WO2004091515A2 - iRNA CONJUGATES - Google Patents

Abstract

Therapeutic iRNA agents and methods of making and using are enclosed.

Description

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. 60/506,341, 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. 60/510,246, filed October 9, 2003; U.S. Provisional Application No. 60/510,318, 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.

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 : 806-811, 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 subjects. The advantages ofthe current invention include practical, uncomplicated methods of administration and therapeutic applications, e.g., at dosages of less than 2 mg/kg.

SUMMARY

Aspects ofthe invention relate to compositions and methods for silencing genes expressed in the liver, e.g., to freat disorders of or related to the liver. An iRNA agent composition ofthe invention can be one which has been modified to alter distribution in favor of the liver. A composition ofthe 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 substantially identical to a region ofthe 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, h one embodiment, the iRNA is 21 nucleotides in length, and the duplex region ofthe iRNA is 19 nucleotides. hi 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 ofthe 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 prefened 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 prefened embodiment the iRNA agent will include regions complementary to the palindromic pairs of Tables 9 or 10 as a duplex region. hi a preferred embodiment the duplex region ofthe 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 1, 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 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 ofthe disorder or a symptom ofthe disorder. These disorders include HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired 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 of serum LDL-C and/or HDL-C and/or total cholesterol in a subject. 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 subject.

In a prefened embodiment the iRNA agent is administered repeatedly. Adminisfration 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 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 adminisfration ofthe 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. 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- O(hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(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. hi another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a polymeric carrier 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 and or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells ofthe liver (see Table 1). In a prefened 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 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 ofthe 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 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. 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 incoφorated 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 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 that has a sequence that is substantially identical to a sequence ofthe glucose-6-phosphatase gene. Attorney's Docket No.: 14174-072W01

In a prefened embodiment, the subject is treated with an iRNA agent that targets one of the sequences listed in Table 11. In a prefened embodiment it targets both sequences of a palindromic pair provided in Table 11. The most prefened targets are listed in descending order of preferability, in other words, the more prefened targets are listed earlier in Table 11. In a prefened embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 11. In a prefened embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 11 as a duplex region.

In a prefened embodiment the duplex region ofthe 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 prefened 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 prefened 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 experiments with rats or cultured rat cells. h a prefened 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 occurring 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-phosρhatase 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 risk for the disorder to delay onset ofthe disorder or a symptom ofthe 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- 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 ofthe targeted genes are decreased in the liver following administration ofthe iRNA agent. 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 ofthe iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length. 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 ofthe beta-catenin 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 ofthe iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In a prefened embodiment, the subject is treated with an iRNA agent which targets one ofthe sequences listed in Table 13. In a prefened embodiment it targets both sequences of a palindromic pair provided in Table 13. The most prefened targets are listed in descending order of prefenability, in other words, the more prefened targets are listed earlier in Table 13.

In a prefened embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 13. In a prefened embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 13 as a duplex region. In a prefened embodiment the duplex region ofthe 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 hi a prefened 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 prefened 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 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 ofthe disorder or a symptom ofthe disorder hi a prefened 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 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 ofthe beta-catenin 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. 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 ofthe liver. A proliferation disorder ofthe 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 ofthe liver can include dyslipidemias, and iπegularities in glucose regulation. Viral diseases ofthe 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 ofthe 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 substantially identical to a sequence of a gene ofthe 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 ofthe invention can target any one ofthe nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

In a prefened embodiment, the subject is treated with an iRNA agent which targets one ofthe sequences listed in Table 14. In a prefened embodiment it targets both sequences of a palindromic pair provided in Table 14. The most prefened targets are listed in descending order of prefenability, in other words, the more prefened targets are listed earlier in Table 14. hi a prefened embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 14. In a prefened embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 14 as a duplex region.

In a prefened embodiment the duplex region ofthe iRNA agent will target a sequence listed in Table 14, 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 Attorney's Docket No.: 14174-072W01

In a prefened 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 prefened 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 prefened 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, hi 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 prefened 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 ofthe 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 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 freatment 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 prefened embodiment, an iRNA agent administered to treat a liver disorder is 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 ofthe genes discussed herein and in particular for any of apoB-100, glucose-6-phosphatase, beta-catenin, factor V, or any ofthe HVC genes discussed herein. The methods and compositions ofthe invention, e.g., the methods and compositions to treat diseases and disorders ofthe liver described herein, can be used with any ofthe iRNA agents described, hi addition, the methods and compositions ofthe 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 ofthe 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 ofthe 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 conespondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage ofthe 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 present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 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 ofthe molecule.

The details of one or more embodiments ofthe invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of 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 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 pynoline-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- based RRMSs. RI 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 injected on Day 7.

FIG. 6B is a graph depicting blood glucose levels in mice treated with siRNA targeting glucose 6-phosphatase. Mice freated 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. 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. 8 A is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (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. 8 A. FIG. 9 is a graph depicting the pharmacokinetics of cholesterol-conjugated and unconjugated siRNA. The diamonds represent the amount of unconjugated P-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. "LI 163" is equivalent to ALN3000; "L1163Chol" is equivalent to ALN-3001. 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. "LI 163" is equivalent to ALN3000 (light bars); "L1163Chol" is equivalent to ALN-3001 (dark bars). 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 as ug/g liver tissue.

FIG. 12 is a gel of U/U siRNA (see Table 19) detected in the liver of Balbc mice at increasing time points following hydrodynamic (hd) tail vein injection. U/U 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 control samples isolated from livers of mice that were not injected with U/U siRNA. The confrol samples were further used in parallel RNAse protection assays.

FIG. 13 is a gel comparing different siRNA species detected in the livers of Balbc mice at increasing time points following hydrodynamic (hd) or nonhydrodynamic (iv) tail vein injection. U/U siRNA was injected by hd and by iv injection. 3'C/3'C and 3'C/U (see Table 19) were each 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 confrol 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 inj ected 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 by theory, it may be that a molecular signal, which may be merely the specific length ofthe 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 for specifically inactivating gene function. The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides ofthe same or 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 ofthe iRNA agent strands in an sRNA 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 ofthe interferon response, e.g., double stranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can he 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.

hr 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 prefened 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 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 adminisfration, e.g., once per week or once per month.

A number of exemplary routes of delivery are described that can be used to administer an iRNA agent to a subject, hi 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 ofthe invention are liver-based diseases. Disorders involving the liver include, but are not limited to, hepatic injury; jaundice and cholestasis, such as bilirubin and bile formation; hepatic failure and cinhosis, such as cinhosis, 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 enors of metabolism and pediatric liver disease, such as hemochromatosis, Wilson disease, al-antitrypsin deficiency, and neonatal hepatitis; intrahepatic biliary tract disease, such as secondary biliary cinhosis, primary biliary cinhosis, primary sclerosing cholangitis, and anomalies ofthe biliary 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, including hepatic vein thrombosis (Budd-Chiari syndrome) and veno-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 tumorous conditions, such as nodular hyperplasias, adenomas, and malignant tumors, including primary carcinoma ofthe liver and metastatic tumors.

An iRNA agent can also be administered to inhibit Factor V expression in the liver. Two to five percent ofthe United States population is heterozygous for an allele ofthe Factor V gene that encodes a single amino acid change at position 1961. These heterozygous individuals have a 3-8 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 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 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 ofthe invention can be targeted to any gene whose overexpression is associated with the liver diseases.

Targeting to the Liver The iRNA agents ofthe 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. Prefened 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, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-

O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, 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 ofthe liver (see Table 1). 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 semm 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 Targeting agents (Ligands) and their associated receptors

Liver Cells Ligand Receptor

1) Parenchymal Cell (PC) Galactose ASGP-R (Hepatocytes) (Asiologlycoprotein receptor)

Gal Ac 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

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 sunogate, all of which are defined herein (see, e.g., the section below entitled RNA Agents). While numerous modified RNAs and nucleoside sunogates are described, prefened examples Attorney's Docket No.: 14174-072W01

include those which have greater resistance to nuclease degradation than do unmodified RNAs. Prefened 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 refened to in the art as RNAi, or pre-franscriptional 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 prefened that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

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 ofthe 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 ofthe term "ribonucleotide" or "nucleotide", herein can, in the case of a modified RNA or nucleotide sunogate, also refer to a modified nucleotide, or sunogate 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. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the conespondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage ofthe 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 ofthe 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 ofthe molecule.

As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside sunogates in addition to the RRMS. Single stranded regions of an iRNA agent will often be modified or include nucleoside sunogates, 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 sunogates. 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, ahasic, 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 trigger the interferon response (which molecules can also be cleaved by Dicer and/or 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. Prefened 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 target gene, an iRNA agent will preferably have one or more ofthe following properties:

(1) it will be ofthe 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 phosphate groups or one or more analogs of a phosphate group;

(3) it will, despite modifications, even to a very large number, or all ofthe 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 conect base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation ofthe target, e.g., by cleavage of the target RNA;

(4) it will, despite modifications, even to a very large number, or all ofthe 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 in which all ofthe nucleotide sugars contain e.g., 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 Cy-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 prefened that a modified moiety at the 2' sugar position will be able to enter into Attorney's Docket No.: 14174-072W01

H-bόnding which is more characteristic ofthe OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A prefened iRNA agent will: exhibit a Cy-endo pucker in all, or at least 50, 75,80, 85, 90, or 95 % of its sugars; exhibit a Cy-endo 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 Cy-endo pucker structure. These limitations are particularly preferably in the antisense strand;

(5) regardless ofthe nature ofthe modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is prefened that DNA molecules, or any molecule in which more than 50, 60, or 70 % ofthe nucleotides in the molecule, or more than 50, 60, or 70 % ofthe 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 be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In prefened 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 ((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'-phosphorothiolate ((HO)2(O)P-S-5*); any additional combination of oxygen/sulfiir 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'-alkylρhosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, (OH)2(O)P-5*-CH2-), 5*-alkyletherphosphonates Attorney's Docket No.: 14174-072W01

(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(0)-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 50nucleotides 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. Prefened ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3', and preferably ofthe antisense side ofthe hairpin. Prefened 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: Prefened ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length.

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. Prefened 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. Prefened ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. Attorney's Docket No.: 14174-072W01

hi 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 sfrand, 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 sR A/protein or RISC. This can be accomplished by a modification which prevents 5'-phosphorylation ofthe 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 phosphodiesterases can cleave such a linkage and release a functional sRNA 5'-end. Antisense strand modifications include 5' phosphorylation as well as any ofthe other 5' modifications discussed herein, particularly the 5' modifications discussed above in the section on single stranded iRNA molecules.

It is prefened that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends ofthe 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. Prefened 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 ofthe same length being staggered. 5' ends are preferably phosphorylated.

Prefened 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 ofthe 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 refened to herein as mRNA to be silenced. Such a gene is also refened 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.

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 used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound ofthe invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding ofthe 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 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 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 sunogates. Unmodified RNA refers to a molecule in which the components ofthe 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 refened to rare or unusual, 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 ofthe components ofthe nucleic 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 refened to as modified "RNAs," they will of course, because ofthe modification, include molecules which are not RNAs. Nucleoside sunogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the conect spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics ofthe ribophosphate backbone. Examples of all ofthe above are discussed herein.

Much ofthe 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 required or prefened. 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 ofthe single stranded structures described below are within the invention. Prefened lengths are described elsewhere herein. Attorney's Docket No.: 14174-072W01

As nucleic acids are polymers of subunits or monomers, many ofthe 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 ofthe subject 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 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 prefened, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide sunogates, 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 ofthe 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 ofthe ribose sugar, e.g., the use of 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 sunogates are discussed below.

FORMULA 1

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 ofthe 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 sunogates can include one or more of:

(i) alteration, e.g., replacement, of one or both ofthe non-linking (X and Y) phosphate oxygens and/or of one or more ofthe linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one ofthe positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of 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 ofthe ribose sugar, e.g., ofthe 2' hydroxyl on the ribose sugar, or wholesale replacement ofthe ribose sugar with a structure other than ribose, e.g., as described herein;

(iii) wholesale replacement ofthe phosphate moiety (bracket I) with "dephospho" linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification ofthe ribose-phosphate backbone (bracket II);

(vi) modification ofthe 3' end or 5' end ofthe RNA, e.g., removal, modification or 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" ofthe 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 ofthe embodiments ofthe 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

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 ofthe oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance ofthe oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it 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 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 conesponding 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 prefened.

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 prefened.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some ofthe sugar groups ofthe ribonucleic acid. E.g., the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. While not being bound 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 nucleophilic 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.

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(CH₂CH₂O)_nCH₂CH₂OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, e.g., by a methylene bridge, to the 4' carbon ofthe same ribose sugar; O-AMLNE (AMINE = NH ; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH₂)_nAMINE, (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), (OCH₂CH₂OCH₃, a PEG Attorney's Docket No.: 14174-072 01

derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

"Deoxy" modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_nCH₂CH₂-AMINE (AMINE =NH₂; 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 maybe optionally substituted with e.g., an amino functionality. Prefened substitutents are 2'-methoxyethyl, 2'- OCH3, 2'-O-allyl, 2'-C- allyl, and 2'-fluoro.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that ofthe conesponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.

Modified RNAs can also include "abasic" sugars, which lack a nucleobase at C-l'. These abasic sugars can also be further contain modifications at one or more ofthe 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" 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 ofthe 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, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Prefened replacements include the methylenecarbonylamino and methylenemethylimino groups.

Candidate modifications can be evaluated as described below.

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 sunogates (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 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 sunogate backbone.

Examples include the mophilino, cyclobutyl, pynolidine and peptide nucleic acid (PNA) nucleoside sunogates. A prefened sunogate is a PNA sunogate.

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 ofthe molecule. They can include modification or replacement of an entire terminal phosphate or of one or more ofthe atoms ofthe 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, TAMRA, 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 ofthe spacer can connect to or replace the linking atom ofthe phosphate group or the C-3' or C-5' O, N, S or C group ofthe sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide sunogate (e.g., PNAs). These spacers or linkers can include e.g., - (CH₂)_n-, -(CH₂)_nN-, -(CH₂)_nO-, -(CH₂)_nS-, O(CH₂CH₂O)_nCH₂CH₂OH (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 molecular entity-spacer/phosphate anay is interposed between two strands of iRNA agents, this anay 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 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, l,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, Attorney's Docket No.: 14174-072W01

bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles) .

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 ofthe 5' end with phosphate or phosphate analogs. E.g., in prefened embodiments iRNA agents, especially antisense strands, 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'-monophosρhate ((HO)2(O)P-O-5'); 5'-diρhosphate ((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'- 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'-alkyletherphosρhonates (R=alkyletheι=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-).

Terminal modifications can also be useful for monitoring distribution, and in such cases the prefened groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross- 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 uracil 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 nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one ofthe above modifications. Alternatively, substituted or modified analogs of any ofthe 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- 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-nitropynole, 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, IS^-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, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less prefened 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 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 ofthe 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 Cy5. Confrol and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, ofthe 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.

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 ofthe 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 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 ofthe candidate agent on Attorney's Docket No.: 14174-072W01

gene expression can be assessed by comparing cell fluorescence in the presence ofthe 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-mos, can be injected into an immature mouse oocyte to assess the ability ofthe agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). A phenotype ofthe oocyte, e.g., the ability to maintain anest 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 anest and initiate parthenogenetic development (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect ofthe modified agent on target RNA levels can be verified by Northern blot to assay for a decrease in the level of target 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.

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 Approach", Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modem 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, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chirn. Ada, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223- 2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references refened to therein. Attorney's Docket No.: 14174-072W01

Modification described in WO 00/44895, WO01/75164, or WO02/44321 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 5,177,198. 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 found in the following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36, 831- 841), 2^*-MOE (Martin, P. Helv. Chim. Ada 1996, 79, 1930-1938), "LNA" (Wengel, J. Ace. Chem. Res. 1999, 32, 301-310).

Replacement ofthe 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.: 14174-072W01

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/US92/04294 and PCT/US92/04305 (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. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E.P. Nucleic Acids Res. 1989, 17, 6129.

Replacement ofthe Phosphate-Ribose Backbone References

Cyclobutyl sugar sunogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pynolidine sugar sunogate can be prepared as is described in U.S. Pat. No. 5,519,134. Morpholino sugar sunogates 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 ofthe various procedures refened 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 and Nucleic Acid Drug Development 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.: 14174-072W01

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

Prefened iRNA Agents

Prefened RNA agents have the following structure (see Formula 2 below):

FORMULA 2

Referring to Formula 2 above, R¹, R², 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-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5- Attorney's Docket No.: 14174-072W01

trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrirnidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-proρynylcytosine, 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-nitropynole, 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, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N- methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_mCH₂CH₂OR⁸; O(CH₂)_nR⁹; O(CH₂)_nOR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_mCH₂CH₂NHR⁹; NHC(O)R⁸; ; cyano; mercapto, SR⁸; alkyl-thio-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, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or 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:

; H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

(a prefened Al , 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)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 stracture (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.), 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'- Attorney's Docket No.: 14174-072W01

alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-)).

A² is:

A³ is:

and

Attorney's Docket No.: 14174-072W01

A⁴ is:

; H; Z ; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_nR¹⁰, (CH₂)_nNHR¹⁰, (CH₂)_n OR¹⁰, (CH₂)_n SR¹⁰; O(CH₂)_nR¹⁰;

O(CH₂)_nOR .1^ι0^υ, O(CH₂)_nNR .1^ι0^υ, O(CH₂)_nSR , 1^ι0^υ; O(CH₂)_nSS(CH₂)„OR 1^ι0^υ, O(CH₂)_nC(O)OR , 10 NH(CH₂)_nR¹⁰; NH(CH₂)_nNR¹⁰ ;NH(CH₂)_nOR¹⁰, NH(CH₂)_nSR¹⁰; S(CH₂)_nR¹⁰, S(CH₂)_nNR¹⁰, S(CH₂)_nOR¹⁰, S(CH₂)_nSR¹⁰ O(CH₂CH₂O)_mCH₂CH₂OR¹⁰; O(CH₂CH₂O)_mCH₂CH₂NHR¹⁰ , NH(CH₂CH₂NH)_mCH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰ N-Q-R¹⁰, S-Q-R¹⁰ or -O-. W⁴ is O, CH₂, NH, or S.

X¹, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O^", OR⁸, S, Se, BH₃ ^", H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH, (CH₂)_nR¹⁰,

(CH₂)„NHR¹⁰, (CH₂)_n OR¹⁰, (CH₂)_n SR¹⁰; O(CH₂)_nR¹⁰; O(CH₂)_nOR¹⁰, O(CH₂)_nNR¹⁰, Attorney's Docket No.: 14174-072W01

O(CH₂)_nSR¹⁰, O(CH₂)_nSS(CH₂)_nOR¹⁰, O(CH₂)_nC(O)OR¹⁰; NH(CH₂)_nR¹⁰; NH(CH₂)„NR¹⁰ ;NH(CH₂)_nOR¹⁰, NH(CH₂)_nSR¹⁰; S(CH₂)_nR¹⁰, S(CH₂)_nNR¹⁰, S(CH₂)_nOR¹⁰, S(CH₂)_nSR¹⁰ O(CH₂CH₂O)_mCH₂CH₂OR¹⁰, O(CH₂CH₂O)_mCH₂CH₂NHR¹⁰ , NH(CH₂CH₂NH)_mCH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R¹⁰ N-Q-R¹⁰, S-Q-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent described herein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [-O-CH₂-] covalently bound bridge between the sugar 2' and 4' carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R¹⁰ 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), porphyriiis (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 acid, dihydrotestosterone, l,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, bistamine, 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, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents. Attorney's Docket No.: 14174-072W01

Prefened RNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):

FORMULA 3

Referring to Formula 3, A¹⁰- A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ 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(CH₂)_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, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino . Attorney's Docket No.: 14174-072W01

R¹⁰, R²⁰, and R³⁰ 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-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-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-nifroindole, 3-nitropynole, 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, N6-methyladenine, N6-isopentyladenine, 2- methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_mCH₂CH₂OR⁸; O(CH₂)_nR⁹; O(CH₂)_nOR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_mCH₂CH₂R⁹; NHC(O)R⁸;; cyano; mercapto, SR⁷; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which maybe optionally substituted with halo, hydroxy, oxo, nifro, 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, and ureido groups; or R⁴⁰, R⁵⁰, or R⁶⁰ together combine with R⁷⁰ 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

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰, or R⁶⁰ to form an [-O-CH₂-] 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.

Prefened nucleoside sunogates have the following structure (see Formula 4 below):

SLR¹⁰⁰-(M-SLR²⁰⁰)_X-M-SLR³⁰⁰

FORMULA 4

S is a nucleoside sunogate selected from the group consisting of mophilino, cyclobutyl, pynolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH₂(CH₂)_g; N(CH₂)_g; O(CH₂)_g; S(CH₂)_g; -C(O)(CH₂)_n-or maybe absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ 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-ρroρynyl 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-aminoρropyladenine, 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- pyridinones, 5-nitroindole, 3-nitropynole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5- Attorney's Docket No.: 14174-072 W01

methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5- methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5- methylcytosine, IN^-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/US04/07070, 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 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.

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 refened 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., serum albumin, or a member ofthe 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. : 14174-072 WO 1

While not wishing to be bound by theory, it is believed that modifications ofthe 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 ofthe functional components ofthe RISC complex. Prefened modifications are those that increase exonuclease and endonuclease resistance and thus prolong the half-life ofthe 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 ofthe modifications at or near the 3' and/or 5' end of antisense strands can result in iRNA agents that meet the prefened nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement ofthe 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 comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more ofthe modifications described herein. The anti sense strand may include modifications at the 3' end and/or 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 ofthe sfrand. The sense strand may include modifications at the 3' end and/or the 5' end and/or at any one ofthe intervening positions between the two ends ofthe sfrand. 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 ofthe modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3' end and/or 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 ofthe strand. Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet the prefened nuclease resistance criteria delineated above can include one or more ofthe following chemical and or stereochemical modifications ofthe sugar, base, and/or phosphate backbone: Attorney's Docket No.: 14174-072W01

(i) chiral (S_P) thioates. Thus, prefened 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 prefened. Enriched means at least 70, 80, 90, 95, or 99% ofthe prefened 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, prefened NRMs include monomers at the terminal position derivatized at a cationic group. As the 5' end of an antisense sequence should have a tenuinal -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 sfrand, e.g, at the 5' position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, prefened 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-CH2-5' and 3' CH2-NH-(O=)-CH2-5'.;

(iv) 3 '-bridging thiophosphates and 5 '-bridging thiophosphates. Thus, prefened NRM's can included these structures;

(v) L-RNA, 2'-5' linkages, inverted linkages, a-nucleosides. Thus, other prefened

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 described herein having an alpha linkage;

(vi) conjugate groups. Thus, prefened 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, prefened 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, prefened NRM's include monomers, preferably at the terminal position, e.g., the 5' position, in which one or more atoms ofthe phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result ofthe 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 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 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 sfrand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.

Chiral Sp Thioates

A modification can include the alteration, e.g., replacement, of one or both ofthe non- linking (X and Y) phosphate oxygens and/or of one or more ofthe linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar sunogate-base moieties, SB_Ϊ and SB₂. Attorney's Docket No. : 14174-072W01

FORMULA X

hr certain embodiments, one ofthe non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one ofthe following: S, Se, BR (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphoras atom in an unmodified phosphate group is achiral. However, replacement of one ofthe non-linking oxygens with one ofthe above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphoras atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphoras 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 phosphoras 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 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 R_P 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 phosphoras 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 phosphoras 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 phosphoras 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.).

In a prefened 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 prefened embodiment, the population of phosphorothioate stereogenic phosphoras atoms may have the S configuration and be substantially free of stereogenic phosphorus atoms having the Rp configuration.,

Phosphorothioates may be incoφorated 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

solid phase reagent

X-l 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 ofthe sfrand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, R₂> 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-l and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphoras atoms having essentially only the R_P configuration (i.e., being substantially free ofthe Sp configuration) or only the Sp configuration (i.e., being substantially free ofthe Rp configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphoras atoms in which about Attorney's Docket No.: 1 174-072 WO 1

50% ofthe atoms have the R_P configuration and about 50% ofthe atoms have the S_P configuration. Dimers having stereogenic phosphoras 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, base, and/or the phosphoras 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 prefened 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 sunogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O- AMINE (AMINE = NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH₂)_nAMINE, (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 diheteroaryl amino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphate linkages at the 5' and or 3' end of a sfrand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Prefened replacements include the methyl phosphonate and hydroxylamino groups. Attorney's Docket No.: 14174-072W01

3 '-bridging thiophosphates and 5 '-bridging thiophosphates; locked-RNA, 2 '-5 ' likages, inverted linkages, a-nucleosides; conjugate groups; abasic linkages; and 5 '-phosphonates and 5 '-phosphate prodrugs

Referring to formula X above, modifications can include replacement of one ofthe 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 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-l'. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that ofthe conesponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar, hi another subset of this modification, the natural, unusual, or universal base may have the α-confϊguration. 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]₂CH₂C^5'-sugar. In the latter case, the prodrag groups may be decomposed via reaction first with carboxy 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 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 prefenable 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 subject sequence or gene.. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences ofthe 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 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 stracture with the other sequence ofthe iRNA agent — it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence ofthe 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 refened to as an anti-sense sequence) or will not target a sequence in the subject (often refened to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which 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 ofthe first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side ofthe 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 ofthe 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 ofthe terminus, of a sequence which targets or a sequence 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 sfrand 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 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 1, 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 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 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 Attorney's Docket No. : 14174-072 WO 1

from 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).

An iRNA agent can also target two sequences and can have a first and second strand chosen from:

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, 2, 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 1, 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 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 1, 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); Attorney's Docket No. : 14174-072 WO 1

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 sfrand 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 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).

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 No. 60/454,962, filed on March 13, 2003, and International Application No. PCT/US04/07070, 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 targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or stracture 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.

Thus, an aspect ofthe 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 ofthe nucleic acid. The secondary hydroxy group confers Attorney's Docket No.: 14174-072 WO 1

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 phosphate group phosphoras ofthe iRNA agent. In prefened embodiments, there are five intervening bonds between the terminal phosphate group phosphoras and the secondary hydroxyl group.

Prefened iRNA agent delivery modules with five intervening bonds between the tenninal phosphate group phosphorus and the secondary hydroxyl group have the following stracture (see formula Y below):

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" ofthe phosphate group. These spacers or linkers can include e.g., -(CH2)_n-, -(CH₂)_nN-, -

(CH₂)_nO-, -(CH₂)_nS-, O(CH₂CH₂O)_nCH₂CH₂OH (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, 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, BH₃ ^", Cι-C₆ alkyl, C_ό-C^ aryl, H, O, O^", alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is S.

R_\ and R₃ are each, independently, hydrogen; or -Cioo alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R is hydrogen; -doo 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, R₂ may be taken together with with R₄ or R₆ to form a ring of 5-12 atoms.

Rt is hydrogen; C₁-C₁₀₀ 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, _t may be taken together with with R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C1₀₀ 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, R₅ maybe taken together with with _t to form a ring of 5-12 atoms.

R is hydrogen, C1-C100 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, R₆ maybe taken together with with R₂ to form a ring of 6-10 atoms; Attorney's Docket No.: 14174-072W01

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_qC(O)NHR₉; T is hydrogen or a functional group; n and q are each independently 1-100; R₈ is Ci- o alkyl or C₆-C₁₀ aryl; andR₉ is hydrogen, C1-C10 alkyl, C6-C10 aryl or a solid support agent.

Prefened embodiments may include one of more ofthe 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 ofthe RNA agent.

hi 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 taken together to form a ring containing six atoms and R₄ and R₅ are taken together to form a ring containing six atoms. Preferably, the ring system is a trarcs-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-l):

The functional group can be, for example, a targeting group (e.g., a steroid or a 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, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin. Attorney's Docket No.: 14174-072 W01

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.G.M. Wuts, Protective Groups in Organic 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 Replacement Monomer Subunits

iRNA agents can be modified in a number of ways which can optimize one or more characteristics ofthe 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. 60/493,986, filed on August 8, 2003, which is hereby incoφorated by reference; United States Provisional Application Serial No. 60/494,597, filed on August 11, 2003, which is hereby incoφorated by reference; United States Provisional Application Serial No. 60/506,341, filed on September 26, 2003, which is hereby incoφorated by reference; United States Provisional Application Serial No. 60/158,453, filed on November 7, 2003, which is hereby incoφorated by reference; and International Application No. PCT/US04/07070, filed March 8, 2004, which is hereby incoφorated 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 incoφorates 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) carrier. A ribonucleotide subunit in which the ribose sugar ofthe subunit has been so replaced is refened to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The 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 functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incoφoration ofthe 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 ofthe cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a 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 incoφoration or tethering of another chemical entity, e.g., a ligand to the constituent ring.

Incoφoration 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. Incoφoration 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 ofthe duplex form ofthe iRNA agent, alter distribution or target the iRNA agent to a particular part ofthe 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 sfrand, wherein at least one subunit having a formula (R-l) is incoφorated into at least one of said strands.

(R-l)

Referring to formula (R-l), X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰, or absent; and Z is CR R or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^a, OR , (CH₂)_nOR^a, or (CH₂)_nOR , provided that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^a or OR^b and that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_nOR^a, or (CH₂)_nOR (when the RRMS is terminal, one of R¹ , R², R³, R⁴, R⁹, and R¹⁰ will include R^a and one will include R^b; when the RRMS is internal, two of R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R ); further provided that preferably OR^a may only be present with (CH )_nOR^b and (CH₂)_nOR^a may only be present with OR^b.

Each of R , R , R »π , and R , 12 is, independently, H, Cι-C₆ alkyl optionally substituted with 1-3 R » 1¹3^J, or C(O)NHR >7';. orR^D and R , 11 together are C₃-C₈ cycloalkyl optionally substituted with

R 14 Attorney's Docket No.: 14174-072W01

R⁷ is C₁-C₂0 alkyl substituted with NR^cR^d; R⁸ is Cι-C₆ alkyl; R¹³ is hydroxy, C C₄ alkoxy, or halo; and R¹⁴ is NR^CR⁷.

R^a is:

; and

R is:

A

-O Strand

Each of A and C is, independently, O or S.

B is OH, O^", or Attorney's Docket No.: 14174-072 WO 1

R^c is H or C1-C6 alkyl; R^d is H or a ligand; and n is 1-4.

h a prefened embodiment the ribose is replaced with a pyπoline scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

In other prefened embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR^πR¹².

In other prefened embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR^πR¹².

In other prefened embodiments the ribose is replaced with a moφholino scaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR^{1 l}Rⁿ .

In other prefened embodiments the ribose is replaced with a decalin scaffold, and X isCH₂; Y is CR⁹R¹⁰; and Z is CR^πR¹²; and R⁵ and R¹¹ together are C⁶ cycloalkyl.

hi other prefened embodiments the ribose is replaced with a decalin/indane scafold and , and X is CH₂; Y is CR⁹R¹⁰; and Z is CR^{1 ]}R¹²; and R⁵ and R^{1 !} together are C⁵ cycloalkyl.

In other prefened embodiments, the ribose is replaced with a hydroxyproline scaffold.

RRMSs described herein may be incoφorated 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 sfrand, in which the antisense strand and/or the sense strand may include one or more ofthe 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.: 14174-072 W01

RRMS can be placed at or near (within 1, 2, or 3 positions) ofthe 3' or 5' end ofthe sense strand or at near (within 2 or 3 positions of) the 3' end ofthe antisense strand, hi some embodiments it is prefened to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end ofthe antisense sfrand. 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 ofthe antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end ofthe antisense strand and at (or within 1, 2, or 3 positions of) the 3' end ofthe sense strand, i an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end ofthe antisense strand and an RRMS at the 5' end ofthe sense strand, in which both ligands are located at the same end ofthe 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 ofthe hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3' end ofthe antisense strand and an RRMS at the 5' end ofthe 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 refened to herein as a "haiφin ligand."

In other embodiments, an iRNA agent may have an RRMS at the 3 ' end ofthe sense strand and an RRMS at an internal position ofthe sense strand. An iRNA agent may have an RRMS at an internal position ofthe sense strand; or may have an RRMS at an internal position ofthe antisense strand; or may have an RRMS at an internal position ofthe sense strand and an RRMS at an internal position ofthe antisense strand.

In prefened 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 prefened 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 ofthe 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. Prefened 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 ofthe same length being staggered. 5' ends are preferably phosphorylated.

An RNA agent, e.g., an iRNA agent, containing a prefened, 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 ofthe RNA molecule, i.e., one ofthe 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.

The modified RNA molecule of formula (R-2) can be obtained using oligonucleotide synthetic methods known in the art. In a prefened embodiment, the modified RNA molecule of formula (II) can be prepared by incoφorating one or more ofthe conesponding 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 RRMS monomers generally include two differently functionalized hydroxyl groups

(OFG and OFG 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 (OFG¹) 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 B, 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 incoφorated 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 incoφorated 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 incoφorated into the strand.

The (OFG¹) protecting group may be selected as desired, e.g., from T.W. Greene and

P.G.M. Wuts, Protective Groups in 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 groups in which the hydrogen has been replaced with a protecting group, e.g., a triaryhnethyl 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 prefened protecting group is the dimethoxytrityl group.

When the OFG 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 OFG¹ 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 oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG². Subsequent to this operation, OFG¹ 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 prefened embodiments, R' is methyl, allyl or 2-cyanoethyl. R" may a Cι-C₁₀ alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl. Attorney's Docket No.: 14174-072W01

OFG² 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 chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the sunounding 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 ofthe 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- stable polystyrene based supports or PEG are prefened.

Prefened carriers have the general formula (R-3) provided below. (In that structure prefened backbone attachment points can be chosen from R¹ or R²; R³ or R⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions are chosen to give two backbone attachment points, e.g., R¹ and R⁴, or R⁴ and R⁹. Prefened tethering attachment points include R⁷; R⁵ or R⁶ when X is CH₂. The carriers are described below as an entity, which can be incoφorated 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) ofthe attachment points, e.g., R¹ or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Y is CR⁹R¹⁰), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one ofthe above-named R groups can be - 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-3)

X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰; and Z is CR^πR¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR\ or (CH₂)_nOR , provided that at least two of R¹, R², R³, R⁴, R⁹, and R¹⁰ are OR^a and/or (CH₂)_nOR .

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, -C_ό alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is H, a ligand, or C₁-C₂₀ alkyl substituted with NR°R^d; R⁸ is H or C C₆ alkyl; R¹³ is hydroxy, C C₄ alkoxy, or halo; R¹⁴ is NR^CR⁷; R¹⁵ is C C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl; R is Ci-Cio alkyl; and R is a liquid or solid phase support reagent.

L is -C(O)(CH₂)_qC(O)-, or -C(O)(CH₂)_qS-; R^a is CAr₃; R^b is P(O)(O^")H, P(OR¹⁵)N(R¹⁶)₂ or L-R¹⁷; R^c is H or -Ce alkyl; and R^d is H or a ligand.

Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄ alkoxy; n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR^{1 !}R¹²; or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR^πR¹²; or X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR^πR¹²; or X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R^{1 x} together form C₆ cycloalkyl (H, z = 2), or the indane ring Attorney's Docket No. : 14174-072W01

system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CRⁿR¹², and R⁵ and R¹¹ together form C₅ cycloalkyl (H, z = l).

In certain embodiments, the carrier maybe based on the pyπoline ring system or the 3- hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent (D). OFG¹ is preferably attached to a primary carbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one ofthe carbons in the five-membered ring (-

1 J

CH₂OFG in D). OFG is preferably attached directly to one ofthe carbons in the five- membered ring (-OFG² in D). For the pynoline-based carriers, -CH₂OFG^! may be attached to C- 2 and OFG² may be attached to C-3; or -C^OFG¹ may be attached to C-3 and OFG² may be attached to C-4. . In certain embodiments, CH2OFG¹ and OFG² may be geminally substituted to one ofthe above-referenced carbons.For the 3-hydroxyproline-based carriers, -CH₂OFG¹ maybe attached to C-2 and OFG² may be attached to C-4. The pyπoline- and 3-hydroxyproline-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, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any ofthe pairings delineated above Accordingly, all cis/trans 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 ofthe 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)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR^πR¹². OFG¹ is preferably

E

attached to a primary carbon, e.g., an exocychc alkylene group, e.g., a methylene group (n=l) or ethylene group (n=2), connected to one ofthe carbons in the six-membered ring [-(CH₂)_nOFG¹ in E]. OFG is preferably attached directly to one ofthe carbons in the six-membered ring (-OFG in E). -(CH^_nOFG¹ and OFG² maybe 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, or C-4. Alternatively, -

1

(CH₂)_nOFG and OFG may be disposed m a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., -(CH₂)_nOFG¹ may be attached to C-2 and OFG² may be attached to C-3; -(CH₂)_nOFG¹ may be attached to C-3 and OFG² may be attached to C-2; -(CH₂)_nOFG¹ may be attached to C-3 and OFG² may be attached to C-4; or -(CH₂)_nOFG¹ 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)_nOFG¹ and OFG² may be cis or trans with respect to one another in any ofthe pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also Attorney's Docket No.: 14174-072W01

contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms ofthe 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)R⁷ or NR⁷, Y is NR⁸, and Z is CR^πR¹², or the moφholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR^πR¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocychc alkylene group, e.g., a methylene group,

1 9 connected to one of the carbons in the six-membered ring (-CH₂OFG in F or G). OFG is preferably attached directly to one ofthe carbons in the six-membered rings (-OFG² in F or G). For both F and G, -CH^OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or vice

1 9 versa. In certain embodiments, CH₂OFG and OFG may be geminally substituted to one ofthe above-referenced carbons.The piperazine- and moφholine-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, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any ofthe pairings delineated above. Accordingly, all cis/trans 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., C C₆ alkyl, preferably CH . 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 CH₂; Y is CR⁹R¹⁰; Z is CR^{1 l}R^u, and R⁵ and R^{1 !} together form C₆ cycloalkyl (H, z = 2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR^πR¹², and R⁵ and R¹¹ together form C₅ cycloalkyl (H, z = 1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocychc methylene group (n=l) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [-(CH₂)_nOFG¹ in H]. OFG² is preferably attached directly to one of C-2, C-3, C-4, or C-5 (-OFG² in H). -(CH₂)_nOFG¹ and OFG² 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, -(CH₂)_nOFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups maybe attached to adjacent ring carbon atoms, e.g., -(CH^_nOFG¹ maybe attached to C-2 and OFG² may be attached to C-3; -(CH^_nOFG¹ may be attached to C-3 and OFG² may be attached to C-2; -(CH^_nOFG¹ may be attached to C-3 and OFG² may be attached to C-4; or -

(CH₂)_nOFG¹ maybe attached to C-4 and OFG² maybe attached to C-3; -(CH^_nOFG¹ maybe attached to C-4 and OFG² maybe attached to C-5; or -(CH^_nOFG¹ maybe attached to C-5 and

OFG² may be attached to C-4. The decalin or indane-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)_πOFG¹ and OFG² may be cis or trans with respect to one another in any ofthe pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more Attorney's Docket No. : 14174-072 WO 1

asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms ofthe monomers are expressly included. In a prefened embodiment, the substituents at C-l and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus, -(CH nOFG¹ and OFG may be cis or trans with respect to one another. Accordingly, all cis/trans 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 forms ofthe monomers are expressly included. The tethering attachment point is preferably nitrogen. '■

Representative carriers are shown in FIG. 5.

In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any Ci-Cioo carbon-containing moiety, (e.g. -C75, C\- C₅o, C₁-C₂₀, - o, Cι-C₆), preferably having at least one nitrogen atom. In prefened embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Prefened tethers (underlined) include TAP- (CH₂ nNH₂: TAP-C(O)fCH7)nNH₂; or TAP-NR""(CH_?)_nNH_z. in which n is 1-6 and R"" is C C₆ alkyl. and R^d is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., -ONH₂, or hydrazino group, -NHNH₂. 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. Prefened tethered ligands may include, e.g., TAP-fCH₂) NHfLIGAND),

TAP-C(O)(CH₂)_nNH(LIGAND). or TAP-NR' ' ' '(CH₂)nNH(LIGAND ;

TAP-(CH_z nONH(LIGAND). TAP-C(O (CH₂ nONH(LIGAND . or

TAP-NR' ' ' ' (CH₂)nONH(LIGAND); TAP-(CH₂)nNHNH₂(LIGAND .

TAP-C(O (CH₂)nNHNH₂(LIGAND . or TAP-NR' ' "(CH₂ nNHNΗ₂(LIGAND .

In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position ofthe tether. Prefened 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. Prefened tethers (underlined) include TAP- (CH^CHO; TAP-αθ fCH₂) CHO; or TAP-NR" "fCH CHO. in which n is 1-6 and R"" is d-Ce alkyl; or TAP-(CH₂)nC(O)ONHS; TAP-CfO)(CH₂)„C(O)ONHS; or

TAP-NR' ' ' '(CH?) _πC(O)ONHS, in which n is 1-6 and R"" is C C₆ alkyl;

TAP-(CH₂ (O OCgF : TAP-C(O (CH_{2 J1}C(O OC^; or TAP-NR" "(CH₂\ (O OQ , in which n is 1-6 and R"" is d-C₆ alkyl; or -(CH₂)_aCH₂LG; TAP-C(O)fCH₂)_nCH LG; or TAP- NR""(CH )nCH₂LG. in which n is 1-6 and R"" is d-C₆ alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.

Tethered Entities

A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only prefened, entities can be coupled at other points to an iRNA agent. Prefened entities are those which target to the liver. Attorney's Docket No.: 14174-072W01

Prefened moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In prefened embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incoφorated into the growing strand. In some embodiments, the ligand may be incoφorated into a "precursor" RRMS after a "precursor" RRMS monomer has been incoφorated 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-(CH₂)_nNH₂ may be incoφorated into a growing sense or antisense strand. In a subsequent operation, i.e., after incoφoration ofthe 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 ofthe ligand with the terminal nucleophilic group ofthe precursor RRMS tether.

In prefened embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incoφorated. hi prefened embodiments a ligand provides an 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 ofthe body, as, e.g., compared to a species absent such a ligand. Prefened ligands will not take part in duplex pairing in a duplexed nucleic acid.

Prefened ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance ofthe resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), 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 apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, speπnidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic poφhyrin, 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), poφhyrins (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, l,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3- (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytiityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport absoφtion 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-/cB.

The ligand can be a substance, e.g, a drag, which can increase the uptake ofthe iRNA agent into the cell, for example, by disrapting the cell's cytoskeleton, e.g., by disrapting the cell's microtubules, microfilaments, and/or intermediate filaments. The drag can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latranculin A, phalloidin, swinholide A, indanocine, or myoservin.

The ligand can increase the uptake ofthe 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 ofthe 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 ofthe 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 ofthe conjugate, (b) increase targeting or transport into a target cell or cell membrane, and or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding ofthe 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 prefened 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 prefened 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., ofthe 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, B12, 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 amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also refened to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three- dimensional stracture similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution ofthe iRNA, such as by enhancing cellular recognition and absoφtion. 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

Attorney's Docket No.: 14174-072W01

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Tφ 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 JD NO:6715). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:6716)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can cany large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:6717)) and the Drosophila Antennapedia protein (RQLKiWFQNRRMKWKK (SEQ ID NO: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 incoφorated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peρtide, 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 ofthe structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al, Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing otyβ₃ (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 α_vβ₃ integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the α_v-β₃ integrin ligand. Generally, such ligands can be used Attorney's Docket No.: 14174-072 WO 1

to control proliferating cells and angiogeneis. Prefened 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 α-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a -defensin, /?-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al, Nucl. Acids Res. 31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic - helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis- 1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H- bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i ± 3, or i ± 4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, omithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides. 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. 60/463,772, filed on April 17, 2003, which is hereby incoφorated 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 incoφorated 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 amphipathic delivery agent described herein, an iRNA associated with a drag delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incoφorates a modified or non-naturally occuring base.

The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trafert et al, Tetrahedron, 52:3005, 1996; and Manoharan, "Oligonucleotide Conjugates in Antisense Technology," in Antisense Drag Technology, ed. S.T. Crooke, Marcel Dekker, Inc., 2001.

In one embodiment ofthe invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific prefened conformation, which can increase the potency and selectivity ofthe peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the c-carbon of an amino acid with a nitrogen atom without changing the structure ofthe 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 ofthe 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 Applications, 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.

In one embodiment ofthe invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a /3-peptide. /3-peptides form stable secondary structures such as helices, pleated sheets, turns and haiφins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state. 3-peptides are resistant to degradation by proteolytic enzymes. /3-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a /3-peptide.

In one embodiment ofthe 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.

In one embodiment ofthe 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 o the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea 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 ofthe 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, such as in the loop of a short haiφin iRNA agent, hi 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 carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside ofthe carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior ofthe 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 ofthe agents to the interior ofthe 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 variety 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 ofthe invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incoφoration of an RRMS containing a ligand that targets the liver. For example, a liver-targeting agent can be a lipophilic moiety. Prefened 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, l,3-Bis-O(hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytiityl, 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 incoφorates a sugar, e.g., galactose and or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells ofthe 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(WAFl/DLPl),

P27(KLP1), the α-fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer ofthe liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of Attorney's Docket No.: 14174-072W01

an HDL/LDL 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, hi another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR- Ga ma 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 ofthe invention can target any one ofthe nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

Prefened 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 chain, containing the indicated number of carbon atoms. For example, Cι-C₁₂ 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 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 sp³ carbons may optionally serve as the point of attachment ofthe 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" refers to an -S-alkyl radical.

The term "alkylene" refers to a divalent alkyl (i.e., -R-), e.g., -CH₂-, -CH₂CH₂-, and - CH₂CH₂CH -. The term "alkylenedioxo" refers to a divalent species ofthe structure -O-R-O-, in which R represents an alkylene.

The term "aryl" refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring 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 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 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, moφholino, pynolinyl and pynolidinyl.

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, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)_nalkyl (where n is 0-2), S(O)_n aryl (where n is 0-2), S(O)„ heteroaryl (where n is 0-2), S(O)_n 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, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, xmsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset ofthe 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 ofthe 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-foπnylcytosinyl,

Attorney's Docket No.: 14174-072W01

N4-methylcytosinyl,

5-hydroxymethylcytosinyl,

1 -methylguaninyl,

N2-methylguaninyl,

7-methylguaninyl,

N2,N2-dimethylguaninyl,

Attorney's Docket No.: 14174-072W01

N2,7-dimethylguaninyl,

N2,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-methoxycarbonylmethyluracilyl,

5-methoxycarbonylmethyl-2-thiouracilyl,

5-aminomethyl-2-thiouracilyl,

5-methylaminomethyluracilyl,

5-methylaminomethyl-2-thiouracilyl,

5-methylaminomethyl-2-selenouracilyl,

5-carbamoylmethyluracilyl,

5 -carboxymethylaminomethyluracilyl,

5-carboxymethylaminomethyl-2-thiouracilyl,

3-methyluracilyl,

l-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl, 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 stracture 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. 60/462,894, filed April 14,2003; and International Application No. PCT/US 04/07070, filed March 8, 2004, all of which are hereby incoφorated by reference. The iRNA agents ofthe 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 ofthe iRNA agent can be on different RNA strands, and the mismatch between the first and second sequences can be less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%. The first and second sequences ofthe 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 foim. The first and second sequences ofthe 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 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 mutations, or polymoφhisms, 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 ofthe 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 conespond to hot-spots for genetic variation.

The compositions ofthe 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 ofthe target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. The target RNA regions can conespond to genetic hot- spots.

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 conesponds to a different variant or mutant ofthe gene (e.g., a region including the codon encoding p53 248Q and/or p53 249S). The iRNA agent can be constructed such that a first sequence is complementary to a first ofthe plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second ofthe plurality of variant RNA targets (e.g., encoding 249S), 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 ofthe 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.

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 conespond 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 polymoφhisms of the target gene. The first and second variant RNA target regions can conespond 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 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. PCT/US04/07070, filed March 8, 2004, both of which are hereby incoφorated by reference.

The use of "other than canonical Watson-Crick pairing" between monomers of a duplex can be used to confrol, 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 ofthe monomers, the position ofthe monomers in the iRNA agent, and the target sequence (sometimes refened 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 ofthe duplex iRNA agent in the context ofthe 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 ofthe 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. hi this case one or more ofthe monomers, the position ofthe monomers in the iRNA agent, and an off-target sequence, 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 ofthe duplex formed by the sense strand and the off-target sequence).

Thus, inherent in the stracture ofthe iRNA agent is the property of having a first stability for the intra-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 ofthe monomers at a selected or constrained position, the selection ofthe position in the duplex to place the selected or constrained position, and selection ofthe 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 refened herein as constrained sequences. Exercise ofthe constraint or selection parameters can e, e.g., by inspection, or by computer assisted methods. Exercise ofthe 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 ofthe 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, hi a duplex region ofthe 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 conesponding positions. One, and preferably both monomers are selected such that the stability ofthe pairing ofthe monomers contribute to a duplex between the first and second sequence will differ form the stability ofthe pairing between the first or second sequence with a target sequence.

Usually, the monomers will be selected (selection ofthe target sequence may be required as well) such that they form a pairing in the iRNA agent duplex w ich has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring ofthe 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 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 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 middle ofthe 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 ofthe 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 prefened overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a haiφin, 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 foπn 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 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-Atran_s, G-A_Ci_S, and GU.

The monomer in one or both ofthe sequences is selected such that, it does not pair, or forms a pair with its conesponding 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 conesponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) ofthe sequences with the respective target sequence. The monomer is one or both strands is also chosen to promote stability in one or both ofthe duplexes made by a strand and its target sequence. E.g., one or more ofthe 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 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 ofthe following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes refened 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.

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 foπn 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 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, A_ci_s, A_trans, 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 Attorney's Docket No.: 14174-072W01

will foi 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, hi 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 occuπing monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that ofthe pairing ofthe 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 ofthe 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-thio pyrimidine analog of U or T.

When placed in complementary positions ofthe 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. This is shown in the FIG. 1. Attorney's Docket 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 pynole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incoφorated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the conesponding position ofthe 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 ofthe 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 unwinding enzyme. In a duplex region ofthe 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 conesponding 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 conesponding site in the anti-sense strand are less stable than the H bonds fonned by the monomer ofthe 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 ofthe modified base and its canonical Watson-Crick partner);

The monomer is in the conesponding 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 conesponding 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 conesponding 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 ofthe following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes refened 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 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 middle ofthe 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 ofthe 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, 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 prefened overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a haiφin, 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 Attorney's Docket No.: 14174-072 W01

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-Atnms- G-Aci_S, and GU.

The monomer in one or both ofthe sequences is selected such that, it does not pair, or forms a pair with its conesponding monomer in the other sequence which minimizes stability (e.g., the H bonding fonned between the monomer at the selected site in the one sequence and its monomer at the conesponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) ofthe sequences with the respective target sequence. The monomer is one or both strands is also chosen to promote stability in one or both ofthe duplexes made by a strand and its target sequence. E.g., one or more ofthe 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 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 ofthe following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes refened 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. 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, hi 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 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, A_ci_s, A_trans, 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.

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, lh 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 Attorney's Docket No.: 14174-072W01

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 ofthe pairing ofthe 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 ofthe 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-thio pyrimidine analog of U or T.

When placed in complementary positions ofthe 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 a monomer which includes 3-nitro pynole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incoφorated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the conesponding position ofthe 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 ofthe 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 ofthe 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 conesponding monomer in the anti-sense sfrand 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 conesponding site in the anti-sense strand are less stable than the H bonds formed- by the monomer ofthe 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 ofthe modified base and its canonical Watson-Crick partner);

The monomer is in the conesponding 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 conesponding 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 conesponding 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 ofthe following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes refened 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 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 ofthe 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 prefened 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 sfrand 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. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region ofthe iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

hi some embodiment the duplex region ofthe 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, 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 prefened overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a haiφin, or by other non-base linkers.

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 occurring ribonucleotides, or modified ribonucleotides having naturally occurring 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 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 ofthe 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 ofthe 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.

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 conesponding 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 which pairs with A), the conesponding monomer in the target is an A, and the sense sfrand 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 conesponding monomer in the target is a G, and the sense strand is chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, A_ci_s, Atans, 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 conesponding monomer in the target is a C, and the sense sfrand is chosen from a monomer which will not pair or which will form a non-canonical pairing.

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

ofthe pairing ofthe 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-thio pyrimidine analog of U or T. As is discussed above, when placed in complementary positions ofthe 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 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 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 pynole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incoφorated by reference. In these cases the monomer at the conesponding position ofthe 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.

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 subject, 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 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 ofthe anti-sense strand ofthe iRNA agent is selected such that at one, some, or all ofthe positions which conespond 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 prefened example the anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pynole, 2-amino A, 2-thio U or 2-thio T, or other universal base refened to herein.

hi a prefened 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 conesponding 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 subject. This analysis can be performed successively to refine or desing optimized iRNA agents.

In another aspect, the invention features, a kit which inlcudes one or more ofthe folowing an iRNA described herein, a sterile container in which the iRNA agent is discolsed, and instructions for use.

hi another aspect, the invention features, an iRNA agent containing a constrained sequence made by a method described herein. The iRNA agent can target one or more ofthe genes refened 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 ofthe methods described herein and to target any ofthe genes described herein or to treat any ofthe disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incoφorated into any ofthe 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 ofthe 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 conesponding position in a second sequence of a duplex in which one or more ofthe 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 ofthe duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurring 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 ofthe monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less strong than the bonds fonned 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 silenced.

Universal Bases: "wild-cards" ; shape-based complementarity

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by 'inverse' sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol, 1997, 4, 919-926)

(Importance of terminal base pair hydrogen-bonding in 3 '-end proofreading by the Klenow fragment of DNA polymerase I. Morales, J. C; Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

(Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.; Kool, E. T. J. Am. Chem. 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. Chem. 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. Chem., 1998, 63, 9652-9656)

Attorney's Docket No.: 14174-072W01

Nθ2

5-nitroindole

(

(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)

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. Am. Chem. Soc, 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc, 2000, 122, 8803-8804)

7AI

(Efforts toward expansion ofthe genetic alphabet: replication of DNA with three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc, 2001, 123, 7439-7440)

(1. Efforts toward expansion ofthe 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. Am. Chem. Soc, 1999, 121, 11585-11586)

(A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple: Interstrand stacking as file stability determining factor. Brotschi, C; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014)

(2,2'-Bipyridine Ligandoside: A novel building block for modifying DNA with intra-duplex metal complexes. Weizman, H.; Tor, Y. J. Am. Chem. Soc, 2001, 123, 3375-3376) Attorney's Docket No.: 14174-072W01

(Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc, 2000, 122, 6512-13)

(Effect ofthe 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 ofthe l-(2'-deoxy-β-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: l-(2'-deoxy-β-D-ribofuranosyl)-3-mtropyπ:ole. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C; Nichols, R. J. Am. Chem. Soc, 1995, 117, 1201-1209)

(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. Am. Chem. Soc, 199S, 117, 10769-10770) Attorney's Docket No.: 14174-072W01

(A universal, photocleavable DNA base: nitropiperonyl 2'-deoxyriboside. J. Org. Chem., 2001, 66, 2067-

2071)

(Recognition of a single guanine bulge by 2-acylamino- 1,8-naphthyridine. Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. 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)

Other universal bases can have the following formulas: Attorney's Docket No.: 14174-072W01

wherein:

Q is N or CR >4""4;.

Q' is N or CR⁴⁵;

Q"isNorCR 47. Attorney's Docket No.: 14174-072W01

Q'" is or CR⁴⁹;

Q^iv is N or CR⁵⁰;

R⁴⁴ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR , or NR^bR^c, Cι-C₆ alkyl, C₆-Cιo aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁵ forms -OCH₂O-;

R⁴⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, Ci-Q; alkyl, C₆-Cι₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁴ or R⁴⁶ forms -OCH₂O-;

R⁴⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR , or NR R^c, d-C₆ alkyl, C₆-Cι₀ aryl, C₆-C₁₀ heteroaryl, C -C₈ heterocyclyl, or when taken together with R⁴⁵ or R⁴⁷ forms -OCH₂O-;

R⁴⁷ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, Cι-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁶ or R⁴⁸ forms -OCH2O-;

R⁴⁸ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, d-C₆ alkyl, C₆-Cιo aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁷ forms -OCH2O-; τ>49 τ>50 TI 51 -n52 τ>53 _O54 τ>57 -r>58 r»59 τ>_{60 Ώ} 61 _Ώ 62 τ>₆3 τ_>64 -nδ5 τ>66 -n67 r>68 r,69 is. is. , is. , iv , is. , Jtv. , 1s. , 1s. , is. , is , is. , 1s. , 1s. , 1s. , 1s. , is. , is. , 1s. , Jts. ,

R⁷⁰, R⁷¹, and R⁷² are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR R^c, d-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃- C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R°;

R⁵⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR R^c, Cι-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R°, or when taken together with R⁵⁶ forms a fused aromatic ring which may be optionally substituted; Attorney's Docket No.: 14174-072W01

R⁵⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR R^c, d-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(0)R¹⁷, or NC(O)R°, or when taken together with R⁵⁵ forms a fused aromatic ring which may be optionally substituted;

R¹⁷ is halo, NH₂, NHR^b, or NR^bR^c;

R is C C₆ alkyl or a nitrogen protecting group;

R^c is Cι-C₆ alkyl; and

R° is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^b, or NR^bR^c, d-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, orNC(O)R°.

Attorney's Docket No.: 14174-072W01

Examples of xmiversal bases include:

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/US04/07070, filed March 8, 2004, which is hereby incoφorated 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 stracture 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 incoφorates an asymmetrical modification.

An asymmetrically modified iRNA agent is one in which a sfrand has a modification which is not present on the other strand. An asymmetrical modification is a modification found 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 ofthe 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; 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 refened to as one which promotes stabilization might also enhance targeting.

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 ofthe different or "asymmetrical" functions ofthe 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 ofthe 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 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 ofthe sense strand ofthe iRNA agent. In a prefened example, a P ofthe 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 ofthe sense strand of the iRNA agent.

In a prefened 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: 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.

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 sfrand.

In an embodiment one strand has an asymmetrical 2' modification, e.g., a 2' OMe modification, and the other strand has an asymmetrical modification ofthe 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 prefened embodiment the RNAi agent will have Attorney's Docket No.: 14174-072W01

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, ofthe subunits ofthe sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, ofthe subunits ofthe antisense sfrand 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 sfrand. All ofthe subunits on one or both strands can be so modified. A particularly prefened embodiment of multiple asymmetric modification on both strands has a duplex region 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, fri prefened embodiments the modification on the antisense strand is one which will not interfere with silencing ofthe target, e.g., one which will not interfere with cleavage ofthe 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 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 ofthe backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement ofthe U with a C5 amino linker; replacement ofthe A with a G (sequence changes are prefened to be located on the sense strand and not the antisense strand); and modification ofthe at the 2', 6', 7', or 8' position. Prefened 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. 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 prefened embodiments the modification on the antisense strand is one which will not interfere with silencing ofthe target, e.g., one which will not interfere with cleavage ofthe 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 ofthe molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); modification ofthe 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' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification ofthe 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' 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. Prefened 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.

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 ofthe sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications ofthe sense strand. Targeting modifications which introduce 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 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 stracture, 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 ofthe iRNA agent defined by the 5' end ofthe sense strand and the 3 'end ofthe antisense sfrand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3' overhang. In prefened embodiments asymmetrical modifications which result in one or more ofthe following are used: modifications ofthe 5' end ofthe sense strand which inhibit kinase activation ofthe 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" stracture at this end ofthe molecule.

The end region ofthe iRNA agent defined by the 3' end ofthe sense strand and the 5'end ofthe antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3 ' overhang. Prefened 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" stracture at this end ofthe molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification ofthe antisense sfrand which inhibit kinase activation are avoided in prefened 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 ofthe sense to the antisense strand, the 3' modifications can be used at the 5' end ofthe sense strand to avoid sense sfrand 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 ofthe sense to the antisense strand, or can be used at the 5' end ofthe sense strand to avoid sense strand activation by RISC;

(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 ofthe sense to the antisense strand, or can be used at the 5' end ofthe sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding ofthe 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 resistance or to enhance binding ofthe 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 ofthe sense sfrand 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 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 prefened to have a "loose" structure in the 5' region, additionally, they may interfere with kinase activity;

(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 ofthe sugar; as nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 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 prefened.

The 5'-OH ofthe antisense strand should be kept free to promote activity. In some prefened embodiments modifications that promote nuclease resistance should be included at the 3' end, particularly in the 3' overhang.

hi 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 ofthe asymmetric modification optimizes a property ofthe 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 prefened 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 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 prefened 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.

hi a prefened embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and 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 ofthe sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all ofthe monomers of a sequence can be modified.

Table 3 shows examples having strand I with a selected modification and strand II with a selected modification.

Table 3. Exemplary strand I- and strand II-modifications

Strand I Strand II

Nuclease Resistance (e.g., 2'-OMe) Biodistribution (e.g., P=S)

Biodistribution conjugate Protein Binding Functionality (e.g., Lipophile) (e.g., Naproxen)

Tissue Distribution Functionality Cell Targeting Functionality (e.g., Carbohydrates) (e.g., Folate for cancer cells)

Tissue Distribution Functionality Fusogenic Functionality (e.g., liver Cell Targeting moieties) (e.g., Polyethylene imines)

Cancer Cell Targeting Fusogenic Functionality (e.g., RGD peptides and imines) (e.g., peptides)

Increase in binding Affinity (5-Me-C, 5-Me-U, 2-

Nuclease Resistance (e.g., 2 '-OMe) thio-U, 2-amino-A, G-clamp, LNA)

Tissue Distribution Functionality

RISC activity improving Functionality

Helical conformation changing Tissue Distribution Functionality Attorney's Docket No.: 14174-072W01

Functionalities (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 incoφorated by reference, copending, co-owned United States Provisional Application Serial No. 60/510,318, filed on October 10, 2003, which is hereby incoφorated by reference, and copending, co-owned International Application No. PCT/US 04/07070, filed March 8, 2004.

hi 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 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 drag delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incoφorates a Z-X-Y architecture.

Thus, an iRNA agent can have a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:

Z— X— Y.

It may be desirable to modify subunits in one or both of ZandVor Y on one hand and X on i the other hand. In some cases they will have the same modification or the same class of 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 ofthe 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 embodiments self associate, e.g., to form a loop stracture. Such structures can be formed by the Attorney's Docket No.: 14174-072W01

end of a strand looping back and forming an mtrastrand duplex. E.g., 2, 3, 4, 5 or more infra- 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 ofthe 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 prefened that it is a 3- overhang. Typical inter-subunit bonds in the Z region include: P=O; P=S; S-P=S; P-NR₂; 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 prefened 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 conesponding region ofthe single strand, or in the case of a double stranded iRNA agent, with the conesponding region ofthe other strand. The length ofthe X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly prefened 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 prefened while phophorothioate or non-phosphate bonds are absent. Other modifications prefened in the 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 prefened 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. 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.

hi a prefened 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 ofthe regions, Y, X, and Z. However, it is prefened that their occureceis minimized and in particular it is prefened that consecutive modified linkages be avoided.

In a prefened 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 any ofthe regions, Y, X, and Z. However, it is prefened that they are minimized and in particular it is prefened that consecutive modified linkages be avoided.

Thus, in a prefened embodiment, not all ofthe subunit linkages ofthe 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 prefened iRNA agents 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.

It is particularly prefened to minimize the occunence in region X. Thus, in prefened embodiments each ofthe nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or 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 prefened 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 prefened 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 prefened embodiment Y and /or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications ofthe subunit linkages.

In a prefened 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 prefened 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 prefened embodiment one or more phosphorothioate linkages (or other modifications ofthe 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 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 prefened 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, 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' 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 ofthe above recited limitation on adjacent 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 ofthe 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 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 and/or Z 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 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 prefened embodiment more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a prefened embodiment the frequency ofthe modification will differ between Yand /or Z and X, e.g., the modification will be present one of Y and/or 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 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 prefened embodiment more than 1 of such subunits can b present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.

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 1, 2, or 3 positions) ofthe 3' or 5' end ofthe sense strand or at near (within 2 or 3 positions of) the 3' end ofthe antisense strand. In some embodiments it is prefened to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end ofthe antisense strand. An Attorney's Docket No. : 14174-072 WO 1

RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area ofthe antisense strand not critical for 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 architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drag delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incoφorates DMTDS.

iRNA agents can be optimized by increasing the propensity ofthe duplex to disassociate or melt (decreasing the free energy of duplex association), in the region ofthe 5' end ofthe antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity ofthe duplex to disassociate or melt in the region ofthe 5' end ofthe antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity ofthe duplex to disassociate of melt in the region ofthe 5'end . While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect ofthe enzyme in the proximity ofthe 5' end ofthe antisense strand.

The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity ofthe duplex to disassociate or melt (increasing the free energy of duplex association), in the region ofthe 3' end ofthe antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity ofthe duplex to disassociate or melt in the region ofthe 3' end ofthe antisense strand. It can also be Attorney's Docket No.: 14174-072W01

accomplished by the attachment of ligand that decreases the propensity ofthe duplex to disassociate of melt in the region ofthe 5'end.

Modifications which increase the tendency ofthe 5' end ofthe duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency ofthe 3' end ofthe duplex to dissociate. Likewise, modifications which decrease the tendency ofthe 3' end ofthe duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency ofthe 5' end ofthe duplex to dissociate.

Decreasing the stability ofthe AS 5 ' end ofthe 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), hi terms of promoting dissociation:

A:U is prefened over G:C;

G:U is prefened over G:C;

I:C is prefened over G:C (I=inosine);

mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are prefened over canonical (A:T, A:U, G:C) pairings;

pairings which include a universal base are prefened over canonical pairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5' RJ NI

N NS [N] N-5 K4 N_-3 N_-2 N.i R₂ 3'

AS 3' R3 N1 N2N3N4N5 [N] N-5 N-4 N.3 N-2 N.i Rt 5'

S:AS Pj P₂ P₃ P₄ P₅ [N] P_5P-4P.3P.2P-1 5' Attorney's Docket No.: 14174-072W01

S indicates the sense strand; AS indicates antisense strand;

indicates an optional (and nonprefened) 5' sense strand overhang; R₂ indicates an optional (though prefened) 3' sense overhang; R₃ indicates an optional (though prefened) 3' antisense sense overhang; R₄ indicates an optional (and nonprefened) 5' antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and P_x, indicates a paring of sense N_x and antisense N_x. Overhangs are not shown in the P diagram. In some embodiments a 3' AS overhang conesponds to region Z, the duplex region conesponds to region X, and the 3' S strand overhang conesponds 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.)

It is prefened that pairings which decrease the propensity to form a duplex are used at 1 or more ofthe positions in the duplex at the 5' end ofthe AS strand. The terminal pair (the most 5' pair in terms ofthe AS strand) is designated as P._1? and the subsequent pairing positions (going in the 3' direction in terms ofthe AS strand) in the duplex are designated, P_-2, P..₃, P^, P_-5, and so on. The prefened region in which to modify to modulate duplex formation is at P.₅ through P_ι , more preferably P_-4 through P._! , more preferably P-₃ through P._! . Modification at P. ₁, is particularly prefened, alone or with modification(s) other position(s), e.g., any ofthe positions just identified. It is prefened that at least 1, and more preferably 2, 3, 4, or 5 ofthe pairs of one ofthe recited regions be chosen independently from the group of:

A:U

G:U

LC

mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base.

In prefened embodiments the change in subunit needed to achieve a pairing which 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 prefened embodiment the at least 2, or 3, ofthe pairs in P-i, through P_-4, are pairs which promote disociation.

In a prefened embodiment the at least 2, or 3, ofthe pairs in P._l5 through P.₄, are A:U.

In a prefened embodiment the at least 2, or 3, ofthe pairs in P._l5 through P.₄, are G:U.

In a prefened embodiment the at least 2, or 3, ofthe pairs in P._l5 through P_₄, are I:C.

In a prefened embodiment the at least 2, or 3, ofthe pairs in P._l5 through P.₄, are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.

In a prefened embodiment the at least 2, or 3, ofthe pairs in P_ι, tlirough P_-4, are pairings which include a universal base.

Increasing the stability ofthe AS 3 ' end ofthe 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), hi terms of promoting duplex stability:

G:C is prefened over A:U

Watson-Crick matches (A:T, A:U, G:C) are prefened over non-canonical or other than canonical pairings

analogs that increase stability are prefened over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U is prefened over A:U

2-thio U or 5 Me-thio-U: A are prefened over U:A

G-clamp (an analog of C having 4 hydrogen bonds):G is prefened over C:G Attorney's Docket No.: 14174-072W01

guanadinium-G-clamp : G is prefened over C : G

psuedo uridine:A is prefened over U:A

sugar modifications, e.g., 2' modifications, e.g., 2'F, ENA, or LNA, which enhance binding are prefened over non-modified moieties and can be present on one or both strands to enhance stability ofthe duplex. It is prefened that pairings which increase the propensity to form a duplex are used at 1 or more ofthe positions in the duplex at the 3' end ofthe AS strand. The terminal pair (the most 3' pair in tenns ofthe AS strand) is designated as P_ls and the subsequent pairing positions (going in the 5' direction in terms ofthe AS strand) in the duplex are designated, P₂, P₃, P₄, P₅, and so on. The prefened region in which to modify to modulate duplex formation is at P₅ through Pi, more preferably P₄ through Pi , more preferably P₃ through Pi. Modification at Pi, is particularly prefened, alone or with mdification(s) at other position(s), e.g.,any ofthe positions just identified. It is prefened that at least 1, and more preferably 2, 3, 4, or 5 ofthe pairs ofthe 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

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 prefened embodiment the at least 2, or 3, ofthe pairs in P_-l5 through P_-4, are pairs which promote duplex stability.

In a prefened embodiment the at least 2, or 3, ofthe pairs in Pi, through P , are G:C.

In a prefened embodiment the at least 2, or 3, ofthe pairs in Pi, through P , are a pair having an analog that increases stability over Watson-Crick matches.

In a prefened embodiment the at least 2, or 3, ofthe pairs in Pi, through P₄, are 2-amino- A:U.

In a prefened embodiment the at least 2, or 3, ofthe pairs in Pi, through P₄, are 2-thio U or 5 Me-thio-U:A.

In a prefened embodiment the at least 2, or 3, ofthe pairs in Pi, through P₄, are G- clamp:G.

In a prefened embodiment the at least 2, or 3, ofthe pairs in Pi, through P₄, are guanidinium-G-clamp : G.

In a prefened embodiment the at least 2, or 3, ofthe pairs inPi, through P , are psuedo uridine:A.

In a prefened embodiment the at least 2, or 3, ofthe pairs in Pi, 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 and Gait, "The Synthesis of 2'-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition ofthe HIV-1 Tat-TAR Interaction," Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al, "Steric inhibition of human imnmnodefϊciency 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. : 14174-072 O 1

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. : 14174-072 WO 1

Simultaneously decreasing the stability ofthe AS 5'end ofthe duplex and increasing the stability ofthe AS 3' end ofthe duplex

As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5'end ofthe duplex and increase the stability ofthe AS 3' end ofthe duplex. This can be effected by combining one or more ofthe stability decreasing modifications in the AS 5' end of the duplex with one or more ofthe stability increasing modifications in the AS 3' end ofthe duplex. Accordingly a prefened embodiment includes modification in P.₅ through P.i, more preferably P^ through P_ι and more preferably P_-3 through P._t. Modification at P_-l5 is particularly prefened, alone or with other position, e.g., the positions just identified. It is prefened that at least 1, and more preferably 2, 3, 4, or 5 ofthe pairs of one ofthe recited regions ofthe AS 5' end ofthe duplex region be chosen independently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and

a modification in P₅ through Pi, more preferably P₄ through Pi and more preferably P₃ through Pi. Modification at Pi, is particularly prefened, alone or with other position, e.g., the positions just identified. It is prefened that at least 1, and more preferably 2, 3, 4, or 5 ofthe pairs of one ofthe recited regions ofthe AS 3' end ofthe 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)

2-amino-A:U Attorney's Docket No. : 14174-072 WO 1

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 iRNA 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._! and/or at least one P in P₅ through ?ι to provide a DMTDS iRNA agent.

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 incoφorate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of 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 intrasfrand 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 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 ofthe 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 ofthe 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 vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity ofthe cyclodextrin. hi 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 incoφorated by reference, are suitable for use in methods ofthe 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 incoφorated by reference, are suitable for use in methods ofthe invention. An iRNA agent can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both ofthe Attorney's Docket No.: 14174-072W01

cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.

A composition for iRNA delivery can employ an "inclusion complex," a molecular compound having the characteristic stracture of an adduct. hi 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 ofthe host molecule without affecting the framework structure ofthe host. A "host" is preferably cyclodextrin, but can be any ofthe molecules suggested in U.S. Patent Publ. 2003/0008818, herein incoφorated 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 ofthe host and guest molecules should be complementary in the stereoelecfronic sense. A composition ofthe invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite ofthe polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer ofthe 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 ofthe particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer ofthe particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer ofthe particulate composite can also contain both host and guest functionalities and foπn 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 ofthe particulate composite, the activity ofthe 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 ofthe 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 ofthe iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, ofthe non-human mammal and the human. By determining the toxicity ofthe iRNA agent in the non-human mammal, one can extrapolate the toxicity ofthe 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 conelate 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 drag 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. PCT/US04/07070, filed March 8, 2004, both of which are hereby incoφorated 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 drag delivery conjugate or module.

The iRNA agents can be complexed to a delivery agent that features a modular complex.

The complex can include a carrier 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 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.

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 agent ofthe 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 subject having a disorder, e.g., a disorder described herein, such as a disease or disorder ofthe liver.

The fusogenic agent and the condensing agent can be different agents or the one and the 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 carrier agent linked to one or more of (preferably two or more, more preferably all three of): Attorney's Docket No. : 14174-072 WO 1

(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, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.

Carrier agents

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 prefened. It would also be prefened that the carrier molecule be biodegradable..

The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dexfran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier 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-maleic acid anhydride copolymer, poly(L- lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. 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 carrier 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 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 ofthe iRNA agent in 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 ofthe 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 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 rapture) 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.

In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. hi some embodiment, the fusogenic agent can have an alpha helical stracture. 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 ofthe following characteristics: (a) is at least IDa 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 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 ofthe iRNA. The ability ofthe fusogenic agent to respond to, e.g., change charge 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 compound to respond to, e.g., change charge depending on, the pH environment.

Condensing 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 ofthe 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 poφhyrin, quarternary salt of a polyamine, or an alpha helical peptide.

A condensing agent can have the following characteristics: (a) at least IDa 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 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 ofthe 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 is identified as a condensing agent if it changes the mass and/or charge (preferably both) ofthe 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.

Amphipathic 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. PCT/US04/07070, filed March 8, 2004, which is hereby incoφorated by reference.

h 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 prefened amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs descriebd herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary stracture.

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: Grell et al. (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(l):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; Comut 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(l):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 afropisomerism, 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 ofthe 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.

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 amphipathic 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), hi some embodiments, an amphipathic polymer can contain both amino acid 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 refened 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 ofthe 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 ofthe composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule ofthe 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 ofthe hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face ofthe polymer is a hydrophilic face and the other face ofthe polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-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 and/or one or more hydrophobic groups), hi 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 afropisomerism, e.g., a 2,2'-bis(substituted)-l-l '-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 refened to herein as "amphipathic iRNA constracts." 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 constracts 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 ofthe 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 ofthe composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule ofthe 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.: 14174-072W01

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 ofthe hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face ofthe polymer is a hydrophilic face and the other face ofthe polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary stracture.

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 and/or 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 afropisomerism, e.g., a 2,2'-bis(substituted)-l-l '-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

Referring to Formula M, Ri is CpCioo alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR^; or C1-C₁₀₀ 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, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

R₃ is hydrogen, or when taken together with R froms a fused phenyl ring.

R is hydrogen, or when taken together with R₃ froms a fused phenyl ring.

R₅ is C1-C100 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C₁-C₁₀₀ perfluoroalkyl; and R is Ci- C₁₀₀ 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, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

Increasing cellular uptake of dsRNAs

A method ofthe invention that can include the administration of an iRNA agent and a drag that affects the uptake ofthe iRNA agent into the cell. The drag can be administered before, after, or at the same time that the iRNA agent is administered. The drag can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The drag can have a transient effect on the cell.

The drug can increase the uptake ofthe iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrapting the cell's microtubules, microfilaments, and/or intermediate filaments. The drag can be, for example, taxon, vincristine, vinblastine, Attorney's Docket No.: 14174-072W01

cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The drug can also increase the uptake ofthe 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 conjugates

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 directed to the treatment ofthe 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 Synthesis

An iRNA can be made by separately synthesizing each respective strand of a double- stranded RNA molecule. The component strands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA sfrand for a given iRNA. The

OligoPilotll reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA sfrand, 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 ofthe species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymoφhism, e.g., a single nucleotide polymoφhism. Further the location ofthe polymoφhism can be precisely defined. In some embodiments, the polymoφhism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both ofthe 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 be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe™ 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 generated for T7 transcription ofthe two complementary strands for the dsRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsRNAis produced. Similar methods using PCR and/or other RNApolymerases (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 ofthe recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or comparable RNAse Ill-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 αl. Genes Dev 2001 Oct 15;15(20):2654-9. and Hammond Science 2001 Aug 10;293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, each being a particular

21 to 23 nt fragment of a source dsRNA molecule. 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 ofthe 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 sunogate 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 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 than 80, 50, 30, 20, or 10% water). In another example, the iRNAis in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., be incoφorated 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 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 ofthe following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

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 lRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the 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 freatment 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 freatment of a cancer might further comprise a chemo therapeutic agent.

Exemplary formulations are discussed below:

Liposomes

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 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 ananged 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 ofthe cellular membranes. As the merging ofthe 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 ofthe 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 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 carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Further description of methods for producing stable polynucleotide delivery vehicles, which incoφorate a polynucleotide/cationic lipid complex as structural components ofthe delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Feigner, P. L. et al, Proc. Natl. Acad. Sci, USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Ada 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Ada 775:169, 1984; Kim, et al. Biochim. Biophys. Ada 728:339, 1983; and Fukunaga, 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. Biochim. Biophys. Ada 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Ada 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 ofthe exogenous gene was detected in the target cells (Zhou et al, Journal of Controlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylchohne. Neufral liposome compositions, for example, can be fonned from dimyristoyl phosphatidylchohne (DMPC) or dipalmitoyl phosphatidylchohne (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 fonned from phosphatidylchohne (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylchohne and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able 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: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incoφorate 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 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume ofthe liposomes.

A positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)ρropyl]-N,N,N- trimethylammonium chloride (DOTMA) can be used to foπn small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids ofthe cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Feigner, 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, l,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, l,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") (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization ofthe 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 DMRfE 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 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absoφtion ofthe 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 enhance the penefration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drags formulated as liposomes to the skin has been documented (see, e.g., Weiner et al, Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al, Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl 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 ofthe 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, 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 common way of classifying and ranking the properties ofthe many different types of surfactants, both natural and synthetic, is by the use ofthe hydrophile/lipophile balance (HLB). The nature ofthe hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms," Marcel Dekker, Inc., New York, NY, 1988, p. 285). 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 ethoxylated propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members ofthe nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members ofthe anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drag 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. : 14174-072 WO 1

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 ananged in a spherical stracture such that all the hydrophobic portions ofthe molecules are directed inward, leaving the hydrophilic portions in contact with the sunounding aqueous phase. The converse anangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution ofthe iRNA composition, an alkali metal C₈ to C₂₂ 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, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition ofthe alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing ofthe ingredients but vigorous mixing is prefened 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 another method, the micellar composition is prepared by mixing the iRNA composition, the alkali metal alkyl sulphate and at least one ofthe micelle forming compounds, followed by addition ofthe 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 ofthe mixed micellar composition.

For delivery ofthe 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 ofthe 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 ofthe contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

The prefened propellants are hydrogen-containing chlorofluorocarbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether. Even more prefened is HFA 134a (1,1,1,2 tetrafluoroethane).

The specific concentrations ofthe essential ingredients can be determined by relatively straightforward experimentation. For absoφtion through the oral cavities, it is often desirable to increase, e.g. at least double or triple, the dosage for through injection or adminisfration 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

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 incoφorated 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 stracture 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 ofthe 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 μm and preferably larger than 20 μm). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of 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 μm rather than 10 μm. 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 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 spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstractures 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 prefened 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 prefened 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 be subj ected to further formulation. A pharmaceutical composition of iRNA can be prepared by spray drying a homogeneous aqueous mixture that includes a iRNA 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 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.

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 ofthe 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 sprayeα into a cunent 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 Bucbi Ltd. or Niro Coφ. 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 maybe some degree of iπegularity in shape depending upon the incoφorated 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 (or blowing agent) is used in their production. Particularly prefened 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 incoφorated surfactant, typically 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 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 microstractures 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.: 14174-072 WO 1

an aqueous dispersion ofthe medicament and surfactant(s) 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 microstractures 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 microstractures are contemplated as being within the scope ofthe invention. By "associated with or comprise" it is meant that the structural matrix or perforated microstructure may incoφorate, 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 ofthe 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. Particularly prefened 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 concenfrations 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 incoφorated 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 phosphatidylchohne, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl-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 polyvinylpynolidone; 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 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 (Spans™ 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.). Prefened block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic.RTM. F68), poloxamer 407 (Pluronic.RTM. F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In prefened embodiments, the microstractures may comprise oleic acid or its alkali salt.

In addition to the aforementioned surfactants, cationic surfactants or lipids are prefened especially in the case of delivery of an iRNA agent, e.g., a double-sfranded 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-sfranded 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, l,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 ofthe devices used in these processes include "Parabisu [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 Kakoki 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 ofthe inlet ofthe gas used to dry the sprayed materials such that it does not cause heat deactivation ofthe sprayed 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 ofthe 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 amoφhous powder 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 ofthe resulting powder is such that more than about 98% ofthe mass is in particles having a diameter of about 10 μm or less with about 90% ofthe mass being in particles having a diameter less than 5 μm. Alternatively, about 95% ofthe mass will have particles with a diameter of less than 10 μm with about 80% ofthe mass ofthe particles having a diameter of less than 5 μm.

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 patient, but may also serve to enhance the stability ofthe iRNA compositions and to improve the dispersibility ofthe powder within a powder dispersion device in order to provide more efficient and reproducible delivery ofthe iRNA and to improve handling characteristics ofthe 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 carrier material to the purified bulk solution, hi that way, the carrier particles will be formed simultaneously with the drag particles to produce a homogeneous powder. Alternatively, the earners may be separately prepared in a dry powder form and combined with the dry powder drag by blending. The powder carriers will usually be crystalline (to avoid water absoφtion), but might in some cases be amoφhous or mixtures of crystalline and amoφhous. The size ofthe carrier particles may be selected to improve the flowability ofthe drag powder, typically being in the range from 25 μm to 100 μm. A prefened carrier material is crystalline lactose having a size in the above-stated range.

Powders prepared by any ofthe above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other puφoses, 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 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 U.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 chlorthalidone (lipophilic drags) and a hydrophilic adjuvant (pentaerythritol) in azeofropic 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 combinations include FR 2594693; DE 2209477; and WO 88/07870.

LYOPHILIZATION.

An iRNA agent, e.g., a double-sfranded 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-sfranded 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 incoφoration of nucleic acids in perforated microstractures 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 microstractures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstractures having the desired porosity and size, they are conformance with the teachings herein and are expressly contemplated as being within the scope ofthe instant invention.

Targeting

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-sfranded 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-sfranded 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 ofthe 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 ofthe 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.

GENES AND DISEASES

hi one aspect, the invention features, a method of treating a subject at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmahgnant cell proliferation. The method includes:

providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA having a stracture 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 subject,

thereby treating the subject.

In a prefened 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 prefened 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 prefened 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 prefened 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 prefened 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 prefened 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 prefened embodiment the iRNA agent silences the RAS gene, and thus can be used to freat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.

In another prefened 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 prefened embodiment the iRNA agent silences the JNK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JNK expression, e.g., pancreatic or breast cancers.

In a prefened 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 prefened 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 Erkl/2 expression, e.g., lung cancer. Attorney's Docket No.: 14174-072W01

In another prefened 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 prefened 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 prefened 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.

In another prefened 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 prefened embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted FOS expression, e.g., skin or prostate cancers.

In a prefened 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 prefened embodiment the iRNA agent silences the Cyclin D gene, and thus can be 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 prefened embodiment the iRNA agent silences the VEGF gene, and thus can be used to freat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers.

In a prefened 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 prefened 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.

hi another prefened 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 prefened 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 prefened 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 prefened 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 prefened embodiment the iRNA agent silences the PKC gene, and thus can be used to freat a subject having or at risk for a disorder characterized by unwanted PKC expression, e.g., breast cancer.

In a prefened 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 prefened 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 prefened embodiment the iRNA agent silences the survivin gene, and thus can be used to freat 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 prefened 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 xmwanted Her2/Neu expression, e.g., breast cancer.

In another prefened 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 prefened 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.

In a prefened 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 prefened embodiment the iRNA agent silences mutations in the p21(WAFl/CIPl) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p21(WAFl/CIPl) expression, e.g., liver cancer.

In a prefened embodiment the iRNA agent silences mutations in the p27(KIPl) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIPl) expression, e.g., liver cancer.

In prefened embodiments the iRNA agent silences mutations in tumor suppressor genes, 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:

providing an iRNA agent, e.g., an iRNA agent having a stracture 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 viras 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 stracture 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 subj ect.

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.

In a prefened embodiment, the expression of a HPV gene is reduced. In another prefened embodiment, the HPV gene is one ofthe group of E2, E6, or E7.

In a prefened 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 Immunodeficiency Viras (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (ALDS).

In a prefened embodiment, the expression of a HIV gene is reduced. In another prefened embodiment, the HIV gene is CCR5, Gag, or Rev.

In a prefened embodiment the expression of a human gene that is required for HIV replication is reduced, i another prefened embodiment, the gene is CD4 or TsglOl . Attorney's Docket No. : 14174-072 WO 1

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., cinhosis and heptocellular carcinoma.

In a prefened embodiment, the expression of a HBV gene is reduced, hi another prefened embodiment, the targeted HBV gene encodes one ofthe group ofthe tail region ofthe HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region. In another prefened embodiment, a targeted HBV-RNA sequence is comprised ofthe poly(A) tail.

In prefened 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.

hi a prefened 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 Viras (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cinhosis

In a prefened embodiment, the expression of a HCV gene is reduced.

In another prefened 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 ofthe 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 prefened embodiment, the expression of a Hepatitis, D, E, F, G, or H gene is reduced.

In another prefened 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 ofthe invention also provide for treating patients infected by the Respiratory Syncytial Viras (RS V) or at risk for or afflicted with a disorder mediated by RS V, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.

hi a prefened embodiment, the expression of a RSV gene is reduced. In another prefened embodiment, the targeted HBV gene encodes one ofthe group of genes N, L, or P.

In a prefened embodiment the expression of a human gene that is required for RSV replication is reduced.

Methods ofthe invention provide for treating patients infected by the Heφes Simplex Viras (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g, genital heφes and cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients.

In a prefened embodiment, the expression of a HSV gene is reduced. In another prefened embodiment, the targeted HSV gene encodes DNA polymerase or the helicase- primase.

In a prefened 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 heφes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital viras infections and morbidity in immunocompromised patients.

In a prefened embodiment, the expression of a CMV gene is reduced.

In a prefened embodiment the expression of a human gene that is required for CMV replication is reduced.

Methods ofthe invention also provide for a method of treating patients infected by the heφes Epstein Ban Viras (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 prefened embodiment, the expression of a EBV gene is reduced.

In a prefened embodiment the expression of a human gene that is required for EBV replication is reduced.

Methods ofthe invention also provide for treating patients infected by Kaposi's Sarcoma- associated Heφes Viras (KSHV), also called human heφesvirus 8, 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 prefened embodiment, the expression of a KSHV gene is reduced.

In a prefened 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 Viras (JCV) or a disease or disorder associated with this viras, e.g., progressive multifocal leukoencephalopathy (PML).

In a prefened embodiment, the expression of a JCV gene is reduced.

In prefened embodiment the expression of a human gene that is required for JCV replication is reduced.

Methods ofthe invention also provide for freating patients infected by the myxoviras or at risk for or afflicted with a disorder mediated by myxoviras, e.g., influenza.

In a prefened embodiment, the expression of a myxoviras gene is reduced.

In a prefened embodiment the expression of a human gene that is required for myxoviras replication is reduced.

Methods ofthe invention also provide for treating patients infected by the rhinoviras or at risk for of afflicted with a disorder mediated by rhinoviras, e.g., the common cold.

In a prefened embodiment, the expression of a rhinoviras gene is reduced. Attorney's Docket No.: 14174-072W01

hi prefened embodiment the expression of a human gene that is required for rhinoviras replication is reduced.

Methods ofthe 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.

In a prefened embodiment, the expression of a coronavirus gene is reduced.

In prefened embodiment the expression of a human gene that is required for coronavirus replication is reduced.

Methods ofthe invention also provide for treating patients infected by the flaviviras West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.

In a prefened embodiment, the expression of a West Nile Virus gene is reduced. In another prefened embodiment, the West Nile Virus gene is one ofthe group comprising E, NS3, orNS5.

In a prefened embodiment the expression of a human gene that is required for West Nile Viras replication is reduced.

Methods ofthe invention also provide for freating patients infected by the St. Louis

Encephalitis flaviviras, or at risk for or afflicted with a disease or disorder associated with this viras, e.g., viral haemonhagic fever or neurological disease.

In a prefened embodiment, the expression of a St. Louis Encephalitis gene is reduced.

h a prefened embodiment the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced.

Methods ofthe invention also provide for treating patients infected by the Tick-borne encephalitis flaviviras, or at risk for or afflicted with a disorder mediated by Tick-bome encephalitis virus, e.g., viral haemonhagic fever and neurological disease.

In a prefened embodiment, the expression of a Tick-borne encephalitis virus gene is reduced. Attorney's Docket No.: 14174-072W01

In a prefened embodiment the expression of a human gene that is required for Tick- borne encephalitis viras replication is reduced.

Methods ofthe invention also provide for methods of treating patients infected by the Munay Valley encephalitis flaviviras, which commonly results in viral haemonhagic fever and neurological disease.

hi a prefened embodiment, the expression of a Munay Valley encephalitis viras gene is reduced.

In a prefened embodiment the expression of a human gene that is required for Munay Valley encephalitis viras replication is reduced.

The invention also includes methods for treating patients infected by the dengue flaviviras, or a disease or disorder associated with this viras, e.g., dengue haemonhagic fever.

In a prefened embodiment, the expression of a dengue viras gene is reduced.

In a prefened embodiment the expression of a human gene that is required for dengue virus replication is reduced.

Methods ofthe invention also provide for treating patients infected by the Simian Viras

40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., rumorigenesis.

In a prefened embodiment, the expression of a SV40 gene is reduced.

In a prefened embodiment the expression of a human gene that is required for SV40 replication is reduced.

The invention also includes methods for treating patients infected by the Human T Cell

Lymphotropic Viras (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.

In a prefened embodiment, the expression of a HTLV gene is reduced. In another prefened embodiment the HTLV1 gene is the Tax transcriptional activator. Attorney's Docket No.: 14174-072 W01

In a prefened embodiment the expression of a human gene that is required for HTLV replication is reduced.

Methods ofthe invention also provide for freating patients infected by the Moloney- Murine Leukemia Viras (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo- MuLV, e.g., T-cell leukemia.

In a prefened embodiment, the expression of a Mo-MuLV gene is reduced.

hi a prefened embodiment the expression of a human gene that is required for Mo-MuLV replication is reduced.

Methods ofthe invention also provide for treating patients infected by the encephalomyocarditis viras (EMC V) 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 viras is therefore a concern for patients undergoing xenotransplantation.

In a prefened embodiment, the expression of a EMCV gene is reduced.

In a prefened embodiment the expression of a human gene that is required for EMCV replication is reduced.

The invention also includes a method for freating patients infected by the measles viras (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles.

In a prefened embodiment, the expression of a MV gene is reduced.

hi a prefened embodiment the expression of a human gene that is required for MV replication is reduced.

The invention also includes a method for freating 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 prefened embodiment, the expression of a VZV gene is reduced. Attorney's Docket No.: 14174-072W01

In a prefened 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 adenoviras or at risk for or afflicted with a disorder mediated by an adenoviras, e.g. respiratory tract infection.

In a prefened embodiment, the expression of an adenoviras gene is reduced.

In a prefened embodiment the expression of a human gene that is required for adenoviras replication is reduced.

The invention includes a method for treating patients infected by a yellow fever viras (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection.

In a prefened embodiment, the expression of a YFV gene is reduced. In another prefened embodiment, the prefened gene is one of a group that includes the E, NS2A, or NS3 genes.

In a prefened embodiment the expression of a human gene that is required for YFV replication is reduced.

Methods ofthe invention also provide for treating patients infected by the polioviras or at risk for or afflicted with a disorder mediated by polioviras, e.g., polio.

In a prefened embodiment, the expression of a polioviras gene is reduced.

In a prefened embodiment the expression of a human gene that is required for polioviras replication is reduced.

Methods ofthe 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 prefened embodiment, the expression of a poxvirus gene is reduced. Attorney's Docket No. : 14174-072 WO 1

In a prefened 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 freating the subject.

The target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.

Thus, the present invention provides for a method of freating patients infected by a plasmodium that causes malaria.

In a prefened embodiment, the expression of a plasmodium gene is reduced. In another prefened embodiment, the gene is apical membrane antigen 1 (AMAl).

In a prefened 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. Burali ulcers.

In a prefened embodiment, the expression of a Mycobacterium ulcerans gene is reduced.

hi a prefened 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 prefened embodiment, the expression of a Mycobacterium tuberculosis gene is reduced.

In a prefened 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 prefened embodiment, the expression of a Mycobacterium leprae gene is reduced.

In a prefened 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 prefened embodiment, the expression of a Staphylococcus aureus gene is reduced.

hi a prefened embodiment the expression of a human gene that is required for Staphylococcus aureus replication is reduced.

The invention also includes methods for freating 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 prefened embodiment, the expression of a Sfreptococcus pneumoniae gene is reduced.

In a prefened 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 or Scarlet fever. Attorney's Docket No.: 14174-072W01

In a prefened embodiment, the expression of a Sfreptococcus pyogenes gene is reduced.

In a prefened 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 prefened embodiment, the expression of a Chlamydia pneumoniae gene is reduced.

In a prefened embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced.

The invention also includes methods for freating 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 prefened embodiment, the expression of a Mycoplasma pneumoniae gene is reduced.

hi a prefened embodiment the expression of a human gene that is required for 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 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 ofthe invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent ofthe invention. The iRNA agent is selected such that it targets the single allele ofthe 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 polymoφhisms, 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 gene 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 SI 4, eukaryotic initiation factor 5 A, 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 ofthe allele of a gene in the region of LOH and preferably determining the genotype of both alleles ofthe 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 freating the disorder.

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 polymoφhic 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 AAGCTGGCCCTGGACATGGAGAT (SEQ LD NO:6719) is conserved between mouse lamin

Bl, 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.

The iRNA molecules ofthe invention can be incoφorated into pharmaceutical compositions suitable for adminisfration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incoφorated into the compositions.

The phannaceutical compositions ofthe present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral adminisfration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of adminisfration 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 adminisfration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral adminisfration 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, carriers 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 adminisfration 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 adminisfration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be 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 methylcellulose or poly(vinyl 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 O 1

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 prefened 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-sfranded 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 ofthe 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 ofthe 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 common topical 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 ofthe skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical adminisfration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery ofthe 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 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 ofthe skin is called the epidermis. The epidermis is comprised ofthe sfratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum corneum being at the surface ofthe skin and the stratum basale being the deepest portion ofthe epidermis. The epidermis is between 50 μm 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 ofthe major functions ofthe skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier ofthe skin is provided by the sfratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces between cells in the stratum corneum is filled with different lipids ananged 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 larger molecules to cross the skin's permeability barrier, mechanisms other than normal osmosis must be used.

Several factors determine the permeability ofthe skin to administered agents. These factors include the characteristics ofthe freated skin, the characteristics ofthe delivery agent, interactions between both the drag and delivery agent and the drag and skin, the dosage ofthe drag applied, the form of treatment, and the post freatment regimen. To selectively target the epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration ofthe drag 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 absoφtion is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsoφtion. Absoφtion via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penefration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration ofthe skin and the use of controlled release topical patches. The fransdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or 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

absoφtion of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al, Critical Reviews in Therapeutic Drag 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 Drag 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 in 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 ofthe invention 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, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections ofthe skin.

Pulmonary 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 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-sfranded iRNA agent, or sRNA agent, or precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably iRNA, 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 ofthe 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 prefened. One ofthe 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 drags 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 incoφorated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during adminisfration ofthe aerosol 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 less than about 10 μm in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μm and most preferably less than about 5.0 μm. Usually the particle size distribution is between about 0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5 μm.

The term "dry" means that the composition has a moisture content below about 10% by 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 drag in the subject to be treated to give the anticipated 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 carriers may be in a crystalline or amoφhous form or may be a mixture ofthe 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, frehalose, 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 prefened group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being prefened.

Additives, which are minor components ofthe composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility ofthe 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 bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is prefened.

Pulmonary adminisfration 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.

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 ofthe drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drag can be applied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface ofthe oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface ofthe tongue and the floor of the mouth or the buccal mucosa which constitutes the lining ofthe cheek. The sublingual mucosa is relatively permeable thus giving rapid absoφtion and acceptable bioavailability of many drags. 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 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 absoφtion occurs when molecules are un-ionized or neutral in electrical charges. Therefore charged molecules present the biggest challenges to absoφtion through the oral mucosae.

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.

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-sfranded 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-sfranded 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 ofthe vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature ofthe lung, heart, or leg.

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.

hi one embodiment, unit doses or measured doses of a composition that includes iRNA are dispensed by an implanted device. The device can include a sensor that momtors 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-sfranded 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) 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 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 manow 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 barrier 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-sfranded 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-sfranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include condoms, diaphragms, IUD (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.

DOSAGE

In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-sfranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose ofthe iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-sfranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (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, 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 freat 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 prefened dosages are less than 2, 1, or 0.1 mg/kg of body weight.

In a prefened 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 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-sfranded 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-sfranded 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 ofthe initial dose. A maintenance regimen can include freating the subject with a dose or doses ranging from 0.01 μg 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 5, 10, or 30 days. Further, the freatment regimen may last for a period of time which will vary depending upon the nature ofthe particular disease, its severity and the overall condition ofthe patient. In prefened 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 ofthe symptoms ofthe disease state. The dosage ofthe compound may either be increased in the event the patient does not respond significantly to cunent dosage Attorney's Docket No.: 14174-072W01

• levels, or the dose maybe decreased if an alleviation ofthe symptoms ofthe 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 stent (e.g., intravenous, infraperitoneal, 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 non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

In some cases, a patient is freated 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 ofthe disease in conjunction with a drag known to inhibit activity ofthe target gene product. For example, a patient being treated for a cancer ofthe 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 maintenance therapy to prevent the recunence ofthe disease state, wherein the compound ofthe invention is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight (see US 6,107,094).

The concentration ofthe 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 concenfration 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 ofthe 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 freat a subject, including but not limited to the severity ofthe disease or disorder, previous treatments, the general health and/or age ofthe subject, and other diseases present. Moreover, freatment 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-sfranded iRNA agent, or sRNA agent, or precursor thereof) can include a single treatment or, preferably, can include a series of freatments. It will also be appreciated that the effective dosage of a iRNA agent such as a sRNA agent used for freatment 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 ofthe iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness ofthe disease condition to be treated,, with the course of freatment 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 drag accumulation in the body ofthe patient. Persons of ordinary skill can 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 EC50s found to be effective in in vitro 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 conesponding 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 ofthe 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, infraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Adminisfration can be provided by the subject 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. 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-sfranded 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 freatment 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 ofthe colon or removal of polyps, which includes means for delivery ofthe medication.

The rectal administration ofthe iRNA agent is by means of an enema. The iRNA agent ofthe 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 hydropropyhnethylcellulose.

Any ofthe 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 ofthe oral cavity.

Any ofthe 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 ofthe buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inlialation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

Any ofthe iRNA agents described herein can be administered to ocular tissue. For example, the medications can be applied to the surface ofthe eye or nearby tissue, e.g., the inside ofthe eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject 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 ofthe eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or stracture. Ocular treatment is particularly desirable for treating inflammation ofthe eye or nearby tissue.

Any ofthe iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer ofthe skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue. Administration ofthe 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 fransdermal 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. : 14174-072 WO 1

molecular assembly. The transdermal adminisfration can be applied with at least one penefration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.

Any ofthe 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 prefened 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 ofthe iRNA agents described herein can be administered nasally. Nasal adminisfration 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 adminisfration to a surface ofthe nasal cavity. The medication can be provided in a dispenser with delivery ofthe 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.

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 stracture derived therefrom. Attorney's Docket No.: 14174-072 W01

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 ofthe 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-sfranded 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 freated 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, 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-sfranded 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 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-sfranded 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/defined amount of an iRNA agent, e.g., a double-sfranded 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-sfranded 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 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 subject. 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 offranscri.pt levels, a measure of a protein level, a disease or disorder symptom or 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-sfranded 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-sfranded iRNA agent, or sRNA 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 prefened embodiment the method includes administering a second amount Attorney's Docket No.: 14174-072W01

ofthe composition, wherein the timing of administration or dosage ofthe second amount is a function ofthe 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-sfranded 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-sfranded 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) 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 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. hi a prefened embodiment the liposomes are cationic liposomes.

In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-sfranded 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, l-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a Ci-io alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

In another embodiment, the topical penefration 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, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.

In another embodiment, the penefration 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 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 penefration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti- inflammatory agents and mixtures thereof, hi yet another embodiment the penetration enhancer can be a glycol, a pynol, an azone, or a teφenes.

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-sfranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region ofthe gasfro-intestinal tract, e.g., small intestine, colon (e.g., to freat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent ofthe 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 retrovirases. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution ofthe tablets, capsules or gel capsules in a mammalian stomach. In a prefened 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 ofthe pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxychohc acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form ofthe pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form ofthe pharmaceutical composition includes a plasticizer. The plasticizer 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 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- sfranded 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-sfranded 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 prefened 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-sfranded 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-sfranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form ofthe pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a prefened 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-sfranded 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 ofthe tablets, capsules or gel capsules in a mammalian stomach. In a prefened 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 ofthe pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer, described herein.

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-sfranded 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-sfranded 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-sfranded 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-sfranded 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-sfranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incoφorated 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-sfranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject 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.

hi one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a prefened embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another prefened embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and frehalose.

In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a prefened 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 prefened 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, α-lactalbumin, trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) 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-sfranded 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-sfranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components ofthe pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components ofthe pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier 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 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-sfranded 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 ofthe composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, 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 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 tofreat these disorders. 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 ofthe protein and the RNA using the Western blotting and RT-PCR methods, respectively. siRNA molecules that efficiently inhibit apoB expression will be tested for similar effects in vivo. ii) In vivo trials using an apoB transgenic mouse model (apoBlOO 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 ofthe siRNA on gene expression in vivo can be measured by monitoring the HDL/LDL cholesterol level in serum. The Attorney's Docket No. : 14174-072W01

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 ofthe 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 of approximately 95% triglycerides. The chylomicrons fransport 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 lipoprotein receptors.

Apolipoproteins coat lipoprotein particles, and include the A-I, A-II, A-IV, B, CI, CII, CIH, 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 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 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-1, A-2, C-l, 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 acyl-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 ofthe plasma triglyceride) and low density lipoprotein (LDL, which carry most ofthe 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 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 TDL and LDL particles. After the removal of apolipoproteins A, E and C, apoB is incoφoration into VLDL by hepatocytes. ApoB-48 is present in chylomicrons and plays an essential role in the intestinal absoφtion 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 ofthe 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 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 ofthe 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 nonnalipidemic subjects.

Attorney's Docket No. : 14174-072 WO 1

Table 4. Reference Range Lipid level in the Blood

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 models. Future experiments will facilitate the identification of human ortho logous 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 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 Hypercholesterolemia (liigh level ofapo B) A common genetic disorder of lipid 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 (iigh level ofapo 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 coronary artery disease (CAD) (high level ofapo 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. 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).

Model for 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). Cunent therapies, however, have little or no effect on apo(a) levels and the homology between apo(a) and plasminogen presents barriers to drag development. Lρ(a) particles consist of apo(a) and apoB-100 proteins, and they are found only in primates and the hedgehog. The development of LPA fransgenic 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 ofthe 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- 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 Cholesteryl ester transfer protein over expression The apoB-100 fransgenic 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 fransgenic mice express both CETP and apoB- 100, resulting in mice with a human like semm 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.

ApoBlOO Transgenic 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 and within the media, and have significantly more complex atherosclerotic lesions than control animals.

Double Transgenic Mice, CETP/ApoBlOO (Order Model #: 1007 -TT) These mice express both CETP and apoB-100, resulting in a human-like serum HDL/LDL distribution. These mice are 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 LDL 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 ofthe LDL receptors, have familial hypercholesterolemia and 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 intestinally synthesized chylomicrons. It is also a constituent of a subclass of high density lipoproteins (HDLs) involved in cholesterol transport activity among cells. One ofthe 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 fransport preventing the 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 ofthe apoE gene that is defective in binding to the LDL receptor and are at risk for early development 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 Replacement 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 murine regulatory sequences. The E4 isoform occurs in approximately 14% ofthe 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 in vivo study ofthe human apoE isoforms.

CETP Transgenic Mice (Order Model #: 1003-T) These animals express the human plasma 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

Transgene/Promoter: 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 (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 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. Cre/loxP tissue-specific recombination techniques were also used to generate liver-specific Mttp knockout mice. Inactivation ofthe 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. Cunently 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 (apolipoprotein B) Transgene mice show apo B locus may have a causative role male infertility 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% ofthe animals from the second backcross generation produced any offspring within the 4-month test period. Ofthe animals that produced progeny, litters resulted from < 50% of documented matings. In confrast, all wild-type mice (616— 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 markedly reduced in the apoB (+/-) animals compared with that ofthe wild-type controls. Ofthe sperm from apoB (+/-) mice, 20% (i.e., 4.9% ofthe initial 20% motile sperm) remained motile after 6 hr of incubation, whereas 45% (i.e., 33.6% ofthe initial 69.5%) ofthe 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 (4-/-) mice fertilized 84% of eggs once the zona pellucida had been removed. Numerous sperm from apoB (+/-) mice were seen binding to zona-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 zona pellucida 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 ofthe 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

Introduction The regulation of hepatic gluconeogenesis is an important process in the adjustment ofthe blood glucose level. Pathological changes in the glucose production ofthe 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 ofthe two rate-limiting gluconeogenic enzymes, phosphoenolpyravate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase). Insulin is known to repress both PEPCK 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 phosphoenolpyravate from oxaloacetate), G6Pase catalyzes the terminal step of both, gluconeogenesis and glycogenolysis, the cleavage of glucose-6-ρhosphate 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 ofthe metabolic abenations associated with diabetes. Hepatic glucose production can be reduced by an IRNA-based reduction of PEPCK and G6Pase enzymatic activity in subjects with type-2-diabetes.

Targets for iRNA

Glucose-6-phosphatase (G6Pase)

GόPase mRNA is expressed principally in liver and kidney, and in lower amounts in the 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 ofthe 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 increase in G6Pase mRNA. Attorney's Docket No.: 14174-072W01

Phosphoenolpyruvate carboxykinase (PEPCK)

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 (LRS)-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression.

Table 5. Other targets to inhibit hepatic glucose production

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 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 G6Pase, 2 sequences, 5 animals per sequence

1 nonspecific sequence, 5 animals 1 confrol group (only injected, 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. Assays:

G6P enzyme assays and PEPCK enzyme assays are used to measure the activity ofthe enzymes. Northern blotting is used to detect levels of G6Pase and PEPCK mRNA. Antibody-based techniques (e.g., immunoblotting, immimofluorescence) 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; GenBank® No: U00445,Mus musculus glucose-6-phosphatase mRNA, complete eds 1..2259,

ORF 83..1156

GenBank® No: BC013448

PEPCK

GenBank® No: NM_011044, Mus musculus phosphoenolpyravate carboxykinase 1, cytosolic (Pckl), nιRNA.1..2618, ORF 141..2009

GenBank® No: AF009605.1

Administration of iRNA: iRNA conesponding to the genes described above could be administered to mice with hydrodynamic injection. One control group of animals would be treated with Metformin as a positive control for reduction in hepatic glucose levels.

Experimental Protocol

Mice could be housed in a facility in which there is light from 7:00 AM to 7:00 PM. Mice would be fed ad libidum 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 O: 7:00 PM: Approximately 100 μl 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 μl; 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 in vivo iRNA targeted to the Glucose-6-Phosphatase (G6P) gene was used to examine the effects of inhibition of G6P expression on glucose metabolism in vivo.

Female mice, 10 weeks of age, strain BKS.Cg-m +/+ Lepr db (The Jackson Laboratory) were used for in vivo analysis of enzymes ofthe 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 lOOμl 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 3-5 μl) 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 μg) and siRNAs (100 μg 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 injected are shaded.

Table 6. Blood glucose levels in mice

plasmid GL3 GL3 GL3 GL3 GL3 GL3 GL3 GL3 siRNA G6P4 G6P4 G6P4 no G6P4 RL RL no mouse 03 mouse 04 mouse 05 mouse 07 mouse 09 mouse 14 mouse 15 mouse 17 day BG BG BG BG BG BG BG BG

1 512 250 537 241 196 275 538 437

2 555 437 339 556 408 315 524 386

3 483 446 356 567 283 491 600 459

4 579 543 552 423 404 457 548 375

5 600 501 600 277 198 441 533 430

6 464 600 408 454 461 412 490 301

7 '_21 _ A A so A 11 -245Α, " ^"260^* ' 494 " * A 800 429^{" l} taj£pigri_

8 _lr- 600 , 566 246 521 277 600 576 404

"i; 448 438 536 600 459 [iηjectkm

10 369 600 446 average day 1 to 6 532 463 465 420 325 399 539 398

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

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 injected 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.

Example 4: Selected Palindromic Sequences Tables 9-14 below provide selected palindromic sequences from the following genes: human ApoB, human glucose-6-phosphatase, rat glucose-6-phosphatase, β-catenin, and hepatitis C viras (HCV).

EQ ID NO 2026|gaaggctgactctgtggtt 4283 4302SEQ ID NO:2332aacctatgccttaatcttc 13161 13180 EQ ID NO 2027ratggttgacctgctttcc 4295 4314SEQ ID NO: 2333 ggaaagttaaaacaacaca 6957 6976 EQ ID NO 2028 cctgctttcctacaatgtg 4304 4323SEQ ID NO 2334 cacaccttgacattgcagg 11080 11099 EQ ID NO 2029 ctgctttcctacaatgtgc 4305 4324SEQ ID NO: 2335 gcacaccttgacattgcag 11079 11098 EQ ID NO 2030 tcctacaatgtgcaaggat 431 1 4330SEQ ID NO 2336 atccgctggctctgaagga 8569 8588 EQ ID NO 2031 ttatgaccacaagaatacgt 4344 4363SEQ ID NO: 2337 acgtccgtgtgccttcata 9976 9995 EQ ID NO 2032 atgaccacaagaatacgtc 4345 4364SEQ ID NO 2338 gacgtccgtgtgccttcat 9975 9994 EQ ID NO 2033 gaatacgtctacactatca 4355 4374SEQ ID NO: 2339rtgattatctgaattcattc 6479 6498 EQ ID NO 2034ptctagattcgaatatca 4398 4417SEQ ID NO 2340rtgatttacatgatttgaaa 6677 6696 EQ ID NO 2035 gattcgaatatcaaattca 4404 4423SEQ ID NO: 2341 ftgaagtagctgagaaaatc 7094 7113 EQ ID NO 2036 gaaacaacccagtctcaaa 4441 4460SEQ ID NO 2342tttgaaaaattctcttttc 9206 9225 EQ ID NO 2037 cccagtctcaaaaggttta 4448 4467SEQ ID NO 2343fcaaattcattactcctggg 11294 11313 EQ ID NO 2038 ctcaaaaggtttactaata 4454 4473SEQ ID NO 2344 tattcaaaactgagttgag 12223 12242 EQ ID NO 2039ρaaaaggtttactaatat 4455 4474SEQ ID NO: 2345 atattcaaaactgagttga 12222 12241 EQ ID NO 2040aaaaggtttactaatattc 4457 4476SEQ ID NO 2346 gaatttgaaagttcgtttt 9272 9291 EQ ID NO 2041 gaaacagcatttgtttgtc 4535 4554SEQ ID NO: 2347 gacagcatcttcgtgtttc 11206 11225 EQ ID NO 2042 atttgtttgtcaaagaagt 4543 4562SEQ ID NO 2348 acttaaaaaatataaaaat 8014 8033 EQ ID NO 2043 caagattgatgggcagtt 4561 4580SEQ ID NO 2349 aactctcaagtcaagttga 13414 13433 EQ ID NO 2044 cagagtctcttcgttct 4578 4597SEQ ID NO 2350 agaagatggcaaatttgaa 11987 12006 EQ ID NO 2045 cagagtctcttcgttctat 4580 4599SEQ ID NO 2351 atagcatggacttcttctg 8865 8884 EQ ID NO 2046atgctaaaggcacatatgg 4597 4616SEQ ID NO 2352 ccatttgagatcacggcat 9237 9256 EQ ID NO 2047 gcacatatggcctgtcttg 4606 4625SEQ ID NO 2353 caagttggcaagtaagtgc 9364 9383 EQ ID NO 2048 gagtccaacctgaggttta 4659 4678SEQ ID NO 2354ftaaagtgccacttttactc 6182 6201 EQ ID NO 2049 agtccaacctgaggtttaa 4660 4679SEQ ID NO: 2355 ttaacagggaagatagact 9300 9319 EQ ID NO 2050 cctacctccaaggcaccaa 4684 4703SEQ ID NO [2356 ttggcaagtaagtgctagg 9368 9387 EQ ID NO 2051 gaagatggaaccctctccc 4722 4741 SEQ ID NO 2357 gggaagaagaggcagcttc 12283 12302 EQ ID NO 2052røatctgcaaagtggcatc 4754 4773SEQ ID NO 2358 gatgaggaaactcagatca 12255 12274 EQ ID NO 2053 gatctgcaaagtggcatca 4755 4774SEQ ID NO 2359røatgaggaaactcagatc 12254 12273 EQ ID NO 2054gcttccctaaagtatgaga 4785 4804SEQ ID NO: 2360ftctcgtgtctaggaaaagc 5969 5988 EQ ID NO 2055 gtatgagaactacgagctg 4796 4815SEQ ID NO 2361 cagcttaagagacacatac 6912 6931 EQ ID NO 2056 aacaagatggatatga 4860 4879SEQ ID NO 2362teattttccaactaataga 13024 13043 EQ ID NO 2057 ctgctgcgttctgaatatc 4899 4918SEQ ID NO 2363 gatacaagaaaaactgcag 6893 6912 EQ ID NO 2058 attgaggttcttcagcc 4932 4951 SEQ ID NO: 2364 ggctcatatgctgaaatga 5340 5359 EQ ID NO 2059 ctggatcactaaattcc 4955 4974SEQ ID NO [2365|ggaaggacaaggcccagaa| 12541 12560 EQ ID NO 2060ccatggtcttgagttaaat 4973 4992SEQ ID NO 2366 atttttattcctgccatgg 10095 10114 EQ ID NO 2061 ftcttaggcactgacaaaat 4999 5018SEQ ID NO 2367 attttttgcaagttaaaga 14011 14030 EQ ID NO 2062 acaaggcgacactaaggat 5032 5051 SEQ ID NO 2368 atccatgatctacatttgt 6786 6805 EQ ID NO 2063røcaacgaccaacttgaag 5075 5094SEQ ID NO 2369 cttcagggaacacaatgca 5177 5196 EQ ID NO 2064 caacttgaagtgtagtctc 5084 5103SEQ ID NO: 2370 gagatgagagatgccgttg 6231 6250 EQ ID NO 2065 gctggagaatgagctgaat 5108 5127SEQ ID NO 2371 attctcttttcttttcagc 9214 9233 EQ ID NO 2066|gcagagcttggcctctctg 5127 5146SEQ ID NO 2372 cagatacaagaaaaactgc 6891 6910 EQ ID NO 2067 ctggggcatctatgaa 5140 5159SEQ ID NO 2373ttcattcaattgggagaga 6491 6510 EQ ID NO 2068Mggggcatctatgaaat 5142 5161 SEQ ID NO 2374 atttgtaagaaaatacaga 6428 6447 EQ ID NO 2069 aacacaatgcaaaattcag 5185 5204SEQ ID NO 2375 ctgaagcattaaaactgtt 7498 7517 EQ ID NO 2070ptcacagagctatcactgg 5223 5242SEQ ID NO 2376 ccagatgctgaacagtgag 8141 8160 EQ ID NO 2071|tgggaagtgcttatcaggc 5239 5258SEQ ID NO 2377 gcctacgttccatgtccca 1 1348 11367 EQ ID NO 2072pcaaggtcagtcaagaag 5295 5314SEQ ID NO: 2378 cttcagtgcagaatatgaa 11969 11988 EQ ID NO 2073aatgacatgatgggctcat 5328 5347SEQ ID NO 2379 atgattatctgaattcatt 6478 6497 EQ ID NO 2074 gctcatatgctgaaatgaa 5341 5360SEQ ID NO: 2380π:tcagccattgacatgagc 5738 5757 EQ ID NO 2075 atatgctgaaatgaaattt 5345 5364SEQ ID NO 2381 aaatagctattgctaatat 6694 6713 EQ ID NO 2076 ^aacatt9^ca gctta 5378 5397SEQ ID NO 2382raagaaccagaagatcaga 10988 1 1007 EQ ID NO 2077 gaacattgcaggcttatca 5381 5400SEQ ID NO: 2383 tgatatcgacgtgaggttc 12482 12501 EQ ID NO 2078tgcaggcttatcactggac 5387 5406SEQ D NO 2384 gtcctggattccacatgca 11844 11863 1 3 EQ ID NO 2079|tcaaaacttgacaacattt 5412 5431 SEQ D NO 2385 aaattccttgacatgttga 7362 7381 EQ ID NO 2080 atttacagctctgacaagt 5427 5446SEQ D NO 2386 acttaaaaaatataaaaat 8014 8033 EQ ID NO 2081 ctctgacaagttttataag 5435 5454SEQ D NO 2387 cttacttgaattccaagag 10666 10685 EQ ID NO 2082 gttaatttacagctacagc 5460 5479SEQ D NO 2388 gctgcatgtggctggtaac 5570 5589 EQ ID NO 2083|ttctctggtaactacttta 5483 5502SEQ D NO 2389raaaagattactttgagaa 7267 7286 EQ ID NO 2084 cctaaaaggagcctaccaa 5588 5607SEQ D NO 2390Mggcaagtaagtgctagg 9368 9387 EQ ID NO 2085 aaaaggagcctaccaaaat 5591 5610SEQ D NO 2391 atttacaattgttgctttt 6263 6282 EQ ID NO 2086 aggagcctaccaaaataat 5594 5613SEQ D NO 2392attacctatgatttctcct 10119 10138 EQ ID NO 2087 ataatgaaataaaacacat 5608 5627SEQ D NO 2393atgtcaaacactttgttat 7057 7076 EQ ID NO 2088 aaaacacatctatgccatc 5618 5637SEQ D NO 2394gatgaagatgacgactttt 12150 12169 EQ ID NO: 2089røctaaggttcagggtgtg 5678 5697SEQ D NO 2395 cacaagtcgattcccagca 9079 9098 EQ ID NO: 2090 gagtttagccatcggctca 5697 5716SEQ D NO 2396 :gaggtgactcagagactc 7442 7461 EQ ID NO 2091 gctggcttcagccattgac 5732 5751 SEQ D NO 2397 gtcagtgaagttctccagc 8588 8607 EQ ID NO: 2092 atttcagcaatgtcttccg 5782 5801 SEQ D NO 2398 cggagcatgggagtgaaat 8620 8639 EQ ID NO 2093ptcagcaatgtcttccgt 5783 5802SEQ D NO 2399 acggagcatgggagtgaaa 8619 8638 EQ ID NO 2094pcagcaatgtcttccgtt 5784 5803SEQ D NO 2400aacggagcatgggagtgaa 8618 8637 EQ ID NO 2095 cagcaatgtcttccgttct 5786 5805SEQ D NO 2401 agaagtgtcttcaaagctg 12404 12423 EQ ID NO: 2096 gtcttccgttctgtaatg 5792 5811 SEQ D NO 2402 cattcaattgggagagaca 6493 6512 EQ ID NO 2097 gtcttccgttctgtaatgg 5793 5812 SEQ D NO 2403ccattcagtctctcaagac 12967 12986 EQ ID NO 2098atgggaaactcgctctctg 5851 5870SEQ D NO 2404 cagataaaaaactcaccat 12205 12224 EQ ID NO 2099 ggagaacatactgggcagc 5871 5890SEQ D NO 2405gctgttttgaagactctcc 1080 1099 EQ ID NO 21 OOgttgaaagcagaacctctg 5906 5925SEQ D NO 2406cagaattcataatcccaac 8266 8285 EQ ID NO 2101 gtctaggaaaagcatcagt 5975 5994SEQ D NO 2407 actgcaagatttttcagac 13604 13623 EQ ID NO 2102[agcatcagtgcagctcttg 5985 6004SEQ D NO 2408 caagaacctgttagttgct 13343 13362 EQ ID NO 2103Pgaacacaaagtcagtgc 6001 6020SEQ D NO 2409 gcacatcaatattgatcaa 6410 6429 EQ ID NO 2104 gcagacaggcacctggaaa 6038 6057SEQ D NO 241 Oftttcagatggcattgctgc 11602 11621 EQ ID NO 21 Oδgaaactcaagacccaattt 6053 6072SEQ D NO 241 1 aaatcccatccaggttttc 8029 8048 EQ ID NO 21 Oδacaatgaatacagccagga 6076 6095SEQ D NO: 2412ftcctttggctgtgctttgt 9674 9693 EQ ID NO 2107cttggatgcttacaacact 6095 6114SEQ D NO 2413agtgaagttctccagcaag 8591 8610 EQ ID NO 21 Oδ ggcgtggagcttactgg 6124 6143SEQ D NO 2414ccagaattcataatcccaa 8265 8284 EQ ID NO 21 Oθcacttttactcagtgagcc 6190 6209SEQ D NO 241 δggctattgatgttagagtg 6980 6999 EQ ID NO 2110Ptagagatgagagatgcc 6227 6246SEQ D NO 241 θggcatgatgctcatttaaa 9169 9188 EQ ID NO 2111 gagaagccccaagaattta 6249 6268SEQ D NO 2417π:aaagccattcagtctctc 12962 12981 EQ ID NO 2112caattgttgcttttgtaaa 6268 6287SEQ D NO 241 δπtaaccagtcagatattg 10179 10198 EQ ID NO 2113fttttgtaaagtatgataaa 6278 6297SEQ D NO 2419πtattgctgaatccaaaa 13647 13666 EQ ID NO 2114πgtaaagtatgataaaaa 6280 6299SEQ D NO 2420πttgagaggaatcgacaa 6350 6369 EQ ID NO 211 δpcactccattaacctccc 6307 6326SEQ D NO 2421 gggaaaaaacaggcttgaa 9δ68 9687 EQ ID NO 2116pttgagaccttgcaagaa 6329 6348SEQ D NO 2422rttctctctatgggaaaaaa 9558 9577 EQ ID NO 211 accttgcaagaatattttg 6336 6356SEQ D NO 2423 caaaagaagcccaagaggt 12940 12969 EQ ID NO 2118ftcaatattgatcaatttgt 6416 6434SEQ D NO 2424 acaaagcagattatgttga 11821 11840 EQ ID NO 2119cagagcagccctgggaaaa 6443 6462SEQ D NO 242δrttttcagaccaactctctg 13614 13633 EQ ID NO 2120pctgggaaaactcccacag 6452 6471 SEQ D NO 2426 ctgtctctggtcagccagg 7716 7735 EQ ID NO 2121 actcccacagcaagctaat 6461 6480SEQ D NO 2427 attacacttcctttcgagt 12861 12880 EQ ID NO 2122aattcattcaattgggaga 6489 6508SEQ D NO 2428H:ctcttcctccatggaatt 10471 10490 EQ ID NO 2123pcaattgggagagacaag 6495 6514 SEQ D NO 2429cttggagtgccagtttgaa 11800 11819 EQ ID NO 2124 aggagaaactgactgctct 6526 6546SEQ D NO 2430agagcttatgggatttcct 11155 11174 EQ ID NO 2125actgactgctctcacaaaa 6533 6552SEQ D NO: 2431 pttggcaagctatacagt 8372 8391 EQ ID NO 2126 gactgctctcacaaaaaag 6536 6555SEQ D NO 2432ctttgtgagtttatcagtc 9687 9706 EQ ID NO 2127 cagacatatatgatacaat 6633 6652SEQ D NO: 2433 attggatatccaagatctg 1925 1944 EQ ID NO: 2128 aatttgatcagtatattaa 6649 6668SEQ D NO I2434fttaaaagaaatcttcaatt 13807 13826 EQ ID NO: 2129 tgatttacatgatttga 6675 6694SEQ D NO 2435 tcaatgattatatcccata 13120 13139 Q ID NO 2130Ptgaaaatagctattgct 6689 6708SEQ D NO 2436 agcacagaaaaaattcaaa 13856 13875 1 3 Q ID NO 2131 ptgaaaatagctattgcta 6690 6709SEQ D NO 2437 tagcacagaaaaaattcaa 13855 13874 Q ID NO 2132[aatagctattgctaatatt 6695 6714 SEQ D NO: 2438 aataaatggagtctttatt 14076 14095 Q ID NO 2133 attattgatgaaatcattg 6711 6730SEQ D NO: 2439 caataccagaattcataat 8260 8279 Q ID NO 2134aaagtcttgatgagcacta 6739 6758SEQ D NO 2440rtagtgattacacttccttt 12856 12875 Q ID NO 2135aagtcttgatgagcactat 6740 6759SEQ D NO 2441 atagcaacactaaatactt 8761 8780 Q ID NO 2136Pgatgagcactatcatat 6745 6764SEQ D NO 2442 atatccaagatgagatcaa 13093 13112 Q ID NO 2137 taattttagtaaaaacaat 6769 6788SEQ D NO 2443 attgagattccctccatta 11694 1 1713 Q ID NO 2138pttagtaaaaacaatcca 6772 6791 SEQ D NO 2444 tggagtgccagtttgaaaa 11802 11821 Q ID NO 2139acatttgtttattgaaaat 6797 6816SEQ D NO: 2445 atttcctaaagctggatgt 11167 11186 Q ID NO 2140attgattttaacaaaagtg 6816 6835SEQ D NO 2446|cactgttccagttgtcaat 9863 9882 Q ID NO 2141 attttaacaaaagtggaag 6820 6839SEQ D NO 2447[cttcaaagacttaaaaaat 8006 8025 Q ID NO 2142aaatcagaatccagataca 6880 6899SEQ D NO 2448 tgtaccataagccatattt 10080 10099 Q ID NO 2143 gaatccagatacaagaaaa 6886 6905SEQ D NO 2449 ttttctaaacttgaaattc 9057 9076 Q ID NO 2144paagagacacatacagaa 6916 6935SEQ D NO 2450fttcttaaacattcctttaa 9483 9502 Q ID NO: 2145 atccagcacctagctggaa 6942 6961 SEQ D NO 2451 mccaatttccctgtggat 3680 3699 Q ID NO 2146røagcatgtcaaacacttt 7052 7071 SEQ D NO: 2452 aaagtgccacttttactca 6183 6202 Q ID NO 2147gagcatgtcaaacactttg 7053 7072SEQ D NO 2453 caaatgacatgatgggctc 5326 5345 Q ID NO 2148aaacactttgttataaatc 7062 7081 SEQ D NO 2454 gattatatcccatatgttt 13125 13144 Q ID NO 2149raagaaaatcaatgccttc 7103 7122SEQ D NO 2455 gaaggaaaagcgcacctca 12021 12040 Q ID NO 215θ gaagtagaccaacaaa 7152 7171 SEQ D NO 2466 tttgtggagggtagtcata 10323 10342 Q ID NO 2151 aagtagaccaacaaatcca 7156 7175SEQ D NO: |2457 tggatgaagatgacgactt 12148 12167 Q ID NO: 2152aagttgaaggagactattc 7215 7234SEQ D NO: 2458 gaataccaatgctgaactt 10160 10179 Q ID NO 2153acaagttaagataaaagat 7256 7275SEQ D NO 2459 atctaaattcagttcttgt 11326 11345 Q ID NO 2154aagataaaagattactttg 7263 7282SEQ D NO 2460 caaaatagaagggaatctt 2069 2088 Q ID NO 2155gattactttgagaaattag 7272 7291 SEQ D NO 2461 ctaaacttgaaattcaatc 9061 9080 Q ID NO 2156røagaaattagttggattt 7280 7299SEQ D NO: 2462 aaatccgtgaggtgactca 7435 7454 Q ID NO 21 δ aaattagttggatttattg 7284 7303SEQ D NO: 2463 caattttgagaatgaattt 10411 10430 Q ID NO 2158røgatttattgatgatgct 7292 7311 SEQ D NO: 2464 agcatgcctagtttctcca 9945 9964 Q ID NO: 2159p:cattgaagatgttaacaa 7345 7364SEQ D NO 2465πgtagatgaaaccaatga 7414 7433 Q ID NO 21 ΘOcattgaagatgttaacaaa 7346 7365SEQ D NO 2466 ttgtagatgaaaccaatg 7413 7432 Q ID NO 2161 attgaagatgttaacaaat 7347 7366SEQ D NO: 2467 atttaagtatgatttcaat 10487 10506 Q ID NO 2162pgaagatgttaacaaatt 7348 7367SEQ D NO 2468 aatttaagtatgatttcaa 10486 10505 Q ID NO 2163røaagatgttaacaaattc 7349 7368SEQ D O 2469 gaatttaagtatgatttca 10485 10504 Q ID NO 2164acatgttgataaagaaatt 7372 7391 SEQ D NO: 2470 aattccctgaagttgatgt 11479 11498 Q ID NO 2165Ptgattaccaccagtttg 7398 7417SEQ D NO: 2471 caaattgaacatccccaaa 8783 8802 Q ID NO 2166caaaatccgtgaggtgact 7433 7452SEQ D NO: 2472 agtccccctaacagatttg 7964 7983 Q ID NO: 2167aaaatccgtgaggtgactc 7434 7453SEQ D NO 2473gagtgaaatgctgtttttt 8630 8649 Q ID NO: 2168aggtgactcagagactcaa 7444 7463SEQ D NO 2474fttgatgatatctggaacct 10723 10742 Q ID NO 2169gtgaaattcaggctctgga 7465 7484SEQ D NO 2475ftccaatctcctcttttcac 8401 8420 Q ID NO 2170gttgcagtgtatctggaaa 7539 7558SEQ D NO: 2476ptcaagcaaatgcacaac 8532 8551 Q ID NO 2171 raaagttcagcatctttgg 7608 7627SEQ D NO 2477 ccaatgctgaactttttaa 10165 10184 Q ID NO 2172røaaggccaaattccgaga 7633 7652SEQ D NO 2478 cctttcttcatcttca 10205 10224 Q ID NO: 2173aatgtatcaaatggacatt 7676 7695SEQ D NO 2479 aatgaagtccggattcatt 11013 11032 Q ID NO: 2174attcagcaggaacttcaac 7692 7711 SEQ D NO 2480 gttgagaagccccaagaat 6246 6265 Q ID NO 2175acctgtctctggtcagcca 7714 7733SEQ D O: |2481|tggcaagtaagtgctaggt 9369 9388 Q ID NO: 2176cctgtctctggtcagccag 7715 7734SEQ D NO: 2482 ctggacttctctagtcagg 8802 8821 Q ID NO: 2177ggtcagccaggtttatagc 7724 7743SEQ D NO 2483 gctaaaggagcagttgacc 10527 10546 Q ID NO: 2178ccaggtttatagcacactt 7730 7749SEQ D NO [2484|aagtccggattcattctgg 11017 11036 Q ID NO: 2179gtttatagcacacttgtca 7734 7753SEQ D NO 2485raacctgtccattcaaaac 13673 13692 Q ID NO: 2180acttgtcacctacatttct 7745 7764SEQ D NO: 2486 agaaaaaggggattgaagt 10275 10294 Q ID NO 2181 ctgattggtggactcttgc 7762 7781 SEQ D NO 2487 gcaagttaaagaaaatcag 14018 14037 Q ID NO 2182atgaaagcattggtagagc 7839 7858SEQ D NO β4δδ gctcatctcctttctteat 10200 10219 Q ID NO 2183røaaagcattggtagagca 7840 7859SEQ D NO 2489ftgctcatctcctttcttca 10199 10218 Q ID NO 2184 gggttcactgttcctgaaa 7660 7δ79_SEQ D NO 2490tttcaccatagaaggaccc 8951 8970 Q ID NO 21 δδFaagaccatccttgggac 7879 7898 SEQ D NO 2491 gtccccctaacagatttga 7965 7984 Q ID NO 2186 ccttgggaccatgcctgcc 7889 7908SEQ D NO: 2492 ggcaccagggctcggaagg 13970 13989 Q ID NO 2187|ttcaggctcttcagaaagc 7921 7940SEQ D NO: [2493|gcttgaaggaattcttgaa 9580 9599 Q ID NO 2188|ttcagataaacttcaaaga 7996 8015SEQ D NO 2494p:cttcataagttcaatgaa 13175 13194 Q ID NO 21 δθacttcaaagacttaaaaaa 8005 8024SEQ D NO 2495ltttaacaaaagtggaagt 6821 6840 Q ID NO 2190atcccatccaggttttcca 8031 δ050SEQ D NO: 2496røgagaagcaaatctggat 9464 9483 Q ID NO 2191 gaatttaccatccttaaca 8055 8074 SEQ D NO 2497ftgttgaagtgtctccattc 9881 9900 Q ID NO: 2192 cattccttcctttacaatt 8081 8100SEQ D NO 2498 aattccaattttgagaatg 10406 10425 Q ID NO 2193røaccagatgctgaacag 8137 8156SEQ D NO 2499 ctgttgaaagatttatcaa 12924 12943 Q ID NO 2194aatcaccctgccagacttc 8225 6244SEQ D NO 2500 gaagttctcaattttgatt 8514 8533 Q ID NO 2195røaccttcacataccagaa 8312 8331 SEQ D NO 2501 πcttctggaaaagggtca 8876 8695 Q ID NO 2196pccagcttccccacatct 8331 8350SEQ D NO 2502agattctcagatgagggaa 8913 6932 Q ID NO: 21 gηaagctatacagtattctga 8379 8398SEQ D NO 2503 tcagatggcattgctgctt 11604 11623 Q ID NO 2198attctgaaaatccaatctc 8391 δ410SEQ D NO 2504 gagataaccgtgcctgaat 11544 11563 Q ID NO: 2199rtttcacattagatgcaaat 8414 8433SEQ D NO:l 2505attttgaaaaaaacagaaa 9730 9749 Q ID NO 2200 caaatgctgacatagggaa 8428 6447SEQ D NO 2506ttccatcacaaatcctttg 9662 9681 Q ID NO 2201 gagagtccaaattagaagt 6500 8519SEQ D NO 2507 actttacttcccaactctc 13402 13421 Q ID NO: 2202 agagtccaaattagaagtt 8501 8520SEQ D NO 2508 aactttacttcccaactct 13401 13420 Q ID NO 2203Mcaattttgattttcaa 8519 8538SEQ D NO 2509ttgattcccttttttgaga 11529 11548 Q ID NO 2204paattttgattttcaagca 8522 8541 SEQ D NO 251 Orøctgaatccaaaagattg 13652 13671 Q ID NO 2205 aatgcacaactctcaaacc 8541 8560SEQ D NO 2511 ggtttatcaaggggccatt 12452 12471 Q ID NO 2206 agttctccagcaagtacct 8596 8615SEQ D NO 2512aggttccatcgtgcaaact 11380 11399 Q ID NO 2207 agtacctgagaacggagca 8608 8627SEQ D NO 2513røctccaggagaacttact 13772 13791 Q ID NO: 220δFaaacacagtggcaagtt 8670 8689SEQ D NO 2514 aactctcaagtcaagttga 13414 13433 Q ID NO 2209 acaatcagcttaccctgga 6743 8762SEQ D NO 251 δftccattctgaatatattgt 13372 13391 Q ID NO 221 Octggatagcaacactaaat 8757 8776SEQ D NO 251 δattttctgaacttccccag 12694 12713 Q ID NO 2211 ctgacctgcgcaacgagat 8821 8840SEQ D NO 2517atctgatgaggaaactcag 12261 12270 Q ID NO: 2212 agatgagggaacacatgaa 8921 8940SEQ D NO 2δ1 δfttcatgtccctagaaatct 10030 10049 Q ID NO 2213 ^aacttttctaaacttga 9062 9071 SEQ D NO 2519ftcaaggataacgtgtttga 12610 12629 Q ID NO 2214rttctaaacttgaaattcaa 9059 9078SEQ D NO 2520ttgatgatgctgtcaagaa 7300 7319 Q ID NO 221 δgaaattcaatcacaagtcg 9069 9088SEQ D NO 2521 cgacgaagaaaataatttc 13558 13577 Q ID NO 221 θcactgtttggagaagggaa 9133 9152SEQ D NO: 2522πccagaaagcagccagtg 12498 12517 Q ID NO 221 actgtttggagaagggaag 9134 9153SEQ D NO 2523 cttccccaaagagaccagt 2890 2909 Q ID NO: 2218 aattctcttttcttttcag 9213 9232SEQ D NO 2524ctgattactatgaaaaatt 13630 13649 Q ID NO 221 θpcttttcagcccagccat 9222 9241 SEQ D NO 2525atggaaaagggaaagagaa 13486 13505 Q ID NO 2220Ptgaaagttcgttttcca 9275 9294SEQ D NO 2526H:ggaagtgtcagtggcaaa 10372 10391 Q ID NO 2221 cagggaagatagacttcct 9304 9323SEQ D NO: 2527 aggacctttcaaattcctg 9840 9859 Q ID NO: 2222 ataagtacaaccaaaattt 9397 9416SEQ D NO: 2528 aaatcaggatctgagttat 14030 14049 Q ID NO: 2223acaacgagaacattatgga 9427 9446SEQ D NO 2529pcattctgaatatattgt 13372 13391 Q ID NO: 2224 aggaataaatggagaagca 9455 9474SEQ D NO: 2530 ctggaattgtcattcct 11726 11745 Q ID NO 2225 agcaaatctggatttctta 9470 9489SEQ D NO: 2531 ftaagttctctgtacctgct 1 171 1 11730 Q ID NO 2226 ^{ctttaacaattcct}9^aa 9494 9513SEQ D NO: 2532fttcaaaacgagcttcagga 13198 13217 Q ID NO 2227ptaacaattcctgaaatg 9497 9516 SEQ D NO: 2533 catttgatttaagtgtaaa 9613 9632 Q ID NO: 2228 acacaataatcacaactcc 9526 9545SEQ D NO 2534 ggagacagcatcttcgtgt 11203 11222 Q ID NO: 2229 aagatttctctctatggga 9553 9572SEQ D NO 2535tcccagaaaacctcttctt 3928 3947 Q ID NO:[ 2230 gaaaaaacaggcttgaagg 9570 9589SEQ D NO 2536ccttttacaattcattttc 13013 13032 Q ID NO 2231 ttgaaggaattcttgaaaa 9582 9601 SEQ D NO: 2537fttttgagaatgaatttcaa 10414 10433 Q ID NO: 2232røaaggaattcttgaaaac 9583 9602SEQ D NO 2538 gttttggctgataaattca 11283 11302 Q ID NO 2233 agctcagtataagaaaaac 9632 9651 SEQ D NO 2539 gtttgataagtacaaagct 9797 9816 Q ID NO 2234[tcaaatcctttgacaggca 9712 9731 SEQ ID NO 2540 tgcctgagcagaccattga 1 1680 11699 Q ID NO 2235 atgaaacaaaaattaagtt 9781 9800SEQ ID NO 2541 aactttgcactatgttcat 12754 12773 Q ID NO 2236 aattcctggatacactgtt 9851 9870SEQ ID NO 2542 aacacatgaatcacaaatt 8930 8949 Q ID NO 2237 ccagttgtcaatgttga 9868 9887SEQ ID NO 2543fcaaaacgagcttcaggaa 13199 13218 Q ID NO 2238 aagtgtctccattcaccat 9886 9905SEQ ID NO 2544atgggaagtataagaactt 4834 4853 Q ID NO 2239 gtcagcatgcctagtttct 9942 9961 SEQ ID NO 2545 agaaaaggcacaccttgac 11072 11091 Q ID NO 2240 ctgccatgggcaatattac 0105 10124SEQ ID NO 2546 gtaagaaaatacagagcag 6432 6451 Q ID NO 2241 røaataccaatgctgaact 0159 10178SEQ ID NO 2547agttgaaggagactattca 7216 7235 Q ID NO 2242 tattgttgctcatctcctt 0193 10212SEQ ID NO 2548 aaggaaacataaactaata 12881 12900 Q ID NO 2243 tgttgctcatctcctttct 0196 10215SEQ ID NO 2549 agaagaaatctgcagaaca 12423 12442 Q ID NO 2244|tctg^tcattgatgcactgc 0224 10243SEQ ID NO 2550gcagtagactataagcaga 13920 13939 Q ID NO 2245 ccacagctctgtctctgag 0297 10316SEQ ID NO 2551 ctcagggatctgaaggtgg 8187 8206 Q ID NO 2246 atttgtggagggtagtcat 0322 10341 SEQ ID NO 2552 atgaagtagaccaacaaat 7153 7172 Q ID NO 2247 atatggaagtgtcagtggc 0369 10388SEQ ID NO 2553 gccacactccaacgcatat 10770 10789 Q ID NO 2248 tggaaataccaagtcaaaa 0445 10464SEQ ID NO 2554fttttacaattcattttcca 13015 13034 Q ID NO 2249 aagtcaaaacctactgtct 0455 10474SEQ ID NO 2555agacctagtgattacactt 12851 12870 Q ID NO 2250 actgtctcttcctccatgg 0467 10486SEQ ID NO 2556 ccatgcaagtcagcccagt 10916 10935 Q ID NO 2251 cttcctccatggaatttaa 0474 10493SEQ ID NO 2557rctaatcgagaggtatgaag 7140 7159 Q ID NO 2252 attcttcaatgctgtactc 0504 10523SEQ ID NO 2558 gagttgagggtccgggaat 12234 12253 Q ID NO 2253|ttgaccacaagcttagctt 0540 10559SEQ ID NO 2559 aagcgcacctcaatatcaa 12028 12047 Q ID NO 2254 cctcacctcttacttttcc 0565 10584SEQ ID NO 2560ggaactattgctagtgagg 10641 10660 Q ID NO 2255 agctgcagggcacttccaa 0702 10721 SEQ ID NO 2561 fttgggaagaagaggcagct 12281 12300 Q ID NO 2256pccaaaattgatgatatc 0715 10734SEQ ID NO 2562 gatatacactagggaggaa 12737 12756 Q ID NO 2257 gagaacatacaagcaaagc 0852 10871 SEQ ID NO 2563 gcttggttttgccagtctc 2459 2478 Q ID NO 2258 atggcaaatgtcagctctt 0889 10908SEQ ID NO 2564 aagaggtatttaaagccat 12952 12971 Q ID NO 2259røgcaaatgtcagctcttg 0890 10909SEQ ID NO 2565 caagaggtatttaaagcca 12951 12970 Q ID NO 2260 ttgttcaggtccatgcaag 0906 10925SEQ ID NO 2566 cttgggggaggaggaacaa 14058 14077 Q ID NO 2261 røttcaggtccatgcaagt 0907 10926SEQ ID NO 2567 acttgggggaggaggaaca 14057 14076 Q ID NO 2262 agttccttccatgatttcc 0932 10951 SEQ ID NO 2568 ggaatctgatgaggaaact 12248 12267 Q ID NO 2263røctaacactaagaaccag 0979 10998SEQ ID NO:l 2569 ctggatgtaaccaccagca 11178 11197 Q ID NO 2264actaagaaccagaagatca 0986 11005SEQ ID NO 2570raatcaagaacctgttagt 13339 13358 Q ID NO 2265 ctaagaaccagaagatcag 0987 11006SEQ ID O 2571 ctgatcaagaacctgttag 13338 13357 Q ID NO 2266 cagaagatcagatggaaaa 0995 11014SEQ ID NO 2572fttttcagaccaactctctg 13614 13633 Q ID NO 2267 aaaaatgaagtccggattc 1010 11029SEQ ID NO 2573gaatttgaaagttcgtttt 9272 9291 Q ID NO 2268 gattcattctgggtctttc 1024 11043SEQ ID NO 2574 gaaaacctatgccttaatc 13158 13177 Q ID NO 2269 aagaaaaggcacaccttga 1071 11090SEQ ID NO: 2575Faaaacctactgtctctt 10458 10477 Q ID NO 2270 aaggacacctaaggttcct 1107 11126SEQ ID NO 2576 aggacaccaaaataacctt 7564 7583 Q ID NO 2271 ccagcattggtaggagaca 1191 11210SEQ ID NO 2577 tgtcaacaagtaccactgg 12362 12381 Q ID NO 2272 ctttgtgtacaccaaaaac 1231 11250SEQ ID NO 2578gtttttaaattgttgaaag 13140 13159 Q ID NO 2273 ccatccctgtaaaagtttt 1269 11288SEQ ID NO 2579 aaaagggtcatggaaatgg 8885 8904 Q ID NO 2274røatctaaattcagttctt 1324 11343SEQ ID NO 2580 aagatagtcagtctgatca 13326 13345 Q ID NO 2275 aagaagctgagaacttcat 1424 11443SEQ ID NO 2581 atgagatcaacacaatctt 13102 13121 Q ID NO 2276Ptgccctcaacctaccaa 1445 11464SEQ ID NO 2582mggtacgagttactcaaa 12633 12652 Q ID NO 2277 cttgattcccttttttgag 1528 I11547SEQ ID NO: 2583 ctcaattttgattttcaag 8520 8539 Q ID NO 2278Pcacgcttccaaaaagtg 1583 11602SEQ ID NO: 2584 cactcattgattttctgaa 12685 12704 Q ID NO 2279[tgtttcagatggcattgct 1600 11619SEQ ID NO 2585 agcagattatgttgaaaca 11825 11844 Q ID NO 2280 aatgcagtagccaacaaga 1631 11650SEQ ID NO 2586pttttcagcccagccatt 9223 9242 Q ID NO 2281 ctgagcagaccattgagat 1683 11702SEQ ID NO: 2587 atctgatgaggaaactcag 12251 12270 Q ID NO 2282|tgagcagaccattgagatt 1684 11703SEQ ID NO: 2588 aatctgatgaggaaactca 12250 12269 Q ID NO 2283|ttgagattccctccattaa 1695 11714SEQ ID NO: 2589κaatcttcataagttcaa 13171 13190 Q ID NO 2284 acttggagtgccagtttga 1799 11818SEQ ID NO: 2590ftcaattgggagagacaagt 6496 6515 Q ID NO 2285 caaatttgaaggacttcag 1996 12015SEQ ID NO 2591 ctgagaacttcatcatttg 1 1430 11449 Q ID NO 2286 agcccagcgttcaccgatc 1204812067SEQ ID O 2592 gatccaagtatagttggct 13278 13297 1 3 Q ID NO 2287 cagcgttcaccgatctcca 12052 12071 SEQ ID NO 2593 gacctgcaccaaagctg 13952 13971 3 Q ID NO 2288 ctccatctgcgctaccaga 12066 12085SEQ ID NO 2594tctgatatacatcacggag 13703 13722 3 Q ID NO 2289 atgaggaaactcagatcaa 12256 12275SEQ ID NO 2595 ttgagttgcccaccatcat 11659 11678 Q ID NO 2290 aggcagcttctggcttgct 12292 1231 1 SEQ ID NO 2596 agcaagtctttcctggcct 3010 3029 Q ID NO 2291 røaaagacaacgtgcccaa 12319 12338SEQ ID NO |2597[ttgggagagacaagtttca 6500 6519 Q ID NO 2292 gattatgtcaacaa gt 12354 12373 SEQ ID NO 2598 actttgcactatgttcata 12755 12774 Q ID NO 2293 cattaggcaaattgatgat 12467 12486SEQ ID NO 2599 atcaacacaatcttcaatg 13107 13126 Q ID NO 2294Pgactcaggaaggccaag 12576 12595SEQ ID NO 2600 cttggtacgagttactcaa 12632 12651 Q ID NO 2295 gaaacctgggatatacact 12728 12747SEQ ID NO 2601 agtgattacacttcctttc 12857 12876 Q ID NO 2296F^ctltc9^a9^ttaa99^aaa 12869 12888SEQ ID NO 2602ftttctgccactgctcagga 13516 13535 Q ID NO 2297 gccattcagtctctcaaga 12966 12985SEQ ID NO 2603pttccgttctgtaatggc 5794 5813 Q ID NO 2298 gtgctacgtaatcttcagg 12993 13012SEQ ID NO 2604 cctgcaccaaagctggcac 13956 13975 Q ID NO 2299 agctgaaagagatgaaatt 13057 13076SEQ ID NO 2605 aatttattcaaaacgagct 13192 1321 Q ID NO 2300 aatttacttatcttattaa 13072 13091 SEQ ID NO 2606rcaaaagaaatcttcaatt 13807 13826 Q ID NO 2301 fttttaaattgttgaaagaa 13142 13161 SEQ ID NO 2607πctctctatgggaaaaaa 9558 9577 Q ID NO 2302 atcttcataagttcaat 13172 13191 SEQ ID NO 2608 attgagattccctccatta 11694 11713 Q ID NO 2303 atattttgatccaagtata 13271 13290SEQ ID NO 2609 taagcagaagcacatat 13929 13948 Q ID NO 2304toaaatattatgaacttga 13303 13322SEQ ID NO 261 O aaccttaatgattttca 8287 8306 Q ID NO 2305 caatttctgcacagaaata 113434 13453SEQ ID O 2611ftattcttcttttccaattg 13826 13845 Q ID NO 2306agaagattgcagagctttc 13501 13520SEQ ID NO 2612gaaatcttcaatttattct 13813 13832 Q ID NO 2307gaagaaaataatttctgat 13562 13581 SEQ ID NO 2613 atcagttcagataaacttc 7991 8010 Q ID NO 2308Pgacctgtccattcaaaa 13672 13691 SEQ ID NO 2614 ttttgagaatgaatttcaa 10414 10433 Q ID NO 2309 ^{aaaactaccacacattt} 13685 13704SEQ ID NO 261 δaaattccttgacatgttga 7362 7381 Q ID NO 231 OFtttaaaagaaatcttc 13803 13822SEQ ID NO 2616gaagtgtcagtggcaaaaa 10374 10393 Q ID NO 2311 aggatctgagttattttgc 1403514054SEQ ID NO 2617 gcaagggttcactgttcct 7856 7875 Q ID NO 2312Ptgctaaacttgggggag 14049 14068 SEQ ID NO 261 δctccccaggacctttcaaa 9834 9853

# = Match Number

B = Middle Matching Bases

Table 10. Selected palindromic sequences from human ApoB

Q ID NO: 2665^aag^{tta t}g^aaag^aag^ttc 1955 1974 SEQ D NO 3994 gaacctctggcatttactt 5924 59431 Q ID NO: 2666 aaagaagttctgaaagaat 1964 1983 SEQ D NO 3995 attctctggtaactacttt 5490 5509 Q ID NO: 2667F99^cta*^accaaa9^atg 2330 2349 SEQ D NO: 3996 catcttaggcactgacaaa 5005 50241 Q ID NO: 2668F9^tt9^a9^aa9^ct9^attaaa 2389 2408 SEQ D NO 3997 ttagccatcggctcaaca 5708 572711 Q ID NO: 2669 caggaagggctcaaagaat 2569 2588 SEQ D NO 3998 attcctttaacaattcctg 9500 95191 Q ID NO: 2670 aggaagggctcaaagaatg 2570 2589 SEQ D NO 3999 cattcctttaacaattcct 9499 95181 Q ID NO: 2671 gaagggctcaaagaatgac 2572 2591 SEQ D NO W000 gtcagtcttcaggctcttc 7922 7941 1 Q ID NO: 2672 caaagaatgacttttttct 2580 2599 SEQ D NO: 001 agaaggatggcattttttg 14008 14027 Q ID NO: 2673 catggagaatgcctttgaa 2611 2630 SEQ D NO r4002Ftcagagccaaagtccatg 7127 7146 1 Q ID NO: 26749g^a9^ccaagg^ctgg^{a taa} 2687 2706 SEQ D NO W003 tactccaacgccagctcc 3058 30771 Q ID NO: b675 cattccttccccaaagag 2892 2911 SEQ D NO J4004 ctctctggggcatctatga 5147 5166 Q ID NO: 2676 acctatgagctccagagag 3173 3192_SEQ D NO: 14005 ctctcaagaccacagaggt 12984 13003 Q ID NO: 2677 gggcaaaacgtcttacaga 3373 3392 SEQ D NO: W006FCtgaaagacaacgtgccc 12325 12344 1 Q ID NO: 2678 accctggacattcagaaca 3395 3414 SEQ D O: W007ftgttgctaaggttcagggt 5683 5702 Q ID NO: 2679 atgggcgacctaagttgtg 3437 3456 SEQ D NO: WOOδ cacaaattagtttcaccat 8949 89681 Q ID NO: 2680 gatgaagagaagattgaat 3626 3645_SEQ D NO W009 attccagcttccccacatc 8338 83571 Q ID NO: 2681 caatgtagataccaaaaaa 3664 3683 SEQ D NO: W010 ftttttggaaatgccattg 8651 8670 1 Q ID NO: P-682gt^a9^{ataccaaaaaaat}9^a 3668 3687 SEQ D NO: W01 i catgtgatgggtctctac 4379 43981 Q ID NO: 2683 gcttcagttcatttggact 4517 4536 SEQ D NO: r4012agtcaagaaggacttaagc 5312 5331 1 Q ID NO: 2684F9 ttgtcaaagaagtc 4552 4571 SEQ D O W013 gacttcagagaaatacaaa 11408 11427 1 Q ID NO: 2685F^t9^ttt9^tcaaa9^aa9^tca 4553 4572 SEQ D NO WOMftgacttcagagaaatacaa 11407 11426 Q ID NO 2686F9g^caatggg^aaactc9^ct 5854 5873 SEQ D O W015 agcgagaatcaccctgcca 8227 8246 Q ID NO 2687 aacctctggcatttacttt 5925 5944 SEQ D NO W016 aaaggagatgtcaagggtt 10607 10626 1 Q ID NO: 2688 catttactttctctcatga 5934 5953 SEQ D NO: W01 Trtcatttgaaagaataaatg 7034 7053 Q ID NO: 2689 aaagtcagtgccctgctta 6017 6036SEQ D NO W-018Faagaaccttactgacttt 7792 7811 Q ID NO: 2690F^cccatttt 9^a9^acctt 6330 6349 SEQ D NO W019 aaggacttcaggaatggga 12012 12031 Q ID NO 2691 catcaatattgatcaattt 6421 6440_SEQ D NO l020aaattaaaaagtcttgatg 6740 6759 Q ID NO: 2692 ^aaa9^ata9^ttat9^attta 6673 6692_SEQ D NO W021 Faaaccaaaacttggttta 9027 90461 Q ID NO: 2693F^att9^at9^aaatcattgaa 6721 6740 SEQ D NO ]4022Ftcaaagacttaaaaaata 8015 8034 Q ID NO: 2694 atgatctacatttgtttat 6798 6817SEQ D NO: W023 ataaagaaattaaagtcat 7388 7407 Q ID NO: 2695 agagacacatacagaatat 6927 6946 SEQ D NO: ]4024 atatattgtcagtgcctct 13390 13409 1 Q ID NO: 26969^acacat^aca9^aax^ax^a9^a 6930 6949 SEQ D NO W025H:ctaaattcagttcttgtc 11335 11354 1 Q ID NO: beg agcatgtcaaacactttgt 7062 7081 SEQ D NO W026 acaaagtcagtgccctgct 6015 6034 Q ID NO: 2698 ftttttagaggaaaccaagg 7523 7542 SEQ D NO W027 cctttgtgtacaccaaaaa 11238 11257 1 Q ID NO: b699Ftttagaggaaaccaaggc 7524 7543SEQ D NO: W028 gcctttgtgtacaccaaaa 11237 11256 1 Q ID NO: 2700 ggaagatagacttcctgaa 9315 9334 SEQ D NO r4029 tcagaaatactgttttcc 12832 12851 :1 Q ID NO: 2701 cactgtttctgagtcccag 9342 9361 SEQ D NO μ4030 ctgggacctaccaagagtg 12531 12550 Q ID NO: 2702 cacaaatcctttggctgtg 9676 9695 [SEQ D NO: 031 cacatttcaaggaattgtg 10071 10090 1 Q ID NO: 2703F^tc;ctggatacactgttcc 9861 9880 SEQ D O W032ggaactgttgactcaggaa 12577 12596 i1 Q ID NO: 2704 gaaatctcaagctttctct 10050 10069 SEQ D NO 033 agagccaggtcgagctttc 11052 11071 Q ID NO 2705FCttcatcttcatctgt 10218 10237 SEQ D NO: I4034 acagctgaaagagatgaaa 13063 13082 Q ID NO: b706Fctaccgctaaaggagcag 10529 10548 SEQ D O r4035 ctgcacgctttgaggtaga 11769 11788 !1 Q ID NO: 2707ptaccgctaaaggagcagt 10530 10549 SEQ D NO W036 actgcacgctttgaggtag 11768 11787 Q ID NO: 2708 agggcctctttttcaccaa 10839 10858 SEQ D NO: W037mggccaggaagtggccct 10965 10984 Q ID NO: b7ogpctccatccctgtaaaag 11273 11292 SEQ D O: W038 ctttttcaccaacggagaa 10846 10865 Q ID NO: 2710Θ^aaaaacaaa9^cag^attat 11824 11843 SEQ D NO 4039 ataaactgcaagatttttc 13608 13627r Q ID NO: 2711 actcactcattgattttct 12690 12709 SEQ DNO: W040agaaaatcaggatctgagt 14035 140541 Q ID NO: 2712 taaactaatagatgtaatc 12898 12917 SEQ D NO W041 gattaccaccagcagttta 13586 136051 Q ID NO: 2713 caaaacgagcttcaggaag 13208 13227 SEQ D O: W042 cttcgtgaagaatattttg 13268 13287 Q ID NO: 2714 tggaataatgctcagtgtt 2374 2393 SEQ D NO W043 aacacttacttgaattcca 10670 10689 - Q ID NO: 2715 gatttgaaatccaaagaag 2408 2427SEQ D NO 14044 cttcagagaaatacaaatc 11410 114293 Q ID NO: 2716 atttgaaatccaaagaagt 2409 2428 SEQ D NO: 14045 acttcagagaaatacaaat 1 1409 114283 Q ID NO: 2717 atcaacagccgcttctttg 998 1017 SEQ D NO W046 caaagaagtcaagattgat 4561 45802 Q ID NO: 2718 tgttttgaagactctccag 1090 09 SEQ D NO J4047 ctggaaagttaaaacaaca 6963 69822 Q ID NO: 271 g cccttctgatagatgtggt 1332 1351 SEQ D NO: 14048 accaaagctggcaccaggg 13969 139882 Q ID NO: 2720 tgagcaagtgaagaacttt 1876 1895 SEQ D NO: W049 aaagccattcagtctctca 12971 129902 Q ID NO: 2721 atttgaaatccaaagaagt 2409 2428 SEQ D NO W050 acttttctaaacttgaaat 9063 90822 Q ID NO: 2722 atccaaagaagtcccggaa 2416 2435 SEQ D NO ]405lFtccggggaaacctgggat 12729 12748 Q ID NO: 2723 agagcctacctccgcatct 2438 2457SEQ D NO W052 agatggtacgttagcctct 11929 119482 Q ID NO: 2724 aatgcctttgaactcccca 2618 2637_SEQ D NO M-053Fgggaactacaatttcatt 7020 70392 Q ID NO: 2725 gaagtccaaattccggatt 3305 3324 SEQ D NO 054aatcttcaatttattcttc 13823 138422 23 Q ID NO: 2₇26 tgcaagcagaagccagaag 3504 35 SEQ D NO W055 cttcaggttccatcgtgca 11384 11403 _Q ID NO: 2727 gaagagaagattgaatttg 3629 3648 SEQ D NO 14056 caaaacctactgtctcttc 10467 104862 Q ID NO: 2728 atgctaaaggcacatatgg 4605 4624 SEQ D NO W057fccatatgaaagtcaagcat 12664 12683. Q ID NO: 2729 tccctcacctccacctctg 4745 4764 SEQ D NO W058 cagattctcagatgaggga 8920 89392 Q ID NO: 730 atttacagctctgacaagt 5435 5454_SEQ D NO 059 acttttctaaacttgaaat 9063 90822 Q ID NO: 2731 aggagcctaccaaaataat 5602 5621 SEQ D NO 14060 attatgttgaaacagtcct 11838 1 18572 Q ID NO: 2732 aaagctgaagcacatcaat 6409 6428_SEQ D NO W061 attgttgctcatctccttt 10202 102212 Q ID NO: 2733 ctgctggaaacaacgagaa 9426 9445 SEQ D NO W062rctctgattaccaccagcag 13582 13601 Q ID NO: 2734 tg^aagg^aattcttg^; aaaa 9590 9609SEQ D NO; μ063 ptttaaaagaaatcttcaa 13813 138322 Q ID NO: 2735 gaagtaaaagaaaattttg 10751 10770 SEQ D NO 14064 caaaacctactgtctcttc 10467 104862 Q ID NO: 2736 tgaagaagatggcaaattt 11992 12011 SEQ D O |4065 aaatgtcagctcttgttca 10902 10921 : _Q ID O. 2737 aggatctgagttattttgc 14043 14062SEQ D NO: 14066 gcaagtcagcccagttcct 10928 109472 Q ID NO: 2738 gtgcccttctcggttgctg 26 45 SEQ D NO: |4067pagccattgacatgagcac 5748 5767 Q ID NO: 2739 ggcgctgcctgcgctgctg 154 1 3 SEQ D NO 14068 cagctccacagactccgcc 3070 3089 ' Q ID NO: 2740 ctgcgctgctgctgctgct 162 181 SEQ D NO W069 agcagaaggtgcgaagcag 3232 3251 Q ID NO: 2741 gctgctggcgggcgccagg 178 197 SEQ D NO: W070 cctggattccacatgcagc 11854 11873 Q ID NO: 2742 aagaggaaatgctggaaaa 201 220 [SEQ D NO K071 tttttcttcactacatctt 259 2611 Q ID NO: 2743 ctggaaaatgtcagcctgg 212 231 SEQ D NO W072 ccagacttccacatcccag 3923 39421 Q ID NO: 2744 tggagtccctgggactgct 304 323_SEQ D NO: W073 agcatgcctagtttctcca 9953 99721 Q ID NO: |2745 ggagtccctgggactgctg 305 324_SEQ D NO W074 cagcatgcctagtttctcc 9952 9971 ' Q ID NO: b_746 tgggactgctgattcaaga 313 32SEQ D NO r4075Fcttccatcacttgaccca 2050 2069 Q ID NO: 2747 ctgctgattcaagaagtgc 318 337SEQ D NO W076gcacaccttgacattgcag 11087 11106 _Q ID NO: [2748 tgccaccaggatcaactgc 334 353_SEQ D NO W07 gcaggctgaactggtggca 2725 2744 Q ID NO: [2749 gccaccaggatcaactgca 335 354SEQ D NO _r4078ftgcaggctgaactggtggc 2724 27431 Q ID NO: 2750 tgcaaggttgagctggagg 350 369SEQ D NO W079 cctccacctctgatctgca 4752 4771 Q ID NO: 2751 caaggttgagctggaggtt 352 371 SEQ D NO W080 aacccctacatgaagcttg 13763 13782 _Q ID Nθ 2752 ctctgcagcttcatcctga 377 396SEQ D O: W081 paggaagcttctcaagag 13219 13238 Q ID NO: 2753 cagcttcatcctgaagacc 382 401 SEQ D NO:r⁰⁸²99^tctt9^a9^ttaaat9^ct9 4985 5004 Q ID NO: 2754 gcttcatcctgaagaccag 384 403 SEQ D NO: °⁸3pggacgctaagaggaagc 863 8821 Q ID NO: 2755tcatcctgaagaccagcca 387 406 SEQ D NO W084Fggcatggcattatgatga 3612 3631 - Q ID NO: 2756 gaaaaccaagaactctgag 460 479 SEQ D NO: W085 ctcaaccttaatgattttc 8294 83131 Q ID NO: 2757 agaactctgaggagtttgc 468 487_SEQ D NO:r⁰⁸⁶9^caa9^ctataca9^tattct 8385 8404 Q ID NO: 2758 tctgaggagtttgctgcag 473 492SEQ D NO: ⁰⁸ 1^ctg^cagg g^ateccccaga 2534 25531 Q ID NO: |2806|^ctcattg^ag^aac^aggcagt 29841 30031 SEQ D O r 35|actgactgcacgctttgag 11764 117831 Q ID NO: |2807|^ttg^ag^cag^tatt^ct cag 3150| 3169| SEQ D NO: 36|ctgagagaagtgtcttcaa 12407 12426 Q ID NO: |2808| jaccttgtccagtgaagtcc 3293| 33121 SEQ D NO 37|ggacggtactgtcccaggt 12792 12811 Q ID NO: |2809|^cca9^tg^aagt^ccaaattec 3300I 33191 SEQ D O:| 38|ggaaggcagagtttactgg 9156 9175 1 Q ID NO: [281 ojacattcagaacaagaaaat 3402| 3421 SEQ D NO 39]atttcctaaagctggatgt 11175 11194 Q ID N0: |2811 |gaaaaatcaagggtgttat 3471 3490| SEQ D O 40|ataaactgcaagatttttc 13608 13627 Q ID NO: [2812|^aaatcaa9Sgtgttatttc 3474| 3493I SEQ D NO:| 41 jgaaacaatgcattagattt 9753 9772 1 Q ID NO: |2813|tggcattatgatgaagaga 36171 3636| SEQ D NO 42|tctcccgtgtataatgcca 11789 11808 Q ID NO: |2814|^aa9^a9^aagattgaatttga 3630| 3649|_SEQ D O:| 43|tcaaaacctactgtctctt 10466 10485 1 Q ID NO: [2815|aaatgacttccaatttccc 3681 3700| SEQ D NO:| 44|gggaactacaatttcattt 7021 7040 1 Q ID NO: [2816|^atg^acttccaatttccctg 3683| 702|SEQ D O 45|caggctgattacgagtcat 4925 49441 Q ID NO: J2817|acttccaatttccctgtgg 3686| 3 05|SEQ D NO:| 46|ccacgaaaaatatggaagt 10368 10387 11 Q ID NO: |2818l agttgcaatgagctcatgg 3811 3830| SEQ D NO:| 47|ccatcagttcagataaact 7997 8016 Q ID NO: |281 g[tttgcaagaccacctcaat 3868 3887|sEQ D NO 48|attgacctgtccattcaaa 13679 13698 Q ID NO: |282θ|9^aagg^a9^ttcaacctcca9 3892| 3911 SEQ D NO:| 49|ctggaattgtcattccttc 11736 11755 1 Q ID NO: 12821 [acttccacatcccagaaaa 3927| 3946I_SEQ D O:| 50 [ttttaacaaaagtggaagt 6829 6848

|2822|^ct<Λtcttaaaaagcgatg Q ID NO: 3947| 3966] SEQ D NO: 51 [catcactgccaaaggagag 8494 8513 Q ID NO: |2823|^aaaa9^{c at} g^ccgg ^tca 3956| 3975| SEQ D NO:| 52|tgactcactcattgatttt 12688 12707 Q ID NO: |2824F^tcctttg^ccttttggtgg 4011 4030|_SEQ D NO:| 53|ccacaaacaatgaagggaa 9264 9283 Q ID NO: |2825l^caa9^tct9^tgg ^attGcat 4087| 4106| SEQ D NO 54|atgggaaaaaacaggcttg 9574 95931 Q ID NO: |2826|^aa9^{tccc ac}tttt^accat 41251 41441 SEQ D O:| 55|atgggaagtataagaactt 4842 4861 - Q ID NO: 2827t^tgcctctcctg99^t9^ttct 41671 41861 SEQ D NO 56|agaaaaacaaacacaggca| 9651 96701 Q ID NO: |2828|^accaS^caca9^accatttca 42501 4269| SEQ D O: 57|tgaagtgtagtctcctggt 5097 51161 Q ID NO: |2829|^cca9^caca9^accattt^ca9 4251 4270I SEQ D O 58|ctgaaatacaatgctctgg 5519 55381 Q ID NO: |283o|^actatcat9^tg^atgggtet 4375| 4394|SEQ D NO 59|agacacctgattttatagt 7956 79751 Q ID NO: £831 accacagatgtctgcttca 4504| 4523|SEQ D NO: 60|tgaaggctgactctgtggt 4290 4309 1 Q ID NO: |2832l^ccacag^atgtetgcttcag 4505] 4524|sEQ D O:| 61 |ctgagcaacaaatttgtgg 10319 10338 1 Q ID NO: |2833|tttggactccaaaaagaaa 4528| 4547|SEQ D NO 62|tttctctcatgattacaaa 5941 5960 |1 Q ID NO: P_834|tc^aaagaagtcaagattga 4560| 4579|SEQ D NO 63|tcaaggataacgtgtttga 12618 12637 1 Q ID NO: |2835|^atg^ag^aactacg^agctgac 4806I 4825| SEQ D NO 64|gtcagatattgttgctcat 10195 10214 1 Q ID NO: |2836F^taaaa*^c*9^acaccaatg 4826| 4845|sEQ D NO:|4 65|cattcattgaagatgttaa 7350 7369 Q ID NO: |2837|gaagtataagaactttgcc 4846 4865| SEQ D NO: 66|ggcaaatttgaaggacttc 12002 12021 1 Q ID NO: |2838|^aag^tataag^aacttt9^cca 4847| 4866| SEQ D NO:| 67[tggcaaatttgaaggactt 12001 12020 Q ID NO: |2839F^cttcagcctgctttctg 4949! 4968|SEQ D NO:| 68|cagaatccagatacaagaa 6892 6911 Q ID NO: |2840| [ctggatcactaaattccca 4965| 4984|SEQ D NO 69|tgggtctttccagagccag 11041 11060 Q ID NO: 12841 aaattaatagtggtgctca 5022| 5041 SEQ D O: 70|tgagaagccccaagaattt 6256 6275 Q ID NO: |2842|^agtgcaacgaccaacttga 5081 5100| [SEQ D NO:|4 71|tcaaattcctggatacact 9856 98751 Q ID NO: |2843| |ctgggaagtgcttatcagg 52461 5265|_SEQ D NO:| 72|cctgaccttcacataccag 8318 8337 Q ID NO: |2844|9^caaaaacatttt^caactt: 5286 5305|_SEQ D NO:| 73|aagtaaaagaaaattttgc 10752 10771 Q ID NO: |2845| aaaaacattttcaacttca 5288! 5307|SEQ D NO: 74|tgaagtaaaagaaaatttt 10750 10769 1 Q ID NO: |2846p^a9^tcaa9^aa99^acttaa 5310| 5329|sEQ D NO:! 75|ttaaggacttccattctga 13371 13390 1 Q ID NO: |2847F^caaat9^{acat at}gg ct 5333 5352|SEQ D NO: 76|agcccatcaatatcattga 6213 6232 1 Q ID NO: |2848|^cacacaaaca9^tctg^aaca 5375| 5394| SEQ D NO:!⁴ 77|tgtttcaactgcctttgtg 11227 11246 11 Q ID NO: |284g]t^cttcaaaacttgacaaca 5417| 5436|SEQ D NO:!⁴ 78|tgttttcctatttccaaga 12843 12862 1 Q ID NO: |285θ|^caa9^ttttataa9^caaact 5449| 5468| SEQ D NO:| 79|agttattttgctaaacttg 14051 14070 |1 Q ID NO: |2851 |tggtaactactttaaacag 5496! 55151 SEQ D NO: 80|ctgtttttagaggaaacca 7520 7539 1 Q ID NO: |2852|^aacag^tg^acctg^{aaa a}ca 55101 5529|SEQ D NO:|4 81 Fgtatagcaaattcctgtt 5898 59171 Q ID NO: 2853pgg^aaactacggctagaac 5552 5571 SEQ D NO W182gttccttccatgatttccc 10941 109601 QIDNO: 2854 aacacatctatgccatctc 5628 5647 SEQ D NO W183gagacagcatcttcgtgtt 11212 11231 Q ID NO: 2855F^ca9^caa9^ctataaa9^ca9 5660 5679 SEQ D O: W184ctgctaagaaccttactga 7788 7807 Q ID NO: 2856g^ca9^acactgttgctaagg 5675 5694 SEQ D NO W185cctttcaagcactgactgc 11754 11773 Q ID NO: 2857Ftggggagaacatactgg 5874 5893 SEQ D NO: m 86 ccaggttttccacaccaga 8046 8065 Q ID NO: 2858F^ctctcat9^attacaaa9 5942 5961 SEQ D NO W187ptttttcaccaacggagaa 10846 10865 Q ID NO: 859^ctg^{a ca}g^ac^aggcacctg 6042 6061 [SEQ D NO: W188 caggaggctttaagttcag 7607 7626 Q ID NO: 2860 caatttaacaacaatgaat 6074 6093 SEQ D NO W189attccttcctttacaattg 8090 8109 Q ID NO: 2861 ragacgaactctggctgac 6148 6167 SEQ D NO W190 gtcagcccagttccttcca 10932 10951 Q ID NO: 2862 cttttactcagtgagccca 6200 6219 SEQ D NO: W19lFgggctaaacgtatgaaag 7835 7854 Q ID NO: 2863F^catt9^at9^cttta9^a9^at 6225 6244 SEQ D NO: 14192atcttcataagttcaatga 13182 13201 Q ID NO: 2864 ^aaaaccaagatgttcactc 6303 6322 SEQ D NO: W193gagtgaaatgctgtttttt 8638 8657 Q ID NO: 2865 aggaatcgacaaaccatta 6365 6384 SEQ D NO: 14194ftaatgattttcaagttcct 8302 8321 Q ID NO: 866F^ag^tt9^tactgg^aaaac9^t 6384 6403 SEQ D NO: 14195 acgttagcctctaagacta 11936 11955 Q ID NO: 286 gg^aaaacg^tacag^ag^aaa9 6394 6413 SEQ D NO W196 cttttacaattcattttcc 13022 13041 Q ID NO: 2868gaaaacgtacagagaaagc 6395 6414 SEQ D NO: m θηgctttctcttccacatttc 10060 10079 Q ID NO: 2869 aaagctgaagcacatcaat 6409 6428 SEQ D NO: W198attgatgttagagtgcttt 6992 7011 Q ID NO: 2870 aagctgaagcacatcaata 6410 6429 SEQ D NO: 99Kattgatgttagagtgctt 6991 70101 Q ID NO: b₈71 9 ^agc^ac^atcaatattga 6414 6433 SEQ D NO W200Kcaaccttaatgattttca 8295 83141 Q ID NO: 2872 atcaatattgatcaatttg 6422 6441 SEQ D NO W201 caaagccatcactgatgat 1668 1687 1 Q ID NO: b873F^aat9^att^atctgaattca 6484 6503SEQ D NO W202ftgaaatcattgaaaaatta 6727 67461 Q ID NO: 2874 gattatctgaattcattca 6488 6507SEQ D NO W203Fgaagtagctgagaaaatc 7102 7121 - Q ID NO: 2875 aattgggagagacaagttt 6506 6525 SEQ D NO 204 aaacattcctttaacaatt 9496 95151 Q ID NO: 2876 aaaatagctattgctaata 6701 6720 SEQ D NO W205ftattgaaaatattgatttt 6814 68331 Q ID NO: b877iaaaattaaaaagtcttgat 6739 6758 SEQ D NO W206 atcatatccgtgtaatttt 6765 6784 1 Q ID NO: 2878F^tg^aaaat^attgattttaa 6816 6835 SEQ D NO r4207Ftaatcttcataagttcaa 13179 13198 1 Q ID NO: 2879 agacatccagcacctagct 6946 6965 SEQ D NO W208 agcttggttttgccagtct 2466 2485 Q ID NO: 2880 caatttcatttgaaagaat 7029 7048 SEQ D NO W209 attccttcctttacaattg 8090 8109 Q ID NO: 2881 aggttttaatggataaatt 7182 7201 SEQ D NO W210 aattgttgaaagaaaacct 13155 13174 Q ID NO: 2882^ca9^aag^ctaa9^caat9^tcc 7241 7260 SEQ D NO W211 ggacaaggcccagaatctg 12553 12572 Q ID NO: 2883 ^aa9^ataaaa9^attacttt 7270 7289 SEQ D NO wλ 12 aaagaaaacctatgcctta 13163 13182 Q ID NO: 2884 aaagattactttgagaaat 7277 7296 SEQ D NO W213atttcttaaacattccttt 9489 9508 Q ID NO: 2885g^ag^aaatta9^tt9 ^attta 7289 7308 SEQ D NO 214rtaaagccattcagtctctc 12970 12989 Q ID NO: 2886 atttattgatgatgctgtc 7303 7322 SEQ D NO W215 gacatgttgataaagaaat 7379 7398 Q ID NO: 2887P^aattatcttttaaaacat 7334 7353 SEQ D NO W216 atgtatcaaatggacattc 7685 7704 Q ID NO: 2888F^taccaccag^ttt9^ta9^at 7411 7430 SEQ D NO W21 tctggaaccttgaagtaa 10739 10758 Q ID NO: 2889F^t9^ca9^t9^tatct99^aaa9 7548 7567SEQ D O W218 cttttcacattagatgcaa 8420 8439 1 Q ID NO: |289θl^cattca9^cagg^aactl:caa 7699 7718 SEQ D O: W21 gptgaaggacttcaggaatg 12009 12028 1 Q ID NO: 2891 acacctgattttatagtcc 7958 97 SEQ D NO W220 ggactcaaggataacgtgt 12614 12633 1 Q ID NO: b892Θg^attccatcag^ttcag^at 7992 8011 SEQ D NO W221 atcttcaatg attatatcc 13124 13143 1 Q ID NO: 893Ftgtagaaatgaaagtaaa 8112 8131 SEQ D NO: W222rttt^atgattatgtcaacaa 12360 12379 1 Q ID NO: 2894Pg^aacag^tg^a9^ct9^ca9^t 8156 8175 SEQ D NO W223 actggacttctctagtcag 8809 8828 Q ID NO: 2895 aatccaatctcctcttttc 8407 8426 SEQ D NO W224baaaaatgaagtccggatt 11017 11036 1 Q ID NO: 2896 attttgattttcaagcaaa 8532 8551 SEQ D NO: W225Fttgcaagttaaagaaaat 14023 14042 1 Q ID NO: 28971 Ftttgattttcaagcaaat 8533 8552 ISEQ D NO: W226 atttgatttaagtgtaaaa 9622 9641 1 Q ID NO: 2898 g^atttt^caa9^caaatg^ca 8536 8555 SEQ D NO W227Fgcaagttaaagaaaatca 14025 14044 Q ID NO: 2899^atgctgttttttggaaatg 8645 8664 SEQ D NO: W228 cattggtaggagacagcat 11203 11222 1 Q ID NO: 29ooF^{gctgttttttggaaatgc} 8646 8665fc_EQ D NO: W229 gcattggtaggagacagca 11202 11221 1 Q ID NO: 2901 aaaaaaatacactggagct 8706 8725 SEQ D NO: W230 agctagagggcctcttttt 10833 10852 Q ID NO: 2902 actggagcttagtaatgga 8716 8735 ISEQ D O: W231 Fccactcacatcctccagt 1289 1308 Q ID NO: 2903 cttctggaaaagggtcatg 8886 8905 SEQ D NO W232 catgaacccctacatgaag 13759 13778 Q ID NO: 290499^{aaaa tcat}gg^aaat 8891 8910 [SEQ D NO W233 atttgaaagttcgttttcc 9282 9301 Q ID NO: 290599gcctgccccagattctc 8910 8929 SEQ D NO: tø234gagaacattatggaggccc 9440 9459 Q ID NO: 2906F^tcag^atg^aggg^aacac 8924 8943_SEQ D NO 14235 gtgtcttcaaagctgagaa 12416 12435 1 Q ID NO: 2907fø^at9^ag g^aacacatg^aat 8930 8949 [SEQ D NO W236 attccagcttccccacatc 8338 83571 Q ID NO: 2908 ctttggactgtccaataag 8986 9005SEQ D NO W237pttatgggatttcctaaag 11167 11186 1 Q ID NO: bgog røtccacaaacaatgaag 9260 9279SEQ D NO: W238 cttcatctgtcattgatgc 10227 10246 !1 Q ID NO: 291 o ^acaaacaatg^aag g^aat 9265 9284 SEQ D NO: 14239 attccctgaagttgatgtg 11488 11507 1 Q ID NO: 2911 ccaaaatttctctgctgga 9415 9434SEQ D NO W24θFccatcacaaatcctttgg 9671 96go 1 Q ID NO: _9i caaaatttctctgctggaa 9416 9435 SEQ D NO: 14241 Ftccatcacaaatcctttg 9670 9689 1 Q ID NO: 2913F^tg^ctgg^aaacaacg^ag^a 9425 9444_SEQ D NO: j4242 ctcaagagttacagcaga 13229 13248 Q ID NO: 291 ^ctgctggaaacaacgagaa 9426 9445 SEQ D NO ]4243Ftctcaagagttacagcag 13228 13247 Q ID NO: 2915 agaacattatggaggccca 9441 9460 SEQ D NO j4244Fgggcctgccccagattct 8909 8928 1 Q ID NO: 2916 agaagcaaatctggatttc 9475 9494_SEQ D NO: W245 gaaatcttcaatttattct 13821 13840 Q ID NO: 29 7ftttctctctatgggaaaaa 9565 9584_SEQ D NO: W246 ftttttgcaagttaaagaaa 14021 14040 Q ID NO: 2918F^ag^agcatcaaatccttt 9712 9731 SEQ D NO W247taaagaaaatcaggatctga 14033 14052 1 Q ID NO: 291 g cagaaacaatgcattagat 9751 9770_SEQ D NO: W248 atctatgccatctcttctg 5633 5652 Q ID NO: 2920F^acacatt^aatc;ctgccat 10001 0020 SEQ D NO: W249 atggagtctttattgtgta 1408 14108 Q ID NO: 2921 agtcagatattgttgctca 10194 0213 SEQ D O l4250Fgagaactacgagctgact 4807 4826 1 Q ID NO: 2922 gg^agggtagtcataacagt 10336 0355SEQ D NO W251 actggtggcaaaaccctcc 2734 2753 1 Q ID NO: 2923paaaagccgaaattccaat 10404 0423SEQ D NO W252 attgaagtacctacttttg 8366 83851 Q ID NO: 2924 ^aaaagccgaaattccaatt 10405 0 24SEQ D O W253 aattgaagtacctactttt 8365 8384 1 Q ID NO: 2925Ftcaagcaagaacttaatg 10436 0455SEQ D O W254 cattatggcccttcgtgaa 13258 13277 1 Q ID NO: 2926^cct^ctt^acttttccattga 10578 0597_SEQ D NO: W255Fcaaaagaagcccaagagg 12947 12966 Q ID NO: 2927F9^a99^{ccaacacttactt}g 10663 0682_SEQ D NO: W256 caagcatctgattgactca 12676 126951 Q ID NO: 2928^cactt^acttgaattccaag 10672 0691 SEQ D NO W257pttgaacacaaagtcagtg 6008 6027 1 Q ID NO: 29299^aa9^taaaa9^aaaatttt9 10751 0770SEQ D NO K258 caaaaacattttcaacttc 5287 5306 1 Q ID NO: 2930 cctggaactctctccatgg 10882 0901 SEQ D NO: W259 ccatttacagatcttcagg 11372 11,391 QID NO:2931 agctggatgtaaccaccag 11184 1203 SEQ D NO:r²⁶⁰F^{ggattccacatgca}9^ct 11855 11874 Q ID NO: 2932 aaaattccctgaagttgat 11485 1504 SEQ D NO W261 atcatatccgtgtaatttt 6765 6784 Q ID NO: 2933^ca9^atgg^cattg^ctg^cttt 11613 1632 SEQ D NO: _r4262aaagctgagaagaaatctg 12424 12443 Q ID NO: _934 agatggcattgctgctttg 11614 1633 _SEQ D NO: kt263caaagctgagaagaaatct 12423 12442 Q ID NO: 2935 9^ttS^aaacagtcct9^sat 11842 1861 SEQ D NO:r²64^atccaagatgagatcaaca 13103 13122 1 Q ID NO: b_936 catattcaaaactgagttg 12229 2248_SEQ D NO: W265 caactctctgattactatg 13631 13650 Q ID NO: 2937paagatttatcaaaagaag 12938 2957SEQ D NO: . 266cttcaatttattcttcttt 13826 13845 Q ID NO: 2938 attttccaactaatagaag 13034 3053 SEQ D NO:r²67pttcaaagacttaaaaaat 8014 8033 1 Q ID NO: 2939 aattatatccaagatgaga 13097 3116 SEQ D NO:p268Fctcttcctccatggaatt 10479 10498 !1 Q ID NO: 2940Ftcaggaagcttctcaaga 13218 3237SEQ D NO: W269 Cttcataagttcaatgaa 13183 13202 Q ID NO: 2941 toagcaatttctg cacag 13437 3 56SEQ D NO:r²70 tgttgaaagatttatcaa 12932 12951 1 Q ID NO: 2942 ctgatatacatcacggagt 13712 3731 SEQ D NO: W271 actcaatggtgaaattcag 7465 7484 Q ID NO:2943 acatcacggagttactgaa 13719 3738 SEQ D NO: ²72Ftcagaagctaagcaatgt 7239 72581 Q ID NO: 2944 actgcctatattgataaaa 13882 3901 SEQ D O: W273 jttttggcaagctatacagt 8380 83991 Q ID NO: 2945pgg^atg ^{catt ttt c:aa} 14011 4030 SEQ D NO:r²74Ftgcaagcaagtctttcct 3013 3032 1 Q ID NO: 2946 Ftttttgcaagttaaagaa 14020 4039 SEQ D NO:r²⁷⁵F^{ctctctatgggaaaaaa} 9566 95851

Q ID NO: 2993Fctgcagcttcatcctgaa 378 397SEQ D NO W322Ktcaaatataatcggcaga 3269 32881 Q ID NO: bggφccagtgcaccctgaaaga 402 421 SEQ D O: W323pttccgttctgtaatggc 5802 5821 11 Q ID NO: 2995ptctgaggagtttgctgca 472 491 SEQ D O W324H:gcaagaatattttgagag 6348 6367 1 Q ID NO: 2996 aggtatgagctcaagctgg 500 519 SEQ D NO: W325 ccagtttccggggaaacct 12724 12743 1 Q ID NO: bggTFcctttacccggagaaaga 543 562_SEQ D NO W326 ctttttgggaagcaagga 2227 2246 Q ID NO: b_998 catcaagaggggcatcatt 583 602SEQ D NO: W32 atggtcaagttcctgatg 2285 2304 Q ID NO: bgggFctggttcccccagagac 609 628_SEQ D NO W328 gtctctgaactcagaagga 13996 14015 Q ID NO: 3000 aagaagccaagcaagtgtt 630 649 SEQ D NO W329 aacaaataaatggagtctt 14080 14099 Q ID NO: 3001 aagcaagtgttgtttctgg 638 657SEQ D NO W-330 ccagagccaggtcgagctt 11050 11069 Q ID NO: 3002F^{tggatacGgtgtatgga} 652 671 SEQ D NO: 331 ccatgtcccatttacaga 11364 11383 Q ID NO: 3003 ccactcactttaccgtcaa 678 697 SEQ D NO W332pgattttaacaaaagtgg 6825 6844 :1 Q ID NO: 3oo4^agg^aag g^caat gg^caa 701 720 SEQ D NO l4333Ftgcaagcaagtctttcct 3013 30321 Q ID NO: !3oo5g^caatg^tgg^caaca9^aaat 708 727 SEQ D NO: 14334 atttccataccccgtttgc 3488 3507 Q ID NO [3006^caatg^tg9^caaca9^aaata 709 728SEQ D NO: H335Fttcttcttttccaattg 13834 13853 Q ID NO: 3007F99^caaca9^aaat^atccac 714 733 SEQ D NO: 14336 gtggcttcccatattgcca 1895 1914 Q ID NO: 3008 agεigεicctgggccagtgtg 737 756 SEQ D NO W337teacattacatttggtctct 2938 29571 Q ID NO: 3009F^{gtgatcgcttGaa}9^CCGa 752 771 SEQ D NO l338taggaaagccgccctcaca 5218 5237" Q ID NO: 3010 gtgatcgcttcaagcccat 753 772_SEQ D NO: 14339 atgggaaagccgccctcac 5217 5236 - Q ID NO: 3011 cagcccacttgctctcatc 784 803SEQ D NO: 14340 gatgctgaacagtgagctg 8152 8171 Q ID NO: 30 agctctcatcaaaggcatga 794 813 SEQ D NO: ]434l cataacagtactgtgagc 10345 10364 Q ID NO: 3013 ccttgtcaactctgatcag 819 838 SEQ D O W342 ctgagtgggtttatcaagg 12453 12472 Q ID NO: 3014pttgtcaactctgatcagc 820 839 SEQ D O W343 gctgagtgggtttatcaag 12452 12471 Q ID NO: 301 gccatctgcaaggagcaa 892 911 SEQ D O: ]4344pgcaatgagctcatggct 3813 3832 Q ID NO: 3016 gccatctgcaaggagcaac 893 912 SEQ D NO W345 gttgcaatgagctcatggc 3812 3831 - Q ID NO: 3017pttcctgcctttctcctac 916 935 SEQ D NO w-346 gtaggaataaatggagaag 9461 9480 Q ID NO: 30 δptttctcctacaagaataa 924 943 SEQ D NO W347Ftattgctgaatccaaaag 13656 13675 1 Q ID NO: 301 ggatcaacagccgcttcttt 997 016 SEQ D NO w-348 aaagccatcactgatgatc 1669 1688 1 Q ID NO: 3020 atcaacagccgcttctttg 998 017 SEQ D NO W349 caaagccatcactgatgat 1668 16871 Q ID NO: 3021 acagccgcttctttggtga 1002 021 SEQ D NO W35θFcacaaatcctttggctgt 9675 96941 Q ID NO: 3022 aagatgggcctcgcatttg 1031 050 [SEQ D NO 351 caaaatagaagggaatctt 2077 20961 Q ID NO: 3023Fgttttgaagactctccag 1090 109 SEQ D NO W352 ctggtaactactttaaaca 5495 5514 Q ID NO: 3024pg^aag^actctccaggaac 1094 113SEQ D NO W353 gttcaatgaatttattcaa 13192 13211 Q ID NO: 3025^aaGtg^aaaaaactaaccat 1110 129 SEQ D O 4354 atggcattttttgcaagtt 14014 14033 Q ID NO: 3026 ptgaaaaaactaaccatct 1112 131 SEQ D O 4355agattgatgggcagttcag 4572 4591 Q ID NO: 3027 ^aaaGtaaccatctctgag 1117 136 SEQ D NO 4356 ctcaaagaatgactttttt 2578 25971 Q ID NO: 3028F^{gagcaaaatatcca aga} 1132 151 SEQ D NO 4357ftctccagataaaaaactca 12209 12228 Q ID NO 3029paataagctggttactgag 1162 181 SEQ D NO 4358 ctcagatcaaagttaattg 12273 12292 Q ID NO: 3030P^Gt9^agGtga9^a9^gcctG 1174 193 SEQ D NO 4359 gagggtagtcataacagta 10337 10356 Q ID NO: 3031 gcctcagtgatgaagcagt 1188 207SEQ D NO W360 actgttgactcaggaaggc 12580 12599 Q ID NO: 3032 agtcacatctctcttgcca 1204 223 SEQ D NO: 36lFggccacatagcatggact 8866 8885 Q ID NO: 3033 atctctcttgccacagctg 1210 229 SEQ D NO:r³ 2^Ga9^Gtg^{acc Ga}tcg^ag^at 2169 21881 Q ID NO: 3034Ftctcttgccacagctga 1211 230 SEQ D NO:r³63F^Gagctgacctcatcgaga 2168 2187 Q ID NO: 3035Fs^ccacag^{ct attga}g ^t 1218 237 SEQ D NO: 364acctgcaccaaagctggca 13963 13982 Q ID NO: 3036g^GGacag^Gt9^attg^a99^tg 1219 238 SEQ D NO:r³65ρ^accaaaaaccccaatggc 11248 11267 Q ID NO 3037Fcactttacaagccttggt 1248 267 SEQ D NO: 366^accagatgctgaacagtga 8148 8167 1 Q ID NO: 3038 cccttctgatagatgtggt 1332 351 SEQ D NO: ³67^accacttacagctagaggg 10824 10843 Q ID NO: 303ggtcacctacctggtggccc 1349 368 SEQ D NO;W368gggcgacctaagttgtgac 3439 34581 Q ID NO: 3040 cttgtatgcgctgagcca 1440 1459 SEQ D NO W369Fggctggtaacctaaaagg 5586 56051 Q ID NO: 3041 gacaaaccctacagggacc 1480 1499 [SEQ D NO 4370ggtcctttatgattatgtc 12355 12374 Q ID NO: 3042F^gGtaattaCGt9^atg9^aa 1516 1535 SEQ D NO 4371 pcccaaaagcagtcagca 9938 9957 Q ID NO: 3043F9^actgcact 9^g9^atg^aa 1546 1565 SEQ D NO: W372pcaggtccatgcaagtca 10917 10936 Q ID NO: 3044 actgcactggggatgaaga 1548 1567 SEQ D NO W373 cttgaacacaaagtcagt 6007 6026 Q ID NO: 3045 atgaagattacacctattt 1560 1579 SEQ D NO: 14374 aaatgaaagtaaagatcat 8118 81371 Q ID NO: 3046 accatggagcagttaactc 1610 1629 SEQ D NO: W375 gagtaaaccaaaacttggt 9024 9043 1 Q ID NO: 304τ cagttaactccagaactc 1618 1637 SEQ D NO: 14376 gagttactgaaaaagctgc 13727 13746 1 Q ID NO: 3048 cagaactcaagtcttcaat 1629 1648 SEQ D NO ]437ηattggatatccaagatctg 1933 1952 1 Q ID NO: 3049 caggctctgcggaaaatgg 1703 1722 SEQ D NO: 14378 ccatgacctccagctcctg 2485 2504 1 Q ID NO: 3050 ccaggaggttcttcttcag 1738 1757SEQ D NO 4379 ctgaaatacaatgctctgg 5519 55381 Q ID NO: 3051 ggttcttcttcagactttc 1744 1763SEQ D O w-380 gaaaaacttggaaacaacc 4439 4458 Q ID NO: 3052F^Gcttgatgatgcttct 1759 1778 SEQ D NO: W381 agaatccagatacaagaaa 6893 69121 Q ID NO: 30539g^ag^ataa9^G9^aGtgg^Gtg 1781 1800 SEQ D NO: 14382 cagcatgcctagtttctcc 9952 9971 1 Q ID NO: [30549^Gt9^{GGtatcttatgtt}9^a 1796 1S15SEQ D NO W383Fcaatatcaaaagcccagc 12045 12064 1 Q ID NO: 3055 actttgtggcttcccatat 1890 1909 SEQ D NO 14384 atatctggaaccttgaagt 10737 10756 1 Q ID NO: 3056 gccaatatcttgaactcag 1910 1929 SEQ D NO 14385 ctgaactcagaaggatggc 14000 14019 Q ID NO: 3057patatcttgaactcagaag 1913 1932 SEQ D NO W386 cttccattctgaatatatt 13378 13397 Q ID NO: 3058 tcagaagaattggatatc 1924 1943 SEQ D NO W387taataaaagattactttgag 7273 7292 Q ID NO: 3059 aagaattggatatccaaga 1929 1948 SEQ D NO 388rtcttcaatttattcttctt 13825 13844 Q ID NO: 3060 agaattggatatccaagat 1930 1949 [SEQ D NO 389atcttcaatttattcttct 13824 13843 Q ID NO: 3061F9^{gatatccaagatctgaa} 1935 1954 SEQ D NO W39θFtcacataccagaattcca 8325 8344 Q ID NO: 3062 atatccaagatctgaaaaa 1938 1957SEQ D NO 4391 ittttaaccagtcagatat 10185 10204 1 Q ID NO: 3063ptccaagatctgaaaaag 1939 1958 SEQ D NO 4392 ctttttaaccagtcagata 10184 10203 1 Q ID NO: 3064 caagatctgaaaaagttag 1943 1962 SEQ D NO 4393 ctaaattcccatggtcttg 4973 4992 11 Q ID NO: 3065^aag^atctg^aaaaag^ttag^t 1944 1963 SEQ D NO 4394 actaaattcccatggtctt 4972 4991 11 Q ID NO: 3066 g^aaaaag^ttag^tg^{aaa a} 1950 1969 SEQ D NO W395ptttctcgggaatattca 10630 10649 Q ID NO: 3067F^GaaGtgtcatggacttc 1990 2009 SEQ D NO W396baagcacatatgaactgga 13945 13964 1 Q ID NO: 3068F^ca9^aaaattGtGtGg9^aa 2007 2026 SEQ D O: W397μcctttaacaattcctga 9501 9520 1 Q ID NO: 3069pccatcacttgacccagc 2052 2071 SEQ DNO: 14398gctgacatagggaatggaa 8441 8460" Q ID NO: 3070cccagcctcagccaaaata 2065 2084 SEQ DNO t399Fattctatccaagattggg 7820 7839 " Q ID NO: 3071 agcctcagccaaaatagaa 2068 087SEQ D NO I4400pctatccaagattgggct 7822 7841 Q ID NO: 3072 atcttatatttgatccaaa 2091 2110 SEQ D NO: I4401mtgaaaaacaaagcagat 11821 11840 Q ID NO: 3073Fcttatatttgatccaaat 2092 2111 SEQ D NO: 4402 attttttgcaagttaaaga 14019 14038 Q ID NO: 3074pttcctaaagaaagcatgc 2117 2136 SEQ D NO: 14403 gcatggcattatgatgaag 3614 3633 Q ID NO: 3075 ctaaagaaagcatgctgaa 2121 2140 SEQ D NO 4404pcagggtgtggagtttag 5694 57131 Q ID NO: 3076F^aaagaaagcatgctgaaa 2122 2141 SEQ D NO 4405ftttcttaaacattccttta 9490 9509 1 Q ID NO: 3077 agattggcttggaaggaa 2183 2202 SEQ D NO ]4406Ftccctccattaagttctc 11709 11728 1 Q ID NO: 3078 ctttgagccaacattggaa 2206 2225 SEQ D NO 4407fttccaatgaccaagaaaag 11068 11087 11 Q ID NO: 3079 cagacagtgtcaacaaagc 2253 2272 SEQ D O 4408bcttactggacgaactctg 6142 6161 1 Q ID NO: 3080 cagtgtcaacaaagctttg 2257 2276 SEQ D O W409 caaattcctggatacactg 9857 9876" Q ID NO: 3081 agtgtcaacaaagctttgt 2258 2277 SEQ D NO W410 acaagaatacgtctacact 4359 4378 " Q ID NO: 3082F9^atgg^tg^{tctGtaag t} 2298 2317 SEQ D NO 411 acctcggaacaatcctcag 3333 33521 Q ID NO: 3083F^gat 9tgtctctaaggtc 2299 2318 [SEQ D NO 4412 gacctgcgcaacgagatca 8831 8850 " Q ID NO: 3084paacatgagcaggatatgg 2351 2370 SEQ D NO: 4413 ccatgatctacatttgttt 6796 6815 Q ID NO: 30859 ^ag^Gtg^attaaagatttg 2395 2414 SEQ D NO: W414 caaaaacattttcaacttc 5287 53061 Q ID NO: 3086 aaagatttgaaatccaaag 2405 2424 SEQ D NO:r41^{5 ctttaagttcagcatcttt} 7614 76331 Q ID NO: Bos ^ ggt c∞gcacte 2518 2537_SEQ D NO W416cagatttgaggattccatc 7983 80021 Q ID NO: 3088 gggatcccccagatgattg 2540 559SEQ D NO 441 caatcacaagtcgattccc 9083 9102 1 Q ID NO: 3089 pttcttcactacatcttc 2593 2612 SEQ D NO W41 δgaagtgtcagtggcaaaaa 10382 10401¹ ii Q ID NO: BOΘO ^⁰⁸⁰*³⁰³*⁰^⁰³^ 2596 2615 SEQ D O: 4419 catggcattatgatgaaga 3615 3634 1 Q ID NO: 309 Facatcttcatggagaatg 2603 2622 SEQ D NO 14420 cattatggaggcccatgta 9445 94641 Q ID NO: 3092pcatggagaatgcctttg 2609 2628SEQ D NO: 4421' caaaatcaactttaatgaa 6607 6626 |1 Q ID NO: 3093Fcatggagaatgcctttga 2610 2629 SEQ D NO W422Fcaacacaatcttcaatga 13116 13135 1 Q ID NO: 3094Fttgaactccccactggag 2624 2643SEQ D NO: 14423 ctccccaggacctttcaaa 9842 9861 Q ID NO: 3095pgaactccccactggagc 2625 644SEQ D NO 4424 gctccccaggacctttcaa 9841 98601 Q ID NO: 3096F^{gaactcCGGactg}g^ag^Gt 2626 2645 SEQ D NO: 14425 agctccccaggacctttca 9840 9859 ' Q ID NO: 3097 actggagctggattacag 2635 2654SEQ D NO 4426 ctgtttctgagtcccagtg 9344 9363 ' Q ID NO: 3098 actggagctggattacagt 2636 2655 SEQ D NO W427tactgtttctgagtcccagt 9343 9362 Q ID NO: 3099 agttgcaaatatcttcatc 2652 2671 [SEQ D NO: 14428 gatgatgccaaaatcaact 6599 6618 Q ID NO: oo gttgcaaatatcttcatct 2653 2672SEQ D NO 14429 agatgatgccaaaatcaac 6598 6617 1 Q ID NO: 01 [aaatatcttcatctggagt 2658 2677 [SEQ D NO: 4430actcagaaggatggcattt 14004 14023 Q ID NO: 02F^aaaact9g^aa9^taS^ccaa 2703 2722_SEQ D NO: 443lFtggttacaggaggcttta 7600 7619 Q ID NO: 03 ggctgaactggtggcaaaa 2728 2 47SEQ D O 4432 rctttcttttcagcccagcc 9228 92471 Q ID NO: 04Fgtggagtttgtgacaaat 2758 2777 [SEQ D NO 4433 attttcaagcaaatgcaca 8538 85571 Q ID NO: Oδpgtgacaaatatgggcat 2766 2785SEQ D NO W434 atgcgtctaccttacacaa 9521 95401 Q ID NO: 06 atgaacaccaacttcttcc 2819 2838SEQ D NO; 4435 ggaagctgaagtttatcat 2877 2896 Q ID NO: OTicttccacgagtcgggtctg 2833 2852SEQ D NO W436 cagagctatcactgggaag 5235 52541 Q ID NO: Oβgagtcgggtctggaggctc 2840 2859SEQ D NO 443 agcttactggacgaactc 6140 6159 11 Q ID NO: øg cctaaaagctgggaagctg 2866 2885SEQ D NO: W438 cagcctccccagccgtagg 12120 12139 Q ID NO: loagctgggaagctgaagttt 2872 2891 SEQ D NO 4439 aaactgttaatttacagct 5463 54821 Q ID NO: 11 ccagattagagctggaact 3114 3133 SEQ D NO 4440 agtttccggggaaacctgg 12726 12745 Q ID NO: 12 ggataccctgaagtttgta 3208 3227SEQ D NO: W44lFacagtattctgaaaatcc 8393 8412 Q ID NO: 13 ctgaggctaccatgacatt 3252 3271 SEQ D NO W442 aatgagctcatggcttcag 3817 3836 1 Q ID NO: 14tgtccagtgaagtccaaat 3297 3316 SEQ D NO; 4443 attttgagaggaatcgaca 6357 6376 " Q ID NO: 15 aattccggattttgatgtt 3313 3332SEQ D NO: 444^aacacatgaatcacaaatt 8938 8957 Q ID NO: 16pccggattttgatgttga 3315 3334SEQ D NO 445Fcaaaacgagcttcaggaa 13207 132261 Q ID NO: 1 cggaacaatcctcagagtt 3337 3356SEQ D NO: W446 aacttgtacaactggtccg 4211 4230 Q ID NO: 8F^Gctcagagttaatgatga 3345 3364SEQ D NO:r447F^Gatcaattggttacagga 7593 7612 Q ID NO: 1 g ctcaccctggacattcaga 3392 3411 SEQ D O 4448FCtgcagaacaatgctgag 12439 12458" Q ID NO: 20 cattcagaacaagaaaatt 3403 3422 SEQ D NO:r449^aattg^actttgtagaaatg 8104 8123 Q ID NO: 21 actgaggtcgccctcatgg 3422 3441 SEQ D NO: 450p^Gatg^Gaagtcagcccagt 10924 10943 1 Q ID NO: 22F^atttccataccccgttt 3486 3505 SEQ D NO. 451 aaactgcctatattgataa 13880 13899 1 Q ID NO: 23 gtttgcaagcagaagccag 3501 3520SEQ D NO:p452 tggacttctcttcaaaac 5408 5427 1 Q ID NO: 24ptgcaagcagaagccaga 3502 3521 SEQ D NO: ⁴53Fctgggtgtcgacagcaaa 5272 5291 ' Q ID NO: 25pgcaagcagaagccagaa 3503 3522 SEQ D NO: 454ttctgggtgtcgacagcaa 5271 52901 Q ID NO: 26 ptgcttctccaaatggact 3554 3573SEQ D NO: 45qagtcaagattgatgggcag 4567 45861 Q ID NO: 27Fgctacagcttatggctcc 3577 3596SEQ D NO: 456bgaggctttaagttcagca 7609 76281 Q ID NO: 28 acagcttatggctccacag 3581 3600 SEQ D NO:r457ptgt^atagcaaattcctgt 5897 59161 Q ID NO: 29F^ccaag^ag g^t g^catg 3600 3619 SEQ D NO:r458patggacttcttctggaaa 8877 88961 Q ID NO: 30 ccaagagggtggcatggca 3603 3622 SEQ D NO:r⁴59 g^GGGag^Gaag^Gaag tgg 9361 9380 " Q ID NO: 31 gtggcatggcattatgatg 3611 3630 SEQ D NO: 4460 catccttaacaccttccac 8071 80901 Q ID NO: 32Fgatgaagagaagattgaa 3625 3644 SEQ D NO:r461pcactgttcctgaaatca 7871 7890 Q ID NO: 33 gaagagaagattgaatttg 3629 3648 SEQ D NO: W462 caaaaacattttcaacttc 5287 53061

Q ID NO 3228^aaaagt^ata ^aattacaga 6558 6577_SEQ D NO 14557Fctggctccctcaactttt 9050 90691 Q ID NO: 3229 atcaactttaatgaaaaac 6611 6630_SEQ D NO: 4558 gtttattgaaaatattgat 6811 6830 Q ID NO: 3230F^gatttgaaaatag^Gtatt 6694 6713SEQ D NO: 4559 aatattattgatgaaatca 6716 6735 Q ID NO: 3231 atttgaaaatagctattgc 6696 6715 SEQ D NO: 560gcaagaacttaatggaaat 10441 10460r Q ID NO: 3232 attgctaatattattgatg 6710 6729 SEQ D NO W561 catcacactgaataccaat 10159 10178 Q ID NO: 3233 gaaaaattaaaaagtcttg 6737 6756 SEQ D NO: 14562 caagagcttatgggatttc 11161 11180 1 Q ID NO: 3234 actatcatatccgtgtaat 6762 6781 SEQ D NO W563 attactttgagaaattagt 7281 7300 1 Q ID NO: 3235pttgattttaacaaaagt 6823 6842_SEQ D NO: 4564acttgacttcagagaaata 11404 11423 1 Q ID NO: 3236p^{tgGa ca}g^Gttaag^a9^aG 6914 6933 SEQ D NO: 4565 gtcttcagtgaagctgcag 10699 10718 11 Q ID NO: 3237 aaaacaacacattgaggct 6973 6992SEQ D NO: 14566 agcctcacctcttactttt 10571 10590 1 Q ID NO: 3238P^{gagGat tGaaaGactt} 7059 7078 [SEQ D NO r4567taagtagctgagaaaatcaa 7104 7123 " Q ID NO: 3239Fg^aa9^tag^Gtg^ag^aaaa 7100 7119 SEQ D NO: 4568 pttcacattagatgcaaa 8421 8440 ' Q ID NO: 3240P^agtagag^ttggcccaGG 7199 7218 SEQ D NO 4569 ggtggactcttgctgctaa 7776 7795¹ Q ID NO: 3241 raaaggagactattcagaa 7227 7246SEQ D O: 4570fttctcaattttgattttca 8526 8545 1 Q ID NO: 3242 gagectattcagaagctaa 7232 7251 SEQ D NO 457lFtagccacagctctgtctc 10301 10320 Q ID NO: 3243aattagttggatttattga 7293 7312 SEQ D NO j4572Fcaagaagcttaatgaatt 7320 7339 Q ID NO: 3244 gcttaatgaattatctttt 7327 7346 SEQ D NO: 4573 aaaacgagcttcaggaagc 13209 13228 11 Q ID NO: I3245paacaaattccttgacat 7365 7384SEQ D NO: 4574 atgtcctacaacaagttaa 7254 7273" Q ID NO: 3246 aaattaaagtcatttg att 7394 7413 SEQ D NO 4575 aatcctttgacaggcattt 9723 97421 Q ID NO: 3247gactcaatggtgaaattca 7464 7483 SEQ D NO: j4576 gaaattcaatcacaagtc 9076 90951 Q ID NO: 32489^aaattca99^ctctgg^aaG 7475 7494 SEQ D NO: 4577tattctcaattttgattttc 8525 8544 1 Q ID NO: 3249 actaccacaaaaagctgaa 7492 7511 SEQ D NO 4578ρcaggaactattgctagt 10645 10664 1 Q ID NO: 3250 pcaaaataaccttaatcat 7578 7597 SEQ D NO: 4579 atgatttccctgaccttgg 10950 10969 1 Q ID NO: 3251 aaataaccttaatcatcaa 7581 7600SEQ D NO: 458θFtgaagtaaaagaaaattt 10749 10768 1 Q ID NO: 3252F^aag^^ca9^ca^^G^9 7615 7634_SEQ D NO 4581 caaatctggatttcttaaa 9480 9499 1 Q ID NO: 3253p^ag^gtttatag^GacaGtt9 7739 7758SEQ D NO 4582caagggttcactgttcctg 7865 7884 " Q ID NO 3254g^ttGaGt9^ttGGtg^aaatc 7870 7889SEQ D NO: 4583 gattctcagatgagggaac 8922 8941 - Q ID NO: 3255 actgttcctgaaatcaag 7873 7892SEQ D NO: 4584 cttgaacacaaagtcagtg 6008 60271 Q ID NO: 3256 actgttcctgaaatcaaga 7874 7893 SEQ D O 4585 cttgaacacaaagtcagt 6007 6026 Q ID NO: 3257 gcctgcctttgaagtcagt 7909 79 8SEQ D NO 4586 actgttgactcaggaaggc 12580 12599 Q ID NO: 3258p^acagatttgaggattcc 7980 7999 [SEQ D NO: 4587tagaagcttctcaagagtta 13222 13241 1 Q ID NO: 3259 gttttccacaccagaattt 8050 8069 SEQ D NO 4588 aaatttctctgctggaaac 9418 9437 ii Q ID NO: 3260 ^Ga9^aaGGattgacGa9^a* 8136 8155 SEQ D NO 4589atctgcagaacaatgctga 12438 12457 Q ID NO: 3261pgcg^ag^aatc^accctgcc 8226 8245SEQ D NO: 4590 ggcagcttctggcttgcta 12301 12320 1 Q ID NO: 3262 cttaatgattttcaagtt 8299 8318 SEQ D NO: 4591 aactgttgactcaggaagg 12579 12598 i1 Q ID NO: 3263 acataccagaattccagct 8328 8347SEQ D NO:4592agctgccagtccttcatgt 10026 10045 1 Q ID NO: 3264^aatgctgacatagggaatg 8438 8457SEQ D NO: 4593 cattaatcctgccatcatt 10005 10024 1 Q ID NO: 3265^a gc g^{aGa a}ggg^aatgg 8439 8458 SEQ D NO: 4594 ccatttgagatcacggcat 9245 9264 1 Q ID NO: 3266 aaccacctcagcaaacgaa 8458 8477SEQ D NO:r595pcgttttccattaaggtt 9291 9310 1 Q ID NO: 32671^agcaggtatcgcagcttcc 8476 8495 SEQ D NO:r⁵96gg^aagtggccctgaatgct 10972 10991 1 Q ID NO: 3268 ^gcacaactct^caaaccct 8551 8570 SEQ D NO:f*597pggg^aaag^agaagattgca 13501 13520 Q ID NO: 3269 aggagtcagtgaagttctc 8592 8611 SEQ D NO; 4598raagaacttactatcatcct 13788 13807 1 Q ID NO: 3270 ptttggaaatgccattga 8652 8671 SEQ D O:r59 F^{aat aattta} tcaaaa 13194 13213 1 Q ID NO: 3271 aatggagtgattgtcaaga 8729 8748 SEQ D NO: WδOOFcttttcagcccagccatt 9231 9250 Q ID NO: 32729tcaagataaacaatcagc 8741 8760 SEQ D NO:4601 gctgactttaaaatctgac 4819 4838 Q ID NO: 3273 tccacaaattgaacatccc 8787 8806_SEQ D NO: 4602gggatttcctaaagctgga 11172 11191 1 Q ID NO: 3274Ftgaacatccccaaactgg 8795 8814 SEQ D NO: 4603 ccagtttccagggactcaa 12603 12622 Q ID NO: 3275 acatccccaaactggactt 8799 8818 SEQ D NO W604 aagtcgattcccagcatgt 9090 91091 Q ID NO: 3276 acttctctagtcaggctga 8814 8833 SEQ D NO w-δOδrtcagatggaaaaatgaagt 11010 11029 1 Q ID NO: 3277Fgaatcacaaattagtttc 8944 8963 SEQ D NO W606 gaaagtccataatggttca 12817 12836 1 Q ID NO: 3278 agaaggacccctcacttcc 8968 8987 SEQ D NO 4607bgaagaagaggcagcttct 12292 12311 Q ID NO: 3279pggactgtccaataagat 8988 9007SEQ D NO 4608 atctaaatgcagtagccaa 11634 11653 Q ID NO: 3280 actgtccaataagatcaat 8992 9011 SEQ D NO W609 attgataaaaccatacagt 13891 13910 Q ID NO 3281 ctgtccaataagatcaata 8993 9012 SEQ D NO: 1461 OFattgataaaaccatacag 13890 13909 Q ID NO: 3282 gtttatgaatctggctccc 9041 9060 SEQ D NO W611 gggaatctgatgaggaaac 12255 12274 Q ID NO: 3283 atgaatctggctccctcaa 9045 9064 SEQ D O: 14612 tgagttgcccaccatcat 11667 11686 Q ID NO: 3284 ctcaacttttctaaacttg 9059 9078 SEQ D NO: 14613 caagatcgcagactttgag 11653 11672 Q ID NO: 3285p^taaagg^cat9^gcactgtt 9129 9148 SEQ D NO: 14614 aacagaaacaatgcattag 9749 9768 Q ID NO: 3286 aaggcatggcactgtttgg 9132 9151 SEQ D NO: 4615 ccaagaaaaggcacacctt 11077 11096 1 Q ID NO: 3287pccacaaacaatgaaggg 9262 9281 SEQ D NO 14616 ccctaacagatttgaggat 7977 7996 Q ID NO: 3288 ggaatttgaaagttcgttt 9279 9298 SEQ D NO 4617taaacaaacacaggcattcc 9655 96741 Q ID NO: 3289 aataactatgcactgtttc 9332 9351 SEQ D NO 4618gaaatactgttttcctatt 12836 128551 Q ID NO: 329ogaaacaacgagaacattat 9432 9451 SEQ D O 4619 ataaactgcaagatttttc 13608 13627 Q ID NO: 329ipcttgaaaacgacaaagc 9599 9618 SEQ D NO 4620 gctttccaatgaccaagaa 11065 11084 Q ID NO: 3292 ataagaaaaacaaacacag 9648 9667SEQ D NO 4621 ctgtgctttgtgagtttat 9690 9709 Q ID NO: 3293 aaaacaaacacaggcattc 9654 9673 SEQ D NO 4622gaatttgaaagttcgtttt 9280 9299 Q ID NO: 3294 gcattccatcacaaatcct 9667 9686 SEQ DNO 4623aggaagtggccctgaatgc 10971 10990 Q ID NO: 3295pt9^aaaaaaaca9^aaaca 9740 9759 SEQ D NO: l4624Fgttgaaagatttatcaaa 12933 12952 Q ID NO: l3296paatgcattagattttgtc 975 9776 SEQ D NO: 14625 gacaagaaaaaggggattg 10279 10298 Q ID NO: 3297paaagctgaaaaatctcag 9817 9836 SEQ D NO 4626 ctgagaacttcatcatttg 11438 11457 1 Q ID NO: 3298 pctggatacactgttccag 9863 9882 SEQ D NO: I4627ptggacttctctagtcagg 8810 8829 Q ID NO: 3299 gttgaagtgtctccattca 9890 9909 SEQ D NO: l4628Fgaatctggctccctcaac 9046 90651 Q ID NO: 330optctccatcctaggttct 9964 9983 SEQ D NO: 14629 agaatccagatacaagaaa 6893 69121 Q ID NO: 3301 pctccatcctaggttctg 9965 9984SEQ D NO W630 cagaatccagatacaagaa 6892 6911 Q ID NO: 3302F^attagagctgccagtcc 10019 0038 SEQ D NO 4631 ggacagtgaaatattatga 13305 13324 1 Q ID NO: 3303Fgctgaactttttaaccag 10177 0196 SEQ D NO 4632 ctggatgtaaccaccagca 11186 11205 Q ID NO: 3304 ctcctttcttcatcttcat 10214 0233SEQ D NO 4633 atgaagcttgctccaggag 13772 13791 Q ID NO: 3305 g^tcattg^atgcactgcag 10234 0253 SEQ D NO 4634 ctgcgctaccagaaagaca 12080 12099 Q ID NO: 3306 g^{atgcactgcagtacaaa} 10240 0259 SEQ D NO 4635ptgagttgcccaccatca 11666 11685 Q ID NO: 3307Θgctctgtctctgagcaac 10309 0328 SEQ D NO 4636 gttgaccacaagcttagct 10547 10566 Q ID NO: 3308 agccgaaattccaattttg 10408 0427 SEQ D O 4637paaagctggcaccagggct 13971 13990 Q ID NO: 3309pgagaatgaatttcaagc 10424 0443 SEQ D NO: 4638 gcttcaggaagcttctcaa 13216 13235 Q ID NO: 3310 ^aaacctactgtctcttcct 10469 0488 SEQ D NO: 4639 aggaaggccaagccagttt 12591 12610 Q ID NO: 331 Facttttccattgagtcat 10583 0602 SEQ D NO: W640 atgattatgtcaacaagta 12363 12382 1 Q ID NO: 3312F^cagg^tccat9^caag^tcag 10918 0937SEQ D NOJ46 1 ctgacatcttaggcactga 5001 5020 1 Q ID NO: 3313 atgcaagtcagcccagttc 10926 0945 SEQ D NO:r^{642gaactcagaaggat}gg^cat 14002 14021 ^;1 Q ID NO: 3314F9^aatgctaacactaagaa 10983 1002 SEQ D NO: 643 tctcaattttgattttca 8526 8545 1 Q ID NO 3315agaagatcagatggaaaaa 11004 1023 SEQ DNO: W644 pttctaaatggaacttct 12173 12192 1 Q ID NO: 331 βΘQctattcattctccatcc 11264 1283 SEQ D NO: 645ggatctaaatgcagtagcc 11632 11651 Q ID NO: 3317teaagttttggctgataaat 11288 1307 SEQ D NO: 646atttcttaaacattccttt 9489 9508 Q ID NO: 331 δagttttggctgataaattc 11290 1309 SEQ D NO:r647gaatctggctccctcaact 9047 9066 Q ID NO: 3319 ctgggctgaaactaaatga 11316 1335 SEQ D NO:r648ftcattctgggtctttccag 11035 11054 Q ID NO: 3320 cagagaaatacaaatctat 11413 1432 SEQ D NO:r649^atagcatggacttcttctg 8873 88921 Q ID NO: 3321 gaggtaaaattccctgaag 11480 1499 SEQ D NO: 650 cttctggcttgctaacctc 12306 123251 Q ID NO: 3322 cttttttgagataaccgtg 11545 11564 SEQ D NO 4651 cacggagttactgaaaaag 13723 137421 Q ID NO: 3323 gctggaattgtcattcctt 11735 11754 SEQ D NO 14652 aaggcatctccacctcagc 12102 12121 Q ID NO: 3324 gtgtataatgccacttgga 11795 11814 SEQ D NO |4653Fccaagatgagatcaacac 13104 13123 Q ID NO: 3325 attccacatgcagctcaac 11859 11878 SEQ D NO: 4654gttgagaagccccaagaat 6254 62731 Q ID NO: 3326F9^aa9^aa9^at99^caaattt 11992 12011 SEQ D O 4659aaattctcttttcttttca 9220 9239 1 Q ID NO: 332 atcaaaagcccagcgttca 12050 12069 SEQ D NO 4656Fgaaagtcaagcatctgat 12669 12688 1 Q ID NO: 3328gtgggc^atggatatggatg 12143 12162_SEQ D NO 4657batccttaacaccttccac 8071 8090 1 Q ID NO: 3329 aaatggaacttctactaca 12179 12198 SEQ D NO 4658Fgtaccataagccatattt 10088 10107 ^■1 Q ID NO: 3330 aaaaactcaccatattcaa 12219 12238_SEQ D NO: 4659Ktgatgttagagtgctttt 6993 7012 1 Q ID NO: 3331 ctgagaagaaatctgcaga 12428 12447 SEQ D NO: 4660π:ctgcacagaaatattcag 13447 13466 1 Q ID NO: 3332 acaatgctgagtgggttta 12447 12466 SEQ D NO 4661paatggagtctttattgt 14086 14105 1 Q ID NO: 3333 caatgctgagtgggtttat 12448 467SEQ D NO 4662 ataaatggagtctttattg 14085 14104 1 Q ID NO: 3334paggcaaattgatgatat 12477 12496 SEQ D NO 4663 atattgtcagtgcctctaa 13392 13411 1 Q ID NO: 3335 ataaactaatagatgtaat 12897 12916 SEQ D O 4664 attactatgaaaaatttat 13641 13660 1 Q ID NO: 3336 ccaactaatagaagataac 13039 13058 SEQ D O 4665 gttattttgctaaacttgg 14052 14071 1 Q ID NO: 3337paattatatccaagatga 13095 13114 SEQ D NO 4666patcctctaattttttaa 13800 13819 Q ID NO: 3338F^{aaattgttgaaagaaa} 13151 13170 [SEQ D NO 466ηtttcatttgaaagaataaa 7032 7051 Q ID NO: 3339 aagttcaatgaatttattc 13190 13209 SEQ D NO 4668 gaataccaatgctgaactt 10168 10187 Q ID NO: 3340P^{gaagaaaagatagtca}g 13326 13345 SEQ D NO 4669 ctgagagaagtgtcttcaa 12407 12426 Q ID NO: 3341 acttccattctgaatatat 13377 13396 SEQ D NO 4670 atatctggaaccttgaagt 10737 10756 Q ID NO: 33₄2cacagaaatattcaggaat 13451 13470 SEQ D NO: W671 attccctgaagttgatgtg 11488 11507 Q ID NO: 3343 ccattgcgacgaagaaaat 13560 13579 SEQ D NO: 4672 atttttattcctgccatgg 10103 10122 1 Q ID NO: 3344ptaaactgcaagattttt 13607 13626 SEQ D NO: 14673 aaaattcaaactgcctata 13873 13892 1 Q ID NO: 3345 ctgattactatgaaaaat 13637 13656 SEQ D NO: 14674 atttgtaagaaaatacaga 6436 6455 11 Q ID NO: 3346gg^{a ttact}g^aaaaag^ctg 13726 13745 SEQ D NO 14675 cagcatgcctagtttctcc 9952 9971 1 Q ID NO: 3347 gaagcttgctccaggaga 13773 13792 SEQ D NO 4676ftctcctttcttcatcttca 10213 10232 1 Q ID NO: 3348 ^gaactgg^acctg^caccaa 13955 13974 SEQ D NO: 677pggtagagcaagggttca 7856 7875 Q ID NO: 3349F9^ctaaacttgggg^gagg 14058 1 077SEQ D NO: 14678 cctcctacagtggtggcaa 4230 4249 ' Q ID NO: 3350 gattcgaatatcaaattca 4412 4431 SEQ D NO ]4679Fgaaaacgacaaagcaatc 9603 96223 Q ID NO: 3351 atttgtttgtcaaagaagt 4551 4570 SEQ D O W680 acttttctaaacttgaaat 9063 90823 Q ID NO: 3352ptcggttgctgccgctga 33 52SEQ D NO 468lFcagcccagccatttgaga 9236 92552 Q ID NO: 3353 gctgaggagcccgcccagc 47 66 SEQ D NO 4682gctggatgtaaccaccagc 11185 112042 Q ID NO: 3354^ctgg^tct9^tccaaaag^atg 227 246 SEQ D NO W683 catcagaaccattgaccag 8134 81532 Q ID NO: 3355F ^a9^{a ttcca}g^tgg^ag^t 291 310 SEQ D NO W684 actcaatggtgaaattcag 7465 74842 Q ID NO: 3356p^agtg^caccctg^aaa9^a 404 423 SEQ D NO W685 cctcacttcctttggactg 8977 89962 Q ID NO: 3357^ctctgaggagtttgctgca 472 491 SEQ D NO; r4686Fgcaaacttgacttcagag 11399 11418. Q ID NO: 3358 acatcaagaggggcatcat 582 601 SEQ D NO: W687ktgacgttcttgagcatgt 7050 70692 Q ID NO: 3359 tgatcagcagcagccagt 830 849 SEQ D NO: W688 actggacttctctagtcag 8809 88282 Q ID NO: 3360 ggacgctaagaggaagcat 865 884 SEQ D NO: W689 atgcctacgttccatgtcc 11354 113732 Q ID NO: 3361 agctgttttgaagactctc 1087 1106 SEQ D NO:r6 °g^ag^aagtg^tcttcaaagct 12411 124302 Q ID NO: 3362 g^aaaaaactaaccatctc 1113 1132 SEQ D NO: W691 gagatcaacacaatcttca 13112 13131 ; Q ID NO: 3363^{ctgagctgagag cctca}g 1176 1195 SEQ D O:r69 F9^aatt^actgcacctcag 3035 30542 Q ID NO: 3364F^gaaacg^tg^tgcatgccaa 1311 1330 SEQ D NO: 693pggtagagcaagggttca 7856 78752 Q ID NO: 3365 cttgtatgcgctgagcca 1440 1459 SEQ D NO: 694 ggcactgtttggagaagg 9138 91572 Q ID NO: 3366 aggagctgctggacattgc 1500 1519 SEQ D NO:r695gcaagtcagcccagttcct 10928 109472 Q ID NO: 3367ptttgattctgcgggtcat 1575 1594 [SEQ D NO: W696 atgaaaccaatgacaaaat 7428 74472 Q ID NO: 3368pc^ag^aactcaagtcttca 1627 1646 SEQ D NO: W697Fgaaatacaatgctctgga 5520 55392 Q ID NO: 3369 ggttcttcttcagactttc 1744 1763 SEQ D NO W698 gaaataccaagtcaaaacc 10455 104742 Q ID NO: 3370 gttgatgaggagtccttca 1810 1829 SEQ D NO: 4699rcgaaaaagctgcaatcaac 13734 137532 Q ID NO: 337lFccaagatctgaaaaagtt 1941 1960 SEQ D NO K700 aactgcttctccaaatgga 3552 3571 Q ID NO: 3372 agttagtgaaagaagttct 1956 1975 SEQ D NO 4701 agaattcataatcccaact 8275 82942 Q ID NO: 3373 gaagggaatcttatatttg 2084 2103 SEQ D NO r4702 caaaacctactgtctcttc 10467 104862 Q ID NO: 3374 ggaagctctttttgggaag 2221 2240 [SEQ D NO: 14703 cttcacataccagaattcc 8324 83432 Q ID NO: 3375 gg^aataatg^{ctca t tt} 2374 2393 SEQ D NO 4704 aacaaacacaggcattcca 9656 96752 Q ID NO: 3376gatttgaaatccaaagaag 2408 27SEQ D O: 14705 cttcatgtccctagaaatc 10037 100562 Q ID NO: 3377Fccaaagaagtcccggaag 2417 2436 SEQ D NO 14706 cttcagcctgctttctgga 4951 49702 Q ID NO: 3378 aggaagggctcaaagaatg 2570 2589 SEQ D NO I4707pattagagctgccagtcct 10020 100392 Q ID NO: 3379 agaatgacttttttcttca 2583 2602 SEQ D NO 4708Fgaagatgacgacttttct 12160 121792 Q ID NO: 338o g^tg^acaaatatg g^ca 2765 2 84SEQ D NO W709Fgccagtttgaaaaacaaa 11815 118342 Q ID NO 3381 ctgaggctaccatgacatt 3252 3271 SEQ D NO: W710 aatgtcagctcttgttcag 10903 109222 Q ID NO: ₃₃₈ gtagataccaaaaaaatga 3668 3687SEQ D NO 471 i catttgccctcaacctac 11450 114692 Q ID NO: 3383 aaatgacttccaatttccc 3681 3700 SEQ D NO 4712gggaactgttgaaagattt 12927 129462 Q ID NO: 3384 atgacttccaatttccctg 3683 3702 SEQ D NO: 4713 caggagaacttactatcat 13785 138042 Q ID NO: 3385 atctgccatctcgagagtt 4104 4123_SEQ D NO 4714 aactcctccactgaaagat 9547 95662 Q ID NO: 3386 atttgtttgtcaaagaagt 4551 4570 SEQ D O: 1471 δacttccgtttaccagaaat 8247 8266 Q ID NO 338τιg^cag^ag^cttgg^cctctctg 5135 5154 SEQ D NO 4716 cagagctttctgccactgc 13518 135372 Q ID NO: 3388 atatgctgaaatgaaattt 5353 5372 SEQ D NO 471 aattcaaactgcctatat 13874 138932 Q ID NO: 33₈g caaaacttgacaacattt 5420 5439_SEQ D NO: 1471 δaaatacttccacaaattga 8780 87992 Q ID NO: 3390 cagtgacctgaaatacaat 5512 5531 SEQ D NO: 14719 attgaacatccccaaactg 8794 88132 Q ID NO: 3391pcaaatggcaatgggaaa 5848 5867SEQ D NO: 4720ρtcaactgcctttgtgta 11229 112482 Q ID NO 3392 [cttttgtaaagtatgataa 6285 6304 SEQ D NO 4721 pattgctgaatccaaaag 13656 136752 Q ID NO: 3393Ftgtaaagtatgataaaaa 6288 6307_SEQ D NO: W722 ttttcaagcaaatgcacaa 8539 8558 Q ID NO: 3394Fccattaacctcccatttt 6320 6339 SEQ D NO: 14723 aaaagaaaattttgctgga 10756 107752 Q ID NO: 3395 gattatctgaattcattca 6488 6507SEQ D NO 14724H:gaagtagaccaacaaatc 7162 7181E Q ID NO: 3396 aattgggagagacaagttt 6506 6525 SEQ D NO 4725 aaactaaatgatctaaatt 11324 113432 Q ID NO: 3397tøtttgaaaatagctattgc 6696 6715 SEQ D O W726 gcaatttctgcacagaaat 13441 13460 Q ID NO: 3398F9^agcatg^tcaaacacttt 7060 7079_SEQ D NO: W72ηaaagccattcagtctctca 12971 129902 Q ID NO: 339gpgaagatgttaacaaatt 7356 7375SEQ D NO: W728 aattccatatgaaagtcaa 12660 126792 Q ID NO: 3400 acttgtcacctacatttct 7753 7772SEQ D NO W729 agaatattttgatccaagt 13276 13295 Q ID NO: 3401 gttttccacaccagaattt 8050 8069 SEQ D NO r4730 aaatctggatttcttaaac 9481 95002 Q ID NO: 3402 ataagtacaaccaaaattt 9405 9424SEQ D NO 4731 aaataaatggagtctttat 14083 14102 Q ID NO: 3403 ggg^{acct c}gggg^ctg^ag 27SEQ D NO I4732 ctcagttaactgtgtcccg 11571 11590 Q ID NO: 3404 agtgcccttctcggttgct 25 44 SEQ D NO W733 agcatctgattgactcact 12678 12697 Q ID NO: 3405g^ctg^a g^{a ccc}g^cccagc 47 66 SEQ D NO J734gctgattgaggtgtccagc 1225 1244 Q ID NO: 3406 gaggagcccgcccagccag 50 69SEQ D NO 4735 ctggatcacagagtccctc 3752 3771 - Q ID NO: 3407lgggccgcgaggccgaggcc 72 91 SEQ D NO: 4736 ggccctgatccccgagccc 1363 13821 Q ID NO: 3408pcaggccgcagcccaggag 89 108 SEQ D NO: W737ptcccggagccaaggctgg 2682 2701 - Q ID NO: J3409 ggagccgccccaccgcagc 104 123 SEQ D O 4738 gctgttttgaagactctcc 1088 1107" Q ID NO: 341 og^{aa a}gg^aaatg^ctgg^aaa 200 219 SEQ D NO 4739ptcaagttcctgaccttc 8309 8328 ' Q ID NO: 3411 caaaagatgcgacccgatt 237 256SEQ D NO: 4740 aatcttattggggattttg 7085 7104 Q ID NO: [3412 attcaagcacctccggaag 253 72SEQ D NO: 741 cttccacatttcaaggaat 10067 10086 Q ID NO: 3413 gttccagtggagtccctgg 297 316 SEQ D NO: 742 ccagcaagtacctgagaac 8610 8629 :ι Q ID NO: 341 actgctgattcaagaagt 316 335 SEQ D NO: 43^acttgaagaaaagatagtc 13324 13343 Q ID NO: 341 δgtgccaccaggatcaactg 333 352SEQ D NO:r⁷⁴4pagtgaagctgcagggcac 10704 107231

Q ID NO: 3557pagtgttgagaagctgat 2385 2404 SEQ D NO 14886 atcacaactcctccactga 9542 9561 1 Q ID NO: 3558 cagtgttgagaagctgatt 2386 2405 SEQ D O W887katcacaactcctccactg 9541 9560 Q ID NO: 3559 agtgttgagaagctgatta 2387 2406 SEQ D NO 888ftaatcacaactcctccact 9540 95591 Q ID NO: 3560^gattaaa9^atttgaaatcc 2401 2420 SEQ D NO: W889 ggatactaagtaccaaatc 6874 68931 Q ID NO: 3561 gatttgaaatccaaagaag 2408 2427 feEQ D NO 4890 cttccgtttaccagaaatc 8248 82671 Q ID NO: 3562 atttgaaatccaaagaagt 2409 2428 SEQ D NO 4-891 acttccgtttaccagaaat 8247 82661 Q ID NO: 3563 atccaaagaagtcccggaa 2416 2435 SEQ D NO W892pccaatttccctgtgg at 3688 37071 Q ID NO: 3564pcaaagaagtcccggaag 2417 2436 SEQ D NO W893 cttccaatttccctgtgga 3687 3706 - Q ID NO: 3565 agagcctacctccgcatct 2438 2457_SEQ D NO 4894 agattaatccgctggctct 8571 8590 - Q ID NO: 3566 gagcctacctccgcatctt 2439 2458 SEQ D NO 4895 aagattaatccgctggctc 8570 8589 Q ID NO: 3567p^ttgg ^a9^agg^ag^cttg9^t 2455 ²474_SEQ D NO 4896 accactgggacctaccaag 12527 12546 Q ID NO: 3568g ^ag^cttgg^{tttt cca}g^t 2464 2483 SEQ D O 4897kctggtggcaaaaccctcc 2734 2753 Q ID NO '3569pg^gttttg^{cca tctcca} 2469 2488 SEQ D NO tø898tagagaagccacactccaa 10771 10790 1 Q ID NO: 3570 cagtctccatgacctccag 2479 2498 SEQ D NO: W899 ctggtcgcctgccaaactg 3538 3557 1 Q ID NO 3571 ctccatgacctccagctcc 2483 502_SEQ D NO: 14900 ggagtcattgctcccggag 2672 2691' Q ID NO 3572 ctgggaaagctgcttctga 2501 2520SEQ D NO: I4901π:cagaaagctaccttccag 7939 79581 Q ID NO 3573 gaggtcatcaggaagggct 2561 2580 SEQ D NO 14902 agccagaagtgagatcctc 3514 35331 Q ID NO: 3574 aagaatgacttttttcttc 2582 2601 SEQ D NO 4903gaaggcatctgggagtctt 3835 38541 Q ID NO: [3575F ;ttttttcttcactacatc 2590 2609 |SEQ D NO 4904gatgcttacaacactaaag 6107 6126 ι1 Q ID NO: 3576 catcttcatggagaatgcc 2605 2624SEQ D NO W-905 ggcacttccaaaattgatg 10718 10737 11 Q ID NO: 3577 cttcatggagaatgccttt 2608 ²627_SEQ D NO 14906 aaagttaattgggaagaag 12281 12300 1 Q ID NO: 3578 aatgcctttgaactcccca 2618 2637SEQ D NO l4907Fgggctggcttcagccatt 5737 5756 1 Q ID NO: 3579 gcctttgaactccccactg 2621 2640 SEQ D NO: 14908 cagtctgaacattgcaggc 5383 5402 1 Q ID NO: 358opaaggctggagtaaaactg 2692 2711 SEQ D O: I4909pagtgcaacgaccaacttg 5080 5099 Q ID NO: 3581 ragagtaaaactggaagta 2698 2717 SEQ D NO 491 OFactccaacgccagctcca 3059 3078 - Q ID NO: 3582 ggaagtagccaacatgcag 2710 2729_SEQ D NO: W911 ctgccatctcgagagttcc 4106 4125 Q ID NO: 3583Fg^tg^acaaatatgg^gca 2765 2784 SEQ D NO: }4912 gcctttgtgtacaccaaa 11236 11255 Q ID NO 3584F^{gtgacaaatatgggcatc} 2767 2786SEQ D NO 4913gatgggtctctacgccaca 4385 4404 Q ID NO 3585gg^{acttc ctag}g^{a t}gg 2794 2813 SEQ D NO 4914 cccaaggccacaggggtcc 12341 12360 Q ID NO: 3586 ^tgggg^{tccagatgaacac} 2808 2827 SEQ D NO 4915 gtgttctagacctctccac 4179 4198 Q ID NO: 3587pccacgagtcgggtctgg 2834 2853 SEQ D NO 4916 ccagaatctgtaccaggaa 12562 12581 1 Q ID NO: 3588 agtcgggtctggaggctca 2841 2860 SEQ D NO 4917Fgagaactacgagctgact 4807 4826 11 Q ID NO: 3589pggg^tctggag^gctcatg 2843 2862 SEQ D NO: 4918 catgaaggccaaattccga 7639 7658 1 Q ID NO: 3590 aaaagctgggaagctgaag 2869 2888 SEQ D NO: 4919 cttccagacacctgatttt 7951 79701 Q ID NO: 3591 aagctgaagtttatcattc 2879 2898 SEQ D No_:W920gaatttacaattgttgctt 6269 6288 1 Q ID NO: 35929^ag^accag^tcaag^ctg^ctc 2908 2927 SEQ D No_:M-921bagcttcaggaagcttctc 13214 13233 Q ID NO: 3593bcaacacattacatttggt 2934 2953 SEQ D NO 4922 accagtcagatattgttgc 10191 10210 1 Q ID NO: 3594 acattacatttggtctcta 2939 2958 SEQ D NO 4923π:agaatatgaactaaatgt 11889 11908 Q ID NO: 3595 cattacatttggtctctac 2940 2959SEQ D O 4924 gtagctgagaaaatcaatg 7106 7125 -1 Q ID NO: 3596 aaacggaggtgatcccacc 2964 2983 SEQ D O 4925 ggtggataccctgaagttt 3205 3224 1 Q ID NO: 359₇attgagaacaggcagtcct 2987 3006 SEQ D NO 4926aggaaaagcgcacctcaat 12031 12050 Q ID NO: 3598F^gagaacaggca9^tcctgg 2989 3008_SEQ D NO: 4927bcagcttccccacatctca 8341 8360 Q ID NO: [3599 ctgcacctcaggcgcttac 3043 3062 SEQ D NO:r928 gtaagaaaatacagagcag 6440 6459 ' Q ID NO: 3600p^caca9^ac'^:ccgcc*^cct 3074 3093 SEQ D NO:r^{929 a}gg^aca9^a9^ccttgg^tgg^a 3192 3211 Q ID NO: 3601 ctgaccggggacaccagat 3101 3120 SEQ D NO:r930atctgatgaggaaactcag 12259 12278 Q ID NO 3602pg^a9^ctgg^aact9^aggcc 3120 3139 [SEQ D O: 4931 ggcctctctggggcatcta 5144 5163 Q ID NO: 3603F^at9^ag^{ctcca a a}g^ag 3175 3194 SEQ D NO:r⁹32ptctcacaaaaaagtatag 6549 65681 Q ID NO 3604^cttgg^tgg^ataccctg^aag 3202 3221 SEQ D NO: 14933cttcaggaagcttctcaag 13217 132361 Q ID NO 3605P^gtaactcaagcagaagg 3222 3241 SEQ D NO 14934 ccttacacaataatcacaa 9530 9549 1 Q ID NO: 3606F^aactcaa9^caS^aagg^tgc 3225 3244 SEQ D NO: W935gcacctagctggaaagtta 6955 69741 Q ID NO 36oτig^cag^aagg^tg^cg^aag^cag^a 3233 3252 SEQ D NO: r4936rcctgtgggattccatctgc 4091 4110 ,1 Q ID NO 3608 ^cag^aaggtgcgaagcagac 3234 3253 SEQ D NO 4-937tatctgtgggattccatctg 4090 4109 1 Q ID NO: 3609 gtatgaccttgtccagtga 3288 3307SEQ D NO r4938Fcaccaacggagaacatac 10851 10870 1 Q ID NO: 361 optgβccttgtccagtgaa 3289 3308_SEQ D NO: 4939rctcaccaacggagaacata 10850 10869 Q ID NO: 3611 gaagtccaaattccggatt 3305 3324SEQ D NO W940 aatctcaagctttctcttc 10052 10071 Q ID NO: 3612 gβgggcaaaacgtcttaca 3371 3390_SEQ D NO 4941 rtgtacaactggtccgcctc 4215 4234 Q ID NO: 3613 agggcaaaacgtcttacag 3372 3391 SEQ D NO 4942 ctgttaggacaccagccct 4062 4081 - Q ID NO 3614 gactcaccctggacattca 3390 3409 [SEQ D NO W943Fgaaattcaatcacaagtc 9076 90951 Q ID NO: 361 δptggacattcagaacaaga 3398 3417 SEQ D NO: W944Fcttttcttttcagcccag 9226 92451 Q ID NO: 3616patgggcgacctaagttg 3435 3454 SEQ D NO: 4945 caactgcagacatatatga 6635 66541 Q ID NO: 3617 gggcgacctaagttgtga 3438 3457_SEQ D NO 946n:cactccattaacctccca 6316 6335 1 Q ID NO: 3618 agttgtgacacaaaggaag 3449 3468 SEQ D NO 4947 cttcttttccaattgaact 13838 13857 11 Q ID NO: 361 gtaacacaaaggaagaaaga 3454 3 73SEQ D NO 4948pttcatcttcatctgtca 10220 10239 1 Q ID NO: 3620 gacacaaaggaagaaagaa 3455 3474 SEQ D NO: W949ttcttcatcttcatctgtc 10219 10238 Q ID NO: [3621 ggaagaaagaaaaatcaag 3463 3482 SEQ D NO: W950 cttgtcatgcctacgttcc 11348 11367 1 Q ID NO: 3622 aaaatcaagggtgttattt 3473 3492 SEQ D NO: 4951 aaatcttattggggatttt 7084 7103 1 Q ID NO: 3623pcataccccgtttgcaag 3491 3510 SEQ D NO W952 cttggattcaaaatgtgga 6858 6877- Q ID NO: 3624F9^caagcagaag^ccag^aag 3504 3523 SEQ D NO 4953 cttcagggaacacaatgca 5185 52041 Q ID NO: 3625p^ag^aag^ccagaagtg^agat 3510 3529SEQ D NO 4954 atctatgccatctcttctg 5633 56521 Q ID NO: 3626Fg^a9^{atcctcgcccactgg} 3523 3542 SEQ D NO 4955 ccagcttccccacatctca 8341 8360 Q ID NO: 3627p9^tc9^cct9^ccaaact9^ct 3540 3559 SEQ D NO 4956 agcacatatgaactggacc 13947 13966 Q ID NO: 3628F9^cttc*^ccaaatggac*^c 3555 3574_SEQ D NO 495 agtttatcagtcagagca 9701 9720 Q ID NO: l3629F99^actcatct9^ctaca9^c 3567 3586 SEQ D NO W958bctgcagtggcccgttcca 8167 81861 Q ID NO: 36309^ctaca9^cttatggctcca 3578 3597 SEQ D O:r959Fgg^a g^acattcctctagc 8211 82301 Q ID NO: 3631 ggtggcatggcattatgat 3610 3629 SEQ D NO 4960 atcacaaattagtttcacc 8947 89661 Q ID NO: 3632^a9^a9^aag^attg^aattt9^aa 3631 3650 SEQ D NO 4961pcaacgatacctgtctct 7713 77321 Q ID NO: 3633 paggcaccaatgtagatac 3657 3676 SEQ D NO: 4962 gtatgctaatagactcctg 3736 37551 Q ID NO: 3634 gacttccaatttccctgtg 3685 3704 SEQ D NO: 963 acaatgcaaaattcagtc 5195 5214 Q ID NO: j3635 tccctcaaacagacatga 3764 3783 SEQ D NO 4964pataagggaggtagggac 12777 12796 1 Q ID NO: 3636 caaacagacatgactttcc 3770 3789 SEQ D NO: 4965ggaactacaatttcatttg 7022 7041 1 Q ID NO: 363/latagttgcaatgagctcat 3809 3828 SEQ D NO: 966^atgatttgaaaatagctat 6693 6712 Q ID NO: ^|3638g^cttcag^aaggcatGtggg 3829 3848SEQ D NO: 4967pccaagaggtatttaaagc 12957 12976 Q ID NO: 3639 gg agttcaacctccagaac 3895 3914 SEQ D NO:r968gttcactccattaacctcc 6314 6333 Q ID NO: 3 40 agaaaacctcttcttaaaa 3940 3959 SEQ D NO: 4969 tttctaaatggaacttct 12173 12192 Q ID NO: 3641 aaaacctcttcttaaaaag 3942 3961 SEQ D NO:4970ctttgaaaaattctctttt 9213 9232 Q ID NO: 3642 aaaaagcgatggccgggtc 3955 3974 SEQ D NO: 4971 gaccttgcaagaatatttt 6343 63621 Q ID NO: 3643 gtcaaatataccttgaaca 3971 3990 SEQ D NO:r972Fgttaacaaattccttgac 7363 73821 Q ID NO: 3644p:gaacaagaacagtttgaa 3984 4003 SEQ D NO:r973pcaagttcctgaccttca 8310 83291 Q ID NO: 3645 agtttgaaaattgagattc 3995 4014 SEQ D NO: 4974 gaatctggctccctcaact 9047 9066 Q ID NO: 36460^ttt9^aaaattg^a9^attcc 3996 4015 SEQ D NO: 4975 ggaaataccaagtcaaaac 10454 10473 1 Q ID NO: l3647pgaaaattgagattcctt 3998 4017 SEQ D NO:r97^{6 a}aggaaaagcgcacctcaa 12030 12049 1 Q ID NO: 3648 ptaaagatgttagagactg 4046 4065 SEQ D NO:r977p^agttg^accacaagcttag 10545 10564 1 Q ID NO: [3649 atgttagagactgttagga 4052 4071 SEQ D O: 4978Fcttaacaccttccacat 8073 8092 1 Q ID NO: 3650 cagccctccacttcaagtc 4074 4093 SEQ D NO:4979gacttctctagtcaggctg 8813 88321

Q ID NO 3698Fgcaacgaccaacttgaag 5083 5102 SEQ D NO 5027 cttcagggaacacaatgca 5185 52041 Q ID NO 3699 caacttgaagtgtagtctc 5092 5111 SEQ D NO 5028gagatgagagatgccgttg 6239 6258' Q ID NO 37oo^gct g^{a aat}g^ag^ctg^aat 5116 5135 SEQ D NO 5029attctcttttcttttcagc 9222 9241 - Q ID NO 3701 gcagagcttggcctctctg 5135 5154 SEQ D NO 5030 cagatacaagaaaaactgc 6899 69181 Q ID NO 3702ptctggggcatctatgaa 5148 5167 SEQ D NO 5031 fttcattcaattgggagaga 6499 65181 Q ID NO 3703ptggggcatctatgaaat 5150 5169 SEQ D NO 5032atttgtaagaaaatacaga 6436 64551 Q ID NO 3704 aacacaatgcaaaattcag 5193 5212 [SEQ D NO 5033 ctgaagcattaaaactgtt 7506 7525 1 Q ID NO 3705 ctcacagagctatcactgg 5231 5250 SEQ D NO 5034 ccagatgctgaacagtgag 8149 81681 Q ID NO 3706 ggg^aag^{t cttatca}g ^c 5247 5266 SEQ D O 5035 gcctacgttccatgtccca 11356 11375 1 Q ID NO 3707pcaaggtcagtcaagaag 5303 5322SEQ D NO 5036 cttcagtgcagaatatgaa 11977 11996 Q ID NO 3708 aatgacatgatgggctcat 5336 5355SEQ D O 5037 atgattatctgaattcatt 6486 6505 1 Q ID NO 3709 gctcatatgctgaaatgaa 5349 5368 SEQ D NO 5038pcagccattgacatgagc 5746 57651 Q ID NO 3710 atatgctgaaatgaaattt 5353 5372_SEQ D NO 5039 aaatagctattgctaatat 6702 6721 1 Q ID NO 371 iptgaacattgcaggctta 5386 5405 SEQ D NO 504θFaagaaccagaagatcaga 10996 110151 Q ID NO 3712 gaacattgcaggcttatca 5389 5408 SEQ D O 5041rtgatatcgacgtgaggttc 12490 12509 1 Q ID NO 3713Fgcaggcttatcactggac 5395 5 14 SEQ D O 5042 gtcctggattccacatgca 11852 11871 Q ID NO 3714Fcaaaacttgacaacattt 5420 5439 SEQ D NO 5043 aaattccttgacatgttga 7370 7389 Q ID NO 3715 atttacagctctgacaagt 5435 5454_SEQ D NO 5044 acttaaaaaatataaaaat 8022 8041 Q ID NO 3716 ctctgacaagttttataag 5443 5462_SEQ D NO 5045 cttacttgaattccaagag 10674 10693 Q ID NO 371 T^gttaatttacagctacagc 5468 5 87SEQ D NO 5046 gctgcatgtggctggtaac 5578 5597 Q ID NO 3718p^ctctgg^taactac tt^a 5491 5510 SEQ D NO 5047Faaaagattactttgagaa 7275 72941 Q ID NO 371 g cctaaaaggagcctaccaa 5596 5615 SEQ D NO 5048pggcaagtaagtgctagg 9376 93951 Q ID NO 3720 aaaaggagcctaccaaaat 5599 5618 SEQ D NO 5049atttacaattgttgctttt 6271 6290 1 Q ID NO 3721 aggagcctaccaaaataat 5602 5621 SEQ D O 5050 attacctatgatttctcct 10127 10146 1 Q ID NO 3722 ataatgaaataaaacacat 5616 5635SEQ D O 5051 atgtcaaacactttgttat 7065 7084 1 Q ID NO 3723 aaaacacatctatgccatc 5626 5645 SEQ D NO 5052gatgaagatgacgactttt 12158 12177 1 Q ID NO 3724 ^ctaag ^ttcagg ^tg^t9 5686 5705SEQ D NO 5053 cacaagtcgattcccagca 9087 9106 1 Q ID NO 3725 g^agtttagccatcggctca 5705 5 24SEQ D NO 5054Fgaggtgactcagagactc 7450 7469 " Q ID NO 3726 gctggcttcagccattgac 5740 5759 SEQ D NO 5055 gtcagtgaagttctccagc 8596 86151 Q ID NO 3727p.tttcagcaatgtcttccg 5790 5809 SEQ D NO 5056 cggagcatgggagtgaaat 8628 8647 Q ID NO 3728ptcagcaatgtcttccgt 5791 5810 SEQ D NO 5057tøcggagcatgggagtgaaa 8627 8646 Q ID NO 372gpcagcaatgtcttccgtt 5792 5811 [SEQ D NO 5058 aacggagcatgggagtgaa 8626 8645 Q ID NO 3730pagcaatgtcttccgttct 5794 5813 SEQ D NO 5059 agaagtgtcttcaaagctg 12412 12431 Q ID NO 3731 rtgtcttccgttctgtaatg 5800 5819 SEQ D NO pOδOcattcaattgggagagaca 6501 6520" Q ID NO 3732 gtcttccgttctgtaatgg 5801 5820 SEQ D NO 5061 ccattcagtctctcaagac 12975 12994 Q ID NO 3733 atgggaaactcgctctctg 5859 5878 SEQ D O 5062 cagataaaaaactcaccat 12213 12232 1 Q ID NO 373499^ag^aac^atactgggcagc 5879 5898SEQ D NO 5063 gctgttttgaagactctcc 1088 11071 Q ID NO 3735 gttgaaagcagaacctctg 5914 5933 SEQ D NO 5064 cagaattcataatcccaac 8274 8293 11 Q ID NO 3736 ptctaggaaaagcatcagt 5983 6002 SEQ D NO 5065 actgcaagatttttcagac 13612 13631 1 Q ID NO 3737tøgcatcagtgcagctcttg 5993 6012 SEQ D NO 5066 caagaacctgttagttgct 13351 13370 1 Q ID NO 3738P^{gaacacaaagtcagt}9^c 6009 6028 SEQ D NO 5067bcacatcaatattgatcaa 6418 6437 1 Q ID NO 3739 gcagacaggcacctggaaa 6046 6065 SEQ D NO 5068Fttcagatggcattgctgc 11610 11629 1 Q ID NO 3740paaactcaagacccaattt 6061 6080 SEQ D NO 5069 aaatcccatccaggttttc 8037 8056 Q ID NO 3741 acaatgaatacagccagga 6084 6103 SEQ D NO 5070ttcctttggctgtgctttgt 9682 9701 - Q ID NO 3742 cttggatgcttacaacact 6103 6122 SEQ D O 5071 agtgaagttctccagcaag 8599 8618 " Q ID NO 3743pgg^{cgtggagcttact}9g 6132 6151 SEQ D NO 5072 ccagaattcataatcccaa 8273 8292 1 Q ID NO 3744 cacttttactcagtgagcc 6198 6217 SEQ D NO [5073|ggctattgatgttagagtg 6988 70071

Q ID NO: 3₇g2 aaattagttggatttattg 7292 7311 SEQ D NO:⁵ 21 caattttgagaatgaattt 10419 104381 Q ID NO: 3793Fggatttattgatgatgct 7300 7319 SEQ D NO 22 agcatgcctagtttctcca 9953 9972 Q ID NO: 3794Fcattgaagatgttaacaa 7353 7372 [SEQ D NO 23Ftgtagatgaaaccaatga 7422 7441 - Q ID NO: 3795 cattgaagatgttaacaaa 7354 7373 SEQ D O 24Fttgtagatgaaaccaatg 7421 7440 Q ID NO: 3796 attgaagatgttaacaaat 7355 7374 SEQ D NO 25 atttaagtatgatttcaat 10495 10514 1 Q ID NO: 3797Ftgaagatgttaacaaatt 7356 7375 SEQ D NO 26 aatttaagtatgatttcaa 10494 10513 1 Q ID NO: 3798Fgaagatgttaacaaattc 7357 7376 SEQ D NO 27taaatttaagtatgatttca 10493 10512 1 Q ID NO: 3799 acatgttgataaagaaatt 7380 7399 SEQ D NO 28 aattccctgaagttgatgt 11487 11506 1 Q ID NO 3800F^{gattaccaccagtttg} 7406 7425 SEQ D NO 29 caaattgaacatccccaaa 8791 8810 1 Q ID NO: 3801 caaaatccgtgaggtgact 7441 7460_SEQ D NO 30 agtccccctaacagatttg 7972 7991 - Q ID NO: 3802aaaatccgtgaggtgactc 7442 7461 SEQ D NO 31 gagtgaaatgctgtttttt 8638 8657 Q ID NO: 3803aggtgactcagagactcaa 7452 7471 SEQ D NO: 32Ftgatgatatctggaacct 10731 10750 Q ID NO: 38049^tg^aaattcag^gctct9^ga 7473 7492_SEQ D NO 33π:ccaatctcctcttttcac 8409 8428 Q ID NO: 3805P^ttgcagt9^tatotgg^aaa 7547 7566SEQ D NO 34Fttcaagcaaatgcacaac 8540 8559 Q ID NO: 3806F^taa9^ttcag^catctttgg 7616 7635 SEQ D NO: 35 ccaatgctgaactttttaa 10173 10192 Q ID NO: 3807F^{gaa ccaaattccga}9^a 7641 7660 SEQ D NO 36ptcctttcttcatcttca 10213 10232 Q ID NO: 3808 aatgtatcaaatggacatt 7684 7703 SEQ D NO 37taatgaagtccggattcatt 11021 11040 1 Q ID NO: 3809 attcagcaggaacttcaac 7700 7719 SEQ D NO 38gttgagaagccccaagaat 6254 6273 1 Q ID NO: 381 o acctgtctctggtcagcca 7722 7741 SEQ D NO 39rtggcaagtaagtgctaggt 9377 93961 Q ID NO: 3811 cctgtctctggtcagccag 7723 7742 SEQ D NO 40 ctggacttctctagtcagg 8810 8829 1 Q ID NO: 38i2gg^tcag^cca99^tttatag^c 7732 7751 SEQ D NO 41 gctaaaggagcagttgacc 10535 10554 1 Q ID NO: 3813 ccaggtttatagcacactt 7738 7757 SEQ D NO 42 aagtccggattcattctgg 11025 11044 1 Q ID NO: 3814 tttatagcacacttgtca 7742 7761 SEQ D NO 43Fgacctgtccattcaaaac 13681 13700 1 Q ID NO: 3815 acttgtcacctacatttct 7753 7772 SEQ D NO 44agaaaaaggggattgaagt 10283 10302 Q ID NO: 3816 ctgattggtggactcttgc 7770 7789 SEQ D NO 45gcaagttaaagaaaatcag 14026 14045 Q ID NO: 3817atgaaagcattggtagagc 7847 7866 SEQ D NO 46 gctcatctcctttcttcat 10208 10227 Q ID NO: 3818F9^aaagcattggtagagca 7848 7867_SEQ D O 47 tgctcatctcctttcttca 10207 10226 Q ID NO: 3819 gggttcactgttcctgaaa 7868 7887SEQ D NO 48Fttcaccatagaaggaccc 8959 8978 Q ID NO: 3820p^{aagaccatccttg}9g^ac 7887 7906 SEQ D NO 49 gtccccctaacagatttga 7973 7992 Q ID NO: 3821 ccttgggaccatgcctgcc 7897 7916 SEQ D NO 50 ggcaccagggctcggaagg 13978 13997 Q ID NO: 3822P^cagg^ctcttcag^aaag^c 7929 7948 SEQ D NO 51 gcttgaaggaattcttgaa 9588 9607 Q ID NO: 3823pcagataaacttcaaaga 8004 8023 SEQ D NO 52FCttcataagttcaatgaa 13183 13202 Q ID NO: 3824 acttcaaagacttaaaaaa 8013 8032 SEQ D NO 53 pttaacaaaagtggaagt 6829 6848 Q ID NO: 3825 atcccatccaggttttcca 8039 8058 SEQ D NO 54Fggagaagcaaatctggat 9472 9491 - Q ID NO: 3826 P^aatrt^{accatccttaaca} 8063 8082 SEQ D NO: 55Fgttgaagtgtctccattc 9889 9908 1 Q ID NO: 382 cattccttcctttacaatt 8089 8108 SEQ D NO 56 aattccaattttgagaatg 10414 10433 1 Q ID NO: 3828P^gaccagatg^ctgaaca 8145 8164 [SEQ D NO 57ptgttgaaagatttatcaa 12932 12951 1 Q ID NO: 3829 aetcaccctgccagacttc 8233 8252 SEQ D O 58 gaagttctcaattttgatt 8522 8541 1 Q ID NO: 3830F9^accttcacataccagaa 8320 8339 SEQ D NO 59ρcttctggaaaagggtca 8884 89031 Q ID NO: 3831 ttccagcttccccacatct 8339 8358 SEQ D NO 60 agattctcagatgagggaa 8921 8940 Q ID NO: 3832 aagctatacagtattctga 8387 8406 SEQ D NO δl cagatggcattgctgctt 11612 11631 Q ID NO: 3833 attctgaaaatccaatctc 8399 8418 SEQ D NO 62gagataaccgtgcctgaat 11552 11571 Q ID NO: 3834ptcacattagatgcaaat 8422 8441 SEQ D NO 63 attttgaaaaaaacagaaa 9738 9757 Q ID NO: 3835paaatgctgacatagggaa 8436 8455 SEQ D NO 64pccatcacaaatcctttg 9670 9689 Q ID NO: 3836P^ag^ag^tccaaattag^aag^t 8508 8527 SEQ D NO 65 actttacttcccaactctc 13410 13429 Q ID NO: 3837agagtccaaattagaagtt 8509 8528 SEQ D NO 66 aactttacttcccaactct 13409 13428 1 Q ID NO: 3838ptcaattttgattttcaa 8527 8546 SEQ D NO:⁵ δTpgattcccttttttgaga 11537 115561 Q ID NO: 3839 caattttgattttcaagca 8530 8549 SEQ D NO 68Fgctgaatccaaaagattg 13660 136791 Q ID NO: 3840 aatgcacaactctcaaacc 8549 8568 SEQ D NO 69 ggtttatcaaggggccatt 12460 12479 1 Q ID NO: 3841 agttctccagcaagtacct 8604 8623 SEQ D NO 70 aggttccatcgtgcaaact 11388 11407 Q ID NO: 3842^a9^tacctgagaacggagca 8616 8635 SEQ D NO 7lK:gctccaggagaacttact 13780 13799 Q ID NO: 3843p^aaacaca9^tgg^caa9^tt 8678 8697_SEQ D NO 72 aactctcaagtcaagttga 13422 13441 Q ID NO 3344 acaatcagcttaccctgga 8751 8770 SEQ D NO 73pcattctgaatatattgt 13380 13399 Q ID NO: 3₈45ptggatagcaacactaaat 8765 8784 SEQ D NO 74 attttctgaacttccccag 12702 12721 Q ID NO: 3846^ctg^acctg^{c caac}g^ag^at 8829 8848 SEQ D NO 75 atctgatgaggaaactcag 12259 12278 Q ID NO: 3847i^ag^atg^a gg^aacacatg^aa 8929 8948SEQ D NO 76pcatgtccctagaaatct 10038 10057 Q ID NO: 384₈paacttttctaaacttga 9060 9079 SEQ D NO 77Fcaaggataacgtgtttga 12618 12637 Q ID NO: 384gpctaaacttgaaattcaa 9067 9086 SEQ D NO 78pgatgatgctgtcaagaa 7308 7327 1 Q ID NO: 3850 p aaattcaatcacaagtcg 9077 9096 SEQ D NO 79 cgacgaagaaaataatttc 13566 13585 !1 Q ID NO: 3851 cactgtttggagaagggaa 9141 9160 SEQ D NO δOpccagaaagcagccagtg 12506 12525 1 Q ID NO: 3852^actg^tttg ^ag^aaggg^aag 9142 9161 SEQ D NO 81 cttccccaaagagaccagt 2898 2917 Q ID NO: 3353 aattctcttttcttttcag 9221 9240 SEQ D NO 82 ctgattactatgaaaaatt 13638 13657 Q ID NO: 3354pcttttcagcccagccat 9230 9249_SEQ D NO 83 atggaaaagggaaagagaa 13494 13513 1 Q ID NO: 3855F^{gaaagttcgttttcca} 9283 9302_SEQ D O 84rtggaagtgtcagtggcaaa 10380 10399 Q ID NO: 3856p^aggg^aa9^atag^acttcct 9312 9331 SEQ D NO 85 aggacctttcaaattcctg 9δ48 9867 Q ID NO: 3857 ataagtacaaccaaaattt 9405 9424 SEQ D NO 86 aaatcaggatctgagttat 14038 14057 Q ID NO: 3858 acaacgagaacattatgga 9435 9454 SEQ D NO 87Fccattctgaatatattgt 13330 13399 Q ID NO: 3859aggaataaatggagaagca 9463 94δ2_SEQ DNO δδFgctggaattgtcattcct 11734 11753 Q ID NO: 3860 agcaaatctggatttctta 9478 9497_SEQ D NO 89Faagttctctgtacctgct 11719 11738 1 Q ID NO: 336ipctttaacaattcctgaa 9502 9521 SEQ D NO 9θFtcaaaacgagcttcagga 13206 13225 1 Q ID NO: 3862F^aacaattcctgaaatg 9505 9524 SEQ D NO 91 catttgatttaagtgtaaa 9621 9640 1 Q ID NO: 3863 acacaataatcacaactcc 9534 9553 SEQ D NO 92 ggagacagcatcttcgtgt 11211 11230 |1 Q ID NO: 3864 aagatttctctctatggga 9561 9580SEQ D NO 93Fcccagaaaacctcttctt 3936 3955 Q ID NO: 3865paaaaaacaggcttgaagg 9578 9597 SEQ D NO 94 ccttttacaattcattttc 13021 13040 1 Q ID NO: 3866P^{gaag aattctt}g^aaaa 9590 9609 SEQ D NO 95 Ftttgagaatgaatttcaa 10422 10441 11 Q ID NO: 3867Fg^aag^{gaattcttgaaaac} 9591 9610 SEQ D NO 96 gttttggctgataaattca 11291 11310 1 Q ID NO: 3368agctcagtataagaaaaac 9640 9659 SEQ D NO 97btttgataagtacaaagct 9805 98241 Q ID NO: 3869p^aaatcctttgacaggca 9720 9739 SEQ D NO 98fejcctgagcagaccattga 11688 11707 Q ID NO: 3₈70atgaaacaaaaattaagtt 9789 9808 SEQ D NO 99 aactttgcactatgttcat 12762 12781 Q ID NO: 3871 aattcctggatacactgtt 9859 9876 SEQ D O 5200 aacacatgaatcacaaatt 893δ δ957 Q ID NO: J3372pccagttgtcaatgttga 9876 9895SEQ D NO 5201π:caaaacgagcttcaggaa 13207 13226 Q ID NO: 3873 aagtgtctccattcaccat 9894 9913 SEQ D NO 5202 atgggaagtataagaactt 4842 4861 Q ID NO: 3874 gtcagcatgcctagtttct 9950 9969 SEQ D NO 5203 agaaaaggcacaccttgac 11080 11099 Q ID NO: 3875ptgccatgggcaatattac 10113 10132 SEQ D NO 5204 gtaagaaaatacagagcag 6440 6459 Q ID NO: 3876raaataccaatgctgaact 10167 10186 SEQ D NO 5205agttgaaggagactattca 7224 7243 Q ID NO: 3877 ttgttgctcatctcctt 10201 10220 SEQ D NO 5206 aaggaaacataaactaata 128δ9 1290δ Q ID NO: 3878F9ttgctcatctcctttct 10204 10223 SEQ D NO 5207pgaagaaatctgcagaaca 12431 12450 1 Q ID NO: 3₈7gftctgtcattgatgcactgc 10232 10251 SEQ D NO 5208gcagtagactataagcaga 13928 13947 1 Q ID NO: l38₈opcacagctctgtctctgag 10305 10324 SEQ D NO 5209 ctcagggatctgaaggtgg 8195 6214 1 Q ID NO: 3881 atttgtggagggtagtcat 10330 10349 SEQ D NO 5210 atgaagtagaccaacaaat 7161 7180 Q ID NO: 3882 atatggaagtgtcagtggc 10377 10396 SEQ D NO 5211 gccacactccaacgcatat 10778 10797 Q ID NO: 33₈3Fggaaataccaagtcaaaa 10453 10472 SEQ D NO 5212 Ftttacaattcattttcca 13023 13042 1 Q ID NO: 38₈4aagtcaaaacctactgtct 10463 10482SEQ D NO 5213 agacctagtgattacactt 12δ59 12878 Q ID NO: 3335 actgtctcttcctccatgg 10475 10494 SEQ D NO 5214 ccatgcaagtcagcccagt 10924 109431 Q ID NO: 3886 cttcctccatggaatttaa 10432 0501 SEQ D NO 521 δttaatcgagaggtatgaag 7148 71671 Q ID NO: 388 attcttcaatgctgtactc 10512 0531 SEQ D NO 5216 gagttgagggtccgggaat 12242 12261 Q ID NO: 3888pg^accacaag^cttagctt 10548 056 SEQ D NO 521 aagcgcacctcaatatcaa 12036 12055 Q ID NO: 38₈9pctcacctcttacttttcc 10573 0592 SEQ D NO 5218 ggaactattgctagtgagg 10649 106681 Q ID NO: 3890 agctgcagggcacttccaa 10710 0729 SEQ D NO 5219ρgggaagaagaggcagct 12289 12308 1 Q ID NO: 3₈9ipccaaaattgatgatatc 10723 0742 SEQ D NO 5220 gatatacactagggaggaa 12745 12764 1 Q ID NO: 3392 gagaacatacaagcaaagc 10860 0879 SEQ D NO 5221 gcttggttttgccagtctc 2467 2486 1 Q ID NO: 3893 atggcaaatgtcagctctt 10897 0916 SEQ D NO 5222aagaggtatttaaagccat 12960 12979 1 Q ID NO: 3S94Fggcaaatgtcagctcttg 10898 0917 SEQ D NO 5223 caagaggtatttaaagcca 12959 12978 1 Q ID NO: 3₈95rttgttcaggtccatgcaag 10914 0933 SEQ D NO 5224 cttgggggaggaggaacaa 14066 14085 1 Q ID NO: 3896F^gttcagg^tccatg^caagt 10915 0934 [SEQ D NO 5225 acttgggggaggaggaaca 14065 14084 11 Q ID NO: 389 agttccttccatgatttcc 10940 0959SEQ D NO 5226 ggaatctgatgaggaaact 12256 12275 Q ID NO: 3898Fgctaacactaagaaccag 10987 1006 SEQ D NO 5227ptggatgtaaccaccagca 11186 11205 Q ID NO: 3899 actaagaaccagaagatca 10994 1013 SEQ D NO 5228Fgatcaagaacctgttagt 13347 13366 Q ID NO: kgooptaagaaccagaagatcag 10995 1014 SEQ D NO 5229 ctgatcaagaacctgttag 13346 13365 Q ID NO: 3901 cagaagatcagatggaaaa 11003 1022 SEQ D NO: 5230 pttcagaccaactctctg 13622 13641 Q ID NO: 3902 aaaaatgaagtccggattc 11018 037SEQ D NO 5231 gaatttgaaagttcgtttt 9280 9299 Q ID NO: 3903 gattcattctgggtctttc 11032 1051 SEQ D NO 5232 gaaaacctatgccttaatc 13166 13185 Q ID NO; 3904 aagaaaaggcacaccttga 11079 1098 SEQ D O 5233 caaaacctactgtctctt 10466 10485 Q ID NO: 3905 aaggacacctaaggttcct 11115 1134 SEQ D O 5234aggacaccaaaataacctt 7572 7591 1 Q ID NO: 3906 pcagcattggtaggagaca 11199 1218 SEQ D NO 5235ratcaacaagtaccactgg 12370 12389 1 Q ID NO: 3907 tttgtgtacaccaaaaac 11239 1258 SEQ D NO 5236gtttttaaattgttgaaag 13148 13167 1 Q ID NO: 3908pcatccctgtaaaagtttt 11277 1296 SEQ D NO 5237taaaagggtcatggaaatgg 8893 8912 1 Q ID NO: 3909Fg^atctaaattcagttctt 11332 1351 SEQ D NO 5238 aagatagtcagtctgatca 13334 13353 1 Q ID NO: gi 0 aagaagctgagaacttcat 11432 1451 SEQ D NO 5239 atgagatcaacacaatctt 13110 13129 1 Q ID NO: 3911 Fttgccctcaacctaccaa 11453 1472 SEQ D NO 524θFtggtacgagttactcaaa 12641 12660 1 Q ID NO: 3912 cttgattcccttttttgag 11536 1555 SEQ D NO 5241 ctcaattttgattttcaag 8528 8547 i Q ID NO: 3913Ftcacgcttccaaaaagtg 11591 1610 SEQ D NO 5242 cactcattgattttctgaa 12693 12712 1 Q ID NO: 3914rtgtttcagatggcattgct 11608 1627SEQ D NO 5243 agcagattatgttgaaaca 11833 11852 1 Q ID NO: 391 atgcagtagccaacaaga 11639 1658 SEQ D NO 5244FCttttcagcccagccatt 9231 9250 1 Q ID NO: 391 eptgagcagaccattgagat 11691 1710 SEQ D NO 5245 atctgatgaggaaactcag 12259 12278 1 Q ID NO: 3917 :gagcagaccattgagatt 11692 1711 SEQ D NO 5246 aatctgatgaggaaactca 12258 12277 1 Q ID NO; 391 epgagattccctccattaa 11703 1722 SEQ D NO 5247paatcttcataagttcaa 13179 13198 1 Q ID NO: 391 g acttggagtgccagtttga 11807 1826 SEQ D NO 5248Fcaattgggagagacaagt 6504 6523 1 Q ID NO: 3920paaatttgaaggacttcag 12004 2023 SEQ D NO 5249ctgagaacttcatcatttg 11438 11457 Q ID NO: 3921 agcccagcgttcaccgatc 12056 2075SEQ D NO 5250 gatccaagtatagttggct 13286 13305 Q ID NO: 3922 pagcgttcaccgatctcca 12060 2079 SEQ D NO 525lFggacctgcaccaaagctg 13960 13979 Q ID NO: 3923 ctccatctgcgctaccaga 12074 2093 SEQ D NO 5252Ftgatatacatcacggag 13711 13730 Q ID NO: 3924 atgaggaaactcagatcaa 12264 2283 SEQ D NO 5253πgagttgcccaccatcat 11667 11686 Q ID NO: 3925 aggcagcttctggcttgct 12300 2319 SEQ D NO: 5254 agcaagtctttcctggcct 3018 3037 Q ID NO; 3926F^gaaaS^acaacgt^gcccaa 12327 2346 SEQ D NO 5255Ftgggagagacaagtttca 6508 6527 Q ID NO; 3g27ptgattatgtcaacaagt 12362 2381 SEQ D NO: 5256 actttgcactatgttcata 12763 12782 Q ID NO: 392₈ cattaggcaaattgatgat 12475 2494 SEQ D NO 525ηatcaacacaatcttcaatg 13115 13134 1 Q ID NO: 3929pgactcaggaaggccaag 12584 2603 SEQ D NO 5258 cttggtacgagttactcaa 12640 12659 1 Q ID NO: 3930 gaaacctgggatatacact 12736 2755 SEQ D NO 5259 agtgattacacttcctttc 12865 12884 1 Q ID NO: 3931pctttcgagttaaggaaa 12877 2896 SEQ D NO 526θFttctgccactgctcagga 13524 13543 1 Q ID NO: 3932 gccattcagtctctcaaga 12974 2993 SEQ D NO 526lH:cttccgttctgtaatggc 5802 5821 1

Table 11. Selected palindromic sequences from human glucose-6-phosphatase

Source Start End Match Start End

Indexlndex Index Index

SEQ ID NO 5291 tccatcttcaggaagctgt 222 241 [SEQ ID NO 5369 acagactctttcagatgga 1340 1359

SEQ ID NO: 5292 ccatcttcaggaagctgtg 223 242SEQ ID NO: 5370 cacagactctttcagatgg 1339 1358

SEQ ID NO: 5293 cctctggccatgccatggg 417 436SEQ ID NO 5371 cccattttgaggccagagg 1492 1511

SEQ ID NO: 5294 ctctggccatgccatgggc 418 437SEQ ID NO 5372 gcccattttgaggccagag 1491 1510

SEQ ID NO: 529δpgaatgtcattttgtggt 621 540SEQ ID NO: 5373 accatacattatcattcaa 2945 2964

SEQ ID NO: 5296Fagtaatgggggaccagc 1886 1905 SEQ ID NO 5374 gctggtctcgaactcctga 2731 2750

SEQ ID NO: δ297pttactgtgcatacatgt 1966 1975 SEQ ID NO: 5375 acatctttgaaaagaaaaa 2983 3002

SEQ ID NO 5298røaggtgccaaggaaatga 50 69SEQ ID NO: 5376 catgtctcagcctcctca 2620 2639

SEQ ID NO 5299 gaggtgccaaggaaatgag 51 70SEQ ID NO 5377 ctcatgtctcagcctcctc 2619 2638

SEQ ID NO: 5300 gggaaagataaagccgacc 487 506SEQ ID NO 5378 ggtcgcctggcttattccc 1295 1314

SEQ ID NO: 5301 pttcctcatcaagttgtt 598 617SEQ ID NO 5379 aacatctttgaaaagaaaa 2982 3001

SEQ ID NO: 5302 ctttcagccacatccacag 651 670SEQ ID NO 5380 ctgtggactctggagaaag 773 792

SEQ ID NO: 5303Fggactctggagaaagccc 776 795SEQ ID NO 5381 gggctggctctcaactcca 884 903

SEQ ID NO: 5304 agcctcctcaagaacctgg 848 867SEQ ID NO 5382 ccagattcttccactggct 2107 2126

SEQ ID NO: 5305 ggcctggggctggctctca 878 897 SEQ ID NO 5383π:gagccaccgcaccgggcc 2801 2820

SEQ ID NO: 5306 gagctcactcccactggaa 1439 1458 SEQ ID NO 5384H:tccaggtagggccagctc 1676 1695

SEQ ID NO 5307 agctaatgaagctattgag 1572 1591 SEQ ID NO; 5385 ctcagcctcctcagtagct 2626 2645

SEQ ID NO 5308 gctaatgaagctattgaga 1573 1592 SEQ ID NO 5386Jtctcagcctcctcagtagc 2625 2644

SEQ ID NO: 5309 ctaaatggctttaattata 1854 1873 SEQ ID NO [5387ftatatttttagaattttag 2683 2702

SEQ ID NO: 5310 ctgcttttctttttttttc 2509 2528SEQ ID NO: 5388 gaaaaatatatatgtgcag 2996 3015

SEQ ID NO 5311 caatcaccaccaagcctgg 19SEQ ID NO: 5389 ccagaatgggtccacattg 812 831

SEQ ID NO 5312 agcctggaataactgcaag 12 31 SEQ ID NO: 5390 cttggatttctgaatggct 1987 2006

SEQ ID NO. 5313 gttccatcttcaggaagct 220 239SEQ ID NO: 5391 agctcactcccactggaac 1440 1459

SEQ ID NO: 5314røgtgggttttggatactg 326 345SEQ ID NO: 5392 cagtcctcccaccctacca 2425 2444

SEQ ID NO 5315 acctgtgagactggaccag 392 411 SEQ ID NO: 5393 ctggagaaagcccagaggt 782 801 4

SEQ ID NO 5316 gctgttacagaaactttca 638 657SEQ ID NO 5394rtgaatggtcttctgccagc 1474 1493 ^' 4

SEQ ID NO 5317 acagcatctataatgccag 666 685SEQ ID NO: 5395 ctgggtgtagacctcctgt 758 777 4

SEQ ID NO: 531 δgggtgtagacctcctgtgg 760 779SEQ ID NO: 5396 ccacattgacaccacaccc 823 δ42 4

SEQ ID NO: 5319 ggtgtagacctcctgtgga 761 760SEQ ID NO: 5397 tccacattgacaccacacc δ22 641

SEQ ID NO 5320 gtgtagacctcctgtggac 762 7δ1 SEQ ID NO: 539δ [gtccacattgacaccacac 821 840

SEQ ID NO 5321 gacctcctgtggactctgg 767 786SEQ ID NO 5399 ccagatattgcactaggtc 2014 2033

SEQ ID NO 5322 cctgggcacgctctttggc δ62 δδ1 SEQ ID NO: 5400 gccagctcacaagcccagg 1667 1706

SEQ ID NO 5323 ctgggcacgctctttggcc δ63 882SEQ ID NO: 5401 ggccagctcacaagcccag 1686 1705 4

SEQ ID NO: 5324 ctggtcttctacgtcttgt 1028 1047SEQ ID NO: 5402 acaaaagcaagacttccag 1663 1682 4

SEQ ID NO 5325 agagtgcggtagtgcccct 1056 1075 SEQ ID NO: 5403 agggccaggattcctctct 2229 2248

SEQ ID NO 5326 tgggcactggtatttggag 1217 1236SEQ ID NO: 5404 ctcccactggaacagccca 1446 1465

SEQ ID NO 5327 gaattaaatcacggatggc 1267 1266SEQ ID NO: 5405 gccaaccaagagcacattc 2311 2330

SEQ ID NO: 532δrøttgctagaagttgggtt 159δ 1617SEQ ID NO 5406 aaccatcctgctcataaca 2967 29δ6

SEQ ID NO 5329 aggagctctgaatctgata 1764 1783 SEQ ID NO 5407rtatcacattacatcatcct 2063 2082

SEQ ID NO 5330Faaatggctttaattatat 1855 1874 SEQ ID NO 5408 atatatgtgcagtatttta 3003 3022

SEQ ID NO 5331 aaaatgacaaggggagggc 2215 2234SEQ ID NO: 5409 gccctccttgcctgttttt 2817 2836

SEQ ID NO: 5332ttaaaggaaaagtcaacat 2330 2349 SEQ ID NO 5410 atgtgcagtattttattaa 3007 3026

SEQ ID NO: 5333 acatcttctctcttttttt 2345 2364SEQ ID NO 5411 aaaagaaaaatatatatgt 2992 3011

SEQ ID NO: 5334 ttctacgtcctcttcccca 197 216 SEQ ID NO 5412 tgggccagccgcacaagaa 1116 1135 SEQ ID NO 5335 tgggtagctgtgattggag 257 276SEQ ID NO [5413 ctcccactggaacagccca 1446 14651 3

SEQ ID NO 5336 gctgtgattggagactggc 263 282SEQ ID NO 5414[gccatgccatgggcacagc 423 442 "

SEQ ID NO [5337 cacttccgtgcccctgata 358 377SEQ ID NO 5415 tatcacccaggctggagtg 2548 2567 -

SEQ ID NO 5338 acatctactctttccatct 464 483SEQ ID NO 5416|agatgggatttcatcatgt 2705 2724'

SEQ ID NO: 5339 ctactctttccatctttca 468 487SEQ ID NO 5417Kgaatactctcacaagtag 1419 1438-

SEQ ID NO: 5340 agataaagccgacctacag 492 511 SEQ ID NO 5418 ctgtttttcaatctcatct 2828 2847-

SEQ ID NO: 5341 fetgcagctgaatgtctgt 553 572SEQ ID NO 5419 acagaaactttcagccaca 644 663 -

SEQ ID NO: 5342 atgtctgtctgtcacgaat 564 583SEQ ID NO 5420 attcaggtatagctgacat 2038 2057

SEQ ID NO 5343 ctgtcacgaatctaccttg 572 591 SEQ ID NO 5421 [caaggtgctaggattacag 2779 2798

SEQ ID NO: 5344 atcaagttgttgctggagt 606 625SEQ ID NO: 5422 actcctgacctcaagtgat 2742 2761

SEQ ID NO: 5345 cagaaactttcagccacat 645 664SEQ ID NO 5423 atgtttcaattaggctctg 2185 2204

[SEQ ID NO: 5346 actttcagccacatccaca 650 669SEQ ID NO 5424Fgtggcgtatcatgcaagt 1818 1837-

|SEQ ID NO: 5347 atgccagcctcaagaaata 678 697SEQ ID NO |5425ttattttttttactgtgcat 1950 1969'

SEQ ID NO: 5348 agaaatattttctcattac 690 709 SEQ ID NO 5426 gtaaatatgactcctttct 2283 2302

SEQ ID NO: 5349 gaaatattttctcattacc 691 710SEQ ID NO 5427 ggtaaatatgactcctttc 2282 2301

SEQ ID NO 535o g^ctg^ctcaaggg^actg g 744 763 SEQ ID NO: 5428 cccaagccaaccaagagca 2306 2325 '

SEQ ID NO 5351 cctgtggactctggagaaa 772 791 SEQ ID NO: 5429 tttcatcatgttggccagg 2713 2732 -

SEQ ID NO 5352 ggagaaagcccagaggtgg 784 803 SEQ ID NO 5430 ccaccgcaccgggccctcc 2805 2824"

SEQ ID NO 5353pgaaacccccatcccaag 1004 1023 SEQ ID NO 5431 cttgaattcctgggctcaa 2405 2424'

SEQ ID NO 5354 cagatggaggtgccatatc 1351 1370 SEQ ID NO: 5432 gatatgcagagtatttctg 2847 2866 '

SEQ ID NO: 5355 ggagctcactcccactgga 1438 1457SEQ ID NO 5433Fccacctgccttggcctcc 2760 2779 '

SEQ ID NO: 5356pgggtaatgtttttgaaa 1553 1572SEQ ID NO 5434ptctctatcccaagccaa 2297 2316"

SEQ ID NO 5357gaagttgggttgttctgga 1606 1625SEQ ID NO 5435 Ccaccccactggatcttc 2131 2150-

SEQ ID NO 5358 aaaagaaggctgcctaagg 1785 1804 SEQ ID NO 5436ccttgcctgcttttctttt 2503 2522

SEQ ID NO 5359aaagaaggctgcctaagga 1786 1805SEQ ID O 5437FCttgcctgcttttcttt 2502 2521

SEQ ID NO 5360aagaaggctgcctaaggag 1787 1806SEQ ID NO; 5438ctccttgcctgcttttctt 2501 2520-

SEQ ID NO 5361 agaaggctgcctaaggagg 1788 1807SEQ ID NO: 5439 cctccttgcctgcttttct 2500 2519 '

SEQ ID NO 5362 atttccttggatttctgaa 1982 2001 SEQ ID NO: 5440 ttcaattaggctctgaaat 2189 2208 -

SEQ ID NO 5363F^cttataag^cccag^ctct 2081 2100SEQ ID NO: 5441 [agagcacattcttaaagga 2319 2338 '

SEQ ID NO 5364 ataagcccagctctgcttt 2086 2105 SEQ ID NO: 5442 aaagctgaagcctatttat 2889 2908-

SEQ ID NO 5365 ggccaggattcctctctca 2231 2250 SEQ ID NO: 5443Fgagccaccgcaccgggcc 2801 2820-

SEQ ID NO 5366 gccaactcctccttgcctg 2493 2512SEQ ID NO: 5444 caggctggagtggagtggc 2555 2574"

SEQ ID NO 5367ttttttttctttttttgag 2519 2538SEQ ID NO: ,5445 ctcataacatctttgaaaa 2977 2996-

SEQ ID NO: 5368pcggcgtgcaccacc^'atgc 2652 2671 SEQ ID NO: 5446 gcatgagccaccgcaccgg 2798 2817"

Table 12. Selected palindromic sequences from rat glucose-6-phosphatase

Source Start End Match Start End Index Index Index Index m Q ID NO: [5447 ctgactattacagcaacag 301 320SEQ ID NO 5471 ctgtggctgaaactttcag 598 617 - Q ID NO: [5448 ctcttggggttggggctgg 831 850 SEQ ID NO 5472 ccagcatgtaccgcaagag 859 878 Q ID NO [5449 tgcaaaggagaactgcgca 879 898SEQ ID NO 5473 tgcgaccgtcccctttgca 1019 1038 Q ID NO: 5450 cctcgggccatgccatggg 376 395SEQ ID NO 5474 cccagtgtggggccagagg 1171 1190 - Q ID NO 5451 pgagcaaaccatatgcaa 1478 1497 SEQ ID NO 5475pgcagagtgtgtcttcaa 2057 2076 ' Q ID NO: 5452 cagcttcctgaggtaccaa 21 SEQ ID NO 5476 ttggtgtctgtgatagctg 123 142 ' Q ID NO 5453 ggtaccaaggaggaaggati 13 32 SEQ ID NO 5477 atccagtcgactcgctacc 66 85 ' Q ID NO 5454 ctccacgactttgggatcc 51 70 SEQ ID NO 5478 ggatcgggaggagggggag 1448 1467 ' Q ID NO 5455 caggactggtttgtcttgg 108 127 SEQ ID NO 5479 ccaagcccgactgtgcctg 2018 2037 4 Q ID NO: 5456pttctatgtcctctttccc 155 174SEQ ID NO 5480 gggacagacacacaagaag 1076 1095 4 Q ID NO: 5457 ttctatgtcctctttccca 156 175 SEQ ID NO: 5481 rcgggacagacacacaagaa 1075 1094 - Q ID NO 5458Fggttccacattcaagaga 177 196 SEQ ID NO 5482 tctcaataatgatagacca 1549 1568 - Q ID NO:[ 5459p:gcctctgataaaacagtt 325 344SEQ ID NO 5483 aactctgagatcttgggca 1868 1887- Q ID NO: 5460 agcccggctcctgggacag 1064 1083 SEQ ID NO: 5484 [ctgtcctccagcctgggct 2034 2053 ' Q ID NO: 5461 agtctctgacacaagtcag 1111 1130 SEQ ID NO 5485 ctgaatggtaatggtgact 1659 1678 - Q ID NO 5462 aaaaaggtgaatttttaaa 1237 1256 SEQ ID NO: 5486rtttattaaaacgacatttt 2201 2220 Q ID NO 5463 acactctcaataatgatag 1545 1564SEQ ID NO 5487 ctatgaatgatgcctgtgt 2121 2140 ' Q ID NO: 5464 aaagaatgaacgtgctcca 37 56 SEQ ID NO 5488p:ggacctcctgtggacttt 724 743 ' Q ID NO 5465ρtttgggatccagtcgact 59 78 SEQ ID NO 5489 agtcagcggccgtgcaaag 1124 1143 " Q ID NO 5466 gtgatcgctgacctcagga 132 151 SEQ ID NO 549θ Cctctctccaaaggtcac 1911 1930 - Q ID NO 5467 ggaacgccttctatgtcct 148 167SEQ ID O: 5491 aggactcatcactgcttcc 1748 1767 - Q ID NO: 5468 gactgtgggcatcaatctc 194 213 SEQ ID NO 5492 gagactggaccagggagtc 357 376 ' Q ID NO: [5469 ggacactgactattacagc 296 315 SEQ ID NO 5493 gctgaacgtctgtctgtcc 518 537 " Q ID NO 5470 aagcccccgtcccagattg 966 985 SEQ ID NO: 5494 caattgtttgctggtgctt 1833 1852 -

Table 13. Selected palindromic sequences from human B-catenin

Source Start End Match Start End Index Index Index Index m Q ID NO 5495 agcagcttcagtccccgcc 70 89 SEQ ID NO 5542 ggcgacatatgcagctgct 2152 2171 Q ID NO: 5496 ccattctggtgccactacc 304 323SEQ ID NO 5543 ggtatggaccccatgatgg 2387 2406 - Q ID NO 5497rtccttctctgagtggtaaa 328 347SEQ ID NO 5544ptattacatcaagaagga 985 1004 - Q ID NO 5498[tctgagtggtaaaggcaat 334 353SEQ ID NO 5545 attgtacgtaccatgcaga 791 810 ' Q ID NO 5499 cagagggtacgagctgcta 473 492 SEQ ID NO 5546hiagctgcaggggtcctctg 2037 2056 - Q ID NO: 5500 ctaaatgacgaggaccagg 677 696SEQ ID NO 5547 cctgtaaatcatcctttag 2539 2558 ' Q ID NO: 5501 [taaatgacgaggaccaggt 678 697 SEQ ID NO 5548 acctgtaaatcatccttta 2538 2557 1 Q ID NO: 5502 gtcctgtatgagtgggaac 383 402SEQ ID NO 5549 gttccgaatgtctgaggac 2176 21952 - Q ID NO: 5503 cccagcgccgtacgtccat 1839 1858 SEQ ID NO 5550 atgggctgccagatctggg 2451 24702 - Q ID NO: 5504 CCcctgagggtatttgaa 143 162SEQ ID NO 5551 pcacatcctagctcggga 1929 1948 1 Q ID NO: 5505 gggtatttgaagtatacca 151 170SEQ ID NO 5552 tggttaagctcttacaccc 1680 1699 - Q ID NO 5506 gctgttagtcactggcagc 260 279 SEQ ID NO 5553 gctgcctccaggtgacagc 2494 2513 - Q ID NO: 5507 gtcctgtatgagtgggaac 383 402SEQ ID NO 5554 gttcgccttcactatggac 1652 1671 Q ID NO: 5508 Ctgtatgagtgggaaca 384 403SEQ ID NO 5555hgttccgaatgtctgagga 2175 2194- Q ID NO: 5509 gtatgcaatgactcgagct 454 473SEQ ID NO 5556 agctggcctggtttgatac 2517 2536 ' Q ID NO 551 Ogtccagcgtttggctgaac 563 582SEQ ID NO 5557|gttcgccttcactatggac 1652 1671 Q ID NO 5511natcaagatgatgcagaac 623 642SEQ ID NO 5558 gttcgtgcacatcaggata 1820 1839 - Q ID NO 5512K:atggtccatcagctttct 718 3 SEQ ID NO 5559 agaaagcaagctcatcata 1126 1145 Q ID NO: 5513 ccctggtgaaaatgcttgg 915 934 SEQ ID NO 5560 ccaaagagtagctgcaggg 2029 2048 Q ID NO: 5514 agctttaggacttcacctg 1291 1310SEQ ID NO 5561 caggtgacagcaatcagct 2502 2521 Q ID NO 551 δggaatctttcagatgctgc 1356 1375SEQ ID NO 5562 gcagctgctgttttgttcc 2162 2181 Q ID NO: 5516K:gtccttcgggctggtgac 1549 1568 SEQ ID NO 5563 gtcatctgaccagccgaca 1605 1624 ' Q ID NO 5517 cacagctcctctgacagag 2107 2126SEQ ID NO 5564 ctctaggaatgaaggtgtg 2134 2153 - Q ID NO 5518 ccagacagaaaagcggctg 245 264SEQ ID NO 5565 cagctcgttgtaccgctgg 828 8472 Q ID NO: 5519 cagcagcgttggcccggcc 23 SEQ ID NO 5566 ggccaccaccctggtgctg 2420 2439 - Q ID NO 5520 aggtctgaggagcagcttc 60 79SEQ ID NO 5567 gaagaggatgtggatacct 359 378 ' Q ID NO: 5521 actgttttgaaaatccagc 174 193SEQ ID NO 5568 gctgatattgatggacagt 437 456 " Q ID NO 5522 ctgatttgatggagttgga 213 232 SEQ ID NO 5569 tccaggtgacagcaatcag 2500 2519 Q ID NO 5523 ccagacagaaaagcggctg 245 264 SEQ ID NO 5570 cagcaacagtcttacctgg 275 294 Q ID NO: 5524 acagctccttctctgagtg 323 342SEQ ID NO: 5571 cactgagcctgccatctgt 1579 1598 - Q ID NO: 5525Fggatacctcccaagtcct 369 388 SEQ ID NO 5572 aggactaaataccattcca 1972 1991 Q ID NO 5526 tcaagaacaagtagctgat 424 443 SEQ ID NO 5573 atcagctggcctggtttga 2514 2533 ' Q ID NO 5527 agctcagagggtacgagct 469 488SEQ ID NO 5574 agctggtggaatgcaagct 1276 1295 - Q ID NO 5528 gcatgcagatcccatctac 516 535SEQ ID NO 5575 gtagaagctggtggaatgc 1271 1290 ' Q ID NO 5529 ccacacgtgcaatccctga 645 664SEQ ID NO 5576 tcagatgatataaatgtgg 1430 1449 - Q ID NO 5530 cacacgtgcaatccctgaa 646 665 SEQ ID NO 5577n:tcagatgatataaatgtg 1429 1448 ' Q ID NO 5531 ggaccttgcataacctttc 846 865SEQ ID NO: 5578 gaaatcttgccctttgtcc 1743 1762 ' Q ID NO 5532 ctccacaaccttttattac 974 993SEQ ID NO 5579 gtaaatcatcctttaggag 2542 2561 Q ID NO 5533 cagagtgctgaaggtgcta 1222 1241 SEQ ID NO 5580 tagctgcaggggtcctctg 2037 2056 - Q ID NO 5534 ggactctcaggaatctttc 1347 1366 SEQ ID NO 5581 gaaatcttgccctttgtcc 1743 1762 ' Q ID NO 5535Fgatataaatgtggtcacc 1435 1454SEQ ID NO: 5582 ggtgacagggaagacatca 1562 1581 ' Q ID NO 5536 cccagcgccgtacgtccat 1839 1858 SEQ ID NO 5583 atggccaggatgccttggg 2370 2389 Q ID NO 5537 gtccatgggtgggacacag 1852 1871 SEQ ID NO 5584 ctgtgaacttgctcaggac 2053 2072 Q ID NO 5538 ttgtaccggagcccttcac 1915 1934 SEQ ID NO: 5585|gtgaacttgctcaggacaa 2055 2074 Q ID NO; 5539pgttatcagaggactaaa 1962 1981 SEQ ID NO: 5586ptaggagtaacaatacaa 2553 2572 Q ID NO: 5540 gaagctattgaagctgagg 2084 2103 SEQ ID NO 5587 cctctgacagagttacttc 2114 2133 - Q ID NO 5541 H:cagaacagagccaatggc 2247 2266 SEQ ID NO: 5588 gccaccaccctggtgctga 2421 2440 - Table 14. Selected palindromic sequences from human hepatitis C viras (HCV)

Source Start End Match Start Endrø Index Index Indexflndex^

SEQ ID NO 5589 cagcacctgggtgctggta 5314 5333 SEQ D NO 35rcaccatcacccagctgctg 6196 6215 1

SEQ ID NO 5590 aactcgtccggatgcccgg 1682 1701 SEQ D NO 36 ccgggcagcgggtcgagtt 8202 8221 1

SEQ ID NO 5591 cgctgctgggtagcgctca 1049 1068 SEQ D NO 37 tgagagcgacgccgcagcg 6151 6170 1

SEQ ID NO 5592 ctccggatcccacaagccg 1352 1371 SEQ D NO 38 cggcatgtgggcccgggag 6053 6072 1

SEQ ID NO 5593 tgtaacatcgggggggtcg 2048 2067 SEQ D NO 39 cgacccctcccacattaca 6871 6890

SEQ ID NO 5594 gtaacatcgggggggtcgg 2049 2068 SEQ D O 40 ccgacccctcccacattac 6870 6889

SEQ ID NO 5595 cagccaccaagcaggcgga 5556 5575SEQ D NO 41 tccggctggttcgttgctg 9254 9273 7

SEQ ID NO 5596 ctcaccacccagaacaccc 5744 5763SEQ D NO 42 gggtgtgcacggtgttgag 6291 6310 7

SEQ ID NO 5597 ccagccttaccatcaccca 6189 6208SEQ D NO 43rtgggcgctggtatcgctgg 5832 5851 7

SEQ ID NO 5598 ctacgccgtgttccggctc 6249 6268 SEQ D NO 44 gagcccgaaccggacgtag 6830 6849 7

SEQ ID NO 5599 tacgccgtgttccggctcg 6250 6269 SEQ D NO 45 cgagcccgaaccggacgta 6829 6848

SEQ ID NO 5600 gagttcctggtaaaagcct 8216 8235 SEQ D NO 46 aggctatgactaggtactc 8634 8653

SEQ ID NO 5601 atggcggggaactgggcta 1430 1449 SEQ D O 47 agcgcattttcactccat 9019 90382

SEQ ID NO 5602 aaccaaacgtaacaccaac 370 389SEQ D NO 48 gttgccgctaccttaggtt 4115 4134

SEQ ID NO 5603 ggtggtcagatcgttggtg 419 438 SEQ D O 49 caccagcccgctcaccacc 5734 5753

SEQ ID NO 5604 ccttggcccctctatggca 584 603SEQ D O 50n:gccaacgtgggtacaagg 6374 6393

SEQ ID NO 5605 taccccggccacgcgtcag 1265 1284 SEQ D NO 51 ctgacgactagctgcggta 8465 8484

SEQ ID NO 5606 gggcacgctgcccgcctca 1508 1527SEQ D NO 52 tgagacgacgaccgtgccc 4759 4778

SEQ ID NO 5607 ctgcaatgactccctccag 1624 1643SEQ D NO 53 ctggtggccctcaatgcag 2594 2613

SEQ ID NO 5608 aaccgatcgtctcggcaac 1897 1916SEQ D NO 54 gttgccgctaccttaggtt 4115 4134

SEQ ID NO 5609 gtgcggggcccccccgtgt 2032 2051 SEQ D NO 55 acaccacgggcccctgcac 6537 6556

SEQ ID NO 5610 atgtggggggcgtggagca 2238 2257SEQ D NO 56 tgctcaatgtcctacacat 7610 7629

SEQ ID NO 5611 ggagagcgttgcaacttgg 2288 2307SEQ D NO 57 ccaagctcaaactcactcc 9207 9226 6

SEQ ID NO 5612 cgtccgttgccggagcgca 2613 2632 SEQ D NO 58 tgcgagcccgaaccggacg 6827 6846 6

SEQ ID NO 5613 gtctggcattattgacctt 2817 2836 SEQ D NO 59 aaggtcacctttgacagac 7763 7782 6

SEQ ID NO 5614H:ctttgatatcaccaaact 2997 3016 SEQ D NO 60 agttcgatgaaatggaaga 5454 5473 6

SEQ ID NO 561 δcttctgattgccatactcg 3014 3033 SEQ D NO 61 cgagcaattcaagcagaag 5δ18 6637

SEQ ID NO 6616 gcggcgtgtggggacatca 3314 3333 SEQ D O 62 tgatcacgccatgcgccgc 7641 7660

SEQ ID NO 5617gggacatcatcctgggcct 3324 3343SEQ D O 63 aggcggtggattttgtccc 391 δ 3934

SEQ ID NO 6618 gggcgtcttccgggccgct 3874 3893 SEQ D NO 64 agcggcacggcgaccgccc 7439 7458

SEQ ID NO 5619 ggcgtcttccgggccgctg 3875 3894SEQ D NO 65 cagcggcacggcgaccgcc 7438 7457

SEQ ID NO 5620 gcgtcttccgggccgctgt 3876 3895 SEQ D NO 66 acaggtgccctgatcacgc 7631 7650

SEQ ID NO 6621 gtccccggtcttcacagac 3961 3980 SEQ D NO 67 gtcttggaagaacccggac 7262 7271

SEQ ID NO 6622 catcaggactggggtaagg 4174 4193 SEQ D NO 68 ccttcctcaagccgtgatg 81 δδ 8174

SEQ ID NO 6623 ccgacggtggttgctccgg 4246 4264 SEQ D NO 69 ccgggggaacggccctcgg 4863 4872

SEQ ID NO 6624 ggggggaaggcacctcatt 4501 4520SEQ D NO 70 aatgttgtgacttggcccc 8334 8353

SEQ ID NO 5625 ccgagcaattcaagcagaa 5517 5536SEQ D NO 71 ttctgattgccatactcgg 3015 3034

SEQ ID NO [5626 agatgaaggcaaaggcgtc 7821 7840SEQ D NO 72 [gacgaccttgtcgttatct 8564 8583

SEQ ID NO [5627 cccctagggggcgctgcca 767 786 SEQ D NO 73rtggccggcgccccccgggg 3674 3693

SEQ ID NO 5628 ctcccggcctagttggggc 646 665SEQ D NO 74 gcccccccttgagggggag 7519 75382

SEQ ID NO 5629 ttccgctcgtcggcggccc 750 769 SEQ D NO 75 gggcaaaggacgtccggaa 7923 79422

SEQ ID NO 5630 cccctagggggcgctgcca 767 786 SEQ D NO 76rtggcgggggcccactgggg 1383 14022

SEQ ID NO 5631 gccccgccggcatgcgaca 1222 1241 SEQ D NO 77 tgtcccagggggggagggc 9147 91662

SEQ ID NO [5632 aggacgaccgggtcctttc 178 197 SEQ D O 78 gaaaaaggacggttgtcct 7341 7360

SEQ ID NO 5633 ggacgaccgggtcctttct 179 198 SEQ D NO 79 agaaaaaggacggttgtcc 7340 7359 &^' ID NO la^'aa^'accaa^'acgtaacacca 368 387SEQ ID NO 6180 tggtttttttttttttttt 9443946215

SEQ ID NO 5635caaccgccgcccacaggac 385 404 SEQ ID NO 81 gtcctgaacccgtctgttg 4100 4119 -

SEQ ID NO 5636 cggtggtcagatcgttggt 418 437SEQ ID NO 82 accattgagacgacgaccg 4754 4773 '

SEQ ID NO 5637 acctgttgccgcgcagggg 444 463SEQ ID NO 83 ccccggccacgcgtcaggt 1267 1286 -

SEQ ID NO: 5638 tgccgcgcaggggccccag 450 469SEQ ID NO 84 ctgggcgcgctgacgggca 3164 3183 -

SEQ ID NO 5639 gggccccaggttgggtgtg 460 479 SEQ ID NO 85 cacagcctgtctcgtgccc 9296 9315 -

SEQ ID NO 5640 gttggggccccacggaccc 657 676SEQ ID NO 86 gggtgggtagccgcccaac 5783 5802 '

SEQ ID NO: 5641 ggggccccacggacccc 658 677SEQ ID NO 87 ggggtgggtagccgcccaa 5782 5801

SEQ ID NO 5642 tggggccccacggaccccc 659 678SEQ ID NO 88 gggggtgggtagccgccca 5781 5800 -

SEQ ID NO 5643 cctcacatgcggcctcgcc 715 734 SEQ ID NO 89 ggcggggcgacaatagagg 3774 3793 '

SEQ ID NO 5644 cacatgcggcctcgccgac 718 737SEQ ID NO 90 gtcgtcggagtcgtgtgtg 6020 6039 -

SEQ ID NO 5645 tccgctcgtcggcggcccc 751 770 SEQ ID NO 91 ggggcaaaggacgtccgga 7922 7941

SEQ ID NO 5646 ggcgctgccagggccttgg 776 795SEQ ID NO 92 ccaagccacagtgtgcgcc 5110 5129 "

SEQ ID NO: 5647 ccatgtcacgaacgactgc 943 962SEQ ID NO 93 gcagcaacacgtggcatgg 6498 6517 -

SEQ ID NO 5648 gtgccctgcgttcgggagg 1019 1038 SEQ ID NO 94 cctcacaacgggggggcac 1495 1514 -

SEQ ID NO: 5649 gccctgcgttcgggaggg 1020 1039 SEQ ID NO 95 ccctcacaacgggggggca 1494 1513 -

SEQ ID NO 5650 gccctgcgttcgggagggt 1021 1040 SEQ ID NO 96 accctcacaacgggggggc 1493 1512 -

SEQ ID NO 5651 aggaatgctaccatcccca 1085 1104SEQ ID NO 97n:gggcatcggcacagtcct 4323 4342

SEQ ID NO 5652 tccccactacgacaatacg 1098 1117SEQ ID NO 98 cgtattcccagatttggga 8092 8111

SEQ ID NO 5653 atacgacaccacgtcgatt 1112 1131 SEQ ID NO 6199 aatcaatgctgtagcgtat 4576 4595

SEQ ID NO 5654 atttgctcgttggggcggc 1128 1147SEQ ID NO 6200 gccgccacttgcggcaaat 9164 9183

SEQ ID NO 5655 ccttctcgccccgccggca 1215 1234 SEQ ID NO 6201 rtgccaacgtgggtacaagg 6374 6393 '

SEQ ID NO: 5656 accccggccacgcgtcagg 1266 1285SEQ ID NO 6202 cctgccgcggttaccgggt 6340 6359

SEQ ID NO 5657 gccctcgtagtgtcgcagt 1331 1350SEQ ID NO 6203 actgcgtcggcatgtgggc 6046 6065

SEQ ID NO: 5658 gccgtctcagagaatccag 1558 1577SEQ ID NO 6204 ctggtatcgctggtgcggc 5838 5857

SEQ ID NO 5659 ctgaactgcaatgactccc 1619 1638 SEQ ID NO 6205 gggacagatcggagctcag 2313 2332 '

SEQ ID NO 5660 agactgggtttcttgccgc 1641 1660 SEQ ID NO 6206 gcggcgagcctacgagtct 8609 8628 '

SEQ ID NO 5661π:cgtccggatgcccggagc 1685 1704SEQ ID NO 6207 gctccgggggcgcttacga 4257 4276 '

SEQ ID NO 5662 ccagggatggggtcctatc 1738 1757SEQ ID NO 6208 gataacttcccctacctgg 5084 5103 -

SEQ ID NO 5663 gacaaccgatcgtctcggc 1894 1913SEQ ID NO 6209 gccgcggttaccgggtgtc 6343 6362 '

SEQ ID NO 5664 caagacgtgcggggccccc 2026 2045SEQ ID NO 6210 ggggtctcccccctccttg 6919 6938 -

SEQ ID NO 5665 acgtgcggggcccccccgt 2030 2049 SEQ ID NO 6211 acgggcgcccccattacgt 4202 4221

SEQ ID NO 5666 ccggaagcaccccgaggcc 2101 2120 SEQ ID NO 6212 ggccgctgtatgcacccgg 3886 3905 '

SEQ ID NO 5667 aggccacgtactcaaaatg 2115 2134SEQ ID NO 6213 cattatgtccaaatggcct 3137 3156 -

SEQ ID NO 5668K:gtatgtggggggcgtgga 2235 2254SEQ ID NO 6214π:ccaagtggcccatctaca 4011 4030 '

SEQ ID NO 5669 gagtggcaggttctgccct 2354 2373SEQ ID NO 621 δagggcaggggtggcgactc 3400 3419 -

SEQ ID NO: 5670π:cctttgcaatcaaatggg 2474 2493SEQ ID NO 6216 cccaccttatgggcaagga 8861 8880 '

SEQ ID NO: 5671 agcccaggccgaggccgcc 2566 2585SEQ ID NO 6217 ggcgtccacagtcaaggct 7834 7853 '

SEQ ID NO: 5672 ggcggcatatgctttctat 2698 2717 SEQ ID NO 6218 atagaagaagcctgccgcc 7865 7884 '

SEQ ID NO [5673 gcggcatatgctttctatg 2699 2718 SEQ ID NO 6219 catagaagaagcctgccgc 7864 7883'

SEQ ID NO 5674 cggcatatgctttctatgg 2700 2719SEQ ID NO 6220ccatagaagaagcctgccg 7863 7882'

SEQ ID NO 5675 gcatgtgtgggttccccc 2913 2932SEQ ID NO 6221 ggggggacggcatcatgca 6402 6421

SEQ ID NO 5676 cccccctcaacgtccgggg 2928 2947SEQ ID NO 6222 ccccaatcgatgaacgggg 9376 9395

SEQ ID NO 5677 gggcaggggtggcgactcc 3401 3420 SEQ ID NO 6223 ggaggccgcaagccagccc 8066 8085 "

SEQ ID NO 5678 atgttggactgtctaccat 3574 3593 SEQ ID NO 6224 atggtaccgaccctaacat 4158 4177-

SEQ ID NO 5679Fgttggactgtctaccatg 3575 3594 SEQ ID NO 6225 catggtaccgaccctaaca 4157 4176 -

SEQ ID NO 5680 cgttccctgacaccatgca 3695 3714SEQ ID NO 6226tejcacgatgctcgtgaacg 8543 8562 '

SEQ ID NO: 5681 acaccatgcacctgtggca 3704 3723SEQ ID NO 6227rtgccgcggttaccgggtgt 6342 6361

SEQ ID NO: 5682 caccatgcacctgtggcag 3705 3724SEQ ID NO 6228 ctgccgcggttaccgggtg 6341 6360

SEQ ID NO: 5683 ggcatcggcacagtcctgg 4325 4344 SEQ ID NO 6229 ccaggattgcccgtttgcc 4979 4998 ' ,„ .. . feE. "ΪD"NO^a; Sa^gcgg^'agacggctggagc 4347 4366SEQ ID NO 6230 gctccccccagcgctgctt 5804 5823 1 5

SEQ ID NO 5685 ggagcgcggcttgtcgtgc 4361 4380_SEQ ID NO 6231 gcacggcgaccgcccctcc 7443 7462

SEQ ID NO; 5686 cgaagccatcaagggggga 4489 4508 SEQ ID NO 6232K:ccccccagcgctgcttcg 5806 5825

SEQ ID NO 5687rcggaagtgtctcatacggc 5165 5184SEQ ID O 6233 gccggattacaatcctcca 7225 7244

SEQ ID NO 5688 gggtgctggtaggcggagt 5322 5341 SEQ ID NO 6234 actcgcgatcccaccaccc 8765 8784

SEQ ID NO 5689 gtgggtaggatcatcttgt 5390 5409SEQ ID NO 6235 acaacatggtctacgccac 7713 7732

SEQ ID NO 5690 cgccgagcaattcaagcag 5515 5534SEQ ID NO 6236 ctgcacgccttccccggcg 6550 6569

SEQ ID NO 5691 tggagtccaagtggcgagc 5592 5611 SEQ ID NO 6237 gctcctcatacggattcca 8175 8194

SEQ ID NO 5692 tggcgagctttggagacct 5603 5622 SEQ ID NO 6238 aggtgccctgatcacgcca 7633 7652

SEQ ID NO 5693 gcccgctcaccacccagaa 5739 5758SEQ ID NO 6239H:tctggcgggctatggggc 5895 5914

SEQ ID NO 5694κgagtgacttcaagacctg 6306 6325SEQ ID NO 6240 caggctataaaatcgctca 8363 8382

SEQ ID NO: 5695 atgtcaaaaacggttccat 6456 6475SEQ ID NO 6241 atggtaccgaccctaacat 4158 4177

SEQ ID NO: 5696 ccgaaaacctgcagcaaca 6488 6507SEQ ID NO 6242rtgttcctccaatgtgtcgg 8708 8727

SEQ ID NO: 5697 ggcgccaaactattccaag 6565 6584SEQ ID NO 6243 cttgaaagcctctgccgcc 8500 8519

SEQ ID NO: 5698 gccctccttgagggcgaca 6967 6986SEQ ID NO 6244 tgtctcctacttgaagggc 3814 3833

SEQ ID NO 5699 cacccgcgtggagtcggag 7078 7097SEQ ID NO 6245 ctccggtggtacacgggtg 7278 7297

SEQ ID NO 5700 ggagggggatgagaatgaa 7138 7157 SEQ ID NO 6246ntcatgctgtgcctactcc 9326 9345

SEQ ID NO: 5701 gcggcgatacccatatggg 7202 7221 SEQ ID NO 6247 cccagggggggagggccgc 9150 9169

SEQ ID NO 5702Ktgccacctgtcaaggccc 7301 7320 SEQ ID NO: 6248 gggccgccacttgcggcaa 9162 9181

SEQ ID NO; 5703 cccccccttgagggggagc 7520 7539SEQ ID NO 6249 gctcccggcctagttgggg 645 664

SEQ ID NO: 5704 ctgctgctcaatgtcctac 7606 7625SEQ ID NO 6250 gtaggactggcaggggcag 4809 4828

SEQ ID NO: 5705 catggacaggtgccctgat 7626 7645SEQ ID NO 6251 atcattgaacgactccatg 8996 9015

SEQ ID NO: 5706 atggacaggtgccctgatc 7627 7646SEQ ID NO 6252 gatcattgaacgactccat 8995 9014

SEQ ID NO: 5707 ggctatgactaggtactcc 8635 8654SEQ ID NO: 6253 ggagcaacttgaaaaagcc 8920 8939

SEQ ID NO 5708 caccatagatcactcccct 27 46 SEQ ID NO: 6254 agggccttggcacatggtg 785 8042

SEQ ID NO 5709 agctgttcaccttctcgcc 1206 1225 SEQ ID NO 6255 ggcgtgctgacgactagct 8459 84782

SEQ ID NO: 5710 ctgcaatgactccctccag 1624 1643 SEQ ID NO 6256 ctggtgcggctgttggcag 5847 58662

SEQ ID NO 5711 atgtggggggcgtggagca 2238 2257SEQ ID NO: 6257H:gctgcgccatcacaacat 7701 77202

SEQ ID NO 5712røgggacatcatcctgggc 3322 3341 SEQ ID NO 6258 gcccaactcgctcccccca 5795 58142

SEQ ID NO: 5713 gggacatcatcctgggcct 3324 3343 SEQ ID NO: 6259 aggcaggagataacttccc 5076 50952

SEQ ID NO: 5714 gggagatactcctggggcc 3366 3385SEQ ID NO: 6260 ggcccctgcacgccttccc 6545 65642

SEQ ID NO 5715atgttggactgtctaccat 3574 3593SEQ ID NO: 6261 atggtctacgccacgacat 7718 77372

SEQ ID NO:¹ 5716ccagccttaccatcaccca 6189 6208SEQ ID NO: 6262 tgggtacaagggagtctgg 6382 6401

SEQ ID NO: 5717 gccctccttgagggcgaca 6967 6986SEQ ID NO 6263rtgtcccagggggggagggc 9147 91662

SEQ ID NO; 5718 ccagcccccgattgggggc 1 20 SEQ ID NO: 6264 gcccgagggcagggcctgg 550 569

SEQ ID NO 5719 accatagatcactcccctg 28 47SEQ ID NO 6265 cagggccttggcacatggt 784 803

SEQ ID NO 5720 atgagtgtcgtgcagcctc 95 114SEQ ID NO 6266 gaggccgcgatgccatcat 2946 2965

SEQ ID NO: 5721 gtgcagcctccaggacccc 104 123SEQ ID NO 6267 gggggacggcatcatgcac 6403 6422

SEQ ID NO: 5722 tgcagcctccaggaccccc 105 124SEQ ID NO: 6268 ggggggacggcatcatgca 6402 6421

SEQ ID NO: 5723 ccaggaccccccctcccgg 113 132SEQ ID NO 6269 ccggctggttcgttgctgg 9255 9274

SEQ ID NO: 5724 accccccctcccgggagag 118 137 SEQ ID NO 6270 ctctcatgccaacgtgggt 6368 6387

SEQ ID NO: 5725 ccccctcccgggagagcca 121 140SEQ ID NO 6271 rtggcaatgagggcatgggg 598 617

SEQ ID NO: 5726 agactgctagccgagtagt 243 262 SEQ ID NO 6272 actatgcggtccccggtct 3953 3972

SEQ ID NO: 5727 agccgagtagtgttgggtc 251 270 SEQ ID NO 6273 gaccaggatctcgtcggct 3656 3675

SEQ ID NO: 5728 ggtgcttgcgagtgccccg 299 318 SEQ ID NO: 6274 cggggccttggttgacacc 2139 2158

SEQ ID NO: 5729 gcgagtgccccgggaggtc 306 325 SEQ ID NO: 6275 gacccccggcgtaggtcgc 671 690

SEQ ID NO 5730 accgtgcaccatgagcacg 331 350 SEQ ID NO: 6276 cgtgcaatacctgtacggt 2437 2456

SEQ ID NO [5731 cccgggcggtggtcagatc 412 431 SEQ ID NO [6277 gatcatgcatactcccggg 997 1016

SEQ ID NO 5732 gccgcgcaggggccccagg 451 470SEQ ID NO 6278 cctgcacgccttccccggc 6549 6568

SEQ ID NO 5733 [accccgtggaaggcgacag 511 530 SEQ ID NO 6279 ctgtatgcacccggggggt 3891 3910 fe ' ID NO m [ccccg ggaag^'gcgacagc 512 531 SEQ ID NO 6280 gctgtatgcacccgggggg 3890 39091 4

SEQ ID NO 5735 agcctatccccaaggctcg 528 547SEQ ID NO 6281 cgagggcagggcctgggct 553 572 1

SEQ ID NO 5736 ctatccccaaggctcgccg 531 550 SEQ ID NO 6282 cggctgtcgttcccgatag 5418 5437 '

SEQ ID NO 5737Fatccccaaggctcgccgg 532 551 SEQ ID NO 6283 ccggctgtcgttcccgata 5417 5436

5738 cgggtatccttggcccctc

SEQ ID NO 577 596 SEQ ID NO 6284 gaggccgcaagccagcccg 8067 8086

SEQ ID NO 5739 gcatggggtgggcaggatg 609 628 SEQ ID NO 6285[catcgataccctcacatgc 706 725

SEQ ID NO: 574θFcctgtcaccccgcggctc 630 649 SEQ ID NO 6286 gagctgcaaagctccagga 8523 8542

SEQ ID NO [5741 gggccccacggacccccgg 661 680SEQ ID NO: 6287 cggccgcatatgcggccc 4064 4083 4

SEQ ID NO 5742 ggccccacggacccccggc 662 681 SEQ ID NO 6288 gccggccgcatatgcggcc 4063 4082 ^' 4

SEQ ID NO: 5743 cggcctcgccgacctcatg 724 743SEQ ID NO 6289 catgaggatcatcgggccg 6472 6491

SEQ ID NO 5744 ggcctcgccgacctcatgg 725 744SEQ ID NO 6290 ccatgaggatcatcgggcc 6471 6490

SEQ ID NO 5745 ggccccctagggggcgctg 764 783 SEQ ID NO 6291 cagctccgaattgtcggcc 7414 7433 4

SEQ ID NO: 5746H:ggcacatggtgtccgggt 792 811 SEQ ID NO 6292 acccacgctgcacgggcca 5188 5207 1 4

SEQ ID NO: 5747 cttcctcttggctctgctg 868 887SEQ ID NO 6293 cagcataggtcttgggaag 5863 5882 4

SEQ ID NO 5748 catgtcacgaacgactgct 944 963 SEQ ID NO 6294 agcagtgctcacttccatg 6847 6866 ^' 4

SEQ ID NO 5749 gaggcggcggacttgatca 983 1002 SEQ ID NO 6295 gatggcattcacagcctc 5712 5731

SEQ ID NO:| 5750 catccccactacgacaata 1096 1115SEQ ID NO |6296|tattaccggggtcttgatg 4592 4611

SEQ ID NO 5751 gctgttcaccttctcgccc 1207 1226 SEQ ID NO 6297 gggctgcgtgggaaacagc 8793 8812'

SEQ ID NO 5752 gccccgccggcatgcgaca 1222 1241 SEQ ID NO 6298H:gtctcctacttgaagggc 3814 3833'

SEQ ID NO: 5753Fggcctgggacatgatgat 1293 1312 SEQ ID NO 6299 atcaatttgctccctgcca 5981 6000 4

SEQ ID NO 5754 cacaagccgtcatcgacat 1362 1381 SEQ ID NO 6300 atgtttgggactgggtgtg 6279 6298 4

SEQ ID NO 5755 agccgtcatcgacatggtg 1366 1385SEQ ID NO 6301 caccaagcaggcggaggct 5560 55791

SEQ ID NO 5756 ggtggcgggggcccactgg 1381 1400 SEQ ID NO 6302 ccagggctcaggccccacc 5127 5146 '

SEQ ID NO: 5757 gggggcccactggggagtc 1387 1406 SEQ ID NO 6303 gactaggtactccgccccc 8641 8660'

SEQ ID NO 5758 atggcggggaactgggcta 1430 1449 SEQ ID NO 6304 agcagtgctcacttccat 6846 6865

SEQ ID NO 5759rttgattgtgatgctacttt 1454 1473 SEQ ID NO ,6305 aaagcaagctgcccatcaa 7665 7684

SEQ ID NO: 5760 caacgggggggcacgctgc 1500 1519 SEQ ID NO 6306 gcagaaggcgctcgggttg 5530 5549

SEQ ID NO 5761 acgctgcccgcctcaccag 1512 1531 SEQ ID NO 6307 ctggacccgaggagagcgt 2278 2297

SEQ ID NO 5762 agagaatccagcttata 1564 1583 SEQ ID NO 6308 tatatcgggggtcccctga 8393 8412

SEQ ID NO 5763 accaatggcagttggcaca 1586 1605 SEQ ID NO 6309 tgtggctcggggccttggt 2132 2151

SEQ ID NO: 5764 ccaatggcagttggcacat 1587 1606 SEQ ID NO 631 Oatgtggctcggggccttgg 2131 2150

SEQ ID NO 5765 gtcctatcacttatgctga 1749 1768 SEQ ID NO 6311 tcaggactggggtaaggac 4176 4195

SEQ ID NO 5766 ctgagcctacaaaagaccc 1764 1783 SEQ ID NO 6312 gggtggcttcatgcctcag 9063 9082

SEQ ID NO 5767 caggtgtgtggtccagtgt 1844 1863 SEQ ID NO 6313 acactccagttaactcctg 8817 8836 '

SEQ ID No ⁷⁶⁸ 9^tsg^tccag^tg^tattg^ct 1850 1869 SEQ ID NO 6314 agcagggccatcaaccaca 7949 7968 '

SEQ ID NO 5769 gcttcaccccaagtcctgt 1866 1885 SEQ ID NO 631 δacagcagaggcggctaagc 6887 6906 -

SEQ ID NO 5770 ctgttgtcgtggggacaac 1881 1900SEQ ID NO 6316 gttgcaacttggacgacag 2295 2314

SEQ ID NO 5771 [gccgccgcaaggcaactgg 1972 1991 SEQ ID NO: 6317 ccagttggacttatccggc 9241 9260 -

SEQ ID NO 5772 ggcaactggttcggctgta 1982 2001 SEQ ID NO: 6318pacacgggtgcccattgcc 7287 7306 '

SEQ ID NO: 5773 gcaactggttcggctgtac 1983 2002SEQ ID NO: 6319 gtacacgggtgcccattgc 7286 7305"

SEQ ID NO: 5774 ccccgtgtaacatcggggg 2043 2062SEQ ID NO 6320 ccccaatcgatgaacgggg 9376 9395'

SEQ ID NO 5775 ggactgcttccggaagcac 2092 2111 SEQ ID NO: 6321 gtgctggtaggcggagtcc 5324 5343 "

SEQ ID NO 5776 gactgcttccggaagcacc 2093 2112 SEQ ID NO: 6322 ggtgctggtaggcggagtc 5323 5342'

SEQ ID NO 5777Fcggaagcaccccgaggc 2100 2119 SEQ ID NO 6323 gcctacgagtcttcacgga 8616 86351

SEQ ID NO 5778 actcaaaatgtggctcggg 2124 2143SEQ ID NO: 6324 cccgggcagcgggtcgagt 8201 8220 '

SEQ ID NO 5779 ggccttggttgacacctag 2142 2161 SEQ ID NO: 6325 ctagccggcccaaaaggcc 3611 3630 4

SEQ ID NO: 5780 aggagagcgttgcaacttg 2287 2306 SEQ ID NO 6326 caagccgtgatgggctcct 8162 8181 ^' 4

SEQ ID NO 5781 ggacagatcggagctcagc 2314 2333SEQ ID NO 6327 gctgggggtcattatgtcc 3128 3147-

SEQ ID NO: 5782 cagatcggagctcagcccg 2317 2336 SEQ ID NO: ι6328 cgggtggcccactgctctg 3837 3856'

SEQ ID NO 5783 ggagctcagcccgctgctg 2323|2342lSEQ ID NO: 6329 cagctgctgaagaggctcc 6206 6225' ISEQl -W) [57^'84^'| caccctaccggctctgtcc 2383 2402SEQ D NO: |6330[ggactgggtgtgcacggtg 6286 63051 4

SEQ ID NO: [5785 cggctctgtccactggctt 2391 2410 SEQ D NO 6331 aagcaggcggaggctgccg 5564 5583 1

SEQ ID NO: 5786 ccatcagaacatcgtggac 2419 2438SEQ D NO 6332 jgtccccgttgagtccatgg 3929 3948 '

SEQ ID NO: [5787 ggtcagcggttgtctcctt 2460 2479SEQ D NO: 6333 aaggatgattctgatgacc 8875 8894'

SEQ ID NO 5788 gccgccttagagaacctgg 2579 2598SEQ D NO 6334 ccagttggacttatccggc 9241 9260-

SEQ ID NO 5789 gccttagagaacctggtgg 2582 2601 SEQ D NO 6335 ccaccaagcaggcggaggc 5559 5578 "

SEQ ID NO 5790 gccggagcgcacggcatcc 2621 2640SEQ D NO 6336 ggattgggcccacgccggc 3214 3233 '

SEQ ID NO 5791 gctgcatcgtgcggaggcg 2786 2805SEQ D NO 6337 cgccacgacatcccgcagc 7726 7745'

SEQ ID NO: 5792 attattgaccttgtcgcca 2824 2843SEQ D NO: 6338rtggcaacagacgctctaat 4647 4666'

SEQ ID NO 5793Fgccatattacaaggtgt 2837 2856 SEQ D NO 6339 acacaatctttcctggcga 3539 3558 '

SEQ ID NO 5794 cgccatattacaaggtgtt 2838 2857SEQ D NO 6340 aacacaatctttcctggcg 3538 3557 '

SEQ ID NO 5795 gtccggggaggccgcgatg 2939 2958 SEQ D NO 6341 catcggcacagtcctggac 4327 4346"

SEQ ID NO 5796 tcaccccactgcgggattg 3201 3220SEQ D NO 6342 caatttaccaatgttgtga 8325 8344'

SEQ ID NO: 5797 ttgggcccacgccggccta 3217 3236SEQ D NO 6343rcaggctaggggccgtccaa 5221 5240 -

SEQ ID NO 5798 ctacgggaccttgcggtag 3233 3252SEQ D NO 6344 ctactcctactttctgtag 9338 9357 '

SEQ ID NO 5799 cctgtcgtcttctctgaca 3260 3279SEQ D NO: 6345n:gtcctacacatggacagg 7617 7636

SEQ ID NO: 5800 ctgtcgtcttctctgacat 3261 3280SEQ D O: 6346 atgtcctacacatggacag 7616 7635

SEQ ID NO: 5801 cctggggggcagacaccgc 3297 3316 SEQ D NO 6347 gcggggtaggactggcagg 4804 4823

SEQ ID NO: 5802 gggggcagacaccgcggcg 3301 3320 SEQ D NO 6348 cgcccaactcgctcccccc 5794 5813

SEQ ID NO 5803 ggcgtgtggggacatcatc 3316 3335SEQ D NO 6349 gatgttattccggtgcgcc 3755 3774-

SEQ ID NO: 5804 tggggccggccgatagtct 3378 3397SEQ D NO 6350 agacgacgaccgtgcccca 4761 4780 '

SEQ ID NO: [5805 gaaccaggtcgagggggag 3499 3518SEQ D NO; 6351 ctccacctatggcaagttc 4222 4241

SEQ ID NO: 5806 gagggggaggttcaagtgg 3509 3528SEQ D NO 6352 ccacctgtcaaggcccctc 7304 7323 '

SEQ ID NO: 5807 aggcccaatcgcccagatg 3625 3644SEQ D NO: 6353 catcccgcagcgcgggcct 7734 7753 4

SEQ ID NO: 5808 ggcccaatcgcccagatgt 3626 3645SEQ D NO: 6354 acatcccgcagcgcgggcc 7733 7752 ^" 4

SEQ ID NO 5809 caggatctcgtcggctggc 3659 3678SEQ D NO: 6355 gccaataggccatttcctg 9410 9429 '

SEQ ID NO: 581 Oaggatctcgtcggctggcc 3660 3679SEQ D NO: 6356 ggccaataggccatttcct 9409 9428 '

SEQ ID NO: 5811 gccccccggggcgcgttcc 3682 3701 SEQ D NO 6357 ggaacctatccagcagggc 7938 7957-

SEQ ID NO: 5812 gcacctgtggcagctcgga 3711 3730SEQ D NO 6358h:ccggtggtacacgggtgc 7279 7298 '

SEQ ID NO: 5813 ctgtggcagctcggacctt 3715 3734 SEQ D NO 6359 aaggcaaaggcgtccacag 7826 7845 -

SEQ ID NO 5814 gcggggcgacaatagaggg 3775 3794SEQ D NO 6360 ccctgcctgggaaccccgc 5682 5701

SEQ ID NO: 5815 ggagcttgctctcccccag 3792 3811 SEQ D NO: 6361 ctggttgggtcacagctcc 6806 6825

SEQ ID NO: 5816 gagcttgctctcccccagg 3793 3812SEQ D NO: 6362 cctggttgggtcacagctc 6805 6824

SEQ ID NO: 5817acttgaagggctcttcggg 3822 3841 SEQ D NO 6363 cccgtggtggagtccaagt 5585 5604

SEQ ID NO: 581 δtgtccccgttgagtccatg 3928 3947SEQ D NO 6364 catggtctacgccacgaca 7717 7736 '

SEQ ID NO 5819 gaaactactatgcggtccc 3947 3966 SEQ D NO: 6365 gggaaggcacctcattttc 4504 4523"

SEQ ID NO: 5820 aaactactatgcggtcccc 3948 3967SEQ D NO 6366 ggggggcatatacaggttt 4828 4847'

SEQ ID NO 5821 ctcccactggcagcggcaa 4032 4051 SEQ D NO: 6367K:tgccaggaccatctggag 4993 5012 -

SEQ ID NO: 5822 ggcgtatatgtctaaagca 4138 4157 SEQ D NO: 6368 gctcgccaccgctacgcc 4377 4396 '

SEQ ID NO 5823 gcgtatatgtctaaagcac 4139 4158 SEQ D O: 6369 gtgctcgccaccgctacgc 4376 4395'

SEQ ID NO 5824h:ggggtaaggaccattacc 4183 4202 SEQ D NO 6370 ggtaaccatgtctccccca 6119 6138 '

SEQ ID NO: 5825 accattaccacgggcgccc 4193 4212 SEQ D NO 6371 gggcgctggtatcgctggt 5833 5852 '

SEQ ID NO: 5826 cgtactccacctatggcaa 4218 4237 SEQ D NO: 6372pgccccaaccagaatacg 8669 8688-

SEQ ID NO: 5827 cagtcctggaccaagcgga 4335 4354SEQ D NO 6373 cgtgagccgcatgactg 9560 9579-

SEQ ID NO 5828 aggggggaaggcacctcat 4500 4519SEQ D NO 6374 atgagcggcgaggcgccct 5948 5967'

SEQ ID NO: 5829 cactccaagaagaagtgcg 4526 4545SEQ D O 6375 cgcatgactgcagagagtg 9569 9588 '

SEQ ID NO: 5830 atcaatgctgtagcgtatt 4577 4596 SEQ D NO 6376 aatacgacttggagttgat 8682 8701

SEQ ID NO: 5831 cataccgaccagcggagac 4618 4637SEQ D NO: 6377 gtctcccccacgcactatg 6128 6147 -

SEQ ID NO 5832 aggactggcaggggcaggg 4811 4830SEQ D NO: 6378 ccctgccatcctctctcct 5992 6011

SEQ ID NO: 5833 gggaacggccctcgggcat 4857 4876SEQ D NO: 6379 atgctcaccgacccctccc 6863 6882 ' SEQ ID NO 5834 cgggcatgttcgattcctc 4869 4888_SEQ D O: 6380 gaggccgcaagccagcccg 8067 8086 '

SEQ ID NO 5835 tggtacgagctcacccccg 4922 4941 SEQ D NO 6381 cggggacttgccccaacca 8662 8681

SEQ ID NO 5836 gggcttacctaaatacacc 4962 4981 SEQ D NO 6382lggtggctccatcttagccc 9518 9537

SEQ ID NO 5837 ggcttacctaaatacacca 4963 4982 SEQ D NO 6383 tggtggctccatcttagcc 9517 9536

SEQ ID NO 5838 gagataacttcccctacct 5082 5101 SEQ D NO: 6384]aggttggccagggggtctc 6908 6927

SEQ ID NO 5839 cccacctccatcgtgggat 5140 5159SEQ D NO: 6385 atccaagtttggctatggg 7906 7925¹

SEQ ID NO 5840 catggcatgcatgtcggcc 5278 5297SEQ D NO 6386 ggcctctctgcagatcatg 9596 9615 '

SEQ ID NO 5841 ggccgacctggaagtcgtc 5293 5312SEQ D NO 6387 gacgcccccacattcggcc 7885 7904 -

SEQ ID NO 5842 gccgacctggaagtcgtca 5294 5313SEQ D NO: 6388κgacgcccccacattcggc 7884 7903

SEQ ID NO 5843 tggaagtcgtcaccagcac 5301 5320SEQ D NO: 6389 gtgcccatgtcaggttcca 6676 6695

SEQ ID NO 5844 gcacctgggtgctggtagg 5316 5335SEQ D NO 6390 cctacacatggacaggtgc 7620 7639'

SEQ ID NO 5845 ggttatcgtgggtaggatc 5383 5402SEQ D NO 6391 gatcatcgggccgaaaacc 6478 6497 '

SEQ ID NO 5846 cccgatagggaagtcctct 5429 5448 SEQ D NO [6392 agagcggctttatatcggg 8383 8402 '

SEQ ID NO 5847røaaatggaagaatgcgcc 5461 5480SEQ D NO 6393 ggcgcgctcgtggccttca 5924 5943'

SEQ ID NO 5848 ccaagtggcgagctttgga 5598 5617 SEQ D NO 6394rtccattgttagagtcttgg 7240 7259

SEQ ID NO 5849 ttcatcagcgggatacagt 5645 5664SEQ D NO 6395 actgcacgatgctcgtgaa 8541 8560

SEQ ID NO 5850 agcgggcttatccaccctg 5668 5687SEQ D NO 6396 caggggtggctggcgcgct 5913 5932

SEQ ID NO 5851 ccagcccgctcaccaccca 5736 5755SEQ D NO: 6397rtgggcgctggtatcgctgg 5832 5851

SEQ ID NO 5852 gtgggcgctggtatcgctg 5831 5850 SEQ D NO 6398 cagcagggccatcaaccac 7948 7967'

SEQ ID NO 5853 ggaaggtgctagtggacat 5877 5896SEQ D NO: 6399 atgtggtctccacccttcc 8142 8161 '

SEQ ID NO 5854 ggtcatgagcggcgaggcg 5944 5963SEQ D NO: 6400 cgcccctcctgaccagacc 7453 7472 '

SEQ ID NO 5855 catgtgggcccgggagagg 6056 6075SEQ D NO 6401 cctccttgagggcgacatg 6969 6988 '

[SEQ ID NO 5856 atgtgggcccgggagaggg 6057 6076SEQ D NO: 6402 ccctccttgagggcgacat 6968 6987'

SEQ ID NO 5857 ggggccgtgcagtggatga 6074 6093SEQ D NO 6403Fcatgctcctctatgcccc 7505 7524-

SEQ ID NO 5858 gcgttcgcttcgcggggta 6104 6123 SEQ D NO 6404paccaccacgagcttacgc 2751 2770 -

SEQ ID NO 5859 ggggtaaccatgtctcccc 6117 6136 SEQ D NO 6405 gggggagccgggggacccc 7531 7550'

SEQ ID NO 5860 catcacccagctgctgaag 6199 6218 SEQ D NO [6406 cttcgagcggagggggatg 7130 7149 -

SEQ ID NO 5861 aggactgttctacgccgtg 6240 6259SEQ D NO 6407 cacggcgaccgcccctcct 7444 7463 "

SEQ ID NO 5862pcaagacctggctccagt 6314 6333 SEQ D NO 6408 actgcacgatgctcgtgaa 8541 8560-

SEQ ID NO 5863 ctcctgccgcggttaccgg 6338 6357SEQ D NO; 6409 ccgggacgtgcttaaggag 7804 7823"

SEQ ID NO 5864 jcaccacgggcccctgcacg 6538 6557SEQ D NO: 6410 cgtggaggtcacgcgggtg 6613 6632 '

SEQ ID NO 5865 ggaggtcacgcgggtgggg 66.16 6635SEQ D NO 6411 cccctccaataccacctcc 7317 7336 '

SEQ ID NO 5866 gaggtcacgcgggtggggg 6617 6636 SEQ D NO: 6412 cccctcctgaccagacctc 7455 7474'

SEQ ID NO 5867 atgtcaggttccagctcct 6682 6701 SEQ D NO: 6413 aggagatgggcggaaacat 7059 7078 -

SEQ ID NO 5868 atgaaatatccattgcggc 7152 7171 SEQ D NO: 6414 gccgtgatgggctcctcat 8165 8184 '

SEQ ID NO 5869 ctccattgttagagtcttg 7239 7258SEQ D NO: 6415 caagtggcgagctttggag 5599 5618 -

SEQ ID NO 5870røcccattgccacctgtca 7295 7314 SEQ D NO: 641 θrøactaattcaaaagggca 8409 8428'

SEQ ID NO 5871 accacctccacggagaaaa 7327 7346SEQ D NO: 6417rttttttccctctttatggt 9502 9521

SEQ ID NO 5872ccacctccacggagaaaaa 7328 7347SEQ DNO: 6418H:tttccctctttatggtgg 9504 9523

SEQ ID NO 5873 acctccacggagaaaaagg 7330 7349 SEQ D NO: 6419 [cctttgacagactgcaggt 7770 7789 '

SEQ ID NO 5874 ggttgtcctgacggactcc 7351 7370SEQ D NO: 6420 ggagctcgctaccaaaacc 7390 7409'

SEQ ID NO 5875 cctgaccagacctccgaca 7460 7479SEQ D NO: 6421 gtcctacacatggacagg 7617 7636'

SEQ ID NO 5876 agcaagctgcccatcaacg 7667 7686SEQ D NO 6422 cgttgagcaactctttgct 7686 7705 '

SEQ ID NO 5877 ggatgaccattaccgggac 7792 7811 SEQ D NO: 6423 gtcccagttggacttatcc 9238 9257 4

SEQ ID NO 5878røgcaaagaatgaggtttt 8028 8047SEQ D NO: 6424 aaaaagccctggattgcca 8931 8950 ^' 4

SEQ ID NO 5879 ggcaaagaatgaggttttc 8029 8048SEQ D O: 6425 gaaaaagccctggattgcc 8930 8949

SEQ ID NO 5880 gggcagcgggtcgagttcc 8204 8223 SEQ D NO: 6426 ggaagaaagcaagctgccc 7660 7679

SEQ ID NO 5881 gactagctgcggtaatacc 8470 8489 SEQ D O 6427 ggtaccgcccttgcgagtc 9091 9110

SEQ ID NO 5882 ctcgcgatcccaccacccc 8766 8785SEQ D NO 6428 ggggtaccgcccttgcgag 9089 9108 -

SEQ ID NO 5883 aggatgattctgatgaccc 8876 8895SEQ D NO [6429 gggtcagcggttgtctcct 2459 2478 ' SEQ ID NO 5884 agccacttgacctacctca 8976 8995SEQ D NO [6430|tgagatcaatagggtggct 9052 9071 1 4

SEQ ID NO 5885 gggtaccgcccttgcgagt 9090 9109 SEQ D NO 6431 actcgcgatcccaccaccc 8765 8784

SEQ ID NO [5886 ctgcaatgactccctccag 1624 1643 SEQ D NO 6432 [ctggcgggctatggggcag 5897 59163

SEQ ID NO 5887 ccagcccccgattgggggc 20 SEQ D NO 6433|gcccactggggagtcctgg 1391 14102

SEQ ID NO 5888 aaggcgacagcctatcccc 520 539 SEQ D NO 6434 gggggtctcccccctcctt 6918 69372

SEQ ID NO 5889 ggccccacggacccccggc 662 681 SEQ D O |6435|gccgcaaagctgtcaggcc 4553 45722

SEQ ID NO 5890 gaggcggcggacttgatca 983 1002 SEQ D NO 6436H:gataacatcatgttcctc 8697 87162

SEQ ID NO 5891 ctgcaattgttcgatctac 1249 1268 SEQ D NO |6437|gtaggcggagtcctcgcag 5330 53492

SEQ ID NO 5892 ctccagactgggtttcttg 1637 1656 SEQ D NO: [6438 caagtggcgagctttggag 5599 56182

SEQ ID NO 5893 tcgtacctgcgtcgcaggt 1830 1849 SEQ D NO 6439 acctcagatcattgaacga 8989 90082

SEQ ID NO 5894 caagacgtgcggggccccc 2026 2045SEQ D NO 6440|gggggagggccgccacttg 9156 91752

SEQ ID NO 5895 aatgctgcatgcaactgga 2264 2283 SEQ D NO 6441 ccaggccaataggccatt 9405 94242

SEQ ID NO 5896 caccctaccggctctgtcc 2383 2402 SEQ D O 6442 ggactacgtccctccggtg 7267 72862

SEQ ID NO 5897 cgccatattacaaggtgtt 2838 2857 SEQ D O 6443 aacagccaccaagcaggcg 5554 55732

SEQ ID NO 5898 cgaagccatcaagggggga 4489 4508SEQ D NO 6444 cccagatttgggagttcg 8097 81162

SEQ ID NO 5899 ccagcccgctcaccaccca 5736 5755SEQ D NO: 6445ftgggtacaagggagtctgg 6382 6401

SEQ ID NO 5900 ggctatgactaggtactcc 8635 8654SEQ D NO 6446 ggagacatatatcacagcc 9284 93032

SEQ ID NO 5901 ctccaccatagatcactcc 24 43 SEQ D NO 6447 ggagacatcgggccaggag 9111 9130

SEQ' ID NO 5902 tccaccatagatcactccc 25 44SEQ D NO 6448 gggagttcgatgaaatgga 5451 5470

SEQ ID NO 5903 caccatagatcactcccct 27 46 SEQ D NO 6449 aggggccccaggttgggtg 458 477

SEQ ID NO 5904 tcactcccctgtgaggaac 36 55SEQ D O 6450 gttctggaggacggcgtga 809 828

SEQ ID NO 5905 cgttagtatgagtgtcgtg 88 107 SEQ D O: 6451 cacgctgcacgggccaacg 5191 5210

SEQ ID NO 5906 tgtcgtgcagcctccagga 100 119SEQ D O 6452Fctgttgtcgtggggaca 1879 1898

SEQ ID NO 5907 ccccccctcccgggagagc 119 138 SEQ D NO: 6453 gctcccggcctagttgggg 645 664

SEQ ID NO 5908 ggagagccatagtggtctg 131 150 SEQ D NO 6454 cagatcattgaacgactcc 8993 9012

SEQ ID NO 5909 gagccatagtggtctgcgg 134 153 SEQ D NO: 6455 ccgctgctgggtagcgctc 1048 1067

SEQ ID NO 591 Ogtggtctgcggaaccggtg 142 161 SEQ D NO: 6456 cacccatatagatgcccac 5038 5057

SEQ ID NO 5911 agtacaccggaattgccag 161 180 SEQ D NO: 6457 ctggcgggccttgcctact 1406 1425

SEQ ID NO 5912 ggtcctttcttggatcaac 188 207SEQ D O 6458 gttgagtgacttcaagacc 6304 6323

SEQ ID NO 5913pcttggatcaacccgctc 194 213 SEQ D O 6459 gagcggagggggatgagaa 7134 7153

SEQ ID NO 5914 ctcaatgcctggagatttg 210 229SEQ D NO 6460 caaagactccgacgctgag 7486 7505

SEQ ID NO 5915H:gcctggagatttgggcgt 215 234SEQ D NO 6461 acgcggccgccgcaaggca 1967 1986

SEQ ID NO 5916gcctggagatttgggcgtg 216 235 SEQ D NO 6462 cacgcggccgccgcaaggc 1966 1985

SEQ ID NO 5917gagatttgggcgtgccccc 221 240SEQ D NO: 6463 ggggacaaccgatcgtctc 1891 1910

SEQ ID NO 5918 aaaggccttgtggtactgc 273 292 SEQ D NO 6464 gcagaagaaggtcaccttt 7756 7775

SEQ ID NO 5919 aaggccttgtggtactgcc 274 293SEQ D NO 6465 ggcagaagaaggtcacctt 7755 7774

SEQ ID NO 5920 gtggtactgcctgataggg 282 301 SEQ D O 6466 ccctaccggctctgtccac 2385 2404

SEQ ID NO 5921 cctgatagggtgcttgcga 291 310 SEQ D O 6467 gccggcccgagggcagg 544 563

SEQ ID NO 5922 cgagtgccccgggaggtct 307 326SEQ D NO 6468 agacgcagtgtcgcgctcg 4780 4799

SEQ ID NO 5923 gccccgggaggtctcgtag 312 331 SEQ D NO: 6469 ctaccttaggttttggggc 4122 4141

SEQ ID NO [5924|ttacctgttgccgcgcagg 442 461 SEQ D NO 6470 cctgcgttcgggagggtaa 1023 1042

SEQ ID NO 5925 tacctgttgccgcgcaggg 443 462SEQ D NO 6471 ccctgcgttcgggagggta 1022 1041

SEQ ID NO 5926 cctgttgccgcgcaggggc 445 464 SEQ D NO 6472 gcccccgaagccagacagg 8348 8367

SEQ ID NO 5927 ctgttgccgcgcaggggcc 446 465 SEQ D NO 6473 ggcccccgaagccagacag 8347 8366

SEQ ID NO 592δFcgagcggtcgcaacccc 497 516 SEQ D NO 6474 ggggcaaaggacgtccgga 7922 7941

SEQ ID NO 5929 [ggtcgcaaccccgtggaag 504 523SEQ D O 6475 cttctctgacatggagacc 3268 3287

SEQ ID NO 5930 gtcgcaaccccgtggaagg 505 524SEQ D NO 6476 ccttcaccattgagacgac 4749 4768

SEQ ID NO 5931 aaggcgacagcctatcccc 520 539 SEQ D NO 6477 ggggcgctgccagggcctt 774 793

SEQ ID NO 5932 cagcctatccccaaggctc 527 546 SEQ D NO: 6478 gagcacaggcttaatgctg 2252 2271 SEQ ID NO 5933 gagggcagggcctgggctc 554 573 [SEQ D NO 6479 gagcgtcttcacaggcctc 5020 50391 3

SEQ ID NO 5934 cagggcctgggctcagccc 559 578 SEQ D NO 6480 gggcatcggcacagtcctg 4324 4343 '

SEQ ID NO 5935 gggcctgggctcagcccgg 561 580SEQ D NO 6481 ccggccgcatatgcggccc 4064 4083 '

SEQ ID NO 5936 cctgggctcagcccgggta 564 583SEQ D NO 6482 taccgaccctaacatcagg 4162 4181

SEQ ID NO 5937 cccctctatggcaatgagg 590 609 SEQ D NO 6483 cctcgccgacctcatgggg 727 746

SEQ ID NO 5938 gagggcatggggtgggcag 605 624SEQ D NO 6484 ctgcggatctgttttcctc 1 180 1199 '

SEQ ID NO 5939 agggcatggggtgggcagg 606 625 SEQ D NO 6485 cctgctctttcaccaccct 2370 2389 '

SEQ ID NO 5940aggatggctcctgtcaccc 622 641 SEQ D NO 6486 gggtcagcggttgtctcct 2459 2478 '

SEQ ID NO 5941 gatggctcctgtcaccccg 624 643SEQ D NO 6487 cgggggcgcttacgacatc 4261 4280 '

SEQ ID NO 5942 gtcaccccgcggctcccg 633 652 SEQ D NO 6488 cggggcgcgttccctgaca 3688 3707-

SEQ ID NO 5943 gtcaccccgcggctcccgg 634 653SEQ D NO 6489 ccggggcgcgttccctgac 3687 3706 '

SEQ ID NO 5944 gcggctcccggcctagttg 642 661 SEQ D NO 6490 caacgtccggggaggccgc 2935 2954 '

SEQ ID NO 5945 ctcccggcctagttggggc 646 665SEQ D NO 6491 gccctgtcgaacactggag 4439 4458

SEQ ID NO 5946 ataccctcacatgcggcct 711 730 SEQ D NO 6492 aggcaacattatcatgtat 8839 8858

SEQ ID NO 5947pccgctcgtcggcggccc 750 769 SEQ D NO 6493 gggcaaagcacatgtggaa 5625 5644

SEQ ID NO 5948 cccctagggggcgctgcca 767 786 SEQ D NO 6494togcaatgagggcatgggg 598 617

SEQ ID NO 5949κgcaacagggaacctgccc 832 851 SEQ D NO 6495 gggctcattcgtgcatgca 3092 3111

SEQ ID NO 5950 gcgtaacgcgtccggggta 922 941 SEQ D NO 6496 accaccacgagcttacgc 2751 2770

SEQ ID NO 5951 aagcattgtgtttgagg 968 987 SEQ D NO 6497 cctctatgcccccccttga 7512 7531

SEQ ID NO 5952 cccacgctcgcggccagga 070 1089 SEQ D NO 6498Fcctgtttaacatcttggg 5763 5782 '

SEQ ID NO 5953 cggccaggaatgctaccat 080 1099 SEQ D NO 6499 atggcatgcatgtcggccg 5279 5298 '

SEQ ID NO 5954 acgacaatacgacaccacg 106 1125 SEQ D NO 6500 cgtggggacaaccgatcgt 1888 1907'

SEQ ID NO 5955 gggcggctgctctctgctc 140 1159 SEQ D O 6501 gagcaacttgaaaaagccc 8921 8940 '

SEQ ID NO 5956 cgtgggggacctctgcgga 168 1187SEQ D NO 6502H:ccgttgccggagcgcacg 2615 26341

SEQ ID NO 5957 agctgttcaccttctcgcc 206 1225 SEQ D NO 6503 ggcgacaatagagggagct 3779 3798 '

SEQ ID NO 5958 ctgttcaccttctcgcccc 208 1227 SEQ D NO 6504 ggggagacatatatcacag 9282 9301

SEQ ID NO 5959 ctgcaattgttcgatctac 249 1268 SEQ D NO: 6505 gtaggactggcaggggcag 4809 4828

SEQ ID NO 5960 attgttcgatctaccccgg 254 1273 SEQ D NO 6506 ccggcccaaaaggcccaat 3615 3634 '

SEQ ID NO 5961 atctaccccggccacgcgt 262 1281 SEQ D NO 6507 acgccatggaccgggagat 2766 2785'

SEQ ID NO 5962 cggccacgcgtcaggtcac 270 1289 SEQ D NO 6508 gtgatgctactttttgccg 1460 1479 -

SEQ ID NO 5963 ccgcatggcctgggacatg 288 1307 SEQ D NO 6509 catggaaactactatgcgg 3943 3962 '

SEQ ID NO 5964 cgcagttactccggatccc 344 1363 SEQ D NO 6510 gggaacccaggaggatgcg 8593 8612 -

SEQ ID NO 5965cccacaagccgtcatcgac 360 1379 SEQ D NO 6511 gtcgtcaccagcacctggg 5306 5325 '

SEQ ID NO 5966 ctggggagtcctggcgggc 396 1415 SEQ D NO 6512 gcccggagcgcatggccag 1695 1714 "

SEQ ID NO 5967 ggcgggccttgcctactat 408 1427 SEQ D NO 6513atagaagaagcctgccgcc 7865 7884-

SEQ ID NO 5968Fttgccggcgttgacgggc 472 1491 SEQ D NO 6514 gcccccacattcggccaaa 7888 7907

SEQ ID NO 5969 caccctcacaacggggggg 492 1511 SEQ D NO 651 δccccaatatcgaggaggtg 4420 4439 '

SEQ ID NO 5970 gggggggcacgctgcccgc 504 1523 SEQ D O 6516 gcggcacggcgaccgcccc 7440 7459

SEQ ID NO 5971 ggggcacgctgcccgcctc 507 1526 SEQ D NO 6517 gagggagcttgctctcccc 3789 3808

SEQ ID NO 5972 gcccgcctcaccagcgggt 517 1536 SEQ D NO 651 δaccctcacaacgggggggc 1493 1512

SEQ ID NO 5973 atccagcttataaacacca 571 1590 SEQ D NO 6519ttggttatcgtgggtaggat 5382 5401

SEQ ID NO 5974 ctccagactgggtttcttg 637 1656 SEQ D NO 6620 caagcggagacggctggag 4346 4366 '

SEQ ID NO 5975 cccggagcgcatggccagc 696 1715SEQ D NO 6521 gctgtgggcgtcttccggg 3869 3888 '

SEQ ID NO 5976 ctgccgctccattgacaag 714 1733 SEQ D NO 6522 cttggtacatcaagggcag 2667 2686 '

SEQ ID NO 5977 aagttcgaccagggatggg 730 1749 SEQ D NO 6523 cccaaccagaatacgactt 6673 3692 -

SEQ ID NO 597δ ggggtcctatcacttatgc 746 1765 SEQ D NO: 6524 gcatgtgtgggttcccccc 2914 2933 '

SEQ ID NO 5979 ccagaggccttattgctgg 766 1805SEQ D NO 6525 ccaggatctcgtcggctgg 365δ 3677 '

SEQ ID NO 59δ0 cccacctcaacaatgtggt 810 1829 SEQ D NO: 6526 accaagatcatcacctggg 3284 3303 '

SEQ ID NO 5981 Hicgtacctgcgtcgcaggt 830 1849 SEQ D NO: 6527 accttcaccattgagacga 4748 4767 '

SEQ ID NO 5982 tgcgtcgcaggtgtgtggt 837 1856 SEQ D NO: 6528 accatgtctcccccacgca 6123 6142 - SEQ ID NO 59δ3Fggggacaaccgatcgtct 1390 1909 SEQ D O 6529 agacgacgaccgtgcccca 4761 47801

SEQ ID NO 59δ4 cagctggggggagaacgat 1924 1943 SEQ D NO 6530|atcggagctcagcccgctg 2320 2339

SEQ ID NO 59δ5 cgccgcaaggcaactggtt 1974 1993 SEQ D NO 6531 aacccaggaggatgcggcg 6596 6615

SEQ ID NO 5986 gccgcaaggcaactggttc 1975 1994SEQ D O 6532|gaacccaggaggatgcggc 8595 8614

SEQ ID NO 5987 ctgtacatggatgaatagc 1996 2015 SEQ D NO 6533 gctataaaatcgctcacag 8366 6335

SEQ ID NO 5988 tgtacatggatgaatagca 1997 2016SEQ D NO 6534 gctgctcaatgtcctaca 7607 7626'

SEQ ID NO 5989 gttcaccaagacgtgcggg 2020 2039SEQ D NO 6535 cccgctcaccacccagaac 5740 5759 '

SEQ ID NO 5990 agacgtgcggggccccccc 2028 2047SEQ D NO 6536 ggggaggttcaagtggtct 3512 3531 3

SEQ ID NO 5991 cccccgtgtaacatcgggg 2042 2061 SEQ D NO 6537 ccccaatcgatgaacgggg 9376 9395 3

SEQ ID NO 5992rcaacaccttgacctgcccc 2071 2090SEQ D NO 6538 ggggacgaccttgtcgtta 8561 8580 '

SEQ ID NO 5993 ggctctggcactacccctg 2184 2203 SEQ D NO 6539 caggaggatgcggcgagcc 8600 8619 '

SEQ ID NO I5994h:gcactgtcaacttctcca 2201 2220 SEQ D NO: |6540|tggatggggtgcggttgca 6717 6736 '

SEQ ID NO 5995 caggcttaatgctgcatgc 2257 2276 SEQ D NO: 6541 gcatcatgcacaccacctg 6411 6430

SEQ ID NO 5996 aatgctgcatgcaactgga 2264 2283 SEQ D NO 6542 tccatggtcttagcgcatt 9009 9028

SEQ ID NO 5997 ctgcatgcaactggacccg 2268 2287 SEQ D NO 6543 cgggaccttgcggtagcag 3236 3255'

SEQ ID NO 5998 caactggacccgaggagag 2275 2294 SEQ D NO: 6544 ctcttacgggatgaggttg 6761 6780 '

SEQ ID NO 5999 gacagggacagatcggagc 2309 2328 SEQ D NO 6545 gctctcccccaggcctgtc 3799 3818 '

SEQ ID NO 6000 gacagatcggagctcagcc 2315 2334 SEQ D NO 6546 ggctggagcgcggcttgtc 4357 4376 '

SEQ ID NO 6001 acagatcggagctcagccc 2316 2335SEQ D NO: 6547 gggccaacgcccctgctgt 5201 5220

SEQ ID NO 6002 actggcttgatccacctcc 2402 2421 SEQ D NO 6548 ggagagggggccgtgcagt 6068 6087

SEQ ID NO 6003 ggcttgatccacctccatc 2405 2424 SEQ D NO 6549 gatgatgctgctgatagcc 2551 2570

SEQ ID NO 6004 gtcagcggttgtctccttt 2461 2480 SEQ D NO 6550 aaaggacggttgtcctgac 7344 7363 '

SEQ ID NO 6005 gagtatgtcgtgttgcttt 2492 2511 SEQ D NO 6551 aaagaccaagctcaaactc 9202 9221

SEQ ID NO 6006rcgtggatgatgctgctgat 2547 2566 SEQ D NO 6552atcactgatggcattcaca 5707 5726

SEQ ID NO 6007 ccgaggccgccttagagaa 2574 2593 SEQ D NO 6553pctgattgccatactcgg 3015 3034

SEQ ID NO 6008 agaacctggtggccctcaa 2589 2608 SEQ D NO 6554pgatatcaccaaacttct 3000 3019

SEQ ID NO 6009H:acatcaagggcaggctgg 2672 2691 SEQ D NO: 6555 ccagatgtacactaatgta 3637 3656 '

SEQ ID NO 6010 caagggcaggctggtccct 2677 2696SEQ D NO: 6556 aggggtaggcatctacttg 9355 93741

SEQ ID NO 6011 gcatggccgctgctcctgc 2720 2739SEQ D NO 6557 gcagtgctcacttccatgc 6848 6867 '

SEQ ID NO 6012 catggccgctgctcctgct 2721 2740SEQ D NO 6558 agcagtgctcacttccatg 6847 6866 '

SEQ ID NO 6013 gccgctgctcctgctcctc 2725 2744SEQ D NO 6559 gagggccgccacttgcggc 9160 9179 -

SEQ ID NO 6014 ggagatggctgcatcgtgc 2779 2798 SEQ D NO: 6560 gcacggcgaccgcccctcc 7443 7462'

SEQ ID NO 601 δatggctgcatcgtgcggag 2783 2802 SEQ D NO: 6561 ctccaggccaataggccat 9404 9423'

SEQ ID NO 6016 ggcgcggtttttgtgggtc 2801 2820 SEQ D NO: 6562 gaccattaccacgggcgcc 4192 4211

SEQ ID NO 6017H:cttatcaccagagctgag 2887 2906 SEQ D NO: 6563 ctcacaggccgggacaaga 3482 3501

SEQ ID NO 6018 gtgtgggttccccccctca 2918 2937SEQ D NO: 6564 gaggtcaccctcacacac 5242 5261

SEQ ID NO 6019p:ccccccctcaacgtccgg 2926 2945SEQ D NO 6665 ccggctcgtggctgaggga 6261 6280

SEQ ID NO 6020 ctcaacgtccggggaggcc 2933 2952SEQ D NO: [6566 ggcctgttactccattgag 8959 8978

SEQ ID NO 6021 accaaacttctgattgcca 3008 3027SEQ D NO 6567Fggctctctacgatgtggt 8130 8149

SEQ ID NO 6022 caaacttctgattgccata 3010 3029 SEQ D NO 6568Fatgacacccgctgttttg 8267 8286

SEQ ID NO 6023 ggaccgctcatggtgctcc 3032 3051 SEQ D NO 6569 ggagatcctgcggaagtcc 7171 71901

SEQ ID NO 6024 gaccgctcatggtgctcca 3033 3052 SEQ D NO 657θ ggaaactactatgcggtc 3945 39641

SEQ ID NO 6025 atgcatgttagtgcggaaa 3106 3125SEQ D NO 6571 rtttctgtaggggtaggcat 9348 9367'

SEQ ID NO 6026 ttatgtccaaatggccttc 3139 3158 SEQ D NO 6572 gaagccagacaggctataa 8354 83731

SEQ ID NO 6027 ccaaatggccttcatgaga 3145 3164SEQ D NO: 6573 tcagcgacgggtcttgg 7552 7571

SEQ ID NO 6028 ccttcatgagactgggcgc 3153 3172 SEQ D NO: 6574 gcgctcgtggccttcaagg 5927 5946'

SEQ ID NO 6029 ccttgcggtagcagtggag 3241 3260 SEQ D NO: 6575 ctccgcccgaaggggaagg 3349 3368'

SEQ ID NO 6030|tgtcgtcttctctgacatg 3262 3281 SEQ D NO 6576 catggtctacgccacgaca 7717 7736'

SEQ ID NO 6031 tggggggcagacaccgcgg 3299 3318SEQ D NO 6577 ccgccttatcgtattccca 8083 8102'

SEQ ID NO 6032ggggggcagacaccgcggc 3300 3319[SEQ D NO 6578 gccgcccaactcgctcccc 5792 5811 SEQ ID NO 6033 gtggggacatcatcctggg 3321 3340SEQ D O 6579|cccatctacacgctcccac 4020 40391 3

SEQ ID NO 6034p:ggggacatcatcctgggc 3322| 3341 SEQ D NO 6580 gcccatctacacgctccca 4019 4038] 1

SEQ ID NO 6035 ggggacatcatcctgggcc 3323 3342SEQ D NO 6581 ggccagggggtctcccccc 6913 6932 '

SEQ ID NO 6036 acctgtctccgcccgaagg 3343 3362SEQ D NO |6582|cctttgacagactgcaggt 7770 7789 '

SEQ ID NO 6037rcgtctccgcccgaagggga 3346 3365SEQ D NO 6583 CCccggtcttcacagaca 3962 3981

SEQ ID NO 6038 gggagatactcctggggcc 3366 3385SEQ D NO 6584 ggcccatctacacgctccc 4018 4037 -

SEQ ID NO 6039 ctcccaacagacccggggc 3439 3458SEQ D NO |6585[gcccccccttgagggggag 7519 7538 '

SEQ ID NO 604θ caccgcaacacaatctt 3530 3549SEQ D NO 6586 aagaggctccaccagtgga 6215 62341

SEQ ID NO 6041 cacaatctttcctggcgac 3540 3559SEQ D NO 6587 gtcgtcggagtcgtgtgtg 6020 6039-

SEQ ID NO 6042 ggctggccggcgccccccg 3671 3690SEQ D NO 6588 cgggttgttgcaaacagcc 5542 5561

SEQ ID NO 6043 ccccggggcgcgttccctg 3685 3704SEQ D NO 6589 caggtttgtaactccgggg 4840 4859 '

SEQ ID NO 6044 tccctgacaccatgcacct 3698 3717SEQ D NO 6590 aggtcacgcgggtggggga 6618 6637'

SEQ ID NO 6045rttccggtgcgccggcgggg 3762 3781 SEQ D NO 6591 ccccgttgagtccatggaa 3931 3950 1

SEQ ID NO 6046 ctcccccaggcctgtctcc 3802 3821 SEQ D NO 6592 ggagacatcgggccaggag 9111 9130 1

SEQ ID NO 6047 gggggttgcaaaggcggtg 3904 3923SEQ D NO 6593 caccctgcctgggaacccc 5680 5699 1

SEQ ID NO 6048ptgtccccgttgagtcca 3926 3945SEQ D NO 6594 ggagaccttctgggcaaa 5613 5632 '

SEQ ID NO 6049 ccgtaccgcaaacattcca 3996 4015SEQ D NO 6595 ggattgccaaatctacgg 8940 8959 '

SEQ ID NO 6050 caagtggcccatctacacg 4013 4032SEQ D NO 6596 cgtgggtaggatcatcttg 5389 5408 '

SEQ ID NO 6051 cacgctcccactggcagcg 4028 4047SEQ D NO: 6597 cgctgcttcggctttcgtg 5815 5834

SEQ ID NO 6052 ccgcatatgcggcccaagg 4068 4087SEQ D NO 6598 ccttcaaggtcatgagcgg 5937 5956

SEQ ID NO 6053 cgtatatgtctaaagcaca 4140 4159SEQ D NO 6599røtggaagtgtctcatacg 5163 5182-

SEQ ID NO 6054 gtatatgtctaaagcacat 4141 4160SEQ D NO 6600 atgtggaagtgtctcatac 5162 5181

SEQ ID NO 6055 ggaccattaccacgggcgc 4191 4210SEQ D NO 6601 gcgcgtgtcactcaggtcc 6167 6186'

SEQ ID NO 6056 cccccattacgtactccac 4209 4228 SEQ D NO 6602 gtgggcccgggagaggggg 6059 6078"

SEQ ID NO 6057 agttccttgccgacggtgg 4236 4255SEQ D NO 6603 ccacagtcaaggctaaact 7839 7858"

SEQ ID NO 6058 gagacggctggagcgcggc 4352 4371 SEQ D NO 6604 gccgggggaccccgatctc 7537 75561

SEQ ID NO 6059 caccgctacgcctccagga 4384 4403SEQ D NO 6605Fcctacacatggacaggtg 7619 7638'

SEQ ID NO 6060Fggagagatccccttctac 4453 4472SEQ D NO 6606 gtagcagtgctcacttcca 6845 68641

SEQ ID NO 6061 agccatccccatcgaagcc 4477 4496SEQ D NO 6607 ggctggttcgttgctggct 9257 9276 '

SEQ ID NO 6062Fccccatcgaagccatcaa 4482 4501 SEQ D NO 6608 ttgagggggagccggggga 7527 7546 -

SEQ ID NO 6063 ccccatcgaagccatcaag 4483 4502SEQ D NO 6609 cttgagggggagccggggg 7526 7545'

SEQ ID NO 6064 ggcctcggaatcaatgctg 4568 4587SEQ D NO 661 Ocagctccgaattgtcggcc 7414 7433 '

SEQ ID NO 6065 gtccgtcataccgaccagc 4612 4631 SEQ D NO: 6611 gctgagggatgtttgggac 6271 6290-

SEQ ID NO 6066 gtcataccgaccagcggag 4616 4635SEQ D NO 6612 ctccattgagccacttgac 8968 8987"

SEQ ID NO 6067 cgggctataccggtgactt 4668 4687SEQ D NO 6613 aagtccaagaagttccccg 7184 7203-

SEQ ID NO 6068 ctttgattcagtgatcgac 4684 4703SEQ D NO 6614 gtcgagttcctggtaaaag 8213 8232'

SEQ ID NO 6069 acagtcgacttcagcttgg 4724 4743SEQ D NO 6615 ccaaatctacggggcctgt 8947 8966"

SEQ ID NO 6070 cttggaccccaccttcacc 4738 4757SEQ D NO: 6616ggtgttgagtgacttcaag 6301 6320"

SEQ ID NO 6071 gagacgacgaccgtgcccc 4760 4779 SEQ D NO 6617ggggacaaccgatcgtctc 1891 1910-

SEQ ID NO 6072 ggggtaggactggcagggg 4806 4825SEQ D NO 661 δccccccggggacttgcccc 8657 86761

SEQ ID NO 6073 gggcatatacaggtttgta 4831 4850SEQ D NO 661 θFacacatggacaggtgccc 7622 7641

SEQ ID NO 6074 gggggaacggccctcgggc 4855 4874SEQ D NO 6620gcccctgcacgccttcccc 6546 6565

SEQ ID NO [6075toacgcgggctgtgcttgg 4906 4925SEQ D NO; 6621 ccaattgacaccaccgtca 8009 8028

SEQ ID NO 6076 gacgcgggctgtgcttggt 4907 4926SEQ D NO: 6622 accaattgacaccaccgtc 8008 8027

SEQ ID NO 6077 gcttggtacgagctcacc 4918 4937SEQ D NO; 6623 ggtgcggctgttggcagca 5849 5868 '

SEQ ID NO 6078Kgcccacttcctgtcccag 5050 5069 SEQ D NO: 6624 ctgggcgcgctgacgggca 3164 3183 -

SEQ ID NO 6079 ggtggcataccaagccaca 5101 5120 SEQ D NO 6625Fgtgacaccaattgacacc 8002 8021

SEQ ID NO 6080 gggctcaggccccacctcc 5130 5149 SEQ D NO 6626 ggaggccgcaagccagccc 8066 8085 '

SEQ ID NO 6081 ccatcgtgggatcaaatgt 5147 5166 SEQ D NO: 6627 acattctggcgggctatgg 5892 5911

SEQ ID NO 6082Fcatacggctaaaacccac 5175 5194SEQ D NO: 6628 gtggccttcaaggtcatga 5933 5952" SEQ ID NO 6083tgctgtataggctaggggc 5214 5233 SEQ D NO 6629 gcccgaaccggacgtagca 6832 6851

SEQ ID NO 6084 ccaaatacatcatggcatg 5268 5287SEQ D NO [6630|catgcctcaggaaacttgg 9072 9091

SEQ ID NO 6085 ggagtcctcgcagctctgg 5336 5355SEQ D NO 6631 ccagctgtctgcgccctcc 6955 6974 '

SEQ ID NO 6086 gcctgacaacaggcagtgt 5364 5383SEQ D NO 6632 acactccaggccaataggc 9401 9420 -

SEQ ID NO 6087 agccaccaagcaggcggag 5557 5576SEQ D NO: 6633|ctccagttaactcctggct 8820 8839

SEQ ID NO 6088 catgtggaatttcatcagc 5635 5654SEQ D NO 6634 gctgcgccatcacaacatg 7702 7721

SEQ ID NO 6089 ctctatcaccagcccgctc 5728 5747SEQ D NO 6635 gagccgcatgactgcagag 9565 9584

SEQ ID NO 6090 cccagaacaccctcctgtt 5751 5770SEQ D NO 6636 aacatcttgggggggtggg 5771 5790

SEQ ID NO 6091 ctcctgtttaacatcttgg 5762 5781 SEQ D NO: 6637 ccaatcgatgaacggggag 9378 9397 '

SEQ ID NO 6092rttgggggggtgggtagccg 5777 5796SEQ D NO 6638 cggcgccaaactattccaa 6564 6583 '

SEQ ID NO 6093p:gcttcggctttcgtgggc 5818 5837 SEQ D NO 6639 gcccgaaccggacgtagca 6832 6851

SEQ ID NO 6094H:cgtgggcgctggtatcgc 5829 5848SEQ D NO 6640 gcgagcggcgtgctgacga 8453 8472

SEQ ID NO 6095 cgctggtgcggctgttggc 5845 5864_SEQ D NO 6641 gccacgacatcccgcagcg 7727 7746 '

SEQ ID NO 6096 cggctgttggcagcatagg 5853 5872SEQ D NO 6642 cctagactctttcgagccg 7111 7130 -

SEQ ID NO 6097 ggggcaggggtggctggcg 5909 5928SEQ D NO 6643 cgcccaactcgctcccccc 5794 5813

SEQ ID NO 6098 ctggcgcgctcgtggcctt 5922 5941 SEQ D NO 6644 aagggaggccgcaagccag 8063 8082

SEQ ID NO 6099 tggcgcgctcgtggccttc 5923 5942 SEQ D NO 6645 gaagggaggccgcaagcca 8062 8081

SEQ ID NO 00 gagcggcgaggcgccctct 5950 5969SEQ D NO 6646 agagcgtcgtctgctgctc 7596 7615 '

SEQ ID NO Olttgggcccgggagagggggc 6060 6079SEQ D NO 6647 gcccatctacacgctccca 4019 4038 '

SEQ ID NO 02 cggctgatagcgttcgctt 6095 6114SEQ D NO 6648 aagcaggcggaggctgccg 5564 5583

SEQ ID NO 03 gtgcctgagagcgacgccg 6146 6165 SEQ D NO 6649 cggccgccgacagcggcac 7428 7447

SEQ ID NO 04 atgaggactgttctacgcc 6237 6256 SEQ D NO 6650 ggcggggggacggcatcat 6399 6418

SEQ ID NO 05 gtccaagctcctgccgcgg 6331 6350SEQ D NO 6651 ccgctccgtgtgggaggac 7969 7988

SEQ ID NO 06 acagatcgccggacatgtc 6442 6461 SEQ D NO: 6652 gacatatatcacagcctgt 9287 9306

SEQ ID NO 07 acgtggcatggaacattcc 6506 6525SEQ D NO 6653 ggaagaacccggactacgt 7257 7276

SEQ ID NO 08 gggcccctgcacgccttcc 6544 6563 SEQ D NO 6654 ggaagaaagcaagctgccc 7660 7679 '

SEQ ID NO 09 agtgcccatgtcaggttcc 6675 6694SEQ D NO 6655 ggaaacagctagacacact 8803 8822 '

SEQ ID NO 10π:gcccatgtcaggttccag 6677 6696SEQ D NO 6656 ctgggcgcgctgacgggca 3164 3183 -

SEQ ID NO 11 cagctcctgagtttttcac 6693 6712 SEQ D NO ^!6657 gtgagagcgtcgtctgctg 7593 7612 '

SEQ ID NO 12rtcacggaggtggatggggt 6708 6727SEQ D NO 6658 acccttcctcaagccgtga 8153 8172"

SEQ ID NO 13 cacggaggtggatggggtg 6709 6728SEQ D NO 6659 cacccttcctcaagccgtg 8152 8171

SEQ ID NO 14 gacccctcccacattacag 6872 6891 SEQ D NO 6660 ctgttttgactcaacggtc 8278 8297 "

SEQ ID NO 1 δrttggccagggggtctcccc 6911 6930SEQ D NO 6661 ggggtgggtagccgcccaa 5782 5801

SEQ ID NO 16 ccttgagggcgacatgcac 6972 6991 SEQ D NO 6662 gtgcttaaggagatgaagg 7811 7830"

SEQ ID NO 17 ggagatgggcggaaacatc 7060 7079SEQ D NO 6663 gatgacccatttcttctcc 8887 8906 '

SEQ ID NO 18 gagatgggcggaaacatca 7061 7080SEQ D NO; 6664p:gatgacccatttcttctc 8886 8905

SEQ ID NO 19 ctagactctttcgagccgc 7112 7131 SEQ D NO 6665 gcggcgtgctgacgactag 8457 8476

SEQ ID NO 2θFagactctttcgagccgct 7113 7132 SEQ D NO 6666 agcgacgggtcttggtcta 7556 7575

SEQ ID NO 21 agaatgaaatatccattgc 7149 7168SEQ D NO 6667 gcaaagaatgaggttttct 8030 8049"

SEQ ID NO 22rttgcggcggagatcctgcg 716 7183 SEQ D NO 6668 cgcacgatgcatctggcaa 8730 8749 '

SEQ ID NO 23 agcgaggaggctggtgaga 7580 7599SEQ D NO: 6669Ftcgtgcccgaccccgct 9305 9324 '

SEQ ID NO 24rtgagagcgtcgtctgctgc 7594 7613SEQ DNO: 6670gcagtaaagaccaagctca 9197 9216'

SEQ ID NO 25 gtcgtctgctgctcaatgt 7601 7620SEQ D NO: 6671 acatggtctacgccacgac 7716 7735 '

SEQ ID NO 26 gcgccatcacaacatggt 7704 7723SEQ D NO 6672 accatgtctcccccacgca 6123 6142 '

SEQ ID NO 27 cagaagaaggtcacctttg 7757 7776SEQ D NO: 6673 caaagaatgaggttttctg 8031 8050 -

SEQ ID NO 28 cctggatgaccattaccgg 7789 7808SEQ D NO: 6674 ccggaacctatccagcagg 7936 7955 '

SEQ ID NO 29 ggacgtgcttaaggagatg 7807 7826SEQ D O: 6675 catcgggccaggagcgtcc 9116 9135 -

SEQ ID NO 30 aaagaatgaggttttctgc 8032 8051 SEQ D NO: 6676 gcagaagaaggtcaccttt 7756 7775 '

SEQ ID NO 31 agttcgtgtatgcgagaag 8110 8129 SEQ D NO: 6677 cttcatgcctcaggaaact 9069 9088 -

SEQ ID NO 6132 ggctataaaatcgctcaca 8365 8384|SEQ D NO 6678 gtgaaaggtccgtgagcc 9551 9570 - SEQ ID NO 6133Ftctccatccttctagctc 8900 8919 SEQ ID NO: 6679 gagcggagggggatgagaa 7134 7153 '

SEQ ID NO 6134 gtctcgtgcccgaccccg 9303 9322SEQ ID NO [6680|cggggcgcgttccctgaca 3688 3707 '

Table 15. Sequences from human hepatitis C viras (HCV) (Direct Match Type)

Table 16. Sequences of Exemplary Gene Targets

gi I 4502152 | ref | NM_000384 . 1 1 Homo sapiens apolipoprotein B (including Ag (x) antigen) (APOB) , mRNA

ATTCCCACCGGGACCTGCGGGGCTGAGTGCCCTTCTCGGTTGCTGCCGCTGAGGAGCCCGCCCAGCCAGC CAGGGCCGCGAGGCCGAGGCCAGGCCGCAGCCCAGGAGCCGCCCCACCGCAGCTGGCGATGGACCCGCCG AGGCCCGCGCTGCTGGCGCTGCTGGCGCTGCCTGCGCTGCTGCTGCTGCTGCTGGCGGGCGCCAGGGCCG AAGAGGAAATGCTGGAAAATGTCAGCCTGGTCTGTCCAAAAGATGCGACCCGATTCAAGCACCTCCGGAA GTACACATACAACTATGAGGCTGAGAGTTCCAGTGGAGTCCCTGGGACTGCTGATTCAAGAAGTGCCACC AGGATCAACTGCAAGGTTGAGCTGGAGGTTCCCCAGCTCTGCAGCTTCATCCTGAAGACCAGCCAGTGCA CCCTGAAAGAGGTGTATGGCTTCAΆCCCTGAGGGCAAΆGCCTTGCTGAAGAAAACCAAGAACTCTGAGGA GTTTGCTGCAGCCATGTCCAGGTATGAGCTCAAGCTGGCCATTCCAGAAGGGAAGCAGGTTTTCCTTTAC CCGGAGAAAGATGAACCTACTTACATCCTGAACATCAAGAGGGGCATCATTTCTGCCCTCCTGGTTCCCC CAGAGACAGAAGAAGCCAAGCAAGTGTTGTTTCTGGATACCGTGTATGGAAACTGCTCCACTCACTTTAC CGTCAAGACGAGGAAGGGCAATGTGGCAACAGAAATATCCACTGAAAGAGACCTGGGGCAGTGTGATCGC TCAAGCCCATCCGCACAGGCATCAGCCCACTTGCTCTCATCAAAGGCATGACCCGCCCCTTGTCAACTC TGATCAGCAGCAGCCAGTCCTGTCAGTACACACTGGACGCTAAGAGGAAGCATGTGGCAGAAGCCATCTG CAAGGAGCAACACCTCTTCCTGCCTTTCTCCTACAACAΆTAAGTATGGGATGGTAGCACAAGTGACACAG CTTTGAAACTTGAAGACACACCAAAGATCAACAGCCGCTTCTTTGGTGAAGGTACTAAGAAGATGGGCC CGCATTTGAGAGCACCAAATCCACATCACCTCCAAAGCAGGCCGAAGCTGTTTTGAAGACTCTCCAGGA ACTGAΆAAAACTAACCATCTCTGAGCAAAATATCCAGAGAGCTAATCTCTTCAATAAGCTGGTTACTGAG CTGAGAGGCCTCAGTGATGAAGCAGTCACATCTCTCTTGCCACAGCTGATTGAGGTGTCCAGCCCCATCA CTTTACAΆGCCTTGGTTCAGTGTGGACAGCCTCAGTGCTCCACTCACATCCTCCAGTGGCTGAAACGTGT GCATGCCAACCCCCTTCTGATAGATGTGGTCACCTACCTGGTGGCCCTGATCCCCGAGCCCTCAGCACAG CAGCTGCGAGAGATCTTCAACATGGCGAGGGATCAGCGCAGCCGAGCCACCTTGTATGCGCTGAGCCACG CGGTCAACAACTATCATAAGACAAACCCTACAGGGACCCAGGAGCTGCTGGACATTGCTAATTACCTGAT GGAACAGATTCAAGATGACTGCACTGGGGATGAAGATTACACCTATTTGATTCTGCGGGTCATTGGAAAT ATGGGCCAAACCATGGAGCAGTTAACTCCAGAACTCAAGTCTTCAATCCTCAAATGTGTCCAAAGTACAA GCCATCACTGATGATCCAGAAAGCTGCCATCCAGGCTCTGCGGAAAATGGAGCCTAAAGACAAGGACCA ^■ GGAGGTTCTTCTTCAGACTTTCCTTGATGATGCTTCTCCGGGAGATAAGCGACTGGCTGCCTATCTTATG TTGATGAGGAGTCCTTCACAGGCAGATATTAACAAAATTGTCCAAATTCTACCATGGGAACAGAATGAGC AAGTGAAGAACTTTGTGGCTTCCCATATTGCCAATATCTTGAACTCAGAAGAATTGGATATCCAAGATCT GAAAAAGTTAGTGAAAGAAGCTCTGAAAGAATCTCAACTTCCAACTGTCATGGACTTCAGAAAATTCTCT CGGAACTATCAACTCTACAAATCTGTTTCTCTTCCATCACTTGACCCAGCCTCAGCCAAAATAGAAGGGA ATCTTATATTTGATCCAAATAACTACCTTCCTAAAGAAAGCATGCTGAAAACTACCCTCACTGCCTTTGG ATTTGCTTCAGCTGACCTCATCGAGATTGGCTTGGAAGGAAAAGGCTTTGAGCCAACATTGGAAGCTCTT TTTGGGAAGCAAGGATTTTTCCCAGACAGTGTCAACAAAGCTTTGTACTGGGTTAATGGTCAAGTTCCTG ATGGTGTCTCTAAGGTCTTAGTGGACCACTTTGGCTATACCAAAGATGATAAACATGAGCAGGATATGGT AAATGGAATAATGCTCAGTGTTGAGAAGCTGATTAAAGATTTGAAATCCAAAGAAGTCCCGGAAGCCAGA GCCTACCTCCGCATCTTGGGAGAGGAGCTTGGTTTTGCCAGTCTCCATGACCTCCAGCTCCTGGGAAAGC TGCTTCTGATGGGTGCCCGCACTCTGCAGGGGATCCCCCAGATGATTGGAGAGGTCATCAGGAAGGGCTC AAAGAATGACTTTTTTCTTCACTACATCTTCATGGAGAATGCCTTTGAACTCCCCACTGGAGCTGGATTA CAGTTGCAAATATCTTCATCTGGAGTCATTGCTCCCGGAGCCAAGGCTGGAGTAAAACTGGAAGTAGCCA ACATGCAGGCTGAACTGGTGGCAAAACCCTCCGTGTCTGTGGAGTTTGTGACAAATATGGGCATCATCAT TCCGGACTTCGCTAGGAGTGGGGTCCAGATGAACACCAACTTCTTCCACGAGTCGGGTCTGGAGGCTCAT GTTGCCCTAAAAGCTGGGAAGCTGAAGTTTATCATTCCTTCCCCAAAGAGACCAGTCAAGCTGCTCAGTG GAGGCAACACATTACATTTGGTCTCTACCACCAAAACGGAGGTGATCCCACCTCTCATTGAGAACAGGCA GTCCTGGTCAGTTTGCAAGCAAGTCTTTCCTGGCCTGAATTACTGCACCTCAGGCGCTTACTCCAACGCC AGCTCCACAGACTCCGCCTCCTACTATCCGCTGACCGGGGACACCAGATTAGAGCTGGAACTGAGGCCTA CAGGAGAGATTGAGCAGTATTCTGTCAGCGCAACCTATGAGCTCCAGAGAGAGGACAGAGCCTTGGTGGA TACCCTGAAGTTTGTAACTCAAGCAGAAGGTGCGAAGCAGACTGAGGCTACCATGACATTCAAATATAAT CGGCAGAGTATGACCTTGTCCAGTGAAGTCCAAATTCCGGATTTTGATGTTGACCTCGGAACAATCCTCA GAGTTAATGATGAATCTACTGAGGGCAAAACGTCTTACAGACTCACCCTGGACATTCAGAACAAGAAAAT TACTGAGGTCGCCCTCATGGGCCACCTAAGTTGTGACACAAAGGAAGAAAGAAAAATCAAGGGTGTTATT TCCATACCCCGTTTGCAAGCAGAAGCCAGAAGTGAGATCCTCGCCCACTGGTCGCCTGCCAAACTGCTTC TCCAAATGGACTCATCTGCTACAGCTTATGGCTCCACAGTTTCCAAGAGGGTGGCATGGCATTATGATGA AGAGAAGATTGAATTTGAATGGAACACAGGCACCAATGTAGATACCAAAAAAATGACTTCCAATTTCCCT GTGGATCTCTCCGATTATCCTAAGAGCTTGCATATGTATGCTAATAGACTCCTGGATCACAGAGTCCCTG AAACAGACATGACTTTCCGGCACGTGGGTTCCAAATTAATAGTTGCAATGAGCTCATGGCTTCAGAAGGC ATCTGGGAGTCTTCCTTATACCCAGACTTTGCAAGACCACCTCAATAGCCTGAAGGAGTTCAACCTCCAG AACATGGGATTGCCAGACTTCCACATCCCAGAAAACCTCTTCTTAAAAAGCGATGGCCGGGTCAAATATA CCTTGAACAAGAACAGTTTGAAAATTGAGATTCCTTTGCCTTTTGGTGGCAAATCCTCCAGAGATCTAAA GATGTTAGAGACTGTTAGGACACCAGCCCTCCACTTCAAGTCTGTGGGATTCCATCTGCCATCTCGAGAG TTCCAAGTCCCTACTTTTACCATTCCCAAGTTGTATCAACTGCAAGTGCCTCTCCTGGGTGTTCTAGACC TCTCCACGAATGTCTACAGCAACTTGTACAACTGGTCCGCCTCCTACAGTGGTGGCAACACCAGCACAGA CCATTTCAGCCTTCGGGCTCGTTACCACATGAAGGCTGACTCTGTGGTTGACCTGCTTTCCTACAATGTG CAAGGATCTGGAGAAACAACATATGACCACAAGAATACGTTCACACTATCATGTGATGGGTCTCTACGCC ACAAATTTCTAGATTCGAATATCAAATTCAGTCATGTAGAAAAACTTGGAAACAACCCAGTCTCAAAAGG TTTACTAATATTCGATGCATCTAGTTCCTGGGGACCACAGATGTCTGCTTCAGTTCATTTGGACTCCAAA AAGAAACAGCATTTGTTTGTCAAAGAAGTCAAGATTGATGGGCAGTTCAGAGTCTCTTCGTTCTATGCTA AAGGCACATATGGCCTGTCTTGTCAGAGGGATCCTAACACTGGCCGGCTCAATGGAGAGTCCAACCTGAG GTTTAACTCCTCCTACCTCCAAGGCACCAACCAGATAACAGGAAGATATGAAGATGGAACCCTCTCCCTC

ACCTCCACCTCTGATCTGCAAAGTGGCATCATTAAAAATACTGCTTCCCTAAAGTATGAGAACTACGAGC

TGACTTTAAAATCTGACACCAATGGGAAGTATAAGAACTTTGCCACTTCTAACAAGATGGATATGACCTT

^' CTCTAAGCAAAATGCACTGCTGCGTTCTGAATATGAGGCTGATTACGAGTCATTGAGGTTCTTCAGCCTG

CTTTCTGGATCACTAAATTCCCATGGTCTTGAGTTAAATGCTGACATCTTAGGCACTGACAAAATTAATA GTGGTGCTCACAAGGCGACACTAAGGATTGGCCAAGATGGAATATCTACCAGTGCAACGACCAACTTGAA GTGTAGTCTCCTGGTGCTGGAGAATGAGCTGAATGCAGAGCTTGGCCTCTCTGGGGCATCTATGAAATTA ACAACAAATGGCCGCTTCAGGGAACACAATGCAAAATTCAGTCTGGATGGGAAAGCCGCCCTCACAGAGC TATCACTGGGAAGTGCTTATCAGGCCATGATTCTGGGTGTCGACAGCAAAAACATTTTCAACTTCAAGGT CAGTCAAGAAGGACTTAAGCTCTCAAATGACATGATGGGCTCATATGCTGAAATGAAATTTGACCACACA AACAGTCTGAACATTGCAGGCTTATCACTGGACTTCTCTTCAAAACTTGACAACATTTACAGCTCTGACA AGTTTTATAAGCAAACTGTTAATTTACAGCTACAGCCCTATTCTCTGGTAACTACTTTAAACAGTGACCT GAAATACAATGCTCTGGATCTCACCAACAATGGGAAACTACGGCTAGAACCCCTGAAGCTGCATGTGGCT GGTAACCTAAAAGGAGCCTACCAAAATAATGAAATAAAACACATCTATGCCATCTCTTCTGCTGCCTTAT CAGCAAGCTATAAAGCAGACACTGTTGCTAAGGTTCAGGGTGTGGAGTTTAGCCATCGGCTCAACACAGA CATCGCTGGGCTGGCTTCAGCCATTGACATGAGCACAAACTATAATTCAGACTCACTGCATTTCAGCAAT GTCTTCCGTTCTGTAATGGCCCCGTTTACCATGACCATCGATGCACATACAAATGGCAATGGGAAACTCG CTCTCTGGGGAGAACATACTGGGCAGCTGTATAGCAAATTCCTGTTGAAAGCAGAACCTCTGGCATTTAC TTTCTCTCATGATTACAAAGGCTCCACAAGTCATCATCTCGTGTCTAGGAAAAGCATCAGTGCAGCTCTT GAACACAAAGTCAGTGCCCTGCTTACTCCAGCTGAGCAGACAGGCACCTGGAAACTCAAGACCCAATTTA ACAACAATGAATACAGCCAGGACTTGGATGCTTACAACACTAAAGATAAAATTGGCGTGGAGCTTACTGG ACGAACTCTGGCTGACCTAACTCTACTAGACTCCCCAATTAAAGTGCCACTTTTACTCAGTGAGCCCATC AATATCATTGATGCTTTAGAGATGAGAGATGCCGTTGAGAAGCCCCAAGAATTTACAATTGTTGCTTTTG TAAAGTATGATAAAAACCAAGATGTTCACTCCATTAACCTCCCATTTTTTGAGACCTTGCAAGAATATTT TGAGAGGAATCGACAAACCATTATAGTTGTAGTGGAAAACGTACAGAGAAACCTGAAGCACATCAATATT GATCAATTTGTAAGAAAATACAGAGCAGCCCTGGGAAAACTCCCACAGCAAGCTAATGATTATCTGAATT CATTCAATTGGGAGAGACAAGTTTCACATGCCAAGGAGAAACTGACTGCTCTCACAAAAAAGTATAGAAT TACAGAAAATGATATACAAATTGCATTAGATGATGCCAAAATCAACTTTAATGAAAAACTATCTCAACTG CAGACATATATGATACAATTTGATCAGTATATTAAAGATAGTTATGATTTACATGATTTGAAAATAGCTA TTGCTAATATTATTGATGAAATCATTGAAAAATTAAAAAGTCTTGATGAGCACTATCATATCCGTGTAAA TTTAGTAAAAACAATCCATGATCTACATTTGTTTATTGAAAATATTGATTTTAACAAAAGTGGAAGTAGT ACTGCATCCTGGATTCAAAATGTGGATACTAAGTACCAAATCAGAATCCAGATACAAGAAAAACTGCAGC AGCTTAAGAGACACATACAGAATATAGACATCCAGCACCTAGCTGGAAAGTTAAAACAACACATTGAGGC TATTGATGTTAGAGTGCTTTTAGATCAATTGGGAACTACAATTTCATTTGAAAGAATAAATGATGTTCTT GAGCATGTCAAACACTTTGTTATAAATCTTATTGGGGATTTTGAAGTAGCTGAGAAAATCAATGCCTTCA

GAGCCAAAGTCCATGAGTTAATCGAGAGGTATGAAGTAGACCAACAAATCCAGGTTTTAATGGATAAATT AGTAGAGTTGACCCACCAATACAAGTTGAAGGAGACTATTCAGAAGCTAAGCAATGTCCTACAACAAGTT . AAGATAAAAGATTACTTTGAGAAATTGGTTGGATTTATTGATGATGCTGTGAAGAAGCTTAATGAATTAT 5 CTTTTAAAACATTCATTGAAGATGTTAACAAATTCCTTGACATGTTGATAAAGAAATTAAAGTCATTTGA TTACCACCAGTTTGTAGATGAAACCAATGACAAAATCCGTGAGGTGACTCAGAGACTCAATGGTGAAATT CAGGCTCTGGAACTACCACAAAAAGCTGAAGCATTAAAACTGTTTTTAGAGGAAACCAAGGCCACAGTTG CAGTGTATCTGGAAAGCCTACAGGACACCAAAATAACCTTAATCATCAATTGGTTACAGGAGGCTTTAAG TTCAGCATCTTTGGCTCACATGAAGGCCAAATTCCGAGAGACTCTAGAAGATACACGAGACCGAATGTAT

10 CAAATGGACATTCAGCAGGAACTTCAACGATACCTGTCTCTGGTAGGCCAGGTTTATAGCACACTTGTCA

CCTACATTTCTGATTGGTGGACTCTTGCTGCTAAGAACCTTACTGACTTTGCAGAGCAATATTCTATCCA

, AGATTGGGCTAAACGTATGAAAGCATTGGTAGAGCAAGGGTTCACTGTTCCTGAAATCAAGACCATCCTT

^' GGGACCATGCCTGCCTTTGAAGTCAGTCTTCAGGCTCTTCAGAAAGCTACCTTCCAGACACCTGATTTTA

TAGTCCCCCTAACAGATTTGAGGATTCCATCAGTTCAGATAAACTTCAAAGACTTAAAAAATATAAAAAT

^•15 . CCCATCCAGGTTTTCCACACCAGAATTTACCATCCTTAACACCTTCCACATTCCTTCCTTTACAATTGAC TTTGTCGAAATGAAAGTAAAGATCATCAGAACCATTGACCAGATGCAGAACAGTGAGCTGCAGTGGCCCG TTCCAGATATATATCTCAGGGATCTGAAGGTGGAGGACATTCCTCTAGCGAGAATCACCCTGCCAGACTT CCGTTTACCAGAAATCGCAATTCCAGAATTCATAATCCCAACTCTCAACCTTAATGATTTTCAAGTTCCT GACCTTCACATACCAGAATTCCAGCTTCCCCACATCTCACACACAATTGAAGTACCTACTTTTGGCAAGC

20 TATACAGTATTCTGAAAATCCAATCTCCTCTTTTCACATTAGATGCAAATGCTGACATAGGGAATGGAAC CACCTCAGCAAACGAAGCAGGTATCGCAGCTTCCATCACTGCCAAAGGAGAGTCCAAATTAGAAGTTCTC AATTTTGATTTTCAAGCAAATGCACAACTCTCAAACCCTAAGATTAATCCGCTGGCTCTGAAGGAGTCAG TGAAGTTCTCCAGCAAGTACCTGAGAACGGAGCATGGGAGTGAAATGCTGTTTTTTGGAAATGCTATTGA GGGAAAATCAAACACAGTGGCAAGTTTACACACAGAAAAAAATACACTGGAGCTTAGTAATGGAGTGATT

25 GTCAAGATAAACAATCAGCTTACCCTGGATAGCAACACTAAATACTTCCACAAATTGAACATCCCCAAAC TGGACTTCTCTAGTCAGGCTGACCTGCGCAACGAGATCAAGACACTGTTGAAAGCTGGCCACATAGCATG GACTTCTTCTGGAAAAGGGTCATGGAAATGGGCCTGCCCCAGATTCTCAGATGAGGGAACACATGAATCA CAAATTAGTTTCACCATAGAAGGACCCCTCACTTCCTTTGGACTGTCCAATAAGATCAATAGCAAACACC TAAGAGTAAACCAAAACTTGGTTTATGAATCTGGCTCCCTCAACTTTTCTAAACTTGAAATTCAATCACA

30 ^' AGTCGATTCCCAGCATGTGGGCCACAGTGTTCTAACTGCTAAAGGCATGGCACTGTTTGGAGAAGGGAAG GCAGAGTTTACTGGGAGGCATGATGCTCATTTAAATGGAAAGGTTATTGGAACTTTGAAAAATTCTCTTT CTTTTCAGCCCAGCCATTTGAGATCACGGCATCCACAAACAATGAAGGGAATTTGAAAGTTCGTTTTCC ATTAAGGTTAACAGGGAAGATAGACTTCCTGAATAACTATGCACTGTTTCTGAGTCCCAGTGCCCAGCAA GCAAGTTGGCAAGTAAGTGCTAGGTTCAATCAGTATAAGTACAACCAAAATTTCTCTGCTGGAAACAACG

35 AGAACATTATGGAGGCCCATGTAGGAATAAATGGAGAΆGCAAATCTGGΆTTTCTTAAACATTCCTTTAAC AATTCCTGAAATGCGTCTACCTTACACAATAATCACAACTCCTCCACTGAAAGATTTCTCTCTATGGGAA AAAACAGGCTTGAAGGAATTCTTGAAAACGACAAAGCAATCATTTGATTTAAGTGTAAAAGCTCAGTATA AGAAAAACAAACACAGGCATTCCATCACAAATCCTTTGGCTGTGCTTTGTGAGTTTATCAGTCAGAGCAT •CAAATCCTTTGACAGGCATTTTGAAAAAAACAGAAACAATGCATTAGATTTTGTCACCAAATCCTATAAT

40 GAAACAAAAATTAAGTTTGATAAGTACAAAGCTGAAAAATCTCACGACGAGCTCCCCAGGACCTTTCAAA TTCCTGGATACACTGTTCCAGTTGTCAATGTTGAAGTGTCTCCATTCACCATAGAGATGTCGGCATTCGG CTATGTGTTCCCAAAAGCAGTCAGCATGCCTAGTTTCTCCATCCTAGGTTCTGACGTCCGTGTGCCTTCA TACACATTAATCCTGCCATCATTAGAGCTGCCAGTCCTTCATGTCCCTAGAAATCTCAAGCTTTCTCTTC CACATTTCAAGGAATTGTGTACCATAAGCCATATTTTTATTCCTGCCATGGGCAATATTACCTATGATTT

45 CTCCTTTAAATCAAGTGTCATCACACTGAATACCAATGCTGAACTTTTTAACCAGTCAGATATTGTTGCT CATCTCCTTTCTTCATCTTCATCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGA CAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTCATAA CAGTACTGTGAGCTTAACCACGAAAAATATGGAAGTGTCAGTGGCAAAAACCACAAAAGCCGAAATTCCA ATTTTGAGAATGAATTTCAAGCAAGAACTXAATGGAAATACCAAGTCAAAACCTACTGTCTCTTCCTCCA

50 TGGAATTTAAGTATGATTTCAATTCTTCAATGCTGTACTCTACCGCTAAAGGAGCAGTTGACCACAAGCT TAGCTTGGAAAGCCTCACCTCTTACTTTTCCATTGAGTCATCTACCAAAGGAGATGTCAAGGGTTCGGTT CTTTCTCGGGAATATTCAGGAACTATTGCTAGTGAGGCCAACACTTACTTGAATTCCAAGAGCACACGGT CTTCAGTGAAGCTGCAGGGCACTTCCAAAATTGATGATATCTGGAACCTTGAAGTAAAAGAAAATTTTGC TGGAGAAGCCACACTCCAACGCATATATTCCCTCTGGGAGCACAGTACGAAAAACCACTTACAGCTAGAG

55 GGCCTCTTTTTCACCAACGGAGAACATACAAGCAAAGCCACCCTGGAACTCTCTCCATGGCAAATGTCAG CTCTTGTTCAGGTCCATGCAAGTCAGCCCAGTTCCTTCCATGATTTCCCTGACCTTGGCCAGGAAGTGGC CCTGAATGCTAACACTAAGAACCAGAAGATCAGATGGAAAAATGAAGTCCGGATTCATTCTGGGTCTTTC CAGAGCCAGGTCGAGCTTTCCAATGACCAAGAAAAGGCACACCTTGACATTGCAGGATCCTTAGAAGGAC ACCTAAGGTTCCTCAAAAATATCATCCTACCAGTCTATGACAAGAGCTTATGGGATTTCCTAAAGCTGGA TGTAACCACCAGCATTGGTAGGAGACAGCATCTTCGTGTTTCAACTGCCTTTGTGTACACCAAAAACCCC AATGGCTATTCATTCTCCATCCCTGTAAAAGTTTTGGCTGATAAATTCATTACTCCTGGGCTGAAACTAA ATGATCTAAATTCAGTTCTTGTCATGCCTACGTTCCATGTCCCATTTACAGATCTTCAGGTTCCATCGTG CAAACTTGACTTCAGAGAAATACAAATCTATAAGAAGCTGAGAACTTCATCATTTGCCCTCAACCTACCA ACACTCCCCGAGGTAAAATTCCCTGAAGTTGATGTGTTAACAAAATATTCTCAACCAGAAGACTCCTTGA TTCCCTTTTTTGAGATAACCGTGCCTGAATCTCAGTTAACTGTGTCCCAGTTCACGCTTCCAAAAAGTGT TTCAGATGGCATTGCTGCTTTGGATCTAAATGCAGTAGCCAACAAGATCGCAGACTTTGAGTTGCCCACC ATCATCGTGCCTGAGCAGACCATTGAGATTCCCTCCATTAAGTTCTCTGTACCTGCTGGAATTGTCATTC CTTCCTTTCAAGCACTGACTGCACGCTTTGAGGTAGACTCTCCCGTGTATAATGCCACTTGGAGTGCCAG TTTGAAAAACAAAGCAGATTATGTTGAAACAGTCCTGGATTCCACATGCAGCTCAACCGTACAGTTCCTA GAATATGAACTAAATGTTTTGGGAACACACAAAATCGAAGATGGTACGTTAGCCTCTAAGACTAAAGGAA CACTTGCACACCGTGACTTCAGTGCAGAATATGAAGAAGATGGCAAATTTGAAGGACTTCAGGAATGGGA AGGAAAAGCGCACCTCAATATCAAAAGCCCAGCGTTCACCGATCTCCATCTGCGCTACCAGAAAGACAAG AAAGGCATCTCCACCTCAGCAGCCTCCCCAGCCGTAGGCACCGTGGGCATGGATATGGATGAAGATGACG ACTTTTCTAAATGGAACTTCTACTACAGCCCTCAGTCCTCTCCAGATAAAAAACTCACCATATTCAAAAC TGAGTTGAGGGTCCGGGAATCTGATGAGGAAACTCAGATCAAAGTTAATTGGGAAGAAGAGGCAGCTTCT GGCTTGCTAACCTCTCTGAAAGACAACGTGCCCAAGGCCACAGGGGTCCTTTATGATTATGTCAACAAGT ACCACTGGGAACACACAGGGCTCACCCTGAGAGAAGTGTCTTCAAAGCTGAGAAGAAATCTGCAGAACAA TGCTGAGTGGGTTTATCAAGGGGCCATTAGGCAAATTGATGATATCGACGTGAGGTTCCAGAAAGCAGCC AGTGGCACCACTGGGACCTACCAAGAGTGGAAGGACAAGGCCCAGAATCTGTACCAGGAACTGTTGACTC ^■ AGGAAGGCCAAGCCAGTTTCCAGGGACTCAAGGATAACGTGTTTGATGGCTTGGTACGAGTTACTCAAAA ATTCCATATGAAAGTCAAGCATCTGATTGACTCACTCATTGATTTTCTGAACTTCCCCAGATTCCAGTTT CCGGGGAAACCTGGGATATACACTAGGGAGGAACTTTGCACTATGTTCATAAGGGAGGTAGGGACGGTAC TGTCCCAGGTATATTCGAAAGTCCATAATGGTTCAGAAATACTGTTTTCCTATTTCCAAGACCTAGTGAT TACACTTCCTTTCGAGTTAAGGAAACATAAAC AATAGATGTAATCTCGATGTATAGGGAACTGTTGAAA GATTTATCAAAAGAAGCCCAAGAGGTATTTAAAGCCATTCAGTCTCTCAAGACCACAGAGGTGCTACGTA ATCTTCAGGACCTTTTACAATTCATTTTCCAACTAATAGAAGATAACATTAAACAGCTGAAAGAGATGAA ATTTACTTATCTTATTAATTATATCCAAGATGAGATCAACACAATCTTCAATGATTATATCCCATATGTT TTTAAATTGTTGAAAGAAAACCTATGCCTTAATCTTCATAAGTTCAATGAATTTATTCAAAACGAGCTTC AGGAAGCTTCTCAAGAGTTACAGCAGATCCATCAATACATTATGGCCCTTCGTGAAGAATATTTTGATCC AAGTATAGTTGGCTGGACAGTGAAATATTATGAACTTGAAGAAAAGATAGTCAGTCTGATCAAGAACCTG TTAGTTGCTCTTAAGGACTTCCATTCTGAATATATTGTCAGTGCCTCTAACTTTACTTCCCAACTCTCAA GTCAAGTTGAGCAATTTCTGCACAGAAATATTCAGGAATATCTTAGCATCCTTACCGATCCAGATGGAAA AGGGAAAGAGAAGATTGCAGAGCTTTCTGCCACTGCTCAGGAAATAATTAAAAGCCAGGCCATTGCGACG

AAGAAAATAATTTCTGATTACCACCAGCAGTTTAGATATAAACTGCAAGATTTTTCAGACCAACTCTCTG

ATTACTATGAAAAATTTATTGCTGAATCCAAAAGATTGATTGACCTGTCCATTCAAAACTACCACACATT

^■ TCTGATATACATCACGGAGTTACTGAAAAAGCTGCAATCAACCACAGTCATGAACCCCTACATGAAGCTT

GCTCCAGGAGAACTTACTATCATCCTCTAATTTTTTAAAAGAAATCTTCATTTATTCTTCTTTTCCAATT GAACTTTCACATAGCACAGAAAAAATTCAAACTGCCTATATTGATAAAACCATACAGTGAGCCAGCCTTG CAGTAGGCAGTAGACTATAAGCAGAAGCACATATGAACTGGACCTGCACCAAAGCTGGCACCAGGGCTCG GAAGGTCTCTGAACTCAGAAGGATGGCATTTTTTGCAAGTTAAAGAAAATCAGGATCTGAGTTATTTTGC TAAACTTGGGGGAGGAGGAACAAATAAATGGAGTCTTTATTGTGTATCATA (SEQ ID NO: 6681)

>gi 14557442 | ref |NM_000078.11 Homo sapiens cholesteryl ester transfer protein, plasma (CETP) , mRNA

GTGAATCTCTGGGGCCAGGAAGACCCTGCTGCCCGGAAGAGCCTCATGTTCCGTGGGGGCTGGGCGGACA TACATATACGGGCTCCAGGCTGAACGGCTCGGGCCACTTACACACCACTGCCTGATAACCATGCTGGCTG CCACAGTCCTGACCCTGGCCCTGCTGGGCAATGCCCATGCCTGCTCCAAAGGCACCTCGCACGAGGCAGG CATCGTGTGCCGCATCACCAAGCCTGCCCTCCTGGTGTTGAACCACGAGACTGCCAAGGTGATCCAGACC GCCTTCCAGCGAGCCAGCTACCCAGATATCACGGGCGAGAAGGCCATGATGCTCCTTGGCCAAGTCAAGT ATGGGTTGCACAACATCCAGATCAGCCACTTGTCCATCGCCAGCAGCCAGGTGGAGCTGGTGGAAGCCAA GTCCATTGATGTCTCCATTCAGAACGTGTCTGTGGTCTTCAAGGGGACCCTGAAGTATGGCTACACCACT GCCTGGTGGCTGGGTATTGATCAGTCCATTGACTTCGAGATCGACTCTGCCATTGACCTCCAGATCAACA CACAGCTGACCTGTGACTCTGGTAGAGTGCGGACCGATGCCCCTGACTGCTACCTGTCTTTCCATAAGCT GCTCCTGCATCTCCAAGGGGAGCGAGAGCCTGGGTGGATCAAGCAGCTGTTCACAAATTTCATCTCCTTC ACCCTGAAGCTGGTCCTGAAGGGACAGATCTGCAAAGAGATCAACGTCATCTCTAACATCATGGCCGATT TTGTCCAGACAAGGGCTGCCAGCATCCTTTCAGATGGAGACATTGGGGTGGACATTTCCCTGACAGGTGA TCCCGTCATCACAGCCTCCTACCTGGAGTCCCATCACAAGGGTCATTTCATCTACAAGAATGTCTCAGAG GACCTCCCCCTCCCCACCTTCTCGCCCACACTGCTGGGGGACTCCCGCATGCTGTACTTCTGGTTCTCTG AGCGAGTCTTCCACTCGCTGGCCAAGGTAGCTTTCCAGGATGGCCGCCTCATGCTCAGCCTGATGGGAGA CGAGTTCAAGGCAGTGCTGGAGACCTGGGGCTTCAACACCAACCAGGAAATCTTCCAAGAGGTTGTCGGC GGCTTCCCCAGCCAGGCCCAAGTCACCGTCCACTGCCTCAAGATGCCCAAGATCTCCTGCCAAAACAAGG GAGTCGTGGTCAATTCTTCAGTGATGGTGAAATTCCTCTTTCCACGCCCAGACCAGCAACATTCTGTAGC TTACACATTTGAAGAGGATATCGTGACTACCGTCCAGGCCTCCTATTCTAAGAAAAAGCTCTTCTTAAGC CTCTTGGATTTCCAGATTACACCAAAGACTGTTTCCAACTTGACTGAGAGCAGCTCCGAGTCCATCCAGA GCTTCCTGCAGTCAATGATCACCGCTGTGGGCATCCCTGAGGTCATGTCTCGGCTCGAGGTAGTGTTTAC AGCCCTCATGAACAGCAAAGGCGTGAGCCTCTTCGACATCATCAACCCTGAGATTATCACTCGAGATGGC TTCCTGCTGCTGCAGATGGACTTTGGCTTCCCTGAGCACCTGCTGGTGGATTTCCTCCAGAGCTTGAGCT AGAAGTCTCCAAGGAGGTCGGGATGGGGCTTGTAGCAGAAGGCAAGCACCAGGCTCACAGCTGGAACCCT GGTGTCTCCTCCAGCGTGGTGGAAGTTGGGTTAGGAGTACGGAGATGGAGATTGGCTCCCAACTCCTCCC TATCCTAAAGGCCCACTGGCATTAAAGTGCTGTATCCAAG (SEQ ID NO: 6682)

>gi|414668 | emb |X75500.1 | HSMTP H. sapiens mRNA for microsomal triglyceride transfer protein

TGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATT CAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTC CACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAAC GTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGG ATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAA AATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTC AAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTAT TTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCA GGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACG ACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCT TTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAA AATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAG GCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGA GCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAA CCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAA GAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTG CTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCT CCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATT AGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTG TCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGG AGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTT CCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCA CTGCTCTCCAGAGATATGATCTCCCTTTCATAACTGATGAGGTGAAGAAGACCTTAAACAGAATATACCA CCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCCA TCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATGC TCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGGA AATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGAA CGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGAA GTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCCA AGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTATG TCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCCA AAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTCA GGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTCA GGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTAA TAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAGC AGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAAG GATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCTC AGAAAΆGAΆAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCAA AGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTAC TTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGTT TACΆTATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGTA TATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTAC TATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGTT AAAAΆCAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAΆCAATTTTTGATCAATGTA TATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAAA AAAAAAAAAAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGGAG AGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGTTA GTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATC (SEQ ID NO: 6683)

>gi|l217638]emb|X91148.l]HSMTTP H. sapiens mRNA for microsomal triglyceride transfer protein

TGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATT CAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTC CACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAAC GTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGG ATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAA AATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTC AAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTAT TTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCA GGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACG ACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCT TTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAA AATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAG GCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGA GCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAA CCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAA GAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTG CTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCT CCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATT AGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTG TCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGG AGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTT CCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCA CTGCTCTCCAGAGATATGATGCTCCCTTTCATAACTGATGAGGTGAAGAAGACCTTAAACAGAATATACC ACCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCC ATCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATG CTCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGG AAATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGA ACGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGA AGTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCC AAGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTAT GTCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCC AAAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTC AGGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTC AGGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTA ATAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAG CAGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAA GGATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCT CAGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCA AAGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTA CTTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGT TTACATATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGT ATATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTA CTTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGT TAAAAACAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGT ATATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAA AAAAAAAAAAGAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGG AGAAGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGT TAGTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATCTCATCTTTCTGCTGACCATTTTAGTGAG GTTGTTCCAAAGAGCATTCAGGTCTCTACCTCCAGCCCTGCAAAAATATTGGACCTAGCACAGAGGAATC AGGAAAATTAATTTCAGAAACTCCATTTGATTTTTCTTTTGCTGTGTCTTTTTTGAGACTGTAATATGGT ACACTGTCCTCTAAGGACATCCTCATTTTATCTCACCTTTTTGGGGGTGAGAGCTCTAGTTCATTTAACT GTACTCTGCACAATAGCTAGGATGACTAAGAGAACATTGCTTCAAGAAACTGGTGGATTTGGATTTCCAA AATATGAAATAAGGAGAAAAATGTTTTTATTTGTATGAATTAAAAGATCCATGTTGAACATTTGCAAATA TTTATTAATAAACAGATGTGGTGATAAACCCAAAACAAATGACAGGTGCTTATTTTCCACTAAACACAGA CACATGAAATGAAAGTTTAGCTAGCCCACTATTTGTTGTAAATTGAAAACGAAGTGTGATAAAATAAATA TGTAGAAATCAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6684)

>gi | 21361125 | ref |NM_001467.2 | Homo sapiens glucose-6-phosphatase, transport (glucose-6-phosphate) protein 1 (G6PT1) , mRNA GGCACGAGGGGCCACCGAGGCGCTGTCCCTGACCACCAGCACGAGACCCCTTTCTATCGCGCCAGTCCTG TGGTCTCCGCACCTCTCCAGCTCCTGCACCCCCGGCCCCCGTGGTTCCCAGCCGCACAGTAGCGTGTCCT GGGTAGCGTGAGGACCCACGGGGCTGAGCAGGTGCCACGAGCCCGCCGCCTCTTCGCCGCCCGCCGCCTC TCCTCCTCTCCCGCCCGCCGCCTGGCCCTCCCCTACCAGGCTGAGCCTCTGGCTGCCAGAAGCGCGGGGC CTCCGGGAGAATACGTGCGGTCGCCCGCTCCGCGTGCGCCTACGCCTTCTGCTCCAGTTGCTTTCCCAAT TGAGCGGAAAAGCCGGGGCATGTTGCCGGGGCCCTGGGCGGGACGGTTGTGCCCTGCAGCCCGAAGCCCG CCGGGGCACCTTCCCGCCCACGAGCTGCCCAGTCCCTCTGCTTGCGGCCCCTGCCAACGTCCCACAGGAC ACTGGGTCCCCTTGGAGCCTCCCCAGGCTTAATGATTGTCCAGAAGGCGGCTATAAAGGGAGCCTGGGAG GCTGGGTGGAGGAGGGAGCAGAAAAAACCCAACTCAGCAGATCTGGGAACTGTGAGAGCGGCAAGCAGGA ACTGTGGTCAGAGGCTGTGCGTCTTGGCTGGTAGGGCCTGCTCTTTTCTACCATGGCAGCCCAGGGCTAT GGCTATTATCGCACTGTGATCTTCTCAGCCATGTTTGGGGGCTACAGCCTGTATTACTTCAATCGCAAGA CCTTCTCCTTTGTCATGCCATCATTGGTGGAAGAGATCCCTTTGGACAAGGATGATTTGGGGTTCATCAC CAGCAGCCAGTCGGCAGCTTATGCTATCAGCAAGTTTGTCAGTGGGGTGCTGTCTGACCAGATGAGTGCT CGCTGGCTCTTCTCTTCTGGGCTGCTCCTGGTTGGCCTGGTCAACATATTCTTTGCCTGGAGCTCCACAG TACCTGTCTTTGCTGCCCTCTGGTTCCTTAATGGCCTGGCCCAGGGGCTGGGCTGGCCCCCATGTGGGAA GGTCCTGCGGAAGTGGTTTGAGCCATCTCAGTTTGGCACTTGGTGGGCCATCCTGTCAACCAGCATGAAC CTGGCTGGAGGGCTGGGCCCTATCCTGGCAACCATCCTTGCCCAGAGCTACAGCTGGCGCAGCACGCTGG CCCTATCTGGGGCACTGTGTGTGGTTGTCTCCTTCCTCTGTCTCCTGCTCATCCACAATGAACCTGCTGA TGTTGGACTCCGCAACCTGGACCCCATGCCCTCTGAGGGCAAGAAGGGCTCCTTGAAGGAGGAGAGCACC CTGCAGGAGCTGCTGCTGTCCCCTTACCTGTGGGTGCTCTCCACTGGTTACCTTGTGGTGTTTGGAGTAA AGACCTGCTGTACTGACTGGGGCCAGTTCTTCCTTATCCAGGAGAAAGGACAGTCAGCCCTTGTAGGTAG CTCCTACATGAGTGCCCTGGAAGTTGGGGGCCTTGTAGGCAGCATCGCAGCTGGCTACCTGTCAGACCGG GCCATGGCAAAGGCGGGACTGTCCAACTACGGGAACCCTCGCCATGGCCTGTTGCTGTTCATGATGGCTG GCATGACAGTGTCCATGTACCTCTTCCGGGTAACAGTGACCAGTGACTCCCCCAAGCTCTGGATCCTGGT ATTGGGAGCTGTATTTGGTTTCTCCTCGTATGGCCCCATTGCCCTGTTTGGAGTCATAGCCAACGAGAGT GCCCCTCCCAACTTGTGTGGCACCTCCCACGCCATTGTGGGACTCATGGCCAATGTGGGCGGCTTTCTGG CTGGGCTGCCCTTCAGCACCATTGCCAAGCACTACAGTTGGAGCACAGCCTTCTGGGTGGCTGAAGTGAT TTGTGCGGCCAGCACGGCTGCCTTCTTCCTCCTACGAAACATCCGCACCAAGATGGGCCGAGTGTCCAAG AAGGCTGAGTGAAGAGAGTCCAGGTTCCGGAGCACCATCCCACGGTGGCCTTCCCCCTGCACGCTCTGCG GGGAGAAAAGGAGGGGCCTGCCTGGCTAGCCCTGAACCTTTCACTTTCCATTTCTGCGCCTTTTCTGTCA CCCGGGTGGCGCTGGAAGTTATCAGTGGCTAGTGAGGTCCCAGCTCCCTGATCCTATGCTCTATTTAAAA GATAACCTTTGGCCTTAGACTCCGTTAGCTCCTATTTCCTGCCTTCAGACAAACAGGAAACTTCTGCAGT CAGGAAGGCTCCTGTACCCTTCTTCTTTTCCTAGGCCCTGTCCTGCCCGCATCCTACCCCATCCCCACCT GAAGTGAGGCTATCCCTGCAGCTGCAGGGCACTAATGACCCTTGACTTCTGCTGGGTCCTAAGTCCTCTC AGCAGTGGGTGACTGCTGTTGCCAATACCTCAGACTCCAGGGAAAGAGAGGAGGCCATCATTCTCACTGT ACCACTAGGCGCAGTTGGATATAGGTGGGAAGAAAAGGTGACTTGTTATAGAAGATTAAAACTAGATTTG ATACTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6685) gi |4503130 jref |NM_001904.11 Homo sapiens catenin (cadherin-associated protein), beta 1, 88kDa (CTNNB1) , mRNA

AAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCCGGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGAGACGG AGGAAGGTCTGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGGACGGTCGGACTCCCG CGGCGGGAGGAGCCTGTTCCCCTGAGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTG GACAATGGCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTT AGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTC TGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAACAGGG ATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAG AGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGCAGATCCCATCTACACAGTTTGATG CTGCTCATCCCACTAATGTCCAGCGTTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAACTT GATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCCCTGAACTGACAAAACTGCTAAATGAC GAGGACCAGGTGGTGGTTAATAAGGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGAC ACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTACCATGCAGAATACAAATGATGTAGA AACAGCTCGTTGTACCGCTGGGACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATCTTT AAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTTCACCAGTGGATTCTGTGTTGTTTTATGCCA TTACAACTCTCCACAACCTTTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGTGGGCT GCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAA ATTTTAGCTTATGGCAACCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCTTTAGTAA ATATAATGAGGACCTATACTTACGAAAAACTACTGTGGACCACAAGCAGAGTGCTGAAGGTGCTATCTGT CTGCTCTAGTAATAAGCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCACCTGACAGAT CCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAATCTTTCAGATGCTGCAACTAAACAGG AAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAATGTGGTCACCTG TGCAGCTGGAATTCTTTCTAACCTCACTTGCAATAATTATAAGAACAAGATGATGGTCTGCCAAGTGGGT GGTATAGAGGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCT GTGCTCTTCGTCATCTGACCAGCCGACACCAAGAAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTA TGGACTACCAGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATAAAGGCTACTGTTGGA TTGATTCGAAATCTTGCCCTTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGAC TAGTTCAGTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCATGGGTGGGACACAGCAGCA ATTTGTGGAGGGGGTCCGCATGGAAGAAATAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGG GATGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTTGTGCAGCTGCTTTATTCTC CCATTGAAAACATCCAAAGAGTAGCTGCAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGA AGCTATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAGAGTTACTTCACTCTAGGAATGAAGGTGTGGCG^' ACATATGCAGCTGCTGTTTTGTTCCGAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAG TTGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGGAATGAGACTGCTGATCTTGGACTTGA TATTGGTGCCCAGGGAGAACCCCTTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGGT GGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAACATGAGATGGGTGGCCACCACCCTGGTG CTGACTATCCAGTTGATGGGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCCAGG TGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCCTTTAGCTGTATTGTCTGAACTTG CATTGTGATTGGCCTGTAGAGTTGCTGAGAGGGCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGAC ACACTAACCAAGCTGAGTTTCCTATGGGAACAATTGAAGTAAACTTTTTGTTCTGGTCCTTTTTGGTCGA GGAGTAACAATACAAATGGATTTTGGGAGTGACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGA TGGAATTTAGCAAACCCTAGCCTTGCTTGTTAAAATTTTTTTTTTTTTTTTTTTAAGAATATCTGTAATG GTACTGACTTTGCTTGCTTTGAAGTAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTT TTTAAGTCTCTCGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAATTTCTAATTTTTAAGAATTGAGT AATGGTGTAGAACACTAATTAATTCATAATCACTCTAATTAATTGTAATCTGAATAAAGTGTAACAATTG TGTAGCCTTTTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTTTCCTTTTTAATATGCTTAAAA TAAGCAGGTGGATCTATTTCATGTTTTTGATCAAAAACTATTTGGGATATGTATGGGTAGGGTAAATCAG TAAGAGGTGTTATTTGGAACCTTGTTTTGGACAGTTTACCAGTTGCCTTTTATCCCAAAGTTGTTGTAAC CTGCTGTGATACGATGCTTCAAGAGAAAATGCGGTTATAAAAAATGGTTCAGAATTAAACTTTTAATTCA TT (SEQ ID NO: 6686) gi| 18104977 I ref |NM_002827.2 | Homo sapiens protein tyrosine phosphatase, non- receptor type 1 (PTPN1) , mRNA

GTGATGCGTAGTTCCGGCTGCCGGTTGACATGAAGAAGCAGCAGCGGCTAGGGCGGCGGTAGCTGCAGGG GTCGGGGATTGCAGCGGGCCTCGGGGCTAAGAGCGCGACGCGGCCTAGAGCGGCAGACGGCGCAGTGGGC CGAGAAGGAGGCGCAGCAGCCGCCCTGGCCCGTCATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAG TCCGGGAGCTGGGCGGCCATTTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATGTAGAGTGGCCA AGCTTCCTAAGAACAAAAACCGAAATAGGTACAGAGACGTCAGTCCCTTTGACCATAGTCGGATTAAACT ACATCAAGAAGATAATGACTATATCAACGCTAGTTTGATAAAAATGGAAGAAGCCCAAAGGAGTTACATT CTTACCCAGGGCCCTTTGCCTAACACATGCGGTCACTTTTGGGAGATGGTGTGGGAGCAGAAAAGCAGGG GTGTCGTCATGCTCAACAGAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACAAAAAGA AGAAAAAGAGATGATCTTTGAAGACACAAATTTGAAATTAACATTGATCTCTGAAGATATCAAGTCATAT TATACAGTGCGACAGCTAGAATTGGAAAACCTTACAACCCAAGAAACTCGAGAGATCTTACATTTCCACT ATACCACATGGCCTGACTTTGGAGTCCCTGAATCACCAGCCTCATTCTTGAACTTTCTTTTCAAAGTCCG AGAGTCAGGGTCACTCAGCCCGGAGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCT GGAACCTTCTGTCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTGATA TCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAGACAGCCGACCAGCTGCGCTT CTCCTACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGGGGGACTCTTCCGTGCAGGATCAGTGGAAG GAGCTTTCCCACGAGGACCTGGAGCCCCCACCCGAGCATATCCCCCCACCTCCCCGGCCACCCAAACGAA TCCTGGAGCCACACAATGGGAAATGCAGGGAGTTCTTCCCAAATCACCAGTGGGTGAAGGAAGAGACCCA GGAGGATAAAGACTGCCCCATCAAGGAAGAAAAAGGAAGCCCCTTAAATGCCGCACCCTACGGCATCGAA AGCATGAGTCAAGACACTGAAGTTAGAAGTCGGGTCGTGGGGGGAAGTCTTCGAGGTGCCCAGGCTGCCT CCCCAGCCAAAGGGGAGCCGTCACTGCCCGAGAAGGACGAGGACCATGCACTGAGTTACTGGAAGCCCTT CCTGGTCAACATGTGCGTGGCTACGGTCCTCACGGCCGGCGCTTACCTCTGCTACAGGTTCCTGTTCAAC AGCAACACATAGCCTGACCCTCCTCCACTCCACCTCCACCCACTGTCCGCCTCTGCCCGCAGAGCCCACG CCCGACTAGCAGGCATGCCGCGGTAGGTAAGGGCCGCCGGACCGCGTAGAGAGCCGGGCCCCGGACGGAC GTTGGTTCTGCACTAAAACCCATCTTCCCCGGATGTGTGTCTCACCCCTCATCCTTTTACTTTTTGCCCC TTCCACTTTGAGTACCAAATCCACAAGCCATTTTTTGAGGAGAGTGAAAGAGAGTACCATGCTGGCGGCG CAGAGGGAAGGGGCCTACACCCGTCTTGGGGCTCGCCCCACCCAGGGCTCCCTCCTGGAGCATCCCAGGC GGGCGGCACGCCAACAGCCCCCCCCTTGAATCTGCAGGGAGCAACTCTCCACTCCATATTTATTTAAACA ATTTTTTCCCCAAAGGCATCCATAGTGCACTAGCATTTTCTTGAACCAATAATGTATTAAAATTTTTTGA TGTCAGCCTTGCATCAAGGGCTTTATCAAAAAGTACAATAATAAATCCTCAGGTAGTACTGGGAATGGAA GGCTTTGCCATGGGCCTGCTGCGTCAGACCAGTACTGGGAAGGAGGACGGTTGTAAGCAGTTGTTATTTA GTGATATTGTGGGTAACGTGAGAAGATAGAACAATGCTATAATATATAATGAACACGTGGGTATTTAATA AGAAACATGATGTGAGATTACTTTGTCCCGCTTATTCTCCTCCCTGTTATCTGCTAGATCTAGTTCTCAA TCACTGCTCCCCCGTGTGTATTAGAATGCATGTAAGGTCTTCTTGTGTCCTGATGAAAAATATGTGCTTG AAATGAGAAACTTTGATCTCTGCTTACTAATGTGCCCCATGTCCAAGTCCAACCTGCCTGTGCATGACCT GATCATTACATGGCTGTGGTTCCTAAGCCTGTTGCTGAAGTCATTGTCGCTCAGCAATAGGGTGCAGTTT TCCAGGAATAGGCATTTGCCTAATTCCTGGCATGACACTCTAGTGACTTCCTGGTGAGGCCCAGCCTGTC CTGGTACAGCAGGGTCTTGCTGTAACTCAGACATTCCAAGGGTATGGGAAGCCATATTCACACCTCACGC TCTGGACATGATTTAGGGAAGCAGGGACACCCCCCGCCCCCCACCTTTGGGATCAGCCTCCGCCATTCCA AGTCAACACTCTTCTTGAGCAGACCGTGATTTGGAAGAGAGGCACCTGCTGGAAACCACACTTCTTGAAA CAGCCTGGGTGACGGTCCTTTAGGCAGCCTGCCGCCGTCTCTGTCCCGGTTCACCTTGCCGAGAGAGGCG CGTCTGCCCCACCCTCAAACCCTGTGGGGCCTGATGGTGCTCACGACTCTTCCTGCAAAGGGAACTGAAG ACCTCCACATTAAGTGGCTTTTTAACATGAAAAACACGGCAGCTGTAGCTCCCGAGCTACTCTCTTGCCA GCATTTTCACATTTTGCCTTTCTCGTGGTAGAAGCCAGTACAGAGAAATTCTGTGGTGGGAACATTCGAG GTGTCACCCTGCAGAGCTATGGTGAGGTGTGGATAAGGCTTAGGTGCCAGGCTGTAAGCATTCTGAGCTG GGCTTGTTGTTTTTAAGTCCTGTATATGTATGTAGTAGTTTGGGTGTGTATATATAGTAGCATTTCAAAA TGGACGTACTGGTTTAACCTCCTATCCTTGGAGAGCAGCTGGCTCTCCACCTTGTTACACATTATGTTAG AGAGGTAGCGAGCTGCTCTGCTATATGCCTTAAGCCAATATTTACTCATCAGGTCATTATTTTTTACAAT GGCCATGGAATAAACCATTTTTACAAAA (SEQ ID NO: 6687)

gi| 12831192 |gb|AF333324.l| Hepatitis C virus type lb polyprotein mRNA, complete eds GCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCA GAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCA TAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG CTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCC TTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCATCATGAGCACA AATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTTAAGTTCCCGGGCG GTGGTCAGATCGTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAG GAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGGCCCGAGGGT AGGACCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGTATGGGGTGGGCAGGATGGC TCCTGTCACCCCGTGGCTCTCGGCCTAGTTGGGGCCCCACAGACCCCCGGCGTAGGTCGCGTAATTTGGG TAAGGTCATCGATACCCTTACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTTGTCGGCGCCCCC CTAGGAGGCGCTGCCAGGGCCCTGGCGCATGGCGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAG GGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTAGCTTTGCTGTCTTGTTTGACCATCCCAGCTTC CGCTTACGAGGTGCGCAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGTATTGTG TATGAGGCAGCGGACATGATCATGCACACCCCCGGGTGCGTGCCCTGCGTCCGGGAGAGTAATTTCTCCC GTTGCTGGGTAGCGCTCACTCCCACGCTCGCGGCCAGGAACAGCAGCATCCCCACCACGACAATACGACG CCACGTCGATTTGCTCGTTGGGGCGGCTGCTCTCTGTTCCGCTATGTACGTTGGGGATCTCTGCGGATCC GTTTTTCTCGTCTCCCAGCTGTTCACCTTCTCACCTCGCCGGTATGAGACGGTACAAGATTGCAATTGCT CAATCTATCCCGGCCACGTATCAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCACCTACAAC GGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCCGTCGTGGACATGGTGGCGGGGGCCCACTGG GGTGTCCTAGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTGATTGTGATGCTAC TCTTTGCTGGCGTTGACGGGCACACCCACGTGACAGGGGGAAGGGTAGCCTCCAGCACCCAGAGCCTCGT GTCCTGGCTCTCACAAGGGCCATCTCAGAAAATCCAACTCGTGAACACCAACGGCAGCTGGCACATCAAC AGGACCGCTCTGAATTGCAATGACTCCCTCCAAACTGGGTTCATTGCTGCGCTGTTCTACGCACACAGGT TCAACGCGTCCGGATGTCCAGAGCGCATGGCCAGCTGCCGCCCCATCGACAAGTTCGCTCAGGGGTGGGG TCCCATCACTCACGTTGTGCCTAACATCTCGGACCAGAGGCCTTATTGCTGGCACTATGCACCCCAACCG TGCGGTATTGTACCCGCGTCGCAGGTGTGTGGCCCAGTGTATTGCTTCACCCCGAGTCCTGTTGTGGTGG GGACGACCGACCGTTCCGGAGTCCCCACGTATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTACTCAA CAACACGCGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATAGCACCGGGTTCACCAAGACG TGCGGGGGCCCCCCGTGTAACATCGGGGGGGTTGGCAACAACACCTTGATTTGCCCCACGGATTGCTTCC GAAAGCACCCCGAGGCCACTTACACCAAATGCGGCTCGGGTCCTTGGTTGACACCTAGGTGTCTAGTTGA CTACCCATACAGACTTTGGCACTACCCCTGCACTATCAATTTTACCATCTTCAAGGTCAGGATGTACGTG GGGGGCGTGGAGCACAGGCTCAACGCCGCGTGCAATTGGACCCGAGGAGAGCGCTGTGACCTGGAGGACA GGGATAGATCAGAGCTTAGCCCGCTGCTATTGTCTACAACGGAGTGGCAGGTACTGCCCTGTTCCTTTAC CACCCTACCGGCTCTGTCCACTGGATTGATCCACCTCCATCAGAATATCGTGGACGTGCAATACCTGTAC GGTGTAGGGTCAGTGGTTGTCTCCGTCGTAATCAAATGGGAGTATGTTCTGCTGCTCTTCCTTCTCCTGG CGGACGCGCGCGTCTGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCTGAGGCCACCTTAGAGAA CCTGGTGGTCCTCAATGCGGCGTCTGTGGCCGGAGCGCATGGCCTTCTCTCCTTCCTCGTGTTCTTCTGC GCCGCCTGGTACATCAAAGGCAGGCTGGTCCCTGGGGCGGCATATGCTCTCTATGGCGTATGGCCGTTGC TCCTGCTCTTGCTGGCTTTACCACCACGAGCTTATGCCATGGACCGAGAGATGGCTGCATCGTGCGGAGG CGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCATACTATAAGGTGTTCCTCGCTAGGCTCATA TGGTGGTTACAATATTTTATCACCAGGGCCGAGGCGCACTTGCAAGTGTGGGTCCCCCCTCTTAATGTTC GGGGAGGCCGCGATGCCATCATCCTCCTTACATGCGCGGTCCATCCAGAGCTAATCTTTGACATCACCAA ACTCCTGCTCGCCATACTCGGTCCGCTCATGGTGCTCCAAGCTGGCATAACCAGAGTGCCGTACTTCGTG CGCGCTCAAGGGCTCATTCATGCATGCATGTTAGTGCGGAAGGTCGCTGGGGGTCATTATGTCCAAATGG CCTTCATGAAGCTGGGCGCGCTGACAGGCACGTACATTTACAACCATCTTACCCCGCTACGGGATTGGGC CCACGCGGGCCTACGAGACCTTGCGGTGGCAGTGGAGCCCGTCGTCTTCTCCGACATGGAGACCAAGATC ATCACCTGGGGAGCAGACACCGCGGCGTGTGGGGACATCATCTTGGGTCTGCCCGTCTCCGCCCGAAGGG GAAAGGAGATACTCCTGGGCCCGGCCGATAGTCTTGAAGGGCGGGGGTGGCGACTCCTCGCGCCCATCAC GGCCTACTCCCAACAGACGCGGGGCCTACTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAAC CAGGTCGAGGGAGAGGTTCAGGTGGTTTCCACCGCAACACAATCCTTCCTGGCGACCTGCGTCAACGGCG TGTGTTGGACCGTTTACCATGGTGCTGGCTCAAAGACCTTAGCCGGCCCAAAGGGGCCAATCACCCAGAT GTACACTAATGTGGACCAGGACCTCGTCGGCTGGCAGGCGCCCCCCGGGGCGCGTTCCTTGACACCATGC ACCTGTGGCAGCTCAGACCTTTACTTGGTCACGAGACATGCTGACGTCATTCCGGTGCGCCGGCGGGGCG ACAGTAGGGGGAGCCTGCTCTCCCCCAGGCCTGTCTCCTACTTGAAGGGCTCTTCGGGTGGTCCACTGCT CTGCCCTTCGGGGCACGCTGTGGGCATCTTCCGGGCTGCCGTATGCACCCGGGGGGTTGCGAAGGCGGTG GACTTTGTGCCCGTAGAGTCCATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAACTCATCCCCCC CGGCCGTACCGCAGTCATTTCAAGTGGCCCACCTACACGCTCCCACTGGCAGCGGCAAGAGTACTAAAGT GCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTCAATCCGTCCGTTGCCGCTACCTTAGGG TTTGGGGCGTATATGTCTAAGGCACACGGTATTGACCCCAACATCAGAACTGGGGTAAGGACCATTACCA CAGGCGCCCCCGTCACATACTCTACCTATGGCAAGTTTCTTGCCGATGGTGGTTGCTCTGGGGGCGCTTA TGACATCATAATATGTGATGAGTGCCATTCAACTGACTCGACTACAATCTTGGGCATCGGCACAGTCCTG GACCAAGCGGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCG TGCCACACCCAAACATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTATGGCAAAGCCAT CCCCATTGAAGCCATCAGGGGGGGAAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTC GCCGCAAAGCTGTCAGGCCTCGGAATCAACGCTGTGGCGTATTACCGGGGGCTCGATGTGTCCGTCATAC CAACTATCGGAGACGTCGTTGTCGTGGCAACAGACGCTCTGATGACGGGCTATACGGGCGACTTTGACTC AGTGATCGACTGTAACACATGTGTCACCCAGACAGTCGACTTCAGCTTGGATCCCACCTTCACCATTGAG ACGACGACCGTGCCTCAAGACGCAGTGTCGCGCTCGCAGCGGCGGGGTAGGACTGGCAGGGGTAGGAGAG GCATCTACAGGTTTGTGACTCCGGGAGAACGGCCCTCGGGCATGTTCGATTCCTCGGTCCTGTGTGAGTG CTATGACGCGGGCTGTGCTTGGTACGAGCTCACCCCCGCCGAGACCTCGGTTAGGTTGCGGGCCTACCTG AACACACCAGGGTTGCCCGTTTGCCAGGACCACCTGGAGTTCTGGGAGAGTGTCTTCACAGGCCTCACCC ACATAGATGCACACTTCTTGTCCCAGACCAAGCAGGCAGGAGACAACTTCCCCTACCTGGTAGCATACCA AGCCACGGTGTGCGCCAGGGCTCAGGCCCCACCTCCATCATGGGATCAAATGTGGAAGTGTCTCATACGG CTGAAACCTACGCTGCACGGGCCAACACCCTTGCTGTACAGGCTGGGAGCCGTCCAAAATGAGGTCACCC TCACCCACCCCATAACCAAATACATCATGGCATGCATGTCGGCTGACCTGGAGGTCGTCACTAGCACCTG GGTGCTGGTGGGCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACAGGCAGTGTGGTCATTGTG GGTAGGATTATCTTGTCCGGGAGGCCGGCTATTGTTCCCGACAGGGAGCTTCTCTACCAGGAGTTCGATG AAATGGAAGAGTGCGCCACGCACCTCCCTTACATTGAGCAGGGAATGCAGCTCGCCGAGCAGTTCAAGCA GAAAGCGCTCGGGTTACTGCAAACAGCCACCAAACAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAG TGGCGAGCCCTTGAGACATTCTGGGCGAAGCACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAG GCTTATCCACTCTGCCTGGGAACCCCGCAATAGCATCATTGATGGCATTCACAGCCTCTATCACCAGCCC GCTCACCACCCAAAGTACCCTCCTGTTTAACATCTTGGGGGGGTGGGTGGCTGCCCAACTCGCCCCCCCC AGCGCCGCTTCGGCTTTCGTGGGCGCCGGCATCGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGG TGCTTGTGGACATTCTGGCGGGTTATGGAGCAGGAGTGGCCGGCGCGCTCGTGGCCTTTAAGGTCATGAG CGGCGAGATGCCCTCTACCGAGGACCTGGTCAATCTACTTCCTGCCATCCTCTCTCCTGGCGCCCTGGTC GTCGGGGTCGTGTGTGCAGCAATACTGCGTCGGCACGTGGGTCCGGGAGAGGGGGCTGTGCAGTGGATGA ACCGGCTGATAGCGTTCGCCTCGCGGGGTAATCACGTTTCCCCCACGCACTATGTGCCTGAGAGCGACGC CGCAGCGCGTGTTACTCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGGCTCCACCAGTGG ATTAATGAGGACTGCTCCACACCGTGTTCCGGCTCGTGGCTAAGGGATGTTTGGGACTGGATATGCACGG TGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCAGCTACCGGGAGTCCCTTTTTTCTC GTGCCAACGCGGGTACAAGGGAGTCTGGCGGGGAGACGGCATCATGCAAACCACCTGCCCATGTGGAGCA CAGATCACCGGACATGTCAAAAACGGTTCCATGAGGATCGTCGGGCCTAAGACCTGCAGCAACACGTGGC ATGGAACATTCCCCATCAACGCATACACCACGGGCCCCTGCACACCCTCTCCAGCGCCAAACTATTCTAG GGCGCTGTGGCGGGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGGGATTTCCACTACGTGACG GGCATGACCACTGACAACGTAAAGTGCCCATGCCAGGTTCCGGCTCCTGAATTCTTCTCGGAGGTGGACG GAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGGCCTCTCCTACGGGAGGAGGTTACATTCCAGGTCGG GCTCAACCAATACCTGGTTGGGTCACAGCTACCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTCC ATGCTCACCGACCCCTCCCACATCACAGCAGAAACGGCTAAGCGTAGGTTGGCCAGGGGGTCTCCCCCCT CCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCACCATGT CTCTCCGGACGCTGACCTCATCGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGGAACATCACCCGC GTGGAGTCGGAGAACAAGGTGGTAGTCCTGGACTCTTTCGACCCGCTTCGAGCGGAGGAGGATGAGAGGG AAGTATCCGTTCCGGCGGAGATCCTGCGGAAATCCAAGAAGTTCCCCGCAGCGATGCCCATCTGGGCGCG CCCGGATTACAACCCTCCACTGTTAGAGTCCTGGAAGGACCCGGACTACGTCCCTCCGGTGGTGCACGGG TGCCCGTTGCCACCTATCAAGGCCCCTCCAATACCACCTCCACGGAGAAAGAGGACGGTTGTCCTAACAG AGTCCTCCGTGTCTTCTGCCTTAGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGAATCATCGGCCGT CGACAGCGGCACGGCGACCGCCCTTCCTGACCAGGCCTCCGACGACGGTGACAAAGGATCCGACGTTGAG TCGTACTCCTCCATGCCCCCCCTTGAGGGGGAACCGGGGGACCCCGATCTCAGTGACGGGTCTTGGTCTA CCGTGAGCGAGGAAGCTAGTGAGGATGTCGTCTGCTGCTCAATGTCCTACACATGGACAGGCGCCTTGAT CACGCCATGCGCTGCGGAGGAAAGCAAGCTGCCCATCAACGCGTTGAGCAACTCTTTGCTGCGCCACCAT AACATGGTTTATGCCACAACATCTCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTGC AAGTCCTGGACGACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAA ACTCCTATCCGTAGAGGAAGCCTGCAAGCTGACGCCCCCACATTCGGCCAAATCCAAGTTTGGCTATGGG GCAAAGGACGTCCGGAACCTATCCAGCAAGGCCGTTAACCACATCCACTCCGTGTGGAAGGACTTGCTGG AAGACACTGTGACACCAATTGACACCACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGAA AGGAGGCCGTAAGCCAGCCCGCCTTATCGTATTCCCAGATCTGGGAGTCCGTGTATGCGAGAAGATGGCC CTCTATGATGTGGTCTCCACCCTTCCTCAGGTCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTG GGCAGCGAGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAAAACCCCATGGGCTTTTCATATGACAC TCGCTGTTTCGACTCAACGGTCACCGAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGAC TTGGCCCCCGAAGCCAGACAGGCCATAAAATCGCTCACAGAGCGGCTTTATATCGGGGGTCCTCTGACTA ATTCAAAAGGGCAGAACTGCGGTTATCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTAA CACCCTCACATGTTACTTGAAGGCCTCTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACGATGCTC GTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGACGCGGCGAGCCTACGAG TCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCCGGGGACCCGCCCCAACCAGAATACGACTTGGA GCTGATAACATCATGTTCCTCCAATGTGTCGGTCGCCCACGATGCATCAGGCAAAAGGGTGTACTACCTC ACCCGTGATCCCACCACCCCCCTCGCACGGGCTGCGTGGGAAACAGCTAGACACACTCCAGTTAACTCCT GGCTAGGCAACATTATCATGTATGCGCCCACTTTGTGGGCAAGGATGATTCTGATGACTCACTTCTTCTC CATCCTTCTAGCACAGGAGCAACTTGAAAAAGCCCTGGACTGCCAGATCTACGGGGCCTGTTACTCCATT GAGCCACTTGACCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACT CTCCAGGTGAGATCAATAGGGTGGCTTCATGCCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAG ACATCGGGCCAGGAGCGTCCGCGCTAGGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTAC CTCTTCAACTGGGCAGTGAAGACCAAACTCAAACTCACTCCAATCCCGGCTGCGTCCCAGCTGGACTTGT CCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGCTG GTTCATGCTGTGCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGG AGCTAAACACTCCAGGCCAATAGGCCATTTCCTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCT TTTCCTTCTTTTTCCCTTTTTCTTTCTTCCTTCTTTAATGGTGGCTCCATCTTAGCCCTAGTCACGGCTA GCTGTGAAAGGTCCGTGAGCCGCATGACTGCAGAGAGTGCTGATACTGGCCTCTCTGCAGATCATGT (SEQ ID NO: 6688)

gi|306286|gb|M96362.l|HPCUNKCDS Hepatitis C virus mRNA, complete eds

TGCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGC

AGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCC ATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCC GCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGC CTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCAC GAATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGATATTAAGTTCCCGGGC GGTGGTCAGATCGTTGGTGGAGTTTACTTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTA GGAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAGCCTATCCCCAAGGCTCGCCGGCCCGAGGG CAGGGCCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAATGAGGGCTTGGGGTGGGCAGGATGG CTCCTGTCACCCCGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAAGTCGCGTAATTTGG GTAAGGTCATCGACACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCC CCTAGGGGGCGTTGCCAGGGCCCTGGCACATGGTGTCCGGGTGCTGGAGGACGGCGTGAACTATGCAACA GGGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTGGCTCTGCTGTCTTGTTTGACCACCCCAGTTT CCGCTTATGAAGTGCGTAACGCGTCCGGGATGTACCATGTCACGAACGACTGCTCCAACTCAAGCATTGT GTATGAGGCAGCGGACATGATCATGCACACTCCCGGGTGCGTGCCCTGCGTTCGGGAGGACAACTCCTCC CGTTGCTGGGTGGCACTTACTCCCACGCTCGCGGCCAGGAATGCCAGCGTCCCCACTACGACATTGCGAC GCCATGTCGACTTGCTCGTTGGGGTAGCTGCTTTCTGTTCCGCTATGTACGTGGGGGACCTCTGCGGATC TGTTTTCCTTGTTTCCCAGCTGTTCACCTTTTCGCCTCGCCGGCATGAGACGGTACAGGACTGCAACTGC TCAATCTATCCCGGCCGCGTATCAGGTCACCGCATGGCCTGGGATATGATGATGAACTGGTCGCCTACAA CAGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCTGTCGTGGACATGGTGACAGGGTCCCACTG GGGAATCCTGGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTAATTGCGATGCTA CTCTTTGCCGGCGTTGACGGAACCACCCACGTGACAGGGGGGGCGCAAGGTCGGGCCGCTAGCTCGCTAA CGTCCCTCTTTAGCCCTGGGCCGGTTCAGCACCTCCAGCTCATAAACACCAACGGCAGCTGGCATATCAA CAGGACCGCCCTGAGCTGCAATGACTCCCTCAACACTGGGTTTGTTGCCGCGCTGTTCTACAAATACAGG TTCAACGCGTCCGGGTGCCCGGAGCGCTTGGCCACGTGCCGCCCCATTGATACATTCGCGCAGGGGTGGG GTCCCATCACTTACACTGAGCCTCATGATTTGGATCAGAGGCCCTATTGCTGGCACTACGCGCCTCAACC GTGTGGTATTGTGCCCACGTTGCAGGTGTGTGGCCCAGTATACTGCTTCACCCCGAGTCCTGTTGCGGTG GGGACTACCGATCGTTTCGGTGCCCCTACATACAGATGGGGGGCAAATGAGACGGACGTGCTGCTCCTTA ACAACGCCGGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGAC ATGTGGGGGCCCCCCGTGTAACATCGGGGGGGTCGGCAACAATACCTTGACCTGCCCCACGGACTGCTTC CGAAAGCACCCCGGGGCCACTTACACCAAATGCGGTTCGGGGCCTTGGTTAACACCCAGGTGCTTAGTCG ACTACCCGTACAGGCTCTGGCATTACCCCTGCACTGTCAACTTTACCATCTTTAAGGTTAGGATGTACGT GGGGGGCGCGGAGCACAGGCTCGACGCCGCATGCAACTGGACTCGGGGAGAGCGTTGTGACCTGGAGGAC AGGGATAGGTCAGAGCTTAGCCCGCTGCTGCTGTCTACAACAGAGTGGCAGGTACTGCCCTGTTCCTTCA CAACCCTACCGGCTCTGTCCACTGGTTTGATTCATCTCCATCAGAACATCGTGGACATACAATACCTGTA CGGTATAGGGTCGGCGGTTGTCTCCTTTGCGATCAAATGGGAGTATATTGTGCTGCTCTTCCTTCTTCTG GCGGACGCGCGCGTCTGCGCTTGCTTGTGGATGATGCTGCTGGTAGCGCAAGCCGAGGCCGCCTTAGAGA ACCTGGTGGTCCTCAATGCAGCGTCCGTGGCCGGAGCGCATGGCATTCTTTCCTTCATTGTGTTCTTCTG TGCTGCCTGGTACATCAAGGGCAGGCTGGTTCCCGGAGCGGCATACGCCCTCTATGGCGTATGGCCGCTG CTTCTGCTTCTGCTGGCGTTACCACCACGGGCGTACGCCATGGACCGGGAGATGGCCGCATCGTGCGGAG GCGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCACACTATAAAGTGTTCCTTGCCAGGTTCAT ATGGTGGCTACAATATCTCATCACCAGAACCGAAGCGCATCTGCAAGTGTGGGTCCCCCCTCTCAACGTT CGGGGGGGTCGCGATGCCATCATCCTCCTCACATGCGTGGTCCACCCAGAGCTAATCTTTGACATCACAA AATATTTGCTCGCCATATTCGGCCCGCTCATGGTGCTCCAGGCCGGCATAACTAGAGTGCCGTACTTCGT GCGCGCACAAGGGCTCATTCGTGCATGCATGTTGGCGCGGAAAGTCGTGGGGGGTCATTACGTCCAAATG GTCTTCATGAAGCTGGCCGCACTAGCAGGTACGTACGTTTATGACCATCTTACTCCACTGCGAGATTGGG CTCACACGGGCTTACGAGACCTTGCAGTGGCAGTAGAGCCCGTTGTCTTCTCTGACATGGAGACCAAAGT CATCACCTGGGGGGCAGACACCGCGGCGTGCGGGGACATCATCTTGGCCCTGCCTGCTTCCGCCCGAAGG GGGAAGGAGATACTTCTGGGACCGGCCGATAGTCTTGAAGGACAGGGGTGGCGACTCCTTGCGCCCATCA CGGCCTACTCCCAACAAACGCGAGGCCTGCTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAA CCAGGTTGAGGGGGAGGTTCAAGTGGTTTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAATGGC GTGTGTTGGACTGTCTTCCACGGCGCCGGCTCAAAGACCCTAGCCGGCCCAAAGGGTCCAATCACCCAAA TGTACACCAATGTAGACCAGGACCTTGTTGGCTGGCCGGCACCTCCTGGGGCGCGTTCCCTGACACCATG CACTTGCGGCTCCTCGGACCTTTACCTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGGCGGGGT GACGGTAGGGGGAGCCTACTCCCCCCCAGGCCTGTCTCCTACTTGAAGGGCTCCTCGGGTGGTCCACTGC TCTGCCCTTCGGGGCACGCTGTCGGCATACTTCCGGCTGCTGTATGCACCCGGGGGGTTGCCATGGCGGT GGAATTCATACCCGTTGAGTCTATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAATCCGTCTCCC CCGGCTGTACCGCAGACATTCCAAGTGGCCCACTTACACGCTCCCACCGGCAGCGGCAAGAGCACTAGGG TGCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTAAATCCGTCCGTCGCCGCCACCTTGGG TTTTGGGGCGTATATGTCCAAGGCACATGGTATCGACCCCAACCTTAGAACTGGGGTAAGGACCATCACC ACAGGTGCCCCTATCACATACTCCACCTATGGCAAGTTCCTTGCCGACGGTGGCGGCTCCGGGGGCGCCT ATGACATCATAATGTGTGATGAGTGCCACTCAACTGACTCGACTACCATTTATGGCATCGGCACAGTCCT GGACCAAGCGGAGACGGCTGGAGCGCGGCTCGTGGTGCTCTCCACCGCTACGCCTCCGGGATCGGTCACC GTGCCACACCTCAATATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTACGGCAAAGCCA TTCCCATCGAGGCTATCAAGGGGGGAAGGCATCTCATTTTCTGCCATTCCAAGAAGAAGTGTGACGAACT CGCCGCAAAGCTGTCAGGCCTCGGACTCAATGCCGTAGCGTATTACCGGGGTCTTGACGTGTCCGTCATA CCGACCAGCGGAGACGTTGTTGTCGTGGCGACGGACGCTCTAATGACGGGCTTTACCGGCGACTTTGACT CAGTGATCGACTGTAATACGTGTGTCACCCAGACAGTCGATTTCAGCTTGGACCCCACCTTCACCATTGA GACGACGACCGTGCCCCAAGACGCAGTGTCGCGCTCGCAGAGGCGAGGCAGGACTGGTAGGGGCAGGGCT GGCATATACAGGTTTGTGACTCCAGGAGAACGGCCCTCGGGCATGTTCGATTCTTCGGTCCTGTGTGAGT GTTATGACGCGGGTTGTGCGTGGTACGAACTCACGCCCGCTGAGACCTCGGTTAGGTTGCGGGCGTACCT AAACACACCAGGGTTGCCCGTCTGCCAGGACCATCTGGAGTTCTCGGAGGGTGTCTTCACAGGCCTCACC CACATAGATGCCCACTTCTTATCCCAGACTAAACAGGCAGGAGAGAACTTCCCCTACTTGGTAGCATACC AGGCTACAGTGTGCGCCAGGGCTCAAGCCCCACCTCCATCGTGGGATGAAATGTGGAGGTGTCTCATACG GCTGAAACCTACGCTGCACGGGCCAACACCCCTGCTGTATAGGTTAGGAGCCGTCCAAAATGAGGTCACC CTCACACACCCCATAACCAAATTCATCATGACATGTATGTCGGCTGACCTGGAGGTCGTCACCAGCACCT GGGTGCTGGTAGGCGGAGTCCTCGCAGCTCTGGCCGCGTACTGCCTGACAACAGGCAGCGTGGTCATTGT GGGCAGGATCATCCTGTCCGGGAAGCCGGCTATCATCCCCGATAGGGAAGTTCTCTACCAGGAGTTCGAC GAGATGGAGGAGTGTGCCTCACACCTCCCTTACTTCGAACAGGGAATGCAGCTCGCCGAGCAATTCAAAC AGAAGGCGCTCGGGTTGCTGCAAACAGCCACCAAGCAGGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAA GTGGCGAGCCCTTGAGACCTTCTGGGCGAAGCACATGTGGAACTTCATTAGTGGGATACAGTACTTGGCA GGCTTGTCCACTCTGCCTGGGAACCCCGCAATACGATCACCGATGGCATTCACAGCCTCCATCACCAGCC CGCTCACCACCCAGCATACCCTCTTGTTTAACATCTTGGGGGGATGGGTGGCTGCCCAACTCGCCCCCCC CAGCGCTGCCTCAGCTTTCGTGGGCGCCGGCATCGCTGGAGCCGCTGTTGGCACGATAGGCCTTGGGAAG GTGCTTGTGGACATTCTGGCAGGTTATGGAGCAGGGGTGGCGGGCGCACTTGTGGCCTTTAAGATCATGA GCGGCGAGATGCCTTCAGCCGAGGACATGGTCAACTTACTCCCTGCCATCCTTTCTCCCGGTGCCCTGGT CGTCGGGATTGTGTGTGCAGCAATACTGCGTCGGCATGTGGGCCCAGGGGAAGGGGCTGTGCAGTGGATG AACCGGCTGATAGCGTTCGCCTCGCGGGGTAACCACGTCTCCCCCAGGCACTATGTGCCAGAGAGCGAGC CTGCAGCGCGTGTTACCCAGATCCTTTCCAGCCTCACCATCACTCAGCTGTTGAAGAGACTCCACCAGTG GATTAATGAGGACTGCTCTACGCCATGCTCCAGCTCGTGGCTAAGGGAGATTTGGGACTGGATCTGCACG GTGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGATTACCGGGAGTCCCTTTTTTCT CATGCCAACGCGGGTATAAGGGAGTCTGGCGGGGGGACGGCATCATGCACACCACCTGCCCATGCGGAGC ACAGATCACCGGACACGTCAAAAACGGTTCCATGAGGATCGTTGGGCCTAAAACCTGCAGCAACACGTGG TACGGGACATTCCCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCAAACTATTCCA AGGCATTGTGGAGAGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGAGATTTTCACTACGTGAC GGGCATGACCACTGACAACGTGAAGTGTCCATGCCAGGTTCCGGCCCCCGAATTCTTCACGGAGGTGGAT GGAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGACCTCTCCTACGGGAGGAGGTCGTATTCCAGGTCG GGCTCCACCAGTACCTGGTCGGGTCACAGCTCCCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTC CATGCTCACTGACCCCTCCCACATTACAGCAGAGACGGCTAAGCGTAGGCTGGCCAGGGGGTCTCCCCCC TCCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCATCATG ACTCCCCGGACGCTGACCTCATTGAGGCCAACCTCTTGTGGCGGCAAGAGATGGGCGGGAACATCACCCG CGTGGAGTCAGAGAATAAGGTGGTAATCCTGGACTCTTTCGACCCGCTCCGAGCGGAGGATGATGAGGGG GAAATATCCGTTCCGGCGGAGATCCTGCGGAAATCCAGGAAATTCCCCCCAGCGCTGCCCATATGGGCGC CGCCGGATTACAACCCTCCGCTGCTAGAGTCCTGGAAGGACCCGGACTACGTTCCTCCGGTGGTACACGG GTGCCCGTTGCCGCCCACCAAGGCCCCTCCAATACCACCTCCACGGAGGAAGAGGACGGTTGTCCTGACA GAATCCACCGTGTCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGGATCGTCGGCCA TCGACAGCGGTACGGCGACCGCCCCTCCTGACCAAGCCTCCGGTGACGGCGACAGAGAGTCCGACGTTGA GTCGTTCTCCTCCATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGATCTCAGCGACGGATCTTGGTCC ACCGTGAGCGAGGAGGCTAGTGAGGACGTCGTCTGCTGTTCGATGTCCTACACATGGACAGGCGCCCTGA TCACGCCATGCGCTGCGGAGGAAAGCAAGTTGCCCATCAACCCGTTGAGCAATTCTTTGCTACGTCACCA CAACATGGTCTATGCTACAACATCCCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTG CAAGTCCTGGACGACCACTACCGGGACGTGCTTAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTA AACTTCTATCTGTAGAAGAAGCCTGCAAACTGACGCCCCCACATTCGGCCAAATCCAAATTTGGCTACGG GGCGAAGGACGTCCGGAGCCTATCCAGCAGGGCCGTTACCCACATCCGCTCCGTGTGGAAGGACCTGCTG GAAGACACTGAAACACCAATTAGCACTACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGA AGGGAGGCCGCAAGCCAGCTCGCCTTATCGTGTTCCCAGATCTGGGAGTTCGTGTATGCGAGAAGATGGC CCTTTATGACGTGGTCTCCACCCTTCCTCAGGCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCT AAGCAGCGGGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAATGCCCCATGGGCTTCTCATATGACA CCCGCTGTTTTGACTCAACGGTCACTGAGAATGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGA CTTGGCCCCCGAAGCCAAACTGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACT AATTCAAAAGGGCAGAACTGCGGTTACCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTA ATACCCTCACATGTTACCTGAAAGCCACTGCGGCCTGTCGAGCTGCGAAGCTCCGGGACTGCACGATGCT CGTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGATGCGGCGAGCCTACGA GTCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCTGGGGACCCGCCTCAACCGGAATACGACTTGG AGTTGATAACATCATGTTCCTCCAATGTGTCGGTCGCACACGATGCATCTGGTAAAAGGGTGTACTACCT CACCCGTGACCCTACCACCCCCCTTGCACGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCC TGGCTAGGCAACATCATCATGTATGCGCCCACCTTATGGGCAAGGATGATTCTGATGACTCATTTCTTCT CCATCCTTCTAGCTCAGGAGCAACTTGAAAAAACCCTAGATTGTCAGATCTACGGGGCCTGTTACTCCAT TGAACCACTTGATCTACCTCAGATCATTGAGCGACTCCATGGTCTTAGCGCATTTTCACTCCATAGTTAC TCTCCAGGCGAGATCAATAGGGTGGCTTCATGCCTCAGAAAACTTGGGGTACCACCCTTGCGAGCCTGGA GACATCGGGCCAGAAGTGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTA CCTCTTCAACTGGGCGGTGAGGACCAAGCTCAAACTCACTCCAATCCCAGCCGCGTCCCGGTTGGACTTG TCCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGCT GGTTCATGTTGTGCCTACTCCTACTTTCCGTGGGGGTAGGCATCTACCTGCTCCCCAACCGATGAATGGG GAGCTAAACACTCCAGGCCAATAGGCCGTTTCTC (SEQ ID NO: 6689)

gi| 329739 |gb|L02836.l|HPCCGENOM Hepatitis C China virus complete genome ATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTA GCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGC GGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTG GAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTG CCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGAATCCTAAACC TCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTCAAGTTCCCGGGCGGTGGTCAGATC GTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAGGAAGACTTCCG AGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGACCCGAGGGCAGGACCTGGGC TCAGCCCGGGTATCCTTGGCCCCTCTATGGCAATGAGGGCTTTGGGTGGGCAGGATGGCTCCTGTCACCC CGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAGGTCGCGTAATTTGGGTAAGGTCATCG ATACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCCCTTGGGGGGCGC TGCCAGGGCCCTGGCACATGGTGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAGGGAATTTGCCC GGTTGCTCTTTCTCTATCTTCCTTTTAGCCTTGCTATCCTGTTTGACCACCCCAGCTTCCGCTTACGAAG TGCGTAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGCATTGTGTATGAGGCAGC GGACCTGATCATGCATACCCCTGGGTGCGTGCCCTGCGTTCGGGAAGGCAACTCCTCCCGTTGCTGGGTA GCGCTCACTCCCACGCTCGCGGCCAGGAACGCCACGATCCCCACTGCGACAGTACGACGGCATGTCGATC TGCTCGTTGGGGCGGCTGCTTTCTCTTCCGCCATGTACGTGGGGGATCTCTGCGGATCTGTTTTCCTTGT CTCTCAGCTGTTCACCTTCTCGCCTCGCCGGTATGAGACAATACAGGACTGCAATTGCTCAATCTATCCC GGCCACGTAACAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCGCCTACAACAGCTCTAGTGG TGTCGCAGTTACTCCGGATCCCTCAAGCCGTCATGGACATGGTGGTGGGGGCCCACTGGGGAGTCCTGGC GGGCCTTGCCTACTATGCCATGGTGGGGAATTGGGCTAAGGTTTTGATTGTGATGCTACTCTTCGCCGGC GTTGATGGGGATACCTACGCGTCTGGGGGGGCGCAGGGCCGCTCCACCCTCGGGTTCACGTCCCTCTTTA CACCTGGGGCCTCTCAGAAGATCCAGCTTATAAATACCAATGGTAGCTGGCATATCAACAGGACTGCCCT GAACTGCAATGACTCCCTCAATACTGGGTTTCTTGCCGCGCTGTTCTATACACACAGGTTCAACGCGTCC GGATGCGCAGAGCGCATGGCCAGCTGCCGCCCCATTGATACATTCGATCAGGGCTGGGGCCCCATCACTT ATACTGAGCCTGATAGCTCGGACCAGAGGCCTTATTGCTGGCACTACGCGCCTCGAAAGTGCGGCATCGT ACCTGCGTCGGAGGTGTGCGGTCCAGTGTATTGTTTCACCCCAAGCCCTGTCGTCGTGGGGACGACCGAT CGTTTCGGTGTCCCCACATATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTCCTCAACAACACGCGGC CGCCGCAAGGCAACTGGTTTGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGACGTGCGGGGGGCC TCCGTGTAACATCGGGGGGGTCGGCAACAACACTTTGACTTGCCCCACGGATTGCTTTCGGAAGCACCCC GAGGCTACGTATACAAGGTGTGGTTCGGGGCCTTGGCTGACACCTAGGTGCTTAGTTGACTACCCATACA GGCTCTGGCACTACCCCTGCACTGTCAACTTTGCCATCTTCAAAGTTAGGATGTATGTGGGGGGCGTGGA GCACAGGCTCGATGCTGCATGCAACTGGACTCGAGGAGAGCGCTGTAACTTGGAGGACAGGGATAGATCA GAACTCAGCCCGCTGCTACTGTCTACAACAGAGTGGCAGATACTACCCTGCGCCTTCACCACCCTACCGG CTCTGTCCACTGGTTTAATCCATCTCCATCAGAACATCGTGGACGTGCAATACCTGTACGGTATAGGGTC AGCGGTTGCCTCCTTTGCAATTAAATGGGAGTATGTCTTGTTGCTTTTCCTTCTACTAGCAGACGCGCGC GTATGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCCGAGGCCGCCTTAGAGAACCTGGTGGTCC TCAATGCGGCGTCCGTGGCCGACGCGCATGGCATCCTCTCCTTCCTTGTGTTCTTTTGTGCCGCCTGGTA CATTAAGGGCAGGCTGGTCCCCGGGGCAGCATACGCTTTCTACGGCGTGTGGCCGCTGCTCCTGCTCCTG CTGACATTACCACCACGAGCTTACGCCATGGACCGGGAGATGGCTGCATCGTGCGGAGGCGCGGTTTTTG TAGGTCTGGTATTCCTGACTTTGTCACCATACTACAAGGTGTTCCTCGCTAGGCTCATATGGTGGTTGCA ATACTTCCTCACCATAGCCGAGGCGCACCTGCAAGTGTGGATCCCCCCTCTCAACATTCGAGGGGGCCGC GATGCCATCATCCTCCTCACGTGTGCAATCCACCCAGAGTCAATCTTTGACATCACCAAACTCCTGCTCG CCACGCTCGGTCCGCTCCTGGTGCTTCAGGCTGGCATAACTAGAGTGCCGTACTTTGTGCGCGCTCATGG GCTCATTCGCGCGTGCATGCTATTGCGGAAAGTTGCTGGGGGTCATTATGTCCAAATGGCCTTCATGAAG CTGGGCGCACTGACAGGTACGTACGTCTATAACCATCTTACTCCGCTGCAGTATTGGCCACGCGCGGGTT TACGAGAACTCGCGGTGGCAGTAGAGCCCGTCATCTTCTCTGACATGGAGACCAAGATTATCACCTGGGG GGCAGACACTGCAGCGTGTGGAGACATCATCTTGGGTTTACCCGTCTCCGCCCGAAGGGGAAAGGAGATA CTCCTGGGGCCGGCCGATAGTCTTGAAGGGCAGGGGTGGCGACTCCTTGCGCCCATCACGGCCTACTCCC AACAGACGCGGGGCTTACTTGGTTGCATCATCACTAGCCTCACAGGCCGAGACAAGAACCAGGTCGAGGG GGAGGTTCAAGTGGTCTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAACGGTGTGTGTTGGACT GTCTATCATGGCGCCGGCTCAAAAACCTTAGCCGGCCCAAAGGGCCCAATCACCCAAATGTACACCAATG TAGACCAGGACCTCGTCGGCTGGCACCGGCCCCCCGGGGCGCGTTCCCTAACACCATGCACCTGCGGCAG CTCGGACCTTTACTTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGTCGAGGCGACAGTAGGGGG AGTTTACTCTCCCCCAGGCCTGTCTCCTACCTGAAGGGCTCGTCGGGGGGCCCACTGCTCTGCCCCTTCG GGCACGTTGCAGGCATCTTCCGGGCTGCTGTGTGCACCCGGGGGGTTGCGAAGGCGGTGGATTTTATACC CGTTGAGACCATGGAAACTACCATGCGGTCCCCGGTCTTCACGGACAACTCATCCCCTCCTGCCGTACCG CAGACATTCCAAGTGGCCCATCTACACGCTCCCACTGGCAGCGGCAAAAGCACCAAGGTGCCGGCTGCAT ATGCAGCCCAAGGGTACAAGGTACTTGTCTTGAACCCGTCTGTTGCCGCCACTTTAGGTTTTGGGGCGTA TATGTCTAAGGCACATGGTGTCGACCCCAACATTAGAACCGGGGTAAGGACCATCACCACGGGCGCCCCC ATCACATACTCTACCTATGGCAAGTTCCTTGCTGATGGTGGTTGCTCTGGGGGTGCCTATGACATTATAA TATGTGATGAGTGCCATTCAACTGACTCGACTACCATCTTGGGCATCGGCACGGTCCTGGACCAAGCGGA GACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCGTGCCACATCCA AACATCGAGGAGGTGGCCCTGTCCAATACTGGAGAGATCCCCTTCTATGGTAAAGCCATCCCCATCGAAG CCATCAGGGGGGGAAGGCATCTCATTTTCTGCCACTCCAAGAAGAAGTGTGACGAGCTTGCTGCAAAGCT ATCATCGCTCGGGCTCAACGCTGTGGCGTACTACCGGGGGCTTGATGTGTCCGTCATACCATCTAGCGGA GACGTCGTTGTCGTGGCAACGGACGCTCTAATGACGGGCTTTACGGGCGACTTTGACTCAGTGATCGACT GTAACACATGTGTTACCCAAACAGTCGATTTCAGCTTGGACCCCACCTTCACCATCGAGACAACGACCGT GCCCCAAGACGCGGTGTCGCGCTCGCAGCGGCGAGGTAGGACTGGCAGGGGTAGGGAAGGCATCTACAGG TTTGTTACTCCAGGAGAACGGCCCTCGGGCATGTTCGACTCCTCAGTCCTGTGTGAGTGCTATGACGCGG GCTGTGCTTGGTACGAGCTCACGCCGGCTGAGACCACGGTTAGGTTGCGGGCTTACCTAAATACACCAGG GTTGCCCGTCTGCCAGGACCATCTGGAGTTCTGGGAGGGCGTCTTCACAGGTCTCACCCATATAGACGCT CACTTTCTGTCCCAGACCAAGCAAGCAGGAGACAACTTCCCCTACCTGGTAGCATACCAAGCTACAGTGT GTGCCAAGGCTCAGGCCCCACCTCCATCGTGGGATCAAATGTGGAAGTGCCTCACACGGCTAAAGCCTAC GCTGCAGGGACCAACACCCCTGCTGTATAGGCTAGGAGCCGTCCAAAATGAGGTCACCCTCACACACCCC ATAACTAAATACATCATGACATGCATGTCGGCTGACCTGGAGGTCGTCACCAGCACCTGGGTGCTGGTGG GCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACGGGCAGCGTGGTCATTGTGGGTAGGATTGT CTTGTCCGGAAGTCCGGCTATTGTTCCTGACAGGGAAGTTCTTTACCAAGACTTCGACGAGATGGAAGAG TGTGCCTCACACCTCCCTTACATCGAACAGGGAATGCAGCTCGCCGAGCAGTTCAAGCAGAAGGCGCTCG GGTTGCTGCAAACAGCCACCAAGCAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAGTGGCGAGCCCT CGAGACATTTTGGGAAAAACACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAGGCTTATCCACT CTGCCTGGGAACCCCGCAATGGCATCACTGATGGCATTCACAGCTTCTATCACCAGCCCGCTCACTACCC AACACACCCTCCTGTTTAACATCTTGGGTGGATGGGTGGCTGCCCAACTCGCTCCCCCCAGCGCCGCTTC GGCCTTTGTGGGCGCCGGCATTGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGGTGCTTGTGGAC ATCCTGGCGGGTTATGGGGCGGGGGTGGCTGGCGCACTCGTGGCCTTTAAGGTCATGAGTGGCGAAATGC CCTCCACTGAGGACCTGGTTAATTTACTCCCTGCCATCCTCTCTCCTGGTGCCCTAGTCGTCGGGGTCGT GTGCGCAGCAATACTGCGCCGACACGTGGGCCCGGGAGAGGGGGCTGTGCAGTGGATGAACCGGCTGATA GCGTTCGCTTCGCGGGGTAACCATGTCTCCCCCACGCACTATGTGCCTGAAAGTGACGCCGCAGCGCGTG TTACCCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGACTTCACCAGTGGATTAATGAGGA CTGTTCCACACCATGCTCCGGCTCGTGGCTAAGGGATGTTTGGGATTGGATATGCACGGTGTTGACCGAT TTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGGTTGCCCGGAGTCCCTTTCCTCTCATGCCAACGCG GGTACAAGGGAGTCTGGCGGGGGGACGGTATTATGCAAACCACCTGTCCATGTGGAGCACAGATTACTGG ACATGTCAAAAACGGTTCCATGAGAATCGTTGGGCCTAAGACTTGTAGCAACACGTGGCATGGAACATTC CCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCGAACTATTCCAGGGCGCTGTGGC GGGTGGCTCCTGAGGAGTACGTGGAGGTTACGCGGGTGGGGGATTTCCACTACGTGACGGGCATGACCAC CGACAACGTGAAATGCCCATGCCAAGTCCCGGCCCCTGAATTCTTCACGGAGGTGGATGGAGTACGGCTG CACAGGTACGCTCCGGCGTGCAAACCTCTCCTACGGGAGGAGGTCGTGTTCCAGGTCGGGCTCAACCAAT ACCTGGTTGGATCACAGCTCCCATGCGAGCCCGAGCCGGACGTAACAGTGCTCACTTCCATGCTTACCGA CCCCTCCCACATCACAGCAGAGACGGCCAAGCGTAGGCTGGCCAGGGGGTCTCCCCCCTCCTTGGCCAGC TCTTCAGCTAGCCAATTGTCTGCGCCTTCTTTGAAGGCGACATGTACTACCCATCATGACTCCCCGGACG CCGACCTCATTGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGAAACATCACCCGTGTGGAGTCAGA AAATAAGGTAGTGATCCTGGACTCTTTCGACCCGCTTCGGGCGGAGGAGGACGAGAGGGAAGTATCCGTT GCGGCGGAGATCCTGCGGAAATCCAGGAAGTTCCCCTCAGCGCTGCCCATATGGGCACGCCCAGACTACA ACCCTCCACTGCTAGAGTCCTGGAAGGACCCAGATTATGTCCCTCCGGTGGTACACGGGTGCCCGTTGCC GCCTACCACGGCCCCTCCAGTACCACCTCCACGGAGAAAAAGGACGGTCGTCCTAACAGAGTCATCCGTG TCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCTGAATCGTCGGCCGTCGACAGCGGCA CGGCGACTGCCCCTCCTGACGAGGCCTCCGGCGGCGGCGACAAAGGATCCGACGTTGAGTCGTACTCCTC CATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGACCTCAGCGACGGGTCCTGGTCTACCGTGAGTGAG GAGGCCAGTGAGGACGTCGTCTGCTGCTCAATGTCCTATACATGGACAGGCGCCTTGATCACGCCATGTG CTGCGGAGGAGAGCAAGCTGCCCATCAACCCGCTGAGCAACTCCTTGCTGCGTCACCACAACATGGTCTA TGCTACAACATCCCGCAGTGCAAGCCTACGGCAGAAGAAGGTCGCTTTTGACAGAATGCAAGTCCTGGAC GACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAAACTCCTATCCA TAGAAGAGGCCTGCAAGCTGACGCCCCCACATTCAGCCAAATCCAAATTTGGCTATGGGGCAAAAGACGT CCGGAACCTATCCAGCAAGGCCGTTAACCACATCCGCTCCGTGTGGAAGGACTTGTTGGAAGACAATGAG ACACCAATCAATACCACCATCATGGCAAAAAATGAGGTTTTCTGCGTCCAACCAGAGAAAGGAGGCCGTA AGCCAGCTCGCCTTATCGTATTCCCAGACTTGGGAGTCCGTGTGTGCGAGAAGATGGCCCTTTATGACGT GGTCTCCACCCTTCCTCAGCCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTGGGCAGCGGGTC GAATTCCTGCTAAATGCCTGGAAATCAAAGGAAAACCCTATGGGCTTCTCATATGACACCCGCTGTTTTG ACTCAACGGTCACTCAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGACTTGGCCCCCGA GGCCAGACGGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACTAATTCAAAAGGG CAGAACTGCGGTTATCGCCGGTGCCGCGCAAGTGGCGTGCTGACGACCAGCTGCGGTAATACCCTTACAT GTTACTTGAAGGCCTCTGCGGCCTGTCGAGCTGCGAAGCTGCAGGACTGCACGATGCTCGTGAACGGAGA CGACCTTGTCGTTATCTGTGAAAGCGCGGGAACTCAAGAGGATGCGGCGAGCCTACGAGTCTTCACGGAG GCTATGACTAGGTACTCTGCCCCCCCTGGGGACCTGCCCCAACCAGAATACGACTTGGAGCTAATAACAT CATGCTCCTCCAATGTGTCAGTCGCCCACGATGCATCTGGCAAAAGGGTGTACTACCTCACCCGTGACCC CACCATCCCCCTCGCGCGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCCTGGCTAGGCAAC ATCATCATGTATGCGCCCACTCTATGGGCAAGGATGATTCTGATGACTCACTTCTTCTCCATCCTTCTAG CTCAGGAGCAACTTGAGAAAGCCCTGGATTGCCAAATCTACGGGGCCTACTACTCCATTGAGCCACTTGA CCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACTCTCCAGGTGAG ATCAATAGGGTGGCGTCATGTCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAGACATCGGGCCA GAAGCGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTACCTCTTCAACTG GGCAGTAAAGACCAAGCTTAAACTCACTCCAATCCCGGCTGCGTCCCGGTTGGACTTGTCCGGCTGGTTC GTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGTTGGTTCATGTTGT GCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGGAGATAAACACT CCAGGCCAATAGGCCATCCC (SEQ ID NO: 6690)

gi 115422182 |gb|AY051292.l| Hepatitis C virus from India polyprotein mRNA, complete eds

GCCAGCCCCCTGATGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCA GAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCA TAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG

CTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCC

TTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACG

- AATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGACGCCCACAGAACGTTAAGTTCCCGGGTG

GCGGCCAGATCGTTGGCGGAGTTTGCTTGTTGCCGCGCAGGGGTCCCAGAGTGGGTGTGCGCGCGACGAG GAAGACTTCCGAGCGGTCACAACCTCGCGGAAGGCGTCAGCCTATTCCCAAGGCCCGCCGACCCGAGGGC AGGTCCTGGGCGCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGCTGTGGGTGGGCAGGATGGC TCTTGTCCCCCCGCGGCTCCCGGCCTAGTCGGGGCCCCTCTGACCCCCGGCGCAGGTCACGCAATTTGGG TAAGGTCATCGATACCCTCACGTGTGGCTTCGCCGACCTCATGGGGTACATCCCGCTCGTCGGTGCTCCT CTAGGGGGCGCTGCTAGGGCTCTGGCACATGGTGTTAGGGTTCTAGAAGACGGCGTAAATTACGCAACAG GGAACCTTCCTGGTTGCTCTTTTTCTATCTTCTTGCTTGCTCTTCTCTCCTGCTTGACAGTCCCTGCTTC GGCCGTCGAAGTGCGCAACTCTTCGGGGATCTACCATGTCACCAATGATTGCCCCAATGCGTCTGTTGTG TACGAGACAGATAGCTTGATCATACATCTGCCCGGGTGTGTGCCCTGCGTACGCGAGGGCAACGCTTCGA GGTGCTGGGTCTCCCTTAGTCCTACTGTTGCCGCTAAGGATCCGGGCGTCCCCGTCAACGAGATTCGGCG TCACGTCGACCTGATTGTCGGGGCCGCTGCATTCTGTTCGGCTATGTATGTAGGGGACTTATGCGGTTCC ATCTTCCTCGTTGGCCAGCTTTTCACCCTCTCCCCTAGGCGCCACTGGACAACACAAGACTGTAATTGCT CCATCTACCCAGGACATGTGACAGGCCATCGAATGGCTTGGGACATGATGATGAATTGGTCACCTACTGG CGCTTTGGTGGTAGCGCAGCTACTCCGGATCCCACAAGCCGTCTTGGATATGATAGCCGGTGCCCACTGG GGTGTCCTAGCGGGCCCGGCATACTACTCCATGGTGGGGAACTGGGCTAAGGTTTTGGTTGTGCTACTGC TCTTCGCTGGCGTCGATGCAACCACCCAAGTCACAGGTGGCACCGCGGGCCGTAATGCATATAGATTGGC TAGCCTCTTCTCCACCGGCCCCAGCCAAAATATCCAGCTCATAAACTCCAATGGCAGCTGGCACATTAAC AGGACTGCCCTGAATTGCAATGACAGCCTGCACACCGGCTGGGTAGCAGCGCTGTTCTACTCCCACAAGT TCAACTCTTCGGGGCGTCCTGAGAGGATGGCTAGTTGTCGGCCTCTTACCGCCTTCGACCAAGGGTGGGG GCCCATCACTTACGGGGGGAAAGCTAGTAACGACCAGCGGCCGTATTGCTGGCACTATGCCCCACGCCCG TGCGGTATCGTGCCGGCGAAAGAGGTTTGCGGGCCTGTATACTGTTTCACACCCAGTCCCGTGGTAGTGG GGACGACGGACAAGTACGGCGTTCCTACCTACACATGGGGCGAGAATGAGACGGATGTACTGCTCCTTAA CAACTCTAGGCCGCCAATAGGGAATTGGTTCGGGTGTACGTGGATGAATTCCACTGGTTTCACCAAGACG TGCGGGGCTCCTGCCTGTAACGTCGGCGGGAGCGAGACCAACACCCTGTCGTGCCCCACAGATTGCTTCC GCAGACATCCGGACGCAACATACGCTAAGTGCGGCTCTGGCCCTTGGCTTAACCCTCGATGCATGGTGGA CTACCCTTACAGGCTCTGGCACTATCCCTGCACAGTCAATTACACCATATTCAAGATCAGGATGTTCGTG GGCGGGATTGAGCACAGGCTCACCGCCGCGTGCAACTGGACGCGGGGAGAGCGCTGCGACTTGGACGACA GGGATCGTGCCGAGTTGAGCCCGCTGTTGCTGTCCACCACGCAATGGCAGGTCCTCCCCTGCTCATTCAC AACGCTGCCCGCCCTGTCAACTGGCCTAATACATCTCCACCAGAACATCGTGGACGTGCAGTACCTCTAC GGGTTGAGCTCGGTAGTTACATCCTGGGCCATAAGGTGGGAGTATGTCGTGCTCCTTTTCTTGCTGTTAG CAGATGCCCGCATTTGTGCCTGCCTTTGGATGATGCTTCTCATATCCCAGGTAGAGGCGGCGCTGGAGAA CCTGATAGTCCTCAACGCTGCTTCCCTGGCTGGGACACACGGCATCGTCCCTTTCTTCATCTTTTTTTGT GCAGCCTGGTATCTGAAAGGCAAGTGGGCCCCTGGACTCGTCTACTCCGTCTACGGAATGTGGCCGCTGC TCCTGCTTCTCCTGGCGTTGCCCCAACGGGCGTACGCCTTGGATCAGGAGTTGGCCGCGTCGTGTGGGGC CGTGGTCTTCATCAGCCTAGCGGTACTTACCCTGTCGCCGTACTACAAACAGTACATGGCCCGCGGCATC TGGTGGCTGCAGTACATGCTGACCAGAGCGGAGGCGCTCCTGCACGTCTGGGTCCCCTCGCTCAACGCCC GGGGAGGGCGTGATGGTGCCATACTGCTCATGTGTGTGCTCCACCCGCACTTGCTCTTTGACATCACCAA AATCATGCTGGCCATTCTCGGGCCCCTGTGGATCTTGCAGGCCAGTCTGCTCAGGGTGCCGTACTTCGTG CGCGCCCACGGTCTCATTAGGCTCTGCATGCTGGTGCGCAAAACAGCGGGCGGTCACTATGTGCAGATGG CTCTGTTGAAGCTGGGGGCACTTACTGGCACTTACATTTACAACCACCTTTCCCCACTCCAAGACTGGGC TCATGGCAGCTTGCGTGATCTAGCGGTGGCCACCGAGCCCGTCATCTTCTCCCGGATGGAGATCAAGACT ATCACCTGGGGGGCAGACACCGCGGCCTGTGGAGACATCATCAACGGGCTGCCTGTTTCTGCTCGGAGGG GGAGAGAGGTGTTGTTGGGACCAGCCGATGCCCTGACTGACAAGGGATGGAGGCTTTTAGCCCCCATCAC AGCTTACGCCCAACAGACACGAGGTCTCTTGGGCTGTATTGTCACCAGCCTCACCGGTCGGGACAAAAAT CAAGTGGAGGGGGAAATCCAGATTGTGTCTACCGCAACCCAGACGTTCTTGGCCACTTGCATCAACGGAG CTTGCTGGACTGTTTATCATGGGGCCGGATCGAGGACCATCGCTTCGGCGTCGGGTCCTGTGGTCCGGAT GTACACCAATGTGGACCAGGATTTGGTGGGCTGGCCAGCGCCTCAGGGAGCGCGCTCCCTGACGCCGTGC ACGTGCGGTGCCTCGGATCTGTACTTGGTCACGAGGCACGCGGATGTCATCCCAGTGCGGCGTCGAGGCG ATAACAGGGGAAGCTTGCTTTCTCCCCGGCCCATCTCATACCTAAAAGGATCCTCGGGAGGCCCTCTGCT CTGCCCCATGGGACATGTCGCGGGCATTTTTAGGGCCGCGGTGTGCACCCGTGGGGTTGCAAAGGCGGTC GACTTTGTGCCCGTTGAGTCCTTAGAGACCACCATGAGGTCCCCAGTGTTTACTGACAATTCCAGCCCTC CAACAGTGCCCCAGAGTTACCAGGTGGCACATCTACATGCACCCACTGGGAGTGGCAAGAGCACGAAGGT GCCGGCCGCTTACGCAGCTCAAGGGTACAAGGTACTTGTGCTGAACCCGTCTGTTGCTGCCACCTTAGGG TTCGGTGCTTATATGTCAAAGGCCCATGGGATTGACCCAAACGTCAGGACCGGCGTGAGGACCATTACCA CAGGCTCCCCCATCACCTACTCCACCTACGGGAAATTTTTGGCTGATGGCGGATGCCCAGGAGGTGCGTA CGACATCATAATATGTGACGAATGTCACTCAGTGGACGCCACCTCGATTCTGGGCATAGGGACCGTCTTG GACCAAGCGGAGACGGCGGGGGTTAGGCTCACTGTCCTTGCCACCGCTACACCACCTGGCTTGGTCACCG TGCCACATTCCAACATCGAGGAAGTTGCACTGTCCGCTGACGGGGAGAAACCATTTTATGGTAAGGCCAT CCCCCTAAACTACATCAAGGGGGGGAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTC GCTGCAAAGCTGGTCGGTCTGGGCGTCAACGCGGTGGCCTTTTACCGTGGCCTCGACGTATCTGTCATTC CAACTACAGGAGACGTCGTTGTTGTAGCGACCGACGCCTTGATGACTGGCTTCACCGGCGATTTCGACTC TGTGATAGACTGCAACACCTGTGTCGTCCAGACAGTCGACTTCAGCCTAGACCCTATATTCTCTATTGAG ACTTCCACCGTGCCCCAGGACGCCGTGTCCCGCTCCCAACGGAGGGGTAGGACCGGTCGAGGGAAGCATG GTATTTACAGATATGTGTCACCCGGGGAGCGGCCGTCTGGCATGTTCGACTCCGTGGTCCTCTGTGAGTG CTATGACGCGGGTTGTGCTTGGTACGAGCTTACACCCGCCGAGACCACAGTCAGGCTACGGGCATACCTT AACACCCCAGGATTGCCCGTGTGCCAGGACCACTTGGAGTTCTGGGAGAGTGTCTTCACCGGCCTCACCC ACATAGATGCCCACTTCCTGTCCCAGACGAAACAGAGTGGGGAGAACTTCCCCTACCTAGTCGCATACCA AGCCACCGTGTGCGCTAGAGCTAGAGCTCCTCCCCCGTCATGGGACCAAATGTGGAAGTGCCTGATACGG CTCAAGCCCACCCTCACTGGGGCTACCCCATTACTATACAGACTGGGTAGTGTACAGAATGAGATCACCT TAACACACCCAATCACCCAATACATCATGGCTTGCATGTCGGCGGACCTGGAGGTCGTCACTAGCACGTG GGTGTTGGTGGGCGGCGTCCTAGCCGCTTTGGCCGCTTACTGCCTGTCCACAGGCAGCGTGGTCATAGTG GGCAGGATAATCCTAGGTGGGAAGCCGGCAGTCATACCTGACAGGGAGGTTCTCTACCGAGAGTTTGATG AGATGGAGGAGTGCGCCGCCCACGTCCCCTACCTCGAGCAGGGGATGCATTTGGCTGGACAGTTCAAGCA GAAAGCTCTCGGGTTGCTCCAGACAGCATCCAAGCAAGCGGAGACGATCACTCCCACTGTCCGCACCAAC TGGCAGAAACTCGAGTCCTTCTGGGCTAAGCACATGTGGAACTTCGTTAGCGGGATACAATACCTGGCGG GCCTGTCAACGCTGCCCGGGAACCCCGCTATAGCGTCGCTGATGTCGTTTACGGCCGCGGTGACGAGTCC ACTAACCACCCAGCAAACCCTCTTCTTTAACATCTTAGGGGGGTGGGTGGCGGCCCAGCTTGCTTCCCCA GCTGCCGCTACTGCTTTTGTCGGTGCTGGTATTACTGGCGCCGTTGTTGGCAGTGTGGGCCTAGGGAAGG TCCTAGTGGACATTATTGCTGGCTACGGGGCTGGTGTGGCGGGGGCCCTCGTGGCTTTCAAAATCATGAG CGGGGAGACCCCCACCACCGAGGATCTAGTCAACCTTCTGCCTGCCATCCTATCGCCAGGAGCTCTCGTT GTCGGCGTGGTGTGCGCAGCAATACTACGCCGGCACGTGGGCCCTGGCGAGGGCGCCGTGCAGTGGATGA ACCGGCTGATAGCGTTTGCTTCTCGGGGTAACCACGTCTCCCCTACACACTACGTGCCGGAGAGCGACGC GTCGGCTCGTGTCACACAAATTCTCACCAGCCTCACTGTTACTCAGCTTCTGAAAAGGCTCCACGTGTGG ATAAGCTCGGATTGCATCGCCCCGTGTGCTAGTTCTTGGCTTAAAGATGTCTGGGACTGGATATGCGAGG TGCTGAGCGACTTCAAGAATTGGCTGAAGGCCAAACTTGTACCACAACTGCCCGGGATCCCATTCGTATC CTGCCAACGCGGGTACCGTGGGGTCTGGCGGGGCGAGGGCATCGTGCACACTCGTTGCCCGTGTGGGGCC AATATAACTGGACATGTCAAGAACGGTTCGATGAGAATCGTCGGGCCTAAGACTTGCAGCAACACCTGGC GTGGGTCGTTCCCCATTAACGCTTACACTACAGGCCCGTGCACGCCCTCCCCGGCGCCGAACTATACGTT CGCGCTATGGAGGGTGTCTGCAGAGGAGTATGTGGAGGTAAGGCGGCTGGGGGACTTCCATTACGTCACG GGGGTGACCACTGATAAACTCAAGTGTCCATGCCAGGTCCCCTCACCCGAGTTCTTCACAGAGGTGGACG GGGTGCGCCTGCATAGGTACGCCCCCCCCTGCAAACCCCTGCTGCGAGAAGAGGTGACGTTTAGCATCGG GCTCAATGAATACTTGGTGGGGTCCCAGTTGCCCTGCGAGCCCGAGCCAGACGTAGCTGTACTGACATCA ATGCTTACAGACCCCTCCCACATCACTGCAGAGACGGCAGCGCGTAGGCTGAAGCGGGGGTCTCCCCCCT CCCTGGCCAGCTCTTCCGCCAGCCAGCTGTCCGCGCCGTCACTGAAGGCAACATGCACCACTCACCACGA CTCTCCAGACGCTGACCTCATAGAAGCCAACCTCCTGTGGAGACAGGAGATGGGGGGGAACATCACTAGG GTGGAGTCGGAGAACAAGATTGTCGTTCTGGATTCTTTCGACCCGCTCGTAGCGGAGGAGGATGATCGGG AGATCTCTATTCCAGCTGAGATTCTGCGGAAGTTCAAGCAGTTTCCTCCCGCTATGCCCATATGGGCACG GCCAGATTATAATCCTCCCCTTGTGGAACCGTGGAAGCGCCCGGACTATGAGCCACCCTTAGTCCACGGG TGCCCCCTACCACCTCCCAAGCCAACTCCGGTGCCGCCACCCCGGAGAAAGAGGACGGTGGTGCTGGACG AGTCTACAGTATCATCTGCTCTGGCTGAGCTTGCCACTAAGACCTTCGGCAGCTCTACAACCTCAGGCGT GACAAGTGGTGAAGCGACTGAATCGTCCCCGGCGCCCTCCTGCGGCGGTGAGCTGGACTCCGAAGCTGAA TCTTACTCCTCCATGCCCCCTCTCGAGGGGGAGCCGGGGGACCCCGATCTCAGCGACGGGTCTTGGTCTA CCGTGAGCAGTGATGGTGGCACGGAAGACGTTGTGTGCTGCTCGATGTCTTACTCGTGGACGGGCGCTTT AATCACGCCCTGTGCCTCAGAGGAAGCCAAGCTCCCTATCAACGCATTGAGCAACTCGCTGCTGCGCCAC CACAACTTGGTGTATTCCACCACCTCTCGCAGCGCTGGCCAGAGACAGAAAAAAGTCACATTTGACAGAG TGCAAGTCCTGGACGACCATTACCGGGACGTGCTCAAGGAGGCTAAGGCCAAGGCATCCACGGTGAAGGC TAGACTGCTATCCGTTGAGGAAGCGTGTAGCCTGACGCCCCCACACTCCGCCAGATCAAAATTTGGCTAT GGGGCGAAGGATGTCCGAAGCCATTCCAGTAAGGCTATACGCCACATCAACTCCGTGTGGCAGGACCTTC TGGAGGACAATACAACACCCATAGACACTACCATCATGGCAAAGAATGAGGTCTTCTGTGTGAAGCCCGA AAAGGGGGGCCGCAAGCCCGCTCGTCTTATCGTGTACCCCGACCTGGGAGTGCGCGTATGCGAGAAGAGG GCTTTGTATGACGTAGTCAAACAGCTCCCCATTGCCGTGATGGGAGCCTCCTACGGGTTCCAGTACTCAC CAGCGCAGCGGGTCGACTTCCTGCTTAAAGCGTGGAAATCTAAGAAAGTCCCCATGGGGTTTTCCTATGA CACCCGTTGCTTTGACTCAACAGTCACTGAGGCTGATATCCGTACGGAGGAAGACCTCTACCAATCTTGT GACCTGGCCCCTGAGGCTCGCATAGCCATAAGGTCCCTCACAGAGAGGCTTTACATCGGGGGCCCACTCA CCAATTCTAAGGGACAAAACTGCGGCTATCGGCGATGCCGCGCAAGCGGCGTGCTGACCACTAGCTGCGG TAACACCATAACCTGCTTCCTCAAAGCCAGTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACCATG CTCGTGTGCGGCGACGACCTCGTCGTTATCTGTGAGAGCGCCGGTGTCCAGGAGGACGCTGCGAGCCTGA GAGCCTTCACGGAGGCTATGACCAGGTACTCCGCCCCCCCGGGAGACCCGCCTCAACCAGAATACGACTT GGAGCTTATAACATCCTGCTCCTCCAATGTGTCGGTCGCGCGCGACGGCGCTGGCAAAAGGGTCTATTAT CTGACCCGTGACCCTGAGACTCCCCTCGCGCGTGCCGCTTGGGAGACAGCAAGACACACTCCAGTGAACT CCTGGCTAGGCAACATCATCATGTTTGCCCCCACTCTGTGGGTACGGATGGTCCTCATGACCCATTTTTT CTCCATACTCATAGCTCAGGAGCACCTTGGAAAGGCTCTAGATTGTGAAATCTATGGAGCCGTACACTCC GTCCAACCGTTGGACTTACCTGAAATCATCCAAAGACTCCACAGCCTCAGCGCGTTTTCGCTCCACAGTT ACTCTCCAGGTGAAATCAATAGGGTGGCTGCATGCCTCAGGAAGCTTGGGGTTCCGCCCTTGCGAGCTTG GAGACACCGGGCCCGGAGCGTTCGCGCCACACTCCTATCCCAGGGGGGGAAAGCCGCTATATGCGGTAAG TACCTCTTCAACTGGGCGGTGAAAACCAAACTCAAACTCACTCCATTACCGTCCATGTCTCAGTTGGACT TGTCCAACTGGTTCACGGGCGGTTACAGCGGGGGAGACATTTATCACAGCGTGTCTCATGCCCGGCCCCG TTTGTTCCTCTGGTGCCTACTCCTACTTTCAGTAGGGGTAGGCATCTATCTCCTTCCCAACCGATAGACG GNTGGGCAACCACTCCGGGTCTTTAGGCCCTATTTAAACACTCCAGGCCTTTAGGCCCCGT (SEQ ID NO: 6691)

gi I 23510419| ref |NM_000043.3 I Homo sapiens tumor necrosis factor receptor superfamily, member 6 (TNFRSP6) , transcript variant 1, mRNA CCTACCCGCGCGCAGGCCAAGTTGCTGAATCAATGGAGCCCTCCCCAACCCGGGCGTTCCCCAGCGAGGC TTCCTTCCCATCCTCCTGACCACCGGGGCTTTTCGTGAGCTCGTCTCTGATCTCGCGCAAGAGTGACACA CAGGTGTTCAAAGACGCTTCTGGGGAGTGAGGGAAGCGGTTTACGAGTGACTTGGCTGGAGCCTCAGGGG CGGGCACTGGCACGGAACACACCCTGAGGCCAGCCCTGGCTGCCCAGGCGGAGCTGCCTCTTCTCCCGCG GGTTGGTGGACCCGCTCAGTACGGAGTTGGGGAAGCTCTTTCACTTCGGAGGATTGCTCAACAACCATGC TGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTAGATTATCGTCCAAAAGTGTTAATGC CCAAGTGACTGACATCAACTCCAAGGGATTGGAATTGAGGAAGACTGTTACTACAGTTGAGACTCAGAAC TTGGAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAGGTGAAAGGAAAGCTAGGG ACTGCACAGTCAATGGGGATGAACCAGACTGCGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGC CCATTTTTCTTCCAAATGCAGAAGATGTAGATTGTGTGATGAAGGACATGGCTTAGAAGTGGAAATAAAC TGCACCCGGACCCAGAATACCAAGTGCAGATGTAAACCAAACTTTTTTTGTAACTCTACTGTATGTGAAC ACTGTGACCCTTGCACCAAATGTGAACATGGAATCATCAAGGAATGCACACTCACCAGCAACACCAAGTG CAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTGCCAATTCCACTAATTGTT TGGGTGAAGAGAAAGGAAGTACAGAAAACATGCAGAAAGCACAGAAAGGAAAACCAAGGTTCTCATGAAT CTCCAACCTTAAATCCTGAAACAGTGGCAATAAATTTATCTGATGTTGACTTGAGTAAATATATCACCAC TATTGCTGGAGTCATGACACTAAGTCAAGTTAAAGGCTTTGTTCGAAAGAATGGTGTCAATGAAGCCAAA ATAGATGAGATCAAGAATGACAATGTCCAAGACACAGCAGAACAGAAAGTTCAACTGCTTCGTAATTGGC ATCAACTTCATGGAAAGAAAGAAGCGTATGACACATTGATTAAAGATCTCAAAAAAGCCAATCTTTGTAC TCTTGCAGAGAAAATTCAGACTATCATCCTCAAGGACATTACTAGTGACTCAGAAAATTCAAACTTCAGA AATGAAATCCAAAGCTTGGTCTAGAGTGAAAAACAACAAATTCAGTTCTGAGTATATGCAATTAGTGTTT GAAAAGATTCTTAATAGCTGGCTGTAAΆTACTGCTTGGTTTTTTACTGGGTACATTTTATCATTTATTAG CGCTGAAGAGCCAACATATTTGTAGATTTTTAATATCTCATGATTCTGCCTCCAAGGATGTTTAAAATCT AGTTGGGAAAACAAACTTCATCAΆGAGTAAATGCAGTGGCATGCTAAGTACCCAAATAGGAGTGTATGCA GAGGATGAAAGATTAAGATTATGCTCTGGCATCTAACATATGATTCTGTAGTATGAATGTAATCAGTGTA TGTTAGTACAAATGTCTATCCACAGGCTAACCCCACTCTATGAATCAATAGAAGAAGCTATGACCTTTTG CTGAAATATCAGTTACTGAACAGGCAGGCCACTTTGCCTCTAAATTACCTCTGATAATTCTAGAGATTTT ACCATATTTCTAAACTTTGTTTATAACTCTGAGAAGATCATATTTATGTAAAGTATATGTATTTGAGTGC AGAATTTAAATAAGGCTCTACCTCAAAGACCTTTGCACAGTTTATTGGTGTCATATTATACAATATTTCA ATTGTGAATTCACATAGAAAACATTAAATTATAATGTTTGACTATTATATATGTGTATGCATTTTACTGG CTCAAAACTACCTACTTCTTTCTCAGGCATCAAAAGCATTTTGAGCAGGAGAGTATTACTAGAGCTTTGC CACCTCTCCATTTTTGCCTTGGTGCTCATCTTAATGGCCTAATGCACCCCCAAACATGGAAATATCACCA AAAAATACTTAATAGTCCACCAAAAGGCAAGACTGCCCTTAGAAATTCTAGCCTGGTTTGGAGATACTAA CTGCTCTCAGAGAAAGTAGCTTTGTGACATGTCATGAACCCATGTTTGCAATCAAAGATGATAAAATAGA TTCTTATTTTTCCCCCACCCCCGAAAATGTTCAATAATGTCCCATGTAAAACCTGCTACAAATGGCAGCT TATACATAGCAATGGTAAAATCATCATCTGGATTTAGGAATTGCTCTTGTCATACCCCCAAGTTTCTAAG ATTTAAGATTCTCCTTACTACTATCCTACGTTTAAATATCTTTGAAAGTTTGTATTAAATGTGAATTTTA AGAAATAATATTTATATTTCTGTAAATGTAAACTGTGAAGATAGTTATAAACTGAAGCAGATACCTGGAA CCACCTAAAGAACTTCCATTTATGGAGGATTTTTTTGCCCCTTGTGTTTGGAATTATAAAATATAGGTAA AAGTACGTAATTAAATAATGTTTTTGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6692)

gi|35910 | emb|X12387.1 |HSRCYP3 Human mRNA for cytochrome P-450 (cyp3 locus)

GAATTCCCAAAGAGCAACACAGAGCTGAAAGGAAGACTCAGAGGAGAGAGATAAGTAAGGAAAGTAGTGA TGGCTCTCATCCCAGACTTGGCCATGGAAACCTGGCTTCTCCTGGCTGTCAGCCTGGTGCTCCTCTATCT ATATGGAACCCATTCACATGGACTTTTTAAGAAGCTTGGAATTCCAGGGCCCACACCTCTGCCTTTTTTG GGAAATATTTTGTCCTACCATAAGGGCTTTTGTATGTTTGACATGGAATGTCATAAAAAGTATGGAAAAG TGTGGGGCTTTTATGATGGTCAACAGCCTGTGCTGGCTATCACAGATCCTGACATGATCAAAACAGTGCT AGTGAAAGAATGTTATTCTGTCTTCACAAACCGGAGGCCTTTTGGTCCAGTGGGATTTATGAAAAGTGCC ATCTCTATAGCTGAGGATGAAGAATGGAAGAGATTACGATCATTGCTGTCTCCAACCTTCACCAGTGGAA AACTCAAGGAGATGGTCCCTATCATTGCCCAGTATGGAGATGTGTTGGTGAGAAATCTGAGGCGGGAAGC AGAGACAGGCAAGCCTGTCACCTTGAAAGACGTCTTTGGGGCCTACAGCATGGATGTGATCACTAGCACA TCATTTGGAGTGAACATCGACTCTCTCAACAATCCACAAGACCCCTTTGTGGAAAACACCAAGAAGCTTT TAAGATTTGATTTTTTGGATCCATTCTTTCTCTCAATAACAGTCTTTCCATTCCTCATCCCAATTCTTGA AGTATTAAATATCTGTGTGTTTCCAAGAGAAGTTACAAATTTTTTAAGAAAATCTGTAAAAAGGATGAAA GAAAGTCGCCTCGAΆGATACACAAΆAGCACCGAGTGGATTTCCTTCAGCTGATGATTGACTCTCAGAATT CAAAAGAAACTGAGTCCCACAAAGCTCTGTCCGATCTGGAGCTCGTGGCCCAATCAATTATCTTTATTTT TGCTGGCTATGAAACCACGAGCAGTGTTCTCTCCTTCATTATGTATGAACTGGCCACTCACCCTGATGTC CAGCAGAAACTGCAGGAGGAAATTGATGCAGTTTTACCCAATAAGGCACCACCCACCTATGATACTGTGC TACAGATGGAGTATCTTGACATGGTGGTGAATGAAACGCTCAGATTATTCCCAATTGCTATGAGACTTGA GAGGGTCTGCAAAAAAGATGTTGAGATCAATGGGATGTTCATTCCCAAAGGGTGGGTGGTGATGATTCCA AGCTATGCTCTTCACCGTGACCCAAAGTACTGGACAGAGCCTGAGAAGTTCCTCCCTGAAAGATTCAGCA AGAAGAACAAGGACAACATAGATCCTTACATATACACACCCTTTGGAAGTGGACCCAGAAACTGCATTGG CATGAGGTTTGCTCTCATGAACATGAAACTTGCTCTAATCAGAGTCCTTCAGAACTTCTCCTTCAAACCT TGTAAAGAAACACAGATCCCCCTGAAATTAAGCTTAGGAGGACTTCTTCAACCAGAAAAACCCGTTGTTC TAAAGGTTGAGTCAAGGGATGGCACCGTAAGTGGAGCCTGAATTTTCCTAAGGACTTCTGCTTTGCTCTT CAAGAAATCTGTGCCTGAGAACACCAGAGACCTCAAATTACTTTGTGAATAGAACTCTGAAATGAAGATG GGCTTCATCCAATGGACTGCATAAATAACCGGGGATTCTGTACATGCATTGAGCTCTCTCATTGTCTGTG TAGAGTGTTATACTTGGGAATATAAAGGAGGTGACCAAATCAGTGTGAGGAGGTAGATTTGGCTCCTCTG CTTCTCACGGGACTATTTCCACCACCCCCAGTTAGCACCATTAACTCCTCCTGAGCTCTGATAAGAGAAT CAACATTTCTCAATAATTTCCTCCACAAATTATTAATGAAAATAAGAATTATTTTGATGGCTCTAACAAT GACATTTATATCACATGTTTTCTCTGGAGTATTCTATAGTTTTATGTTAAATCAATAAAGACCACTTTAC AAAAGTATTATCAGATGCTTTCCTGCACATTAAGGAGAATCTATAGAACTGAATGAGAACCAACAAGTAA ATATTTTTGGTCATTGTAATCACTGTTGGCGTGGGGCCTTTGTCAGAACTAGAATTTGATTATTAACATA GGTGAAAGTTAATCCACTGTGACTTTGCCCATTGTTTAGAAAGAATATTCATAGTTTAATTATGCCTTTT TTGATCAGGCACATGGCTCACGCCTGTAATCCTAGCAGTTTGGGAGGCTGAGCCGGGTGGATCGCCTGAG GTCAGGAGTTCAAGACAAGCCTGGCCTACATGGTGAAACCCCATCTCTACTAAAAATACACAAATTAGCT AGGCATGGTGGACTCGCCTGTAATCTCACTACACAGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGA GGCGGATGTTGAAGTGAGCTGAGATTGCACCACTGCACTCCAGTCTGGGTGAGAGTGAGACTCAGTCTTA AAAAAATATGCCTTTTTGAAGCACGTACATTTTGTAACAAAGAACTGAAGCTCTTATTATATTATTAGTT TTGATTTAATGTTTTCAGCCCATCTCCTTTCATATTTCTGGGAGACAGAAAACATGTTTCCCTACACCTC TTGCTTCCATCCTCAACACCCAACTGTCTCGATGCAATGAACACTTAATAAAAAACAGTCGATTGGTCAA AAAAAAAAAAAAAAAAAAAAAAAGAATTC (SEQ ID NO: 6693)

gi | 339549 |gb|M19154.1 | HUMTGFB2A Human transforming growth factor-beta-2 mRNA, complete eds

GCCCCTCCCGTCAGTTCGCCAGCTGCCAGCCCCGGGACCTTTTCATCTCTTCCCTTTTGGCCGGAGGAGC CGAGTTCAGATCCGCCACTCCGCACCCGAGACTGACACACTGAACTCCACTTCCTCCTCTTAAATTTATT TCTACTTAATAGCCACTCGTCTCTTTTTTTCCCCATCTCATTGCTCCAAGAATTTTTTTCTTCTTACTCG CCAAAGTCAGGGTTCCCTCTGCCCGTCCCGTATTAATATTTCCACTTTTGGAACTACTGGCCTTTTCTTT TTAAAGGAATTCAAGCAGGATACGTTTTTCTGTTGGGCATTGACTAGATTGTTTGCAAAAGTTTCGCATC AAAAACAACAACAACAAAAAACCAAACAACTCTCCTTGATCTATACTTTGAGAATTGTTGATTTCTTTTT TTTATTCTGACTTTTAAAAACAACTTTTTTTTCCACTTTTTTAAAAAATGCACTACTGTGTGCTGAGCGC TTTTCTGATCCTGCATCTGGTCACGGTCGCGCTCAGCCTGTCTACCTGCAGCACACTCGATATGGACCAG TTCATGCGCAAGAGGATCGAGGCGATCCGCGGGCAGATCCTGAGCAAGCTGAAGCTCACCAGTCCCCCAG AAGACTATCCTGAGCCCGAGGAAGTCCCCCCGGAGGTGATTTCCATCTACAACAGCACCAGGGACTTGCT CCAGGAGAAGGCGAGCCGGAGGGCGGCCGCCTGCGAGCGCGAGAGGAGCGACGAAGAGTACTACGCCAAG GAGGTTTACAAAATAGACATGCCGCCCTTCTTCCCCTCCGAAACTGTCTGCCCAGTTGTTACAACACCCT CTGGCTCAGTGGGCAGCTTGTGCTCCAGACAGTCCCAGGTGCTCTGTGGGTACCTTGATGCCATCCCGCC CACTTTCTACAGACCCTACTTCAGAATTGTTCGATTTGACGTCTCAGCAATGGAGAAGAATGCTTCCAAT TTGGTGAAAGCAGAGTTCAGAGTCTTTCGTTTGCAGAACCCAAAAGCCAGAGTGCCTGAACAACGGATTG AGCTATATCAGATTCTCAAGTCCAAAGATTTAACATCTCCAACCCAGCGCTACATCGACAGCAAAGTTGT GAAAACAAGAGCAGAAGGCGAATGGCTCTCCTTCGATGTAACTGATGCTGTTCATGAATGGCTTCACCAT AAAGACAGGAACCTGGGATTTAAAATAAGCTTACACTGTCCCTGCTGCACTTTTGTACCATCTAATAATT ACATCATCCCAAATAAAAGTGAAGAACTAGAAGCAAGATTTGCAGGTATTGATGGCACCTCCACATATAC CAGTGGTGATCAGAAAACTATAAAGTCCACTAGGAAAAAAAACAGTGGGAAGACCCCACATCTCCTGCTA * ATGTTATTGCCCTCCTACAGACTTGAGTCACAACAGACCAACCGGCGGAAGAAGCGTGCTTTGGATGCGG CCTATTGCTTTAGAAATGTGCAGGATAATTGCTGCCTACGTCCACTTTACATTGATTTCAAGAGGGATCT AGGGTGGAAATGGATACACGAACCCAAAGGGTACAATGCCAACTTCTGTGCTGGAGCATGCCCGTATTTA- TGGAGTTCAGACACTCAGCACAGCAGGGTCCTGAGCTTATATAATACCATAAATCCAGAAGCATCTGCTT CTCCTTGCTGCGTGTCCCAAGATTTAGAACCTCTAACCATTCTCTACTACATTGGCAAAACACCCAAGAT TGAACAGCTTTCTAATATGATTGTAAAGTCTTGCAAATGCAGCTAAAATTCTTGGAAAAGTGGCAAGACC AAAATGACAATGATGATGATAATGATGATGACGACGACAACGATGATGCTTGTAACAAGAAAACATAAGA GAGCCTTGGTTCATCAGTGTTAAAAAATTTTTGAAAAGGCGGTACTAGTTCAGACACTTTGGAAGTTTGT GTTCTGTTTGTTAAAACTGGCATCTGACACAAAAAAAGTTGAAGGCCTTATTCTACATTTCACCTACTTT GTAAGTGAGAGAGACAAGAAGCAAATTTTTTTTAAAGAAAAAAATAAACACTGGAAGAATTTATTAGTGT TAATTATGTGAACAACGACAACAACAACAACAACAACAAACAGGAAAATCCCATTAAGTGGAGTTGCTGT ACGTACCGTTCCTATCCCGCGCCTCACTTGATTTTTCTGTATTGCTATGCAATAGGCACCCTTCCCATTC TTACTCTTAGAGTTAACAGTGAGTTATTTATTGTGTGTTACTATATAATGAACGTTTCATTGCCCTTGGA AAATAAAACAGGTGTATAAAGTGGAGACCAAATACTTTGCCAGAAACTCATGGATGGCTTAAGGAACTTG AACTCAAACGAGCCAGAAAAAAAGAGGTCATATTAATGGGATGAAAACCCAAGTGAGTTATTATATGACC GAGAAAGTCTGCATTAAGATAAAGACCCTGAAAACACATGTTATGTATCAGCTGCCTAAGGAAGCTTCTT GTAAGGTCCAAAAACTAAAAAGACTGTTAATAAAAGAAACTTTCAGTCAG (SEQ ID NO: 6694)

gi| 186624 |gb| J04111.1 IHUMJUNA Human c-jun proto oncogene (JUN), complete eds, clone hC -1

CCCGGGGAGGGGACCGGGGAACAGAGGGCCGAGAGGCGTGCGGCAGGGGGGAGGGTAGGAGAAAGAAGGG CCCGACTGTAGGAGGGCAGCGGAGCATTACCTCATCCCGTGAGCCTCCGCGGGCCCAGAGAAGAATCTTC TAGGGTGGAGTCTCCATGGTGACGGGCGGGCCCGCCCCCCTGAGAGCGACGCGAGCCAATGGGAAGGCCT TGGGGTGACATCATGGGCTATTTTTAGGGGTTGACTGGTAGCAGATAAGTGTTGAGCTCGGGCTGGATAA GGGCTCAGAGTTGCACTGAGTGTGGCTGAAGCAGCGAGGCGGGAGTGGAGGTGCGCGGAGTCAGGCAGAC AGACAGACACAGCCAGCCAGCCAGGTCGGCAGTATAGTCCGAACTGCAAATCTTATTTTCTTTTCACCTT CTCTCTAACTGCCCAGAGCTAGCGCCTGTGGCTCCCGGGCTGGTGGTTCGGGAGTGTCCAGAGAGCCTTG TCTCCAGCCGGCCCCGGGAGGAGAGCCCTGCTGCCCAGGCGCTGTTGACAGCGGCGGAAAGCAGCGGTAC CCCACGCGCCCGCCGGGGGACGTCGGCGAGCGGCTGCAGCAGCAAAGAACTTTCCCGGCGGGGAGGACCG GAGACAAGTGGCAGAGTCCCGGAGCGAACTTTTGCAAGCCTTTCCTGCGTCTTAGGCTTCTCCACGGCGG TAAAGACCAGAAGGCGGCGGAGAGCCACGCAAGAGAAGAAGGACGTGCGCTCAGCTTCGCTCGCACCGGT TGTTGAACTTGGGCGAGCGCGAGCCGCGGCTGCCGGGCGCCCCCTCCCCCTAGCAGCGGAGGAGGGGACA AGTCGTCGGAGTCCGGGCGGCCAAGACCCGCCGCCGGCCGGCCACTGCAGGGTCCGCACTGATCCGCTCC GCGGGGAGAGCCGCTGCTCTGGGAAGTGAGTTCGCCTGCGGACTCCGAGGAACCGCTGCGCCCGAAGAGC GCTCAGTGAGTGACCGCGACTTTTCAAAGCCGGGTAGCGCGCGCGAGTCGACAAGTAAGAGTGCGGGAGG CATCTTAATTAACCCTGCGCTCCCTGGAGCGAGCTGGTGAGGAGGGCGCAGCGGGGACGACAGCCAGCGG GTGCGTGCGCTCTTAGAGAAACTTTCCCTGTCAAAGGCTCCGGGGGGCGCGGGTGTCCCCCGCTTGCCAG AGCCCTGTTGCGGCCCCGAAACTTGTGCGCGCACGCCAAACTAACCTCACGTGAAGTGACGGACTGTTCT ATGACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGCG GACCTTATGGCTACAGTAACCCCAAGATCCTGAAACAGAGCATGACCCTGAACCTGGCCGACCCAGTGGG GAGCCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTCCTCACCTCGCCCGACGTGGGGCTGCTCAAG CTGGCGTCGCCCGAGCTGGAGCGCCTGATAATCCAGTCCAGCAACGGGCACATCACCACCACGCCGACCC CCACCCAGTTCCTGTGCCCCAAGAACGTGACAGATGAGCAGGAGGGGTTCGCCGAGGGCTTCGTGCGCGC CCTGGCCGAACTGCACAGCCAGAACACGCTGCCCAGCGTCACGTCGGCGGCGCAGCCGGTCAACGGGGCA GGCATGGTGGCTCCCGCGGTAGCCTCGGTGGCAGGGGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGC ACAGCGAGCCGCCGGTCTACGCAAACCTCAGCAACTTCAACCCAGGCGCGCTGAGCAGCGGCGGCGGGGC GCCCTCCTACGGCGCGGCCGGCCTGGCCTTTCCCGCGCAACCCCAGCAGCAGCAGCAGCCGCCGCACCAC CTGCCCCAGCAGATGCCCGTGCAGCACCCGCGGCTGCAGGCCCTGAAGGAGGAGCCTCAGACAGTGCCCG AGATGCCCGGCGAGACACCGCCCCTGTCCCCCATCGACATGGAGTCCCAGGAGCGGATCAAGGCGGAGAG GAAGCGCATGAGGAACCGCATCGCTGCCTCCAAGTGCCGAAAAAGGAAGCTGGAGAGAATCGCCCGGCTG GAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAAC AGGTGGCACAGCTTAAACAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTCATGCTAACGCAGCA GTTGCAAACATTTTGAAGAGAGACCGTCGGGGGCTGAGGGGCAACGAAGAAAAAAAATAACACAGAGAGA CAGACTTGAGAACTTGACAAGTTGCGACGGAGAGAAAAAAGAAGTGTCCGAGAACTAAAGCCAAGGGTAT CCAAGTTGGACTGGGTTCGGTCTGACGGCGCCCCCAGTGTGCACGAGTGGGAAGGACTTGGTCGCGCCCT CCCTTGGCGTGGAGCCAGGGAGCGGCCGCCTGCGGGCTGCCCCGCTTTGCGGACGGGCTGTCCCCGCGCG AACGGAACGTTGGACTTTCGTTAACATTGACCAAGAACTGCATGGACCTAACATTCGATCTCATTCAGTA TTAAAGGGGGGAGGGGGAGGGGGTTACAAACTGCAATAGAGACTGTAGATTGCTTCTGTAGTACTCCTTA AGAACACAAAGCGGGGGGAGGGTTGGGGAGGGGCGGCAGGAGGGAGGTTTGTGAGAGCGAGGCTGAGCCT ACAGATGAACTCTTTCTGGCCTGCTTTCGTTAACTGTGTATGTACATATATATATTTTTTAATTTGATTA AAGCTGATTACTGTCAATAAACAGCTTCATGCCTTTGTAAGTTATTTCTTGTTTGTTTGTTTGGGTATCC TGCCCAGTGTTGTTTGTAAATAAGAGATTTGGAGCACTCTGAGTTTACCATTTGTAATAAAGTATATAAT TTTTTTATGTTTTGTTTCTGAAAATTCCAGAAAGGATATTTAAGAAAATACAATAAACTATTGGAAAGTA CTCCCCTAACCTCTTTTCTGCATCATCTGTAGATCCTAGTCTATCTAGGTGGAGTTGAAAGAGTTAAGAA TGCTCGATAAAATCACTCTCAGTGCTTCTTACTATTAAGCAGTAAAAACTGTTCTCTATTAGACTTAGAA ATAAATGTACCTGATGTACCTGATGCTATGTCAGGCTTCATACTCCACGCTCCCCCAGCGTATCTATATG GAATTGCTTACCAAAGGCTAGTGCGATGTTTCAGGAGGCTGGAGGAAGGGGGGTTGCAGTGGAGAGGGAC AGCCCACTGAGAAGTCAAACATTTCAAAGTTTGGATTGCATCAAGTGGCATGTGCTGTGACCATTTATAA TGTTAGAAATTTTACAATAGGTGCTTATTCTCAAAGCAGGAATTGGTGGCAGATTTTACAAAAGATGTAT CCTTCCAATTTGGAATCTTCTCTTTGACAATTCCTAGATAAAAAGATGGCCTTTGTCTTATGAATATTTA TAACAGCATTCTGTCACAATAAATGTATTCAAATACCAATAACAGATCTTGAATTGCTTCCCTTTACTAC TTTTTTGTTCCCAAGTTATATACTGAAGTTTTTATTTTTAGTTGCTGAGGTT (SEQ ID NO: 6695)

gi | 179982 |gb|M57729.l|HUMCCC5 Human complement component C5 mRNA, complete eds CTACCTCCAACCATGGGCCTTTTGGGAATACTTTGTTTTTTAATCTTCCTGGGGAAAACCTGGGGACAGG AGCAAACATATGTCATTTCAGCACCAAAAATATTCCGTGTTGGAGCATCTGAAAATATTGTGATTCAAGT TTATGGATACACTGAAGCATTTGATGCAACAATCTCTATTAAAAGTTATCCTGATAAAAAATTTAGTTAC TCCTCAGGCCATGTTCATTTATCCTCAGAGAATAAATTCCAAAACTCTGCAATCTTAACAATACAACCAA AACAATTGCCTGGAGGACAAAACCCAGTTTCTTATGTGTATTTGGAAGTTGTATCAAAGCATTTTTCAAA ATCAAAAAGAATGCCAATAACCTATGACAATGGATTTCTCTTCATTCATACAGACAAACCTGTTTATACT CCAGACCAGTCAGTAAAAGTTAGAGTTTATTCGTTGAATGACGACTTGAAGCCAGCCAAAAGAGAAACTG TCTTAACCTTCATAGATCCTGAAGGATCAGAAGTTGACATGGTAGAAGAAATTGATCATATTGGAATTAT CTCTTTTCCTGACTTCAAGATTCCGTCTAATCCTAGATATGGTATGTGGACGATCAAGGCTAAATATAAA GAGGACTTTTCAACAACTGGAACCGCATATTTTGAAGTTAAAGAATATGTCTTGCCACATTTTTCTGTCT CAATCGAGCCAGAATATAATTTCATTGGTTACAAGAACTTTAAGAATTTTGAAATTACTATAAAAGCAAG ATATTTTTATAATAAAGTAGTCACTGAGGCTGACGTTTATATCACATTTGGAATAAGAGAAGACTTAAAA GATGATCAAAAAGAAATGATGCAAACAGCAATGCAAAACACAATGTTGATAAATGGAATTGCTCAAGTCA CATTTGATTCTGAAACAGCAGTCAAAGAACTGTCATACTACAGTTTAGAAGATTTAAACAACAAGTACCT TTATATTGCTGTAACAGTCATAGAGTCTACAGGTGGATTTTCTGAAGAGGCAGAAATACCTGGCATCAAA TATGTCCTCTCTCCCTACAAACTGAATTTGGTTGCTACTCCTCTTTTCCTGAAGCCTGGGATTCCATATC CCATCAAGGTGCAGGTTAAAGATTCGCTTGACCAGTTGGTAGGAGGAGTCCCAGTAATACTGAATGCACA AACAATTGATGTAAACCAAGAGACATCTGACTTGGATCCAAGCAAAAGTGTAACACGTGTTGATGATGGA GTAGCTTCCTTTGTGCTTAATCTCCCATCTGGAGTGACGGTGCTGGAGTTTAATGTCAAAACTGATGCTC CAGATCTTCCAGAAGAAAATCAGGCCAGGGAAGGTTACCGAGCAATAGCATACTCATCTCTCAGCCAAAG TTACCTTTATATTGATTGGACTGATAACCATAAGGCTTTGCTAGTGGGAGAACATCTGAATATTATTGTT ACCCCCAAAAGCCCATATATTGACAAAATAACTCACTATAATTACTTGATTTTATCCAAGGGCAAAATTA TCCATTTTGGCACGAGGGAGAAATTTTCAGATGCATCTTATCAAAGTATAAACATTCCAGTAACACAGAA CATGGTTCCTTCATCCCGACTTCTGGTCTATTATATCGTCACAGGAGAACAGACAGCAGAATTAGTGTCT GATTCAGTCTGGTTAAATATTGAAGAAAAATGTGGCAACCAGCTCCAGGTTCATCTGTCTCCTGATGCAG ATGCATATTCTCCAGGCCAAACTGTGTCTCTTAATATGGCAACTGGAATGGATTCCTGGGTGGCATTAGC AGCAGTGGACAGTGCTGTGTATGGAGTCCAAAGAGGAGCCAAAAAGCCCTTGGAAAGAGTATTTCAATTC TTAGAGAAGAGTGATCTGGGCTGTGGGGCAGGTGGTGGCCTCAACAATGCCAATGTGTTCCACCTAGCTG GACTTACCTTCCTCACTAATGCAAATGCAGATGACTCCCAAGAAAATGATGAACCTTGTAAAGAAATTCT CAGGCCAAGAAGAACGCTGCAAAAGAAGATAGAAGAAATAGCTGCTAAATATAAACATTCAGTAGTGAAG AAATGTTGTTACGATGGAGCCTGCGTTAATAATGATGAAACCTGTGAGCAGCGAGCTGCACGGATTAGTT TAGGGCCAAGATGCATCAAAGCTTTCACTGAATGTTGTGTCGTCGCAAGCCAGCTCCGTGCTAATATCTC TCATAAAGACATGCAATTGGGAAGGCTACACATGAAGACCCTGTTACCAGTAAGCAAGCCAGAAATTCGG AGTTATTTTCCAGAAAGCTGGTTGTGGGAAGTTCATCTTGTTCCCAGAAGAAAACAGTTGCAGTTTGCCC TACCTGATTCTCTAACCACCTGGGAAATTCAAGGCATTGGCATTTCAAACACTGGTATATGTGTTGCTGA TACTGTCAAGGCAAAGGTGTTCAAAGATGTCTTCCTGGAAATGAATATACCATATTCTGTTGTACGAGGA GAACAGATCCAATTGAAAGGAACTGTTTACAACTATAGGACTTCTGGGATGCAGTTCTGTGTTAAAATGT CTGCTGTGGAGGGAATCTGCACTTCGGAAAGCCCAGTCATTGATCATCAGGGCACAAAGTCCTCCAAATG TGTGCGCCAGAAAGTAGAGGGCTCCTCCAGTCACTTGGTGACATTCACTGTGCTTCCTCTGGAAATTGGC CTTCACAACATCAATTTTTCACTGGAGACTTGGTTTGGAAAAGAAATCTTAGTAAAAACATTACGAGTGG TGCCAGAAGGTGTCAAAAGGGAAAGCTATTCTGGTGTTACTTTGGATCCTAGGGGTATTTATGGTACCAT TAGCAGACGAAAGGAGTTCCCATACAGGATACCCTTAGATTTGGTCCCCAAAACAGAAATCAAAAGGATT TTGAGTGTAAAAGGACTGCTTGTAGGTGAGATCTTGTCTGCAGTTCTAAGTCAGGAAGGCATCAATATCC TAACCCACCTCCCCAAAGGGAGTGCAGAGGCGGAGCTGATGAGCGTTGTCCCAGTATTCTATGTTTTTCA CTACCTGGAAACAGGAAATCATTGGAACATTTTTCATTCTGACCCATTAATTGAAAAGCAGAAACTGAAG AAAAAATTAAAAGAAGGGATGTTGAGCATTATGTCCTACAGAAATGCTGACTACTCTTACAGTGTGTGGA AGGGTGGAAGTGCTAGCACTTGGTTAACAGCTTTTGCTTTAAGAGTACTTGGACAAGTAAATAAATACGT AGAGCAGAACCAAAATTCAATTTGTAATTCTTTATTGTGGCTAGTTGAGAATTATCAATTAGATAATGGA TCTTTCAAGGAAAATTCACAGTATCAACCAATAAAATTACAGGGTACCTTGCCTGTTGAAGCCCGAGAGA ACAGCTTATATCTTACAGCCTTTACTGTGATTGGAATTAGAAAGGCTTTCGATATATGCCCCCTGGTGAA AATCGACACAGCTCTAATTAAAGCTGACAACTTTCTGCTTGAAAATACACTGCCAGCCCAGAGCACCTTT ACATTGGCCATTTCTGCGTATGCTCTTTCCCTGGGAGATAAAACTCACCCACAGTTTCGTTCAATTGTTT CAGCTTTGAAGAGAGAAGCTTTGGTTAAAGGTAATCCACCCATTTATCGTTTTTGGAAAGACAATCTTCA GCATAAAGACAGCTCTGTACCTAACACTGGTACGGCACGTATGGTAGAAACAACTGCCTATGCTTTACTC ACCAGTCTGAACTTGAAAGATATAAATTATGTTAACCCAGTCATCAAATGGCTATCAGAAGAGCAGAGGT ATGGAGGTGGCTTTTATTCAACCCAGGACACCATCAATGCCATTGAGGGCCTGACGGAATATTCACTCCT GGTTAAACAACTCCGCTTGAGTATGGACATCGATGTTTCTTACAAGCATAAAGGTGCCTTACATAATTAT AAAATGACAGACAAGAATTTCCTTGGGAGGCCAGTAGAGGTGCTTCTCAATGATGACCTCATTGTCAGTA CAGGATTTGGCAGTGGCTTGGCTACAGTACATGTAACAACTGTAGTTCACAAAACCAGTACCTCTGAGGA AGTTTGCAGCTTTTATTTGAAAATCGATACTCAGGATATTGAAGCATCCCACTACAGAGGCTACGGAAAC TCTGATTACAAACGCATAGTAGCATGTGCCAGCTACAAGCCCAGCAGGGAAGAATCATCATCTGGATCCT CTCATGCGGTGATGGACATCTCCTTGCCTACTGGAATCAGTGCAAATGAAGAAGACTTAAAAGCCCTTGT GGAAGGGGTGGATCAACTATTCACTGATTACCAAATCAAAGATGGACATGTTATTCTGCAACTGAATTCG ATTCCCTCCAGTGATTTCCTTTGTGTACGATTCCGGATATTTGAACTCTTTGAAGTTGGGTTTCTCAGTC CTGCCACTTTCACAGTTTACGAATACCACAGACCAGATAAACAGTGTACCATGTTTTATAGCACTTCCAA TATCAAAATTCAGAAAGTCTGTGAAGGAGCCGCGTGCAAGTGTGTAGAAGCTGATTGTGGGCAAATGCAG GAAGAATTGGATCTGACAATCTCTGCAGAGACAAGAAAACAAACAGCATGTAAACCAGAGATTGCATATG CTTATAAAGTTAGCATCACATCCATCACTGTAGAAAATGTTTTTGTCAAGTACAAGGCAACCCTTCTGGA TATCTACAAAACTGGGGAAGCTGTTGCTGAGAAAGACTCTGAGATTACCTTCATTAAAAAGGTAACCTGT ACTAACGCTGAGCTGGTAAAAGGAAGACAGTACTTAATTATGGGTAAAGAAGCCCTCCAGATAAAATACA ATTTCAGTTTCAGGTACATCTACCCTTTAGATTCCTTGACCTGGATTGAATACTGGCCTAGAGACACAAC ATGTTCATCGTGTCAAGCATTTTTAGCTAATTTAGATGAATTTGCCGAAGATATCTTTTTAAATGGATGC TAAAATTCCTGAAGTTCAGCTGCATACAGTTTGCACTTATGGACTCCTGTTGTTGAAGTTCGTTTTTTTG TTTTCTTCTTTTTTTAAACATTCATAGCTGGTCTTATTTGTAAAGCTCACTTTACTTAGAATTAGTGGCA CTTGCTTTTATTAGAGAATGATTTCAAATGCTGTAACTTTCTGAAATAACATGGCCTTGGAGGGCATGAA GACAGATACTCCTCCAAGGTTATTGGACACCGGAAACAATAAATTGGAACACCTCCTCAAACCTACCACT CAGGAATGTTTGCTGGGGCCGAAAGAACAGTCCATTGAAAGGGAGTATTACAAAAACATGGCCTTTGCTT GAAAGAAAATACCAAGGAACAGGAAACTGATCATTAAAGCCTGAGTTTGCTTTC (SEQ ID NO: 6696)

gi| 189944 |gb| 05144. l|HUMPHOCAR Homo sapiens (clone lamda-hPEC-3) phosphoenolpyruvate carboxykinase (PCK1) mRNA, complete eds TGGGAACACAAACTTGCTGGCGGGAAGAGCCCGGAAAGAAACCTGTGGATCTCCCTTCGAGATCATCCAA AGAGAAGAAAGGTGACCTCACATTCGTGCCCCTTAGCAGCACTCTGCAGAAATGCCTCCTCAGCTGCAAA ACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAAGCCTGGACAGCCTGCCCCAGGCAGTGAGGGAGTT TCTCGAGAATAACGCTGAGCTGTGTCAGCCTGATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAAT GGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTATGACAACTGCTGGT TGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGACGGTTATCGTCACCCAAGAGCAAAG AGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAG AAAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGG GGCCGCTGGGCTCACCTCTGTCGAAGATCGGCATCGAGCTGACGGATTCGCCCTACGTGGTGGCCAGCAT GCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCACTGGGCGATGGGGAGTTTGTCAAATGCCTC CATTCTGTGGGGTGCCCTCTGCCTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGA CGCTCATCGCCCACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCT GCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAGCCGGCTGGCAGAGGAGGAAGGGTGGCTGGCAGAG CACATGCTGATTCTGGGTATAACCAACCCTGAGGGTGAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCG CCTGCGGGAAGACCAΆCCTGGCCATGATGAACCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGA TGACATTGCCTGGATGAΆGTTTGACGCACAAGGTCATTTAAGGGCCATCAΆCCCAGAAAATGGCTTTTTC GGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCAAGACCATCCAGAAGAACACAATCT TTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGG CGTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAAC TCGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGTGTTC CCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTG GCAACATGGAGTCTTTGTGGGGGCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAA ATCATCATGCATGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACT GGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGCCCAAGATCTTCCATGTCAACTGGTTCCGGAAGGA CAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGAGAACTCCAGGGTGCTGGAGTGGATGTTCAACCGG ATCGATGGAAAAGCCAGCACCAACGTCACGCCCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGA AAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAATTCTGGGACAAGGAGGTGGA AGACATCGAGAAGTATCTGGTGGATCAAGTCAATGCCGACCTCCCCTGTGAAATCGAGAGAGAGATCCTT GCCTTGAAGCAAAGAATAAGCCAGATGTAATCAGGGCCTGAGAATAAGCCAGATGTAATCAGGGCCTGAG TGCTTTACCTTTAAAATCATTAAATTAAAATCCATAAGGTGCAGTAGGAGCAAGAGAGGGCAAGTGTTCC CAAATTGACGCCACCTAATAATCATCACCACACCGGGAGCAGATCTGAAGGCACACTTTGATTTTTTTAA GGATAAGAACCACAGAACACTGGGTAGTAGCTAATGAAATTGAGAAGGGAAΆTCTTAGCATGCCTCCAAA AATTCACATCCAATGCATACTTTGTTCAAATTTAAGGTTACTCAGGCATTGATCTTTTCAGTGTTTTTTC ACTTAGCTATGTGGATTAGCTAGAATGCACACCAAAAAGATACTTGAGCTGTATATATATATGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGCATGTATGTGCACATGTGTCTGTGTGATATTTGGTATGTGTATTTGT ATGTACTGTTATTCAAΆATATATTTAATACCTTTGGAAAATCTTGGGCAAGATGACCTACTAGTTTTCCT TGAAAAAAAGTTGCTTTGTTATTAΆTATTGTGCTTAAATTATTTTTATACACCATTGTTCCTTACCTTTA CATAATTGCAATATTTCCCCCTTACTACTTCTTGGAAAAAAATTAGAAAATGAAGTTTATAGAAAAG

(SEQ ID NO: 6697)

gi| 6679892 |ref |NM_008061.l| MUS musculus glucose-6-phosphatase, catalytic (G6pc) , mRNA

AGCAGAGGGATCGGGGCCAACCGGGCTTGGACTCACTGCACGGGCTCTGCTGGCAGCTTCCTGAGGTACC AAGGGAGGAAGGATGGAGGAAGGAATGAACATTCTCCATGACTTTGGGATCCAGTCGACTCGCTATCTCC AAGTGAATTACCAAGACTCCCAGGACTGGTTCATCCTTGTGTCTGTGATTGCTGACCTGAGGAACGCCTT CTATGTCCTCTTTCCCATCTGGTTCCATCTTAAAGAGACTGTGGGCATCAATCTCCTCTGGGTGGCAGTG GTCGGAGACTGGTTCAACCTCGTCTTCAAGTGGATTCTGTTTGGACAACGCCCGTATTGGTGGGTCCTGG ACACCGACTACTACAGCAACAGCTCCGTGCCTATAATAAAGCAGTTCCCTGTCACCTGTGAGACCGGACC AGGAAGTCCCTCTGGCCATGCCATGGGCGCAGCAGGTGTATACTATGTTATGGTCACTTCTACTCTTGCT ATCTTTCGAGGAAAGAAAAAGCCAACGTATGGATTCCGGTGTTTGAACGTCATCTTGTGGTTGGGATTCT GGGCTGTGCAGCTGAACGTCTGTCTGTCCCGGATCTACCTTGCTGCTCACTTTCCCCACCAGGTCGTGGC TGGAGTCTTGTCAGGCATTGCTGTGGCTGAAACTTTCAGCCACATCCGGGGCATCTACAATGCCAGCCTC • CGGAΆGTATTGTCTCATCACCATCTTCTTGTTTGGTTTCGCGCTTGGATTCTACCTGCTACTAAAAGGGC TAGGGGTGGACCTCCTGTGGACTTTGGAGAAAGCCAAGAGATGGTGTGAGCGGCCAGAATGGGTCCACCT TGACACTACACCCTTTGCCAGCCTCTTCAAAAACCTGGGAACCCTCTTGGGGTTGGGGCTGGCCCTCAAC TCCAGCATGTACCGGAAGAGCTGCAAGGGAGAACTCAGCAAGTCGTTCCCATTCCGCTTCGCCTGCATTG TGGCTTCCTTGGTCCTCCTGCATCTCTTTGACTCTCTGAAGCCCCCATCCCAGGTTGAGTTGATCTTCTA CATCTTGTCTTTCTGCAAGAGCGCAACAGTTCCCTTTGCATCTGTCAGTCTTATCCCATACTGCCTAGCC CGGATCCTGGGACAGACACACAAGAAGTCTTTGTAAGGCATGCAGAGTCTTTGGTATTTAAAGTCAACCG CCATGCAAAGGACTAGGAACAACTAAAGCCTCTGAAACCCATTGTGAGGCCAGAGGTGTTGACATCGGCC CTGGTAGCCCTGTCTTTCTTTGCTATCTTAACCAAAAGGTGAATTTTTACAAAGCTTACAGGGCTGTTTG AGGAAAGTGTGAATGCTGGAAACTGAGTCATTCTGGATGGTTCCCTGAAGATTCGCTTACCAGCCTCCTG TCAGATACAGAAGAGCAAGCCCAGGCTAGAGATCCCAACTGAGAATGCTCTTGCGGTGCAGAATCTTCCG GCTGGGAAAAGGAAAAGAGCACCATGCATTTGCCAGGAAGAGAAAGAAGGATCGGGAGGAGGGAGAGTGT' TTTATGTATCGAGCAAACCAGATGCAATCTATGTCTAACCGGCTTCAGTTGTGTCTGCGTCTTTAGATAC GACACACTCAATAATAATAATAGACCAACTAGTGTAATGAGTAGCCAGTTAAAGGCGATTAATTCTGCTT CCAGATAGTCTCCACTGTACATAAAAGTCACACTGTGTGCTTGCATTCCTGTATGGTAGTGGTGACTGTC TCTCACACCACCTTCTCTATCACGTCACAGTTTTCTCCTCCTCAGCCTATGTCTGCATTCCCCAGAATTC TCCACTTGTTCCCTGGCCCTGCTGCTGGACCCTGCTGTGTCTGGTAGGCAACTGTTTGTTGGTGCTTTTG TAGGGTTAAGTTAAACTCTGAGATCTTGGGCAAAATGGCAAGGAGACCCAGGATTCTTCTCTCCAAAGGT ^CACTCCGATGTTATTTTTGATTCCTGGGGCAGAAATATGACTCCTTTCCCTAGCCCAAGCCAGCCAAGAG CTCTCATTCTTAGAAGAAAAGGCAGCCCCTTGGTGCCTGTCCTCCTGCCTCGGCTGATTTGCAGAGTACT TCTTCAAAAAGAAAAAAATGGTAAAGCTATTTATTAAAAATTCTTTGTTTTTTGCTACAAATGATGCATA TATTTTCACCCACACCAAGCACTTTGTTTCTAATATCTTTGATAAGAAAACTACATGTGCAGTATTTTAT TAAAGCAACATTTTATTTA (SEQ ID NO: 6698)

gi | 7110682 | ref |NM_011044.1 | Mus musculus phosphoenolpyruvate carboxykinase 1, cytosolic (Pckl) , mRNA

ACAGTTGGCCTTCCCTCTGGGAACACACCCTCGGTCAACAGGGGAAATCCGGCAAGGCGCTCAGCGATCT CTGATCCAGACCTTCCAAAAGGAAGAAAGGTGGCACCAGAGTTCCTGCCTCTCTCCACACCATTGCAATT ATGCCTCCTCAGCTGCATAACGGTCTGGACTTCTCTGCCAAGGTTATCCAGGGCAGCCTCGACAGCCTGC CCCAGGCAGTGAGGAAGTTCGTGGAAGGCAATGCTCAGCTGTGCCAGCCGGAGTATATCCACATCTGCGA TGGCTCCGAGGAGGAGTACGGGCAGTTGCTGGCCCACATGCAGGAGGAGGGTGTCATCCGCAAGCTGAAG AAATATGACAACTGTTGGCTGGCTCTCACTGACCCTCGAGATGTGGCCAGGATCGAAAGCAAGACAGTCA TCATCACCCAAGAGCAGAGAGACACAGTGCCCATCCCCAAAACTGGCCTCAGCCAGCTGGGCCGCTGGAT GTCGGAAGAGGACTTTGAGAAAGCATTCAACGCCAGGTTCCCAGGGTGCATGAAAGGCCGCACCATGTAT GTCATCCCATTCAGCATGGGGCCACTGGGCTCGCCGCTGGCCAAGATTGGTATTGAACTGACAGACTCGC CCTATGTGGTGGCCAGCATGCGGATCATGACTCGGATGGGCATATCTGTGCTGGAGGCCCTGGGAGATGG GGAGTTCATCAAGTGCCTGCACTCTGTGGGGTGCCCTCTCCCCTTAAAAAAGCCTTTGGTCAACAACTGG GCCTGCAACCCTGAGCTGACCCTGATCGCCCACCTCCCGGACCGCAGAGAGATCATCTCCTTTGGAAGCG GATATGGTGGGAACTCACTACTCGGGAAGAAATGCTTTGCGTTGCGGATCGCCAGCCGTCTGGCTAAGGA GGAAGGGTGGCTGGCGGAGCATATGCTGATCCTGGGCATAACTAACCCCGAAGG^'CAAGAAGAAATACCTG GCCGCAGCCTTCCCTAGTGCCTGTGGGAAGACTAACTTGGCCATGATGAACCCCAGCCTGCCCGGGTGGA AGGTCGAATGTGTGGGCGATGACATTGCCTGGATGAAGTTTGATGCCCAAGGCAACTTAAGGGCTATCAA CCCAGAAAACGGGTTTTTTGGAGTTGCTCCTGGCACCTCAGTGAAGACAAATCCAAATGCCATTAAAACC ATCCAGAAAΆACACCATCTTCACCAACGTGGCCGAGACTAGCGATGGGGGTGTTTACTGGGAAGGCATCG ATGAGCCGCTGGCCCCGGGAGTCACCATCACCTCCTGGAΆGAACAAGGAGTGGAGACCGCAGGACGCGGA ACCATGTGCCCATCCCAACTCGAGATTCTGCACCCCTGCCAGCCAGTGCCCCATTATTGACCCTGCCTGG GAATCTCCAGAAGGAGTACCCATTGAGGGTATCATCTTTGGTGGCCGTAGACCTGAAGGTGTCCCCCTTG TCTATGAAGCCCTCAGCTGGCAGCATGGGGTGTTTGTAGGAGCAGCCATGAGATCTGAGGCCACAGCTGC TGCAGAACACAAGGGCAAGATCATCATGCACGACCCCTTTGCCATGCGACCCTTCTTCGGCTACAACTTC GGCAAATACCTGGCCCACTGGCTGAGCATGGCCCACCGCCCAGCAGCCAAGTTGCCCAAGATCTTCCATG TCAACTGGTTCCGGAAGGACAAAGATGGCAAGTTCCTCTGGCCAGGCTTTGGCGAGAACTCCCGGGTGCT GGAGTGGATGTTCGGGCGGATTGAAGGGGAAGACAGCGCCAAGCTCACGCCCATCGGCTACATCCCTAAG GAAAACGCCTTGAACCTGAAΆGGCCTGGGGGGCGTCAACGTGGAGGAGCTGTTTGGGATCTCTAAGGAGT TCTGGGAGAAGGAGGTGGAGGAGATCGACAGGTATCTGGAGGACCAGGTCAACACCGACCTCCCTTACGA AATTGAGAGGGAGCTCCGAGCCCTGAAACAGAGAATCAGCCAGATGTAAATCCCAATGGGGGCGTCTCGA GAGTCACCCCTTCCCACTCACAGCATCGCTGAGATCTAGGAGAAAGCCAGCCTGCTCCAGCTTTGAGATA GCGGCACAATCGTGAGTAGATCAGAAAAGCACCTTTTAATAGTCAGTTGAGTAGCACAGAGAACAGGCTA GGGGCAAATAAGATTGGGAGGGGAAATCACCGCATAGTCTCTGAAGTTTGCATTTGACACCAATGGGGGT TTTGGTTCCACTTCAAGGTCACTCAGGAATCCAGTTCTTCACGTTAGCTGTAGCAGTTAGCTAAAATGCA CAGAAAACATACTTGAGCTGTATATATGTGTGTGAACGTGTCTCTGTGTGAGCATGTGTGTGTGTGTGTG

TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTACATGCCTGTCTGTCCCATTGTCCACAGTATATTTAA

AACCTTTGGGGAAAAATCTTGGGCAAATTTGTAGCTGTAACTAGAGAGTCATGTTGCTTTGTTGCTAGTA

, TGTATGTTTAAATTATTTTTATACACCGCCCTTACCTTTCTTTACATAATTGAAATTGGTATCCGGACCA

CTTCTTGGGAAAAAAATTACAAAATAAA (SEQ ID NO: 6699)

Example 5. siRNAs decrease mRNA levels in vivo

Male CMV-Luc mice (8-10 weeks old) from Xenogen (Cranbury, NJ) were administered cholesterol conjugated siRNA (see Table 17).

Table 17. Solutions adminstered to mice

Table 18. Test iRNA agents targeting Luciferase siRNA Sequence

5'-GAA CUG UGU GUG AGA GGU CCU-3' (SEQ ID NO: 5277)

ALN-1070 -CG CUϋ GAC ACA CAC UCU CCA GGA-5' (SEQ ID N0:5278)

5'-GAA CUG UGU GUG AGA GGU CCU-GS-3' (SEQ ID NO: 5279)

ALN-1000 -CG CUU GAC ACA CAC UCU CCA GGA-5' (SEQ ID NO: 5280)

5'-GAA CUG UGU GUG AGA GGU CCU-3' (SEQ ID N0:5281)

ALN-3000 S'-CS^S¹ CUU GAC ACA CAC UCU CCA GGA-5' (SEQ ID NO: 5282)

5'-GAA CUG UGU GUG AGA GGU CCU-chol.²- (SEQ ID NO : 5283 )

ALN-3001 -CS^S¹ CUU GAC ACA CAC UCU CCA GGA-5' (SEQ ID N0 : 5284 )

2' O-Me group is attached to the nucleotide and the nucleotides have phosphorothioate linkages (indicated by "s")

2 cholesterol is conjugated to the antisense strand via the linker: U-pyrroline carrier-C(0)-(CH₂)₅-NHC(0)- cholesterol (via cholesterol C-3 hydroxyl).

Animals were injected (tail vein) with a volume of 200-250 μl test solution containing buffer or an siRNA solution. Group 1 received buffer and group 2 received cholesterol conjugated siRNA (ALN-3001) at a dose of 50 mg/kg body weight. Twenty-two hours after injection, animals were sacrificed and livers collected. Organs were snap frozen on dry ice, then pulverized in a mortar and pestle.

For Luciferase mRNA analysis (by the QuantiGene Assay (Genospectra, Inc.; Fremont, CA)), approximately 10 mg of tissue powder was resuspended in tissue lysis buffer, and processed according to the manufacturer's protocol. Samples ofthe lysate were hybridized with probes specific for Luciferase or GAPDH (designed using ProbeDesigner software (Genospectra, Inc., Fremont, CA) in triplicate, and processed for luminometric analysis. Values for Luciferase were normalized to GAPDH. Mean values were plotted with error bars corresponding to the standard deviation ofthe Luciferase measurements. Results indicated that the level of luciferase RNA in animals injected with cholesterol conjugated siRNA was reduced by about 70% as compared to animals injected with buffer (see FIGs. 8A and 8B).

In Vitro Activity

HeLa cells expressing luciferase were transfected with each ofthe siRNAs listed in Table 18. ALN-1000 siRNAs were most effective at decreasing luciferase mRNA levels (~0.6 nM siRNA decreased mRNA levels to about -65% the original expression level, and 1.0 nM siRNA decreased levels to about -20% the original expression level); ALN-3001 siRNAs were least effective (-0.6 nM siRNA had a negligible effect on mRNA levels, and 1.0 nM siRNA decreased levels to about -40% the original expression level).

Pharmacokinetics/Biodistribution

Pharmacokinetic analyses were performed in mice and rats. Test siRNA molecules were radioactively labeled with ³³P on the antisense strand by splint ligation. Labeled siRNAs (5 Omg/kg) were administered by tail vein injection, and plasma levels of siRNA were measured periodically over 24 hrs by scintillation counting. Cholesterol conjugated siRNA (ALN-3001) was discovered to circulate in mouse plasma for a longer period of time than unconjugated siRNA (ALN-3000) (FIG. 9). RNAse protection assays indicated that cholesterol-conjugated siRNA (ALN-3001) was detectable in mouse plasma 12 hours after injection, whereas xmconjugated siRNA (ALN-3000) was not detectable in mouse plasma within two hours after injection. Similar results were observed in rats.

Mouse liver was harvested at varying time points (ranging from 0.08-24 hours) following injection with siRNA, and siRNA localized to the liver was quantified. Over the time period tested, the amount of cholesterol-conjugated siRNA (ALN-3001) detected in the liver ranged from 14.3- 3.55 percent ofthe total dose administered to the mouse. The amount of unconjugated siRNA (ALN-3000) detected in the liver was lower, ranging from 3.91-1.75 percent ofthe total dose administered (FIG. 10).

Detection of siRNA in Different Tissues

Various tissues and organs (fat, heart, kidney, liver, and spleen) were harvested from two

CMV-Luc mice 22 hours following injection with 50 mg/kg ALN-3001. The antisense sfrand ofthe siRNA was detected by RNAse protection assay. The liver contained the greatest concentration of siRNA (-8-10 μg siRNA/g tissue); the spleen, heart and kidney contained lesser amounts of siRNA (-2-7 μg siRNA/g tissue); and fat tissue contained the least amount of siRNA (<~1 μg siRNA/g tissue) (FIG. 11).

Glucose-6-phosphatase siRNA detection by RNAse Protection Assay Balbc mice were injected with U/U, 3 'C/U, or 3 ' C/3 ' C siRNA (4 mg/kg) targeting glucose-6-phosphatase (G6Pase) (see Table 19). Administration was by hydrodynamic tail vein injection (hd) or non-hydrodynamic tail vein injection (iv), and siRNA was subsequently detected in the liver by RNAse protection assay.

Table 19. Test iRNA agents targeting glucose-6-phosphatase

Unconjugated siRNA (U/U) delivered by hd was detected by 15 min. post-injection (the earliest determined time-point) and was still detectable in the liver 18 hours post-injection (FIG. 12).

Delivery by normal iv adminisfration resulted in the greatest concentration of 3 'C/3 'C siRNA (the bis-cholesterol-conjugate) in the liver 1 hour post injection (as compared to the mono- cholesterol-conjugate 3 'C/3 TJ siRNA). At 18 hours post injection, 3 'C/3 'C siRNAs and 3 'C/U siRNA were still detectable in the liver with the bis-conjugate at higher levels compared to the mono-conjugate (FIG. 13).

Example 6. siRNAs decrease protein activity levels in vivo

Male CMV-Luc mice were bred by Charles River Laboratories, Inc. (Wilmington, MA). Mice (6-7 weeks old) were administered cholesterol conjugated siRNA (see Tables 20-22). Table 20. Test rou s for in vivo siRNA assa s-ex eriment 1

Animals were injected (tail vein) with a volume of 200-250 μl test solution containing buffer or an siRNA solution. Group 1 received buffer and group 2 received cholesterol conjugated siRNA

(ALN-3001) at a dose of 75 mg/kg body weight. Nineteen to 22 hours after injection, animals were sacrificed and livers collected. Organs were snap frozen on dry ice, then pulverized in a mortar and pestle.

For Luciferase activity analysis, approximately 50 mg of tissue powder was resuspended in 0.5 ml Cell Lysis Buffer (Promega, Inc.). Samples were vortexed vigorously for three minutes, snap frozen in liquid nitrogen, then thawed in a 37 degree water bath. This process was repeated twice more. After the final thaw, samples were vortexed for three minutes. Insoluble material was removed by cenfrifugation in a microcentrifuge (4 degrees) at full speed for four minutes.

Supernatants were collected. Twenty to 25 microliters of each sample were pipetted into assay tubes in triplicate, and allowed to come to room temperature. For activity measurements, a luminometer

(Berthold, Inc.) was programmed to deliver 200 microliters of "Bright Glow" assay reagent

(Promega, Inc.) to the test sample, and record light emission over a ten second period.

To measure total protein, samples of supernatant were diluted thirty fold, and five microliter samples were measured in triplicate in a Bradford protein microassay (Bio-Rad). Bovine Serum Albumin was used to generate a standard curve. Luciferase activity was determined as the mean ofthe luminometry reading normalized to mean protein content. Mean normalized values were then calculated for the buffer and siRNA- treated groups in each experiment. For each experiment, the normalized Luc level ofthe siRNA treated group is expressed as a percentage ofthe buffer control (which was set to 100%). Error bars indicate standard deviations.

Results indicated that the level of luciferase activity in animals injected with cholesterol conjugated siRNA was reduced by about 55% as compared to animals injected with buffer (see FIG. 14).

OTHER EMBODIMENTS

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope ofthe invention encompassed by the appended claims.

Claims

WHAT IS CLAIMED IS:

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 sfrand.

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 ofthe 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 xmwanted levels of cholesterol, and/or disregulation of lipid metabolism.

9. The method of claim 8, wherein said disorder is chosen from the group of HDL/LDL cholesterol imbalance; dyshpidemias, 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 ofthe 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 freatment 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.