into any of the formulations or preparations, e.g., pharmaceutical or sterile Attorney's Docket No.: 14174-072W01 preparations described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be administered by any of the routes of administration described herein.
The term "other than canonical Watson-Crick pairing" as used herein, refers to a pairing between a first monomer in a first sequence and a second monomer at the corresponding position in a second sequence of a duplex in which one or more of the following is true: (1) there is essentially no pairing between the two, e.g., there is no significant level of H bonding between the monomers or binding between the monomers does not contribute in any significant way to the stability of the duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurnng bases, i.e., they are other than A-T, A-U, or G-C, and they form monomer-monomer H bonds, although generally the H bonding pattern formed is less strong than the bonds formed by a canonical pairing; or(3) at least one of the monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less strong than the bonds formed by a canonical pairing, namely one or more of A-T, A-U, G-C.
The term "off target" as used herein, refers to as a sequence other than the sequence to be ~5 silenced.
Universal Bases: "wild-cards" ; shape-based complementarity Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confincnation by 'inverse' sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926) r G
(Importance of terminal base pair hydrogen-bonding in 3'-end proofreading by the Klenow fragment of 20 DNA polymerase I. Morales, J. C.; Kool, E. T. Biochernistry, 2000, 39, 2626-2632) (Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.;
Kool, E. T. J. Am. Chern.
Soc.,1998, 120, 6191-6192) Attorney's Docket No.: 14174-072W01 (Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA
replication. Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J. Am. Clzem.
Soc.,1997,119, 2056-2057) (Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine. Guckian, K.
M.; Morales, J. C.; Kool, E. T. J. Org. Chenz.,1998, 63, 9652-9656) F CHa H3~ \ N
HO I / F HO N I /
O O
OH F OH _ Attorney's Docket No.: 14174-072W01 HO
N
O
OH
3-nitropyrrole NOZ
HO N ~ /
O
OH
5-nitroindole \ ~\ w ~\
N O N O N O

( (Universal bases for hybridization, replication and chain termination. Berger, M.; Wu. Y.; Ogawa, A. K.;
McMinn, D. L.; Schultz, P.G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914) \
\ / \ / \ / / \ /
ww ..~,~, O .,~"~, O
TM DM ICS PICS
/ \ ~ / \
/ \ / \ / ~ l ~ / \ / \ /
N N
2MN DMN 7A1 2Np 3MN

Attorney's Docket No.: 14174-072W01 (1. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Arrz.
Clzem. Soc., 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Bergen M.; Schultz, P. G.; Romesberg, F. E.
J. Arn. Chem. Soc., 2000, 122, 8803-8804) N
N

(Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. Tae, E. L.;
Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Anz. Clzern. Soc., 2001,123, 7439-7440) I
N
~N
HO
O
OH
(l. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions.
Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F.
E. J. Am. Chem. Soc., 2000, 122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNA polymerase recognition of a highly stable, self pairing hydrophobic base. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.;
Schultz, P. G.; Romesberg, F. E. J. Anz.
Chern. Soc., 1999,121, 11585-11586) (A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple:
Interstrand stacking as the stability determining factor. Brotschi, C.;
Haberli, A.; Leumann, C, 3. Arzgew. Chern. Int.
Ed., 2001, 40, 3012-3014) (2,2'-Bipyridine Ligandoside: A novel building block for modifying DNA with infra-duplex metal complexes. Weizman, H.; Tor, Y. J. Arn. Claern. Soc., 2001, 123, 3375-3376) Attorney's Docket No.: 14174-072W01 NHZ NHZ
N N- ' N
HO I / HO ~
O O
OH OH
d2APy d2APm (Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.;
McLaughlin, L. W. J. Ana.
Claerra. Soc., 2000, 122, 6512-13) NOZ
Ho N
O
OH
(Effect of the Universal base 3-nitropyrrole on the selectivity of neighboring natural bases. Oliver, J. S.;
Parker, K. A.; Suggs, J. W. Organic Lett., 2001, 3, 1977-1980. 2. Effect of the 1-(2'-deoxy-(3-D-ribofuranosyl)-3-nitropyrrol residue on the stability of DNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R. Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure and deoxyribonucleic acid sequencing with a universal nucleosides: 1-(2'-deoxy-(3-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.;
Zhang, P.; Toma, P. H.; Andrews, P.
C.; Nichols, R. J. Am. Chem. Soc.,1995, 117, 1201-1209) OH
H
HO N-H~~,~~~",~~
N-H"~~~~~n ,.. . N
\ \ p ", N
HO~ I I / / N N~Bu Hp O LN O ,.".H_ ~[L >~~ N ' O ~N H H ' H H N- O
OH ,N N Nm.
Bu ~ / / I OH
O \ \
(Model studies directed toward a general triplex DNA recognition scheme: a novel DNA base that binds a CG base-pair in an organic solvent. Zimmerman, S. C.; Schmitt, P. J. Arra.
Chern. Soc.,1995, 117, 10769-10770) Attorney's Docket No.: 14174-072W01 (A universal, photocleavable DNA base: nitropiperonyl 2'-deoxyriboside. J.
Org. Claern., 2001, 66, 2067-2071 ) (Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine.
Nakatani, K.; Sando, S.; Saito, I.
J. Am. Clzem. Soc., 2000, 122, 2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into single guanine bulge as evidenced by photooxidation of GC doublet, Nakatani, K.; Sando, S.;
Yoshida, K.; Saito, I. Bioorg. Med.
Chem. Lett., 2001,11, 335-337) ~z O
N_ ..
N
~~ H
O I N
C OPOO

Other universal bases can have the following formulas:

Attorney's Docket No.: 14174-072W01 ...Q ...Q
Q Q Qiv / \ 52 Qiv \ S R
~N R . \N
R48 ~ ~~ R56 ' R61 / R63 _.
N ~ ~ , and R60 ~ ~ R57 R64 i7 n59 ~ 58 R72 ... ;.. Rss R7~
wherein:
Q is N or CR44;
5 Q' is N or CR4s;
Q" is N or CR47;

Attorney's Docket No.: 14174-072W01 Q"' 1S N Or CR49;
Qi° is N or CRso;
R44 is hydrogen, halo, hydroxy, vitro, protected hydroxy, NH2, NHRb, or NRbR°, Cl-C6 alkyl, C6-Clo aryl, C6-C1° heteroaryl, C3-C8 heterocyclyl, or when taken together with R45 forms s -OCH20-;
R45 is hydrogen, halo, hydroxy, vitro, protected hydroxy, NH2, NHRb, or NRbR~, Cl-C6 alkyl, C6-Clo aryl, C6-C1° heteroaryl, C3-C8 heterocyclyl, or when taken together with R44 or R46 forms -OCH20-;
R46 is hydrogen, halo, hydroxy, vitro, protected hydroxy, NH2, NHRb, or NRbR°, Cl-C6 1o alkyl, C6-Clo aryl, C6-C1° heteroaryl, C3-C8 heterocyclyl, or when taken together with R45 or R47 forms -OCH20-;
R47 is hydrogen, halo, hydroxy, vitro, protected hydroxy, NH2, NHRb, or NRbR°, Cl-C6 alkyl, C6-Clo aryl, C6-Clo heteroaryl, C3-C8 heterocyclyl, or when taken together with R46 or R48 forms -OCH20-;
15 R4g is hydrogen, halo, hydroxy, vitro, protected hydroxy, NH2, NHRb, or NRbR°, C1-C6 alkyl, C6-C1° aryl, C6-C1° heteroaryl, C3-C8 heterocyclyl, or when taken together with R47 forms -OCH20-;
R49 R50' R51' R52' R53~ R54' R57a R58~ R59' R60~ R61' R62' R63' R64' R65' R66' R6T R68' R69' R7°, R71, and R72 are each independently selected from hydrogen, halo, hydroxy, vitro, protected 2o hydroxy, NH2, NHRb, or NRbR°, C1-C6 alkyl, C2-C6 alkynyl, C6-Clo aryl, C6-Clo heteroaryl, C3-C8 heterocyclyl, NC(O)R17, or NC(O)R°;
R55 is hydrogen, halo, hydroxy, vitro, protected hydroxy, NH2, NHRb, or NRbR°, Cl-C6 alkyl, C2-C6 alkynyl, C6-C1° aryl, C6-C1° heteroaryl, C3-C8 heterocyclyl, NC(O)Ri7, or NC(O)R°, or when taken together with R56 forms a fused aromatic ring which may be optionally 25 substituted;

Attorney's Docket No.: 14174-072W01 R56 is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH2, NHRb, or NRbR°, Cl-C6 alkyl, C2-C6 alkynyl, C6-Clo aryl, C6-Clo heteraaryl, C3-C8 heterocyclyl, NC(O)Ri~, or NC(O)R°, or when taken together with R55 forms a fused aromatic ring which may be optionally substituted;
Rl~ is halo, NHZ, NHRb, or NRbR°;
Rb is C1-C6 alkyl or a nitrogen protecting group;
R° is C1-C6 alkyl; and R° is alkyl optionally substituted with halo, hydraxy, nitro, protected hydroxy, NH2, NHRb, or NRbR~, CI-C6 alkyl, C2-C6 alkynyl, C6-Clo aryl, C6-Clo heteroaryl, C3-Cg heterocyclyl, NC(O)R17, or NC(O)R°.

Attorney's Docket No.: 14174-072W01 Examples of universal bases include:
F CHs NH2 NH2 OaN \
HsC ~ \ I \ N~ I \ N , N~N , I ~ \ , i F ' i N , ~ N
i ~ i i N02 \ \ O OI I \ \ ~' \ O
I ~ ~ II , BuHN~ / ~ ' O N I
N N. _ ~O
N N N H~ H N- ~ z CH O CHs O O O CHs ~,' ~,' ~,' HsC I \
\ N~ , I \ N~ , I \ N~ ' I '\ N
I ~ / ~ ~ H C ~ ~ ~ ~ , i CHs 3 , O CHs HsC CHs I \ \~ \
\ N~ ~ ~~~~ ' I ~ ~ , N N , I ~ ~ ~ ~ CHs CHs \ \'''~' \ \ ~''~' - N_ I ~ , I , ~ , and ~ / ~ / .
N
CHs Attorney's Docket No.: 14174-072W01 Asymmetrical Modifications An RNA, e.g., an iRNA agent, can be asymmetrically modified as described herein, and as described in International Application Serial No. PCT/LTS04l07070, filed March 8, 2004, which is hereby incorporated by reference.
In addition, the invention includes iRNA agents having asymmetrical modifications and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an asymmetrical modification.
An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found ~5 on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue;
2o modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.
25 While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or "asymmetrical" functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense stand . In addition, since some Attorney's Docket No.: 14174-072W01 modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of s the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2' modification, e.g., 2' OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5' or 3' end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2'OMe is present in the sense strand, and a targeting moiety is added to either the 5' or 3' end of the sense strand of the iRNA agent.
In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.
Each strand can include one or more asymmetrical modifications. By way of example:
15 one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.
2o In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well.
E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.
2s In an embodiment one strand has an asymmetrical 2' modification, e.g., a 2' OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetrical 2' OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have Attorney's Docket No.: 14171-072W01 asymmetrical 2'-O alkyl, preferably, 2'-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2'-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2'-OMe modifications on the sense strand and multiple phophorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region 1o about 20-21, and preferably 19, subunits in length and one or two 3' overhangs of about 2 subunits in length.
Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the ~ 5 modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some 2o cases it is desirable to provide the UA sequence on both strands with asymmetrical modification.
Examples of modifications which inhibit endonucleolytic degradation can be found herein, Particularly favored modifications include: 2' modification, e.g., provision of a 2' OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate 25 modification, on the U or the A or both, especially on an antisense strand;
replacement of the U
with a CS amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2', 6', 7', or 8' position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has 3o fewer of such modifications.

Attorney's Docket No.: 14174-072W01 Asymmetrical modification can be used to inhibit degradation by exonucleases.
Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3' overhang, e.g., at the 3' terminus, and on the antisense strand, e.g., in a 3' overhang, e.g., at the 3' terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.
Examples of modifications which inhibit exonucleolytic degradation can be found herein.
Particularly favored modifications include: 2' modification, e.g., provision of a ~2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); modification 15 of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorotluoate modification, or the use of a methylated P in a 3' overhang, e.g., at the 3' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate.modification, or the use of a methylated P, in a 3' overhang, e.g., at the 3' 2o terminus; modification with a 3' alkyl; modification with an abasic pyrolidine in a 3' overhang, e.g., at the 3' terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3' terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
2s Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce so moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, Attorney's Docket No.: 14174-072W01 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.
Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical s modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids ,2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand.
iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3' ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA
agent defined by the 5' end of the sense strand and the 3'end of the antisense strand is important for function.
This region can include the terminal 2, 3, or 4 paired nucleotides and any 3' overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following 15 are used: modifications of the 5' end of the sense 'strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5' exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a "tight" structure at this end of the molecule.
2o The end region of the iRNA agent defined by the 3' end of the sense strand and the 5'end of the antiserlse strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3' overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an "open" structure at this end of the 2s molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.

Attorney's Docket No.: 14174-072W01 Exemplary modifications for asymmetrical placement in the sense strand include the following:
(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S-P=S); these modifications can be used to promote nuclease resistance;
(b) 2'-O alkyl, e.g., 2'-OMe, 3'-O alkyl, e.g., 3'-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3' modifications can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;
(c) 2'-5' linkages (with 2'-H, 2'-OH and 2'-OMe and with P=O or P=S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC
15 (d) L sugars (e.g., L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe);
these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC;
(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) 2o and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5' end of the sense. strand to avoid sense strand activation by RISC;
(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease 25 resistance or to enhance binding of the sense to the antisense strand;
(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;

Attorney's Docket No.: 14174-072W01 (h) conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5' end of the sense strand to avoid sense strand activation by RISC.
Exemplary modifications for asymmetrical placement in the antisense strand include the following:
(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S-P=S);
(b) 2'-O alkyl, e.g., 2'-OMe, (at terminal positions);
(c) 2'-5' linkages (with 2'-H, 2'-OH and 2'-OMe) e.g., terminal at the 3' end); e.g., with P=O or P=S preferably at the 3'-end, these modifications are preferably excluded from the 5' end region as they may interfere with RISC enzyme activity such as kinase activity;
(d) L sugars (e.g, L ribose, L-arabinose with 2'-H, 2'-OH and 2'-OMe); e.g., terminal at the 3' end; e.g., with P=O or P=S preferably at the 3'-end, these modifications are preferably ~ 5 excluded from the 5' end region as they may interfere with kinase activity;
(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5' end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a "loose" structure in the 5' region, additionally, they may interfere With kinase activity;
20 (f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);
(g) cationic groups and Zwitterionic groups (preferably at a terminus);
cationic groups and Zwitterionic groups at 2'-position of sugar; 3'-position of the sugar;
as nucleobase modifications (e.g., C-S modified pyrimidines, N-2 modified purines, N-7 25 modified purines, N-6 modified purines);

Attorney's Docket No.: 14174-072W01 conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.
The 5'-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3' end, particularly in the 3' overhang.
In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.
The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50 % activity, as compared with an otherwise similar iRNA lacking the introduced modification.
Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the ~ 5 required maintenance of activity.
The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred 2o asymmetric modifications are 2'-O alkyl modifications, e.g., 2'-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.
In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and 25 provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.

Attorney's Docket No.: 14174-072W01 Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.
Table 3 shows examples having strand I with a selected modification and strand II with a s selected modification.
Table 3. ExemMary strand I- and strand II-modifications Strahd T , ~ Strand II-Nuclease Resistance (e.g., Biodistribution (e.g., P=S) 2'-OMe) Biodistribution conjugate Protein Binding Functionality (e.g., Lipophile) (e.g., Naproxen) Tissue Distribution FunctionalityCell Targeting Functionality (e.g., Carbohydrates) (e.g., Folate for cancer cells) Tissue Distribution FunctionalityFusogenic Functionality (e.g., liver Cell Targeting (e.g., Polyethylene imines) moieties) Cancer Cell Targeting Fusogenic Functionality (e.g., RGD peptides and imines)(e.g., peptides) Nuclease Resistance (e.g., crease in binding Affinity (5-Me-C, 2'-OMe) 5-Me-U, 2-thio-U, 2-amino-A, G-clamp, LNA) Tissue Distribution Functionality RISC activity improving Functionality Helical conformation changingTissue Distribution Functionality Attorney's Docket No.: 14174-072W01 Functionalities I (P=S; lipophile, carbohydrates) Z-X-Y Architecture An RNA, e.g., an iRNA agent, can have a Z-X-Y architecture or structure such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/510,246, filed on October 9, 2003, which is hereby incorporated by reference, copending, co-owned United States Provisional Application Serial No. 60/510,318, filed on October 10, 2003, which is hereby incorporated by reference, and copending, co-owned International Application No. PCT/LTS04/07070, filed March 8, 2004.
In addition, the invention includes iRNA agents having a Z-X-Y structure and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a 1 o palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA
associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent, administered as described herein, or an iRNA
agent formulated as described herein, which also incorporates a Z-X-Y architecture.
15 Thus, an iRNA agent can have a first segment, the Z region, a second segment, the X
region, and optionally a tlurd region, the Y region:
Z-X-Y.
It may be desirable to modify subunits in one or both of Zand/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of 2o modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.
The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some 25 embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the Attorney's Docket No.: 14174-072W01 end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A
typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3' or 5' terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3- overhang. Typical inter-subunit bonds in the Z region include: P=O; P=S; S-P=S; P-NRZ; and P-BR2. Chiral P=X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3'-OR, 3'SR, 2'-OMe, 3'-OMe, and 2'OH
modifications and moieties; alpha configuration bases; and 2' arabino modifications.
The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will ~5 typically be between 10-45 and more preferably between 15 and 35 subunits.
Particularly preferred region X's will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2'-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the 2o X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines.
Some embodiments have 4 or more consecutive 2'OH subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2'OH
subunits.
The Y region will generally conform to the the parameters set out for the Z
regions.
25 However, the X and Z regions need not be the same, different types and numbers of modifications can be present, and infact, one will usually be a 3' overhang and one will usually be a 5' overhang.
In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe or other alkyl; and an X

Attorney's Docket No.: 14174-072W01 region that includes at least four consecutive ribonucleoside subunits in which the 2'-OH
remains unsubstituted.
The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications can be provided bewteen the subunits of any of the regions, Y, X, and Z.
However, it is preferred that their occureceis minimized and in particular it is preferred that consecutive modified linkages be avoided.
In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2'-OH is substituted, e.g., with 2'-OMe; and an X
region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2'-OH
remains unsubstituted.
As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of 15 any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modiFed linkages be avoided.
Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA
agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred iRNA agents will not have four or 2o more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if 25 subunit linkages in region X axe modified, such modifications will be minimized. E.g., although the Y andlor Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of Attorney's Docket No.: 14174-072W01 consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred X regions will not have four or more consecutive subunits, e.g., 2'-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages - i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
In a preferred embodiment Y and for Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.
In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2' moieties.
In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.
In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., a 2'-O-alkyl ~5 moiety. E.g., any adjacent 2'-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosphorothioate linkage.
In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-O-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, 2o having a 2' modification, e.g., 2'-O-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.
In a prefered embodiment each of Y and/orZ independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2' 25 modification, e.g., 2'-O-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2' modification, e.g., 2'-O-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

Attorney's Docket No.: 14174-072W01 In a prefered embodiment one of the above recited limitation on adj acent subunits in Y
and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y
andlor Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2' s modification, e.g., a 2'-O-alkyl moiety. E.g., any adjacent 2'-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2' moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.
A Y andlor Z region can include at least one, and preferably 2, 3 or 4 of a modification 1 o disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than l, 2, 3, or 4 of such 15 modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Yand for Z and X, e.g., the modification will be present one of Y andlor Z or X and absent in the other.
An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described 20 herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some emobodiments it is prefered that no more than l, 2, 3, or 4 of such modifications occur, or occur consecutively.
2s An RRMS (described elswhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y
and/or Z region, at or near (within l, 2, or 3 positions) of the 3' or 5' end of the sense strand or at near (within 2 or 3 positions of) the 3' end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within l, 2, or 3 positions of) the 5' end of the antisense strand. An Attorney's Docket No.: 14174-072W01 RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical fox antisense binding to the target.
Differential Modification of Terminal Duplex Stability In one aspect, the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).
In addition, the invention includes iRNA agents having DMTDS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA
agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA
agent having an 1o architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.
iRNA agents can be optimized by increasing the propensity of the duplex to disassociate ~ 5 or melt (decreasing the free energy of duplex association), in the region of the 5' end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5' end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the Send .
While not wishing to 2o be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5' end of the antisense strand.
The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex 25 association),~in the region of the 3' end of the antisense strand duplex.
This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3' end of the antisense strand. It can also be Attorney's Docket No.: 14174-072W01 accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5'end.
Modifications which increase the tendency of the 5' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3' end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3' end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5' end of the duplex to dissociate.
Decreasing the stability of the AS 5' ehd of the duplex Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:
A:U is preferred over G:C;
~5 G:U is preferred over G:C;
I:C is preferred over G:C (I=inosine);
mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; _ pairings which include a universal base are preferred over canonical pairings.
2o A typical ds iRNA agent can be diagrammed as follows:
S 5' Rl Nl N2 N3 N4 NS [N] N_5 N~ N_3 N_z N_1 Rz 3' AS 3' R3 Nl Nz N3 N4 NS [N] N_5 N_4 N_3 N_z N_1 R4 5' S:AS P1 Pz P3 P4 PS [N] P_SP_4P_3P_zP_1 5' Attorney's Docket No.: 14174-072W01 S indicates the sense strand; AS indicates antisense strand; Rl indicates an optional (and nonpreferred) 5' sense strand overhang; Ra indicates an optional (though preferred) 3' sense overhang; R3 indicates an optional (though preferred) 3' antisense sense overhang; R4 indicates an optional (and nonpreferred) 5' antisense overhang; N indicates subunits;
[N] indicates that additional subunit pairs may be present; and PX, indicates a paring of sense NX and antisense NX.
Overhangs are not shown in the P diagram. In some embodiments a 3' AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3' S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.) 1 o It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5' end of the AS strand. The terminal pair (the most 5' pair in terms of the AS strand) is designated as P_l, and the subsequent pairing positions (going in the 3' direction in terms of the AS strand) in the duplex are designated, P_a, P_3, F~, P_s, and so on. The preferred region in which to modify to modulate duplex formation is at P_5 through P_l, more preferably P_4 through P_1 , more preferably P_3 through P_1. Modification at P_ 1, is particularly preferred, alone or with modifications) other position(s), e.g., any of the positions just identified. It is preferred that at least l, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of A:U
2o G:U
I:C
mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base.
In preferred embodiments the change in subunit needed to achieve a pairing which 2s promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment the at least 2, or 3, of the pairs in P_l, through P_4, are pairs which promote disociation.
In a preferred embodiment the at least 2, or 3, of the pairs in P_l, through P_4, are A:U.
In a preferred embodiment the at least 2, or 3, of the pairs in P_l, through P_4, are G:U.
In a preferred embodiment the at least 2, or 3, of the pairs in P_l, through P_4, are I:C.
In a preferred embodiment the at least 2, or 3, of the pairs in P_l, through P_4, are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.
In a preferred embodiment the at least 2, or 3, of the pairs in P_l, through P_4, are pairings which include a universal base.
1o IncYeasing the stability of the AS 3' end of tlae duplex Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex ~ 5 stability:
G:C is preferred over A:U
Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings analogs that increase stability axe preferred over Watson-Crick matches (A:T, A:U, 2o G:C) 2-amino-A:U is preferred over A:U
2-thio U or 5 Me-thio-U:A are preferred over U:A
G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G

Attorney's Docket No.: 14174-072W01 guanadinium-G-clamp:G is preferred over C:G
psuedo uridine:A is preferred over U:A
sugar modifications, e.g., 2' modifications, e.g., 2'F, ENA, or LNA, which enhance binding axe preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3' end of the AS strand.
The terminal pair (the most 3' pair in teens of the AS strand) is designated as P1, and the subsequent pairing positions (going in the 5' direction in terms of the AS
strand) in the duplex are designated, P2, P3, P4, Ps, and so on. The preferred region in which to modify to modulate 1 o duplex formation is at PS through Pl, more preferably P4 through P1 , more preferably P3 through P1. Modification at Pl, is particularly preferred, alone or with mdification(s) at other position(s), e.g.,any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of G:C
a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C) 2-amino-A:U
2-thio U or 5 Me-thio-U:A
2o G-clamp (an analog of C having 4 hydrogen bonds):G
guanadinium-G-clamp:G
psuedo uridine:A
a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment the at least 2, or 3, of the pairs in P_l, through P_4, are pairs which promote duplex stability.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are G:C.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are a pair having an analog that increases stability over Watson-Crick matches.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are 2-amino-A:U.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are 2-thin U
or 5 Me-thio-U:A.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are G-clamp:G.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are guanidinium-G-clamp:G.
In a preferred embodiment the at least 2, or 3, of the pairs in Pi, through P4, are psuedo uridine:A.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhances binding.
G-clamps and guanidinium G-clamps are discussed in the following references:
Holmes 2o and Gait, "The Synthesis of 2'-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction," Nucleosides, Nucleotides &
Nucleic Acids, 22:1259-1262, 2003; Holmes et al., "Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2'-O-methyl G-clamp ribonucleoside analogues," Nucleic Acids Research, 31:2759-2768, 2003;
Wilds, et al., "Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue:
Guanidinium G-clamp," Helvetica Chimica Acta, 86:966-978, 2003; Rajeev, et al., "High-Attorney's Docket No.: 14174-072W01 Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues," Organic Letters, 4:4395-4398, 2002; Ausin, et al., "Synthesis of Amino- and Guanidino-G-Clamp PNA
Monomers," Organic Letters, 4:4073-4075, 2002; Maier et al., "Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties,"
Biochemistry, 41:1323-7, 2002; Flanagan, et al., "A cytosine analog that confers enhanced potency to antisense oligonucleotides," Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.

Attorney's Docket No.: 14174-072W01 Simultaneously decreasing the stability of the AS 5'end of the duplex and increasing the stability of the AS 3' end of the duplex As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5'end of the duplex and increase the stability of the AS 3' end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5' end of the duplex with one or more of the stability increasing modifications in the AS 3' end of the duplex. Accordingly a preferred embodiment includes modification in P_5 through P_l, more preferably P~ through P_1 and more preferably P_3 through P_l. Modification at P_l, is particularly preferred, alone or with other position, e.g., the positions just identified.
It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5' end of the duplex region be chosen independently from the group of A:U
G:U
I:C
~5 mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and a modification in PS through P1, more preferably P4 through P1 and more preferably P3 through P1. Modification at Pl, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least l, and more preferably 2, 3, 4, or 5 of the 2o pairs of one of the recited regions of the AS 3' end of the duplex region be chosen independently from the group of G:C
a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C) 25 2-amino-A:U

Attorney's Docket No.: 14174-072W01 2-thio U or 5 Me-thio-U:A
G-clamp (an analog of C having 4 hydrogen bonds):G
guanadinium-G-clamp:G
psuedo uridine:A
a pair in which one or both subunits has a sugar modification, e.g., a 2' modification, e.g., 2'F, ENA, or LNA, which enhance binding.
The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iIZNA
agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the AS strand, to provide a desired level of DMTDS.
The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P_5 through P_1 andlor at least one P in PS through P1 to provide a DMTDS
iRNA agent.
~5 DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS
iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of 2o nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3' AS overhangs and/or 3' S strand overhangs) which self associate to form intrastrand duplex structure.
Preferably these iRNA agents will have an architecture described herein.
Other Embodiments An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g., from exogenous 25 DNA templates that are delivered into the cell. For example, the DNA
templates can be inserted Attorney's Docket No.: 14174-072W01 into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No.
5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc. Natl. Acad. Sci. USA
91:3054-3057, 1994).
The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of an iRNA agent and one that produces a transcript that includes the bottom strand of an iRNA agent. When the templates are transcribed, the iRNA agent is produced, and processed into sRNA agent fragments that mediate gene silencing.
In vivo Delivery An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery ~5 vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.
The delivery molecule can include a linear cyclodeXtrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer.
Delivery systems , as described in U.S. Patent No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear cyclodextrin copolymer andlor a linear oxidized cyclodextrin copolymer. Either or both of the Attorney's Docket No.: 14174-072W01 cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer andlor bound to a ligand.
A composition for iRNA delivery can employ an "inclusion complex," a molecular compound having the characteristic structure of an adduct. In this structure, the "host molecule" spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the "guest molecule") is situated in the cavity of the host molecule without affecting the framework structure of the host. A "host" is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 200310008818, herein incorporated by reference.
Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of "host-guest" complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent 2o having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a liver cell), intercellular trafficking, and/or cell entry and release.
Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, from the polymer of the particulate composite, the activity of the iRNA agent is restored.

Attorney's Docket No.: 14174-072W01 Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability.
Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.
Physiological Effects The iRNA agents described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the iRNA agent with both a human and a non-human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey.
The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can be complementary to a human and more than one, e.g., two or three or more, non-human animals.
The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect; such as a toxic effect, or any positive, or desired effect.
Delivery Module An RNA, e.g., an iRNA agent described herein, can be used with a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/454,265, filed on March 12, 2003, and Attorney's Docket No.: 14174-072W01 International Application Serial No. PCTlUS04/07070, filed March ~, 2004, both of which are hereby incorporated by reference.
In addition, the invention includes iRNA agents described herein, e.g., a palindromic iRNA agent, an iRNA agent hving a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such a drug delivery conjugate or module.
The iRNA agents can be complexed to a delivery agent that features a modular complex.
The complex can include a Garner agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fixsogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome ~5 membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a liver cell.
An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing 2o agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e:g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA
agent to a subject in need thereof, e.g., to deliver an iRNA agent to a subj ect having a disorder, e.g., a disorder described herein, such as a disease or disorder of the liver.
The fusogenic agent and the condensing agent can be different agents or the one and the 25 same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent.
The delivery agent can be a modular complex. For example, the complex can include a Garner agent linked to one or more of (preferably two or more, more preferably all three of):

Attorney's Docket No.: 14174-072W01 (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction), (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane), and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a liver cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, 1o multivalent galactose, transfernn, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.
Carrier agerats The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable..
The earner agent can be a naturally occurring substance, such as a protein (e.g., human 2o serum albumin (HSA), low-density lipoprotein (LDL), or globulin);
carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid);
or lipid. The earner molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-malefic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-malefic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (MA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.

Attorney's Docket No.: 14174-072W01 A carrier agent can be characterized by one or more of (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1:1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1:1 Garner agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 carrier agent to targeting agent.
Fusogenic agents A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which 1 o disrupts or increases the permeability of the endosome membrane.
Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in ~ 5 the cell.
In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation.
The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon 2o being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.
The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.
25 In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal.
In some embodiment, the fusogenic agent can have an alpha helical structure.
The fusogenic agent can be a membrane disruptive agent, e.g., mellittin.

Attorney's Docket No.: 14174-072W01 A fusogenic agent can have one or more of the following characteristics: (a) is at least 1Da in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1:1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 fusogenic agent to targeting agent.
Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH
environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A
change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control ~5 cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test:or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge 2o depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH
environment; and a second assay evaluates the ability of a modular complex that includes the test 25 compound to respond to, e.g., change charge depending on, the pH
enviromnent.
Condensiytg agent The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA
agent against Attorney's Docket No.: 14174-072W01 degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide.
A condensing agent can have the following characteristics: (a) at least 1Da in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups;
(c) is present in the complex at a ratio of at least 1:1 condensing agent to carrier agent; (d) is present in the complex 1 o at a ratio of at least 1:1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1:1 condensing agent to targeting agent.
Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent 2o is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic cid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid.
Amuhipathic Delivery Agents An RNA, e.g., an iRNA agent, described herein can be used with an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned United States Provisional Application Serial No. 60/455,050, filed on March 13, 2003, and Attorney's Docket No.: 14174-072W01 International Application Serial No. PCTlUS04/07070, filed March 8, 2004, which is hereby incorporated by reference.
In addition, the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a noncanonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein,,or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such an amphipathic delivery conjugate.
An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region.
Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bylayer of a cell.
As such, they can serve as delivery agent for an associated (e.g., bound) iRNA
(e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs descriebd herein) is a polymer. The 15 polymer may have a secondary structure, e.g., a repeating secondary structure.
One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hybrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance:
2o Grell et a1. (2001) "Protein design and folding: template trapping of self assembled helical ' bundles" J Pept Sci 7(3):146-51; Chen et al. (2002) "Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix" J Pept Res 59(1):18-33;
Iwata et al. (1994) "Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation" J Biol Chem 269(7):4928-33;
25 Cornut et al. (1994) "The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin"
FEBS Lett 349(1):29-33; Negrete et al. (1998) "Deciphering the structural code for proteins:
helical propensities in domain classes and statistical multiresidue information in alpha-helices,"
Protein Sci 7(6):1368-79.

Attorney's Docket No.: 14174-072W01 Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure.
The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell.
In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to , interact with a lipid bilayer, cell membrane or cell.
2o An amphipathic polymer useful in the compositions described herein has at least 2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amplupathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid 2s and cyclic subunits, e.g., aromatic subunits.
The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as "amphipathic iRNA constructs." Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a Attorney's Docket No.: 14174-072W01 cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.
In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.
The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.
1 o The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be ~ 5 such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.
The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an a-helical conformation as its secondary structure.
In one embodiment, the amphipathic polymer includes one or more subunits containing 20 one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups andlor one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit 25 atropisomerism, e.g., a 2,2'-bis(substituted)-1-1'-binaphthyl or a 2,2'-bis(substituted) biphenyl.
A subunit may include an aromatic moiety of Formula (M):

Attorney's Docket No.: 14174-072W01 (M) The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as "amphipathic iRNA constructs." Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e:g., to allow entry of the iRNA agent into a cell.
In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.
The amphipathic molecule of the composition or construct is preferably other than a ~5 phospholipid, e.g., other than a micelle, membrane or membrane fragment.
The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and Attorney's Docket No.: 14174-072WO1 one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.
The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an a-helical conformation as its secondary structure.
In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups andlor one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g.; a 2,2'-bis(substituted)-1-1'-binaphthyl or a 2,2'-bis(substituted) biphenyl.
A subunit may include an aromatic moiety of Formula (M):
(M) Attorney's Docket No.: 14174-072W01 Refernng to Formula M, Rl is C1-Cloo alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo andlor optionally inserted with O, S, alkenyl or alkynyl; C1-Cloo perfluoroalkyl; or ORS.
RZ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR6;
or CI-Cloo alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, caxboxylate, amino carbonyl, or alkoxycarbonyl, andlor optionally inserted with O, NH, S, S(O), SO2, alkenyl, or alkynyl.
1 o R3 is hydrogen, or when taken together with R4 froms a fused phenyl ring.
R4 is hydrogen, or when taken together with R3 froms a fused phenyl ring.
RS is Cl-Cloo alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C1-Cloo perfluoroalkyl; and R6 is C1-Cloo alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl ~ 5 sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), 502, alkenyl, or alkynyl.
Increasing cellular uptake of dsRNAs A method of the invention that can include the administration of an iRNA agent and a 2o drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p3~ MAP kinase, or an activator of NF-KB. The drug can have a transient effect on the cell.
25 The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, Attorney's I?ocket No.: 14174-072W01 cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.
iRNA coniu~ates An iRNA agent can be coupled, e.g., covalently coupled, to a second agent. For example, an iRNA agent used to treat a particular disorder can be coupled to a second therapeutic agent, e.g., an agent other than the iRNA agent. The second therapeutic agent can be one which is 1 o directed to the treatment of the same disorder. For example, in the case of an iRNA used to treat a disorder characterized by unwanted cell proliferation, e.g., cancer, the iRNA agent can be coupled to a second agent which has an anti-cancer effect. For example, it can be coupled to an agent which stimulates the immune system, e.g., a CpG motif, or more generally an agent that activates a toll-like receptor and/or increases the production of gamma interferon.
iRNA Production An iRNA can be produced, e.g.~ in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.
Organic S thesis An iRNA can be made by separately synthesizing each respective strand of a double-2o stranded RNA molecule. The component strands can then be annealed.
A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (LJppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given iRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used.
Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the iRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

Attorney's Docket No.: 14174-072W01 Organic synthesis can be used to produce a discrete iRNA species. The complementary of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.
dsRNA Cleavage iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can 1 o be used:
In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribeTM RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter.
Separate templates are ~ 5 generated for T7 transcription of the two complementary strands for the dsRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsRNA is produced. Similar methods using PCR andlor other RNA polymerases (e.g., T3 or SP6 polymerase) can also be used. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes.
2o Ira vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g. a purified RNAse or RISC complex (RNA-induced silencing complex ). See, e.g., Ketting et al. Genes Dev 2001 Oct 15;15(20):2654-9. and Hammond Science 2001 Aug 10;293(5532):1146-50.
25 dsRNA cleavage generally produces a plurality of iRNA species, each being a particular 21 to 23 nt fragment of a source dsRNAmolecule. For example, iRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsRNA
molecule may be present.

Attorney's Docket No.: 14174-072W01 Regardless of the method of synthesis, the iRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For example, the iRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried iRNA can then be resuspended in a solution appropriate for the intended formulation process.
Synthesis of modified and nucleotide surrogate iRNA agents is discussed below.
FORMULATION
The iRNA agents described herein can be formulated for administration to a subject For ease of exposition the formulations, compositions and methods in this section are 1 o discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.
A formulated iRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less 15 than ~0, 50, 30, 20, or 10% water). In another example, the iRNA is in an aqueous phase, e.g., in a solution that includes water.
The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA
2o composition is formulated in a manner that is compatible with the intended method of administration (see, below) In particular embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and 25 other self assembly.
A iRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to Attorney's Docket No.: 14174-072W01 form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mgz+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
In one embodiment, the iRNA preparation includes another iRNA agent, e.g., a second iRNA that can mediated RNAi with respect to a second gene, or with respect to the same gene.
Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different iRNA species. Such iRNAs can mediated RNAi with respect to a similar number of different genes.
In one embodiment, the iRNA preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, a iRNA composition for the treatment of a viral disease, e.g. HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a iRNA
composition for the treatment of a cancer might further comprise a chemotherapeutic agent.
Exemplary formulations are discussed below:
Li osomes For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents;
e.g., modified iRNA s agents, and such practice is within the invention. An iRNA agent, e.g., a 2o double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA

Attorney's Docket No.: 14174-072W01 composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA
to particular cell types, e.g., to cells of the liver, such as those described herein.
A liposome containing a iRNA can be prepared by a variety of methods.
In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA preparation is then added to the 15 micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA and condense around the iRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA.
If necessary a Garner compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a 2o polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.
Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one 25 or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678;
Bangham, et al. M.
Mol. Biol. 23:238, 1965; Olson, et al. Biochim. BioplZys. Acta 557:9, 1979;
Szoka, et al. Proc.
Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukllnaga, et al. Endocrinol.
115:757, 1984.

Attorney's Docket No.: 14174-072W01 Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Bioclzina.
Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Bioclzina.
Bioplays. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA
preparations into liposomes.
Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., .Iournal of Controlled Release, 19, (1992) 269-274).
One major type of liposomal composition includes phospholipids other than naturally-~ 5 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 2o as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93J24640; WO
91116024;
Felgner, J. Biol. Claem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci.
90:11307, 1993; Nabel, 2s Human Gene Ther. 3:649, 1992; Gershon, Bioclaena. 32:7143, 1993; and Strauss EMBO J.
11:417, 1992.
In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able Attorney's Docket No.: 14174-072W01 to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNAs to macrophages.
Further advantages of liposomes include: liposames obtained from natural phospholipids are biocornpatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms,"
Lieberman, Rieger and Banker (Eds.), 1988, volume l, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge; vesicle size and the aqueous volume of the liposomes.
A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-1o trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles.
LipofectinTM
Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to 2o form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and r efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.
Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine Attorney's Docket No.: 14174-072W01 dioctaoleoylamide ("DOGS") (TransfectamTM, Promega, Madison, Wisconsin) and dipalinitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat.
No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Chol") which has been formulated into liposomes in combination with DOPE
(See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991).
Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions.
Other commercially available cationic lipid products include DMRIE and DMRIE-HP
(Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland).
Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98139359 and WO 96/37194.
Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA, into the skin. In some implementations, liposomes are used for delivering iRNA to epidermal cells and also to 2o enhance the penetration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., .Iourraal ofDrug Targetitag, 1992, vol.
2,405-410 and du Plessis et al., Arativiral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987;
Nicolau, C. et al. Meth.
Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz.
101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl Attorney's Docket No.: 14174-072W01 dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II
(glyceryl distearate/
cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA are useful for treating a dermatological disorder.
Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes.
Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA can be delivered, for example, subcutaneously by infection in order to deliver iRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self optimizing (adaptive to the shape of pores, e.g., in the skin), self repairing, and can frequently reach their targets without fragmenting, and often self loading. The iRNA agents can include an RRMS tethered to a moiety which improves association with a liposome.
Surfactants For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, 2o e.g., modified iRNA agents, and such practice is within the invention.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). iRNA (or a precursor, e.g., a larger dsRNA which can be processed into a iRNA, or a DNA which encodes a iRNA or precursor) compositions can include a surfactant.
In one embodiment, the iRNA is formulated as an emulsion that includes a surfactant.
The most 2s common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms," Marcel Dekker, Inc., New York, N'Y, 1988, p. 285).

Attorney's Docket No.: 14174-072W01 If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.
Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylatedlpropoxylated 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 allcyl 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 irl 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.
2o 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, NY, 1988, p. 285).
Micelles and other Membranous Formulations For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these micelles and other formulations, compositions and methods can Attorney's Docket No.: 14174-072W01 be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) composition can be provided as a micellar formulation. "Micelles"
are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the iRNA composition, an alkali metal C8 to C2a alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, .
~5 monoolein, monooleates, monolaurates, borage oil, evening ofprimrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal 2o alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing is preferred in order to provide smaller size micelles.
In one method a first micellar composition is prepared which contains the iRNA
composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In 25 another method, the micellar composition is prepared by mixing the iRNA
composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be Attorney's Docket No.: 14174-072W01 added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
1o The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).
The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
The iRNA agents can include an RRMS tethered to a moiety which improves association with a micelle or other membranous formulation.
Particles 2o For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these particles, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In another embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent, or a DNA
which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including Attorney's Docket No.: 14174-072W01 lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. See below for further description.
Sustained-Release Formulations. An iRNA agent, e.g., a double-stranded iRNA
agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA
agent, or sRNA
agent, or precursor thereof) described herein can be formulated for controlled, e.g., slow release.
Controlled release can be achieved by disposing the iRNA within a structure or substance which impedes its release. E.g., iRNA can be disposed within a porous matrix or in an erodable matrix, either of which allow release of the iRNA over a period of time.
Polymeric particles, e.g., polymeric in microparticles can be used as a sustained-release reservoir of iRNA that is taken up by cells only released from the microparticle through biodegradation. The polymeric particles in this embodiment should therefore be large enough to preclude phagocytosis (e.g., larger than 10 ~,m and preferably larger than 20 ~,m). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of ~ 5 the first and second emulsions. That is to say, a lower homogenization speed, vortex mixing speed, or sonication setting can be used to obtain particles having a diameter around 100 ~,m rather than 10 ,um. The time of mixing also can be altered.
Larger microparticles can be formulated as a suspension, a powder, or an implantable solid, to be delivered by intramuscular, subcutaneous, intradermal, intravenous, or intraperitoneal 2o injection; via inhalation (intranasal or intrapulmonary); orally; or by implantation. These particles are useful for delivery of any iRNA when slow release over a relatively long term is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation.
Microparticles preferably include pores, voids, hollows, defects or other interstitial 25 spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstructures can be used to form hollow, porous spray dried microspheres.

Attorney's Docket No.: 14174-072W01 Polymeric particles containing iRNA (e.g., a sRNA) can be made using a double emulsion technique, for instance. First, the polymer is dissolved in an organic solvent. A
preferred polymer is polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic acid suspended in aqueous solution is added to the polymer solution and the two solutions are mixed to form a first emulsion. The solutions can be mixed by vortexing or shaking, and in a preferred method, the mixture can be sonicated.
Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. For example, acceptable results can be obtained with a Vibra-cell model VC-250 sonicator with a 1/8" microtip probe, at setting #3.
Spray-Drying. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA
which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof)) can be prepared by spray drying. Spray dried iRNA can be administered to a subject or ~ 5 be subjected to further formulation. A pharmaceutical composition of iRNA
can be prepared by spray drying a homogeneous aqueous mixture that includes a il2NA under conditions sufficient to provide a dispersible powdered composition, e.g., a pharmaceutical composition. The material for spray drying can also include one or more of a pharmaceutically acceptable excipient, or a dispersibility-enhancing amount of a physiologically acceptable, water-soluble protein. The 2o spray-dried product can be a dispersible powder that includes the iRNA.
Spray drying is a process that converts a liquid or slurry material to a dried particulate form. Spray drying can be used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in:
Inhalation Aerosols:
Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996.
25 Spray drying can include atomizing a solution, emulsion, or suspension to form a fine mist of droplets and drying the droplets. The mist can be projected into a drying chamber (e.g., a vessel, tank, tubing, or coil) where it contacts a drying gas. The mist can include solid or liquid pore forming agents. The solvent and pore forming agents evaporate from the droplets into the Attorney's Docket No.: 14174-072W01 drying gas to solidify the droplets, simultaneously forming pores throughout the solid. The solid (typically in a powder, particulate form) then is separated from the drying gas and collected.
Spray drying includes bringing together a highly dispersed liquid, and a sufficient volume of air (e.g., hot air) to produce evaporation and drying of the liquid droplets. The preparation to be spray dried can be any solution, course suspension, slurry, colloidal -dispersion, or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. can effectively produce particles of desired size.
Spray-dried powdered particles can be approximately spherical in shape, nearly uniform in size and frequently hollow. There may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dried microspheres appears to be more effective if an inflating agent ~ 5 (or blowing agent) is used in their production. Particularly preferred embodiments may comprise an emulsion with an inflating agent as the disperse or continuous phase (the other phase being aqueous in nature). An inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 psi.
This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically 2o comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g.
perfluorohexane, perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous 25 aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, freons, and hydrocarbons. Nitrogen gas and carbon dioxide are also contemplated as a suitable blowing agent.
Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required and Attorney's Docket No.: 14174-072W01 an aqueous dispersion of the medicament and surfactants) are spray dried directly. In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that generally lead to the formation of hollow, relatively porous microparticles.
Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques.
The perforated microstructures may optionally be associated with, or comprise, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that the use of surfactants may further increase dispersion stability, simplify formulation procedures or increase bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By "associated with or comprise" it is meant that the structural matrix or perforated microstructure may incorporate, adsorb, absorb, be coated with or be formed by the surfactant.
Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants.
2o Particularly preferred surfactants are substantially insoluble in the propellant, nonfluorinated, and selected from.the group consisting of saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents.
It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e.
biocompatible) fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations.
Lipids, including phospholipids, from both natural and synthetic sources may be used in varying concentrations to form a structural matrix. Generally, compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 40° C. Preferably, the incorporated lipids are relatively long chain (i.e. C6 -C22) saturated lipids and more preferably Attorney's Docket No.: 14174-072W01 comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations comprise egg phosphatidylcholine, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalinitoylphosphatidyl-choline, disteroylphosphatidylcholine, short-chain phosphatidylcholines, phosphatidylethanolamine, dioleylphosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as, polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid;
cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent 1 o biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein.
Compatible nonionic detergents comprise: sorbitan esters including sorbitan trioleate (SpansTM 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and' sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Preferred block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic® F68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In preferred embodiments, the microstructures may comprise oleic acid or its alkali salt.
In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA
agent, or sRNA
agent, or precursor thereof). Examples of suitable cationic lipids include:
DOTMA, N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP,1,2-dioleyloxy-3- .
(trimethylammonio)propane; and DOTB, 1,2-dioleyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol. Polycationic amino acids such as polylysine, and polyarginine are also contemplated.

Attorney's Docket No.: 14174-072W01 For the spraying process, such spraying methods as rotary atomization, pressure atomization and two-fluid atomization can be used. Examples of the devices used in these processes include "Parubisu [phonetic rendering] Mini-Spray GA-32" and "Parubisu Spray Drier DL-41 ", manufactured by Yamato Chemical Co., or "Spray Drier CL-8," "Spray Drier L-8,"
"Spray Drier FL-12," "Spray Drier FL-16" or "Spray Drier FL-20," manufactured by Okawara I~akoki Co., can be used for the method of spraying using rotary-disk atomizer.
While no particular restrictions are placed on the gas used to dry the sprayed material, it is recommended to use air, nitrogen gas or an inert gas. The temperature of the inlet of the gas used to dry the sprayed materials such that it does not cause heat deactivation of the sprayed 1 o material. The range of temperatures may vary between about 50°C to about 200°C, preferably between about 50°C and 100°C. The temperature of the outlet gas used to dry the sprayed material, may vary between about 0°C and about 150°C, preferably between 0°C and 90°C, and even more preferably between 0°C and 60°C.
The spray drying is done under conditions that result in substantially amorphous powder 15 of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the resulting powder is such that more than about 98% of the mass is in particles having a diameter of about 10 ~,m or less with about 90% of the mass being in particles having a diameter less than ,um. Alternatively, about 95% of the mass will have particles with a diameter of less than 10 20 ,um with about 80% of the mass of the particles having a diameter of less than 5 ~.m.
The dispersible pharmaceutical-based dry powders that include the iRNA
preparation may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the iRNA concentration in the powder which is being delivered to a 25 patient, but may also serve to enhance the stability of the iRNA
compositions and to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the iRNA and to improve handling characteristics of the iRNA such as flowability and consistency to facilitate manufacturing and powder filling.

Attorney's Docket No.: 14174-072W01 Such carrier materials may be combined with the drug prior to spray drying, i.
e., by adding the Garner material to the purified bulk solution. In that way, the Garner particles will be formed simultaneously with the drug particles to produce a homogeneous powder.
Alternatively, the carriers may be separately prepared in a dry powder form and combined with the dry powder drug by blending. The powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous. The size of the carrier particles may be selected to improve the flowability of the drug powder, typically being in the range from 25 ~,m to 100 ,um. A preferred carrier material is crystalline lactose having a size in the above-stated range.
Powders prepared by any of the above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other purposes, it will frequently be desirable to disrupt any agglomerates which may have formed by screening or other conventional techniques. For pharmaceutical uses, the dry powder formulations will usually be measured into a single dose, and the single dose sealed into a package. Such packages ~ 5 are particularly useful for dispersion in dry powder inhalers, as described in detail below.
Alternatively, the powders may be packaged in multiple-dose containers.
Methods for spray drying hydrophobic and other drugs and components are described in IJ.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602; and 4,486,435.
Bloch and Speison (1983) Pharm. Acta Helv 58:14-22 teaches spray drying of hydrochlorothiazide and 2o chlorthalidone (lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A number of Japanese Patent application Abstracts relate to spray drying of hydrophilic-hydrophobic product combinations, including JP
806766; JP 7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP
4036233.
Other foreign patent publications relevant to spray drying hydrophilic-hydrophobic product 25 combinations include FR 2594693; DE 2209477; and WO 88107870.
LYOPHILIZATION.
An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA
which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) Attorney's Docket No.: 14174-072W01 preparation can be made by lyophilization. Lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. The particular advantage associated with the lyophilization process is that biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can be dried without elevated temperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems.
With respect to the instant invention such techniques are particularly compatible with the incorporation of nucleic acids in perforated microstructures without compromising physiological activity Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstructures having the desired porosity and size, they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.
Tar~etin~
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNAs. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA
agents, e.g., modified iRNA agents, and such practice is within the invention.
In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
2o agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof) is targeted to a particular cell. For example, a liposome or particle or other structure that includes a iRNA can also include a targeting moiety that recognizes a specific molecule on a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer-specific antigen of the liver or a viral antigen, thus delivering the iRNA to a cancer cell or a virus-infected cell. Exemplary targeting moieties include antibodies (such as IgM, IgG, IgA, Attorney's Docket No.: 14174-072W01 IgD, and the like, or a functional portions thereof), ligands for cell surface receptors (e.g., ectodomains thereof).
An antigen, such as a-feto protein, can be used to target an iRNA to a liver cell.
In one embodiment, the targeting moiety is attached to a liposome. For example, US
Patent 6,245,427 describes a method for targeting a liposome using a protein or peptide. In another example, a cationic lipid component of the liposome is derivatized with a targeting moiety. For example, WO 96/37194 describes converting N-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimide activated ester. The product was then coupled to an RGD peptide.
~ o GENES AND DISEASES
In one aspect, the invention features, a method of treating a subj ect at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmalignant cell proliferation. The method includes:
providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA
having a structure described herein, where the iRNA is homologous to and can silence, e.g., by cleavage, a gene which promotes unwanted cell proliferation;
administering an iRNA agent, e.g., an sRNA or iRNA agent described herein to a subject, preferably a human subj ect, thereby treating the subject.
2o In a preferred embodiment the gene is a growth factor or growth factor receptor gene, a kinase, e.g., a protein tyrosine, serine or threonine kinase gene, an adaptor protein gene, a gene encoding a G protein superfamily molecule, or a gene encoding a transcription factor.
In a preferred embodiment the iRNA agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers.

Attorney's Docket No.: 14174-072W01 In another preferred embodiment the iRNA agent silences the Erb-B gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erb-B
expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences the Src gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Src expression, e.g., colon cancers.
In a preferred embodiment the iRNA agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers.
In a preferred embodiment the iRNA agent silences the GRB2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma.
In another preferred embodiment the iRNA agent silences the RAS gene, and thus can be used to treat a subj ect having or at risk for a disorder characterized by unwanted RAS
expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.
In another preferred embodiment the iRNA agent silences the MEKK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MEKK
expression, e.g., squamous cell carcinoma, melanoma or leukemia.
In another preferred embodiment the iRNA agent silences the JNK gene, and thus can be 2o used to treat a subj ect having or at risk for a disorder characterized by unwanted JNK expression, e.g., pancreatic or breast cancers.
In a preferred embodiment the iRNA agent silences the RAF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAF expression, e.g., lung cancer or leukemia.
In a preferred embodiment the iRNA agent silences the Erkl/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.

Attorney's Docket No.: 14174-072W01 In another preferred embodiment the iRNA agent silences the PCNA(p21) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA
expression, e.g., lung cancer.
In a preferred embodiment the iRNA agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia.
In a preferred embodiment the iRNA agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC
expression, e.g., Burkitt's lymphoma or neuroblastoma.
1o In another preferred embodiment the iRNA agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, e.g., ovarian, prostate or breast cancers.
In another preferred embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subj ect having or at risk for a disorder characterized by unwanted FOS expression, 15 e.g., skin or prostate cancers.
In a preferred embodiment the iRNA agent silences the BCL-2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma.
In a preferred embodiment the iRNA agent silences the Cyclin D gene, and thus can be 2o used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin D
expression, e.g., esophageal and colon cancers.
In a preferred embodiment the iRNA agent silences the VEGF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers.
25 In a preferred embodiment the iRNA agent silences the EGFR gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EGFR expression, e.g., breast cancer.

Attorney's Docket No.: 14174-072W01 In another preferred embodiment the iRNA agent silences the Cyclin A gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin A
expression, e.g., lung and cervical cancers.
In another preferred embodiment the iRNA agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E
expression, e.g., lung and breast cancers.
In another preferred embodiment the iRNA agent silences the WNT-1 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.
In another preferred embodiment the iRNA agent silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta-catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma.
In another preferred embodiment the iRNA agent silences the c-MET gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MET
expression, e.g., hepatocellular carcinoma.
In another preferred embodiment the iRNA agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC
expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences the STAT3 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer.
In another preferred embodiment the iRNA agent silences the survivin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted survivin expression, e.g., cervical or pancreatic cancers.

Attorney's Docket No.: 14174-072W01 In another preferred embodiment the iRNA agent silences the Her2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her2/Neu expression, e.g., breast cancer.
In another preferred embodiment the iRNA agent silences the topoisomerase I
gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase I expression, e.g., ovarian and colon cancers.
In a preferred embodiment the iRNA agent silences the topoisomerase II alpha gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase II expression, e.g., breast and colon cancers.
1 o In a preferred embodiment the iRNA agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma.
In a preferred embodiment the iRNA agent silences mutations in the p21(WAFl/CIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by ~5 unwanted p21(WAF1/CIPl) expression, e.g., liver cancer.
In a preferred embodiment the iRNA agent silences mutations in the p27(I~IP1) gene, and thus can be used to treat a subj ect having or at risk for a disorder characterized by unwanted p27(KIP1) expression, e.g., liver cancer.
In preferred embodiments the iRNA agent silences mutations in tumor suppressor genes, 2o and thus can be used as a method to promote apoptotic activity in combination with chemotherapeutics.
In another aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer. The method includes:
25 providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis;

Attorney's Docket No.: 14174-072W01 administering the iRNA agent to a subject, thereby treating the subject.
In another aspect, the invention features a method of treating a subject infected with a virus or at risk for or afflicted with a disorder or disease associated with a viral infection. The method includes:
providing an iRNA agent, e.g., and iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth;
administering the iRNA agent to a subject, preferably a human subject, thereby treating the subject.
Thus, the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection.
15 In a preferred embodiment, the expression of a HPV gene is reduced. In another preferred embodiment, the HPV gene is one of the group of E2, E6, or E7.
In a preferred embodiment the expression of a human gene that is required for HPV
replication is reduced.
The invention also includes a method of treating patients infected by the Human 2o Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS).
In a preferred embodiment, the expression of a HIV gene is reduced. In another preferred embodiment, the HIV gene is CCRS, Gag, or Rev.
In a preferred embodiment the expression of a human gene that is required for HIV
25 replication is reduced. In another preferred embodiment, the gene is CD4 or Tsg101.

Attorney's Docket No.: 14174-072W01 The invention also includes a method for treating patients infected by the Hepatitis B
Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma.
In a preferred embodiment, the expression of a HBV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region.
In another preferred embodiment, a targeted HBV-RNA sequence is comprised of the poly(A) tail.
In preferred embodiment the expression of a human gene that is required for HBV
replication is reduced.
The invention also provides for a method of treating patients infected by the Hepatitis A
Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV.
In a preferred embodiment the expression of a human gene that is required for HAV
replication is reduced.
The present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis In a preferred embodiment, the expression of a HCV gene is reduced.
In another preferred embodiment the expression of a human gene that is required for HCV replication is reduced.
The present invention also provides for a method of treating patients infected by the any of the group of Hepatitis Viral strains comprising hepatitis D, E, F, G, or H, or patients at risk for or afflicted with a disorder mediated by any of these strains of hepatitis.
In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, or H
gene is reduced.
In another preferred embodiment the expression of a human gene that is required for hepatitis D, E, F, G or H replication is reduced.

Attorney's Docket No.: 14174-072W01 Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.
In a preferred embodiment, the expression of a RSV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of genes N, L, or P.
In a preferred embodiment the expression of a human gene that is required for RSV
replication is reduced.
Methods of the invention provide for treating patients infected by the Herpes Simplex Virus (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g, genital herpes and cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients.
In a preferred embodiment, the expression of a HSV gene is reduced. In another preferred embodiment, the targeted HSV gene encodes DNA polymerase or the helicase-primase.
In a preferred embodiment the expression of a human gene that is required for HSV
replication is reduced.
The invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., 2o congenital virus infections and morbidity in immunocompromised patients.
In a preferred embodiment, the expression of a CMV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for CMV
replication is reduced.
Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment, the expression of a EBV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for EBV
replication is reduced.
Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also called human herpesvirus ~, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.
In a preferred embodiment, the expression of a KSHV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for KSHV
replication is reduced.
The invention also includes a method for treating patients infected by the JC
Virus (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal leukoencephalopathy (PML).
In a preferred embodiment, the expression of a JCV gene is reduced.
~ 5 In preferred embodiment the expression of a human gene that is required for JCV
replication is reduced.
Methods of the invention also provide for treating patients infected by the myxovirus or at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza.
In a preferred embodiment, the expression of a myxovirus gene is reduced.
2o In a preferred embodiment the expression of a human gene that is required for myxovirus replication is reduced.
Methods of the invention also provide for treating patients infected by the rhinovirus or at risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold.
In a preferred embodiment, the expression of a rhinovirus gene is reduced.

Attorney's Docket No.: 14174-072W01 In preferred embodiment the expression of a human gene that is required for rhinovirus replication is reduced.
Methods of the invention also provide for treating patients infected by the coronavirus or at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold.
s In a preferred embodiment, the expression of a coronavirus gene is reduced.
In preferred embodiment the expression of a human gene that is required for coronavirus replication is reduced.
Methods of the invention also provide for treating patients infected by the flavivirus West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.
1 o In a preferred embodiment, the expression of a West Nile Virus gene is reduced. In another preferred embodiment, the West Nile Virus gene is one of the group comprising E, NS3, or NSS.
In a preferred embodiment the expression of a human gene that is required for West Nile , Virus replication is reduced.
15 Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this virus, e.g., viral haemorrhagic fever or neurological disease.
In a preferred embodiment, the expression of a St. Louis Encephalitis gene is reduced.
In a preferred embodiment the expression of a human gene that is required for St. Louis 2o Encephalitis virus replication is reduced.
Methods of the invention also provide for treating patients infected by the Tick-borne encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease.
In a preferred embodiment, the expression of a Tick-borne encephalitis virus gene is 25 reduced.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment the expression of a human gene that is required for Tick-borne encephalitis virus replication is reduced.
Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and s neurological disease.
In a preferred embodiment, the expression of a Murray Valley encephalitis virus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced.
The invention also includes methods fox treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever.
In a preferred embodiment, the expression of a dengue virus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for dengue virus replication is reduced.
15 Methods of the invention also provide for treating patients infected by the Simian Virus 40~(SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis.
In a preferred embodiment, the expression of a SV40 gene is reduced.
In a preferred embodiment the expression of a human gene that is required for replication is reduced.
2a The invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.
In a preferred embodiment, the expression of a HTLV gene is reduced. In another preferred embodiment the HTLV 1 gene is the Tax transcriptional activator.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment the expression of a human gene that is required for HTLV
replication is reduced.
Methods of the invention also provide for treating patients infected by the Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.
In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mo-MuLV
replication is reduced.
Methods of the invention also provide for treating patients infected by the encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation.
In a preferred embodiment, the expression of a EMCV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for EMCV
replication is reduced.
The invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles.
In a preferred embodiment, the expression of a MV gene is reduced.
2o In a preferred embodiment the expression of a human gene that is required for MV
replication is reduced.
The invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g.
chicken pox or shingles (also called zoster).
In a preferred embodiment, the expression of a VZV gene is reduced.

Attorney's DocketNo.: 14174-072W01 In a preferred embodiment the expression of a human gene that is required for VZV
replication is reduced.
The invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g.
respiratory tract infection.
In a preferred embodiment, the expression of an adenovirus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for adenovirus replication is reduced.
The invention includes a method for treating patients infected by a yellow fever virus (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g.
respiratory tract infection.
In a preferred embodiment, the expression of a YFV gene is reduced. In another preferred embodiment, the preferred gene is one of a group that includes the E, NS2A, or NS3 genes.
In a preferred embodiment the expression of a human gene that is required for YFV
15 replication is reduced.
Methods of the invention also provide for treating patients infected by the poliovirus'or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio.
In a preferred embodiment, the expression of a poliovirus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for poliovirus 2o replication is reduced.
Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox In a preferred embodiment, the expression of a poxvirus gene is reduced.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment the expression of a human gene that is required for poxvirus replication is reduced.
In another, aspect the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen. The method includes:
providing a iRNA agent, e.g., a siRNA having a structure described herein, where siRNA
is homologous to and can silence, e.g., by cleavage of a pathogen gene;
administering the iRNA agent to a subject, prefereably a human subject, thereby treating the subject.
The target gene can be one involved in growth, cell wall synthesis, protein synthesis, 1o transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.
Thus, the present invention provides for a method of treating patients infected by a plasmodium that causes malaria.
In a preferred embodiment, the expression of a plasmodium gene is reduced. In another preferred embodiment, the gene is apical membrane antigen 1 (AMAl).
15 In a preferred embodiment the expression of a human gene that is required for plasmodium replication is reduced.
The invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers In a preferred embodiment, the expression of a Mycobacterium ulcerans gene is reduced.
2o In a preferred embodiment the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced.
The invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g.
tuberculosis.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment, the expression of a Mycobacterium tuberculosis gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mycobacterium tuberculosis replication is reduced.
The invention also includes methods for treating patients infected by the Mycobacterium leprae, or a disease or disorder associated with this pathogen, e.g. leprosy.
In a preferred embodiment, the expression of a Mycobacterium leprae gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mycobacterium leprae replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and muscous membranes. .
In a preferred embodiment, the expression of a Staphylococcus aureus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for ~ 5 Staphylococcus aureus replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g.
pneumonia or childhood lower respiratory tract infection.
In a preferred embodiment, the expression of a Streptococcus pneumoniae gene is 2o reduced.
In a preferred embodiment the expression of a human gene that is required for Streptococcus pneumoniae replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat 25 or Scarlet fever.

Attorney's Docket No.: 14174-072W01 In a preferred embodiment, the expression of a Streptococcus pyogenes gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Streptococcus pyogenes replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection In a preferred embodiment, the expression of a Chlamydia pneumoniae gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection In a preferred embodiment, the expression of a Mycoplasma pneumoniae gene is reduced.
In a preferred embodiment the expression of a human gene that is required for ~ 5 Mycoplasma pneumoniae replication is reduced.
The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is 20 heterozygous in euploid cells but is hemizygous in cells having LOH. The regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth. Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent of the invention. The iRNA
25 agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH. In essence, it discriminates between the two alleles, preferentially silencing the selected Attorney's Docket No.: 14174-072W01 allele. In essence polymorphisms, e.g., SNPs of essential genes that are affected by LOH, are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH.
E.g., one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene. The gene encoding the large subunit of human RNA polymerase II, POLR2A, a geiae located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells. Other genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA
associated factor 2H, ribosomal protein 514, eukaryotic initiation factor SA, alanyl tRNA
synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor.
Accordingly, the invention features, a method of treating 'a disorder characterized by LOH, e.g., cancer. The method includes:
optionally, determining the genotype of the allele of a gene in the region of LOH and 15 preferably determining the genotype of both alleles of the gene in a normal cell;
providing an iRNA agent which preferentially cleaves or silences the allele found in the LOH cells;
administerning the iRNA to the subject, thereby treating the disorder.
2o The invention also includes a iRNA agent disclosed herein, e.g, an iRNA
agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene In another aspect, the invention provides a method of cleaving or silencing more than one gene with an iRNA agent. In these embodiments the iRNA agent is selected so that it has sufficient homology to a sequence found in more than one gene. For example, the sequence 2s AAGCTGGCCCTGGACATGGAGAT (SEQ ID N0:6719) is conserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agent targeted to this sequence would effectively silence the entire collection of genes.

Attorney's Docket No.: 14174-072W01 The invention also includes an iRNA agent disclosed herein, which can silence more than one gene.
ROUTE OF DELIVERY
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A
composition that includes a iRNA can be delivered to a subject by a variety of routes.
Exemplary routes include:
intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
1 o The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable Garner. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and ~5 the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a 2o number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
25 The route and site of administration may be chosen to enhance targeting.
For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice.
Lung cells might be targeted by administering the iRNA in aerosol form. The vascular Attorney's Docket No.: 14174-072W01 endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical Garners, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
In the case of tablets, Garners that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
Intraventricular injection may be 2o facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl 2s methylcellulose or polyvinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.

Attorney's Docket No.: 14174-072.W01 Topical Delivery For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In a preferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is delivered to a subject via topical administration. "Topical administration" refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most commontopical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barner and efficient delivery to the target tissue or stratum.
Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to 2o an underlying tissue.
The term "skin," as used herein, refers to the epidermis and/or dermis of an animal.
Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum corneum being at the surface of the skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 ~,m and 0.2 mm thick, depending on its location on the body.
Beneath the epidermis is the dermis, which is significantly thicker than the epidermis.
The dermis is primarily composed of collagen in the form of fibrous bundles.
The collagenous Attorney's Docket No.: 14174-072W01 bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings and immunologically active cells.
One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in vaxious states of differentiation. The spaces between cells in the stratum corneum is filled with different lipids arranged in lattice-like formations that provide seals to further enhance the skins permeability barrier.
The permeability barrier provided by the skin is such that it is largely impermeable to molecules having molecular weight greater than about 750 Da. For laxger molecules to cross the 1 o skin's permeability barrier, mechanisms other than normal osmosis must be used.
Several factors determine the permeability of the skin to administered agents.
These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the ~5 epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum.
Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic 2o conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition 2s disclosed herein for systemic andlor local therapy.
In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the Attorney's Docket No.: 14174-072W01 absorption of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
The compositions and methods provided may also be used to examine the function of various proteins and genes irz vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention 1o can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, ertythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, I~aposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin.
~5 PulmonaryDelivery For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes an 2o iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be administered to a subj ect by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably iRNA, 25 within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved Attorney's Docket No.: 14174-072W01 with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA
composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol 1 o medicament.
The term "powder" means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be "respirable." Preferably the average particle size is ~ 5 less than about 10 ~.m in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 ,um and most preferably less than about 5.0 ~.m. Usually the particle size distribution is between about 0.1 ~m and about 5 ~,m in diameter, particularly about 0.3 ,um to about 5 ,um.
The term "dry" means that the composition has a moisture content below about 10% by 2o weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
The term "therapeutically effective amount" is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated 2s physiological response.
The term "physiologically effective amount" is that amount delivered to a subject to give the desired palliative or curative effect.

Attorney's Docket No.: 14174-072W01 The term "pharmaceutically acceptable carrier" means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.
The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These Garners may be in a crystalline or amorphous form or may be a mixture of the two.
Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like;
alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.
~ 5 Additives, which axe minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.
Suitable pH adjusters or buffers include organic salts prepared from organic acids and 2o bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC
and CFC propellants.
25 ' Oral or Nasal Delivery For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, Attorney's Docket No.: 14174-072W01 that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.
In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
The ability of molecules to permeate through the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules, less than 1000 15 daltons appear to cross mucosa rapidly. As molecular size increases, the permeability decreases rapidly. Lipid soluble compounds are more permeable than non-lipid soluble molecules.
Maximum absorption occurs when molecules are un-ionized or neutral in electrical charges.
Therefore charged molecules present the biggest challenges to absorption through the oral mucosae.
2o A pharmaceutical composition of iRNA may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
25 Devices For ease of exposition the devices, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these devices, formulations, compositions and methods can be practiced with other Attorney's Docket No.: 14174-072W01 iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. An iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be disposed on or in a device, e.g., a device which implanted or otherwise placed in a subject.
Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stems, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stems, can be placed in the vasculature of the lung, heart, or leg.
1 o Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a iRNA, e.g., a device can release insulin.
Other devices include artificial joints, e.g., hip joints, and other orthopedic implants.
In one embodiment, unit doses or measured doses of a composition that includes iRNA
~ 5 are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.
Tissue, e.g., cells or organs, such as the liver, can be treated with an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which 2o can be processed into a sRNA agent, or a DNA which encodes an iRNA agents e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivo and then administered or implanted in a subject.
The tissue can be autologous, allogeneic, or xenogeneic tissue. For example, tissue (e.g., liver) can be treated to reduce graft v. host disease. In other embodiments, the tissue is 25 allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue, such as in the liver. In another example, tissue containing hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.

Attorney's Docket No.: 14174-072W01 Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies.
In some implementations, the iRNA treated cells are insulated from other cells, e.g., by a semi-permeable porous barner that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body.
In one embodiment, the porous barrier is formed from alginate.
In one embodiment, a contraceptive device is coated with or contains an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include condoms, diaphragms, ICTD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices. In one embodiment, the iRNA is chosen to inactive sperm or egg. In another embodiment, the iRNA is chosen to be complementary to a viral or pathogen RNA, e.g., an RNA
of an STD. In some instances, the iRNA composition can include a spermicide.

In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, 20 (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3' overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4 x 1016 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 25 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.
The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA, such as an RNA
present in the liver.

Attorney's Docket No.: 14174-072W01 The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.
In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.
In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral 1 o agent other than an iRNA agent, e.g., other than a double-stranded iRNA
agent, or sRNA agent,.
In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.
In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g:, a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent, or a DNA
which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or 2o doses ranging from 0.01 ,ug to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every S, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage Attorney's Docket No.: 14174-072W01 levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stmt (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are 1o non-overlapping and non-adjacent to another species with respect to a naturally occurnng target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurnng target genes. In another embodiment, the iRNA agent is allele specific.
In some cases, a patient is treated with a iRNA agent in conjunction with other therapeutic modalities. For example, a patient being treated for a liver disease can be administered an iRNA agent specific for a target gene known to enhance the progression of the disease in conjunction with a drug known to inhibit activity of the target gene product. For example, a patient being treated for a cancer of the liver can be administered an iRNA agent specific for a target essential for tumor cell proliferation in conjunction with a chemotherapy Following successful treatment, it may be desirable to have the patient undergo 2o maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 ~,g to 100 g per kg of body weight (see US 6,107,094).
The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary.
For example, nasal formulations tend to require much lower concentrations of some ingredients in order to Attorney's Docket No.: 14174-072W01 avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.
Certain factors may influence the dosage 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 an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof) can include a single treatment or, preferably, can include a series of 1o treatments. It will also be appreciated that the effective dosage of a iRNA
agent such as a sRNA
agent used for treatment may increase or decrease over the course of a particular treatment.
Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA
~5 agent composition can be administered.
Dosing is dependent on severity and responsiveness of the disease condition to be treated,.
with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can 2o easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on ECSOs found to be effective in in vitYO and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g. a gene that produces a target RNA. The transgenic animal can be deficient for the corresponding 25 endogenous RNA. In another embodiment, the composition for testing includes a iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RNA in the animal model and the target RNA in a human.

Attorney's Docket No.: 14174-072W01 The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.
In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA
agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, , topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in 1 o measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.
Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent , or a DNA
which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3' overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.
The medication can be delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.
The rectal administration of the iRNA agent is by means of an enema. The iRNA
agent of the enema can be dissolved in a saline or buffered solution. The rectal administration can also Attorney's Docket No.: 14174-072W01 by means of a suppository, which can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.
Any of the iRNA agents described herein can be administered orally, e.g., in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. Further, the composition can be applied topically to a surface of the oral cavity.
Any of the iRNA agents described herein can be administered buccally. For example, the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one 1o embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.
Any of the iRNA agents described herein can be administered to ocular tissue.
For example, the medications can be applied to the surface of the eye or nearby tissue, e.g., the inside ~5 of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subj ect or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or 2o structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.
Any of the iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably 25 not into the underlying muscle tissue. Administration of the iRNA agent composition can be topical. Topical applications can, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration can be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic Attorney's Docket No.: 14174-072W01 molecular assembly. The transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.
Any of the iRNA agents described herein can be administered to the pulmonary system.
Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication. A preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.
Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.
iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.
Any of the iRNA agents described herein can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g.; by introducing a delivery device which can dispense the medication. Methods of nasal delivery include spray, aerosol, liquid, e.g., by drops, or by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser with delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered 2o dose of medication. The subject, or another person, can administer the medication.
Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.
An iRNA agent can be packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

Attorney's DocketNo.: 14174-072W01 The dosage of a pharmaceutical composition including a iRNA agent can be administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease. A
subject can be treated with the pharmaceutical composition by any of the methods mentioned above.
Gene expression in a subject can be modulated by administering a pharmaceutical composition including an iRNA agent.
A subject can be treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form, e.g., a collection of microparticles, such as crystalline particles. The composition can include a plurality of iRNA
agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.
A subject can be treated by administering a defined amount of an iRNA agent composition that is prepared by a method that includes spray-drying, i. e.
atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs.
The method can include other features described herein.
The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, 2o e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), can be provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery.
This can include providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol 25 composition can be provided in and/or dispensed by a metered dose delivery device.
The subject can be treated for a condition treatable by inhalation, e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA
which encodes Attorney's Docket No.: 14174-072W01 an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.
A subject can be treated by, for example, administering a composition including an effective/defmed amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference ~ 5 value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subj ect. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or 2o characterization (e.g., rate of cell proliferation and/or tumor mass, viral load).
In another aspect, the invention features a method that includes:
administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA
agent, or sRNA
25 agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount Attorney's Docket No.: 14174-072W01 of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.
In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3' overhang 1-5 nt long. In one embodiment, the source releases ds iRNA
agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.
In one aspect, the invention features a pharmaceutical composition that includes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., 15 a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA
agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3' overhang 1-5 nt long. In one 2o embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.
In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic 25 liposomes.
In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration Attorney's Docket No.: 14174-072W01 enhancer. In one embodiment, the topical penetration enhancer is a fatty acid.
The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcartutine, an acylcholine, or a C1_io alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, ~ o polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.
In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.
In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic, 15 surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.
In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic areas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof. In yet another embodiment the penetration enhancer 20 can be a glycol, a pyrrol, an azone, or a terpenes.
In one aspect, the invention features a pharmaceutical composition including an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral 25 delivery. In one embodiment, oral delivery can be used to deliver an iRNA
agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a Attorney's Docket No.: 14174-072W01 cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.
In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhances. The penetration enhances can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.
In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.
~ 5 In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizes. The plasticizes can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.
In one aspect, the invention features a pharmaceutical composition including an iRNA
agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides 20 long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3' overhang 1-5 nucleotides long.
In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded 25 iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration.
The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.In one aspect, the invention features a pharmaceutical Attorney's Docket No.: 14174-072W01 composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent, or a DNA
which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA
agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water;
saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.
In one aspect, the invention features a pharmaceutical composition including an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form: In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer. described herein.
2o In one aspect, the invention features a pharmaceutical composition including an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.
In one aspect, the invention features a pharmaceutical composition including an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form.

Attorney's Docket No.: 14174-072W01 In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.
In one aspect, the invention features a pharmaceutical composition including an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA
agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.
In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA
agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, 15 including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subj ect by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.
In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected 2o from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.
25 In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid.
In a preferred embodiment the amino acid is selected from the group consisting of arginine, Attorney's Docket No.: 14174-072W01 histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.
In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, a-lactalbumin, trypsinogen, and polyalanine.
In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MlVm) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA
agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA
agent; or sRNA
agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a Garner compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to 2o instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.
In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA
agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the Attorney's Docket No.: 14174-072W01 device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion.
Exemplary devices include stems, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.
As used herein, the term "crystalline" describes a solid'having the structure or characteristics of a crystal, i. e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.
The invention is further illustrated by the following examples, which should not be construed as further limiting.
EXAMPLES
Example 1: apoB protein as a therapeutic target for lipid-based diseases Apolipoprotein B (apoB) is a candidate target gene for the development of novel ~5 therapies for lipid-based diseases.
Methods described herein can be used to evaluate the efficacy of a particular siRNA as a therapeutic tool for treating lipid metabolism disorders resulting elevated apoB levels. Use of siRNA duplexes to selectively bind and inactivate the target apoB mRNA is an approach totreat these disorders.
20 Two approaches:
i) Inhibition of apoB in ex-vivo models by transfecting siRNA duplexes homologous to human apoB mRNA in a human hepatoma cell line (Hep G2) and monitor the level of the protein and the RNA using the Western blotting and RT-PCR methods, respectively. siRNA
molecules that efficiently inhibit apoB expression will be tested fox similar effects ifz vivo.
25 ii) In vivo trials using an apoB transgenic mouse model (apoB 100 Transgenic Mice, C57BL/6NTac-TgN (APOB 100), Order Model #'s:1004-T (hemizygotes), B6 (control)). siRNA
duplexes are designed to target apoB-100 or CETP/apoB double transgenic mice which express both cholesteryl ester transfer protein (CETP) and apoB. The effect of the siRNA on gene expression ira vivo can be measured by monitoring the HDL/LDL cholesterol level in serum. The Attorney's Docket No.: 14174-o72W01 results of these experiments would indicate the therapeutic potential of siRNAs to treat lipid-based diseases, including hypercholesterolemia, HDL/LDL cholesterol imbalance, familial combined hyperlipidemia, and acquired hyperlipidemia.
Background Fats, in the form of triglycerides, are ideal for energy storage because they are highly reduced and anhydrous. An adipocyte (or fat cell) consists of a nucleus, a cell membrane, and triglycerides, and its function is to store triglycerides.
The lipid portion of the human diet consists largely of triglycerides and cholesterol (and its esters). These must be emulsified and digested to be absorbed.
Specifically, fats (triacylglycerols) are ingested. Bile (bile acids, salts, and cholesterol), which is made in the liver, is secreted by the gall bladder. Pancreatic lipase digests the triglycerides to fatty acids, and also digests di-, and mono-acylglycerols, which are absorbed by intestinal epithelial cells and then are resynthesized into triacylglycerols once inside the cells. These triglycerides and some cholesterols are combined with apolipoproteins to produce chylomicrons.
Chylomicrons consist ~5 of approximately 95% triglycerides. The chylomicrons transport fatty acids to peripheral tissues.
Any excess fat is stored in adipose tissue.
Lipid transport and clearance from the blood into cells, and from the cells into the blood and the liver, is mediated by the lipoprotein transport proteins. This class of approximately 17 proteins can be divided into three groups: Apolipoproteins, lipoprotein processing proteins, and 20 lipoprotein receptors.
Apolipoproteins coat lipoprotein particles, and include the A-I, A-II, A-IV, B, CI, CII, CIII, D, E, Apo(a) proteins. Lipoprotein processing proteins include lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase and cholesterol ester transfer protein. Lipoprotein receptors include the low density lipoprotein (LDL) receptor, chylomicron-remnant receptor (the' 25 LDL receptor like protein or LDL receptor related protein - LRP) and the scavenger receptor.
Lipoprotein Metabolism Since the triglycerides, cholesterol esters, and cholesterol absorbed into the small intestine are not soluble in aqueous medium, they must be combined with suitable proteins (apolipoproteins) in order to prevent them from forming large oil droplets. The resulting so lipoproteins undergo a type of metabolism as they pass through the bloodstream and certain organs (notably the liver).

Attorney's Docket No.: 14174-072W01 Also synthesized in the liver is high density lipoprotein (HDL), which contains the apoproteins A-l, A-2, C-1, and D; HDL collects cholesterol from peripheral tissues and blood vessels and returns it to the liver. LDL is taken up by specific cell surface receptors into an endosome, which fuses with a lysosome where cholesterol ester is converted to free cholesterol.
The apoproteins (including apo B-100) are digested to amino acids. The receptor protein is recycled to the cell membrane.
The free cholesterol formed by this process has two fates. First, it can move to the endoplasmic reticulum (ER), where it can inhibit HMG-CoA reductase, the synthesis of HMG-CoA reductase, and the synthesis of cell surface receptors for LDL. Also in the ER, cholesterol can speed up the degradation of HMG-CoA reductase. The, free cholesterol can also be converted by ac,yl-CoA and acyl transferase (ACAT) to cholesterol esters, which form oil droplets.
ApoB is the major apolipoprotein of chylomicrons of very low density lipoproteins (VLDL, which carry most of the plasma triglyceride) and low density lipoprotein (LDL, which carry most of the plasma cholesterol). ApoB exists in human plasma in two isoforms, apoB-48 and apoB-100.
ApoB-100 is the major physiological ligand for the LDL receptor. The ApoB
precursor has 4563 amino acids, and the mature apoB-100 has 4536 amino acid residues.
The LDL-binding domain of ApoB-100 is proposed to be located between residues 3129 and 3532.
ApoB-100 is 2o synthesized in the liver and is required for the assembly of very low density lipoproteins VLDL
and for the preparation of apoB-100 to transport triglycerides (TG) and cholesterol from the liver to other tissues. ApoB-100 does not interchange between lipoprotein particles, as do the other lipoproteins, and it is found in IDL and LDL particles. After the removal of apolipoproteins A, E
and C, apoB is incorporation into VLDL by hepatocytes. ApoB-48 is present in chylomicrons and plays an essential role in the intestinal absorption of dietary fats. ApoB-48 is synthesized in the small intestine. It comprises the N-terminal 48% of apoB-100 and is produced by a posttranscriptional apoB-100 mRNA editing event at codon 2153 (C to U'. This editing event is a product of the apoBEC-lb enzyme, which is expressed in the intestine. This editing event creates a stop codon instead of a glutamine codon, and therefore apoB-48, instead of apoB-100 is 3o expressed in the intestine (apoB-100 is expressed in the liver).

Attorney's Docket No.: 14174-072W01 There is also strong evidence that plasma apoB levels may be a better index of the risk of coronary artery disease (CAD) than total or LDL cholesterol levels. Clinical studies have demonstrated the value of measuring apoB in hypertriglyceridemic, hypercholesterolemic and normalipidemic subjects.

Attorney's Docket No.: 14174-072W01 Table 4. Reference Range Lipid level in the Blood Li id Ran a (mmols/
L) Plasma Cholesterol 3.5-6.5 Low density lipoprotein 1.55-4.4 Very low density lipoprotein0.128-0.645 High density lipoprotein/ 0.5-2.1 triglycerides Total lipid 4.0-l Og / L

Molecular genetics of lipid metabolism in both humans and induced mutant mouse models Elevated plasma levels of LDL and apoB are associated with a higher risk for atherosclerosis and coronary heart disease, a leading cause of mortality. ApoB is the mandatory constituent of LDL
particles. In addition to its role in lipoprotein metabolism, apoB has also been implicated as a factor in male infertility and fetal development. Furthermore, two quantitative trait loci regulating plasma apoB levels have been discovered, through the use of transgenic mouse 1o models. Future experiments will facilitate the identification of human orthologous genes encoding regulators of plasma apoB levels. These loci are candidate therapeutic targets for human disorders characterized by altered plasma apoB levels. Such disorders include non-apoB
linked hypobetalipoproteinemia and familial combined hyperlipidemia. The identification of these genetic loci would also reveal possible new pathways involved in the regulation of apoB
~ 5 secretion, potentially providing novel sites for pharmacological therapy.
Diseases and Clinical Pharmacology Familial combined hyperlipemia (FCHL) affects an estimated one in 10 Americans. FCHL can cause premature heart disease.
Familial Hyperclaolesterolemia (laigla level of apo B) A common genetic disorder of lipid 2o metabolism. Familial hypercholesterolemia is characterized by elevated serum TC in association with xanthelasma, tendon and tuberous xanthomas, accelerated atherosclerosis, and early death from myocardial infarction (MI). It is caused by absent or defective LDL cell receptors, resulting in delayed LDL clearance, an increase in plasma LDL levels, and an accumulation of LDL cholesterol in macrophages over joints and pressure points, and in blood vessels.

Attorney's Docket No.: 14174-072W01 Atherosclerosis (laigla level of apo B) Atherosclerosis develops as a deposition of cholesterol and fat in the arterial wall due to disturbances in lipid transport and clearance from the blood into cells and from the cells to blood and the liver.
Clinical studies have demonstrated that elevation of total cholesterol (TC), low- density lipoprotein cholesterol (LDL-C) and apoB-100 promote human atherosclerosis.
Similarly, ' decreased levels of high - density lipoprotein cholesterol (HDL-C) are associated with the development of atherosclerosis.
ApoB may be a factor in the genetic cause of high cholesterol.
The risk of cororaary artery disease (CAD) (high level of apo B) Cardiovascular disease, including coronary heart disease and stroke, is a leading cause of death and disability. The major risk factors include age, gender, elevated low-density lipoprotein cholesterol blood levels, decreased high-density lipoprotein cholesterol levels, cigarette smoking, hypertension, and diabetes. Emerging risk factors include elevated lipoprotein (a), remnant lipoproteins, and C
reactive protein. Dietary intake, physical activity and genetics also impact cardiovascular risk.
15 Hypertension and age are the major risk factors for stroke.
Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP).
2o Model fog human atherosclerosis (Lipoprotein A transgenic mouse) Numerous studies have demonstrated that an elevated plasma level of lipoprotein(a) (Lp(a)) is a major independent risk factor for coronary heart disease (CHD). Current therapies, however, have little or no effect on apo(a) levels and the homology between apo(a) and plasminogen presents barriers to drug development. Lp(a) particles consist of apo(a) and apoB-100 proteins, and they are found only 25 in primates and the hedgehog. The development of LPA transgenic mouse requires the creation of animals that express both human apoB and apo(a) transgenes to achieve assembly of LP(a).
An atherosclerosis mouse model would facilitate the study of the disease process and factors influencing it, and further would facilitate the development of therapeutic or preventive agents.
There are several strategies for gene-oriented therapy For example, the missing or non-so functional gene can be replaced, or unwanted gene activity can be inhibited.

Attorney's Docket No.: 14174-072W01 Model for lipid Metabolism and Atherosclerosis DNX Transgenic Sciences has demonstrated that both CETP/ApoB and ApoB transgenic mice develop atherosclerotic plaques.
Model for apoB-100 overexpression The apoB-100 transgenic mice express high levels of human apoB-100. They consequently demonstrate elevated serum levels of LDL
cholesterol.
After 6 months on a high-fat diet, the mice develop significant foam cell accumulation under the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.
Model for Cholesteyyl ester transfer protein over expressiorz The apoB-100 transgenic mice express the human enzyme, CETP, and consequently demonstrate 'a dramatically reduced level of serum HDL cholesterol.
Model for apoB-100 and CETP overexpression The apoB-100 transgenic mice express both ~ 5 CETP and apoB-100, resulting in mice with a human like serum HDL/LDL
distribution.
Following 6 months on a high-fat diet these mice develop significant foam cell accumulation underlying the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.
2o ApoB100 Transgefaic Mice (Order Model #'s:1004-T (hemizygotes), B6 (control)) These mice express high levels of human apoB-100, resulting in mice with elevated serum levels of LDL cholesterol. These mice are useful in identifying and evaluating compounds to reduce elevated levels of LDL cholesterol and the risk of atherosclerosis. When fed a high fat cholesterol diet, these mice develop significant foam cell accumulation underly the endothelium 2s and within the media, and have significantly more complex atherosclerotic lesions than control animals.
Double Transgenic Mice, CETPlApoB100 (Order Model #: 1007-TT) These mice express both CETP and apoB-100, resulting in a human-like serum HDL/LDL distribution. These mice are 3o useful for evaluating compounds to treat hypercholesterolemia or HDL/LDL
cholesterol imbalance to reduce the risk of developing atherosclerosis. When fed a high fat high cholesterol Attorney's Docket No.: 14174-072W01 diet, these mice develop significant foam cell accumulation underlying the endothelium and within the media, and have significantly more complex atherosclerotic lesions than control animals.
ApoE gene knockout mouse Homozygous apoE knockout mice exhibit strong hypercholesterolemia, primarily due to elevated levels of VLDL and IDL caused by a defect in lipoprotein clearance from plasma. These mice develop atherosclerotic lesions which progress with age and resemble human lesions (Zhang et al., Science 258:46-71, 1992;
Plump et al., Cell 71:343-353, 1992; Nakashima et al., Arterioscler .Thromp. 14:133-140, 1994;
Reddick et al., Arterioscler Tromb. 14:141-147, 1994). These mice are a promising model for studying the effect of diet and drugs on atherosclerosis.
Low density lipoprotein receptor (LDLR) mediates lipoprotein clearance from plasma through the recognition of apoB and apoE on the surface of lipoprotein particles. Humans, who lack or have a decreased number of the LDL receptors, have familial hypercholesterolemia and ~ 5 develop CHD at an early age.
ApoE Knockout Mice (Order Model #: APOE M) The apoE knockout mouse was created by gene targeting in embryonic stem cells to disrupt the apoE gene. ApoE, a glycoprotein, is a structural component of very low density lipoprotein (VLDL) synthesized by the liver and 2o intestinally synthesized chylomicrons. It is also a constituent of a subclass of high density lipoproteins (HDLs) involved in cholesterol transport activity among cells.
One of the most important roles of apoE is to mediate high affinity binding of chylomicrons and VLDL particles that contain apoE to the low density lipoprotein (LDL) receptor. This allows for the specific uptake of these particles by the liver which is necessary for transport preventing the 25 accumulation in plasma of cholesterol-rich remnants. The homozygous inactivation of the apoE
gene results in animals that are devoid of apoE in their sera. The mice appear to develop normally, but they exhibit five times the normal serum plasma cholesterol and spontaneous atherosclerotic lesions. This is similar to a disease in people who have a variant form of the apoE gene that is defective in binding to the LDL receptor and are at risk for early development 30 of atherosclerosis and increased plasma triglyceride and cholesterol levels. There are indications that apoE is also involved in immune system regulation, nerve regeneration and muscle Attorney's Docket No.: 14174-072W01 differentiation. The apoE knockout mice can be used to study the role of apoE
in lipid metabolism, atherogenesis, and nerve injury, and to investigate intervention therapies that modify the atherogenic process.
Apoe4 Targeted Replacenaerat Mouse (Order Model #: 001549-M) ApoE is a plasma protein involved in cholesterol transport, and the three human isoforms (E2, E3, and E4) have been associated with atherosclerosis and Alzheimer's disease. Gene targeting of 129 ES cells was used to replace the coding sequence of mouse apoE with human APOE4 without disturbing the marine regulatory sequences. The E4 isoform occurs in approximately 14% of the human population and is associated with increased plasma cholesterol and a greater risk of coronary artery disease. The Taconic apoE4 Targeted Replacement model has normal plasma cholesterol and triglyceride levels, but altered quantities of different plasma lipoprotein particles. This model also has delayed plasma clearance of cholesterol-rich lipoprotein particles (VLDL), with only half the clearance rate seen in the apoE3 Targeted Replacement model.
Like the apoE3 model, the apoE4 mice develop altered plasma lipoprotein values and atherosclerotic plaques on an atherogenic diet. However, the atherosclerosis is more severe in the apoE4 model, with larger plaques and cholesterol apoE and apoB-48 levels twice that seen in the apoE3 model. The Taconic apoE4 Targeted Replacement model, along with the apoE2 and apoE3 Targeted Replacement Mice, provide an excellent tool for ih vivo study of the human apoE isoforms.
CETP Trarasgefaic Mice (Order Model #: 1003-T) These animals express the human plasma 2o enzyme, CETP, resulting in mice with a dramatic reduction in serum HDL
cholesterol. The mice can be useful in identifying and evaluating compounds that increase the levels of HDL
cholesterol for reducing the risk of developing atherosclerosis TrarzsgenelPronaoter: human apolipoprotein A-I These mice produce mouse HDL
cholesterol particles that contain human apolipoprotein A-I. Transgenic expression is life-long in both sexes 2s (Biochemical Genetics and Metabolism Laboratory, Rockefeller University, NY
City).
A Mouse Model for Abetalipoproteinemia Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). Gene Attorney's Docket No.: 14174-072W01 targeting was used to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp+~ ), the MTP mRNA, protein, and activity levels were reduced by 50% in both liver and intestine. Recent studies with heterozygous MTP knockout mice have suggested that half normal levels of MTP in the liver reduce apoB secretion. They hypothesized that reduced apoB
secretion in the setting of half normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP
interactions. If this hypothesis were true, half normal levels of MTP might have little impact on lipoprotein secretion in the setting of half normal levels of apoB synthesis (since the ratio of MTP to apoB
would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the ratio of MTP to apoB would be even lower). To test this hypothesis; they examined the effects of heterozygous MTP deficiency on apoB
metabolism in the setting of normal levels of apoB synthesis, half normal levels of apoB
synthesis (heterozygous Apob deficiency), and increased levels of apoB
synthesis (transgenic overexpression of human apoB). Contrary to their expectations, half normal levels of MTP
reduced plasma apoB-100 levels to the same extent (~25-35%) at each level of apoB synthesis.
In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum, rather than the MTP:apoB ratio, is the critical determinant of lipoprotein secretion. Finally, heterozygosity for an apoB knockout mutation was found to lower 2o plasma apoB-100 levels more than heterozygosity for an MTP knockout allele.
Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice. CrelloxP tissue-specific recombination techniques were also used to generate liver-specific Mttp knockout mice. Inactivation of the Mttp gene in the liver caused a striking reduction in very low density lipoprotein (VLDL) triglycerides and large reductions in both VLDL/low density lipoproteins (LDL) and high density lipoprotein cholesterol levels. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Currently being tested is the hypothesis that accumulation of triglycerides in the liver renders the liver more susceptible to injury by a second insult (e.g., lipopolysaccharide).
Human apo B (apolipoproteizz B) Trarzsgezze mice show apo B locus zrzay have a causative Bole 3o male izzfe>"tility The fertility of apoB (apolipoprotein B) (+/-) mice was recorded during the course of backcrossing (to C57BL/6J mice) and test mating. No apparent fertility problem was Attorney's Docket No.: 14174-072W01 observed in female apoB (+/-) and wild-type female mice, as was documented by the presence of vaginal plugs in female mice. Although apoB (+/-) mice mated normally, only 40% of the animals from the second backcross generation produced any offspring within the 4-month test period. Of the animals that produced progeny, litters resulted from < 50% of documented matings. In contrast, all wild-type mice (6/6--i. e., 100%) tested were fertile. These data suggest genetic influence on the infertility phenotype, as a small number of male heterozygotes were not sterile. Fertilization in vivo was dramatically impaired in male apoB (+/-) mice. 74% of eggs examined were fertilized by the sperm from wild-type mice, whereas only 3% of eggs examined were fertilized by the sperm from apoB (+/-) mice. The sperm counts of apoB
(+/-) mice were mildly but significantly reduced compared with controls. However, the percentage of motile sperm was maxkedly reduced in the apoB (+/-) animals compared with that of the wild-type controls. Of the sperm from apoB (+/-) mice, 20% (i.e., 4.9% of the initial 20% motile sperm) remained motile after 6 hr of incubation, whereas 45% (i.e., 33.6% of the initial 69.5%) of the motile sperm retained motility in controls after this time. In vitro fertilization yielded no fertilized eggs in three attempts with apo B (+/-) mice, while wild-type controls showed a fertilization rate of 53%. However, sperm from apoB (+/-) mice fertilized 84%
of eggs once the zone pellucida had been removed. Numerous sperm from apoB (+/-) mice were seen binding to zone-intact eggs. However, these sperm lost their motility when observed 4-6 hours after binding, showing that sperm from apoB (+/-) mice were unable to penetrate the zone pellucida 2o but that the interaction between sperm and egg was probably not direct.
Sperm binding to zona-free oocytes'was abnormal. In the apoB (+/-) mice, sperm binding did not attenuate, even after pronuclei had clearly formed, suggesting that apoB deficiency results in abnormal surface interaction between the sperm and egg.
Knockout of the mouse apoB gene resulted in embryonic lethality in homozygotes, protection against diet-induced hypercholesterolemia in heterozygotes, and developmental abnormalities in mice.
Model of insulin resistance, dyslipidemia & overexpression of human apoB It was shown that the livers of apoB mice assemble and secrete increased numbers of VLDL
particles.

Attorney's Docket No.: 14174-072W01 Example 2. Treatment of Diabetes Type-2 with iRNA
IntYOduction The regulation of hepatic gluconeogenesis is an important process in the adjustment of the blood glucose level. Pathological changes in the glucose production of the liver are a central characteristic in type-2-diabetes. For example, the fasting hyperglycemia observed in patients with type-2-diabetes reflects the lack of inhibition of hepatic gluconeogenesis and glycogenolysis due to the underlying insulin resistance in this disease.
Extreme conditions of insulin resistance can be observed for example in mice with a liver-specific insulin receptor knockout ('LIRKO'). These mice have an increased expression of the two rate-limiting gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase). Insulin is known to repress both PEP~CK and G6Pase gene expression at the transcriptional level and the signal transduction involved in the regulation of G6Pase and PEPCK gene expression by insulin is only partly understood. While PEPCK is involved in a very early step of hepatic gluconeogenesis (synthesis of phosphoenolpyruvate from oxaloacetate), G6Pase catalyzes the terminal step of both, 15 gluconeogenesis and glycogenolysis, the cleavage of glucose-6-phosphate into phosphate and free glucose, which is then delivered into the blood stream.
The pharmacological intervention in the regulation of expression of PEPCK and G6Pase can be used for the treatment of the metabolic aberrations associated with diabetes. Hepatic glucose production can be reduced by an iRNA-based reduction of PEPCK and G6Pase 2o enzymatic activity in subjects with type-2-diabetes.
Targets for iRNA
Glucose-6-phosphatase (G6Pase) G6Pase mRNA is expressed principally in liver and kidney, and in lower amounts in the 2s small intestine. Membrane-bound G6Pase is associated with the endoplasmic reticulum. Low activities have been detected in skeletal muscle and in astrocytes as well.
G6Pase catalyzes the terminal step in gluconeogenesis and glycogenolysis. The activity of the enzyme is several fold higher in diabetic animals and probably in diabetic humans.
Starvation and diabetes cause a 2-3-fold increase in G6Pase activity in the liver and a 2-4-fold 3o increase in G6Pase mRNA.

Attorney's Docket No.: 14174-072W01 Phosphoenolpyruvate carboxykinase (PEPCI~
Overexpression of PEPCK in mice results in symptoms of type-2-diabetes mellitus.
PEPCK overexpression results in a metabolic pattern that increases G6Pase mRNA
and results in a selective decrease in insulin receptor substrate (IRS)-2 protein, decreased phosphatidylinositol s 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression.
Table 5. Other targets to inhibit hepatic glucose production Tar et Comment FKHR good evidence for antidiabetic phenotype (Nakae et al., Nat Geraetics 32:245(2002) Glucagon Glucagon receptor Glycogen phosphorylase regulates the cAMP response PGC-1 (PPAR-Gamma (and Coactivator) probably the PKB/FKHR-regulation) on PEPCK/G6Pase Fructose-1,6-bisphosphatase Glucose-6-phospate translocator Glucokinase inhibitory regulatory protein Materials and Methods Animals: BKS.Cg-m +/+ Lepr db mice, which contain a point mutation in the leptin receptor gene are used to examine the efficacy of iRNA for the targets listed above.
BKS.Cg-m +/+ Lepr db are available from the Jackson Laboratory (Stock Number 000642). These animals are obese at 3-4 weeks after birth, show elevation of plasma insulin at 15 10 to 14 days, elevation of blood sugar at 4 to 8 weeks, and uncontrolled rise in blood sugar.
Exogenous insulin fails to control blood glucose levels and gluconeogenic activity increases.
The following numbers of male animals (age>12 weeks) could be tested with the following iRNAs:
PEPCK, 2 sequences, 5 animals per sequence 2o G6Pase, 2 sequences, 5 animals per sequence 1 nonspecific sequence, 5 animals 1 control group (only inj ected, no siRNA), 5 animals 1 control group (not injected, no siRNA), 5 animals Attorney's Docket No.: 14174-072W01 Reagents: Necessary reagents would ideally include a Glucometer Elite XL
(Bayer, Pittsburgh, PA) for glucose quantification, and an Insulin Radioimmunoassay (RIA) kit (Amersham, Piscataway, NJ) for insulin quanitation.
s Assays:
G6P enzyme assays and PEPCK enzyme assays are used to measure the activity of the enzymes.
Northern blotting is used to detect levels of G6Pase and PEPCK mRNA. Antibody-based techniques (e.g., immunoblotting, immunofluorescence) are used to detect levels of G6Pase and PEPCK protein. Glycogen staining is used to detect levels of glycogen in the liver. Histological analysis is performed to analyze tissues.
Gene information:
G6Pase GenBank~ No.: NM 008061,Mus musculus glucose-6-phosphatase, catalytic (G6pc), mRNA 1..2259, ORF 83..1156;
15 GenBank~ No: U00445,Mus musculus glucose-6-phosphatase mRNA, complete cds 1..2259, ORF 83..1156 GenBank~ No: BC013448 PEPCK
GenBank~ No: NM 011044, Mus musculus phosphoenolpyruvate carboxykinase l, cytosolic 20 (Pckl), mRNA.1..2618, ORF 141..2009 GenBank~ No: AF009605.1 Administration of iRNA:
iRNA corresponding to the genes described above could be administered to mice with 2s hydrodynamic injection. One control group of animals would be treated with Metformin as a positive control for reduction in hepatic glucose levels.
Exuerimental Protocol Mice could be housed in a facility in which there is light from 7:00 AM to 7:00 PM.
3o Mice would be fed ad libiduna from 7:00 PM to 7:00 AM and fast from 7:00 AM
to 7:00 PM.

Attorney's Docket No.: 14174-072W01 Day 0: 7:00 PM: Approximately 100 ~,l blood would be drawn from the tail.
Serum could be isolated to measure glucose, insulin, HbAlc (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides.
Day 1-7: Blood glucose could be measured daily at 8:00 AM and 6:00 PM (approx.
3-5 ,ul;
measured with a Haemoglucometer) Day 8: Blood glucose could be measured daily at 8:00 AM and 6:00 PM. iRNA
would be injected between 10:00 AM and 2:00 PM
Day 9-20: Blood glucose could be measured daily at 8:00 AM and 6:00 PM.
Day 21: Mice could be sacrificed after 10 hours of fasting.
Blood would be isolated. Glucose, insulin, HbAlc (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides would be measured. Liver tissue would be isolated for histology, protein assays, RNA assays, glycogen quantitation, and enzyme assays.
Example 3: Inhibition of Glucose-6-Phosphatase iRNA i~a vivo iRNA targeted to the Glucose-6-Phosphatase (G6P) gene was used to examine the effects of inhibition of G6P expression on glucose metabolism iya vivo.
Female mice, 10 weeks of age, strain BKS.Cg-m +/+ Lepr db (The Jackson Laboratory) 2o were used for in vivo analysis of enzymes of the hepatic glucose production. Mice were housed under conditions where it was light from 6:30 am to 6:30 pm. Mice were fed (ad libidum) during the night period and fasted during the day period.
On day 1, approximately 100,1 of blood was collected from test animals by puncturing the retroorbital plexus. On days 1-7, blood glucose was measured in blood obtained from tail veins (approximately 3-S pl) using a Glucometer (Elite XL, Bayer). Blood glucose was sampled daily at 8 am and 6 pm.
On day 7 at approximately 2pm, GL3 plasmid (10 ~,g) and siRNAs (100 ~,g G6Pase specific, Renilla nonspecific or no siRNA control) were delivered to animals using hydrodynamic coinjection.

Attorney's Docket No.: 14174-072W01 On day 8, GL3 expression was analyzed by injection of luceferin (3 mg) after anaesthesia with avertin and imaging. This was done to control for successful hydrodynamic delivery.
On days 8-10, blood glucose was measured in blood obtained from tail veins (approximately 3-5 ml) using a Glucometer (Elite XL, Bayer).
On day 10, mice were sacrificed after 10 hours of fasting. Blood and liver were isolated from sacrificed animals.
Table 6 lists blood glucose levels (mg/dl) for mice injected with GL3 plasmid and G6Pase iRNA (G6P4), Renilla nonspecific iRNA (RL), or no iRNA (no). Days on which nucleic acids were inj ected are shaded.
Table 6. Blood glucose levels in mice plasmidGL3 GL3 GL3 GL3 GL3 GL3 GL3 GL3 siRNAG6P4 G6P4 G6P4no G6P4 RL RL no mouse mouse mouse mouse mouse mouse mouse 17 mouse day BG BG BG BG BG BG BG BG

7 14 . ~ -~ 4~4 ' ' 423 "r ~ 201 . 30J ; ~ iCl 2eyo ~ s j ' ' 600 ~' 'I
~ 245' , r"
Z6' ~

8 , q 566 246 521' _ . 600 576 ~
600 :' 277 ' . .
. _.. _. _ 404 .:

9, 448 438 536 600 459 jyttjeCt'tort ~ :s ~SiT
~

10 _ 600 446 ~

average day 532 463 465 420 325 399 539 398 1to Table 7 lists average blood glucose levels (mg/dl) on days 1-6 or day 7 for mice injected with GL3 plasmid and G6Pase iRNA (G6P4), Renilla nonspecific iRNA (RL), no iRNA (no), or for mice that were not injected, or for which injection failed.
Table 7. Average blood glucose levels G6P4 T ...~RL no RL and no (combined) mouse 03,04,05,09mouse 14, mouse 07, mouse 14, 15, 15 17 07, 17 _ a_~rage 446 469 409 439 (d1-6) stddev (d1-6)124 96 101 101 a~rage (d7)230 547 369 458 stddev (d7)~27 ~ ~ ~ _ ___ __ Attorney's Docket No.: 14174-072W01 FIGS. 6A, 6B, and 6C show graphs depicting blood glucose levels of animals injected with control or no siRNA, G6Pase RNA, or non-injected mice (respectively) at days 1-6 and day 7. FIG. 7 contains a graph of average blood glucose levels for mice inj ected with G6Pase RNA
(solid line) and mice injected with, Renilla nonspecific iRNA (RL) or no iRNA
(no) (dashed line).
Table 8 lists average blood glucose levels for mice injected with G6Pase iRNA
or Renilla nonspecific iRNA (RL) and no iRNA.
Table 8. Average blood glucose levels iRNA _ G6P4 RL an d no (combined) mouse 03,04,05,09_ _ -A ~~~~ _ mouse~~14, 15, 07, 17 average stddev - a~rage stddev day I

2 435 90 445 , 114 _ _ g 475 190 420 106 6 483 82 414 ___ 8 422 187 525 ~ 87 g 412 _ 532 71 ~ ~ 54 Examine 4: Selected Palindromic Seguences Tables 9-14 below provide selected palindromic sequences from the following genes: human ApoB, human glucose-6-phosphatase, rat glucose-6-phosphatase, (3-catenin, and hepatitis C virus (HCV).

Table 9. Selected nalindromic seauence~ from h"rr,an AnnR
Source Start End Match Start End #
B

Index Index Index Index SEQ 1 ggccattccagaagggaag509 528 SEQ 1004cttccgttctgtaatggcc5795 NO: NO:

SEQ 2 tgccatctcgagagttcca099 118 SEQ 1005tggaactctctccatggca10876 NO: NO:

SEQ 3 catgtcaaacactttgtta7056 7075 SEQ 1006taacaaattccttgacatg7358 NO: NO:

SEO 4 tttgttataaatcttattg7068 7087 SEQ 1007caataagatcaatagcaaa8990 NO: NO:

SEQ 5 tctggaaaagggtcatgga8880 $899 SEQ 1008tccatgtcccatttacaga11356 NO: NO:

SEO 6 cagctcttgttcaggtcca10900 10919 SEQ 1009tggacctgcaccaaagctg13952 NO: NO:

SEQ 7 ggaggttccccagctctgc356 375 SEQ 1010gcagccctgggaaaactcc6447 NO: NO:

SEQ 8 ctgttttgaagactctcca1081 1100 SEQ 1011tggagggtagtcataacag10327 NO: NO:

SEQ 9 agtggctgaaacgtgtgca1297 1316 SEQ 1012tgcagagctttctgccact13508 NO: NO:

SEO 10ccaaaatagaagggaatct068 2087 SEQ 1013agattcctttgccttttgg4000 NO: NO:

SEQ 11tgaagagaagattgaattt3620 3639 SEQ 1014aaattctcttttcttttca9212 NO: NO:

SEQ 12agtggtggcaacaccagca230 249 SEQ 1015tgctagtgaggccaacact10649 NO: NO:

SEQ 13aaggctccacaagtcatca5950 5969 SEQ 1016tgatgatatctggaacctt10724 NO: NO:

SEO 14gtcagccaggtttatagca7725 7744 SEQ 1017tgctaagaaccttactgac7781 ID ID ~ 7 NO: NO:

SEO 15tgatatctggaaccttgaa10727 10746 SEQ 1018ttcactgttcctgaaatca7863 NO: NO:

SEQ 16gtcaagttgagcaatttct13423 13442 SEQ 1019agaaaaggcacaccttgac11072 NO: NO:

SEQ 17atccagatggaaaagggaa13480 13499 SEQ 1020ttccaatttccctgtggat3680 NO: NO:

SEQ 18atttgtttgtcaaagaagt543 562 SEQ 1021acttcagagaaatacaaat11401 NO: NO:

SEO 19ctggaaaatgtcagcctgg04 23 SEQ 1022ccagacttccgtttaccag8235 NO: NO:

SEQ 20accaggaggttcttcttca729 1748 SEQ 1023gaagtgtagtctcctggt5089 ID 1 ID t 6 NO: NO:

SEQ 21aaagaagttctgaaagaat956 975 SEQ 1024attccatcacaaatccttt9661 NO: NO:

SEQ 22gctacagcttatggctcca570 3589 SEQ 1025ggatctaaatgcagtagc11623 ID 3 ID t 6 NO: NO:

SEQ 23atcaatattgatcaatttg414 6433 SEQ 1026caaagaagtcaagattgat4553 NO: NO:

SEQ 24gaattatcttttaaaacat326 345 SEQ 027 atgtgttaacaaaatattc1494 NO: . NO:

SEQ 25gaggcccgcgctgctggc30 49 SEQ 028 gccagaagtgagatcctcg3507 ID c 1 1 ID 1 NO: NO:

SEO 6 caactatgaggctgagag71 90 SEQ 029 tctgagcaacaaatttgt0309 0328 ID a 2 ID c 1 1 1 NO: NO:

SEQ ~27gctgagagttccagtggag282 301 SEQ 1030ctccatggcaaatgtcagc10885 ~~~~

NO: NO:

SEQ 28tgaagaaaaccaagaactc48 67 SEQ 1031gagtcattgaggttcttca4929 NO: NO:

SEQ 29cctacttacatcctgaaca558 577 SEQ 1032tgttcataagggaggtagg12766 NO: NO:

SEQ 30ctacttacatcctgaacat559 578 SEQ 1033atgttcataagggaggtag12765 NO: NO:

SEQ 31gagacagaagaagccaagc615 634 SEQ 1034gcttggttttgccagtctc2459 NO: NO:

SEQ 32cactcactttaccgtcaag671 690 SEQ 1035cttgaacacaaagtcagtg6000 NO: NO:

SEQ 33ctgatcagcagcagccagt822 841 SEQ 1036actgggaagtgcttatcag5237 NO: NO:

SEQ 34actggacgctaagaggaag854 873 SEQ 1037cttccccaaagagaccagt2890 NO: NO:

SEQ ID :35agaggaagcatgtggcaga865 884 SEQ ID 1038 tctggcatttactttctct59215940 1 NO:

SEQ ID :36tgaagactctccaggaact10871106SEQ ID 1039 agttgaaggagactattca72167235 1 NO:

SEQ ID 37 ctctgagcaaaatatccag11211140SEQ ID 1040 ctggttactgagctgagag11611180 1 N0: 6 NO:

SEQ ID 38 atgaagcagtcacatctct11891208SEQ ID 1041 agagctgccagtccttcat10016100351 N0: 6 NO:

SEQ ID 39 ttgccacagctgattgagg12091228SEQ ID 1042 cctcctacagtggtggcaa42224241 1 NO: 6 NO:

SEQ ID 40 agctgattgaggtgtccag12161235SEQ ID 1043 ctggattccacatgcagct11847118661 N0: 6 NO:

SEQ ID 41 tgctccactcacatcctcc12781297SEQ ID 1044 ggaggctttaagttcagca76017620 1 N0: 6 NO:

SEQ ID 42 tgaaacgtgtgcatgccaa13031322SEQ ID 1045 ttgggagagacaagtttca65006519 1 N0: 6 NO:

SEQ ID 43 gacattgctaattacctga15031522SEQ ID 1046 tcagaagctaagcaatgtc72327251 1 N0: 6 NO:

SEQ ID 44 ttcttcttcagactttcct17381757SEQ ID 1047 aggagagtccaaattagaa84988517 1 N0: 6 NO:

SEQ ID 45 ccaatatcttgaactcaga19031922SEQ ID 1048 tctgaattcattcaattgg64856504 1 N0: 6 NO:

SEQ ID 46 aaagttagtgaaagaagtt19461965SEQ ID 1049 aactaccctcactgccttt21322151 1 N0: 6 NO:

SEQ ID 47 aagttagtgaaagaagttc19471966SEQ ID 1050 gaacctctggcatttactt59165935 1 N0: 6 NO:

SEQ ID 48 aaagaagttctgaaagaat19561975SEQ ID 1051 attctctggtaactacttt54825501'1 N0: 6 NO:

SEQ ID 49 tttggctataccaaagatg322 2341SEQ ID 1052 catcttaggcactgacaaa49975016 1 N0: 6 NO:

SEQ ID 50 tgttgagaagctgattaaa2381400 SEQ ID 1053 tttagccatcggctcaaca57005719 1 N0: 6 NO:

SEQ ID 51 caggaagggctcaaagaat561 580 SEQ ID 1054 attcctttaacaattcctg94929511 1 N0: 6 NO:

SEQ ID 52 aggaagggctcaaagaatg562 581 SEQ ID 1055 cattcctttaacaattcct94919510 1 N0: 6 NO:

SEQ ID 53 gaagggctcaaagaatgac25642583SEQ ID 1056 gtcagtcttcaggctcttc79147933 1 NO: 6 NO:

SEQ ID 54 caaagaatgacttttttct572 591 SEQ ID 1057 agaaggatggcattttttg14000140191 N0: 6 NO:

SEQ ID 55 catggagaatgcctttgaa603 622 SEQ ID 1058 tcagagccaaagtccatg 7119.7138 1 N0: t 6 NO:

SEQ ID 56 ggagccaaggctggagtaa679 2698SEQ ID 1059 tactccaacgccagctcc N0: t 6 NO:

SEQ ID 57 tcattccttccccaaagag884 903 SEQ ID 1060 ctctctggggcatctatga51395158 1 N0: 6 NO:

SEQ ID 58 acctatgagctccagagag165 3184SEQ ID 1061 ctctcaagaccacagaggt12976129951 N0: 3 6 NO:

SEQ ID 59 gggcaaaacgtcttacaga365 384 SEQ ID 1062 ctgaaagacaacgtgccc N0: 3 3 t 6 NO:

SEQ ID 60 accctggacattcagaaca387 406 SEQ ID 1063 gttgctaaggttcagggt N0: 3 3 t 1 NO:

SEQ ID 61 atgggcgacctaagttgtg429 448 SEQ ID 064 acaaattagtttcaccat N0: 3 3 1 c 1 NO:

SEQ ID 62 gatgaagagaagattgaat618 637 SEQ ID 065 ttccagcttccccacatc N0: 3 3 1 a 1 WO 2004/091515 P('T/TTS2004/011255 NO:

SEQ ID 63 caatgtagataccaaaaaa36563675SEQ ID 1066 ttttttggaaatgccattg8643 N0: 6 NO:

SEQ ID 64 gtagataccaaaaaaatga36603679SEQ ID 1067 tcatgtgatgggtctctac4371 N0: 6 NO:

SEQ ID 65 gcttcagttcatttggact509 528 SEQ ID 1068 agtcaagaaggacttaagc5304 N0: 6 NO:

SEQ ID 66 tttgtttgtcaaagaagtc544 563 SEQ ID 1069 gacttcagagaaatacaaa11400114191 N0: 6 NO:

SEQ ID 67 ttgtttgtcaaagaagtca545 564 SEQ ID 1070 tgacttcagagaaatacaa11399114181 N0: 6 NO:

SEQ ID 68 tggcaatgggaaactcgct58465865SEQ ID 1071 agcgagaatcaccctgcca8219 N0: 6 NO:

SEQ ID 69 aacctctggcatttacttt59175936SEQ ID 1072 aaaggagatgtcaagggtt10599106181 N0: 6 NO:

SEQ ID 70 catttactttctctcatga59265945SEQ ID 1073 tcatttgaaagaataaatg7026 N0: 6 NO:

SEQ ID 71 aaagtcagtgccctgctta60096028SEQ ID 1074 taagaaccttactgacttt7784 N0: 6 NO:

SEQ ID 72 cccattttttgagacctt63226341SEQ ID 1075 aaggacttcaggaatggga12004120231 N0: t 6 NO:

SEQ ID 73 catcaatattgatcaattt64136432SEQ ID 1076 aaattaaaaagtcttgatg6732 N0: 6 NO:

SEQ ID 74 aaagatagttatgattta66656684SEQ ID 1077 taaaccaaaacttggttta9019 N0: t 6 NO:

SEQ ID 75 attgatgaaatcattgaa67136732SEQ ID 1078 ttcaaagacttaaaaaata8007 N0: t 6 NO:

SEQ ID 76 atgatctacatttgtttat67906809SEQ ID 1079 ataaagaaattaaagtcat7380 N0: 6 NO:

SEQ ID 77 agagacacatacagaatat69196938SEQ ID 1080 atatattgtcagtgcctct13382134011 N0: 6 NO:

SEQ ID 78 gacacatacagaatataga69226941SEQ ID 1081 tctaaattcagttcttgtc11327113461 N0: 6 NO:

SEQ ID 79 agcatgtcaaacactttgt70547073SEQ ID 1082 acaaagtcagtgccctgct6007 N0: 6 NO:

SEQ ID 80 ttttagaggaaaccaagg75157534SEQ ID 1083 cctttgtgtacaccaaaaa11230112491 N0: t 6 NO:

SEQ ID 81 tttagaggaaaccaaggc75167535SEQ ID 1084 gcctttgtgtacaccaaaa11229112481 N0: t 6 NO:

SEQ ID 82 gaagatagacttcctgaa93079326SEQ ID 1085 tcagaaatactgttttcc N0: g t 6 NO:

SEQ ID 83 actgtttctgagtcccag93349353SEQ ID 1086 ctgggacctaccaagagtg12523125421 N0: c 6 NO:

SEQ ID 84 acaaatcctttggctgtg96689687SEQ ID 1087 cacatttcaaggaattgtg10063100821 N0: c 6 NO:

SEQ ID 85 tcctggatacactgttcc98539872SEQ ID 1088 ggaactgttgactcaggaa12569125881 N0: t 6 NO:

SEQ ID 86 aaatctcaagctttctct1004210061SEQ ID 089 agagccaggtcgagctttc11044110631 N0: g 1 6 NO:

SEQ ID 87 ttcttcatcttcatctgt1021010229SEQ ID 090 acagctgaaagagatgaaa130551307416 N0: t 1 NO:

SEQ ID 88 ctaccgctaaaggagcag1052110540SEQ ID 091 ctgcacgctttgaggtaga1761 NO: t 1 1 1 NO:

SEQ ID 89 taccgctaaaggagcagt1052210541SEQ ID 092 actgcacgctttgaggtag17601177916 N0: c 1 1 NO:

SEQ ID 90 gggcctctttttcaccaa1083110850SEQ ID 093 tggccaggaagtggccct N0: a 1 t 1 24~

WO 2004/091515... ....... ....... ....... ....... PCT/US2004/011255 NO:

SEQ ID 91 ttctccatccctgtaaaag1126511284SEQ ID 1094 ctttttcaccaacggagaa10838108571 N0: 6 NO:

SEQ ID 92 gaaaaacaaagcagattat1181611835SEQ ID 1095 ataaactgcaagatttttc13600136191 N0: 6 NO:

SEQ ID 93 actcactcattgattttct1268212701SEQ ID 1096 agaaaatcaggatctgagt14027140461 N0: 6 NO:

SEQ ID 94 taaactaatagatgtaatc1289012909SEQ ID 1097 gattaccaccagcagttta13578135971 NO: 6 NO:

SEQ ID 95 caaaacgagcttcaggaag1320013219SEQ ID 1098 cttcgtgaagaatattttg13260132791 N0: 6 NO:

SEQ ID 96 tggaataatgctcagtgtt366 2385SEQ ID 1099 aacacttacttgaattcca10662106813 N0: 5 NO:

SEQ ID 97 gatttgaaatccaaagaag400 2419SEQ ID 1100 cttcagagaaatacaaatc11402114213 NO: 5 NO:

SEQ ID 98 atttgaaatccaaagaagt24012420SEQ ID 1101 acttcagagaaatacaaat11401114203 NO: 5 NO:

SEQ ID 99 atcaacagccgcttctttg990 1009SEQ ID 1102 caaagaagtcaagattgat455345722 NO: 5 NO:

SEQ ID 100 gttttgaagactctccag10821101SEQ ID 1103 ctggaaagttaaaacaaca695569742 N0: t 5 NO:

SEQ ID 101 cccttctgatagatgtggt13241343SEQ ID 1104 accaaagctggcaccaggg13961139802 N0: 5 NO:

SEQ ID 102 gagcaagtgaagaacttt18681887SEQ ID 1105 aaagccattcagtctctca12963129822 N0: t 5 NO:

SEQ ID 103 atttgaaatccaaagaagt24012420SEQ ID 1106 acttttctaaacttgaaat905590742 NO: 5 NO:

SEQ ID 104 atccaaagaagtcccggaa408 2427SEQ ID 1107 ttccggggaaacctgggat12721127402 N0: 5 NO:

SEQ ID 105 agagcctacctccgcatct24302449SEQ ID 1108 agatggtacgttagcctct11921119402 N0: 5 NO:

SEQ ID 106 aatgcctttgaactcccca26102629SEQ ID 1109 tgggaactacaatttcatt701270312 NO: 5 NO:

SEQ ID 107 gaagtccaaattccggatt32973316SEQ ID 1110 aatcttcaatttattcttc13815.138342 N0: 5 NO:

SEQ ID 108 gcaagcagaagccagaag34963515SEQ ID 1111 cttcaggttccatcgtgca113761139525 N0: t NO:

SEQ ID 109 gaagagaagattgaatttg36213640SEQ ID 1112 caaaacctactgtctcttc10459104782 N0: 5 NO:

SEQ ID 110 atgctaaaggcacatatgg597 616 SEQ ID 1113 ccatatgaaagtcaagcat12656126752 N0: 5 NO:

SEQ ID 111 ccctcacctccacctctg737 756 SEQ ID 1114 cagattctcagatgaggga891289312 NO: t 5 NO:

SEQ ID 112 atttacagctctgacaagt54275446SEQ ID 1115 acttttctaaacttgaaat905590742 N0: 5 NO:

SEQ ID 113 aggagcctaccaaaataat55945613SEQ ID 1116 attatgttgaaacagtcct11830118492 N0: 5 NO:

SEQ ID 114 aaagctgaagcacatcaat64016420SEQ ID 1117 attgttgctcatctccttt0194102132 N0: 1 5 NO:

SEQ ID 115 ctgctggaaacaacgagaa94189437SEQ ID 1118 tctgattaccaccagcag N0: t 1 5 NO:

SEQ ID 116 tgaaggaattcttgaaaa95829601SEQ ID 1119 tttaaaagaaatcttcaa N0: t t 1 5 NO:

SEQ ID 117 gaagtaaaagaaaattttg1074310762SEQ ID 1120 caaaacctactgtctcttc0459104782 N0: 1 5 NO:

SEQ ID 118 gaagaagatggcaaattt1198412003SEQ ID 1121 aaatgtcagctcttgttca0894109132 N0: t 1 5 WO 2004/091515 .. PCT/US2004/011255 NO:

SEQ 119 aggatctgagttattttgc1403514054SEQ 1122 gcaagtcagcccagttcct10920109392 N0:

NO:

SEQ 120 gtgcccttctcggttgctg18 37 SEQ 1123 cagccattgacatgagcac5740 NO:

NO:

SEQ 121 ggcgctgcctgcgctgctg146 165 SEQ 1124 cagctccacagactccgcc3062 N0:

NO:

SEQ 122 ctgcgctgctgctgctgct154 173 SEQ 1125 agcagaaggtgcgaagcag3224 N0:

NO:

SEQ 123 gctgctggcgggcgccagg170 189 SEQ 1126 cctggattccacatgcagc11846118651 N0:

NO:

SEQ 124 aagaggaaatgctggaaaa193 212 SEQ 1127 tttttcttcactacatctt2584 N0:

NO:

SEQ 125 ctggaaaatgtcagcctgg04 23 SEQ 1128 ccagacttccacatcccag3915 N0:

NO:

SEQ 126 tggagtccctgggactgct96 315 SEQ 1129 agcatgcctagtttctcca9945 N0:

NO:

SEQ 127 ggagtccctgggactgctg97 316 SEQ 1130 cagcatgcctagtttctcc9944 N0:

NO:

SEQ 128 tgggactgctgattcaaga305 324 SEQ 1131 tcttccatcacttgaccca2042 N0:

NO:

SEQ 129 ctgctgattcaagaagtgc310 329 SEQ 1132 gcacaccttgacattgcag11079110981 N0:

NO:

SEQ 130 tgccaccaggatcaactgc326 345 SEQ 1133 gcaggctgaactggtggca2717 N0:

NO:

SEQ 131 gccaccaggatcaactgca327 346 SEQ 1134 tgcaggctgaactggtggc2716 N0:

NO:

SEQ 132 tgcaaggttgagctggagg342 361 SEQ 1135. cctccacctctgatctgca4744 ID ID ~ 5 N0:

NO:

SEQ 133 caaggttgagctggaggtt344 363 SEQ 1136 aacccctacatgaagcttg13755137741 N0:

NO:

SEQ 134 ctctgcagcttcatcctga369 388 SEQ 1137 tcaggaagcttctcaagag13211132301 N0:

NO:

SEQ 135 cagcttcatcctgaagacc374 393 SEQ 1138 ggtcttgagttaaatgctg4977 N0:

NO:

SEQ 136 gcttcatcctgaagaccag376 395 SEQ 1139 ctggacgctaagaggaagc855 874 N0:

NO:

SEQ 137 tcatcctgaagaccagcca379 398 SEQ 1140 tggcatggcattatgatga3604 N0:

NO:

SEQ 138 gaaaaccaagaactctgag52 71 SEQ 1141 ctcaaccttaatgattttc8286 N0:

NO:

SEQ 139 agaactctgaggagtttgc60 79 SEQ 1142 gcaagctatacagtattct8377 N0:

NO:

SEQ 140 tctgaggagtttgctgcag65 84' SEQ 1143 ctgcaggggatcccccaga2526 N0:

NO:

SEQ 141 tttgctgcagccatgtcca74 93 SEQ 1144 tggaagtgtcagtggcaaa10372103911 N0:

NO:

SEQ 142 caagaggggcatcatttct578 597 SEQ 1145 agaataaatgacgttcttg7035 N0:

NO:

SEQ 143 tcactttaccgtcaagacg674 693 SEQ 1146 cgtctacactatcatgtga4360 N0:

NO:

SEQ 144 tttaccgtcaagacgagga678 697 SEQ 1147 tccttgacatgttgataaa7366 N0:

NO:

SEQ 145 cactggacgctaagaggaa853 872 SEQ 1148 ttccagaaagcagccagtg12498125171 N0:

NO:

~Q ID 146 aggaagcatgtggcagaag867 886 SEQ 1149 cttcatacacattaatcct9988 NO: l ID 5 WO 2004/091515..... ....... ....... ..-.... PCT/US2004/011255 NO:

SEQ ID 147 caaggagcaacacctcttc893 912 SEQ 1150 gaagtagtactgcatcttg N0: ID 5 NO:
' SEQ ID 148 acagactttgaaacttgaa959 978 SEQ 1151 ttcaattcttcaatgctgt N0: ID 5 NO:

SEQ ID 149 tgatgaagcagtcacatct11871206SEQ 1152 agatttgaggattccatca N0: ID 5 NO:

SEQ ID 150 agcagtcacatctctcttg11931212SEQ 1153 caaggagaaactgactgct N0: ID 5 NO:

SEQ ID 151 ccagccccatcactttaca12311250SEQ 1154 tgtagtctcctggtgctgg N0: ID 5 NO:

SEQ ID 152 ctccactcacatcctccag12801299SEO 1155 ctggagcttagtaatggag N0: ID 5 NO:

SEQ ID 153 catgccaacccccttctga13141333SEQ 1156 tcagatgagggaacacatg N0: ID 5 NO:

SEQ ID 154 gagagatcttcaacatggc13901409SEQ 1157 gccaccctggaactctctc N0: ID 5 NO:

SEQ ID 155 tcaacatggcgagggatca13991418SEQ 1158 tgatcccacctctcattga N0: ID 5 NO:

SEQ ID 156 ccaccttgtatgcgctgag14291448SEQ 1159 ctcagggatctgaaggtgg N0: ID 5 NO:

SEQ ID 157 gtcaacaactatcataaga14551474SEQ 1160 tcttgagttaaatgctgac N0: ID 5 NO:

SEQ ID 158 tggacattgctaattacct15011520SEQ 1161 aggtatattcgaaagtcca N0: ID 5 NO:

SEQ ID 159 ggacattgctaattacctg15021521SEQ 1162 caggtatattcgaaagtcc N0: ID 5 NO:

SEQ ID 160 ttctgcgggtcattggaaa15731592SEQ 1163 ttcacatgccaaggagaa 65146533:1 N0: ID t 5 NO:

SEQ ID 161 ccagaactcaagtcttcaa16201639SEQ 1164 tgaagtgtagtctcctgg N0: ID t 5 NO:

SEQ ID 162 agtcttcaatcctgaaatg16301649SEQ 1165 catttctgattggtggact 77577776:1 N0: ID 5 NO:

SEQ ID 163 tgagcaagtgaagaacttt18681887SEQ 1166 aaagtgccacttttactca N0: . ID 5 NO:

SEQ ID 164 agcaagtgaagaactttgt18701889SEQ 1167 acaaagtcagtgccctgct N0: ID 5 NO:

SEQ ID 165 tctgaaagaatctcaactt19641983SEQ 1168 aagtccataatggttcaga N0: ID 5 NO:

SEQ ID 166 actgtcatggacttcagaa1986005 SEQ 1169 tctgaatatattgtcagt N0: ID t 5 NO:

SEQ ID 167 acttgacccagcctcagcc051 070 SEQ 1170 ggctcaccctgagagaagt N0: ID 5 NO:

SEQ ID 168 tccaaataactaccttcct096 115 SEQ 1171 aggaagatatgaagatgga N0: ID 5 NO:

SEQ ID 169 actaccctcactgcctttg133 152 SEQ 1172 caaatttgtggagggtagt N0: ID 5 NO:

SEQ ID 170 ttggatttgcttcagctga149 168 SEQ 1173 cagtataagtacaaccaa N0: ID t 5 N0:

SEQ ID 171 ttggaagctctttttggga211 230 SEQ 1174 cccgattcacgcttccaa N0: ID t 1 5 NO:

SEQ ID 172 ggaagctctttttgggaag213 232 SEQ 1175 cttcagaaagctaccttcc N0: ID 5 NO:

SEQ ID 173 tttttcccagacagtgtca2238257 SEQ 1176 gaccttctctaagcaaaa 876 N0: ID t 4 4 5 NO:

SEQ ID 174 agacagtgtcaacaaagct246 2265SEQ 1177 gcttggttttgccagtct 458 N0: ID a 2 2 5 WO 2004/091515 P('T/TTS2004/011255 NO:

SEQ ID 175 ctttggctataccaaagat321 340 SEQ 1178 atctcgtgtctaggaaaag59685987 1 N0: ID 5 NO:

SEQ ID 176 caaagatgataaacatgag333 2352 SEQ 1179 ctcaaggataacgtgtttg12609126281 N0: ID 5 NO:

SEQ ID 177 gatatggtaaatggaataa23552374 SEQ 1180 ttatcttattaattatatc13079130981 N0: ID 5 NO:

SEQ ID 178 ggaataatgctcagtgttg2367386 SEQ 1181 caacacttacttgaattcc10661106801 N0: ID 5 NO:

SEQ ID 179 tttgaaatccaaagaagtc402 2421 SEQ 1182 gacttcagagaaatacaaa11400114191 N0: ID 5 NO:

SEQ ID 180 gatcccccagatgattgga534 553 SEQ 1183 tccaatttccctgtggatc36813700 1 N0: ID 5 NO:

SEQ ID 181 cagatgattggagaggtca541 560 SEQ 1184 tgaccacacaaacagtctg53635382 1 N0: ID 5 NO:

SEQ ID 182 agaatgacttttttcttca25752594 SEQ 1185 tgaagtccggattcattct11015110341 N0: ID 5 NO:

SEQ ID 183 gaactccccactggagctg619 638 SEQ 1186 cagctcaaccgtacagttc11861118801 N0: ID 5 NO:

SEQ ID 184 atatcttcatctggagtca2652671 SEQ 1187 tgacttcagtgcagaatat11966119851 N0: ID 5 NO:

SEQ ID 185 gtcattgctcccggagcca667 2686 SEQ 1188 tggccccgtttaccatgac58095828 1 N0: ID 5 NO:

SEQ ID 186 gctgaagtttatcattcct2873892 SEQ 1189 aggaggctttaagttcagc76007619 1 N0: ID 5 NO:

SEQ ID 187 attccttccccaaagagac2886905 SEQ 1190 gtctcttcctccatggaat10470104891 N0: ID 5 NO:

SEQ ID 188.ctcattgagaacaggcagt976 995 SEQ 1191 actgactgcacgctttgag11756117751 N0: ID 5 NO:

SEQ ID 189 ttgagcagtattctgtcag31423161 SEQ 1192 ctgagagaagtgtcttcaa12399124181 N0: ID 5 NO:

SEQ ID 190 accttgtccagtgaagtcc32853304 SEQ 1193 ggacggtactgtcccaggt12784128031 N0: ID 5 NO:

SEQ ID 191 ccagtgaagtccaaattcc32923311 SEQ 1194 ggaaggcagagtttactgg91489167 1 N0: ID 5 NO:

SEQ ID 192 acattcagaacaagaaaat33943413 SEQ 1195 atttcctaaagctggatgt11167111861 N0: ID 5 NO:

SEQ ID 193 gaaaaatcaagggtgttat34633482 SEQ 1196 ataaactgcaagatttttc13600136191 N0: ID 5 NO:

SEQ ID 194 aaatcaagggtgttatttc34663485 SEQ 1197 gaaacaatgcattagattt97459764 1 N0: ID 5 NO:

SEQ ID 195 tggcattatgatgaagaga36093628 SEQ 1198 tctcccgtgtataatgcca11781118001 N0: ID 5 NO:

SEQ ID 196 aagagaagattgaatttga36223641 SEQ 1199 tcaaaacctactgtctctt10458104771 N0: ID 5 NO:

SEQ ID 197 aaatgacttccaatttccc36733692 SEQ 1200 gggaactacaatttcattt70137032 1 N0: ID 5 NO:

SEQ ID 198 atgacttccaatttccctg36753694 SEQ 1201 caggctgattacgagtcat49174936 1 N0: ID 5 NO:

SEQ ID 199 acttccaatttccctgtgg36783697 SEQ 1202 ccacgaaaaatatggaagt10360103791 N0: ID 5 NO:

SEQ ID 200 agttgcaatgagctcatgg38033822 SEQ 1203 ccatcagttcagataaact79898008 1 N0: ID 5 NO:

SEQ ID 201 tttgcaagaccacctcaat38603879 SEQ 1204 attgacctgtccattcaaa13671136901 N0: ID 5 NO:

SEQ ID 202 gaaggagttcaacctccag38843903 SEQ 1205 ctggaattgtcattccttc11728117471 N0:1 I 1 1 I ID 5 WO 2004/091515 P('T/TTS2004/011255 NO:

SEQ ID 203 acttccacatcccagaaaa39193938 SEQ 1206 ttttaacaaaagtggaagt68216840 1 N0: ID 5 .

NO:

SEQ ID 204 ctcttcttaaaaagcgatg39393958 SEQ 1207 catcactgccaaaggagag84868505 1 N0: ID 5 NO:

SEQ ID 205 aaaagcgatggccgggtca39483967 SEQ 1208 tgactcactcattgatttt12680126991 N0: ID 5 NO:

SEQ ID 206 ttcctttgccttttggtgg003 022 SEQ 1209 ccacaaacaatgaagggaa92569275 1 N0: ID 5 NO:

SEQ ID 207 caagtctgtgggattccat079 098 SEQ 1210 atgggaaaaaacaggcttg95669585 1 N0: ID 5 NO:

SEQ ID 208 aagtccctacttttaccat117 136 SEQ 1211 atgggaagtataagaactt48344853 1 N0: ID 5 NO:

SEQ ID 209 tgcctctcctgggtgttct159 178 SEQ 1212 agaaaaacaaacacaggca96439662 1 N0: ID 5 NO:

SEQ ID 210 accagcacagaccatttca242 261 SEQ 1213 tgaagtgtagtctcctggt50895108 1 N0: ID 5 NO:

SEQ ID 211 ccagcacagaccatttcag243 262 SEQ 1214 ctgaaatacaatgctctgg55115530 1 N0: ID 5 NO:

SEQ ID 212 actatcatgtgatgggtct367 386 SEQ 1215 agacacctgattttatagt79487967 1 NO: ID 5 NO:

SEQ ID 213 accacagatgtctgcttca496 515 SEQ 1216 tgaaggctgactctgtggt42824301 1 N0: ID 5 NO:

SEQ ID 214 ccacagatgtctgcttcag497 516 SEQ 1217 ctgagcaacaaatttgtgg10311103301 N0: ID 5 NO:

SEQ ID 215 tttggactccaaaaagaaa520 539 SEQ 1218 tttctctcatgattacaaa59335952 1 N0: ID 5 NO:

SEQ ID 216 tcaaagaagtcaagattga552 571 SEQ 1219 tcaaggataacgtgtttga12610126291 NO: ID 5 NO:

SEQ ID 217 atgagaactacgagctgac798 817 SEQ 1220 gtcagatattgttgctcat10187102061 NO: ID 5 NO:

SEQ ID 218 ttaaaatctgacaccaatg818 837 SEQ 1221 cattcattgaagatgttaa73427361 1 N0: ID 5 NO:

SEQ, 219 gaagtataagaactttgcc838 857 SEQ 1222 ggcaaatttgaaggacttc11994120131 ID NO: ID 5 NO:

SEQ ID 220 aagtataagaactttgcca839 858 SEQ 1223 tggcaaatttgaaggactt11993120121 NO: ID 5 NO:

SEQ ID 221 tcttcagcctgctttctg941 960 SEQ 1224 cagaatccagatacaagaa68846903 1 N0: t ID 5 NO:

SEQ ID 222 ctggatcactaaattccca957 976 SEQ 1225 gggtctttccagagccag N0: ID t 5 NO:

SEQ.ID 223 aaattaatagtggtgctca50145033 SEQ 1226 gagaagccccaagaattt N0: ID t 5 NO:

SEQ ID 224 agtgcaacgaccaacttga50735092 SEQ 1227 caaattcctggatacact N0: ID t 5 NO:

SEQ ID 225 ctgggaagtgcttatcagg52385257 SEQ 1228 cctgaccttcacataccag83108329 1 N0: ID 5 NO:

SEQ ID 226 gcaaaaacattttcaactt52785297 SEQ 1229 aagtaaaagaaaattttgc10744107631 N0: ID 5 NO:

SEQ ID 227 aaaaacattttcaacttca52805299 SEQ 1230 gaagtaaaagaaaatttt N0: ID t 5 NO:

SEQ ID 228 cagtcaagaaggacttaa53025321 SEQ 231 taaggacttccattctga N0: t ID t 1 5 NO:

SEQ ID 229 caaatgacatgatgggct53255344 SEQ 232 agcccatcaatatcattga62056224 5 N0: t ID 1 NO:

SEQ ID 230 acacaaacagtctgaaca53675386 SEQ 233 gtttcaactgcctttgtg N0: c ID t 1 NO:

SEQ ID 231 tcttcaaaacttgacaaca5409 5428SEQ 1234 tgttttcctatttccaaga12835128541 N0: ID 5 NO:

SEQ ID 232 caagttttataagcaaact5441 5460SEQ 1235 agttattttgctaaacttg14043140621 N0: ID 5 NO:

SEQ ID 233 tggtaactactttaaacag5488 5507SEQ 1236 ctgtttttagaggaaacca75127531 1 N0: ID 5 NO:

SEQ ID 234 aacagtgacctgaaataca5502 5521SEQ 1237 tgtatagcaaattcctgtt58905909 1 N0: ID 5 NO:

SEQ ID 235 gggaaactacggctagaac5544 5563SEQ 1238 gttccttccatgatttccc10933109521 N0: ID 5 NO:

SEQ ID 236 aacacatctatgccatctc5620 5639SEQ 1239 gagacagcatcttcgtgtt11204112231 N0: ID 5 NO:

SEQ ID 237 tcagcaagctataaagcag5652 5671SEQ 1240 ctgctaagaaccttactga77807799 1 N0: ID 5 NO:

SEQ ID 238 gcagacactgttgctaagg5667 5686SEQ 1241 cctttcaagcactgactgc11746117651 N0: ID 5 NO:

SEQ ID 239 tctggggagaacatactgg5866 5885SEQ 1242 ccaggttttccacaccaga80388057 1 N0: ID 5 NO:

SEQ ID 240 ttctctcatgattacaaag5934 5953SEQ 1243 ctttttcaccaacggagaa10838108571 N0: ID 5 NO:

SEQ ID 241 ctgagcagacaggcacctg6034 6053SEQ 1244 caggaggctttaagttcag75997618 1 N0: ID 5 NO:

SEQ ID 242 caatttaacaacaatgaat6066 6085SEQ 1245 attccttcctttacaattg80828101 1 N0: ID 5 NO:

SEQ ID 243 ggacgaactctggctgac6140 6159SEQ 1246 gtcagcccagttccttcca10924109431 N0: t ID 5 NO:

SEQ ID 244 cttttactcagtgagccca6192 6211SEQ 1247 tgggctaaacgtatgaaag78277846'1 N0: ID 5 NO:

SEQ ID 245 cattgatgctttagagat6217 6236SEQ 1248 atcttcataagttcaatga13174131931 NO: t ID 5 NO:

SEQ ID 246 aaaaccaagatgttcactc6295 6314SEQ 1249 gagtgaaatgctgtttttt86308649r1 N0: ID 5 NO:

SEQ ID 247 aggaatcgacaaaccatta6357 6376SEQ 1250 taatgattttcaagttcct82948313'1 N0: ID 5 NO:

SEQ ID 248 agttgtactggaaaacgt6376 6395SEQ 1251 acgttagcctctaagacta11928119471 N0: t ID 5 NO:

SEQ ID 249 ggaaaacgtacagagaaag6386 6405SEQ 1252 cttttacaattcattttcc13014130331 N0: ID 5 NO:

SEQ ID 250 gaaaacgtacagagaaagc6387 6406SEQ 1253 gctttctcttccacatttc10052100711 N0: ID 5 NO:

SEQ ID 251 aaagctgaagcacatcaat6401 6420SEQ 1254 attgatgttagagtgcttt69847003 1 N0: ID 5 NO:

SEQ ID 252 aagctgaagcacatcaata6402 6421SEQ 1255 attgatgttagagtgctt N0: ID t 5 NO:

SEQ ID 253 gaagcacatcaatattga6406 6425SEQ 1256 caaccttaatgattttca N0: t ID t 5 NO:

SEQ ID 254 atcaatattgatcaatttg6414 6433SEQ 1257 caaagccatcactgatgat16601679 1 N0: ID 5 NO:

SEQ ID 255 aatgattatctgaattca6476 495 SEQ 1258 gaaatcattgaaaaatta N0: t 6 ID t 1 NO:

SEQ ID 256 attatctgaattcattca6480 499 SEQ 259 gaagtagctgagaaaatc N0: g 6 ID t 1 NO:

SEQ ID 257 attgggagagacaagttt6498 517 SEQ 260 aaacattcctttaacaatt488 N0: a 6 ID 9 1 NO:

SEQ ID 258 aaatagctattgctaata6693 712 SEQ 261 attgaaaatattgatttt 806 825 NO:I la 6 ID t 6 6 1 WO 2004/091515 P('T/TTS2004/011255 .. , ..... _ . NO:

SEQ ID 259 aaaattaaaaagtcttgat67316750 SEQ 1262 atcatatccgtgtaatttt6757 N0: ID 5 NO:

SEQ ID 260 ttgaaaatattgattttaa68086827 SEQ 1263 ttaatcttcataagttcaa13171131901 N0: ID 5 NO:

SEQ ID 261 agacatccagcacctagct69386957 SEQ 1264 agcttggttttgccagtct2458 N0: ID 5 NO:

SEQ ID 262 caatttcatttgaaagaat70217040 SEQ 1265 attccttcctttacaattg8082 N0: ID 5 NO:

SEQ ID 263 aggttttaatggataaatt71747193 SEQ 1266 aattgttgaaagaaaacct13147131661 N0: ID 5 NO:

SEQ ID 264 cagaagctaagcaatgtcc72337252 SEQ 1267 ggacaaggcccagaatctg12545125641 N0: ID 5 NO:

SEQ ID 265 taagataaaagattacttt72627281 SEQ 1268 aaagaaaacctatgcctta13155131741 N0: ID 5 NO:

SEQ ID 266 aaagattactttgagaaat72697288 SEQ 1269 atttcttaaacattccttt9481 N0: ID 5 NO:

SEQ ID 267 gagaaattagttggattta72817300 SEQ 1270 taaagccattcagtctctc12962129811 N0: . ID 5 NO:

SEQ ID 268 atttattgatgatgctgtc72957314 SEQ 1271 gacatgttgataaagaaat7371 N0: ID 5 NO:

SEQ ID 269 gaattatcttttaaaacat73267345 SEQ 1272 atgtatcaaatggacattc7677 N0: ID 5 NO:

SEQ ID 270 ttaccaccagtttgtagat74037422 SEQ 1273 atctggaaccttgaagtaa10731107501 N0: ID 5 NO:

SEQ ID 271 ttgcagtgtatctggaaag75407559 SEQ 1274 cttttcacattagatgcaa8412 NO: ID 5 NO:

SEQ ID 272 cattcagcaggaacttcaa76917710 SEQ 1275 ttgaaggacttcaggaatg12001120201 N0: ID 5 NO:

SEQ ID 273 acacctgattttatagtcc79507969 SEQ 1276 ggactcaaggataacgtgt12606126251 N0: ID 5 NO:

SEQ ID 274 ggattccatcagttcagat79848003 SEQ 1277 atcttcaatgattatatcc13116131351 N0: ID 5 NO:

SEQ ID 275 tgtagaaatgaaagtaaa81048123 SEQ 1278 tttatgattatgtcaacaa12352123711 N0: t ID 5 NO:

SEQ ID 276 ctgaacagtgagctgcagt81488167 SEQ 1279 actggacttctctagtcag8801 N0: ID 5 NO:

SEQ ID 277 aatccaatctcctcttttc83998418 SEQ 1280 gaaaaatgaagtccggatt11009110281 N0: ID 5 NO:

SEQ ID 278 attttgattttcaagcaaa85248543 SEQ 1281 ttgcaagttaaagaaaat N0: ID t 5 NO:

SEQ ID 279 tttgattttcaagcaaat85258544 SEQ 1282 atttgatttaagtgtaaaa9614 N0: t ID 5 NO:

SEQ ID 280 gattttcaagcaaatgca85288547 SEQ 1283 gcaagttaaagaaaatca N0: t ID t 5 NO:

SEQ ID 281 atgctgttttttggaaatg86378656 SEQ 284 cattggtaggagacagcat11195112141 N0: ID 5 NO:

SEQ ID 282 gctgttttttggaaatgc86388657 SEQ 285 gcattggtaggagacagca11194112131 N0: t ID 5 NO:

SEQ ID 283 aaaaaaatacactggagct86988717 SEQ 286 agctagagggcctcttttt08251084415 N0: ID 1 NO:

SEQ ID 284 actggagcttagtaatgga87088727 SEQ 287 ccactcacatcctccagt 281 N0: ID t 1 1 1 NO:

SEQ ID 285 ttctggaaaagggtcatg88788897 SEQ 288 atgaacccctacatgaag 3751 N0: c ID c 1 1 NO:

SEQ ID 286 gaaaagggtcatggaaat88838902 SEQ 289 tttgaaagttcgttttcc 274 N0: g ID a 9 9 1 WO 2004/091515 P('T/TTS2004/011255 NO:

SEQ ID 287 gggcctgccccagattctc89028921SEQ ID 1290 gagaacattatggaggccc94329451 1 N0: 5 NO:

SEQ ID 288 ttctcagatgagggaacac89168935SEQ ID 1291 gtgtcttcaaagctgagaa12408124271 N0: 5 NO:

SEQ ID 289 gatgagggaacacatgaat89228941SEQ ID 1292 attccagcttccccacatc83308349 1 N0: 5 NO:

SEQ ID 290 ctttggactgtccaataag89788997SEQ ID 1293 cttatgggatttcctaaag11159111781 N0: 5 NO:

SEQ ID 291 gcatccacaaacaatgaag92529271SEQ ID 1294 cttcatctgtcattgatgc10219102381 N0: 5 NO:

SEQ ID 292 cacaaacaatgaagggaat92579276SEQ ID 1295 attccctgaagttgatgtg11480114991 N0: 5 NO:

SEQ ID 293 ccaaaatttctctgctgga94079426SEQ ID 1296 tccatcacaaatcctttgg96639682 1 N0: 5 NO:

SEQ ID 294 caaaatttctctgctggaa94089427SEQ ID 1297 ttccatcacaaatcctttg96629681 1 N0: 5 NO:

SEQ ID 295 tctgctggaaacaacgaga94179436SEQ ID 1298 tctcaagagttacagcaga13221132401 N0: 5 NO:

SEQ ID 296 ctgctggaaacaacgagaa94189437SEQ ID 1299 ttctcaagagttacagcag13220132391 N0: ~ 5 NO:

SEQ ID 297 agaacattatggaggccca94339452SEQ ID 1300 tgggcctgccccagattct89018920 1 NO: 5 NO:

SEQ ID 298 agaagcaaatctggatttc94679486SEQ ID 1301 gaaatcttcaatttattct13813138321 N0: 5 NO:

SEQ ID 299 tttctctctatgggaaaaa95579576SEQ ID 1302 tttttgcaagttaaagaaa14013140321 N0: 5 NO:

SEQ ID 300 tcagagcatcaaatccttt97049723SEQ ID 1303 aaagaaaatcaggatctga140251404415 N0:

NO:

SEQ ID 301 cagaaacaatgcattagat97439762SEQ ID 1304 atctatgccatctcttctg56255644 1 NO: 5 NO:

SEQ ID 302 tacacattaatcctgccat999310012SEQ ID 1305 atggagtctttattgtgta14081141001 NO: 5 NO:

SEQ ID 303 agtcagatattgttgctca1018610205SEQ ID 1306 tgagaactacgagctgact47994818 1 N0: 5 NO:

SEQ ID 304 ggagggtagtcataacagt1032810347SEQ ID 1307 actggtggcaaaaccctcc27262745 1 N0: 5 NO:

SEQ ID 305 caaaagccgaaattccaat1039610415SEQ ID 1308 attgaagtacctacttttg83588377 1 N0: 5 NO:

SEQ ID 306 aaaagccgaaattccaatt1039710416SEQ ID 1309 aattgaagtacctactttt83578376 1 N0: 5 NO:

SEQ ID 307 tcaagcaagaacttaatg1042810447SEQ ID 1310 cattatggcccttcgtgaa13250132691 N0: t 5 NO:

SEQ ID 308 cctcttacttttccattga1057010589SEQ ID 1311 caaaagaagcccaagagg N0: t 5 NO:

SEQ ID 309 gaggccaacacttacttg1065510674SEQ ID 1312 caagcatctgattgactca12668126871 N0: t 5 NO:

SEQ ID 310 cacttacttgaattccaag1066410683SEQ ID 1313 cttgaacacaaagtcagtg60006019 1 NO: 5 NO:

SEQ ID 311 gaagtaaaagaaaattttg1074310762SEQ ID 314 caaaaacattttcaacttc52795298 1 N0: 1 5 NO:

SEQ ID 312 cctggaactctctccatgg1087410893SEQ ID 315 ccatttacagatcttcagg113641138315 N0: 1 NO:

SEQ ID 313 agctggatgtaaccaccag117611195SEQ ID 316 ctggattccacatgcagct18471186615 NO: 1 1 1 NO:

SEQ ID 314 aaaattccctgaagttgat147711496SEQ ID 317 atcatatccgtgtaatttt67576776 5 NO: 1 1 1 wn ~nnam4i si s prTirrc~nnami i ass .._ _..... .. ... NO: _ .__ SEQ ID 315 cagatggcattgctgcttt1160511624SEQ ID 1318 aaagctgagaagaaatctg12416124351 N0: 5 NO:

SEQ ID 316 agatggcattgctgctttg1160611625SEQ ID 1319 caaagctgagaagaaatct12415124341 N0: 5 NO:

SEQ ID 317 tgttgaaacagtcctggat1183411853SEQ ID 1320 atccaagatgagatcaaca13095131141 N0: 5 NO:

SEQ ID 318 catattcaaaactgagttg1222112240SEQ ID 1321 caactctctgattactatg136231364215 N0:

NO:

SEQ ID 319 aaagatttatcaaaagaag1293012949SEQ ID 1322 cttcaatttattcttcttt13818138371 NO: 5 NO:

SEQ ID 320 attttccaactaatagaag1302613045SEQ ID 1323 cttcaaagacttaaaaaat80068025 1 N0: 5 NO:

SEQ ID 321 aattatatccaagatgaga1308913108SEQ ID 1324 tctcttcctccatggaatt10471104901 N0: 5 NO:

SEQ ID 322 ttcaggaagcttctcaaga1321013229SEQ ID 1325 tcttcataagttcaatgaa13175131941 NO: 5 NO:

SEQ ID 323 ttgagcaatttctgcacag1342913448SEQ ID 1326 ctgttgaaagatttatcaa12924129431 NO: 5 NO:

SEQ ID 324 ctgatatacatcacggagt1370413723SEQ ID 1327 actcaatggtgaaattcag74577476 1 N0: 5 NO:

SEQ ID 325 acatcacggagttactgaa1371113730SEQ ID 1328 ttcagaagctaagcaatgt72317250 1 N0: 5 NO:

SEQ ID 326 actgcctatattgataaaa1387413893SEQ ID 1329 ttttggcaagctatacagt83728391 1 NO: 5 NO:

SEQ ID 327 aggatggcattttttgcaa1400314022SEQ ID 1330 ttgcaagcaagtctttcct30053024 1 N0: 5 NO:

SEQ ID 328 ttttttgcaagttaaagaa1401214031SEQ ID 1331 ttctctctatgggaaaaaa95589577 1 NO: 5 NO:

SEQ ID 329 tccagaactcaagtcttca16191638SEQ ID 1332 tgaaatgctgttttttgga86338652 3 NO: 4 NO:

SEQ ID 330 agttagtgaaagaagttct19481967SEQ ID 1333 agaatctgtaccaggaact12556125753 NO: 4 NO:

SEQ ID 331 atttacagctctgacaagt54275446SEQ ID 1334 acttcagagaaatacaaat11401114203 NO: 4 NO:

SEQ ID 332 gattatctgaattcattca64806499SEQ ID 1335 tgaaaccaatgacaaaatc74217440 3 NO: 4 NO:

SEQ ID 333 gtgcccttctcggttgctg18 37 SEQ ID 1336 cagctgagcagacaggcac60316050 2 NO: 4 NO:

SEQ ID 334 attcaagcacctccggaag45 264SEQ ID 1337 cttcataagttcaatgaat13176131952 NO: 4 NO:

SEQ ID 335 gactgctgattcaagaagt308 327SEQ ID 1338 acttcccaactctcaagtc13407134262 NO: 4 NO:

SEQ ID 336 tgctgcagccatgtccag75 94 SEQ ID 1339 ctgggcagctgtatagcaa58815900 NO: t 4 NO:

SEQ ID 337 agaaagatgaacctactta547 566SEQ ID 1340 aagtatgatttcaattct NO: t 4 NO:

SEQ ID 338 gaagactctccaggaact087 106SEQ ID 1341 agttcaatgaatttattca13183132022 NO: t 1 1 4 NO:

SEQ ID 339 atctctcttgccacagctg202 221SEQ ID 1342 cagcccagccatttgagat92299248 2 NO: 1 1 4 NO:

SEQ ID 340 ctctcttgccacagctga203 222SEQ ID 343 cagcccagccatttgaga 92289247 NO: t 1 1 1 t 4 NO:

SEQ ID 341 gaggtgtccagccccatc223 242SEQ ID 344 gatgggaaagccgccctca52085227 NO: t 1 1 1 4 NO:

SEQ ID 342 cagaactcaagtcttcaa620 639SEQ ID 345 tgaaagcagaacctctgg 59075926 NO: c 1 1 1 t 4 NO:

SEQ ID 343 ctgaaaaagttagtgaaag19411960SEQ 1346 ctttctcgggaatattcag10623106422 NO: ID 4 NO:

SEQ ID 344 tttttcccagacagtgtca2238257 SEQ 1347 tgacaggcattttgaaaaa9722 NO: ID 4 NO:

SEQ ID 345 ttttcccagacagtgtcaa239 258 SEQ 1348 ttgacaggcattttgaaaa9721 NO: ID 4 NO:

SEQ ID 346 cattcagaacaagaaaatt33953414SEQ 1349 aattccaattttgagaatg10406104252 NO: ID 4 NO:

SEQ ID 347 tgaagagaagattgaattt36203639SEQ 1350 aaatgtcagctcttgttca10894109132 NO: ID 4 NO:

SEQ ID 348 tttgaatggaacacaggca36363655SEQ 1351 tgccagtttgaaaaacaaa11807118262 NO: ID 4 NO:

SEQ ID 349 ttctagattcgaatatcaa399 418 SEQ 1352 ttgacatgttgataaagaa7369 NO: ID 4 NO:

SEQ ID 350 gattcgaatatcaaattca404 423 SEQ 1353 tgaagtagaccaacaaatc7154 NO: ID 4 NO:

SEQ ID 351 tgcaacgaccaacttgaag50755094SEQ 1354 cttcaggttccatcgtgca11376113952 NO: ID 4 NO:

SEQ ID 352 ttaagctctcaaatgacat53175336SEQ 1355 atgttgataaagaaattaa7374 NO: ID 4 NO:

SEQ ID 353 caatttaacaacaatgaat60666085SEQ..ID1356 attcaaactgcctatattg13868138872 NO: 4 NO:

SEQ ID 354 tgaatacagccaggacttg60806099SEQ 1357 caagagcacacggtcttca10679106982 NO: ID 4 NO:

SEQ ID 355 catcaatattgatcaattt64136432SEQ 1358 aaattccctgaagttgatg11478114972 NO: ID 4 NO:

SEQ ID 356 ttgagcatgtcaaacactt70517070SEQ 1359 aagtaagtgctaggttcaa9373 NO: ID 4 NO:

SEQ ID 357 gaaggagactattcagaa72197238SEQ 1360 tctgcacagaaatattca NO: t ID t 4 NO:

SEQ ID 358 tcaggctcttcagaaagc79217940SEQ 1361 gcttgctaacctctctgaa12304123232 NO: t ID 4 NO:

SEQ ID 359 ccacaaattgaacatccc87798798SEQ 1362 gggacctaccaagagtgga12525125442 NO: t ID 4 NO:

SEQ ID 360 gaataccaatgctgaact1015910178SEQ 1363 agttcaatgaatttattca13183132022 NO: t ID 4 NO:

SEQ ID 361 aaactaatagatgtaatc1289012909SEQ 1364 gattactatgaaaaattta13632136512 NO: t ID 4 NO:

SEQ ID.NO:362 tgacctgtccattcaaaa1367213691SEQ 1365 tttaaaagaaatcttcaa t ID t 4 NO:

SEQ ID 363 gggctgagtgcccttctcg11 0 SEQ 1366 cgaggccaggccgcagccc76 95 N0: 3 ID 4 NO:

SEQ ID 364 ggctgagtgcccttctcgg12 1 SEQ 1367 ccgaggccaggccgcagcc75 94 NO: 3 ID 4 NO:

SEQ ID 365 ctgagtgcccttctcggtt14 3 SEQ 1368 aaccgtgcctgaatctcag11549115681 N0: 3 ID 4 NO:

SEQ ID 366 ctcggttgctgccgctga5 4 SEQ 1369 cagctgacctcatcgaga 2160 N0: t ID t 4 NO:

SEQ ID 367 caggccgcagcccaggagc82 01 SEQ 1370 ctctgcagcttcatcctg 368 N0: 1 ID g 4 NO:

SEQ ID 368 gctggcgctgcctgcgctg143 62 SEQ 371 agcacagaccatttcagc 4244 N0: 1 ID c 4 NO:

SEQ ID 369 gctgctggcgggcgccag169 88 SEQ 372 tggatgtaaccaccagca N0: t 1 ID c 1 4 NO:

SEQ ID 370 tggtctgtccaaaagatg19 38 SEQ 373 atcctgaagaccagccag 80 99 N0: c ID c 3 3 4 WO 2004/091515..... ......_ ....... ...... ....... PCT/US2004/011255 NO:

SEQ ID 371 ctgagagttccagtggagt83 302 SEQ 1374 actcaccctggacattcag3383 N0: ID 4 NO:

SEQ ID 372 tccagtggagtccctggga291 310 SEQ 1375 tcccggagccaaggctgga2675 N0: ID 4 NO:

SEQ ID 373 aggttgagctggaggttcc346 365 SEQ 1376 ggaaccctctccctcacct4728 N0: ID 4 NO:

SEQ ID 374 tgagctggaggttccccag350 369 SEQ 1377 ctgggaggcatgatgctca9163 NO: ID 4 NO:

SEQ ID 375 tctgcagcttcatcctgaa370 389 SEQ 1378 ttcaaatataatcggcaga3261 N0: ID 4 NO:

SEQ ID 376 gccagtgcaccctgaaaga394 13 SEQ 1379 tcttccgttctgtaatggc5794 N0: ID 4 NO:

SEQ ID 377 ctctgaggagtttgctgca64 83 SEQ 1380 tgcaagaatattttgagag6340 N0: ID 4 NO:

SEQ ID 378 aggtatgagctcaagctgg92 511 SEQ 1381 ccagtttccggggaaacct12716127351 N0: ID 4 NO:

SEQ ID 379 tcctttacccggagaaaga535 554 SEQ 1382 tctttttgggaagcaagga2219 N0: ID 4 NO:

SEQ ID 380 catcaagaggggcatcatt575 594 SEQ 1383 aatggtcaagttcctgatg2277 N0: ID 4 NO:

SEQ ID 381 tcctggttcccccagagac601 620 SEQ 1384 gtctctgaactcagaagga13988140071 N0: ID 4 NO:

SEQ ID 382 aagaagccaagcaagtgtt622 641 SEQ 1385 aacaaataaatggagtctt14072140911 NO: ID 4 NO:

SEQ ID 383 aagcaagtgttgtttctgg630 649 SEQ 1386 ccagagccaggtcgagctt11042110611 N0: ID 4 NO:

SEQ ID 384 tctggataccgtgtatgga644 663 SEQ 1387 tccatgtcccatttacaga1135611375'1 N0: ID 4 NO:

SEQ ID 385 ccactcactttaccgtcaa670 689 SEQ 1388 ttgattttaacaaaagtgg6817 N0: ID 4 NO:

SEQ ID 386 aggaagggcaatgtggcaa693 712 SEQ 1389 ttgcaagcaagtctttcct3005 N0: ID 4 NO:

SEQ ID 387 gcaatgtggcaacagaaat700 719 SEQ 1390 atttccataccccgtttgc3480 N0: ID 4 NO:

SEQ ID 388 caatgtggcaacagaaata701 720 SEQ 1391 tattcttcttttccaattg13826138451 N0: ID 4 NO:

SEQ ID 389 ggcaacagaaatatccac706 725 SEQ 1392 gtggcttcccatattgcca1887 N0: t ID 4 NO:

SEQ ID 390 agagacctgggccagtgtg729 748 SEQ 1393 cacattacatttggtctct2930 NO:

SEQ ID 391 gtgatcgcttcaagccca744 763 SEQ 1394 gggaaagccgccctcaca 5210 N0: t ID t 4 NO:

SEQ ID 392 gtgatcgcttcaagcccat745 764 SEQ 1395 atgggaaagccgccctcac5209 N0: ID 4 NO:

SEQ ID 393 cagcccacttgctctcatc776 795 SEQ 396 gatgctgaacagtgagctg8144 N0: ID 4 NO:

SEQ ID 394 gctctcatcaaaggcatga786 805 SEQ 397 cataacagtactgtgagc N0: ID t 4 NO:

SEQ ID 395 ccttgtcaactctgatcag811 830 SEQ 398 ctgagtgggtttatcaagg12445124641 N0: ID 4 NO:

SEQ ID 396 cttgtcaactctgatcagc812 831 SEQ 399 gctgagtgggtttatcaag24441246314 N0: ID 1 NO:

SEQ ID 397 agccatctgcaaggagcaa884 903 SEQ 400 tgcaatgagctcatggct 805 N0: ID t 3 1 NO:

SEQ ID 398 ccatctgcaaggagcaac885 904 SEQ 401 ttgcaatgagctcatggc 804 N0: g ID g 3 3 1 WO 2004/091515 P('T/TTS2004/011255 NO:

SEQ ID 399 cttcctgcctttctcctac908 927 SEQ ID 1402 gtaggaataaatggagaag94539472 1 N0: 4 NO:

SEQ ID 400 ctttctcctacaagaataa916 935 SEQ ID 1403 ttattgctgaatccaaaag13648136671 N0: 4 NO:

SEQ ID 401 gatcaacagccgcttcttt989 1008SEQ ID 1404 aaagccatcactgatgatc16611680 1 N0: 4 NO:

SEQ ID 402 atcaacagccgcttctttg990 1009SEQ ID 1405 caaagccatcactgatgat16601679 1 N0: 4 NO:

SEQ ID 403 acagccgcttctttggtga994 1013SEQ ID 1406 tcacaaatcctttggctgt96679686 1 N0: 4 NO:

SEQ ID 404 aagatgggcctcgcatttg10231042SEQ ID 1407 caaaatagaagggaatctt20692088 1 N0: 4 NO:

SEQ ID 405 tgttttgaagactctccag10821101SEQ ID 1408 ctggtaactactttaaaca54875506 1 N0: 4 NO:

SEQ ID 406 ttgaagactctccaggaac10861105SEQ ID 1409 gttcaatgaatttattcaa13184132031 N0: 4 NO:

SEQ ID 407 aactgaaaaaactaaccat11021121SEQ ID 1410 atggcattttttgcaagtt14006140251 N0: 4 NO:

SEQ ID 408 ctgaaaaaactaaccatct11041123SEQ ID 1411 agattgatgggcagttcag45644583 1 N0: 4 NO:

SEQ ID 409 aaaactaaccatctctgag11091128SEQ ID 1412 ctcaaagaatgactttttt25702589 1 N0: 4 NO:

SEQ ID 410 tgagcaaaatatccagaga11241143SEQ ID 1413 tctccagataaaaaactca12201122201 N0: 4 NO:

SEQ ID 411 caataagctggttactgag11541173SEQ ID 1414 ctcagatcaaagttaattg12265122841 N0: 4 NO:

SEQ ID 412 tactgagctgagaggcctc11661185SEQ ID 1415 gagggtagtcataacagta10329103481 N0: 4 NO:

SEQ ID 413 gcctcagtgatgaagcagt11801199SEQ ID 1416 actgttgactcaggaaggc12572125911 NO: 4 NO:

SEQ ID 414 agtcacatctctcttgcca11961215SEQ ID 1417 tggccacatagcatggact8858887.71 N0: 4 NO:

SEQ ID 415 atctctcttgccacagctg12021221SEQ ID 1418 cagctgacctcatcgagat21612180 1 N0: 4 NO:

SEQ ID 416 ctctcttgccacagctga12031222SEQ ID 1419 tcagctgacctcatcgaga21602179 1 N0: t 4 NO:

SEQ ID 417 gccacagctgattgaggt12101229SEQ ID 1420 acctgcaccaaagctggca13955139741 N0: t 4 NO:

SEQ ID 418 gccacagctgattgaggtg211 230 SEQ ID 1421 caccaaaaaccccaatggc11240112591 N0: 1 1 4 NO:

SEQ ID 419 cactttacaagccttggt240 259 SEQ ID 1422 accagatgctgaacagtga81408159 1 N0: t 1 1 4 NO:

SEQ ID 420 cccttctgatagatgtggt324 343 SEQ ID 1423 accacttacagctagaggg10816108351 N0: 1 1 4 NO:

SEQ ID 421 gtcacctacctggtggccc341 360 SEQ ID 1424 gggcgacctaagttgtgac34313450 1 N0: 1 1 4 NO:

SEQ ID 422 ccttgtatgcgctgagcca432 451 SEQ ID 1425 ggctggtaacctaaaagg N0: 1 1 t 4 NO:

SEQ ID 423 gacaaaccctacagggacc472 491 SEQ ID 1426 ggtcctttatgattatgtc123471236614 N0: 1 1 NO:

SEQ ID 424 gctaattacctgatggaa508 527 SEQ ID 427 tcccaaaagcagtcagca N0: t 1 1 1 t 1 NO:

SEQ ID 425 gactgcactggggatgaa538 557 SEQ ID 428 tcaggtccatgcaagtca N0: t 1 1 1 t 1 NO:

SEQ ID 426 ctgcactggggatgaaga540 559 SEQ ID 429 cttgaacacaaagtcagt 5999018 N0: a 1 1 1 t 6 1 WO 2004/091515.......... ....... ...... ...... P('T/TTS2004/011255 NO:

SEQ ID 427 atgaagattacacctattt15521571SEQ 1430 aaatgaaagtaaagatcat N0: ID 4 NO:

SEQ ID 428 accatggagcagttaactc16021621SEQ 1431 gagtaaaccaaaacttggt N0: ID 4 NO:

SEQ ID 429 gcagttaactccagaactc16101629SEQ 1432 gagttactgaaaaagctgc N0: ID 4 NO:

SEQ ID 430 cagaactcaagtcttcaat16211640SEQ 1433 attggatatccaagatctg N0: ID 4 NO:

SEQ ID 431 caggctctgcggaaaatgg16951714SEQ 1434 ccatgacctccagctcctg N0: ID 4 NO:

SEQ ID 432 ccaggaggttcttcttcag17301749SEQ 1435 ctgaaatacaatgctctgg N0: ID 4 NO:

SEQ ID 433 ggttcttcttcagactttc17361755SEQ 1436 gaaaaacttggaaacaacc N0: ID 4 NO:

SEQ ID 434 tttccttgatgatgcttct17511770SEQ 1437 agaatccagatacaagaaa N0: ID 4 NO:

SEQ ID 435 ggagataagcgactggctg17731792SEQ 1438 cagcatgcctagtttctcc N0: ID 4 NO:

SEQ ID 436 gctgcctatcttatgttga17881807SEQ 1439 tcaatatcaaaagcccagc N0: ID 4 NO:

SEQ ID 437 actttgtggcttcccatat18821901SEQ 1440 atatctggaaccttgaagt N0: ID 4 NO:

SEQ ID 438 gccaatatcttgaactcag19021921SEQ 1441 ctgaactcagaaggatggc N0: ID 4 NO:

SEQ ID 439 aatatcttgaactcagaag19051924SEQ 1442 cttccattctgaatatatt N0: ID 4 NO:

SEQ ID 440 ctcagaagaattggatatc19161935SEQ 1443 gataaaagattactttgag N0: ID 4 NO:

SEQ ID 441 aagaattggatatccaaga19211940SEQ 1444 tcttcaatttattcttctt N0: ID 4 NO:

SEQ ID 442 agaattggatatccaagat922 1941SEQ 1445 atcttcaatttattcttct N0: 1 ID 4 NO:

SEQ ID 443 tggatatccaagatctgaa927 1946SEQ 1446 ttcacataccagaattcca N0: 1 ID 4 NO:

SEQ ID 444 atatccaagatctgaaaaa930 949 SEQ 1447 tttttaaccagtcagatat N0: 1 1 ID 4 NO:

SEQ ID 445 tatccaagatctgaaaaag931 950 SEQ 1448 ctttttaaccagtcagata N0: 1 1 ID 4 NO:

SEQ ID 446 caagatctgaaaaagttag935 954 SEQ 1449 ctaaattcccatggtcttg N0: 1 1 ID 4 NO:

SEQ ID 447 aagatctgaaaaagttagt936 955 SEQ 1450 actaaattcccatggtctt N0: 1 1 ID 4 NO:

SEQ ID 448 gaaaaagttagtgaaaga942 961 SEQ 1451 ctttctcgggaatattca N0: t 1 1 ID t 1 4 NO:

SEQ ID 449 ccaactgtcatggacttc982 001 SEQ 1452 gaagcacatatgaactgga N0: t 1 2 ID 1 4 NO:

SEQ ID 450 cagaaaattctctcggaa999 018 SEQ 1453 tcctttaacaattcctga 493 N0: t 1 ID t 9 4 NO:

SEQ ID 451 tccatcacttgacccagc044 063 SEQ 454 gctgacatagggaatggaa 433 N0: t 2 2 ID 8 1 NO:

SEQ ID 452 cccagcctcagccaaaata057 076 SEQ 455 attctatccaagattggg 812 N0: 2 ID t 7 1 NO:

SEQ ID 453 agcctcagccaaaatagaa060 079 SEQ 456 tctatccaagattgggct 814 N0: 2 ID t 7 1 NO:

ISEQ 454 atcttatatttgatccaaa083 102 SEQ 457 ttgaaaaacaaagcagat ID N0:1 l 12 ID t 1 WO 2004/091515", """, "..........,. """. PCT/US2004/011255 NO:

SEQ ID 455 tcttatatttgatccaaat084 103 SEQ 1458 attttttgcaagttaaaga14011140301 N0: ID 4 NO:

SEQ ID 456 cttcctaaagaaagcatgc21092128 SEQ 1459 gcatggcattatgatgaag3606 N0: ID 4 NO:

SEQ ID 457 ctaaagaaagcatgctgaa113 2132 SEQ 1460 ttcagggtgtggagtttag5686 N0: ID 4 NO:

SEQ ID 458 taaagaaagcatgctgaaa114 2133 SEO 1461 tttcttaaacattccttta9482 N0: ID 4 NO:

SEQ ID 459 gagattggcttggaaggaa21752194 SEQ 1462 ttccctccattaagttctc11701117201 N0: ID 4 NO:

SEQ ID 460 ctttgagccaacattggaa198 217 SEQ 1463 ttccaatgaccaagaaaag11060110791 N0: ID 4 NO:

SEQ ID 461 cagacagtgtcaacaaagc245 2264 SEQ 1464 gcttactggacgaactctg6134 N0: ID 1 NO:

SEQ ID 462 cagtgtcaacaaagctttg2249268 SEQ 1465 caaattcctggatacactg9849 N0: ID 1 NO:

SEQ ID 463 agtgtcaacaaagctttgt2250269 SEQ 1466 acaagaatacgtctacact4351 NO: ID 1 NO:

SEQ ID 464 ctgatggtgtctctaaggt2290309 SEQ 1467 acctcggaacaatcctcag3325 N0: ID 1 NO:

SEQ ID 465 tgatggtgtctctaaggtc22912310 SEQ 1468 gacctgcgcaacgagatca8823 N0: ID 1 NO:

SEQ ID 466 aaacatgagcaggatatgg343 2362 SEQ 1469 ccatgatctacatttgttt6788 N0: ID 1 NO:

SEQ ID 467 gaagctgattaaagatttg387 406 SEQ 1470 caaaaacattttcaacttc5279 N0: ID 1 NO:

SEQ ID 468 aaagatttgaaatccaaag397 2416 SEQ 1471 ctttaagttcagcatcttt7606 NO: ID 1 NO:

SEQ ID 469 gatgggtgcccgcactctg510 529 SEQ 1472 cagatttgaggattccatc7975 N0: ID 1 NO:

SEQ ID 470 gggatcccccagatgattg532 551 SEQ 1473 caatcacaagtcgattccc9075 N0: ID 1 NO:

SEQ ID 471 ttttcttcactacatcttc585 2604 SEQ 1474 gaagtgtcagtggcaaaaa103741039314 N0: ID

NO:

SEQ ID 472 tcttcactacatcttcatg588 2607 SEQ 1475 catggcattatgatgaaga3607 N0: ID 1 NO:

SEQ ID 473 tacatcttcatggagaatg595 2614 SEQ 1476 cattatggaggcccatgta9437 N0: ID 1 NO:

SEQ ID 474 ttcatggagaatgcctttg601 620 SEQ 1477 caaaatcaactttaatgaa6599 N0: ID ~ 1 NO:

SEQ ID 475 tcatggagaatgcctttga2602621 SEQ 1478 caacacaatcttcaatga N0: ID t NO:

SEQ ID 476 tttgaactccccactggag616 2635 SEQ 1479 ctccccaggacctttcaaa9834 N0: ID 1 NO:

SEQ ID 477 ttgaactccccactggagc617 2636 SEQ 1480 gctccccaggacctttcaa9833 N0: ID 1 NO:

SEQ ID 478 tgaactccccactggagct618 2637 SEQ 1481 agctccccaggacctttca9832 N0: ID 1 NO:

SEQ ID 479 cactggagctggattacag627 2646 SEQ 1482 ctgtttctgagtcccagtg9336 N0: ID 1 NO:

SEQ ID 480 actggagctggattacagt628 2647 SEQ 1483 actgtttctgagtcccagt9335 N0: ID 1 NO:

SEQ ID 481 agttgcaaatatcttcatc644 2663 SEQ 1484 gatgatgccaaaatcaact6591 N0: ID 1 NO:

SEQ ID 482 gttgcaaatatcttcatct2645664 SEQ 1485 gatgatgccaaaatcaac 590 N0: ID a 6 6 1 .WO 2004/091515,., """, ,.",.. ",.". ,~,.,. PCT/US2004/011255 NO:

SEQ ID 483 aaatatcttcatctggagt2650669 SEQ 1486 actcagaaggatggcattt N0: ID 4 NO:

SEQ ID 484 taaaactggaagtagccaa2695714 SEQ 1487 ttggttacaggaggcttta N0: ID 4 NO:

SEQ ID 485 ggctgaactggtggcaaaa720739 SEQ 1488 ttttcttttcagcccagcc N0: ID 4 NO:

SEQ ID 486 gtggagtttgtgacaaat7502769 SEQ 1489 attttcaagcaaatgcaca N0: t ID 4 NO:

SEQ ID 487 tgtgacaaatatgggcat27582777 SEO 1490 atgcgtctaccttacacaa N0: t ID 4 NO:

SEQ ID 488 atgaacaccaacttcttcc811830 SEQ 1491 ggaagctgaagtttatcat N0: ID 4 NO:

SEQ ID 489 cttccacgagtcgggtctg28252844 SEQ 1492 cagagctatcactgggaag N0: ID 4 NO:

SEQ ID 490 gagtcgggtctggaggctc832851 SEQ 1493 gagcttactggacgaactc N0: ID 4 NO:

SEQ ID 491 cctaaaagctgggaagctg858877 SEQ 1494 cagcctccccagccgtagg N0: ID 4 NO:

SEQ ID 492 agctgggaagctgaagttt864883 SEQ 1495 aaactgttaatttacagct N0: ID 4 NO:

SEQ ID 493 ccagattagagctggaact31063125 SEQ 1496 agtttccggggaaacctgg NO: ID 4 NO:

SEQ ID 494 ggataccctgaagtttgta320b3219 SEQ 1497 tacagtattctgaaaatcc N0: ID 4 NO:

SEO ID 495 ctgaggctaccatgacatt32443263 SEQ 1498 aatgagctcatggcttcag N0: ID 4 NO:

SEQ ID 496 gtccagtgaagtccaaat32893308 SEQ 1499 attttgagaggaatcgaca N0: t ID 4 NO:

SEQ ID 497 aattccggattttgatgtt33053324 SEQ 1500 aacacatgaatcacaaatt N0: ID 4 NO:

SEQ ID 498 tccggattttgatgttga33073326 SEQ 1501 tcaaaacgagcttcaggaa N0: t ID 4 NO:

SEQ ID 499 cggaacaatcctcagagtt33293348 SEQ 1502 aacttgtacaactggtccg N0: ID 4 N O:

SEQ ID 500 cctcagagttaatgatga33373356 SEO 1503 tcatcaattggttacagga N0: t ID 4 NO:

SEQ ID 501 ctcaccctggacattcaga33843403 SEQ 1504 tctgcagaacaatgctgag N0: ID 4 NO:

SEQ ID 502 attcagaacaagaaaatt33953414 SEQ 1505 aattgactttgtagaaatg N0: c ID 4 .

NO:

SEQ ID 503 ctgaggtcgccctcatgg34143433 SEQ 1506 ccatgcaagtcagcccagt N0: a ID 4 NO:

SEQ ID 504 tatttccataccccgttt34783497 SEQ 1507 aaactgcctatattgataa N0: t ID 1 4 NO:

SEQ ID 505 tttgcaagcagaagccag34933512 SEQ 1508 ctggacttctcttcaaaac N0: g ID 4 NO:

SEQ ID 506 ttgcaagcagaagccaga34943513 SEQ 1509 ctgggtgtcgacagcaaa N0: t ID t 4 NO:

SEQ ID 507 tgcaagcagaagccagaa34953514 SEQ 1510 tctgggtgtcgacagcaa N0: t ID t 4 NO:

SEQ ID 508 tgcttctccaaatggact35463565 SEQ 1511 agtcaagattgatgggcag N0: c ID 4 4 NO:

SEQ ID 509 gctacagcttatggctcc35693588 SEQ 1512 ggaggctttaagttcagca N0: t ID 7 4 NO:

SEQ ID 510 cagcttatggctccacag35733592 SEQ 1513 ctgtatagcaaattcctgt N0: a ID 5 4 WO 2004/091515 P('T/TTS2004/011255 .. NO:

SEQ ID 511 tttccaagagggtggcatg3592 3611SEQ 1514 catggacttcttctggaaa8869 N0: ID 4 NO:

SEQ ID 512 ccaagagggtggcatggca3595 3614SEQ 1515 tgcccagcaagcaagttgg9353 N0: ID 4 NO:

SEQ ID 513 gtggcatggcattatgatg3603 3622SEQ 1516 catccttaacaccttccac8063 N0: ID 4 NO:

SEQ ID 514 tgatgaagagaagattgaa3617 3636SEQ 1517 ttcactgttcctgaaatca7863 N0: ID 4 NO:

SEQ ID 515 gaagagaagattgaatttg3621 3640SEQ 1518 caaaaacattttcaacttc5279 N0: ID 4 NO:

SEQ ID 516 gagaagattgaatttgaat3624 3643SEQ 1519 attcataatcccaactctc8270 N0: ID 4 NO:

SEQ ID 517 tttgaatggaacacaggca3636 3655SEQ 1520 tgcctttgtgtacaccaaa11228112471 N0: ID 4 NO:

SEQ ID 518 aggcaccaatgtagatacc3650 3669SEQ 1521 ggtaacctaaaaggagcct5583 N0: ID 4 NO:

SEQ ID 519 caaaaaaatgacttccaat3668 3687SEQ 1522 attgaagtacctacttttg8358 N0: ID 1 NO:

SEQ ID 520 aaaaaaatgacttccaatt3669 3688SEQ 1523 aattgaagtacctactttt8357 N0: ID 1 NO:

SEQ ID 521 aaaaaatgacttccaattt3670 3689SEQ 1524 aaatccaatctcctctttt8398 N0: ID 1 NO:

SEQ ID 522 cagagtccctcaaacagac3752 3771SEQ 1525 gtctgtgggattccatctg4082 N0: ID 1 NO:
, SEQ ID 523 aaattaatagttgcaatga_3795 3814SEQ 1526 tcataagttcaatgaattt131781319714 N0: ID

NO:

SEQ ID 524 tcaacctccagaacatgg3891 3910SEQ 1527 ccattgaccagatgctgaa8134 N0: t ID 1 NO:

SEQ ID 525 gggattgccagacttcca3907 3926SEQ 1528 tggaaatgggcctgcccca8895 N0: . ID 1 t NO:

SEQ ID 526 cagtttgaaaattgagatt3986 005 SEQ 1529 aatcacaactcctccactg9533 NO: ID 1 NO:

SEQ ID 527 gaaaattgagattcctttg3992 011 SEQ 1530 caaaactaccacacatttc136861370514 NO: ID

NO:

SEQ ID,N0:528 ttgccttttggtggcaaa007 026 SEQ 1531 tttgagaggaatcgacaaa6351 t : ID 1 NO:

SEQ ID 529 ctccagagatctaaagatg028 047 SEQ 1532 catcaattggttacaggag7586 NO: ID 1 NO:

SEQ ID 530 ctaaagatgttagagact037 056 SEQ 1533 agtccttcatgtccctaga100251004414 N0: t ID

NO:

SEQ ID 531 ctgtgggattccatctgcc084 103 SEQ 1534 ggcattttgaaaaaaacag9727 N0: ID 1 NO:

SEQ ID 532 tctgccatctcgagagtt096 115 SEQ 1535 aactctcaaaccctaagat8548 N0: a ID 1 NO:

SEQ ID 533 ctcgagagttccaagtcc104 123 SEQ 1536 ggacattcctctagcgaga8207 N0: t ID 1 NO:

SEQ ID 534 gtccctacttttaccatt118 137 SEQ 1537 aatgaatacagccaggact6078 N0: a ID 1 NO:

SEQ ID 535 cttttaccattcccaagt125 144 SEQ 1538 actttgtagaaatgaaagt8101 N0: a ID 1 NO:

SEQ ID 536 attcccaagttgtatcaa133 152 SEQ 1539 tgaaggacttcaggaatg 2001 N0: c ID t 1 1 NO:

SEQ ID 537 ccacatgaaggctgactc276 295 SEQ 1540 gagtaaaccaaaacttggt9016 N0: a ID 1 NO:

SEQ ID 538 ttcctacaatgtgcaagg309 328 SEQ 1541 cctttaacaattcctgaaa495 N0: t ID 9 9 1 WO 2004/091515..... ....... ....._ ...... ....... P('T/TTS2004/011255 NO:

SEQ ID 539 ctggagaaacaacatatga330349 SEQ 1542 tcattctgggtctttccag11027110461 N0: ID 4 NO:

SEQ ID 540 atcatgtgatgggtctcta370389 SEQ 1543 tagaattacagaaaatgat65576576 1 N0: ID 4 NO:

SEQ ID 541 catgtgatgggtctctacg372391 SEQ 1544 cgtaggcaccgtgggcatg12125121441 N0: ID 4 NO:

SEQ ID 542 ttctagattcgaatatcaa399418 SEQ 1545 ttgatgatgctgtcaagaa73007319 1 N0: ID 4 NO:

SEQ ID 543 tggggaccacagatgtctg491510 SEQ 1546 cagaattccagcttcccca83268345 1 N0: ID 4 NO:

SEQ ID 544 ctaacactggccggctcaa636655 SEQ 1547 ttgaggctattgatgttag69766995 1 N0: ID 4 NO:

SEQ ID 545 taacactggccggctcaat637656 SEQ 1548 attgaggctattgatgtta69756994 1 N0: ID 4 NO:

SEQ ID 546 aacactggccggctcaatg638657 SEQ 1549 cattgaggctattgatgtt69746993 1 N0: ID 4 NO:

SEQ ID 547 ctggccggctcaatggaga642661 SEQ 1550 tctccatctgcgctaccag12065120841 N0: ID 4 NO:

SEQ ID 548 agataacaggaagatatga705724 SEQ 1551 tcatctcctttcttcatct10202102211 N0: ID 4 NO:

SEQ ID 549 tccctcacctccacctctg737756 SEQ 1552 cagatatatatctcaggga81768195 1 N0: ID 4 NO:

SEQ ID 550 agctgactttaaaatctga810829 SEQ 1553 tcaggctcttcagaaagct79227941 1 N0: ID 4 NO:

SEQ ID 551 ctgactttaaaatctgaca812831 SEQ 1554 tgtcaagataaacaatcag87328751 1 N0: ID 4 NO:

SEQ ID 552 caagatggatatgaccttc865884 SEQ 1555 gaagtagtactgcatcttg68356854 1 N0: ID 4 NO:

SEQ ID 553 gctgcgttctgaatatcag901920 SEQ 1556 ctgagtcccagtgcccagc93429361'1 NO: ID 4 NO:

SEQ ID 554 cgttctgaatatcaggctg905924 SEQ 1557 cagcaagtacctgagaacg86038622 1 N0: ID 4 NO:

SEQ ID 555 aattcccatggtcttgagt968987 SEQ 1558 actcagatcaaagttaatt12264122831 N0: ID 4 NO:

SEQ ID 556 tggtcttgagttaaatgct976995 SEQ 1559 agcacagtacgaaaaacca10801108201 N0: ID 4 NO:

SEQ ID 557 cttgagttaaatgctgaca980999 SEQ 1560 tgtccctagaaatctcaag10034100531 N0: ID 4 NO:

SEQ ID 558 ttgagttaaatgctgacat9815000 SEQ 1561 atgtccctagaaatctcaa10033100521 N0: ID 4 NO:

SEQ ID 559 tgagttaaatgctgacatc9825001 SEQ 1562 gatggaaccctctccctca47254744 1 NO: ID 4 NO:

SEQ ID 560 acttgaagtgtagtctcct50865105 SEQ 563 aggaaactcagatcaaagt12259122781 N0: ID 4 NO:

SEQ ID 561 agtgtagtctcctggtgct0925111 SEQ 564 agcagccagtggcaccact12506125251 N0: 5 ID 4 NO:

SEQ ID 562 gtgctggagaatgagctga1065125 SEQ 565 cagccaggtttatagcac N0: 5 ID t 4 NO:

SEQ ID 563 ctggggcatctatgaaatt1435162 SEQ 566 aatttctgattaccaccag13571135901 N0: 5 ID 4 NO:

SEQ ID 564 atggccgcttcagggaaca1705189 SEQ 567 gttttttggaaatgccat N0: 5 ID t 1 NO:

SEQ ID 565 tcagtctggatgggaaag1995218 EQ ID 568 tttgacaggcattttgaa 97199738 N0: t 5 S 1 c 1 NO:

iSEQ 566 ccatgattctgggtgtcga2575276 EQ ID 569 cgatgcacatacaaatgg ID N0: 5 S 1 t 1 NO:

SEQ ID 567aaaacattttcaacttcaa52815300SEQ ID 1570 ttgatgttagagtgctttt698570041 N0: 4 NO:

SEQ ID 568cttaagctctcaaatgaca53165335SEQ ID 1571 tgtcctacaacaagttaag724772661 N0: 4 NO:

SEQ ID 569ttaagctctcaaatgacat53175336SEQ ID 1572 atgtcctacaacaagttaa724672651 N0: 4 NO:

SEQ ID 570catgatgggctcatatgct53335352SEQ ID 1573 agcatctttggctcacatg761676351 N0: 4 NO:

SEQ ID 571tgggctcatatgctgaaat53385357SEQ ID 1574 atttatcaaaagaagccca12934129531 N0: 4 NO:

SEQ ID 572actggacttctcttcaaaa53995418SEQ ID 1575 ttttggcaagctatacagt837283911 N0: 4 NO:

SEQ ID 573acttctcttcaaaacttga54045423SEQ ID 1576 tcaattgggagagacaagt649665151 N0: 4 NO:

SEQ ID 574ctgacaagttttataagca54375456SEQ ID 1577 tgctttgtgagtttatcag968597041 N0: 4 NO:

SEQ ID 575aagttttataagcaaactg54425461SEQ ID 1578 cagtcatgtagaaaaactt442144401 N0: 4 NO:

SEQ ID 576ctgttaatttacagctaca54585477SEQ ID 1579 tgtactggaaaacgtacag638063991 N0: 4 NO:

SEQ ID 577ttacagctacagccctatt54665485SEQ ID 1580 aatattgatcaatttgtaa641764361 N0: 4 NO:

SEQ ID 578tctggtaactactttaaac54865505SEQ ID 1581 gtttgaaaaacaaagcaga11812118311 N0: 4 NO:

SEQ ID 579tttaaacagtgacctgaaa54985517SEQ ID 1582 tttcatttgaaagaataaa702470431 N0: 4 NO:

SEQ ID 580ttaaacagtgacctgaaat54995518SEQ ID 1583 atttcaagcaagaacttaa0426104451 NO: 1 4 NO:

SEQ ID 581cagtgacctgaaatacaat55045523SEQ ID 1584 attggcgtggagcttactg612361421 N0: 4 NO:

SEQ ID 582tgtggctggtaacctaaaa55765595SEQ ID 1585 ttttgctggagaagccaca0757107761 N0: 1 4 NO:

SEQ ID 583ttatcagcaagctataaag56495668SEQ ID 1586 ctttgcactatgttcataa2756127751 N0: 1 4 NO:

SEQ ID 584ggttcagggtgtggagttt56845703SEQ ID 1587 aaacacctaagagtaaacc900690251 N0: 4 NO:

SEQ ID 585attcagactcactgcattt57675786SEQ ID 1588 aaatgctgacatagggaat429 NO: v 8 4 NO:

SEQ ID 586ttcagactcactgcatttc57685787SEQ ID 1589 gaaatattatgaacttgaa3304133231 N0: 1 4 NO:

SEQ ID 587tacaaatggcaatgggaaa58405859SEQ ID 1590 tttcctaaagctggatgta1168111871 N0: 1 4 NO:

SEQ ID 588gctgtatagcaaattcctg58885907SEQ ID 1591 caggtccatgcaagtcagc091109301 N0: 1 1 4 NO:

SEQ ID 589tgagcagacaggcacctgg60356054SEQ ID 1592 ccagcttccccacatctca333 N0: 8 4 NO:

SEQ ID 590ggcacctggaaactcaaga60456064SEQ ID 1593 tcttcgtgtttcaactgcc1213112321 N0: 1 4 NO:

SEQ ID 591tgaatacagccaggacttg60806099SEQ ID 1594 caagtaagtgctaggttca372 391 N0: 9 9 4 NO:

SEQ ID 592gaatacagccaggacttgg60816100SEQ ID 1595 ccaacacttacttgaattc0660106791 N0: 1 4 NO:

SEQ ID 593ctggacgaactctggctga61396158SEQ ID 1596 tcagaaagctaccttccag931 950 N0: 7 7 4 NO:

IS Q 594ttttactcagtgagcccat61936212SEQ ID 1597 atggacttcttctggaaaa870 889 ID NO:I 8 8 4 WO 2004/091515, ....... ....... ....... ....... PCT/US2004/011255 NO:

SEQ ID 595 gatgagagatgccgttgag62336252SEQ ID 1598 ctcatctcctttcttcatc10201102201 N0: 4 NO:

SEQ ID 596 aattgttgcttttgtaaag62696288SEQ ID 1599 cttttctaaacttgaaatt9056 N0: 4 NO:

SEQ ID 597 cttttgtaaagtatgataa62776296SEQ ID 1600 ttatgaacttgaagaaaag13310133291 N0: 4 NO:

SEQ ID 598 tttgtaaagtatgataaaa62796298SEQ ID 1601 ttttcacattagatgcaaa8413 N0: 4 NO:

SEQ ID 599 tccattaacctcccatttt63126331SEQ ID 1602 aaaattgatgatatctgga10719107381 N0: 4 NO:

SEQ ID 600 ccattaacctcccattttt63136332SEQ ID 1603 aaaagggtcatggaaatgg8885 N0: 4 NO:

SEQ ID 601 cttgcaagaatattttgag63386357SEQ ID 1604 ctcaattttgattttcaag8520 N0: 4 NO:

SEQ ID 602 agaatattttgagaggaat63446363SEQ ID 1605 attccctccattaagttct11700117191 N0: 4 NO:

SEQ ID 603 attatagttgtactggaaa63726391SEQ ID 1606 tttcaagcaagaacttaat10427104461 N0: 4 NO:

SEQ ID 604 gaagcacatcaatattgat64076426SEQ ID 1607 atcagttcagataaacttc7991 N0: 4 NO:

SEQ ID 605 acatcaatattgatcaatt64126431SEQ ID 1608 aattccctgaagttgatgt11479114981 N0: 4 NO:

SEQ ID 606 gaaaactcccacagcaagc64576476SEQ ID 1609 gctttctcttccacatttc10052100711 N0: 4 NO:

SEQ ID 607 ctgaattcattcaattggg64866505SEQ ID 1610 cccatttacagatcttcag11363113821 N0: 4 NO:

SEQ ID 608 tgaattcattcaattggga64876506SEQ ID 1611 tcccatttacagatcttca11362113811 N0: 4 NO:

SEQ ID 609 aactgactgctctcacaaa65326551SEQ ID 1612 tttgaggattccatcagtt7979 N0: 4 NO:

SEQ ID 610.aaaagtatagaattacaga65506569SEQ ID 1613 tctggctccctcaactttt9042 NO: 4 NO:

SEQ ID 611 atcaactttaatgaaaaac66036622SEQ ID 1614 gtttattgaaaatattgat6803 N0: 4 NO:

SEQ ID 612 gatttgaaaatagctatt66866705SEQ ID 1615 aatattattgatgaaatca6708 N0: t 4 NO:

SEQ ID 6~3 atttgaaaatagctattgc66886707SEQ ID 1616 gcaagaacttaatggaaat10433104521 N0: 4 NO:

SEQ ID 614 attgctaatattattgatg67026721SEQ ID 1617 catcacactgaataccaat10151101701 N0: 4 NO:

SEQ ID 615 gaaaaattaaaaagtcttg729 6748SEQ ID 1618 caagagcttatgggatttc11153111721 N0: 6 4 NO:

SEQ ID 616 actatcatatccgtgtaat754 773SEQ ID 1619 attactttgagaaattagt7273 N0: 6 6 4 NO:

SEQ ID 617 attgattttaacaaaagt815 834SEQ ID 1620 acttgacttcagagaaata11396114151 N0: t 6 6 4 NO:

SEQ ID 618 ctgcagcagcttaagagac906 925SEQ ID 1621 gtcttcagtgaagctgcag106911071014 N0: 6 6 NO:

SEQ ID 619 aaaacaacacattgaggct965 984SEQ ID 622 agcctcacctcttactttt105631058214 N0: 6 6 1 NO:

SEQ ID 620 tgagcatgtcaaacactt051 070SEQ ID 623 aagtagctgagaaaatcaa7096 N0: t 7 7 1 1 NO:

SEQ ID 621 ttgaagtagctgagaaaa092 111SEQ ID 624 tttcacattagatgcaaa 8413 432 N0: t 7 7 1 t 8 1 NO:

ISEQ 622 tagtagagttggcccacc191 210SEQ ID 625 gtggactcttgctgctaa 7768 787 ID NO:I t 7 7 1 g 7 1 . WO 2004/091515 ....... ._ . PCT/US2004/011255 NO:

SEQ ID 623 tgaaggagactattcagaa72197238 SEQ 1626 ttctcaattttgattttca 85188537 4'i N0: ID 1 NO:

SEQ ID 624 gagactattcagaagctaa72247243 SEQ 1627 ttagccacagctctgtctc N0: ID

NO:

SEQ ID 625 aattagttggatttattga72857304 SEQ 1628 tcaagaagcttaatgaatt N0: ID 1 NO:

SEQ ID 626 gcttaatgaattatctttt73197338 SEQ 1629 aaaacgagcttcaggaagc N0: ID

NO:

SEQ ID 627 ttaacaaattccttgacat73577376 SEQ 1630 atgtcctacaacaagttaa N0: ID 1 NO:

SEQ ID 628 aaattaaagtcatttgatt73867405 SEQ 1631 aatcctttgacaggcattt N0: ID 1 NO:

SEQ ID 629 gactcaatggtgaaattca74567475 SEQ 1632 tgaaattcaatcacaagtc N0: ID 1 NO:

SEQ ID 630 gaaattcaggctctggaac74677486 SEQ 1633 gttctcaattttgattttc N0: ID 1 NO:

SEQ ID 631 actaccacaaaaagctgaa74847503 SEQ 1634 ttcaggaactattgctagt N0: ID

NO:

SEQ ID 632 ccaaaataaccttaatcat75707589 SEQ 1635 atgatttccctgaccttgg NO: ID 1 NO:

SEQ ID 633 aaataaccttaatcatcaa75737592 SEQ 1636 tgaagtaaaagaaaattt N0: ID t NO:

SEQ ID 634 tttaagttcagcatctttg76077626 SEQ 1637 caaatctggatttcttaaa NO: ID 1 NO:

SEQ ID 635 caggtttatagcacacttg77317750 SEQ 1638 caagggttcactgttcctg N0: ID 1 NO:

SEQ ID 636 gttcactgttcctgaaatc78627881 SEQ 1639 gattctcagatgagggaac NO: ID 1 NO:

SEQ ID 637 cactgttcctgaaatcaag78657884 SEQ 1640 cttgaacacaaagtcagtg N0: ID 1 NO:

SEQ ID 638 actgttcctgaaatcaaga78667885 SEQ 1641 cttgaacacaaagtcagt N0: ID t 1 NO:

SEQ ID 639 gcctgcctttgaagtcagt79017920 SEQ 1642 actgttgactcaggaaggc N0: ID 1 NO:

SEQ ID 640 taacagatttgaggattcc79727991 SEQ 1643 ggaagcttctcaagagtta N0: ID

NO:

SEQ ID 641 gttttccacaccagaattt80428061 SEQ 1644 aaatttctctgctggaaac NO: ID 1 NO:

SEQ ID 642 tcagaaccattgaccagat81288147 SEQ 1645 atctgcagaacaatgctga N0: ID

NO:

SEQ ID 643 tagcgagaatcaccctgcc82188237 SEQ 1646 gcagcttctggcttgcta N0: ID g NO:

SEQ ID 644 ccttaatgattttcaagtt82918310 SEQ 1647 actgttgactcaggaagg N0: ID a NO:

SEQ ID 645 acataccagaattccagct83208339 SEQ 1648 gctgccagtccttcatgt N0: ID a 1 NO:

SEQ ID 646 aatgctgacatagggaatg84308449 SEQ 1649 attaatcctgccatcatt N0: ID c NO:

SEQ ID 647 atgctgacatagggaatgg84318450 SEQ 1650 catttgagatcacggcat N0: ID c 1 NO:

SEQ ID 648 aaccacctcagcaaacgaa84508469 SEQ 1651 tcgttttccattaaggtt 283 N0: ID t 9 1 NO:

SEQ ID 649 agcaggtatcgcagcttcc84688487 SEQ 1652 gaagtggccctgaatgct N0: ID g 1 NO:

SEQ ID 650 gcacaactctcaaaccct85438562 SEQ 1653 gggaaagagaagattgca N0: t ID a 1 26~

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME
NOTE POUR LE TOME / VOLUME NOTE:

Claims

1. A method for reducing apoB-100 levels in a subject comprising administering to a subject an iRNA agent, which targets apoB-100.

2. The method of claim 1, wherein said iRNA agent targets a sequence identical to any one of SEQ ID NOs listed in Tables 9 and 10.

3. The method of claim 1, wherein said iRNA agent comprises a cholesterol moiety.

4. The method of claim 3, wherein said cholesterol moiety is coupled to a sense strand.

5. The method of claim 3, further comprising a second cholesterol moiety.

6. The method of claim 5, wherein said second cholesterol moiety is coupled to a sense strand.

7. The method of claim 1, wherein said iRNA agent is at least 21 nucleotides in length, and the duplex region of the iRNA is about 19 nucleotides in length.

8. The method of claim 1, wherein the subject is suffering from a disorder characterized by elevated or otherwise unwanted expression of apoB-100, elevated or otherwise unwanted levels of cholesterol, and/or disregulation of livid metabolism.

9. The method of claim 8, wherein said disorder is chosen from the group of HDL/LDL
cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia; hypercholesterolemia; statin-resistant hypercholesterolemia;
coronary artery disease (CAD) coronary heart disease (CHD) atherosclerosis

10. The method of claim 9, wherein said iRNA agent is administered to a subject suffering from statin-resistant hypercholesterolemia.

11. A method for reducing glucose-6-phosphatase levels in a subject comprising administering to a subject an iRNA agent that targets glucose-6-phosphatase.

12. The method of claim 11, wherein said iRNA agent is at least 21 nucleotides in length, and the duplex region of the iRNA is about 19 nucleotides in length.

13. The method of claim 12, wherein the iRNA agent is administered to a subject to inhibit hepatic glucose production, or for the treatment of glucose-metabolism-related disorders.

14. The method of claim 12, wherein said disorder is diabetes.

15. The method of claim 12, wherein said disorder is type-2 diabetes.

16. The method of claim 12, wherein said disorder is glitaxzone-resistant diabetes.

17. An iRNA agent comprising a sense sequence and an antisense sequence, wherein the sense sequence comprises one or more cholesterol moeities, and the antisense sequence targets a human gene sequence.

18. The iRNA agent of claim 17, wherein said human gene is an oncogene.

19. The iRNA agent of claim 17, wherein said human gene is apoB 100.

20. The iRNA agent of claim 17, wherein said human gene is glucose-6-phosphatase.

21. The iRNA agent of claim 17, wherein said human gene beta catenin.

22. An iRNA agent, wherein the agent targets apoB 100.

23. An iRNA agent, wherein the agent targets glucose-6-phosphatase.

24. An iRNA agent, wherein the agent targets beta-catenin.