AU2013213756A1 - iRNA agents targeting VEGF - Google Patents

Abstract

The features of the present invention relate to compounds, compositions and methods useful for modulating the expression of vascular endothelial growth factor (VEGF), such as by the mechanism of RNA interference (RNAi). The compounds and compositions include iRNA agents that can be unmodified or chemically-modified. N~~ 0cot o0 0D 0 0 0 0 0 . CL 0. 0. $3 CL 0U a > E 0 I- C" 4c c 77;!iuI~in a CD 0 0 C) coL % IujsjxQJ3 .

Description

AUSTRALIA Patents Act 1990 Alnylam Pharmaceuticals, Inc. COMPLETE SPECIFICATION STANDARD PATENT Invention Title: iRNA agents targeting VEGF The following statement is a full description of this invention including the best method of performing it known to us:iRNA AGENTS TARGETING VEGF RELATED APPLICATIONS This application is a divisional application under S 79B of the Patents Act 1990 of 5 Australian Patent Application No. 2010212443 which is a divisional application of Australian Patent No. 2005222902 filed March 11, 2005, which is the Australian national phase filing of International Application No. PCT/US2005/008182 filed on March 11, 2005 which claims priority from U.S. Provisional Application No. 60/552,620, filed March 12, 2004, U.S. Provisional Application No. 60/559,824, filed April 5, 2004, and U.S. Provisional Application 0 No. 60/647,191, filed January 25, 2005. All three provisional applications are incorporated herein by reference in their entirety. FIELD OF THE INVENTION The present invention is in the filed of iRNA agents that can inhibit expression of vascular 5 endothelial growth factor (VEGF). The invention also relates to the use of siRNA targeting VEGF sequences to treat conditions or disorders related to unwanted expression of VEGF, e.g., age-related macular degeneration or diabetic retinopathy. BACKGROUND .0 VEGF (also known as vascular permeability factor, VPF) is a multifunctional cytokine that stimulates angiogenesis, epithelial cell proliferation, and endothelial cell survival. VEGF can be produced by a wide variety of tissues, and its overexpression or aberrant expression can result in a variety disorders, including retinal disorders such as age-related macular degeneration and diabetic retinopathy, cancer, asthma, and other angiogenic disorders. 25 Macular degeneration is a major cause of blindness in the United States and the frequency of this disorder increases with age. Macular degeneration refers to the group of diseases in which sight-sensing cells in the macular zone of the retina malfunction or loose function and which can result in debilitating loss of vital central or detail vision. Adult macular degeneration (AMD), which is the most common form of macular degeneration, occurs in two 30 main forms. Ninety percent of people with AMD have the form described as "dry" macular degeneration. An area of the retina is affected, which leads to slow breakdown of cells in the macula, and a gradual loss of central vision. The other form of AMD is "wet" macular degeneration. Although only 10% of people with AMD have this type, it accounts for 90% of blindness from the disease. As dry AMD progresses, new blood vessels may begin to grow and 5 cause "wet" AMD. These new blood vessels often leak blood and fluid under the macula. This causes rapid damage to the macula that can lead to loss of central vision in a short time. iRNA agents targeting VEGF can be useful for the treatment of wet and dry macular degeneration. 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 0 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 has been suggested as a method of developing a new class of therapeutic agents. However, to date, these have remained mostly as suggestions with no demonstrate proof that RNAi can be used therapeutically. 5 The present invention advances the art by providing a detailed gene walk across the VEGF gene and a detailed structural analysis of modifications that can be employed to stabilize the molecule against degradation and increase cellular uptake and targeting. SUMMARY OF THE INVENTION .0 The invention provides compounds, compositions and methods useful for modulating the expression of VEGF. The invention provides compounds, compositions and methods useful for modulating the expression of VEGF activity by RNA interference (RNAi) using small nucleic acid molecules, such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hairpin RNA (shRNA) molecules, which collectively fall under 25 the general term of iRNA agents. The iRNA agents can be unmodified or chemically-modified nucleic acid molecules. The iRNA agents can be chemically synthesized or expressed from a vector or enzymatically synthesized. The invention provides various chemically-modified synthetic iRNA agents capable of modulating VEGF gene expression or activity in cells and in a mammal by RNAi. The use of a chemically-modified iRNA agent can improve one or more properties of an iRNA agent through increased resistance to degradation, increased specificity to target moieties, improved cellular uptake, and the like. In one aspect, the invention provides an iRNA agent that down-regulates expression of a VEGF gene. The VEGF gene can include a VEGF encoding sequence and/or VEGF regulatory 5 sequences such as may exist 5' or 3' of a VEGF open reading frame (ORF). In one embodiment, the invention provides an isolated iRNA agent including a sense and antisense sequence, where the sense and antisense sequences can form an RNA duplex. The sense sequence can include a nucleotide sequence that is identical or substantially identical to a target sequence of about 19 to 23 nucleotides of a VEGF sequence. In one embodiment, the 0 VEGF sequence that is targeted includes the sequence of any one of SEQ ID NOs:2-401 (see Table 1). In one embodiment, the sense sequence of the iRNA agent includes a sequence identical or substantially identical to any of the VEGF target sequences, e.g., substantially identical to any of sense sequences provided in Table 1, SEQ ID NOs:2-401. In another embodiment, the 5 antisense sequence of the iRNA agent can include a sequence complementary to or substantially complementary to, any of the target sequences, e.g., complementary to any of SEQ ID NOs:2 401. By "substantially identical" is meant that the mismatch between the nucleotide sequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%. Preferably, no more than 1, 2, 3, 4, or 5 nucleotides differ between the target sequence and sense sequence. Furthermore, sequences that .0 are "complementary" to each other (e.g., sense and antisense sequences) can be fully complementary, or can have no more than 1, 2, 3, 4, or 5 nucleotides that lack full complementarity. In one embodiment, the sense and antisense pairs of sequences of an iRNA agent includes any one of the agents provided in Table 2, or a sequence which differs in the sense strand from 25 the recited sequence by no more than 1, 2, 3, 4, or 5 nucleotides, or in the antisense strand by no more than 1, 2, 3, 4, or 5 nucleotides, or in both strands by no more than 1, 2, 3, 4, or 5 nucleotides. In one preferred embodiment, the sense sequence of an iRNA agent includes a sequence that is selected from the group consisting of SEQ ID NO:456, SEQ ID NO:550, SEQ ID

A

NO:608, and SEQ ID NO:634, or a sequence that differs from the recited sequence by no more than 1, 2, 3, 4, or 5 nucleotides. In another embodiment, the antisense sequence of the iRNA agent includes a sequence fully complementary or substantially complementary to any of the VEGF target sequences, 5 e.g., complementary or substantially complementary to any of SEQ ID NOs:2-401. In another embodiment, the antisense sequence of an iRNA agent includes a sequence selected from the group consisting any of the antisense sequences provided in Table 2, or a sequence which differs from the recited sequence by no more than 1, 2, 3, 4, or 5 nucleotides. In a preferred embodiment, this antisense sequence is fully complementary to a sense sequence or 0 has no more than 1, 2, 3, 4, or 5 nucleotide mismatches with the sense sequence. In a preferred embodiment, the antisense sequence of an iRNA agent includes a sequence selected from the group consisting of SEQ ID NO:457, SEQ ID NO:551, SEQ ID NO:609, and SEQ ID NO:635, or a sequence that differs from the recited sequence by no more than 1, 2, 3, 4, or 5 nucleotides. 5 In another embodiment, the iRNA agent is chemically modified. For example, the iRNA agent can include a non-nucleotide moiety. A chemical modification or other non-nucleotide moiety can stabilize the sense and antisense sequences against nucleolytic degradation. Additionally, conjugates can be used to increase uptake and target uptake of the iRNA agent to particular cell types. Preferred modifications include those specifically provided in the .0 Examples, Tables 6-19. In another embodiment, the iRNA agent includes a 3'-overhang that ranges from 1 to about 6 nucleotides. As used herein, a "3'overhang" refers to at least one unpaired nucleotide extending from the 3' end of an iRNA sequence. The 3' overhang can include ribonucleotides or deoxyribonucleotides or modified ribonucleotides or modified deoxyribonucleotides. The 3' 25 overhang is preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length and most preferably from about 2 to about 4 nucleotides in length. The 3' overhang can occur on the sense or antisense sequence, or on both sequences of an iRNA agent. In one preferred embodiment, the iRNA agent of the invention includes an antisense sequence having 23 nucleotides complementary to the target VEGF sequence and a sense 30 sequence having at least 21 nucleotides. Each sequence can include at least 21 nucleotides that are complementary to each other, and at least the antisense sequence can have a 3' overhang of two nucleotides. In one embodiment, both the sense and antisense sequences of the iRNA agent include a 3' overhang, the length of which can be the same or different for each sequence. In one 5 embodiment, the 3' overhang on each sequence ranges from 1 to about 6 (e.g., from 1 to about 3) nucleotides in length. In a preferred embodiment, the 3' overhang is on both sequences of the iRNA agent and is two nucleotides in length. In another preferred embodiment, the 3' overhang is on both sequences of the iRNA agent and the 3' overhangs include two thymidylic acid residues ("TT"). 0 In one embodiment, an iRNA agent includes an antisense sequence having about 19 to 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides with complementarity to an RNA sequence encoding a VEGF protein. The iRNA agent can further include a sense sequence having about 19 to 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides, and the antisense and sense sequences can have distinct nucleotide sequences with at least about 19, 20, or 21 5 complementary nucleotides. In one embodiment, an iRNA agent of the invention includes an antisense region having about 19 to about 25 (e.g., about 19 to about 23) nucleotides with complementarity to an RNA sequence encoding VEGF, and a sense region having about 19 to 25 (e.g., about 19 to about 23) nucleotides. The sense and antisense regions can be included in a linear molecule with at least .0 about 19 complementary nucleotides. The sense sequence can include a nucleotide sequence that is substantially identical to a nucleotide sequence of VEGF. In one embodiment, the iRNA agent includes an antisense sequence of about 21 nucleotides complementary to the VEGF target sequence and a sense sequence of about 21 nucleotides complementary to the antisense sequence. The iRNA agent can include a non 25 nucleotide moiety. In one embodiment, the sense or antisense sequence of the iRNA agent can include a 2'-O-methyl (2'-OMe) pyrimidine nucleotide, 2'-deoxy nucleotide (e.g., deoxy cytodine), 2'-deoxy-2'-fluoro (2'-F) pyrimidine nucleotide, 2'-O-methoxyethyl (2'-O-MOE), 2' 0-aminopropyl (2'-O-AP), 2'-O-N-methylacetamido (2'-O-NMA), 2'-0 dimethylaminoethlyoxyethyl (2'-DMAEOE), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-0 30 dimethylaminopropyl (2'-O-AP), 2'-hydroxy nucleotide, or a 2'-ara-fluoro nucleotide, or a locked

A;

nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleic acid (HNA), cyclohexene nucleic acid (CeNA), ribo-difluorotoluyl, 5-allyamino-pyrimidines, or 5-Me-2'-modified pyrimidines. A 2' modification is preferably a 2'-OMe modification, and more preferably, a 2' fluoro modification. In a preferred embodiment, one or more 2' modified nucleotides are on the 5 sense strand of the iRNA agent. In one embodiment, an iRNA agent includes a nucleobase modification, such as a cationic modification, such as a 3'-abasic cationic modification. The cationic modification can be, e.g., an alkylamino-dT (e.g., a C6 amino-dT), an allylamino conjugate, a pyrrolidine conjugate, a pthalamido a hydroxyprolinol conjugate or an aminooxy conjugate, on one or more of the 0 terminal nucleotides of the iRNA agent. An alkylamino-dT conjugate is preferably attached to the 3' end of the sense or antisense strand of an iRNA agent. A pyrrolidine linker is preferably attached to the 3' or 5' end of the sense strand, or the 3' end of the antisense strand. An allyl amine uridine is preferably on the 3' or 5' end of the sense strand, and not on the 5' end of the antisense strand. An aminooxy conjugate can be attached to a hydroxyl prolinol and at the 3' or 5 5' end of either the sense or antisense strands. In another embodiment, an iRNA agent that targets VEGF includes a conjugate, e.g., to facilitate entry into a cell or to inhibit exo- or endonucleolytic cleavage. The conjugate can be, for example, a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, a retinoid or a peptide. For example, the conjugate can be naproxen, nitroindole (or another conjugate that .0 contributes to stacking interactions), folate, ibuprofen, retinol or a C5 pyrimidine linker. In other embodiments, the conjugates are glyceride lipid conjugates (e.g. a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. Preferably, conjugates are on the 3' end of the antisense strand, or on the 5' or 3' end of the sense strand, and preferably the conjugates are not on the 3' end of the antisense strand and on the 3' end of the sense strand. 25 In one embodiment, the conjugate is naproxen, and the conjugate is preferably on the 5' or 3' end of the sense or antisense strands. In one embodiment, the conjugate is cholesterol or thiocholesterol, and the conjugate is preferably on the 5' or 3' end of the sense strand and preferably not present on the antisense strand. In some embodiments, the cholesterol is conjugated to the iRNA agent by a pyrrolidine linker, or serinol linker, or hydroxyprolinol linker. 30 In another embodiment, the conjugate is cholanic acid, and the cholanic acid is attached to the 5' -7 or 3' end of the sense strand, or the 3' end of the antisense strand. In one embodiment, the cholanic acid is attached to the 3' end of the sense strand and the 3' end of the antisense strand. In another embodiment, the conjugate is retinol acid, and the retinol acid is attached to the 5' or 3' end of the sense strand, or the 3' end of the antisense strand. In one embodiment, the retinol 5 acid is attached to the 3' end of the sense strand and the 3' end of the antisense strand. In one aspect, an iRNA agent of the invention has RNAi activity that modulates expression of RNA encoded by a VEGF gene. VEGF genes can share some degree of sequence identity with each other, and thus, iRNA agents can target a class of VEGF genes, or alternatively, specific VEGF genes, by targeting sequences that are either shared amongst 0 different VEGF targets or that are unique for a specific VEGF target. Therefore, in one embodiment, an iRNA agent can target a conserved region of a VEGF nucleotide sequence (e.g., RNA sequence). The conserved region can have sequence identity with several different VEGF related sequences (e.g., different VEGF isoforms, splice variants, mutant genes, etc.). Thus, one iRNA agent can target several different VEGF-related sequences. 5 In one embodiment, an iRNA agent is chemically modified. In another embodiment the iRNA agent includes a duplex molecule wherein one or more sequences of the duplex molecule is chemically modified. Non-limiting examples of such chemical modifications include phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, "acyclic" .0 nucleotides, 5'-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue incorporation. These chemical modifications, when used in iRNA agents, can help to preserve RNAi activity of the agents in cells and can increase the serum stability of the iRNA agents. In one embodiment, an iRNA agent includes one or more chemical modifications and the sense and antisense sequences of the double-stranded RNA is about 21 nucleotides long. 25 In a preferred embodiment, the first and preferably the first two internucleotide linkages at the 5' end of the antisense and/or sense sequences are modified, preferably by a phosphorothioate. In a preferred embodiment, the first, and preferably the first two, three, or four internucleotide linkages at the 3' end of a sense and/or antisense sequence are modified, preferably by a phosphorothioate. More preferably, the 5' end of both the sense and antisense 30 sequences, and the 3' end of both the sense and antisense sequences are modified as described.

Q

In another aspect, an iRNA agent that mediates the down-regulation of VEGF expression includes one or more chemical modifications that modulate the binding affinity between the sense and the antisense sequences of the iRNA construct. In one embodiment, the invention features an iRNA agent that includes one or more 5 chemical modifications that can modulate the cellular uptake of the iRNA agent. In another embodiment, the invention features an iRNA agent that includes one or more chemical modifications that improve the pharmacokinetics of the iRNA agent. Such chemical modifications include but are not limited to conjugates, such as ligands for cellular receptors, e.g., peptides derived from naturally occurring protein ligands; protein localization sequences; 0 antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate, retinoids and N acetylgalactosamine; polymers, such as polyethyleneglycol (PEG, e.g. PEG 5 and PEG20); phospholipids; polyamines, such as spermine or spermidine; and others. In one embodiment, the iRNA agent includes a duplex molecule selected from the group consisting of AL-DP-4003, AL-DP-4116, AL-DP-4015, AL-DP-4120, AL-DP-4002, AL-DP 5 4115, AL-DP-4014, AL-DP-4119, AL-DP-4094, AL-DP-4118, AL-DP-4107, AL-DP-4122, AL DP-4004, AL-DP-4117, AL-DP-4016, AL-DP-4121, AL-DP-4127, AL-DP-4128, AL-DP-4129, and AL-DP-4055 (see Tables 2 and 3). In one preferred embodiment, the iRNA agent includes a duplex described as AL-DP 4094, which includes the antisense sequence 5'AAGCUCAUCUCUCCUAUGUGCUG 3' (SEQ .0 ID NO:609) and the sense sequence 5' GCACAUAGGAGAGAUGAGCUU 3' (SEQ ID NO:608). In another preferred embodiment, the iRNA agent includes a duplex described as AL-DP 4004, which includes the antisense sequence 5'CUUUCUUUGGUCUGCAUUCACAU 3' (SEQ ID NO:635) and the sense sequence 5' GUGAAUGCAGACCAAAGAAAG 3' (SEQ ID 25 NO:634). In another preferred embodiment, the iRNA agent includes a duplex described as AL-DP 4015, which includes the antisense sequence 5' GUACUCCUGGAAGAUGUCCTT 3' (SEQ ID NO:551) and the sense sequence 5' GGACAUCUUCCAGGAGUACTT 3' (SEQ ID NO:550). a In another preferred embodiment, the iRNA agent includes a duplex described as AL-DP 4055, which includes the antisense sequence 5' UGCAGCCUGGGACCACUUGTT 3' (SEQ ID NO:457) and the sense sequence 5' CAAGUGGUCCCAGGCUGCATT 3' (SEQ ID NO:456). In one embodiment, the antisense sequence of an iRNA agent described herein does not 5 hybridize to an off-target sequence. For example, the antisense sequence can have less than 5, 4, 3, 2, or 1 nucleotides complementary to an off-target sequence. By "off-target" is meant a sequence other than a VEGF nucleotide sequence. In another embodiment, the sense strand is modified to inhibit off-target silencing. The sense strand can include a cholesterol moeity, such as cholesterol attached to the sense strand by 0 a pyrrolidine linker. In another embodiment, the antisense sequence of an iRNA agent described herein can hybridize to a VEGF sequence in a human and a VEGF sequence in a non-human mammal, e.g., a mouse, rat, or monkey. In another aspect, the invention provides a method of delivering an iRNA agent, e.g., an 5 iRNA agent described herein, to the eye of a subject, e.g., a mammalian subject, such as a mouse, a rat, a monkey or a human. In another aspect, the invention provides a method of delivering an iRNA agent to the eye of a subject, e.g., a mammalian subject, such as a mouse, a rat, a monkey or a human. In one embodiment, the iRNA agent can be delivered to a cell or cells in a choroid region .0 of the eye. In one preferred embodiment, the iRNA agent down-regulates expression of the VEGF gene at a target site within the eye. An iRNA agent delivered to the eye, e.g., choroid cells of the eye, can be an unmodified iRNA agent. In one embodiment, the iRNA agent can be stabilized with phosphorothioate linkages. In another embodiment, the 3' end of the sense or antisense sequences, or both, of the iRNA agent 25 can be modified with a cationic group, such as a 3'-abasic cationic modification. The cationic modification can be, e.g., an alkylamino-dT (e.g., a C6 amino-dT), an allylamine, a pyrrolidine, a pthalamido, a hydroxyprolinol, a polyamine, a cationic peptide, or a cationic amino acid on one or more of the terminal nucleotides of the iRNA agent. The modification can be an external or terminal cationic residue. In preferred embodiments, a pyrrolidine cap is attached to the 3' or 5' 30 end of the sense strand, or the 3' end of the antisense strand. I n In one embodiment, the sense or antisense sequence, or both, of the iRNA agent can be modified with a sugar, e.g., a glycoconjugate or alkylglycoside component, e.g., glucose, mannose, 2-deoxy-glucose, or an analog thereof. In another embodiment, the iRNA agent can be conjugated to an enzyme substrate, e.g., a substrate for which the relative enzyme is present in a 5 higher amount, as compared to the enzyme level in other tissues of the body, e.g., in tissues other than the eye. In one embodiment, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the iRNA agent administered to the subject reaches the eye. In a preferred embodiment, between about 30-90%, 40-80% or 50-70% of the iRNA agent administered to the subject reaches the eye. 0 In another aspect, the invention features a composition, e.g., a pharmaceutical composition that includes an iRNA agent of the present invention in a pharmaceutically acceptable carrier or diluent. The iRNA agent can be any agent described herein. In one embodiment, the iRNA agent is chemically modified, such as with any chemical modification described herein. Preferred modified iRNA agents includes those provided in Tables 2-19. 5 In another aspect, the invention features a method for treating or preventing a disease or condition in a subject. The method can include administering to the subject a composition of the invention under conditions suitable for the treatment or prevention of the disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds. In one embodiment, the iRNA agent is administered at or near the site of unwanted VEGF .0 expression, e.g., by a catheter or other placement device (e.g., a retinal pellet or an implant including a porous, non-porous, or gelatinous material). In one embodiment the iRNA agent is administered via an intraocular implant, which can be inserted, for example, into an anterior or posterior chamber of the eye; or into the sclera, transchoroidal space, or an avascularized region exterior to the vitreous. In another embodiment, the implant is positioned over an avascular 25 region, such as on the sclera, so as to allow for transcleral diffusion of the drug to the desired site of treatment, e.g., to the intraocular space and macula of the eye. Furthermore, the site of transcleral diffusion is preferably in proximity to the macula. In another embodiment, an iRNA agent is administered to the eye by injection, e.g., by intraocular, retinal, or subretinal injection. 1 1 In another embodiment, an iRNA agent is administered topically to the eye, such as by a patch or liquid eye drops, or by iontophoresis. Ointments or droppable liquids can be delivered by ocular delivery systems known in the art such as applicators or eye droppers. In one embodiment, an iRNA is delivered at or near a site of neovascularization. 5 In one embodiment, an iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered hourly, daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending upon 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 0 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 area of the target VEGF nucleotide sequences. In another embodiment, an iRNA agent is delivered to the eye at a dosage on the order of about 0.0000 1 mg to about 3 mg per eye, or preferrably about 0.0001-0.001 mg per eye, about 5 0.03- 3.0 mg per eye, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per eye. In another embodiment, an iRNA agent is administered prophylactically such as to prevent or slow the onset of a disorder or condition that affects the eye. For example, an iRNA can be administered to a patient who is susceptible to or otherwise at risk for a neovascular disorder. .0 In one embodiment one eye of a human is treated with an iRNA agent described herein, and in another embodiment, both eyes of a human are treated. In another aspect, a method of inhibiting VEGF expression is provided. One such method includes administering an effective amount of an iRNA agent of the present invention. In another aspect, a method of treating adult onset macular degeneration is provided. The 25 method includes administering a therapeutically effective amount of an iRNA agent of the present invention. In one embodiment, a human has been diagnosed with dry adult macular degeneration (AMD), and in another embodiment the human has been diagnosed with wet AMD. In one embodiment, a human treated with an iRNA agent described herein is over the age 30 of 50, e.g., between the ages of 75 and 80, and the human has been diagnosed with adult onset 1) macular degeneration. In another embodiment, a human treated with an iRNA agent described herein is between the ages of 30-50, and the human has been diagnosed with late onset macular degeneration. In another embodiment, a human treated with an iRNA agent described herein is between the ages of 5-20, and the human has been diagnosed with middle onset macular 5 degeneration. In another embodiment, a human treated with an iRNA agent described herein is 7 years old or younger, and the human has been diagnosed with early onset macular degeneration. In one aspect, methods of treating any disease or disorder characterized by unwanted VEGF expression are provided. Particularly preferred embodiments include the treatment of 0 disorders of the eye or retina, which are characterized by unwanted VEGF expression. The disease or disorder can be a diabetic retinopathy, neovascular glaucoma, a tumor or metastic cancer (e.g., colon or breast cancer), a pulmonary disease (e.g., asthma or bronchitis), rheumatoid arthritis, or psoriases. Other angiogenic disorders can be treated by the methods featured in the invention. 5 In another aspect, the invention features a kit containing an iRNA agent of the invention. The iRNA agent of the kit can be chemically modified and can be useful for modulating the expression of a VEGF target gene in a cell, tissue or organism. In one embodiment, the kit contains more than one iRNA agent of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same .0 meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features and advantages of the invention will be apparent from the 25 accompanying drawings and description, and from the claims. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. In case of conflict, the present specification, including definitions, will control.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is the nucleotide sequence of the mRNA of the 121 amino acid form of vascular endothelial growth factor, VEGF121. The first nucleotide of the initiator codon is nucleotide 1. The signal peptide is from nucleotide 1 through 78. 5 FIGURE 2 is a graphical representation of a comparative analysis of the activities of single- and double-overhang siRNAs in in vitro assays in HeLa cells. Solid lines with filled symbols represent the single-overhang siRNA, solid lines with open symbols represent the double-overhang siRNAs; dashed lines represent the control siRNAs. The control siRNA hVEGF is described in Reich et al. (Mol. Vis. 9:210, 2003); the control siRNA hrmVEGF is 0 described in Filleur et al. (Cancer Res. 63:3919, 2003). "L2000" refers to Lipofectamine 2000 reagent. hVEGF expression (y-axis) refers to endogenous VEGF expression. FIGURE 3 is a graphical representation of a comparative analysis of the activities of single- and double-overhang siRNAs in ARPE-19 cells. Solid lines with filled symbols represent the single-overhang siRNA; solid lines with open symbols represent the double-overhang 5 siRNAs; dashed lines represent the control siRNAs. The control siRNA hVEGF is described in Reich et al. (Mol. Vis. 9:210, 2003); the control siRNA hrmVEGF is described in Filleur et al. (supra). "L2000" refers to Lipofectamine 2000 reagent. hVEGF expression (y-axis) refers to endogenous VEGF expression. FIGURE 4 is a graphical representation of a comparative analysis of the siRNAs activities .0 in HeLa cells of single-overhang siRNAs with their analogous blunt siRNAs in which the number of base-paired nucleotides is 21. The control siRNA hVEGF is described in Reich et al. (Mol. Vis. 9:210, 2003); the control siRNA hrmVEGF is described in Filleur et al. (supra). "L2000" refers to Lipofectamine 2000 reagent. hVEGF expression (y-axis) refers to endogenous VEGF expression. 25 FIGURE 5 is a graphical representation of a comparative analysis of the siRNAs activities in HeLa cells of double-overhang siRNAs with their analogous blunt siRNAs in which the number of base-paired nucleotides is 19. The control siRNA hVEGF is described in Reich et al. (supra); the control siRNA hrmVEGF is described in Filleur et al. (supra). "L2000" refers to Lipofectamine 2000 reagent. hVEGF expression (y-axis) refers to endogenous VEGF 30 expression. 1A FIGURE 6A is a graphical representation of the activities of single-overhang and double overhang siRNAs targeting ORF 319 (SEQ ID NO:320) (AL-DP-4002 and AL-DP-4014, respectively) and ORF 343 (SEQ ID NO:344) (AL-DP-4094 and AL-DP-4107, respectively) in cells under normal oxygen (normoxia, 20% oxygen). 5 FIGURE 6B is a graphical representation of the activities of single-overhang and double overhang siRNAs targeting ORF 319 (SEQ ID NO:320) (AL-DP-4002 and AL-DP-4014, respectively) and ORF 343 (SEQ ID NO:344) (AL-DP-4094 and AL-DP-4107, respectively) in cells under hypoxic conditions (1% oxygen). FIGURE 6C is a graphical representation of the activities of single-overhang and double 0 overhang siRNAs targeting ORF 319 (SEQ ID NO:320) (AL-DP-4002 and AL-DP-4014, respectively) and ORF 343 (SEQ ID NO:344) (AL-DP-4094 and AL-DP-4107, respectively) in cells under hypoxic conditions (130 pM defoxamine). FIGURE 7 is a graphical representation of the comparative activities of double-overhang (AL-DP-4014) unmodified siRNA and phosphorothioate-modified (AL-DP-4127, AL-DP-4128, 5 AL-DP-4129) siRNAs targeting ORF 319 (SEQ ID NO:320) in HeLa cells. The control siRNA hVEGF is described in Reich et al. (supra); the control siRNA hrmVEGF is described in Filleur et al. (supra). "L2000" refers to Lipofectamine 2000 reagent. hVEGF expression (y-axis) refers to endogenous VEGF expression. FIGURE 8A is a graphical representation of the activities of siRNAs targeting ORF 319 .0 (SEQ ID NO:320) (AL-DP-4014 and AL-DP-4127) and a mutated version AL-DP-4140 (Table 5) in cells under normal oxygen conditions (normoxia, 20% oxygen). The control siRNA Cand5 is identical to the hVEGF control of FIGURE. 7 and is described in Reich et al. (supra). "L2000" refers to Lipofectamine 2000 reagent. VEGF expression (y-axis) refers to endogenous VEGF expression. 25 FIGURE 8B is a graphical representation of the activities of siRNAs targeting ORF 319 (SEQ ID NO:320) (AL-DP-4014 and AL-DP-4127) and a mutated version AL-DP-4140 (Table 5) in cells under normal or hypoxic conditions (hypoxia, 1% Oxygen). The control siRNAs are as described for FIGURE. 8A. FIGURES 9A-9E are graphical representations of the activities of siRNAs having the 30 sequence of AL-DP-4094 but differing in the inclusion of nucleotide modifications (see Table 4). 1 r, The control siRNA "Acuity" is identical to the Cand5 control of FIGURE. 8A and the hVEGF control of FIGURE. 7. The "Filleur" control siRNA is the equivalent of the hrmVEGF control siRNA of FIGURE. 7. FIGURE 10 is a graphical representation of siRNA silencing activity in vitro in HeLa 5 cells. FIGURE 11 is an RP-HPLC scan of AL-DP-4094 siRNA following incubation in human serum. FIGURE 12 is a summary of AL-DP-4094 fragment mapping as determined by LC/MS. The analysis was performed following incubation of the siRNA in human serum. 0 FIGURES 13-29 are graphs of silencing activity of 2'-O-methyl and/or 2'-flouro modified siRNAs in vitro in HeLa cells (Table 6). FIGURE 30 are graphs of silencing activity of alternating 2'-O-methyl and 2'-flouro modified siRNAs in vitro in HeLa cells (Table 7). FIGURES 31-33 are graphs of silencing activity of cholesterol and colonic conjugated 5 siRNAs in vitro in HeLa cells (Table 8). FIGURE 34 is a graph of silencing activity of naproxen conjugated siRNAs in vitro in HeLa cells (Table 9). FIGURE 35 is a graph of silencing activity of biotin conjugated siRNAs in vitro in HeLa cells (Table 10). 20 FIGURE 36 is a graph of silencing activity of 5'-retinal conjugated siRNAs in vitro in HeLa cells (Table 11). FIGURE 37 is a graph of silencing activity of ribo-diflourotoluyl modified siRNAs in vitro in HeLa cells (Table 13).

FIGURE 38 is a graph of silencing activity of 2'-arafluoro-2'deoxy-nucleoside modified siRNAs in vitro in HeLa cells (Table 14). FIGURE 39 5'-O-DMTr-2'-deoxy-2'-fluoro A, C, G and U CPG supports for oligonucleotide synthesis. These supports were used for syntheses of selected sequences listed 5 Tables 6 and 7. FIGURE 40 Cholesterol and 5-cholanic (or cholanic) acid conjugate building blocks for conjugation to oligonucleotides. These building blocks were used for syntheses of selected sequences listed in Table 8. FIGURE 41 5eC and 5MeU RNA building blocks for oligonucleotide synthesis. These 0 building blocks were used for syntheses of selected sequences listed in Table 8. FIGURE 42. Naproxen - trans-4- hydroxy-L-prolinol and naproxen-serinol building blocks for conjugation to oligonucleotides. These building blocks were used for syntheses of selected sequences listed in Table 9. FIGURE 43 Biotin - trans-4- hydroxy-L-prolinol and biotin-serinol building blocks for 5 conjugation to oligonucleotides. These building blocks were used for syntheses of selected sequences listed in Table 10. FIGURE 44 Building blocks for post-synthetic conjugation - Oxime approach. These building blocks were/are used for syntheses of selected sequences listed in Table 11. FIGURE 45 Building blocks for post-synthetic conjugation - Active ester approach. 20 These building blocks were used for syntheses of selected sequences listed in Table 12. FIGURE 46 DFT amidite and CPG for oligonucleotide synthesis. These building blocks were used for syntheses of selected sequences listed in Table 13. FIGURE 47 2'-Deoxy-2'-araf amidite for oligonucleotide synthesis. These building blocks were used for syntheses of selected sequences listed in Table 14. 1-7 FIGURE 48 P-methylphosphonamidite of ribo 5MeU and ribo C(N Ac). These building blocks were used for syntheses of selected sequences listed in Table 15. FIGURE 49 C5-aminoallyl U amidite. These building blocks were used for syntheses of selected sequences listed in Table 16. 5 FIGURE 50 Thiocholesterol conjugate building blocks. BRIEF DESCRIPTION OF THE TABLES Table 1 provides the sequences in the VEGF gene that are targeted by the agents of the present invention. These sequence can also be the sense strand of some of the iRNA agents of 0 the present invention. Table 2 provides 123 iRNA duplexes that target the VEGF gene, the target sequence in the VEGF gene and activity data that is described in the Examples. Table 3 provides iRNA duplexes that are modified to contain phosphorothioate stabilizations and activity data that is described in the Examples. 5 Table 4 provides iRNA duplexes based on the AL-DP-4094 duplex that are modified for stabilization and activity data that is described in the Examples. Table 5 provides iRNA duplexes activity data in HeLa cells for several iRNA agents of the present invention. Table 6 provides iRNA agents with activity data in HeLa cells for agents containing one 20 or more phosporothioate, 2'-O-methyl and 2'-fluoro modifications. Table 7 provides iRNA agents with activity data in HeLa cells for agents containing alternating 2'-O-methyl and 2'-fluoro modifications. Table 8 A and B provides iRNA agents with activity data in HeLa cells for agents containing cholesterol or cholanic acid conjugates. 25 Table 9 provides iRNA agents with activity data in HeLa cells for agents containing naproxen conjugates. Table 10 provides iRNA agents with activity data in HeLa cells for agents containing biotin conjugates. 1 2 Table 11 provides iRNA agents containing aldehydes, retinal and other retinoid conjugates. Table 12 provides iRNA agents containing polyethylene glycol conjugates. Table 13 provides iRNA agents with activity data in HeLa cells for agents containing 5 ribo-difluorotoluyl modifications. Table 14 provides iRNA agents with activity data in HeLa cells for agents containing 2' arafluoro-2'-deoxy-nucleoside modifications. Table 15 provides iRNA agents containing methylphosphonate modifications. Table 16 provides iRNA agents containing C-5 allyamino modifications. 0 Table 17 provides iRNA agents containing a variety and combinations of the modifications as noted in the Table. Table 18 provides physical characterization of iRNA agents containing a variety and combinations of the modifications as noted in the Table. 5 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. .0 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 of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi. Described herein are methods for preparing and administering these 21-23 nt fragments, and 25 other iRNAs agents, and their use for specifically inactivating gene function. The use of iRNA agents (or recombinantly produced or chemically synthesized oligonucleotides of the 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 30 frequently deleterious, siRNAs do not trigger the interferon response, at least not to an extent that is deleterious to the cell and host. In particular, the length of the sense and antisense 10 sequences in an iRNA 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 iRNA agents (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 5 discrete species of iRNA agent can be used to selectively target one allele of a target gene, e.g., in a subject heterozygous for the allele. The target-complementary sequence (the antisense sequence) of an iRNA agent, such as an iRNA duplex, can have a 5' phosphate and ATP may be utilized to maintain the 5'- phosphate moiety on the siRNA (Nykanen et al., Cell 107:309, 2001); however, iRNA agents lacking a 5' 0 phosphate have been shown to be active when introduced exogenously, suggesting that 5' phosphorylation of siRNA constructs may occur in vivo. Vascular endothelial growth factor (VEGF) VEGF, also known as vascular permeability factor, is an angiogenic growth factor. VEGF is a homodimeric 45 kDa glycoprotein that exists in at least three different isoforms. VEGF isoforms are expressed in endothelial cells. The 5 VEGF gene contains 8 exons that express a 189-amino acid protein isoform. A 165-amino acid isoform lacks the residues encoded by exon 6, whereas a 121-amino acid isoform lacks the residues encoded by exons 6 and 7. VEGF145 is an isoform predicted to contain 145 amino acids and to lack exon 7. VEGF can act on endothelial cells by binding to an endothelial tyrosine kinase receptor, .0 such as Flt-1 (VEGFR-1) or KDR/flk-1 (VEGFR-2). VEGFR-2 is expressed in endothelial cells and is involved in endothelial cell differentiation and vasculogenesis. A third receptor, VEGFR-3 has been implicated in lymphogenesis. The various isoforms have different biologic activities and clinical implications. For example, VEGF145 induces angiogenesis and like VEGF189 (but unlike VEGF165) VEGF145 25 binds efficiently to the extracellular matrix by a mechanism that is not dependent on extracellular matrix-associated heparin sulfates. The mRNA corresponding to the coding sequence of human VEGF121 (Genbank Accession Number AF214570, SEQ ID NO:1) is shown in FIG. 1. VEGF displays activity as an endothelial cell mitogen and chemoattractant in vitro and induces vascular permeability and angiogenesis in vivo. VEGF is secreted by a wide variety of cancer cell types 30 and promotes the growth of tumors by inducing the development of tumor-associated vasculature. Inhibition of VEGF function has been shown to limit both the growth of primary experimental tumors as well as the incidence of metastases in immunocompromised mice. VEGF is also expressed at abnormally high levels in inflammatory diseases such as rheumatoid arthritis and psoriasis, and is involved in the inflammation, airway and vascular remodeling that 5 occurs during asthmatic episodes. Elevated VEGF expression is also correlated with several forms of ocular neovascularization that often lead to severe vision loss, including diabetic retinopathy, retinopathy of prematurity, and macular degeneration. iRNA Agents An "RNA agent," as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate. Preferred examples include those which have greater resistance to 0 nuclease degradation than do unmodified RNAs. Preferred examples include those which have a 2' sugar modification, a modification in a single strand overhang, preferably a 3' single strand overhang, or, particularly if single stranded, a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group. An "iRNA agent," as used herein, is an RNA agent which can, or which can be cleaved 5 into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, .0 e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group. 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 25 mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term "ribonucleotide" or "nucleotide," herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA 30 agent is or includes a region which is at least partially, and in some embodiments fully, I1 complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA. 5 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 0 preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5' and/or 3' terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double strand character of the molecule. Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links 5 two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3'- or 5'-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that .0 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., Nature 409:363-366, 2001)) and enter a RISC (RNAi-induced silencing complex); and molecules that are sufficiently short that they do not 25 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 30 or single strand agent, that is sufficiently short that it does not induce a deleterious interferon 1) 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. 5 Each strand of a sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3' overhangs, of 2- 3 nucleotides. 0 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 preferred embodiments single strand iRNA agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5' 5 phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-0-5'); 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)-0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(HO)(O)P-O-(HO)(O)P-O P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap .0 structure (N-0-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-0-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'-alpha thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', 25 (HO)(NH2)(O)P-0-5'), 5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)-O-5'-). (These modifications can also be used with the antisense strand of a double stranded iRNA.) A single strand iRNA agent should be sufficiently long that it can enter the RISC and 30 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 50 nucleotides 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, 20, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 5 100, or 50, in length. Preferred 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 of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length. A "double stranded (ds) iRNA agent" as used herein, is an iRNA agent which includes 0 more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure. Other suitable modifications to a sugar, base, or backbone of an iRNA agent are described in co-owned PCT Application No. PCT/US2004/01193, filed January 16, 2004. An iRNA agent can include a non-naturally occurring base, such as the bases described in co-owned 5 PCT Application No. PCT/US2004/011822, filed April 16, 2004. An iRNA agent can include a non-naturally occurring sugar, such as a non-carbohydrate cyclic carrier molecule. Exemplary features of non-naturally occurring sugars for use in iRNA agents are described in co-owned PCT Application No. PCT/US2004/11829 filed April 16, 2003. An iRNA agent can include an internucleotide linkage (e.g., the chiral phosphorothioate .0 linkage) useful for increasing nuclease resistance. In addition, or in the alternative, an iRNA agent can include a ribose mimic for increased nuclease resistance. Exemplary internucleotide linkages and ribose mimics for increased nuclease resistance are described in co-owned PCT Application No. PCT/US2004/07070 filed on March 8, 2004. An iRNA agent can have a ZXY structure, such as is described in co-owned PCT 25 Application No. PCT/US2004/07070 filed on March 8, 2004. An iRNA agent can be complexed with an amphipathic moiety. Exemplary amphipathic moieties for use with iRNA agents are described in co-owned PCT Application No. PCT/US2004/07070 filed on March 8, 2004. In another embodiment, the iRNA agent can be complexed to a delivery agent that 30 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); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a 5 lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type. iRNA agents complexed to a delivery agent are described in co-owned PCT Application No. PCT/US2004/07070 filed on March 8, 2004. An iRNA agent can have non-canonical pairings, such as between the sense and antisense sequences of the iRNA duplex. Exemplary features of non-canonical iRNA agents are described 0 in co-owned PCT Application No. PCT/US2004/07070 filed on March 8, 2004. Many of these types of modifications are provided in the Examples and are described in Tables 3-18. Design of iRNA 5 The present invention is based on a gene walk of the VEGF gene to identify active iRNA agents that can be used to reduce the level of VEGF mRNA in a cell. Not all potential iRNA agent sequences in the VEGF gene are active, many of which also having significant off-target effects. The present invention advances the art by selecting those sequences which are active and do not have significant off-target effects. Further, the sequence chosen for the iRNA agents .0 of the present invention are conserved amongst multiple species allowing one to use a single agent for animal and toxicological studies as well as using it for therapeutic purposes in humans. Based on these results, the invention specifically provides an iRNA agent that can be used in treating VEGF mediated disorders, particularly in the eye such as AMD, in isolated form and as a pharmaceutical composition described below. Such agents will include a sense strand 25 having at least 15 or more contiguous nucleotides that are complementary to the VEGF gene and an antisense strand having at least 15 or more contiguous nucleotides that are complementary to the sense strand sequence. Particularly useful are iRNA agents that have a sense strand that comprises, consist essentially of or consists of a nucleotide sequence provided in Table 1, such as those agents proved in Table 2, or any of the modifications provided in Tables 3-18.

Candidate iRNA agents can be designed by performing, as done herein, a gene walk analysis of the VEGF gene that will serve as the iRNA target. Overlapping, adjacent, or closely spaced candidate agents corresponding to all or some of the transcribed region can be generated and tested. Each of the iRNA agents can be tested and evaluated for the ability to down regulate 5 the target gene expression (see below, "Evaluation of Candidate iRNA agents"). Preferably, the iRNA agents of the present invention are based on and comprise at least 15 or more contiguous nucleotides from one of the iRNA agents shown to be active in Table 2, or the modified sequences provided in Tables 3-18. In such agents, the agent can comprise, consist of or consist essentially of the entire sequence provided in the Table or can comprise 15 or more 0 contiguous residues along with additional nucleotides from contiguous regions of the target gene. An iRNA agent can be rationally designed based on sequence information and desired characteristics and the information of the target sequence provided in Table 1. For example, an iRNA agent can be designed according to the relative melting temperature of the candidate duplex. Generally, the duplex should have a lower melting temperature at the 5' end of the 5 antisense strand than at the 3' end of the antisense strand. Accordingly, the present invention provides iRNA agents comprising a sense strand and antisense strand each comprising a sequence of at least 15, 16, 17, 18, 19, 20, 21 or 23 nucleotides which is essentially identical to one of the agents provided in Table 1 or 2. The antisense strand of an iRNA agent should be equal to or at least, 15, 16 17, 18, 19, .0 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 50, 40, or 30, nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length. Exemplified iRNA agents include those that comprise 15 or more nucleotides from one of the agents in Table 2 (or are complementary to the target sequence provided in Table 1) but are not longer than 25 nucleotides in length. 25 The sense strand of an iRNA agent should be equal to or at least 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. It should be equal to or less than 50, 40, or 30 nucleotides in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides in length. Exemplified iRNA agents include those that comprise 15 or more nucleotides from one of the agents in Table 2 (or the target sequence in Table 2) but are not longer than 25 nucleotides in 30 length.

The double stranded portion of an iRNA agent should be equal to or at least, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50 nucleotide pairs in length. It should be equal to or less than 50, 40, or 30 nucleotides pairs in length. Preferred ranges are 15-30, 17 to 25, 19 to 23, and 19 to 21 nucleotides pairs in length. 5 The agents provided in Table 2 are 23 nucleotides in length for each strand. The iRNA agents contain a 21 nucleotide double stranded region with a 2 nucleotide overhang on each of the 3' ends of the agent. These agents can be modified as described herein to obtain equivalent agents comprising at least a portion of these sequences (15 or more contiguous nucleotides) and or modifications to the oligonucleotide bases and linkages. Particularly preferred are the 0 modification and agents provided in Tables 3-18. Generally, the iRNA agents of the instant invention include a region of sufficient complementarity to the VEGF gene and are of sufficient length in terms of nucleotides that the iRNA agent, or a fragment thereof, can mediate down regulation of the VEGF gene. The antisense strands of the iRNA agents of the present invention are preferably fully complementary 5 to the mRNA sequences of VEGF gene. However, it is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of a VEGF mRNA. Therefore, the iRNA agents of the instant invention include agents comprising a sense .0 strand and antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical, as defined below, to one of the sequences of the VEGF gene, such as those agent provided in Table 2, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to inhibit VEGF expression. These agents will therefore possess 25 at least 15 or more nucleotides identical to the VEGF gene but 1, 2 or 3 base mismatches with respect to either the VEGF mRNA sequence or between the sense and antisense strand are introduced. Mismatches to the target VEGF mRNA sequence, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of a 5' and/or 3' terminus, most preferably 30 within 6, 5, 4, or 3 nucleotides of the 5'-terminus of the sense strand or the 3'-terminus of the 1 7 antisense strand. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double stranded character of the molecule. It is preferred that the sense and antisense strands be chosen such that the iRNA agent includes a single strand or unpaired region at one or both ends of the molecule, such as those 5 exemplified in Table 2 (as well as Tables 3-18). Thus, an iRNA agent contains sense and antisense strands, preferably paired to contain an overhang, e.g., one or two 5' or 3' overhangs but preferably a 3' overhang of 2-3 nucleotides. Most embodiments will have a 3' overhang. Preferred siRNA agents will have single-stranded overhangs, preferably 3' overhangs, of 1 to 4, or preferably 2 or 3 nucleotides, in length, on one or both ends of the iRNA agent. The 0 overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5'-ends are preferably phosphorylated. Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the siRNA agent range discussed above. Embodiments in which the two strands of the siRNA agent are linked, e.g., covalently linked are 5 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. Synthesis of iRNA Agents Oligonucleotides (e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides) can be synthesized using protocols known .0 in the art, for example as described in Caruthers et al., Methods in Enzymology 211:3, 1992; Thompson et al., International PCT Publication No. WO 99/54459; Wincott et al., Nucleic Acids Res. 23:2677, 1995; Wincott et al., Methods Mol. Bio. 74:59, 1997; Brennan et al., Biotechnol. Bioeng. 61:33, 1998; and Brennan, U. S. Pat. No. 6,001,311. All of these references are incorporated herein by reference. The synthesis of oligonucleotides makes use of common 25 nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. The method of synthesis used for RNA including certain iRNA agents of the invention follows the procedure as described in Usman et al., J. Chem. Soc. 109:7845, 1987; Scaringe et al., Nucleic Acids Res. 18:5433, 1990; Wincott et al., Nucleic Acids Res. 23:2677, 1995; and 30 Wincott et al., Methods Mol. Bio. 74:59, 1997; and makes use of common nucleic acid protecting IQ2 and coupling groups, such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. Detailed descriptions of a variety of synthetic methods to produce modified iRNA agents are provided in the Examples. Alternatively, the nucleic acid molecules of the present invention can be synthesized 5 separately and joined together post-synthetically, for example, by ligation (Moore et al., Science 256:9923, 1992; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., Nucleic Acids Res. 19:4247, 1991; Bellon et al., Nucleosides & Nucleotides 16:951, 1997; Bellon et al., Bioconjugate 8:204, 1997), or by hybridization following synthesis and/or deprotection. 0 An iRNA agent can also be assembled from two distinct nucleic acid sequences or fragments wherein one fragment includes the sense region and the second fragment includes the antisense region of the iRNA agent. iRNA agents can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'- C-allyl, 2'-fluoro, diflurortoluyl, 5 5 allyamino-pyrimidines, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren, Trends in Biochem. Sci. 17:34, 1992). iRNA constructs can be purified by gel electrophoresis using general methods or can be purified by high pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality of which is hereby incorporated herein by reference) and re-suspended in water. .0 In another aspect of the invention, iRNA agents can be expressed from transcription units inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. iRNA agent-expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors capable of expressing the iRNA agents can be delivered as described herein, and persist in target cells. 25 Alternatively, viral vectors can be used that provide for transient expression of iRNA agents. Evaluating iRNA agents Any of the iRNA agents described herein can be evaluated and modified as follows. An iRNA agent may be susceptible to cleavage by an endonuclease or exonuclease, such 30 as when the iRNA agent is introduced into the body of a subject. Methods can be used to 1)0 determine sites of cleavage, e.g., endo- and exonucleolytic cleavage on an iRNA agent and to determine the mechanism of cleavage. An iRNA agent can be modified to inhibit such cleavage. A dsRNA, e.g., an iRNA agent, can be evaluated to identify sites that are susceptible to modification, particularly cleavage, e.g., cleavage by a component found in the body of a subject. 5 The component can be specific for a particular area of the body, such as a particular tissue, organ, or bodily fluid (e.g., blood, plasma, or serum). Sites in an iRNA agent that are susceptible to cleavage, either by endonucleolytic or exonucleolytic cleavage, in certain areas of the body, may be resistant to cleavage in other areas of the body. A method for evaluating an iRNA agent can include: (1) determining the point or points 0 at which a substance present in the body of a subject, and preferably a component present in a compartment of the body into which a therapeutic dsRNA is to be introduced (this includes compartments into which the therapeutic is directly introduced, e.g., the circulation, as well as in compartments to which the therapeutic is eventually targeted, e.g, the liver or kidney; in some cases, e.g, the eye, the two are the same), cleaves a dsRNA, e.g., an iRNA agent; and (2) 5 identifying one or more points of cleavage, e.g., endonucleolytic, exonucleolytic, or both, in the dsRNA. Optionally, the method further includes providing an RNA (e.g., an iRNA agent) modified to inhibit cleavage at such sites. The steps described above can be accomplished by using one or more of the following assays: .0 (i) (a) contacting a candidate dsRNA, e.g., an iRNA agent, with a test agent (e.g., a biological agent), (b) using a size-based assay, e.g., gel electrophoresis to determine if the iRNA agent is cleaved. In a preferred embodiment a time course is taken and a number of samples incubated for different times are applied to the size-based assay. In preferred embodiments, the 25 candidate dsRNA is not labeled. The method can be a "stains all" method. (ii) (a) supplying a candidate dsRNA, e.g., an iRNA agent, which is radiolabeled; (b) contacting the candidate dsRNA with a test agent, (c) using a size-based assay, e.g., gel electrophoresis to determine if the iRNA agent is cleaved. In a preferred embodiment, a time course is taken where a number of samples 30 are incubated for different times and applied to the size-based assay. In preferred embodiments the determination is made under conditions that allow determination of the number of nucleotides present in a fragment. For example, an incubated sample is run on a gel having markers that allow assignment of the length of cleavage products. The gel can include a standard that is a "ladder" digestion. Either the sense or antisense strand can be labeled. Preferably only 5 one strand is labeled in a particular experiment. The label can be incorporated at the 5' end, 3' end, or at an internal position. Length of a fragment (and thus the point of cleavage) can be determined from the size of the fragment based on the ladder and mapping using a site-specific endonuclease such as RNAse T1. (iii) Fragments produced by any method, e.g., one described herein, e.g., one of those 0 above, can be analyzed by mass spectrometry. Following contacting the iRNA with the test agent, the iRNA can be purified (e.g., partially purified), such as by phenol-chloroform extraction followed by precipitation. Liquid chromatography can then be used to separate the fragments and mass spectrometry can be used to determine the mass of each fragment. This allows determination of the mechanism of cleavage, e.g., if by direct phosphate cleavage, such as 5 by 5' or 3' exonuclease cleavage, or mediated by the 2'OH via formation of a cyclic phosphate. In another embodiment, the information relating to a site of cleavage is used to select a backbone atom, a sugar or a base, for modification, e.g., a modification to decrease cleavage. Exemplary modifications include modifications that inhibit endonucleolytic degradation, including the modifications described herein. Particularly favored modifications include: .0 2' modification, e.g., a 2'-O-methylated nucleotide or 2'-deoxy nucleotide (e.g., 2'deoxy cytodine), or a 2'-fluoro, difluorotoluyl, 5-Me-2'-pyrimidines, 5-allyamino-pyrimidines, 2'-0 methoxyethyl, 2'-hydroxy, or 2'-ara-fluoro nucleotide, or a locked nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleic acid (CeNA). In one embodiment, the 2' modification is on the uridine of at least one 5'-uridine-adenine-3' (5'-UA 25 3') dinucleotide, at least one 5'-uridine-guanine-3' (5'-UG-3') dinucleotide, at least one 5' uridine-uridine-3' (5'-UU-3') dinucleotide, or at least one 5'-uridine-cytidine-3' (5'-UC-3') dinucleotide, or on the cytidine of at least one 5'-cytidine-adenine-3' (5'-CA-3') dinucleotide, at least one 5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, or at least one 5'-cytidine-uridine-3' (5'-CU-3') dinucleotide. The 2' modification can also be applied to all the pyrimidines in an 30 iRNA agent. In one preferred embodiment, the 2' modification is a 2'OMe modification on the 21 sense strand of an iRNA agent. In a more preferred embodiment the 2' modification is a 2' fluoro modification, and the 2' fluoro is on the sense or antisense strand or on both strands. Modification of the backbone, e.g., with the replacement of an 0 with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification can be used to inhibit 5 endonuclease activity. In some embodiments, an iRNA agent has been modified by replacing one or more ribonucleotides with deoxyribonucleotides. Preferably, adjacent deoxyribonucleotides are joined by phosphorothioate linkages, and the iRNA agent does not include more than four consecutive deoxyribonucleotides on the sense or the antisense strands. Replacement of the U with a C5 amino linker; replacement of an A with a G (sequence changes 0 are preferred to be located on the sense strand and not the antisense strand); or modification of the sugar at the 2', 6', 7', or 8' position can also inhibit endonuclease cleavage of the iRNA agent. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. 5 Exemplary modifications also include those that inhibit degradation by exonucleases. Examples of modifications that inhibit exonucleolytic degradation can be found herein. In one embodiment, an iRNA agent includes a phosphorothioate linkage or P-alkyl modification in the linkages between one or more of the terminal nucleotides of an iRNA agent. In another embodiment, one or more terminal nucleotides of an iRNA agent include a sugar modification, .0 e.g., a 2' or 3' sugar modification. Exemplary sugar modifications include, for example, a 2'-0 methylated nucleotide, 2'-deoxy nucleotide (e.g., deoxy-cytodine), 2'-deoxy-2'-fluoro (2'-F) nucleotide, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-N methylacetamido (2'-O-NMA), 2'-O-dimethylaminoethlyoxyethyl (2'-DMAEOE), 2'-0 dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-AP), 2'-hydroxy 25 nucleotide, or a 2'-ara-fluoro nucleotide, or a locked nucleic acid (LNA), extended nucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleic acid (CeNA). A 2' modification is preferably 2'OMe, more preferably, 2'fluoro. The modifications described to inhibit exonucleolytic cleavage can be combined onto a single iRNA agent. For example, in one embodiment, at least one terminal nucleotide of an 30 iRNA agent has a phosphorothioate linkage and a 2' sugar modification, e.g., a 2'F or 2'OMe modification. In another embodiment, at least one terminal nucleotide of an iRNA agent has a 5' Me-pyrimidine and a 2' sugar modification, e.g., a 2'F or 2'OMe modification. To inhibit exonuclease cleavage, an iRNA agent can include a nucleobase modification, such as a cationic modification, such as a 3'-abasic cationic modification. The cationic 5 modification can be, e.g., an alkylamino-dT (e.g., a C6 amino-dT), an allylamino conjugate, a pyrrolidine conjugate, a pthalamido or a hydroxyprolinol conjugate, on one or more of the terminal nucleotides of the iRNA agent. An alkylamino-dT conjugate is preferably attached to the 3' end of the sense or antisense strand of an iRNA agent. A pyrrolidine linker is preferably attached to the 3' or 5' end of the sense strand, or the 3' end of the antisense strand. An allyl 0 amine uridine is preferably on the 3' or 5' end of the sense strand, and not on the 5' end of the antisense strand. In another embodiment, the iRNA agent includes a conjugate on one or more of the terminal nucleotides of the iRNA agent. The conjugate can be, for example, a lipophile, a terpene, a protein binding agent, a vitamin, a carbohydrate, a retiniod, or a peptide. For example, 5 the conjugate can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, cholesterol, retinoids, PEG, or a C5 pyrimidine linker. In other embodiments, the conjugates are glyceride lipid conjugates (e.g. a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols. Preferably, conjugates are on the 3' end of the antisense strand, or on the 5' or 3' end of the sense strand, and preferably the conjugates are not .0 on the 3' end of the antisense strand and on the 3' end of the sense strand. In one embodiment, the conjugate is naproxen, and the conjugate is preferably on the 5' or 3' end of the sense or antisense strands. In one embodiment, the conjugate is cholesterol, and the conjugate is preferably on the 5' or 3' end of the sense strand and preferably not present on the antisense strand. In some embodiments, the cholesterol is conjugated to the iRNA agent by a 25 pyrrolidine linker, or serinol linker, aminooxy, or hydroxyprolinol linker. In other embodiments, the conjugate is a dU-cholesterol, or cholesterol is conjugated to the iRNA agent by a disulfide linkage. In another embodiment, the conjugate is cholanic acid, and the cholanic acid is attached to the 5' or 3' end of the sense strand, or the 3' end of the antisense strand. In one embodiment, the cholanic acid is attached to the 3' end of the sense strand and the 3' end of the antisense 30 strand. In another embodiment, the conjugate is PEG5, PEG20, naproxen or retinal.

In another embodiment, one or more terminal nucleotides have a 2'-5' linkage. Preferably, a 2'-5' linkage occurs on the sense strand, e.g., the 5' end of the sense strand. In one embodiment, the iRNA agent includes an L-sugar, preferably at the 5' or 3' end of the sense strand. 5 In one embodiment, the iRNA agent includes a methylphosphonate at one or more terminal nucleotides to enhance exonuclease resistance, e.g., at the 3' end of the sense or antisense strands of the iRNA agent. In one embodiment, an iRNA agent has been modified by replacing one or more ribonucleotides with deoxyribonucleotides. Preferably, adjacent deoxyribonucleotides are joined 0 by phosphorothioate linkages, and the iRNA agent does not include more than four consecutive deoxyribonucleotides on the sense or the antisense strands. In some embodiments, an iRNA agent having increased stability in cells and biological samples includes a difluorotoluyl (DFT) modification, e.g., 2,4-difluorotoluyl uracil, or a guanidine to inosine substitution. 5 The methods described can be used to select and/or optimize a therapeutic dsRNA, e.g., iRNA agent. dsRNAs, e.g., iRNA agents, made by a method described herein are within the invention. The methods can be used to evaluate a candidate dsRNA, e.g., a candidate iRNA agent, which is unmodified or which includes a modification, e.g., a modification that inhibits .0 degradation, targets the dsRNA molecule, or modulates hybridization. Such modifications are described herein. A cleavage assay can be combined with an assay to determine the ability of a modified or non-modified candidate to silence the target. For example, one might (optionally) test a candidate to evaluate its ability to silence a target (or off-target sequence), evaluate its susceptibility to cleavage, modify it (e.g., as described herein, e.g., to inhibit degradation) to 25 produce a modified candidate, and test the modified candidate for one or both of the ability to silence and the ability to resist degradation. The procedure can be repeated. Modifications can be introduced one at a time or in groups. It will often be convenient to use a cell-based method to monitor the ability to silence a target RNA. This can be followed by a different method, e.g, a whole animal method, to confirm activity.

The invention includes using information on cleavage sites obtained by a method described herein to modify a dsRNA, e.g., an iRNA agent. Optimizing the activity of the nucleic acid molecules of the invention Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can 5 prevent their degradation by serum ribonucleases, which can increase their potency (see e.g., Eckstein et al., International Publication No. WO 92/07065; Perrault et al., Nature 344:565, 1990; Phieken et al., Science 253:314, 1991; Usman and Cedergren, Trends in Biochem. Sci. 17:334, 1992; Usman et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. 0 Pat. No. 6,300,074; and Burgin et al., supra ; all of which are incorporated by reference herein). All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar moieties of the nucleic acid molecules described herein. Modifications that enhance their efficacy in cells, and removal of bases from nucleic acid molecules to shorten oligonucleotide synthesis times and reduce chemical requirements are desired. 5 Other suitable modifications to a sugar, base, or backbone of an iRNA agent are described elsewhere herein. There are several examples in the art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules with significant enhancement in their nuclease stability and efficacy. For example, oligonucleotides are modified to enhance stability and/or .0 enhance biological activity by modification with nuclease resistant groups, for example, 2' amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-0- allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, Trends in Biochem. Sci. 17:34, 1992; Usman et al., Nucleic Acids Symp. Ser. 3:163, 1994; Burgin et al., Biochemistry 35:14090, 1996). Sugar modification of nucleic acid molecules have been extensively described in the art (see Eckstein et al., 25 International Publication PCT No. WO 92/07065; Perrault et al., Nature 344:565, 1990; Pieken et al. Science 253:314, 1991; Usman and Cedergren, Trends in Biochem.Sci. 17:334, 1992; Usman et al., International Publication PCT No. W093/15187; Sproat, U.S. Pat. No. 5,334,711, and Beigelman et al., J. Biol. Chem. 270:25702, 1995; Beigelman et al., International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S. Pat. 30 No. 5,627,053; Woolf et al., International PCT Publication No. WO 98/13526; Karpeisky et al., Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48:39, 1998; Verma and Eckstein, Annu. Rev. Biochem. 67:99, 1998; and Burlina et al., Bioorg. Med. Chem. 5:1999, 1997; all of the references are hereby incorporated in their totality by reference herein). Such publications describe general methods and strategies to determine the 5 location of incorporation of sugar, base and/or phosphate modifications and the like into nucleic acid molecules without modulating catalysis, and are incorporated by reference herein. In view of such teachings, similar modifications can be used as described herein to modify the iRNA nucleic acid molecules of the instant invention so long as the ability of iRNA agents to promote RNAi in cells is not significantly inhibited. 0 While chemical modification of oligonucleotide internucleotide linkages with phosphorothioate, phosphorodithioate, and/or 5'-methylphosphonate linkages improves stability, excessive modifications can cause some toxicity or decreased activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. The reduction in the concentration of these linkages should lower toxicity, resulting 5 in increased efficacy and higher specificity of these molecules. The 3' and 5' ends of an iRNA agent can be modified. Such modifications can be at the 3' end, 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. For example, the 3' and 5' ends of an oligonucleotide can be conjugated to other functional molecular entities .0 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 of the spacer can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' 0, N, S or C group of the sugar. Alternatively, the spacer can connect to or 25 replace the terminal atom of a nucleotide surrogate (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 array is interposed between two 30 sequences of an iRNA agent, the array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3' end can be an -OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal 5 modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 0 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03 (oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic 5 acid), and synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles). In some embodiments, conjugates such as retinol or retinoic acid can be attached to the 5' or 3' end, or both ends, of an iRNA agent. Use of such conjugates may improve specific uptake and delivery of iRNA agents to cells that express retinol receptors, such as retinal pigment epithelial cells. .0 Terminal modifications can be added for a number of reasons, such as to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogs. For example, in preferred embodiments iRNA agents, especially antisense sequences, are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus. 5'-phosphate modifications include those 25 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)-0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5'-(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 30 (HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5' 17 monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-0-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen/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-0-5'), 5'-alkylphosphonates 5 (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, (OH)2(O)P-5'-CH2-), 5' alkyletherphosphonates (R=alkylether-methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-). In another embodiment, the invention features conjugates and/or complexes of iRNA agents of the invention. Such conjugates and/or complexes can be used to facilitate delivery of 0 iRNA agents into a biological system, such as a cell. The conjugates and complexes provided by the instant invention can impart therapeutic activity by transferring therapeutic compounds across cellular membranes, altering the pharmacokinetics, and/or modulating the localization of nucleic acid molecules of the invention. The present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not 5 limited to, small molecules, lipids, phospholipids, nucleosides, nucleotides, nucleic acids, antibodies, toxins, negatively charged polymers and other polymers, for example, proteins, peptides, hormones, carbohydrates, polyethylene glycols, or polyamines, across cellular membranes. In general, the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers. These compounds are .0 expected to improve delivery and/or localization of nucleic acid molecules of the invention into a number of cell types originating from different tissues, in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of the molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules. 25 Administration of the iRNA Agents A patient who has been diagnosed with a disorder characterized by unwanted VEGF expression can be treated by administration of an iRNA agent described herein to block the negative effects of VEGF, thereby alleviating the symptoms associated with unwanted VEGF gene expression. For example, the iRNA agent can alleviate 30 symptoms associated with a disease of the eye, such as a neovascular disorder. In other examples, the iRNA agent can be administered to treat a patient who has a tumor or metastatic cancer, such as colon or breast cancer; a pulmonary disease, such as asthma or bronchitis; or an autoimmune disease such as rheumatoid arthritis or psoriasis. The anti-VEGF iRNA agents can be administered systemically, e.g., orally or by intramuscular injection or by intravenous 5 injection, in admixture with a pharmaceutically acceptable carrier adapted for the route of administration. An iRNA agent can comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., Trends in Cell Bio. 2:139, 1992; Delivery Strategies for Antisense 0 Oligonucleotide Therapeutics, ed. Akhtar, 1995; Maurer et al., Mol. Membr Biol., 16:129, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137:165, 1999; and Lee et al., ACS Symp. Ser. 752:184, 2000, all of which are incorporated herein by reference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe the general methods for delivery of nucleic acid molecules. Nucleic acid molecules can be administered to cells by a 5 variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by ionophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins (see for example Gonzalez et al., Bioconjugate Chem. 10:1068, 1999), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722). .0 In the present methods, the iRNA agent can be administered to the subject either as naked iRNA agent, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the iRNA agent. Preferably, the iRNA agent is administered as naked iRNA. The iRNA agent of the invention can be administered to the subject by any means suitable for delivering the iRNA agent to the cells of the tissue at or near the area of unwanted 25 VEGF expression, such as at or near an area of neovascularization. For example, the iRNA agent can be administered by gene gun, electroporation, or by other suitable parenteral administration routes. Suitable enteral administration routes include oral delivery. Suitable parenteral administration routes include intravascular administration (e.g., 30 intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., intraocular injection, intra-retinal injection, or sub-retinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the area at or near the site of neovascularization, for example by a catheter or other placement device 5 (e.g., a retinal pellet or an implant comprising a porous, non-porous, or gelatinous material). It is preferred that injections or infusions of the iRNA agent be given at or near the site of neovascularization. The iRNA agent of the invention can be delivered using an intraocular implant. Such implants can be biodegradable and/or biocompatible implants, or may be non-biodegradable 0 implants. The implants may be permeable or impermeable to the active agent, and may be inserted into a chamber of the eye, such as the anterior or posterior chambers, or may be implanted in the sclera, transchoroidal space, or an avascularized region exterior to the vitreous. In a preferred embodiment, the implant may be positioned over an avascular region, such as on the sclera, so as to allow for transcleral diffusion of the drug to the desired site of treatment, e.g., 5 the intraocular space and macula of the eye. Furthermore, the site of transcleral diffusion is preferably in proximity to the macula. The iRNA agent of the invention can also be administered topically, for example, by patch or by direct application to the eye, or by iontophoresis. Ointments, sprays, or droppable liquids can be delivered by ocular delivery systems known in the art such as applicators or .0 eyedroppers. The compositions can be administered directly to the surface of the eye or to the interior of the eyelid. 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. 25 The iRNA agent of the invention may be provided in sustained release compositions, such as those described in, for example, U.S. Patent Nos. 5,672,659 and 5,595,760. The use of immediate or sustained release compositions depends on the nature of the condition being treated. If the condition consists of an acute or over-acute disorder, treatment with an immediate release form will be preferred over a prolonged release composition. Alternatively, for certain 30 preventative or long-term treatments, a sustained release composition may be appropriate.

An iRNA agent can be injected into the interior of the eye, such as with a needle or other delivery device. The iRNA agent of the invention can be administered in a single dose or in multiple doses. Where the administration of the iRNA agent of the invention is by infusion, the infusion 5 can be a single sustained dose or can be delivered by multiple infusions. Injection of the agent directly into the tissue is at or near the site of neovascularization is preferred. Multiple injections of the agent into the tissue at or near the site of neovascularization are also preferred. Dosage levels on the order of about 1 pg/kg to 100 mg/kg of body weight per administration are useful in the treatment of the neovascular diseases. When administered 0 directly to the eye, the preferred dosage range is about 0.00001 mg to about 3 mg per eye, or preferrably about 0.0001-0.001 mg per eye, about 0.03- 3.0 mg per eye, about 0.1-3.0 mg per eye or about 0.3-3.0 mg per eye. One skilled in the art can also readily determine an appropriate dosage regimen for administering the iRNA agent of the invention to a given subject. For example, the iRNA agent can be administered to the subject once, e.g., as a single injection or 5 deposition at or near the neovascularization site. Alternatively, the iRNA agent can be administered once or twice daily to a subject for a period of from about three to about twenty eight days, more preferably from about seven to about ten days. In a preferred dosage regimen, the iRNA agent is injected at or near a site of unwanted VEGF expression (such as near a site of neovascularization) once a day for seven days. Where a dosage regimen comprises multiple .0 administrations, it is understood that the effective amount of iRNA agent administered to the subject can comprise the total amount of iRNA agent administered over the entire dosage regimen. One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending on a variety of factors, including the specific iRNA agent being administered, the time of administration, the route of administration, the nature of the 25 formulation, the rate of excretion, the particular disorder being treated, the severity of the disorder, the pharmacodynamics of the iRNA agent, and the age, sex, weight, and general health of the patient. Wide variations in the necessary dosage level are to be expected in view of the differing efficiencies of the various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous 30 or intravitreal injection. Variations in these dosage levels can be adjusted using standard A 1 empirical routines of optimization, which are well-known in the art. The precise therapeutically effective dosage levels and patterns are preferably determined by the attending physician in consideration of the above-identified factors. In addition to treating pre-existing neovascular diseases, iRNA agents of the invention 5 can be administered prophylactically in order to prevent or slow the onset of these and related disorders. In prophylactic applications, an iRNA of the invention is administered to a patient susceptible to or otherwise at risk of a particular neovascular disorder. The iRNA agents featured by the invention are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art. 0 Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 18th ed., Mack Publishing Company, Easton, Pa. (1990), and The Science and Practice of 5 Pharmacy, 2003, Gennaro et al., the entire disclosures of which are herein incorporated by reference. The present pharmaceutical formulations comprise an iRNA agent of the invention (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a physiologically acceptable carrier medium. Preferred physiologically acceptable carrier media are water, .0 buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), 25 additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized. Al' For solid compositions, conventional non-toxic solid carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For example, a solid pharmaceutical composition for oral administration can comprise 5 any of the carriers and excipients listed above and 10-95%, preferably 25%-75%, of one or more iRNA agents of the invention. By "pharmaceutically acceptable formulation" is meant a composition or formulation that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity. Non-limiting examples of agents 0 suitable for formulation with the nucleic acid molecules of the instant invention include: P glycoprotein inhibitors (such as PluronicP85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol. 13:16, 1999); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery. Other non limiting examples of delivery strategies for the nucleic acid molecules of the instant invention 5 include material described in Boado et al., J. Pharm. Sci. 87:1308, 1998; Tyler et al., FEBS Lett. 421:280, 1999; Pardridge et al., PNAS USA. 92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73, 1995; Aldrian-Herrada et al., Nucleic Acids Res. 26:4910, 1998; and Tyler et al., PNAS USA 96:7053, 1999. The invention also features the use of the composition comprising surface-modified .0 liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes). These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al., Chem. Rev. 95:2601, 1995; 25 Ishiwata et al., Chem.Phare. Bull. 43:1005, 1995). Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic et al., Science 267:1275, 1995; Oku et al., Biochim. Biophys. Acta 1238:86, 1995). The long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared 30 to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 42:24864, 1995; Choi et al., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390 ; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to 5 avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen. The present invention also includes compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical 0 Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby incorporated by reference herein. For example, preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition, antioxidants and suspending agents can be used. The nucleic acid molecules of the present invention can also be administered to a subject 5 in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects. Alternatively, certain iRNA agents of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, Science 229:345, 1985; McGarry and .0 Lindquist, Proc. Nati. Acad. Sci. USA 83:399, 1986; Scanlon et al., Proc.Natl. Acad. Sci. USA 88:10591, 1991; Kashani-Sabet et al., Antisense Res. Dev. 2:3, 1992; Dropulic et al., J. Virol. 66:1432, 1992; Weerasinghe et al., J. Virol. 65:5531, 1991; Ojwanget al., Proc. Nati. Acad. Sci. USA 89:10802, 1992; Chen et al., Nucleic Acids Res. 20:4581, 1992; Sarver et al., Science 247:1222, 1990; Thompson et al., Nucleic Acids Res. 23:2259, 1995; Good et al., Gene Therapy 25 4:45, 1997). Those skilled in the art realize that any nucleic acid can be expressed in eukaryotic cells from the appropriate DNA/RNA vector. The activity of such nucleic acids can be augmented by their release from the primary transcript by a enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., Nucleic Acids Symp. Ser. 27:156, 1992; Taira et al., Nucleic Acids Res. 19:5125, 1991; Ventura et al., Nucleic 30 Acids Res. 21:3249, 1993; Chowrira et al., J. Biol. Chem. 269:25856, 1994). A A In another aspect of the invention, RNA molecules of the present invention can be expressed from transcription units (see for example Couture et al., Trends in Genetics 12:5 10, 1996) inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids or viral vectors. iRNA agent-expressing viral vectors can be constructed based on, but not limited 5 to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another embodiment, pol III based constructs are used to express nucleic acid molecules of the invention (see for example Thompson, U. S. Pats. Nos. 5,902,880 and 6,146,886). The recombinant vectors capable of expressing the iRNA agents can be delivered as described above, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of nucleic acid 0 molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the iRNA agent interacts with the target mRNA and generates an RNAi response. Delivery of iRNA agent-expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the 5 desired target cell (for a review see Couture et al., Trends in Genetics 12:510, 1996). Additional ophthalmic indications for the iRNA agents of the invention include proliferative diabetic retinopathy (the most severe stage of diabetic retinopathy), uveitis (an inflammatory condition of the eye that often leads to macular edema), cystoid macular edema following cataract surgery, myopic degeneration (a condition in which a patient with a high .0 degree of nearsightedness develops choroidal neovascularization), inflammatory macular degeneration (a condition in which a patient with inflammation in the macular area due to infections or other causes, develops choroidal neovascularization), and iris neovascularization (a serious complication of diabetic retinopathy or retinal vein occlusion involving new blood vessel growth on the surface of the iris). 25 Additional non-ophthalmic indications for the iRNA agents of the invention include cancer, including but not limited to renal and colon cancer, and psoriasis. Solid tumors and their metastases rely on new blood vessel growth for their survival. Psoriasis is a chronic inflammatory skin disease that causes skin cells to grow too quickly, resulting in thick white or red patches of skin. Preclinical and clinical data suggest that VEGF-induced blood vessel growth and blood vessel leakage play a role in the development of this condition. The invention is further illustrated by the following examples, which should not be construed as further limiting. 5 EXAMPLES Example 1: siRNA Design Four hundred target sequences were identified within exons 1-5 of the VEGF-A121 0 mRNA sequence (See Table 1, SEQ ID NOs 2-401) and corresponding siRNAs targeting these subjected to a bioinformatics screen. To ensure that the sequences were specific to VEGF sequence and not to sequences from any other genes, the target sequences were checked against the sequences in Genbank using the BLAST search engine provided by NCBI. The use of the BLAST algorithm is described in 5 Altschul et al., J. Mol. Biol. 215:403, 1990; and Altschul and Gish, Meth. Enzymol. 266:460, 1996. siRNAs were also prioritized for their ability to cross react with monkey, rat and human VEGF sequences. Of these 400 potential target sequences 80 were selected for analysis by experimental .0 screening in order to identify a small number of lead candidates. A total of 114 siRNA molecules were designed for these 80 target sequences 114 (Table 2). Example 2: Synthesis of the siRNA oligonucleotides RNA was synthesized on Expedite 8909, ABI 392 and AB1394 Synthesizers (Applied 25 Biosystems, Applera Deutschland GmbH, Frankfurter Str. 129b, 64293 Darmstadt, Germany) at 1 p mole scale employing CPG solid support and Expedite RNA phosphoramidites (both from Proligo Biochemie GmbH, Georg-Hyken-Str.14, Hamburg, Germany). Ancillary reagents were obtained from Mallinckrodt Baker (Im Leuschnerpark 4:64347 Griesheim, Germany). Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a 30 solution of the Beaucage reagent in acetonitrile (5% weight per volume).

Cleavage of the oligoribonucleotides from the solid support and base deprotection was accomplished with a 3:1 (v/v) mixture of methylamine (41%) in water and methylamine (33%) in ethanol. 2'-Desilylation was carried out according to established procedures (Wincott et al., Nucleic Acids Res. 23:2677-2684, 1995). Crude oligoribonucleotides were purified by anion 5 exchange HPLC using a 22x250 mm DNAPac PA 100 column with buffer A containing 10 mM NaClO 4 , 20 mM Tris, pH 6.8, 6 M urea and buffer B containing 400 mM NaClO 4 , 20 mM Tris, pH 6.8, 6 M Urea. Flow rate was 4.5 mL/min starting with 15% Buffer B which was increased to 55% over 45 minutes. The purified compounds were characterized by LC/ESI-MS (LC: Ettan Micro, Amersham 0 Biosciences Europe GmbH, Munzinger Strasse 9, 79111 Freiburg, Germany, ESI-MS: LCQ, Deca XP, Thermo Finnigan, Im Steingrund 4-6, 63303 Dreieich, Germany) and capillary electrophoresis (P/ACE MDQ Capillary Electrophoresis System, Beckman Coulter GmbH, 85702 UnterschleiBheim, Germany). Purity of the isolated oligoribonucleotides was at least 85%. Yields and concentrations were determined by UV absorption of a solution of the 5 respective RNA at a wavelength of 260 nm using a spectral photometer. Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heating in a water bath at 85 - 90 'C for 3 minutes and cooling to room temperature over a period of 3 - 4 hours. The RNA was kept at -20 'C until use. 0 Example 3: Efficacy Screen of siRNAs Using two efficacy screens, the VEGF siRNA were screened for their ability to become a lead candidate. Table 2 shows the relative efficiencies of some of the siRNAs in their ability to inhibit expression of an endogenous VEGF gene. In this process the number of candidate 25 siRNAs was winnowed. Human HeLa or ARPE- 19 (human retinal pigment epithelial cell line with differentiated properties (Dunn et al., Exp. Eye Res. 62:155, 1996) were plated in 96-well plates (17,000 cells/well) in 100 pl 10% fetal bovine serum in Dulbecco's Modified Eagle Medium (DMEM). When the cells reached approximately 90% confluence (approximately 24 hours later) they were transfected with serial three-fold dilutions of siRNA starting at 20 nM 0.4 30 pl of transfection reagent LipofectamineTM 2000 (Invitrogen Corporation, Carlsbad, CA) was A -7 used per well and transfections were performed according to the manufacturer's protocol. Namely, the siRNA: LipofectamineTM 2000 complexes were prepared as follows. The appropriate amount of siRNA was diluted in Opti-MEM I Reduced Serum Medium without serum and mixed gently. The LipofectamineTM 2000 was mixed gently before use, then for each 5 well of a 96 well plate, 0.4 pl was diluted in 25 pl of Opti-MEM I Reduced Serum Medium without serum and mixed gently and incubated for 5 minutes at room temperature. After the 5 minute incubation, 1 p l of the diluted siRNA was combined with the diluted LipofectamineTM 2000 (total volume is 26.4 p l). The complex was mixed gently and incubated for 20 minutes at room temperature to allow the siRNA: LipofectamineTM 2000 complexes to form. Then 100 pl 0 of 10% fetal bovine serum in DMEM was added to each of the siRNA:Lipofectamine T M 2000 complexes and mixed gently by rocking the plate back and forth. 100 Pl of the above mixture was added to each well containing the cells and the plates were incubated at 37 0 C in a CO 2 incubator for 24 hours, then the culture medium was removed and 100 Pl 10% fetal bovine serum in DMEM was added. Following the medium change, conditioned medium was collected 5 at 24 hours (HeLa cells) or 72 hours (ARPE-19 cells) and a human VEGF ELISA was performed using the DuoSet human VEGF ELISA Development kit (R&D Systems, Inc. Minneapolis, MN 55413). This kit contains the basic component required for the development of sandwich ELISAs to measure natural and recombinant human VEGF in cell culture supernatants and serum. .0 The materials used included: Capture Antibody - 576 pg/ml of goat anti-human VEGF when reconstituted with 0.25 ml PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na 2

HPO

4 , 1.5 mM KH 2

PO

4 , pH 7.2-7.4, 0.2 Pm filtered). After reconstitution, stored at 2-8'C for up to 60 days or aliquoted and stored at -20'C to -70'C in a manual defrost freezer for up to 6 months. Diluted to a working concentration of 25 0.8 pg/ml in PBS without carrier protein. Detection antibody - 4.5 pg/ml of biotinylated goat anti-human VEGF when reconstituted with 1.0 ml of Reagent Diluent (1% bovine serum albumin in PBS, pH 7.2-7.4, 0.2 pm filtered. After reconstitution, stored at 2-8'C for up to 60 days or aliquoted and stored at 20'C to -70'C in a manual defrost freezer for up to 6 months. Diluted to a working concentration 30 of 25 ng/ml in Reagent Diluent. A Q Standard: 110 ng/ml of recombinant when reconstituted with 0.5 ml of Reagent Diluent. Allowed the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. The reconstituted Standard can be stored at 2-8'C for up to 60 days or aliquoted and stored at -20'C to -70'C in a manual defrost freezer for up to 6 months. A seven point standard 5 curve using 2-fold serial dilutions in Reagent Diluent, and a high standard of 4000 pg/ml is recommended. Streptavidin-HRP: 1.0 ml of streptavidin conjugated to horseradish-peroxidase. Stored at 2-8'C for up to 6 months. Diluted to the working concentration specified on the vial label. General ELISA protocol was followed (R&D Systems, Inc., Minneapolis, MN). 0 Controls included no siRNA, human VEGF siRNA (Cand5, (a.k.a., hVEGF5) Reich et al., Mol Vis. 9:210, 2003) and an siRNA matching a 21-nt sequence conserved between the human, rat and mouse VEGF (hrmVEGF, Filleur et al., Cancer Res. 63:3919-3922, 2003). The activities of the siRNAs were compared to the activity of the control human VEGF siRNA of Reich et al. (supra) with "+" representing a lower activity, "++" representing similar 5 activity and "+++" representing a higher activity than the control human VEGF siRNA (Table 2). FIG. 2 shows the activities of single- and double-overhang siRNAs in HeLa cells. Solid lines with filled symbols represent the single-overhang siRNA, solid lines with open symbols represent the double-overhang siRNAs; dashed lines represent the control siRNAs. All of the siRNAs are more active than the control siRNAs and may inhibit expression of VEGF by .0 approximately 80%. In contrast, the siRNA from Reich et al. (supra) reduced the level of endogenous hVEGF by approximately 20% under the same experimental conditions. Similarly, under the same experimental conditions, the siRNA based on consensus sequence hrmVEGF (Filleur et al., supra) reduced the expression level by approximately 45%. FIG. 3 shows the activities of single- and double-overhang siRNAs in ARPE-19 cells. 25 Solid lines with filled symbols represent the single-overhang siRNA, solid lines with open symbols represent the double-overhang siRNAs; dashed lines represent the control siRNAs. All of the siRNAs are more active than the control siRNAs and may inhibit expression of VEGF by approximately 90%. In contrast, the siRNA from Reich et al. (Mol. Vis. 9:210, 2003) reduced the level of hVEGF by approximately 35% under the same experimental conditions. Similarly, under

AO

the same experimental conditions, the siRNA based on consensus sequence hrmVEGF (Filleur et al., supra) reduced the expression level by approximately 70%. FIGs. 4 and 5 show the results of a comparison of single- and double-overhang siRNAs with their analogous blunt-ended siRNAs, respectively in HeLa cells. The results are in 5 agreement with the data of Elbashir et al. (Genes & Development 15:188, 2001) in that the presence of an overhang in an siRNA confers greater efficiency in inhibition of gene silencing. However, it is important to note that the activity of the blunt ended siRNAs are comparable to the results achieved using the control siRNAs. 0 Example 4: In vitro assay for the silencing! of VEGF synthesis under hypoxic conditions Human HeLa cells were plated in 96 well plates at 10,000 cells/well in 100 Pl of growth medium (10% FBS in DMEM). 24 hours post cell seeding when the cells had reached approximately 50% confluence they were transfected with serial three fold dilutions of siRNA 5 starting at 30 nM. 0.2 pl of LipofectamineTM 2000 transfection reagent (Invitrogen Corporation, Carlsbad CA) was used per well and transfections were carried out as described in the Invitrogen product insert. Controls included no siRNA, human VEGF siRNA (Reich et al., Mol. Vis. 9:2 10, 2003) and an siRNA matching a 21-nt sequence conserved between the human, rat and mouse VEGF (hrmVEGF, Filleur et al., Cancer Res. 63:3919-3922, 2003). Transfections were done in .0 duplicate on each plate. Additionally, duplicate plates were transfected so that 24 hours post transfection the growth media could be changed and one plate could be kept in normal oxygen growth conditions (37 0 C, 5% C0 2 , 20% oxygen) and the duplicate plate could be kept in hypoxic conditions (37 0 C, 1% oxygen, balance nitrogen). Hypoxic conditions were maintained by using a Pro-ox Oxygen Controller (BioSpherix, Ltd., Redfield, NY) attached to a Pro-ox in vitro 25 culture chamber. Cells were maintained in either normoxic or hypoxic conditions for 24 hours post media change. Conditioned culture media was then collected from both plates and tested for secreted VEGF levels in a DuoSet VEGF ELISA (R&D Systems, Minneapolis, MN). The assays were performed according to the manufacturer's protocol and as described in Example 2. For deferoxamine chemically induced hypoxia, 130 [tM deferoxamine (Sigma D9533), 30 was used. Deferoxamine was added to the fresh growth media 24 hours post-transfection. Cells treated with deferoxamine were then grown under normal growth conditions (37 0 C, 5% C0 2 , 20% oxygen). FIG. 6 shows the results obtained with siRNAs (both single overhang siRNAs and double overhangs siRNAs) directed against ORF regions having the first nucleotides corresponding to 5 319 and 343 respectively, together with the control siRNAs. Under hypoxic conditions, either 1% oxygen (FIG. 6B) or 130 pM deferoxamine (FIG. 6C), three of the experimental siRNAs achieved almost 95% inhibition of expression of VEGF, namely AL-DP-4094 (single-overhang) directed at ORF 343, and both of the siRNAs (single and double-overhangs) directed at ORF 319. The control siRNAs of Reich et al (supra) and Filleur et al. (supra) demonstrate an ability 0 to inhibit VEGF expression by 45% and 85% respectively. FIGs. 8A and 8B show the results obtained with the siRNAs AL-DP-4014, a phosphorothioate modified version of AL-DP-4014 (AL-DP-4127, see Table 3) and a mutated version of AL-DP-4014 (AL-DP-4140, see Table 5). Under both normal and hypoxic conditions, the unmodified (AL-DP-4014) and the phosphorothioate-modified siRNA (AL-DP-4127) 5 reduced endogenous VEGF expression to less than 20% of its original expression level. Under hypoxic conditions, the phosphorothioate-modified siRNA essentially abolished VEGF expression. Example 5: Modified VEGF siRNA molecules retain full activity and show enhanced .0 stability Phosphorothioate derivatives were made for the AL-DP-4014, targeting ORF 319 of VEGF, and are presented in Table 3. These siRNAs were tested in the HeLa cell assay described in Example 3, and FIG. 7 shows that these derivatives are as active in the HeLa assay as the unmodified siRNA. 25 A panel of siRNAs were synthesized that retained the sequence of the AL-DP-4094 siRNA (Table 1) but included different modifications including phosphorothioate linkages, 0 methyl-modified nucleotides, and 2'-fluoro-modified nucleotides (Table 4). The panel of siRNAs was tested in HeLa cells, and FIGs. 9A-9E demonstrate that all modified versions of the AL-DP-4094 siRNA effectively reduced VEGF expression by greater than 90%, exhibiting 30 greater efficacy than either of the two previously identified VEGF siRNAs ("Acuity" in Reich et r,1 al. (supra), and "Filleur" in Filleur et al. (supra)). FIG. 10 also shows data from in vitro assays in HeLa cells. The graph in FIG. 10 shows that the unmodified AL-DP-4094 siRNA and a phosphorothioate-modified AL-DP-4004 siRNA (AL-DP-4219) reduced VEGF expression by more than 70% (FIG. 10). Scrambled versions of the compound AL-DP-4094 (e.g., AL-DP-4216 5 and AL-DP-4218 (sequences shown below; underlined nucleotides represent mismatched nucleotides as compared to AL-DP-4094)) did not inhibit VEGF expression. An siRNA targeting the firefly luciferase gene (AL-DP-3015; see below) also did not inhibit VEGF expression. AL-DP-4216 AL4094 MI s 5'- GCACAUUGGACAGUUGUGGUU-3' AL4094 MI as '3-GUCGUGUAACCUGUCAACACCAA-'5 AL4094 M5 AL-DP-4218 s 5'- GCACAUAGAAGUGACGCGCUU-3' AL4094 M5 as '3-GUCGUGUAUCUUCACUGCGCGAA-'5 AL-DP-3015 5'- GAACUGUGUGUGAGAGGUCCU-3' '3-CGCUUGACACACACUCUCCAGGA-'5 0 The Stains-All technique (Sigma, St. Louis, MO) was performed to examine the stability of the modified siRNAs. To perform the assay, an siRNA duplex was incubated in 90% human serum at 37'C. Samples of the reaction mix were quenched at various time points (at 0, 0.25, 1, 2, 4, and 24 hours) and subjected to polyacrylamide gel electrophoresis. Cleavage of the RNA 15 over the time course provided information regarding the susceptibility of the siRNA duplex to serum nuclease degradation. 0-methyl and 2'fluoro modifications used in combination with phosphorothioate modifications were found to enhance stability to a greater extent than when phosphorothioate modifications were used alone. For example, modified versions of the AL-DP-4094 siRNA 20 included a phosphorothioate-modified siRNA (AL-DP-4198), a phosphorothioate plus 0-methyl modified siRNAs (e.g., AL-DP-4180, AL-DP-4175, and AL-DP-4220), and phosphorothioate plus 0-methyl plus 2'-fluoro modified siRNAs (e.g., AL-DP-4197 and AL-DP-4221) (Table 4).

The AL-DP-4180, AL-DP-4175, and AL-DP-4197 siRNAs were found to be more stable in human serum than the AL-DP-4198 siRNA. It was determined that the phosphorothioate modification stabilized the siRNAs against exonucleolytic degradation, and the 0-methyl and 2' fluoro modifications stabilized the siRNAs against endonucleolytic degradation. 5 Example 6: In vitro Stability Assay of VEGF siRNAs in Different Rat Serum and Ocular tissues 1. Preparation of Tissue Homogenates Tissues from pooled whole eyes, retinas, vitreous humors from at least three rats were excised 0 and frozen immediately in liquid nitrogen. The frozen tissue was pulverized over dry ice, using instruments that were pre-chilled on dry ice. 1 ml of RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCL, 1mM Na2EDTA, 0.5% Na-deoxycholate deoxycholic acid, 1% IGEPAL CA-630, 0.05% SDS) was added to the frozen tissue powder and the mixture was mixed thoroughly and vigorously. The homogenate was centrifuged at 10,000 x g for 5 min at 4'C and the pellet was 5 discarded. 100 pl aliquots of the supernatant were transferred to pre-chilled microcentrifuge tubes and stored at -70'C or used immediately in the stability assay. 2. 5'-end labeling of single stranded sense or antisense siRNA using T4 polynucleotide kinase and y 32P-ATP The following reagents were used: .0 T4 Polynucleotide Kinase (PNK) 10 units/pIl (New England Biolabs, Beverly, MA) 1oX T4 PNK buffer (700 mM Tris-HCl, 100 mM MgCl 2 , 50 mM Dithiothreitol (DTT), pH7.6) Gamma- 32P-ATP (PerkinElmer, Shelton, CT) 250 pCi, 3000 Ci/mmol (3.3 pM) 10 pM stocks of synthetic RNA oligo diluted in H 2 0 25 Microspin SephadexTM G-25 columns (Amersham Biosciences RNAse-free Water and 0.65 ml microcentrifuge (1.5 ml) tubes A 25 pl kinase reaction contained: 2.5pl from 10 pM stock sense or antisense (1pM final conc.) 2.5pl 1oX PNK Buffer (1X) 30 1.5pl y 32-ATP (Dapproximately 0.2 nM) 1.0pl 10 unit/pl T4 PNK (10 units) 17.5pl dH 2 0 The reaction mix was incubated at 37'C for 1 hour (water bath) prior to fractionating the labeled siRNA through SephadexTM G-25 spin columns (Amersham). 0.5 pL was used to determine the 5 number of counts per minute (cpm)/ml of the radiolabeled sample. 3. Partial alkaline hydrolysis ladder of radiolabeled single-stranded siRNA To generate a sample of size markers a portion of the 5' y32 P-end- labeled siRNA was 0 subjected to alkaline hydrolysis as follows: 30 p l hydrolysis reaction containing 2.5 pl 5' end -labeled siRNA (sense or antisense), 6.0 P l 0.5M Na 2

CO

3 /NaHCO 3 (pH 9.5), 1.5pl 10mg/ml tRNA, and 20.0pl dH 2 0 was incubated at 90'C for 7.5 min, then chilled on ice or at 4'C. 30pl of 90% formamide, 50mM Na 2 EDTA, 10mM DTT, and XC&BB (xylene cyanol and bromophenol blue), of which 1pl+4pl formamide dye was 5 used for the gel electrophoretic analysis. 4. Annealing of radiolabeled 1UM stock siRNA duplexes 30pl 1pM stock of different siRNA duplexes were prepared in which either the sense strand or the antisense strand was radiolabeled. The samples were heated at 90'C for 2 min and then incubated at 37'C for 1 hour and .0 then stored at -20'C until used. 5. Quality control of siRNA duplex: Samples of the siRNA duplex were analyzed by electrophoresis through 15% polyacrylamide in Tris-Borate, EDTA (TBE) Gel. Electrophoresis was performed at 150V for 1 hour prior to running the samples through. Samples were prepared by mixing 0.5-1 pl siRNA 25 duplex or single stranded, 3-3.5 pl 0.5X TBE, 1 pl 5X native loading dye (total volume = 5 Pl). 6. Stability reactions 2 p l siRNA duplex was added to 18 p l serum or tissue lysate or buffer control in PCR tube (0.2ml). A zero time point sample was removed immediately following the addition of the siRNA duplex by removing 2 p l and adding it to 18 pl 90% formamide, 50 mM EDTA, 30 10 mM DTT and xylene cyanol and bromophenol blue (XC & BB). Other samples were removed after 15 min, 30 min, 1 hour, 2 hours, and 4 hours and treated similarly. These samples were stored in a 96-well plate. In some experiments the time points were extended to 8, 24 and 48 hours. Time point samples for the buffer (phosphate buffered saline, PBS, 1X working PBS contains 0.14 M Sodium Chloride, 0.003 M Potassium Chloride, 0.002 M Potassium phosphate, 5 0.01 M Sodium phosphate) were taken at zero and the last time point of the experiment. Samples were analyzed by electrophoresis through 20% polyacrylamide gels (pre-run at 75 W for 1 hour) in 1 X TBE (10 X = 890 mM Tris, 890 mM Boric acid, 20 mM EDTA, pH 8.0). The gel was transferred to a phosphorimager cassette, covered with an enhancer screen and scanned after overnight exposure. 0 Polyacrylamide gel analysis indicated that the ocular environment contains fewer nucleases than human serum. Testing the unmodified form of the VEGF siRNA AL-DP-4014 for stability in rat eye extract revealed only the presence of exonuclease activity. In human serum, experiments with AL-DP-4127 and -4140 (Tables 4 and 5) indicated that terminally modified phosphorothioate modifications protected against exonucleolytic degradation but not 5 against endonucleolytic activity. These results were consistent with experiments performed in rat whole eye extracts. The terminally modified phosphorothioate derivatives AL-DP-4127 and 4140 were stabilized against exonuclease activity as compared to the unmodified AL-DP-4014 siRNA and the unmodified Cand5 siRNA (Reich et al. (supra)). However, the -4127 and -4140 siRNAs were still subject to endonucleolytic degradation. .0 Modifications to the lead compound AL-DP-4094 stabilized the siRNA against exonucleolytic and endonucleolytic degradation. The phosphorothioate-modified siRNA AL DP-4198 was degraded to a similar extent as the unmodified 4094 compound, but the addition of 0-methyl modifications, as in AL-DP-4180 and AL-DP-4220, stabilized the siRNAs in rat whole eye extracts. 25 Notably, the siRNAs were generally more stable in rat retina lysates than in the rat whole eye extracts described above. Neither the unmodified AL-DP-4094, nor the modified AL-DP 4198, -4180, or -4220 siRNAs were degraded in the retina lysates.

Example 7. Endonuclease-sensitive sites were mapped on AL-DP-4094 siRNA. The stability of the AL-DP-4094 siRNA was examined by the Stains-All and radiolabeled techniques following incubation in human serum (see above). These assays revealed susceptibility to exo- and endonucleases. RP-HPLC was used to examine the fragment profile of 5 the siRNA following incubation in serum FIG. 11. Following incubation of the -4094 siRNA in human serum, the fragments were phenol chloroform extracted and precipitated, and then subjected to LC/MS analysis. FIG. 12 describes the identified fragments and associated characteristics. 0 Example 8: Detailed study of modifications to siRNAs targeting! VEGF (Table 6) Eight major different patterns of chemical modification of siRNA duplexes that target the VEGF mRNA were synthesized and evaluated (Table 6). The ribose sugar modifications used were either 2'-O-methyl (2'OMe) or 2'-fluoro (2'F). Both pyrimidines (Py) and purines (Pu) could be modified as provided in Table 6. 5 The first four patterns(A-D) incorporated 2'OMe on both strands at every other position. Four configurations were synthesized: 1) at each even position on the sense strand and at each odd position of the antisense strand, 2) at each odd position on the sense strand and at each even position of the antisense strand, 3) at even positions on both strands, and 4) at odd positions on both strands.; .0 The fifth pattern (E) incorporated the 2'OMe modification at all pyrimidine nucleotides on both the sense and antisense strands of the duplex. Pattern F included duplexes with 2'OMe modifications only on pyrimidines in 5'-PyPu 3' dinucleotides, especially at only at UA, CA, UG sites (both strands). Pattern G duplexes had the 2'F modification on pyrimidines of the antisense strand and 25 2'OMe modifications on pyrimidines in the sense strand. Pattern (H) had antisense strands with 2'F-modified pyrimidines in 5'-PyPu-3' dinucleotides, only at UA, CA, UG sites (both strands) and sense strands with 2'OMe modifications only on pyrimidines in 5'-PyPu-3' dinucleotides, only at UA, CA, UG sites (both strands). 30 A-D: Full Alternating 2'-OMe (both strands) Four configurations: Even/Odd; Odd/Even; Even/Even; Odd/Odd E: 2'-OMe Py (both strands) F: 2'-OMe Py only at UA, CA, UG sites (both strands) G: 2'-OMe All Py (sense) 5 2'-F All Py (anti-sense) H: 2'-OMe Py only at UA, CA, UG sites (sense) 2'-F Py only at UA, CA, UG sites (anti-sense) 17 different parent VEGF duplexes from Table 2 tested 0 1. Evaluation of serum stability of siRNA duplexes 2 pM siRNA duplexes (final concentration) were incubated in 90% pooled human serum at 37'C. Samples were quenched on dry ice after 30 minutes, 4 hours, and 24 hours. For each siRNA sequence, a sample at the same concentration was incubated in the absence of serum (in PBS) at 37'C for 24 hours. After all samples were quenched, RNA was extracted using 5 phenol:chloroform and concentrated by ethanol precipitation. Samples were air dried and resuspended in a denaturing loading buffer. One third of each time point was analyzed on a 20% acrylamide (19:1), 7 M urea, 1XTBE gel run at 60'C. RNA was visualized by staining with stains-all solution. A qualitative assessment of the stability of each modified siRNA was made by comparison to the parent unmodified siRNA for each duplex set. PBS controls served as .0 markers for the quality of the input siRNA. 2. Stability of VEGF modular chemistries Four modular chemistries were screened 1) all pyrimidines substituted with 2'-O-methyl (2'OMe) in both sense and antisense strands, 2) pyrimidines in UA, UG, CA pairs substituted with 2'OMe in both sense and antisense strands, 3) all pyrimidines substituted with 2'OMe in the 25 sense strand and 2'-fluoro (2'F) in the antisense strand, 4) pyrimidines in UA, UG, CA pairs substituted with 2'OMe in the sense strand and 2'F in the antisense strand. In total, 85 siRNAs were screened including the unmodified parent duplexes plus the four modular chemistries. Of the 85 siRNAs screened, 35 were stable for at least 24hours as assessed by visual comparison with the parent unmodified duplexes. These 35 duplexes had 2'OMe pyrimidines in 30 both strands or 2'OMe pyrimidines in the sense strand and 2'F in the antisense strand (chemistries 1 and 3 above). Of the duplexes with fewer modified residues, only five had at least -50% full length material remaining at the 4 hour time point as compared to their unmodified parent. Substitution of all pyrimidines with either 2'OMe or 2'F protects siRNAs from serum 5 nuclease degradation for -24hr in 90% human serum at 37'C. The protected duplexes had roughly 85%-100% full length material remaining at 24 hours as compared to duplex incubated in the absence of serum. Minimal modification of pyrimidines in UA, UG, and CA dinucleotide pairs only stabilized several siRNAs relative to their unmodified parent but did not stabilize sufficiently for long-term nuclease resistance. Some potential RNase A sites were not protected 0 by methylation (YpN, e.g. UC, UU) and this is likely the reason for the lower resistance to serum endonucleases. 3. Analysis of Duplex activity Duplexes were tested for activity in the HeLa cell assay described above. Table 6 and Figures 13-29 provides summary and graphs of duplex activities in HeLa cells for each of the 5 modifications described above. Synthesis of the iRNA agents RNA Synthesis using "fast" deprotection monomers 1. RNA synthesis .0 Oligoribonucleotides were synthesized using phosphoramidite technology on solid phase employing an AKTA 10 synthesizer (Amersham Biosciences) at scales ranging from 35 to 60 pmol. Synthesis was performed on solid supports made of controlled pore glass (CPG, 520, with a loading of 70 [tmol/g) or polystyrene (with a loading of 71 pmol/g). All amidites were dissolved in anhydrous acetonitrile (70 mM) and molecular sieves (3A) were added. 5-Ethyl 25 thiotetrazole (ETT, 600 mM in acetonitrile) was used as the activator solution. Coupling times were 8 minutes. Oxidation was carried out either with a mixture of iodine/water/pyridine (50 mM/10%/90% (v/v)) or by employing a 100 mM solution of 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) in anhydrous acetonitrile in order to introduce phosphorothioate linkages. Standard capping reagents were used. Cholesterol was conjugated to RNA via the either the 5' or the 3' 30 end of the sense strand by starting from a CPG modified with cholesterol (described below) using a hydroxyprolinol linker. The DMT protecting group was removed from cholesterol conjugated RNA, but the DMT was left on unconjugated RNA to facilitate purification. 2. Cleavage and deprotection of support bound oligonucleotide. After solid-phase synthesis, the RNA was cleaved from the support by passing 14 mL of 5 a 3:1 (v/v) mixture of 40% methylamine in water and methylamine in ethanol through the synthesis column over a 30 min time period. For the cholesterol-conjugated RNA, the ratio of methylamine in water to methylamine in ethanol was 1:13. The eluent was divided into four 15 mL screw cap vials and heated to 65'C for additional 30 min. This solution was subsequently dried down under reduced pressure in a speedvac. The residue in each vial was dissolved in 250 0 p L N-methylpyrolidin-2-one (NMP), and 120 p L triethylamine (TEA) and 160 p L TEA- 3HF were added. This mixture was brought to 65'C for 2h. After cooling to ambient temperature, 1.5 mL NMP and 1 mL of ethoxytrimethylsilane were added. After 10 min, the oligoribonucleotide was precipitated by adding 3 mL of ether. The pellets were collected by centrifugation, the supernatants were discarded, and the solids were reconstituted in 1 mL buffer 10 mM sodium 5 phosphate. 3. Purification of oligoribonucleotides Crude oligonucleotides were purified by reversed phase HPLC on an AKTA Explorer system (Amersham Biosciences) using a 16/10 HR column (Amersham Biosciences) packed to a bed height of 10 cm with Source RPC 15. Buffer A was 10 mM sodium phosphate and buffer B .0 contained 65% acetonitrile in buffer A. A flow rate of 6.5 mL/min was employed. UV traces at 260, 280, and 290 nm were recorded. For DMT-on oligoribonucleotides a gradient of 7% B to 45% B within 10 column volumes (CV) was used and for cholesterol-conjugated RNA a gradient of 5% B to 100% B within 14 CV was employed. Appropriate fractions were pooled and concentrated under reduced pressure to roughly 10 mL. DMT-on oligonucleotides were treated 25 with one-third volume 1M NaOAc, pH 4.25 for several hours at ambient temp. Finally, the purified oligonucleotides were desalted by size exclusion chromatography on a column containing Sephadex G-25. The oligonucleotide solutions were concentrated to a volume <15 mL. The concentrations of the solutions were determined by measurement of the absorbance at 260 nm in a UV spectrophotometer. Until annealing the individual strands were 30 stored as frozen solutions at -20'C.

4. Analysis of oligoribonucleotides Cholesterol conjugated RNA was analyzed by CGE and LC/MS. Unconjugated RNA was also analyzed by IEX-HPLC. CGE analysis was performed on a BeckmanCoulter PACE MDQ CE instrument, equipped with a fixed wavelength detector at 254 nm. An eCap DNA capillary 5 (BeckmanCoulter) with an effective length of 20 cm was used. All single stranded RNA samples were analyzed under denaturing conditions containing 6 M urea (eCap ssDNA100 Gel Buffer Kit, BeckmanCoulter) at 40'C. Samples were injected electrokinetically with 10 kV for 5-8 sec. The run voltage was 15 kV. IEX HPLC analysis was performed on a Dionex BioLC system equipped with a fixed 0 wavelength detector (260 and 280 nm), column oven, autosampler, and internal degasser. A Dionex DNAPac P100 column (4*250mm) was used as at a flow rate of 1.0 mL/min and 30'C. Unconjugated RNA (20 [tL, 1 OD/mL concentration) was injected. Eluent A contained 20 mM Na 2

HPO

4 , 10 mM NaBr, 10% acetonitrile, pH 11 and Eluent B was 1 M NaBr in Eluent A. The elution started with 20% B for 1 min and then a linear gradient with a target concentration of 5 80% B over 20 min was employed. LC-MS analysis was performed on an Ettan [tLC-system (Amersham Bioscience) equipped with a Jetstream column heater and a fixed wavelength detector (254nm). A ThermoFinnigan LCQ DecaXP ESI-MS system with micro-spray source and ion trap detector was coupled online to the HPLC. Oligonucleotide samples (25 [tL sample, 1 OD/mL .0 concentration in water for unconjugated RNA and 40 [tL for cholesterol-conjugated RNA) were injected onto a Waters Xterra C8 MS column (2.1 x 50 mm; 2.5 pm particle size) with a flow rate of 200 [tL/min at 60'C. Composition of eluent A was 400 mM hexafluoroisopropanol (HFIP), 16.3 mM TEA in H 2 0, pH 7.9 and eluent B was methanol. For unconjugated RNA elution started with 7% B for 3 min and then a gradient from 7% B to 25% B in 13 min was 25 used. For cholesterol-conjugated material the starting conditions were 35% B for 3 min and then the concentration of eluent B was increased to 75% B in 30 min. Analysis figures are provided in Table 6. 5. Annealing of oligoribonucleotides Complementary strands were annealed by combining equimolar RNA solutions. The 30 mixture was lyophilized and reconstituted with an appropriate volume of annealing buffer (100 mM NaCl, 20 mM sodium phosphate, pH 6.8) to achieve the desired concentration. This solution was placed into a water bath at 95'C and then cooled to ambient temp. within 3h. Extent of duplex formation was monitored by native 10% polyacrylamide gel electrophoresis (PAGE) and bands were visualized by staining with the "stains all" reagent (Sigma). 5 RNA Synthesis using "standard" deprotection monomers including ribo and 2'-O-methyl 0 phosphoramidites. A. RNA/2'OMe (Thioate ends) The chimeric RNA molecules with 2'-OMe nucleotides were synthesized on a 394 ABI machine using the standard cycle written by the manufacturer with modifications to a few wait steps. The solid support was CPG (500A). The monomers were either RNA phosphoramidites or 5 2' OMe RNA phosphoramidites with standard protecting groups and used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated. Specifically the RNA phosphoramidites were 5'-O-Dimethoxytrityl-N 6 -benzoyl-2'-O-tbutyldimethylsilyl-adenosine-3'-O-(3-cyanoethyl N,N'-diisopropyl) phosphoramidite, 5'-O-Dimethoxytrityl-N 2 -isobutyryl-2'-O tbutyldimethylsilyl-guanosine-3'-O-(-cyanoethyl-N,N'-diisopropyl)phosphoramidite, 5'-0 .0 Dimethoxytrityl-N 4 -acetyl-2'-O-tbutyldimethylsilyl-cytidine-3'-O-(-cyanoethyl-N,N' diisopropyl)phosphoramidite and 5'-O-Dimethoxytrityl-2'-O-tbutyldimethylsilyl-uridine-3'-O (-cyanoethyl-N,N'-diisopropyl)phosphoramidite; the 2'OMe RNA phosphoramidites were 5' O-Dimethoxytrityl-N 6 -benzoyl-2' -O-methyl-adenosine-3 '-O-( J-cyanoethyl-N,N' -diisopropyl) phosphoramidite, 5'-O-Dimethoxytrityl-N 2 -isobutyryl-2' -O-methyl-guanosine-3 '-O-( ( 25 cyanoethyl-N,N'-diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-N 4 -acetyl-2'-O-methyl cytidine-3'-O-(-cyanoethyl-N,N'-diisopropyl)phosphoramidite and 5'-O-Dimethoxytrityl-2'-O methyl-uridine-3'-O-(-cyanoethyl-N,N'-diisopropyl)phosphoramidite. The coupling times were 10 min for all monomers. Details of the other reagents are as follows: Activator: 5-(ethylthio) 1H-tetrazole (0.25M); Cap A: 5% acetic anhydride/THF/pyridine; Cap B: 10% N 30 methylimidazole/THF. Phosphate oxidation involved THBP (10% in ACN) for 10 min while A 1 phosphorothioate oxidation utilized 0.05 M EDITH reagent /acetonitrile. Detritylation was achieved with 3% TCA/dichloromethane. The DMT protecting group was removed after the last step of the cycle. After completion of synthesis the controlled pore glass (CPG) was transferred to a screw 5 cap, sterile microfuge tube. The oligonucleotide was cleaved and simultaneously the base and phosphate groups deprotected with 1.0 mL of a mixture of ethanolic methylamine:ammonia (8 M methylamine in ethanol/ 30% aq ammonia) (1:1) for 5 hours at 55C. The tube was cooled briefly on ice and then the solution was transferred to a 5 mL centrifuge tube; this was followed by washing three times with 0.25 mL of 50% acetonitrile . The tubes were cooled at -80C for 15 0 min, before drying in a lyophilizer. The white residue obtained was resuspended in 200 uL of NMP/Et 3 N/Et 3 N-HF and heated at 65C for 1.5h to remove the TBDMS groups at the 2'-position. The oligonucleotides were then precipitated in dry diethyl ether (400 uL) containing Et 3 N (1%). The liquid was removed carefully to yield a pellet at the bottom of the tube. Residual ether was removed in the 5 speed vacuum to give the "crude" RNA as a white fluffy material. Samples were dissolved in lmL RNase free water and quantitated by measuring absorbance at 260 nm. This crude material was stored at -20C. The crude oligonucleotides were analyzed and purified by HPLC. The crude oligonucleotides were analyzed and purified by Reverse Phase IonPair (RP IP) HPLC. The RP .0 HPLC analysis was performed on a Gilson LC system, equipped with a fixed wavelength detector (260 and 280 nm), column oven, autosampler and internal degasser. An XTerra C18 column (4.6*250mm) was used at a flow rate of 1.0 mL/min at 65'C. RNA (20 [tL for analytical run, 1 mL for a preparative run at 1 OD/mL concentration) was injected. Eluent A contained 0.1 M TEAAc, HPLC water, pH 7.0 and Eluent B was 0.1 M TEAAc in HPLC water, 70% 25 acetonitrile, pH 7.0. The elution started with 10% B for 2 min, followed by 25% B in 4 min and then a linear gradient with a target concentration of 50% B over another 30 min was employed. The purified dry oligonucleotides were then desalted using Sephadex G25M . B. Synthesis of oligonucleotides with 2'-Fluoro modifications The RNA molecules were synthesized on a 394 ABI machine using the standard cycle 30 written by the manufacturer with modifications to a few wait steps. The solid support was CPG A 1) (500A, TsT AG 001 from AM Chemicals LLC and the rC and rU were from Prime Synthesis). The monomers were either RNA phosphoramidites or 2' F phosphoramidites with standard protecting groups and used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated. Specifically the RNA phosphoramidites were 5'-O-Dimethoxytrityl-N 6-benzoyl-2'-O 5 tbutyldimethylsilyl-adenosine-3'-O-(-cyanoethyl-N,N'-diisopropyl) phosphoramidite , 5'-0 Dimethoxytrityl-N -isobutyryl-2'-O-tbutyldimethylsilyl-guanosine-3'-O-(J-cyanoethyl-N,N' diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-N 4 -acetyl-2'-O-tbutyldimethylsilyl-cytidine 3'-O-(-cyanoethyl-N,N'-diisopropyl)phosphoramidite, and 5'-O-Dimethoxytrityl-2'-O tbutyldimethylsilyl-uridine-3'-O-(J-cyanoethyl-N,N'-diisopropyl)phosphoramidite; the 2'F RNA 0 phosphoramidites were 5'-O-Dimethoxytrityl-N 4 -acetyl-2'-fluoro-2'-deoxy-cytidine-3'-O-(3 cyanoethyl-N,N'-diisopropyl)phosphoramidite and 5'-O-Dimethoxytrityl-2'-fluoro -2'-deoxy uridine-3'-O-(J-cyanoethyl-N,N'-diisopropyl)phosphoramidite. The coupling times were 10 min for all monomers. Details of the other reagents are as follows: Activator: 5-ethyl thiotetrazole (0.25 M); Cap A: 5% acetic anhydride/THF/pyridine; Cap B: 10% N-methylimidazole/THF; 5 phosphate oxidation involved THBP (10% in ACN) for 10 min while phosphorothioate oxidation utilized 0.05 M EDITH reagent/acetonitrile. Detritylation was achieved with 3% TCA/dichloromethane. The DMT protecting group was removed after the last step of the cycle. After completion of synthesis, CPG was transferred to a screw cap, sterile microfuge tube. The oligonucleotide was cleaved and the base and phosphate groups were simultaneously .0 deprotected with 1.0 mL of a mixture of ethanolic ammonia (1:3) for 7 hours at 55C. The tube was cooled briefly on ice and then the solution was transferred to a 5 mL centrifuge tube; this was followed by washing three times with 0.25 mL of 50% acetonitrile . The tubes were cooled at -80'C for 15 min, before drying in a lyophilizer. The white residue obtained was resuspended in 200 uL of NMP/Et 3 N/Et 3 N-HF and 25 heated at 50'C for 16 h to remove the TBDMS groups at the 2'position. The oligonucleotides were then precipitated in dry diethyl ether (400 uL) containing Et 3 N (1%). The liquid was removed carefully to yield a pellet at the bottom of the tube. Residual ether was removed in the speed vacuum to give the "crude" RNA as a white fluffy material. Samples were dissolved in 1 mL RNase free water and quantitated by measuring the absorbance at 260 nm. This crude 30 material was stored at -20'C.

The crude oligonucleotides were analyzed and purified by HPLC. The purified dry oligonucleotides were then desalted using Sephadex G25M. C. Synthesis of phosphorothioate RNA oligoribonucleotides 5 The oligonucleotides were synthesized on a 394 ABI machine (ALN 0208) using the standard 93 step cycle written by the manufacturer with modifications to a few steps as described below. The solid support was controlled pore glass (CPG, 2 [tmole rA CPG, 520A, or rU CPG, 500A). The monomers were RNA phosphoramidites with standard protecting groups used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated. Specifically the RNA 0 phosphoramidites were 5'-O-Dimethoxytrityl- N -benzoyl-2'-O-tbutyldimethylsilyl- adenosine 3'-O-(-cyanoethyl-N,N'-diisopropyl) phosphoramidite , 5'-0-Dimethoxytrityl- N 2 -isobutyryl 2'-O-tbutyldimethylsilyl- guanosine-3'-O-( -cyanoethyl-N,N'-diisopropyl)phosphoramidite, 5' O-Dimethoxytrityl- N 4 -acetyl-2'-O-tbutyldimethylsilyl- cytidine-3'-O-( -cyanoethyl-N,N' diisopropyl)phosphoramidite and 5'-O-Dimethoxytrityl-2'-O-tbutyldimethylsilyl- uridine-3'-O 5 (-cyanoethyl-N,N'-diisopropyl)phosphoramidite. The coupling times were 10 min. Details of the other reagents are as follows: activator: 5-ethyl thiotetrazole (0.25M); Cap A: 5% acetic anhydride/THF/pyridine; Cap B: 10% N-methylimidazole/THF; PS-oxidation, 0.05M EDITH reagent /acetonitrile. Detritylation was achieved with 3% TCA/dichloromethane. After completion of synthesis the CPG was transferred to a screw cap sterile microfuge .0 tube. The oligonucleotide was cleaved and simultaneously the base and phosphate groups deprotected with 1.0 mL of a mixture of ethanolic methylamine:ammonia (1:1) for 5 hours at 55'C. The tube was cooled briefly on ice and then the solution was transferred to a 5 mL centrifuge tube; this was followed by washing with 3 x 0.25 mL of 50% acetonitrile . The tubes were cooled at -80'C for 15 min, before drying in a lyophilizer. 25 The white residue obtained was resuspended in 200 [tL of TEA-3HF and heated at 65'C for 1.5 h to remove the TBDMS groups at the 2'-position. The oligonucleotides were then precipitated by addition of 400 [tL dry MeOH. The liquid was removed after spinning in a microcentrifuge for 5 minutes on the highest speed available. Residual methanol was removed in speed vacuum. Samples were dissolved in 1 mL RNase free water and quantitated by measuring the absorbance at 260 nm. The crude material was stored at -20'C. The oligonucleotides were analyzed and purified by HPLC and then desalted using Sephadex G25M. Example 9. Synthesis of oligonucleotides with alternating! 2'-F RNA and 2'O-Me RNA 5 (Table 7) A. Synthesis of CPGs for 2'F. CPGs of 5'-O-DMTr-2'-deoxy-2'-fluororibonucleosides with appropriate base protection Bz were synthesized as shown in Scheme A. 5'-O-DMTr-2'-Deoxy-2'-fluoro-N -A and 5'-0 DMTr-2'-Deoxy-2'-fluoro-N iB-G were synthesized as reported (Kawasaki et al., J. Med. Chem., 0 1993, 36, 831). Reaction of compounds 1001 with succinic anhydride in the presence of DMAP in ethylenedichloride yielded compound 1005. Compound 1005 was treated with 2,2' dithiobis(5-nitropyridine) (DTNP) and triphenylphosphine in the presence of DMAP in acetonitrile-ethylenedichloride and subsequently with lcaa CPG as reported by Kumar et al. (Nucleosides & Nucleotides, 1996, 15, 879) yielded the desired CPG 1009. Loading of the CPG 5 was determined as reported in the literature (Prakash et al., J. Org. Chem., 2002, 67, 357). CPGs of suitably protected 2'-deoxy-2'-fluoro A, C and G were obtained as described above (Scheme A). Scheme Aa: lcaa CPG of 2'-deoxy-2'-F A(NBz), C(N Ac), G(N iBu) and U DMTrO - 1 B DMTrO B B DMTrO- 1 O OH F HO O F OF 0H 0 1001 B = U 1005 B = U 1009 B = U; Loading: 97.09 pM/g 1002 B = C(NAc) 1006 B = C(NAc) 1010 B = C(NAc); Loading: 95.30 pM/g 1003 B = A(NBz) 1007 B = A(NBz) 1011 B = A(NBz); Loading: 79.10 pM/g 20 1004 B = G(Nau) 1008 B = G(NiBu) 1012 B = G(Nau); Loading: 91.13 pM/g a (i) Succinic anhydride, DMAP/EDC (ii) DTNP, Ph 3 P, DMAP and lcaa CPG. The chimeric RNA molecules with alternating 2'-F RNA and 2'O-Me RNA were synthesized on a 394 ABI machine using the standard cycle written by the manufacturer with 25 modifications to a few wait steps. The solid support were CPG (500A). The monomers were either 2'-F RNA phosphoramidites or 2' OMe RNA phosphoramidites with standard protecting groups and used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated. Specifically the 2'OMe RNA phosphoramidites were 5'-O-Dimethoxytrityl-N 6-benzoyl-2'-O methyl-adenosine-3'-O-(J-cyanoethyl-N,N'-diisopropyl) phosphoramidite, 5'-0 Dimethoxytrityl-N -isobutyryl-2'-O-methyl-guanosine-3'-O-(J-cyanoethyl-N,N' 5 diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-N 4 -acetyl-2'-O-methyl-cytidine-3'-O-( cyanoethyl-N,N'-diisopropyl)phosphoramidite and 5'-O-Dimethoxytrityl-2'-O-methyl-uridine 3'-O-(J-cyanoethyl-N,N'-diisopropyl)phosphoramidite. The 2'F RNA phosphoramidites 5'-0 Dimethoxytrityl-N 4 -acetyl-2'-fluoro-2'-deoxy-cytidine-3'-O-(J-cyanoethyl-N,N' diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-2'-fluoro-2'-deoxy-uridine-3'-O-( 0 cyanoethyl-N,N'-diisopropyl)phosphoramidite. 5'-O-Dimethoxytrityl-2'-fluoro- N 2 -isobutyryl 2'-deoxy-guanosine-3 -O-(P -cyanoethyl-N,N'-diisopropyl)phosphoramidite and 5'-0 Dimethoxytrityl-2'-fluoro- N 2 -isobutyryl-2'-deoxy-guanosine-3'-O-(-cyanoethyl-N,N' diisopropyl)phosphoramidite. The coupling times were 10 min for all monomers. Details of the other reagents are as follows: Activator: 5-ethyl thiotetrazole (0.25M); Cap A: 5% acetic 5 anhydride/THF/pyridine; Cap B: 10% N-methylimidazole/THF; phosphate oxidation involved 0.02M 1 2

/THF/H

2 0, while PS-oxidation was carried out using EDITH reagent as described above. Detritylation was achieved with 3% TCA/dichloromethane. The final DMT protecting group was removed in the synthesizer. After completion of synthesis the CPG was transferred to a screw cap, sterile microfuge .0 tube. The oligonucleotide was cleaved and the base and phosphate groups were simultaneously deprotected with 1.0 mL of a mixture of ethanolic:ammonia (1:3) for 7 hours at 55C. The tube was cooled briefly on ice and then the solution was transferred to a 5 mL centrifuge tube; this was followed by washing three times with 0.25 mL of 50% acetonitrile. The tubes were cooled at -80'C for 15 min before drying in a lyophilizer to give the "crude" RNA as a white fluffy 25 material. Samples were dissolved in lmL RNase free water and quantitated by measuring the absorbance at 260 nm. This crude material was stored at -20 0 C. The crude oligonucleotides were analyzed and purified by 20% polyacrylimide denaturing gels. The purified dry oligonucleotides were then desalted using Sephadex G25M (Amersham Biosciences). 30 B. Analysis of Duplex activity Duplexes were tested for activity in the HeLa cell assay described above. Table 7 and Figure 30 provides graphs of the activities in HeLa cells for each of the modifications described above. 5 Example 10 Conjug~ated VEGF molecules (Tables 8, 9, 10 and 18) 1. Synthesis: The RNA molecules were synthesized on an ABI-394 machine (Applied Biosystems) using the standard 93 step cycle written by the manufacturer with modifications to a few wait 0 steps as described below. The solid support was controlled pore glass (CPG, lumole, 500 A) and the monomers were RNA phosphoramidites with standard protecting groups (5'-0 dimethoxytrityl-N6-benzoyl-2'-O-t-butyldimethylsilyl-adenosine-3'-O-N,N'-diisopropyl-2 cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-O-t-butyldimethylsilyl-cytidine 3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N2-isobutryl-2'-O-t 5 butyldimethylsilyl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and 5'-0 dimethoxytrityl-2'-O-t-butyldimethylsilyl-uridine-3'-O-N,N'-diisopropyl-2 cyanoethylphosphoramidite. All amidites were used at a concentration of 0. 15M in acetonitrile

(CH

3 CN) and a coupling time of 6 min for unmodified and 2'-O-Me modified monomers and 12 min for modified and conjugated monomers. 5-ethyl thiotetrazole (0.25M) was used as an .0 activator. For the PO-oxidation Iodine/Water/Pyridine and for PS-oxidation Beaucage reagent (2 %) in anhy. acetonitrile was used. The sulfurization time was about 6 min. All syntheses was performed on a 1 umole scale. Reagents Concentration Wait or Coupling step Activator: 0.25M 5-Ethylthio- 1H-tetrazole 720 see PO-oxidation 0.02M Iodine in THF/Water/Pyridine 20 see PO-oxidation 0.02M t-Butyl-hydrogen peroxide 600 sec PS-oxidation 2 % Beaucage reagent /anhy. 360 Sec (200 Acetonitrile sec, wait +30 sec pulse+130 see wait Cap A 5% 5%Phenoxyacetic 20 sec anhydride/THF/pyridine Cap B 10% 10% N-methylimidazole/THF 20 sec Detritylation 3% TCA Trichloro Acetic Acid 70 sec /dichloromethane The following types of modifications were used to perform the synthesis using these protocols: 1. Unmodified phosphodiester backbone (PO) only 5 2. Phosphorothioate (PS) only 3. 2'-O-Me, PS 4. 3'-Naproxen, 2'F- 5Me-U, PS 5. 5'-Cholesterol, PS 6. 3'-Choletserol,PS 0 7. 2'F- 5Me-U, PS 8. 3'-Biotin, 2'F- 5Me-U, PS 9. 3'-cholanic acid, 2'F- 5Me-U, PS 10. Methylphosphonate 11. C-5 allyamino rU 5 2. Deprotection- I (Nucleobase Deprotection) After completion of the synthesis, the controlled pore glass (CPG) was transferred to a screw cap vial or screw cap RNase free microfuge tube. The oligonucleotide was cleaved from the support and the base and phosphate protecting groups were simultaneously removed by using 20 of a mixture of ethanolic ammonia (ammonia (28-30 % : ethanol (3:1))- (1.0 mL) for 15h at 55'C. The vial was cooled briefly on ice and then the ethanolic ammonia mixture was transferred to a new microfuge tube. The CPG was washed with portions of deionized water (2 x 0.1 mL). The supernatant was combined, cooled in dry ice for 10 min and then dried in a speed vac. 25 3. Deprotection-II (Removal of 2'-0- TBDMS group) The white residue obtained was resuspended in a mixture of triethylamine, triethylamine trihydrofluoride (TEA.3HF ca. 24% HF)) and 1-Methyl-2-Pyrrolidinone (NMP) (4:3:7) (400 ul) and heated at 65C for 90 min to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction was then quenched with isopropoxytrimethylsilane (iPrOMe 3 Si, 400 ul) and further incubated on the heating block leaving the caps open for 10min; This causes the volatile isopropoxytrimethylsilylfluoride adduct to vaporize. The residual quenching reagent 5 was removed by drying in a speed vac. 3% Triethylamine in diethyl ether ( 1.5 ml) was added. The mixture was subjected to centrifugation. A pellet of RNA formed. The supernatant was pipetted out without disturbing the pellet. The pellet was dried in a speed vac. The crude RNA was obtained as a white fluffy material in the microfuge tube. 0 4. Quantitation of Crude Oligomer or Raw Analysis Samples were dissolved in deionized water (1.OmL) and quantitated as follows: Blanking was first performed with water alone (lmL). A sample of the RNA solution (20u1) was diluted with water (980 uL) and mixed well in a microfuge tube, then transferred to a cuvette and the 5 absorbance reading was obtained at 260 nm. The crude material was dried down and stored at 20 0 C 5. MS analysis: The crude samples (0.1 OD) analyzed using LC-MS. 0 6. Purification of Oligomers (a) Polyacrylamide Gel Electrophoresis (PAGE) Purification The oligonucleotides were purified by vertical slab polyacrylamide gel electrophoresis (PAGE) using an Owl's Separation Systems (Portsmouth, NH). Electrophoresis grade 25 acrylamide (40%), N,N'-methylene-bis(acrylamide) (BIS), ammonium persulfate (APS, N,N,N'N'-tetramethylenediamine (TEMED), bromophenol blue (BPB), xylene cyanol (XC) 10 x TBE (0.89 M tris-hydroxy-methylaminomethane, borate pH 8.3, 20mM disodium ethylenediaminetetraacetate) were from National Diagnostics (Atlanta, GA). The 12 % denaturing gel was prepared for purification of unmodified and modified oligoribonucleotides.

The thickness of the preparative gels was 1.5 mm. Loading buffer was 80% formamide in lOx TBE. After removal of the glass plates, the gels were covered with Saran Wrap® and placed over a fluorescent TLC plate illuminated by a hand-held UV lamp for visualization. The desired bands were excised and shaken overnight in 2mL of water or 0.03 M Sodium Acetate. The eluent 5 was removed by drying in a speed vac. (b) High Performance Liquid chromatography (HPLC) Purification: Condition A: Purification of unmodified, 2'-O-Me/PS Oligoribonucleotides: Amount of injected sample is about ~100 OD. 0 Column: Dionex PA- 100 Semiprep. Buffer A: Water Buffer B: 0.25 M Tris.Cl pH 8.0 Buffer C: 0.375 M Sod.Perchlorate Heating: 65 C 5 Time Flow Buffer A Buffer B Buffer C TotalYield Purity 0 5.00 88 % 10 % 2.0 % 40-60% 85-98 % 3.0 5.00 88% 10% 2.0% 30.0 5.00 57.0 10 % 33.0 35.0 5.00 88% 10% 2.0% 40.0 5.00 88% 10% 2.0% Condition B: Protocols for Purification of 2'-O-Me/PS Oligoribonucleotides: Column: Dionex PA- 100 Semiprep. Buffer A: Water 20 Buffer B 0.25 M Tris.Cl pH 8.0 Buffer C 0.8 M Sod.Perchlorate Heating: 65 C Time Flow Buffer A Buffer B Buffer C Total Purity Yield 0 5.00 88 % 10 % 2.0 % -40-60% 85-98 % 3.0 5.00 88% 10% 2.0% 30.0 5.00 57.0 10 % 33.0 35.0 5.00 88 % 10 % 2.0 % 40.0 5.00 88% 10% 2.0% 7. Desalting of Purified Oligomer The purified dry oligomer was then desalted using Sephadex G-25 M. The cartridge was conditioned with 10 mL of deionised water thrice. Finally the purified oligomer dissolved 5 thoroughly in 2.5mL RNAse free water was applied to the cartridge with a very slow drop-wise elution. The salt free oligomer was eluted with 3.5 ml deionized water directly into a screw cap vial. The purified RNA material was dried down in speed vac and stored at -20'C. 0 Biotin conjugated siRNAs (Table 10) 1. Synthesis: The RNA molecules were synthesized on an ABI-394 machine (Applied Biosystems) using the standard 93 step cycle written by the manufacturer with modifications to a few wait steps as described below. The solid support was controlled pore glass (CPG, lumole, 500 A) and 5 the monomers were RNA phosphoramidites with standard protecting groups (5'-0 dimethoxytrityl N6-Benzoyl-2'O-t-butyldimethylsilyl- adenosine-3'-O-N,N'-diisopropyl-2 cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-O-t-butyldimethylsilyl-cytidine 3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N2-isobutryl-2'-O-t butyldimethylsilyl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and 5'-0 .0 dimethoxytrityl-2'-O-t-butyldimethylsilyl-uridine-3'-O-N,N'-diisopropyl-2 cyanoethylphosphoramidite. The modified CPG and amidites were synthesized using known methods and as described herein. . All amidites were used at a concentration of 0.15M in acetonitrile (CH 3 CN) and a coupling time of 6 min for unmodified and 2'-O-Me monomers and 12 min for modified and conjugated monomers. 5-Ethylthio-1H-tetrazole (0.25M) was used as an 25 activator. For the PO-oxidation Iodine/Water/Pyridine and for PS-oxidation Beaucage reagent (2 %) in anhy. acetonitrile was used. The sulfurization time is about 6 min. For synthesis of 3' biotin conjugated siRNAs, t-butyl-hydrogen peroxide was used as oxidizing agent (oxidation time 10 min). -71 Reagent Concentration Wait or Coupling step Activator: 0.25M 5-Ethylthio-tetrazole 300 sec for unmodified and 720 sec for modified oligos. PO-oxidation 0.02M Iodine in THF/water/pyridine 20sec PO-oxidation 0.02M t-Butyl-hydrogen peroxide 600 sec PS-oxidation 2 % Beaucage reagent /anhy. 360 Sec (200 Acetonitrile sec, wait +30 sec pulse+130 see wait Cap A 5% 5% Phenoxyacetic 20sec anhydride/THF/pyridine Cap B 10% 10% N- 20sec Methylimidazole/THF Detritylation 3% TCA Trichloro Acetic 70sec Acid/dichloromethane 2. Deprotection- I (Nucleobase Deprotection) After completion of synthesis the controlled pore glass (CPG) was transferred to a screw cap vial or a screw cap RNase free microfuge tube. The oligonucleotide was cleaved from the 5 support with the simultaneous removal of base and phosphate protecting groups with a mixture of ethanolic ammonia [ammonia (28-30%): ethanol (3:1) 1.0 mL] for 15h at 55'C. The vial was cooled briefly on ice and then the ethanolic ammonia mixture was transferred to a new microfuge tube. The CPG was washed with portions of deionized water (2 x 0.1 mL). The combined filtrate was then put in dry ice for 10 min dried in a speed vac. 10 3. Deprotection-II (Removal of 2'-0- TBDMS group) The white residue obtained was resuspended in a mixture of triethylamine, triethylamine trihydrofluoride (TEA.3HF ca, 24% HF) and 1-Methyl-2-Pyrrolidinone (NMP) (4:3:7) (400 ul) and heated at 65'C for 90 min to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' 15 position. The reaction was then quenched with isopropoxytrimethylsilane (iPrOMe 3 Si, 400 ul) and further incubated on the heating block leaving the caps open for 10min; (This causes the volatile isopropxytrimethylsilylfluoride adduct to vaporize). The residual quenching reagent was -71 removed by drying in a speed vac. 3% Triethylamine in diethyl ether (1.5 ml) was added and the mixture was subjected to centrifugation to afford a pellet of RNA. The supernatant was pipetted out without disturbing the pellet. The pellet was dried in a speed vac. The crude RNA was obtained as a white fluffy material in the microfuge tube. 5 4. Quantitation of Crude Oligomer or Raw Analysis Samples were dissolved in deionized water (1.OmL) and quantitated as follows: Blanking was first performed with water alone (lmL). A sample of the RNA solution (20u1) was diluted with water (980 uL) and mixed well in a microfuge tube, then transferred to a cuvette and the 0 absorbance reading was obtained at 260 nm. The crude material was dried down and stored at 20 0 C. 5. MS analysis: Samples of the RNA ( 0.1 OD) were analyzed using MS. 5 6. Purification of Oligomers Polyacrylamide Gel Electrophoresis (PAGE) Purification The oligonucleotides were purified by vertical slab polyacrylamide gel electrophoresis (PAGE) using an Owl's Separation Systems (Portsmouth, NH). Electrophoresis grade .0 acrylamide (40%), N,N'-methylene-bis(acrylamide) (BIS), ammonium persulfate (APS, N,N,N'N'-tetramethylenediamine (TEMED), bromophenol blue (BPB), xylene cyanol (XC) 10 x TBE (0.89 M). Trishydroxy-methylaminomethane, borate (pH 8.3), 20mM disodium ethylenediaminetetraacetate) were from National Diagnostics (Atlanta, GA). The 12 % Denaturing gel was prepared for purification of oligoribonucleotides. The thickness of the 25 preparative gel was 1.5 mm. Loading buffer was 80% formamide in lOx TBE. After removal of the PAGE glass plates, the gels were covered with Saran Wrap® and placed over a fluorescent TLC plate illuminated by a hand-held UV lamp (Upland, CA) for visualization. The desired bands were excised and shaken overnight in water (2mL) or 0.03 M sodium acetate. The eluent was removed and dried in a speed vac. All biotin conjugated sequences were purified by PAGE.

7. Desalting of Purified Oligomer The purified dry oligomer was then desalted using Sephadex G-25 M (Amersham Biosciences). The cartridge was conditioned with of deionized water thrice (10 mL each). Finally the purified oligomer dissolved thoroughly in 2.5mL RNAse free water was applied to 5 the cartridge with very slow drop- wise elution. The salt free oligomer was eluted with deionized water (3.5 ml) directly into a screw cap vial. The purified RNA material was dried down on speed vac and stored at -20'C 8. Quality Control 0 (a) Capillary Gel Electrophoresis (CGE) (b) Electrospray LC/Ms A sample of the oligomer (approx. 0.10 OD) was dissolved in water (50 ul & 100 ml in separate tubes) and then pipetted into special vials for CGE and LC/MS analysis. 5 9. Analysis of Duplex activity Duplexes were tested for activity in the HeLa cell assay described above. Tables 8, 9, 10 and 18 and Figures 31-35 provides data and graphs of the activities in HeLa cells for each of the modifications described above. .0 Example 11 Conjugation of retinoids to RNA (Table 14) Conjugation of all-trans-retinal to Oligonucleotides (RNA): Phoshoramidite 104 was synthesized as shown in Scheme B for retinal conjugation to oligonucleotides. Scheme B: Synthesis of Post-synthetic conjugation building blocks for retinal 25 conjugation - oxime approach 1 for 5'-conjugation. '7A 00 Bn O'H 1,* Bn ~-N HOiO-N 101 02 O 103 0 NO NC O.

0 ~' O0-N 104 a (i) Ph 3 P, DIAD, N-hydroxyphthalimide / MeCN; (ii) H 2 , Pd-C (10 %), 1 atm/ETOAc; (iii) Phosphitylation 5 Step 1: Compound 102:Monobenzylpentan-1,5-diol (15.70 g, 80.82 mmol), Ph 3 P (25.43 g, 96.84 mmol) and and N-hydroxyphthalimide (1 16.0g, 98.08 mmol) were taken in anhydrous

CH

3 CN (100 ml) under argon atm. Neat DIAD(20.0 mL, 103.25 mmol) was added dropwise into the stirring solution over a period of 20 minutes and the stirring was continued for 24h. The reaction was monitored by TLC. Solvents were removed in vacuo; and the residue was triturated 0 with diethyl ether and filtered. Residue was washed with ether, filtered and combined the filtrate. Hexane was added dropwise into the filtrate until it gave turbidity and subsequently the solution was made homogeneous by adding ether into it. The homogeneous solution was stored at 5 C for 24 h. Precipitated Ph 3 PO was filtered off, washed with ether-hexane mixture (1:1). Combined filtrate was evaporated to dryness and the residue was purified by flash silica gel column 5 chromatography (10-15 % EtOAc in Hexane) to obtain 24.5 g (89.3 %) of compound 102 as a viscous pale yellow oil. 1H NMR (400 MHz, CDCl 3 , 25 C): 7.84-7.82 (m, 2H); 7.75-7.73 (m, 2H); 7.34-7.33 (m, 4H); 7.29-7.26 (m, 1H); 4.51 (s, 2H); 4.22-4.18 (t, J(H,H) = 6.71 Hz, 2H); 3.52-3.48 (t, J(H,H) = 6.4 Hz, 2H); 2.04-1.78 (m, 2H); 1.73-1.56 (m, 4H). 1C NMR (100 MHz, CDCl 3 , 25 C): 163.9, 138.8, 134.6,129.2, 128.6, 127.8, 127.7, 123.7, 78.6, 73.1, 70.3, 29.6, 20 28.2, 22.5. Step 2: Compound 103: Compound 102 (23.5 g, 69.29 mmol) was taken in 100 ml of EtOAc/methanol (1:1). The mixture was degassed and purged with argon, to this 2.4 g of Pd-C (10%- wet Degusa type) was added. The mixture was then hydrogenated overnight, filtered 25 through a celite bed over a sintered funnel. The residue was subsequently passed through a -. 7 r, column of silica gel and eluted out using 40 % EtOAc in hexane to obtain compound 103 (15.70 g, 90.9 %) as a white solid. 1H NMR (400 MHz, CDCl 3 , 25 C) 7.83-7.81 (bm. 2H); 7.75-7.73 (bm, 2H); 4.23-4.19 (t, J(H,H) = 6.4 Hz, 2H); 3.70-3.66 (t, J(H,H) = 5.80 Hz, 2H); 1.83-1.79 (m, 2H); 1.67-1.60 (m, 4H). 1C NMR (100 MHz, CDCl 3 , 25 0 C) E 163.9, 134.7, 129.1, 123.7, 78.6, 5 62.7, 32.4, 28.0, 22.0. Step 3: Compound 104: Compound 103 (5.4 g, 21.67 mmol) and triethylamine (4 ml, 28.69 mmol) were taken in anhydrous EtOAc(30 ml) under argon. 2-Cyanoethyl diisopropylchlorophosphoramidite (5.00ml, 21.97 mmol) was added to the reaction mixture 0 dropwise. A white precipitate of Et 3 N.HCl was formed immediately after the addition of the reagent and the reaction was complete in 10 min (monitored by TLC). The precipitate was filtered through a sintered funnel and solvent was removed under reduced pressure. The residue was directly loaded on a silica gel column for purification. Eluted with hexane/EtOAc 9:1 to afford compound 104 as a yellow oil, 8.68g (89.13%). 1 H NMR (400 MHz, CDCl 3 , 25 0 C) D 5 7.85-7.81 (m, 2H); 6 7.77-7.72 (m, 2H); 4.22-4.19 (t. J(H,H) = 6.80 Hz, 2H); 3.91-3.76 (m, 2H); 3.72-3.53 (m, 4H)2.67-2.63 (t, J(H,H) = 6.71 Hz, 2H); 1.86-1.78 (m, 2H); 1.73-1.66 (m, 2H); 1.62-1.56 (m, 2H); 1.19-1.16 (m, 12H). P NMR (162 MHz, CDCl 3 , 25 0 C) 6 145.09. 1C NMR (100 MHz, CDCl 3 , 25 0 C) 6 163.9, 134.7, 129.2, 123.7, 117.9, 78.6, 64.0, 63.4, 58.7, 58.5, 43.2, 43.1, 31.1, 31.0, 28.1, 24.9, 24.8, 24.7, 22.3, 20.6, 20.5. 0 Step 4: Conjugation of all-trans-retinal to Oligonucleotide: All-trans-retinal was conjugated to oligonucleotide as shown in the Scheme C. Compound 104 was coupled to solid bound oligonucleotide 105 under standard solid phase oligonucleotide synthesis conditions to obtain compound 106. Phthalimido protecting group on compound 106 was selectively removed 25 by treating with hydrazinium hydrate as reported by Salo et al. (Bioconjugate Chem. 1999, 10, 815) to obtain compound 107. Treatment of compound 107 with all-trans-retinal under dark condition gave compound 108 as reported in the literature (Bioconjugate Chem. 1999, 10, 815). Standard RNA oligonucleotide deprotection and purification under dark yielded the desired oligonucleotide-retinal conjugate 109. Compound 109 was also obtained from compound 110 as 30 shown in Scheme C. Complete deprotection and purification of compound 106 yielded an -7 A unbound free oligonucleotide 110 which was subsequently reacted with all-trans-retinal to afford the desired compound 109. Scheme C: Conjugation of all-trans-retinal to oligonucleotides C-i ' 5' OH , 0 O O-N 105 106 ON 0 V0 10 11 CG ,N C0 '-NH 2 HO 0 O O-NH 2 H 107 ON 110 iii vi C 108 O' O-N ON 0 V 5 5' 3' N~N-0"OY'03 - OH 5 109 a (i) Phosphoramidite 104, (standard oligonucleotide synthesis cycle); (ii) Hydrazinium hydrate/Py/AcOH (0.124/4/7); (iii) all-trans-retinal in DMF or MeCN; (iv) Oligonucleotide (RNA) deprotection (MeNH 2 , TEA.3HF) and purification; (v) Oligonucleotide (RNA) 10 deprotection (MeNH 2 , TEA.3HF) and purification; (vi) all-trans-retinal in DMSO-H 2 0 Step 4.1. Oligonucleotide Synthesis: All oligonucleotides except AL-3166 were synthesized on an ABI 490 DNA synthesizer. Commercially available controlled pore glass solid supports (dT-CPG and U-CPG, 500A) and 15 RNA phosphoramidites with standard protecting groups, 5'-O-dimethoxytrityl-N6-benzoyl-2'-t butyldimethylsilyl-adenosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-0 dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-0-N,N'-diisopropyl-2 cyanoethylphosphoramidite, 5'-0-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl -7-7 guanosine-3'- O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and 5'-O-dimethoxytrityl-2'-t butyldimethylsilyl-uridine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite were used for the oligonucleotide synthesis. All phosphoramidites were used at a concentration of 0.15M in acetonitrile (CH 3 CN). Coupling time of 10 minutes was used. The activator was 5-ethyl 5 thiotetrazole (0.25M), for the PO-oxidation Iodine/Water/Pyridine was used. Sequence AL-3166 was synthesized on the AKTAoligopilot synthesizer. All phosphoramidites were used at a concentration of 0.2M in acetonitrile (CH 3 CN) except for guanosine which was used at 0.2M concentration in 10% THF/acetonitrile (v/v). 0 Coupling/recycling time of 16 minutes was used. The activator was 5-ethyl thiotetrazole (0.75M), for the PO-oxidation Iodine/Water/Pyridine was used and for the PS-oxidation PADS (2 %) in 2,6-lutidine/ACN (1:1 v/v) was used. The aminooxy-linker phosphoramidite was synthesized as described above and used at a 5 concentration of 0. 15M in acetonitrile. Coupling time for the aminooxy-linker phosphoramidite was 15 minutes. For all sequences, coupling of the aminooxy-linker phosphoramidite was carried out on the ABI 390 DNA synthesizer. Step 4.2. Cleavage of the phthalimido-protecting group from the aminoxy-linker .0 oligonucleotides After coupling of the aminooxy-linker, the CPG was treated with 2.5 ml of 0.5M hydrazinium acetate in pyridine (0.16/4/2 hydrazine anhydrous, pyridine, acetic acid) using the dual syringe method. Every 5 minutes the syringes were pushed back and forth to get new solution on the CPG. After the hydarzinium acetate treatment, the CPG was washed with 2x5 ml 25 of pyridine followed by 3x5ml of acetonitrile. Flushing with dry argon for 30 seconds then dried CPG. Step 4.3. On support conjugation with the aldehydes The 1-pyrene-carboxaldehyde and the all-trans-retinal were from Aldrich and used at concentrations of 0.5M in DMF. The 4-keto-retinol was used at a concentration of 0. 13M in 30 DMF. The CPG from above was added to the aldehyde solutions. Conjugation was carried out -7Q overnight (~16 hrs) at room temperature. After the reaction was complete, the CPG was rinsed with DMF followed by acetonitrile and air dried for 10-15 minutes. For sequence AL-3213, the conjugation with both all-trans-retinal and 1-pyrene-carboxaldehyde was also carried out in acetonitrile. In the case of 1-pyrene-carboxaldehyde, the aldehyde did not fully dissolved at 5 0.5M and the solution was used as is without filtration to get rid of the undissolved aldehyde. Step 4.4. Deprotection- I (Nucleobase Deprotection) of on support conjugated oligonucleotides For on support retinal conjugated oligonucleotides, the support was transferred to a 5 ml 0 tube (VWR). The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 1 mL of 40% aq. methylamine 15 mins at 65'C. The tube was cooled briefly on ice and then the methylamine was filtered into a new 15 ml tube. The CPG was washed with 3 x 1 mL portions of DMSO. 5 Step 4.5. Deprotection-II (Removal of 2' TBDMS group) of on support conjugated oligonucleotides To the above mixture was added 1.5 ml of triethylamine trihydrofluoride (TREAT-HF) and heated at 60'C for 15 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction was then quenched with 5.5 ml of 50mM sodium acetate (pH 5.5) and .0 stored in freezer until purification. Step 4.6. After deprotection conjugation with aldehydes Conjugation with the aldehydes (1-pyrene-carboxaldehyde and all-trans-retinal) after deprotection of the aminooxy-linker oligonucleotides was also carried out as an alternative 25 conjugation strategy. Step 4.7. Deprotection- I (Nucleobase Deprotection) for after deprotection conjugation The support was transferred to a 2 ml screw cap tube. The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 0.5 mL of 30 40% aq. methylamine 15 mins at 65'C. The tube was cooled briefly on ice and then the 70 methylamine was filtered into a new 15 ml tube. The CPG was washed with 2 x 0.5 mL portions of 50% acetonitrile/water. The mixture was then frozen on dry ice and dried under vacuum on a speed vac. 5 Step 4.8. Deprotection-II (Removal of 2' TBDMS group) for after deprotection conjugation The dried residue was resuspended in 0.5 ml of triethylamine trihydrofluoride (TEA.3HF) and heated at 60'C for 15 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction mixture was then cooled to room temperature and RNA 0 precipitated with 2 ml of dry methanol and dried under vacuum on a speed vac. The sample was then dissolved in 2 ml of water and kept frozen in freezer till further analysis. Step 4.9. Quantitation of Crude Oligomer or Raw Analysis For all samples, a lpl, a 10pl or 30pl aliqoute was diluted with 999pl, 990pl or 970pl of 5 deionised nuclease free water (1.0 mL) and absorbance reading obtained at 260 nm. Step 4.10. Purification of conjugated Oligomers (a) Cude LC/MS analysis The crude oligomers were first analyzed by LC/MS, to look at the presence and .0 abundance of the expected final product. (b) Reverse-phase purification The conjugated samples were purified by reverse-phase HPLC on an RPC-Sourcel5 column (21.5 x 1 cm). The buffer system was: A = 20 mM sodium acetate in 10% ACN, pH 8.5 25 and B = 20 mM sodium acetate in 70% ACN, pH 8.5, with a flow rate of 5.0 mL/min, and wavelengths 260 and 375. The fractions containing the full-length oligonucleotides were then individually desalted. Step 4.11. Desalting of purified oligonucleotides 30 The purified oligonucleotide fractions were desalted using the PD-10 Sephadex G-25 columns. First the columns were equilibrated with 25-30 ml of water. The samples were then applied in a volume of 2.5 ml. The samples were then eluted in salt-free fraction of 3.5 ml. The desalted fractions were combined together and kept frozen till needed. 5 Step 4.12. Capillary Gel Electrophoresis (CGE), Ion-Exchange HPLC (IEX) and Electrospray LC/Ms Approximately 0.3 OD of desalted oligonucleotides were diluted in water to 300 Pl and then pipetted in special vials for CGE, IEX and LC/MS analysis. 0 Step 5 Conjugation of all-trans-retinal to 3'-end of Oligonucleotides (RNA): Phoshoramidite 116 for 5'-conjugation and CPG support 115 for 3'-conjugation of retinoids were synthesized as shown in the Scheme D. The CPG support 115 is used for 3' conjugation of retinoids to oligonucleotides Scheme Da. Synthesis of Post-synthetic conjugation building blocks for retinal 5 conjugation - oxime approach 2 for 3' and 5' conjugation. HQ TBDMSQ TBDMSQO ODMTr ODMTr Hi / ODMTr H N N N H O NHCbz O NHCbz N OH 111 112 113 0 HQ ODMTr N, IQI N H V 0 \,ODMTr :_ Ny-_ _ N H 114O-NNC O ON iv 116 0 O O H O CODMTr O O-N 115 0o a (i) TBDMS-Cl, Imidazole/Py, rt; (ii) (a) H 2 , Pd-C (10 %)/EtOAC-MeOH, 4 h and (b) 0 20 Caprolactone, TEA, 55 C, 24 h; (iii) TEA.3HF/THF; (iv) (a) Succinic anhydride, DMAP/EDC, 24 h and (b) DTNP, Ph 3 P, DMAP followed by addition of lcaa CPG; (v) Phosphitylation 21 Step 5.1: Compound 112: Compound 111 (120.0 g, 30.01 mmol) was stirred with TBDMS-Cl (5.43 g, 36.02 mmol) in the presence of imidazole (7.5 g, 110.16 mmol) in anhydrous pyridine (100 mL) overnight. After removing pyridine, the product was extracted into ethyl acetate (300 mL), washed with aqueous sodium bicarbonate, followed by standard workup. 5 Residue obtained was subjected to flash silica gel column chromatography using 1 % methanol in dichloromethane as eluent to afford compound 112as a pale white solid (24.4 g, qunat. 1 H NMR (500 MHz, [D 6 ]DMSO, 25 C): D 7.33-7.13 (bm, 15H, accounted for 14H after D 2 0 exchange); 6.87-6.82 (bm, 4H); 5.01 (s, 0.2H, rotamer minor); 4.99 (s, 1.8H, rotamer major), 4.68-4.64 (m, 0.72 H, major rotamer); 4.14-4.07 (bm, 1H), 3.72 (s, 7H), 3.38-3.36 (m, 0.6H, 0 rotamer minor); 3.26-3.21 (m, 1.4H, rotamer major); 3.08-3.07 (m, 0.3H, rotamer, minor); 2.99 2.89 (m, 2.7H, rotamer, major); 2.22-2.12 (m, 2H), 2.04-1.78 (m, 2H); 1.48-1.23 (m, 6H), 0.84, 0.82 (s, 9H, rotamers major and minor); 0.05 (d, J(H,H) = 1.5 Hz, 4.3H, rotamer major); 0.03 0.02 (d, J(H,H) = 5.5 Hz, 1.7H). Step 5.2: Compound 113: Compound 112 (9.4 g, 14.54 mmol) was suspended in 15 mL 5 of -caprolactone and 10 mL of TEA was added into the suspension. The reaction mixture was stirred under argon at 55 C bath temperature for 24 h. Completion of the reaction was monitored by TLC analysis. TEA was removed form the reaction mixture in vacuo and 150 mL of dichloromethane-hexane (2:1 mixture) was added into the residue. The homogeneous solution thus obtained was directly loaded on a column of silica gel and eluted with dichloromethane .0 hexane (2:1) followed by neat dichloromethane. Elution of the silica column with 4 % methanol in dichloromethane afforded the desired compound 113 as a white solid (8.73 g, 78.9 %). 1 H NMR (400 MHz, [D 6 ]DMSO, 25 C) 6 7.72-7.68 (bm, 1H, exchangeable with D 2 0); 7.33-7.16 (m, 9H); 6.88-6.84 (m, 4H); 4.68-4.62 (m, 0.8H); 4.57-4.52 (m, 0.2H); 4.34-4.31 (t, J(H,H) = 5.18 Hz, 1H, exchangeable with D 2 0); 4.14-4.08 (bm, 1H); 3.74-3.67 (m, 7H); 3.39-3.32 (m, 25 3.3H); 3.25-3.21 (m, 1.7H); 3.09-2.88 (m, 4H) 6. Analysis of Duplex activity Duplexes were tested for activity in the HeLa cell assay described above. Table 14 and Figure 38 provides data and a graph of the activities in HeLa cells for each of the modifications 30 described above. Q1) Example 12 Conjug~ation of Polyethylene glycol to siRNA (Table 12) Amino linker oligonucleotides for PEG Conjugation General. Ion exchange preparative chromatography was performed on TSKgel-SuperQ 5PW (Tosoh). Ion exchange analytical chromatography was performed on a DNAPac PalOO 5 (Dionex). Electron spray ionization mass spectra were recorded with an Agilent 1100 MSD-SL. HPLC Techniques. The RNA was analyzed by ion-exchange chromatography (column, DNAPac PalOO, 4x250mm, analytical), heated to 30'C, flow rate 1.5 mL min , buffer A = 0.020M Na 2

HPO

4 in 10% CH 3 CN, pH 11; buffer B = buffer A + 1 M NaBr in 10% CH 3 CN, pH 11, linear gradient from 0 to 75% in 53 min. The LC/ESI-MS conditions were as follows: 0 column XTerra C8 (2. 1x30 mm, 2.5p m), linear gradient from 5 to 35% in 2 min and from 35 to 70% in 30.5 min, flow rate 0.200 mL min-, buffer A = 400mM HFIP/16.3mM TEA in H 2 0, buffer B = 100% methanol. The RNA was purified by ion-exchange chromatography (5cm in house packed column, TSKgel-SuperQ-5PW, 20pm), heated to 75'C, flow rate 50 mL min- , buffer A = 0.020M Na 2

HPO

4 in 10% CH 3 CN, pH 8.5; buffer B = buffer A + 1 M NaBr in 10% 5 CH 3 CN, pH 8.5, linear gradient from 20 to 55% in 120 min. RNA synthesis. The protected RNA was assembled on an AKTA Oligo Pilot 100 on a 100-150 pmol scale using custom in-house support and phosphoramidite chemistry. Phosphoramidites were used as 0.2 mol L- 1 solutions in dry CH 3 CN, with a 900s coupling time and the manufacturer's recommended synthesis protocols were used. After synthesis, the .0 support-bound RNA was treated with aqueous CH 3

NH

2 (40%)for 90 minutes at 45'C, cooled, filtered and washed with DMSO (3x4OmL). The filtrate was then treated with TEA.3HF (60mL) for 60 minutes at 40'C, and quenched with aq. NaOAc (0.05M, pH 5.5, 200mL). The synthesis was followed by analytical ion-exchange HPLC, preparative HPLC, then desalting on Sephadex G-25. 25 Step 1. Oligonucleotide Synthesis: A general conjugation approach is shown in the Scheme E. All oligonucleotides were synthesized on an AKTAoligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500A) or the phthalimido-hydroxy 30 prolinol solid support and RNA phosphoramidites with standard protecting groups, 5'-0dimethoxytrityl-N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O-N,N'-diisopropyl-2 cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3' O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, 5'-O-dimethoxytrityl-N2-isobutryl-2'-t butyldimethylsilyl-guanosine-3'-O-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and 5'-0 5 dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O-N,N' -diisopropyl-2 cyanoethylphosphoramidite were used for the oligonucleotide synthesis. All phosphoramidites were used at a concentration of 0.2M in acetonitrile (CH 3 CN) except for guanosine which was used at 0.2M concentration in 10% THF/acetonitrile (v/v). Coupling/recycling time of 16 minutes was used. The activator was 5-ethyl thiotetrazole (0.75M), for the PO-oxidation 0 Iodine/Water/Pyridine was used and for the PS-oxidation PADS (2 %) in 2,6-lutidine/ACN (1:1 v/v) was used. The amino-linker phosphoramidite was synthesized and used at a concentration of 0.2M in acetonitrile. Coupling/recycling time for the amino-linker phosphoramidite was 16 minutes. Scheme Ea: Pegylation of RNA Oligonucleotides CN 0 0KOH Q O ONDMTr ON ON 0a 0 X=OorS EH 3' 5' e 3' 5' O. 0 0 OH OK- HO' O110 OH X O0 ON PEG O NH 2 O n Oigonucleotide with amino linker 15 Oigonucleotide - PEG Conjugate a (i) Solid phase Oligonucleotide synthesis; (ii) Deprotection and purification; (iii) PEG NHS ester, NaHCO 3 , pH 8.1, 1 h. 20

Q/A

Step 2. Deprotection- I (Nucleobase Deprotection) After completion of synthesis, the support was transferred to a 100 ml glass bottle. The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 40 mL of a 40% aq. methyl amine 90 mins at 45'C. The bottle was cooled 5 briefly on ice and then the methylamine was filtered into a new 500 ml bottle. The CPG was washed with 3 x 40 mL portions of DMSO. The mixture was then cooled on dry ice. Step 3. Deprotection-II (Removal of 2' TBDMS group) To the above mixture was added 60 ml triethylamine trihydrofluoride (TREAT-HF) and 0 heated at 40'C for 60 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction was then quenched with 220 ml of 50mM sodium acetate (pH 5.5) and stored in freezer until purification. Step 4. Quantitation of Crude Oligomer or Raw Analysis 5 For all samples, a 10pl aliqoute was diluted with 990 pl of deionised nuclease free water (1.0 mL) and absorbance reading obtained at 260 nm. Step 5. Purification of Oligomers (a) HPLC Purification .0 The crude oligomers were first analyzed by HPLC (Dionex PA 100). The buffer system was: A = 20 mM phosphate pH 11, B = 20 mM phosphate, 1.8 M NaBr, pH 11, flow rate 1.0 mL/min, and wavelength 260-280 nm. Injections of 5-15 pl were done for each sample. The samples were purified by HPLC on an TSK-Gel SuperQ-5PW (20) column (17.3 x 5 cm). The buffer system was: A = 20 mM phosphate in 10% ACN, pH 8.5 and B = 20 mM phosphate, 1.0 25 M NaBr in 10% ACN, pH 8.5, with a flow rate of 50.0 mL/min, and wavelength 260 and 294. The fractions containing the fulllength oligonucleotides were then pooled together, evaporated and reconstituted to ~100 ml with deionised water. 30 Step 6. Desalting of Purified Oligomer The purified oligonucleotides were desalted on an AKTA Explorer (Amersham Biosciences) using Sephadex G-25 column. First column was washed with water at a flow rate of 25 ml/min for 20-30 min. The sample was then applied in 25 ml fractions. The eluted salt-free 5 fractions were combined together, dried down and reconstituted in 50 ml of RNase free water. Step 7. Capillary Gel Electrophoresis (CGE) and Electrospray LC/Ms Approximately 0.15 OD of desalted oligonucleotides were diluted in water to 150 P l and then pipetted in special vials for CGE and LC/MS analysis. 0 Step 8. PEG conjugation. A) Initial reaction conditions. The purified and desalted RNA was lyophilized. RNA (1mg) was dissolved in aq.NaHCO 3 (0.1M, 200pL, pH 8.1) and DMF (200pL each). 5K (13 equivalents, 10mg) or 20KPEG (3.4 equivalents, 10mg) was added directly to reaction vial and 5 vortexed thoroughly. The reaction continued overnight at 4'C, and was followed by analytical ion-exchange HPLC. When the reaction reached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pH was ~7. B) Borate buffer conjugation. The purified and desalted RNA was lyophilized. A sample of RNA (1mg) was dissolved in sodium borate buffer (200p L, 0.05M,pHlO). 5KPEG (3mg, 4.5 .0 equivalents Sunbright ME-50HS, NOF Corp.) was dissolved in CH 3 CN (200pL). The RNA solution was added to the PEG solution and vortexed thoroughly. The reaction continued for one hour at room temperature, and was followed by analytical ion-exchange HPLC. When reaction reached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pH was ~7. 25 C) PEG linker (AS and HS) comparison. A sample of RNA (1mg) was dissolved in aq, NaHCO 3 (0.1M, 200p L, pH 8.1) and DMF (200p L). 5KPEG (13.5 eq, 10mg, Sunbright ME 50HS or Sunbright ME-50AS, NOF Corp.) was added directly to the reaction vial and vortexed thoroughly. The reaction continued overnight at 4'C, and was followed by analytical ion exchange HPLC. When the reaction reached >85% completion, it was quenched with aq. 30 NaOAc (0.05M, pH 5.5) until the pH was ~7.

D) Final optimized PEG conjugation. The purified and desalted RNA was lyophilized. A sample of RNA (50mg) was dissolved in aq. NaHCO 3 ( 0.1M,2mLpH 8.1) and DMF (lmL). 20KPEG (approximately 2.7 eq, 400-520mg Sunbright ME-200HS, different amounts for different sequences within this range) was dissolved in CH 3 CN (2mL). The RNA solution was 5 added to the PEG solution and vortexed thoroughly. H 2 0 (250mL) was added to the reaction to decrease turbidity. The reaction continued for one hour at room temperature, and was followed by analytical ion-exchange HPLC. When the reaction reached >85% completion, it was quenched with aq. NaOAc (0.05M, pH 5.5) until the pH was ~7. 0 Step 9. Analysis of Duplex activity Duplexes were tested for activity in the HeLa cell assay described above. Table 12 and Figure 45 provide data and graphs of the activities in HeLa cells for each of the modifications described above. 5 Example 13 Synthesis of oligonucleotides containing! the ribo-difluorotoluvl (DFT) nucleoside (Table 13) The RNA molecules were synthesized on a 394 ABI machine using the standard cycle written by the manufacturer with modifications to a few wait steps. The solid support was 500 dT CPG (2 umole). The monomers were either RNA phosphoramidites or the ribo-difluorotoluyl .0 amidite. All had standard protecting groups and were used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated. Specifically the phosphoramidites were 5'-0 Dimethoxytrityl-N 6-benzoyl-2'-O-tbutyldimethylsilyl-adenosine-3'-O-(-cyanoethyl-N,N' diisopropyl) phosphoramidite, 5'-O-Dimethoxytrityl-N -isobutyryl-2'-O-tbutyldimethylsilyl guanosine-3'-O-( J-cyanoethyl-N,N'-diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-N 4 25 acetyl-2'-O-tbutyldimethylsilyl-cytidine-3'-O-(-cyanoethyl-N,N'-diisopropyl)phosphoramidite, and 5'-O-Dimethoxytrityl-2'-O-tbutyldimethylsilyl-uridine-3'-O-( -cyanoethyl-N,N' diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-difluorotoluyl O-tbutyldimethylsilyl-3'-O (-cyanoethyl-N,N'-diisopropyl)phosphoramidite (0.12 M). The coupling times were 7 min for all RNA monomers and 10 min for the DFT monomer. Details of the other reagents are as 30 follows: Activator: 5-ethylthio-1H-tetrazole (0.25M), Cap A: 5% acetic anhydride/THF/pyridine, Q 7 Cap B: 10% N-methylimidazole/THF; phosphate oxidation involved 0.02M 1 2

/THF/H

2 0. Detritylation was achieved with 3% TCA/dichloromethane. The DMT protecting group was removed after the last step of the cycle. After completion of synthesis the CPG was transferred to a screw cap, sterile microfuge 5 tube. The oligonucleotide was cleaved and the base and phosphate groups were simultaneously deprotected with 1.0 mL of a mixture of ethanolic ammonia (1:3) for 16 hours at 55'C. The tube was cooled briefly on ice and then the solution was transferred to a 5 mL centrifuge tube; this was followed by washing three times with 0.25 mL of 50% acetonitrile . The tubes were cooled at -80'C for 15 min, before drying in a lyophilizer. 0 The white residue obtained was resuspended in 200 uL of triethylamine trihydrofluoride and heated at 65'C for 1.5 h to remove the TBDMS groups at the 2'-position. The oligonucleotides were then precipitated in dry methanol (400 uL). The liquid was removed carefully to yield a pellet at the bottom of the tube. Residual methanol was removed in the speed vacuum to give a white fluffy material. Samples were dissolved in 1 mL RNase free water and 5 quantitated by measuring the absorbance at 260 nm. This crude material was stored at -20'C. The crude oligonucleotides were analyzed and purified by 20% polyacrylamide denaturing gels. The purified dry oligonucleotides were then desalted using Sephadex G25M. Duplexes were tested for activity in the HeLa cell assay described above. Table 13 and Figure 46 provide data and graphs of the activities in HeLa cells for each of the modifications .0 described above. Example 14 Synthesis of RNA modified with 2'-ara-fluoro-2'-deoxy-nucleosides (Table 14) The chimeric RNA molecules were synthesized on a 394 ABI machine using the standard cycle written by the manufacturer with modifications to a few wait steps. The solid support was 25 500 A dT CPG (2 [tmole). The monomers were either RNA phosphoramidites, or 2'-arafluro-2' deoxy (2' ara F) phosphoramidites. All monomers had standard protecting groups and were used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated. Specifically the RNA phosphoramidites were 5'-O-Dimethoxytrityl-N 6-benzoyl-2'-O-tbutyldimethylsilyl adenosine- 3'-O-(a-cyanoethyl-N,N'-diisopropyl) phosphoramidite, 5'-O-Dimethoxytrityl-N2_ 30 isobutyryl-2'-O-tbutyldimethylsilyl-guanosine-3'-O-(-cyanoethyl-N,N' 22 diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-N 4 -acetyl-2'-O-tbutyldimethylsilyl-cytidine 3'-O-(J-cyanoethyl-N,N'-diisopropyl)phosphoramidite, and 5'-O-Dimethoxytrityl-2'-O tbutyldimethylsilyl-uridine-3'-O-(J-cyanoethyl-N,N'-diisopropyl)phosphoramidite; the 2'ara F phosphoramidites were 5'-O-Dimethoxytrityl-N 4 -benzoyl-2'-arafluro-2'-deoxy-cytidine-3'-O 5 (-cyanoethyl-N,N'-diisopropyl)phosphoramidite, and 5'-O-Dimethoxytrityl-2'-arafluoro-2' deoxy-uridine-3'-O-(J-cyanoethyl-N,N'-diisopropyl)phosphoramidite, and 5'-0 Dimethoxytrityl-2'-arafluoro-thymidine-3'-O-(-cyanoethyl-N,N'-diisopropyl)phosphoramidite. The coupling times were 10 min for all monomers. Details of the other reagents are as follows: Activator: 5-ethylthio-1H-tetrazole (0.25M), Cap A: 5% acetic anhydride/THF/pyridine, Cap B: 0 10% N-methylimidazole/THF; phosphate oxidation involved 0.02 M 1 2

/THF/H

2 0. Detritylation was achieved with 3% TCA/dichloromethane. The final DMT protecting group was removed after the last cycle. After completion of synthesis the CPG was transferred to a screw cap, sterile microfuge tube. The oligonucleotide was cleaved and the base and phosphate groups were simultaneously 5 deprotected with 1.0 mL of a mixture of ethanolic ammonia conc (1:3) for 5 hours at 55'C. The tube was cooled briefly on ice and then the solution was transferred to a 5 mL centrifuge tube; this was followed by washing three times with 0.25 mL of 50% acetonitrile . The tubes were cooled at -80'C for 15 min, before drying in a lyophilizer. The white residue obtained was resuspended in 200 [tL of triethylamine trihydrofluoride .0 and heated at 65'C for 1.5h to remove the TBDMS groups at the 2'-OH position. The oligonucleotides were then precipitated in dry methanol (400 [tL). The liquid was removed carefully to yield a pellet at the bottom of the tube. Residual methanol was removed in the speed vacuum to give a white fluffy material. Samples were dissolved in 1 mL RNase free water and quantitated by measuring the absorbance at 260 nm. This crude material was stored at -20'C. 25 The crude oligonucleotides were analyzed and purified by 20% polyacrylamide denaturing gels. The purified dry oligonucleotides were then desalted using Sephadex G25M (Amersham Biosciences). Duplexes were tested for activity in the HeLa cell assay described above. Table 14 and Figure 47 provide data and graphs of the activities in HeLa cells for each of the modifications 30 described above. Qo Example 15 Deprotection of Methylphosphonate Modified siRNAs (Table 15) Deprotection step 1: After completion of the synthesis, the controlled pore glass (CPG) was transferred to a screw cap vial. A solution (0.5 ml) consisting of Acetonitrile/Ethanol/NH 4 0H (45:45:10) was 5 added to the support. The vial was sealed and left at room temperature for 30 min. Ethylenediamine (0,5 mL) was added to the vial and left at room temperature for an additional 6 hours. The supernatant was decanted and the support was washed twice with 1:1 acetonitrile/water (0.5 mL). The combined supernatant was diluted with water (15 mL). The pH was adjusted to 7.0 with 6 M HCl in AcCN/H 2 0 (1:9). The sample was desalted using a Sep 0 pak C 18 cartridge and then dried in a speed vac. Deprotection step 2 (Removal of 2'-0- TBDMS group) The white residue obtained was resuspended in a mixture of triethylamine, triethylamine trihydrofluoride (TEA.3HF ca, 24% HF) and 1-Methyl-2-Pyrrolidinone (NMP) (4:3:7) (400 ul) 5 and heated at 65'C for 90 min to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The reaction was then quenched with isopropoxytrimethylsilane (iPrOMe 3 Si, 400 ul) and further incubated on the heating block leaving the caps open for 10min; (This causes the volatile isopropxytrimethylsilylfluoride adduct to vaporize). The residual quenching reagent was removed by drying in a speed vac. 3% Triethylamine in diethyl ether (1.5 ml) was added and the .0 mixture was subjected to centrifugation to afford a pellet of RNA. The supernatant was pipetted out without disturbing the pellet. The pellet was dried in a speed vac. The crude RNA was obtained as a white fluffy material in the microfuge tube. Purification: 25 All methylphosphonate modified sequences were purified by PAGE Analysis of Duplex activity Duplexes were tested for activity in the HeLa cell assay described above. Table 15 and Figure 48 provide dat and graphs of the activities in HeLa cells for each of the modifications 30 described above. on TABLE 1. Target sequences in VEGF 121 TARGET SEQUENCE IN VEGF121 mRNA SEQ ID NO: ORF 5'to 3' 2 1 AUGAACUUUCUGCUGUCUUGGGU 3 2 UGAACUUUCUGCUGUCUUGGGUG 4 3 GAACUUUCUGCUGUCUUGGGUGC 5 4 AACUUUCUGCUGUCUUGGGUGCA 6 5 ACUUUCUGCUGUCUUGGGUGCAU 7 6 CUUUCUGCUGUCUUGGGUGCAUU 8 7 UUUCUGCUGUCUUGGGUGCAUUG 9 8 UUCUGCUGUCUUGGGUGCAUUGG 10 9 UCUGCUGUCUUGGGUGCAUUGGA 11 10 CUGCUGUCUUGGGUGCAUUGGAG 12 11 UGCUGUCUUGGGUGCAUUGGAGC 13 12 GCUGUCUUGGGUGCAUUGGAGCC 14 13 CUGUCUUGGGUGCAUUGGAGCCU 15 14 UGUCUUGGGUGCAUUGGAGCCUU 16 15 GUCUUGGGUGCAUUGGAGCCUUG 17 16 UCUUGGGUGCAUUGGAGCCUUGC 18 17 CUUGGGUGCAUUGGAGCCUUGCC 19 18 UUGGGUGCAUUGGAGCCUUGCCU 20 19 UGGGUGCAUUGGAGCCUUGCCUU 21 20 GGGUGCAUUGGAGCCUUGCCUUG 22 21 GGUGCAUUGGAGCCUUGCCUUGC 23 22 GUGCAUUGGAGCCUUGCCUUGCU 24 23 UGCAUUGGAGCCUUGCCUUGCUG 25 24 GCAUUGGAGCCUUGCCUUGCUGC 26 25 CAUUGGAGCCUUGCCUUGCUGCU 27 26 AUUGGAGCCUUGCCUUGCUGCUC 28 27 UUGGAGCCUUGCCUUGCUGCUCU 29 28 UGGAGCCUUGCCUUGCUGCUCUA 30 29 GGAGCCUUGCCUUGCUGCUCUAC 31 30 GAGCCUUGCCUUGCUGCUCUACC 32 31 AGCCUUGCCUUGCUGCUCUACCU 33 32 GCCUUGCCUUGCUGCUCUACCUC 34 33 CCUUGCCUUGCUGCUCUACCUCC 35 34 CUUGCCUUGCUGCUCUACCUCCA 36 35 UUGCCUUGCUGCUCUACCUCCAC 37 36 UGCCUUGCUGCUCUACCUCCACC 38 37 GCCUUGCUGCUCUACCUCCACCA 39 38 CCUUGCUGCUCUACCUCCACCAU 40 39 CUUGCUGCUCUACCUCCACCAUG 01 41 40 UUGCUGCUCUACCUCCACCAUGC 42 41 UGCUGCUCUACCUCCACCAUGCC 43 42 GCUGCUCUACCUCCACCAUGCCA 44 43 CUGCUCUACCUCCACCAUGCCAA 45 44 UGCUCUACCUCCACCAUGCCAAG 46 45 GCUCUACCUCCACCAUGCCAAGU 47 46 CUCUACCUCCACCAUGCCAAGUG 48 47 UCUACCUCCACCAUGCCAAGUGG 49 48 CUACCUCCACCAUGCCAAGUGGU 50 49 UACCUCCACCAUGCCAAGUGGUC 51 50 ACCUCCACCAUGCCAAGUGGUCC 52 51 CCUCCACCAUGCCAAGUGGUCCC 53 52 CUCCACCAUGCCAAGUGGUCCCA 54 53 UCCACCAUGCCAAGUGGUCCCAG 55 54 CCACCAUGCCAAGUGGUCCCAGG 56 55 CACCAUGCCAAGUGGUCCCAGGC 57 56 ACCAUGCCAAGUGGUCCCAGGCU 58 57 CCAUGCCAAGUGGUCCCAGGCUG 59 58 CAUGCCAAGUGGUCCCAGGCUGC 60 59 AUGCCAAGUGGUCCCAGGCUGCA 61 60 UGCCAAGUGGUCCCAGGCUGCAC 62 61 GCCAAGUGGUCCCAGGCUGCACC 63 62 CCAAGUGGUCCCAGGCUGCACCC 64 63 CAAGUGGUCCCAGGCUGCACCCA 65 64 AAGUGGUCCCAGGCUGCACCCAU 66 65 AGUGGUCCCAGGCUGCACCCAUG 67 66 GUGGUCCCAGGCUGCACCCAUGG 68 67 UGGUCCCAGGCUGCACCCAUGGC 69 68 GGUCCCAGGCUGCACCCAUGGCA 70 69 GUCCCAGGCUGCACCCAUGGCAG 71 70 UCCCAGGCUGCACCCAUGGCAGA 72 71 CCCAGGCUGCACCCAUGGCAGAA 73 72 CCAGGCUGCACCCAUGGCAGAAG 74 73 CAGGCUGCACCCAUGGCAGAAGG 75 74 AGGCUGCACCCAUGGCAGAAGGA 76 75 GGCUGCACCCAUGGCAGAAGGAG 77 76 GCUGCACCCAUGGCAGAAGGAGG 78 77 CUGCACCCAUGGCAGAAGGAGGA 79 78 UGCACCCAUGGCAGAAGGAGGAG 80 79 GCACCCAUGGCAGAAGGAGGAGG 81 80 CACCCAUGGCAGAAGGAGGAGGG 82 81 ACCCAUGGCAGAAGGAGGAGGGC 83 82 CCCAUGGCAGAAGGAGGAGGGCA 84 83 CCAUGGCAGAAGGAGGAGGGCAG 01) 85 84 CAUGGCAGAAGGAGGAGGGCAGA 86 85 AUGGCAGAAGGAGGAGGGCAGAA 87 86 UGGCAGAAGGAGGAGGGCAGAAU 88 87 GGCAGAAGGAGGAGGGCAGAAUC 89 88 GCAGAAGGAGGAGGGCAGAAUCA 90 89 CAGAAGGAGGAGGGCAGAAUCAU 91 90 AGAAGGAGGAGGGCAGAAUCAUC 92 91 GAAGGAGGAGGGCAGAAUCAUCA 93 92 AAGGAGGAGGGCAGAAUCAUCAC 94 93 AGGAGGAGGGCAGAAUCAUCACG 95 94 GGAGGAGGGCAGAAUCAUCACGA 96 95 GAGGAGGGCAGAAUCAUCACGAA 97 96 AGGAGGGCAGAAUCAUCACGAAG 98 97 GGAGGGCAGAAUCAUCACGAAGU 99 98 GAGGGCAGAAUCAUCACGAAGUG 100 99 AGGGCAGAAUCAUCACGAAGUGG 101 100 GGGCAGAAUCAUCACGAAGUGGU 102 101 GGCAGAAUCAUCACGAAGUGGUG 103 102 GCAGAAUCAUCACGAAGUGGUGA 104 103 CAGAAUCAUCACGAAGUGGUGAA 105 104 AGAAUCAUCACGAAGUGGUGAAG 106 105 GAAUCAUCACGAAGUGGUGAAGU 107 106 AAUCAUCACGAAGUGGUGAAGUU 108 107 AUCAUCACGAAGUGGUGAAGUUC 109 108 UCAUCACGAAGUGGUGAAGUUCA 110 109 CAUCACGAAGUGGUGAAGUUCAU 111 110 AUCACGAAGUGGUGAAGUUCAUG 112 111 UCACGAAGUGGUGAAGUUCAUGG 113 112 CACGAAGUGGUGAAGUUCAUGGA 114 113 ACGAAGUGGUGAAGUUCAUGGAU 115 114 CGAAGUGGUGAAGUUCAUGGAUG 116 115 GAAGUGGUGAAGUUCAUGGAUGU 117 116 AAGUGGUGAAGUUCAUGGAUGUC 118 117 AGUGGUGAAGUUCAUGGAUGUCU 119 118 GUGGUGAAGUUCAUGGAUGUCUA 120 119 UGGUGAAGUUCAUGGAUGUCUAU 121 120 GGUGAAGUUCAUGGAUGUCUAUC 122 121 GUGAAGUUCAUGGAUGUCUAUCA 123 122 UGAAGUUCAUGGAUGUCUAUCAG 124 123 GAAGUUCAUGGAUGUCUAUCAGC 125 124 AAGUUCAUGGAUGUCUAUCAGCG 126 125 AGUUCAUGGAUGUCUAUCAGCGC 127 126 GUUCAUGGAUGUCUAUCAGCGCA 128 127 UUCAUGGAUGUCUAUCAGCGCAG 0I 129 128 UCAUGGAUGUCUAUCAGCGCAGC 130 129 CAUGGAUGUCUAUCAGCGCAGCU 131 130 AUGGAUGUCUAUCAGCGCAGCUA 132 131 UGGAUGUCUAUCAGCGCAGCUAC 133 132 GGAUGUCUAUCAGCGCAGCUACU 134 133 GAUGUCUAUCAGCGCAGCUACUG 135 134 AUGUCUAUCAGCGCAGCUACUGC 136 135 UGUCUAUCAGCGCAGCUACUGCC 137 136 GUCUAUCAGCGCAGCUACUGCCA 138 137 UCUAUCAGCGCAGCUACUGCCAU 139 138 CUAUCAGCGCAGCUACUGCCAUC 140 139 UAUCAGCGCAGCUACUGCCAUCC 141 140 AUCAGCGCAGCUACUGCCAUCCA 142 141 UCAGCGCAGCUACUGCCAUCCAA 143 142 CAGCGCAGCUACUGCCAUCCAAU 144 143 AGCGCAGCUACUGCCAUCCAAUC 145 144 GCGCAGCUACUGCCAUCCAAUCG 146 145 CGCAGCUACUGCCAUCCAAUCGA 147 146 GCAGCUACUGCCAUCCAAUCGAG 148 147 CAGCUACUGCCAUCCAAUCGAGA 149 148 AGCUACUGCCAUCCAAUCGAGAC 150 149 GCUACUGCCAUCCAAUCGAGACC 151 150 CUACUGCCAUCCAAUCGAGACCC 152 151 UACUGCCAUCCAAUCGAGACCCU 153 152 ACUGCCAUCCAAUCGAGACCCUG 154 153 CUGCCAUCCAAUCGAGACCCUGG 155 154 UGCCAUCCAAUCGAGACCCUGGU 156 155 GCCAUCCAAUCGAGACCCUGGUG 157 156 CCAUCCAAUCGAGACCCUGGUGG 158 157 CAUCCAAUCGAGACCCUGGUGGA 159 158 AUCCAAUCGAGACCCUGGUGGAC 160 159 UCCAAUCGAGACCCUGGUGGACA 161 160 CCAAUCGAGACCCUGGUGGACAU 162 161 CAAUCGAGACCCUGGUGGACAUC 163 162 AAUCGAGACCCUGGUGGACAUCU 164 163 AUCGAGACCCUGGUGGACAUCUU 165 164 UCGAGACCCUGGUGGACAUCUUC 166 165 CGAGACCCUGGUGGACAUCUUCC 167 166 GAGACCCUGGUGGACAUCUUCCA 168 167 AGACCCUGGUGGACAUCUUCCAG 169 168 GACCCUGGUGGACAUCUUCCAGG 170 169 ACCCUGGUGGACAUCUUCCAGGA 171 170 CCCUGGUGGACAUCUUCCAGGAG 172 171 CCUGGUGGACAUCUUCCAGGAGU

OA

173 172 CUGGUGGACAUCUUCCAGGAGUA 174 173 UGGUGGACAUCUUCCAGGAGUAC 175 174 GGUGGACAUCUUCCAGGAGUACC 176 175 GUGGACAUCUUCCAGGAGUACCC 177 176 UGGACAUCUUCCAGGAGUACCCU 178 177 GGACAUCUUCCAGGAGUACCCUG 179 178 GACAUCUUCCAGGAGUACCCUGA 180 179 ACAUCUUCCAGGAGUACCCUGAU 181 180 CAUCUUCCAGGAGUACCCUGAUG 182 181 AUCUUCCAGGAGUACCCUGAUGA 183 182 UCUUCCAGGAGUACCCUGAUGAG 184 183 CUUCCAGGAGUACCCUGAUGAGA 185 184 UUCCAGGAGUACCCUGAUGAGAU 186 185 UCCAGGAGUACCCUGAUGAGAUC 187 186 CCAGGAGUACCCUGAUGAGAUCG 188 187 CAGGAGUACCCUGAUGAGAUCGA 189 188 AGGAGUACCCUGAUGAGAUCGAG 190 189 GGAGUACCCUGAUGAGAUCGAGU 191 190 GAGUACCCUGAUGAGAUCGAGUA 192 191 AGUACCCUGAUGAGAUCGAGUAC 193 192 GUACCCUGAUGAGAUCGAGUACA 194 193 UACCCUGAUGAGAUCGAGUACAU 195 194 ACCCUGAUGAGAUCGAGUACAUC 196 195 CCCUGAUGAGAUCGAGUACAUCU 197 196 CCUGAUGAGAUCGAGUACAUCUU 198 197 CUGAUGAGAUCGAGUACAUCUUC 199 198 UGAUGAGAUCGAGUACAUCUUCA 200 199 GAUGAGAUCGAGUACAUCUUCAA 201 200 AUGAGAUCGAGUACAUCUUCAAG 202 201 UGAGAUCGAGUACAUCUUCAAGC 203 202 GAGAUCGAGUACAUCUUCAAGCC 204 203 AGAUCGAGUACAUCUUCAAGCCA 205 204 GAUCGAGUACAUCUUCAAGCCAU 206 205 AUCGAGUACAUCUUCAAGCCAUC 207 206 UCGAGUACAUCUUCAAGCCAUCC 208 207 CGAGUACAUCUUCAAGCCAUCCU 209 208 GAGUACAUCUUCAAGCCAUCCUG 210 209 AGUACAUCUUCAAGCCAUCCUGU 211 210 GUACAUCUUCAAGCCAUCCUGUG 212 211 UACAUCUUCAAGCCAUCCUGUGU 213 212 ACAUCUUCAAGCCAUCCUGUGUG 214 213 CAUCUUCAAGCCAUCCUGUGUGC 215 214 AUCUUCAAGCCAUCCUGUGUGCC 216 215 UCUUCAAGCCAUCCUGUGUGCCC 0) r 217 216 CUUCAAGCCAUCCUGUGUGCCCC 218 217 UUCAAGCCAUCCUGUGUGCCCCU 219 218 UCAAGCCAUCCUGUGUGCCCCUG 220 219 CAAGCCAUCCUGUGUGCCCCUGA 221 220 AAGCCAUCCUGUGUGCCCCUGAU 222 221 AGCCAUCCUGUGUGCCCCUGAUG 223 222 GCCAUCCUGUGUGCCCCUGAUGC 224 223 CCAUCCUGUGUGCCCCUGAUGCG 225 224 CAUCCUGUGUGCCCCUGAUGCGA 226 225 AUCCUGUGUGCCCCUGAUGCGAU 227 226 UCCUGUGUGCCCCUGAUGCGAUG 228 227 CCUGUGUGCCCCUGAUGCGAUGC 229 228 CUGUGUGCCCCUGAUGCGAUGCG 230 229 UGUGUGCCCCUGAUGCGAUGCGG 231 230 GUGUGCCCCUGAUGCGAUGCGGG 232 231 UGUGCCCCUGAUGCGAUGCGGGG 233 232 GUGCCCCUGAUGCGAUGCGGGGG 234 233 UGCCCCUGAUGCGAUGCGGGGGC 235 234 GCCCCUGAUGCGAUGCGGGGGCU 236 235 CCCCUGAUGCGAUGCGGGGGCUG 237 236 CCCUGAUGCGAUGCGGGGGCUGC 238 237 CCUGAUGCGAUGCGGGGGCUGCU 239 238 CUGAUGCGAUGCGGGGGCUGCUG 240 239 UGAUGCGAUGCGGGGGCUGCUGC 241 240 GAUGCGAUGCGGGGGCUGCUGCA 242 241 AUGCGAUGCGGGGGCUGCUGCAA 243 242 UGCGAUGCGGGGGCUGCUGCAAU 244 243 GCGAUGCGGGGGCUGCUGCAAUG 245 244 CGAUGCGGGGGCUGCUGCAAUGA 246 245 GAUGCGGGGGCUGCUGCAAUGAC 247 246 AUGCGGGGGCUGCUGCAAUGACG 248 247 UGCGGGGGCUGCUGCAAUGACGA 249 248 GCGGGGGCUGCUGCAAUGACGAG 250 249 CGGGGGCUGCUGCAAUGACGAGG 251 250 GGGGGCUGCUGCAAUGACGAGGG 252 251 GGGGCUGCUGCAAUGACGAGGGC 253 252 GGGCUGCUGCAAUGACGAGGGCC 254 253 GGCUGCUGCAAUGACGAGGGCCU 255 254 GCUGCUGCAAUGACGAGGGCCUG 256 255 CUGCUGCAAUGACGAGGGCCUGG 257 256 UGCUGCAAUGACGAGGGCCUGGA 258 257 GCUGCAAUGACGAGGGCCUGGAG 259 258 CUGCAAUGACGAGGGCCUGGAGU 260 259 UGCAAUGACGAGGGCCUGGAGUG

()A

261 260 GCAAUGACGAGGGCCUGGAGUGU 262 261 CAAUGACGAGGGCCUGGAGUGUG 263 262 AAUGACGAGGGCCUGGAGUGUGU 264 263 AUGACGAGGGCCUGGAGUGUGUG 265 264 UGACGAGGGCCUGGAGUGUGUGC 266 265 GACGAGGGCCUGGAGUGUGUGCC 267 266 ACGAGGGCCUGGAGUGUGUGCCC 268 267 CGAGGGCCUGGAGUGUGUGCCCA 269 268 GAGGGCCUGGAGUGUGUGCCCAC 270 269 AGGGCCUGGAGUGUGUGCCCACU 271 270 GGGCCUGGAGUGUGUGCCCACUG 272 271 GGCCUGGAGUGUGUGCCCACUGA 273 272 GCCUGGAGUGUGUGCCCACUGAG 274 273 CCUGGAGUGUGUGCCCACUGAGG 275 274 CUGGAGUGUGUGCCCACUGAGGA 276 275 UGGAGUGUGUGCCCACUGAGGAG 277 276 GGAGUGUGUGCCCACUGAGGAGU 278 277 GAGUGUGUGCCCACUGAGGAGUC 279 278 AGUGUGUGCCCACUGAGGAGUCC 280 279 GUGUGUGCCCACUGAGGAGUCCA 281 280 UGUGUGCCCACUGAGGAGUCCAA 282 281 GUGUGCCCACUGAGGAGUCCAAC 283 282 UGUGCCCACUGAGGAGUCCAACA 284 283 GUGCCCACUGAGGAGUCCAACAU 285 284 UGCCCACUGAGGAGUCCAACAUC 286 285 GCCCACUGAGGAGUCCAACAUCA 287 286 CCCACUGAGGAGUCCAACAUCAC 288 287 CCACUGAGGAGUCCAACAUCACC 289 288 CACUGAGGAGUCCAACAUCACCA 290 289 ACUGAGGAGUCCAACAUCACCAU 291 290 CUGAGGAGUCCAACAUCACCAUG 292 291 UGAGGAGUCCAACAUCACCAUGC 293 292 GAGGAGUCCAACAUCACCAUGCA 294 293 AGGAGUCCAACAUCACCAUGCAG 295 294 GGAGUCCAACAUCACCAUGCAGA 296 295 GAGUCCAACAUCACCAUGCAGAU 297 296 AGUCCAACAUCACCAUGCAGAUU 298 297 GUCCAACAUCACCAUGCAGAUUA 299 298 UCCAACAUCACCAUGCAGAUUAU 300 299 CCAACAUCACCAUGCAGAUUAUG 301 300 CAACAUCACCAUGCAGAUUAUGC 302 301 AACAUCACCAUGCAGAUUAUGCG 303 302 ACAUCACCAUGCAGAUUAUGCGG 304 303 CAUCACCAUGCAGAUUAUGCGGA 0-7 305 304 AUCACCAUGCAGAUUAUGCGGAU 306 305 UCACCAUGCAGAUUAUGCGGAUC 307 306 CACCAUGCAGAUUAUGCGGAUCA 308 307 ACCAUGCAGAUUAUGCGGAUCAA 309 308 CCAUGCAGAUUAUGCGGAUCAAA 310 309 CAUGCAGAUUAUGCGGAUCAAAC 311 310 AUGCAGAUUAUGCGGAUCAAACC 312 311 UGCAGAUUAUGCGGAUCAAACCU 313 312 GCAGAUUAUGCGGAUCAAACCUC 314 313 CAGAUUAUGCGGAUCAAACCUCA 315 314 AGAUUAUGCGGAUCAAACCUCAC 316 315 GAUUAUGCGGAUCAAACCUCACC 317 316 AUUAUGCGGAUCAAACCUCACCA 318 317 UUAUGCGGAUCAAACCUCACCAA 319 318 UAUGCGGAUCAAACCUCACCAAG 320 319 AUGCGGAUCAAACCUCACCAAGG 321 320 UGCGGAUCAAACCUCACCAAGGC 322 321 GCGGAUCAAACCUCACCAAGGCC 323 322 CGGAUCAAACCUCACCAAGGCCA 324 323 GGAUCAAACCUCACCAAGGCCAG 325 324 GAUCAAACCUCACCAAGGCCAGC 326 325 AUCAAACCUCACCAAGGCCAGCA 327 326 UCAAACCUCACCAAGGCCAGCAC 328 327 CAAACCUCACCAAGGCCAGCACA 329 328 AAACCUCACCAAGGCCAGCACAU 330 329 AACCUCACCAAGGCCAGCACAUA 331 330 ACCUCACCAAGGCCAGCACAUAG 332 331 CCUCACCAAGGCCAGCACAUAGG 333 332 CUCACCAAGGCCAGCACAUAGGA 334 333 UCACCAAGGCCAGCACAUAGGAG 335 334 CACCAAGGCCAGCACAUAGGAGA 336 335 ACCAAGGCCAGCACAUAGGAGAG 337 336 CCAAGGCCAGCACAUAGGAGAGA 338 337 CAAGGCCAGCACAUAGGAGAGAU 339 338 AAGGCCAGCACAUAGGAGAGAUG 340 339 AGGCCAGCACAUAGGAGAGAUGA 341 340 GGCCAGCACAUAGGAGAGAUGAG 342 341 GCCAGCACAUAGGAGAGAUGAGC 343 342 CCAGCACAUAGGAGAGAUGAGCU 344 343 CAGCACAUAGGAGAGAUGAGCUU 345 344 AGCACAUAGGAGAGAUGAGCUUC 346 345 GCACAUAGGAGAGAUGAGCUUCC 347 346 CACAUAGGAGAGAUGAGCUUCCU 348 347 ACAUAGGAGAGAUGAGCUUCCUA 0)Q 349 348 CAUAGGAGAGAUGAGCUUCCUAC 350 349 AUAGGAGAGAUGAGCUUCCUACA 351 350 UAGGAGAGAUGAGCUUCCUACAG 352 351 AGGAGAGAUGAGCUUCCUACAGC 353 352 GGAGAGAUGAGCUUCCUACAGCA 354 353 GAGAGAUGAGCUUCCUACAGCAC 355 354 AGAGAUGAGCUUCCUACAGCACA 356 355 GAGAUGAGCUUCCUACAGCACAA 357 356 AGAUGAGCUUCCUACAGCACAAC 358 357 GAUGAGCUUCCUACAGCACAACA 359 358 AUGAGCUUCCUACAGCACAACAA 360 359 UGAGCUUCCUACAGCACAACAAA 361 360 GAGCUUCCUACAGCACAACAAAU 362 361 AGCUUCCUACAGCACAACAAAUG 363 362 GCUUCCUACAGCACAACAAAUGU 364 363 CUUCCUACAGCACAACAAAUGUG 365 364 UUCCUACAGCACAACAAAUGUGA 366 365 UCCUACAGCACAACAAAUGUGAA 367 366 CCUACAGCACAACAAAUGUGAAU 368 367 CUACAGCACAACAAAUGUGAAUG 369 368 UACAGCACAACAAAUGUGAAUGC 370 369 ACAGCACAACAAAUGUGAAUGCA 371 370 CAGCACAACAAAUGUGAAUGCAG 372 371 AGCACAACAAAUGUGAAUGCAGA 373 372 GCACAACAAAUGUGAAUGCAGAC 374 373 CACAACAAAUGUGAAUGCAGACC 375 374 ACAACAAAUGUGAAUGCAGACCA 376 375 CAACAAAUGUGAAUGCAGACCAA 377 376 AACAAAUGUGAAUGCAGACCAAA 378 377 ACAAAUGUGAAUGCAGACCAAAG 379 378 CAAAUGUGAAUGCAGACCAAAGA 380 379 AAAUGUGAAUGCAGACCAAAGAA 381 380 AAUGUGAAUGCAGACCAAAGAAA 382 381 AUGUGAAUGCAGACCAAAGAAAG 383 382 UGUGAAUGCAGACCAAAGAAAGA 384 383 GUGAAUGCAGACCAAAGAAAGAU 385 384 UGAAUGCAGACCAAAGAAAGAUA 386 385 GAAUGCAGACCAAAGAAAGAUAG 387 386 AAUGCAGACCAAAGAAAGAUAGA 388 387 AUGCAGACCAAAGAAAGAUAGAG 389 388 UGCAGACCAAAGAAAGAUAGAGC 390 389 GCAGACCAAAGAAAGAUAGAGCA 391 390 CAGACCAAAGAAAGAUAGAGCAA 392 391 AGACCAAAGAAAGAUAGAGCAAG 00 393 392 GACCAAAGAAAGAUAGAGCAAGA 394 393 ACCAAAGAAAGAUAGAGCAAGAC 395 394 CCAAAGAAAGAUAGAGCAAGACA 396 395 CAAAGAAAGAUAGAGCAAGACAA 397 396 AAAGAAAGAUAGAGCAAGACAAG 398 397 AAGAAAGAUAGAGCAAGACAAGA 399 398 AGAAAGAUAGAGCAAGACAAGAA 400 399 GAAAGAUAGAGCAAGACAAGAAA 401 400 AAGAUAGAGCAAGACAAGAAAA 1A Af 0 0 0 uF 0 0 F' 0 0 m' 0 0 u pC2 F 0 0 0 0 0 0 2mC 0 2 mC 2 m 0 0 0 0 0 0 0 0 0 0 00~ 0 0 2m~ 0 0 2 <C 0 0 0 0 2 <C 2 < 0 0 0 0 0 0 0) 0 0 0 4 H 2 0 0 0 2 0C 2 0 L 0 0 m L0 0 0 0 2 0 2 m u 0 0 0 0 2 <C 2 0 0 0 u2~ 0 0 0 0 0 0 0 0 m~ 00 00 u C u C 2 2 00 m0~ 2 mC 0 0 G 2 4 00 004 004 42G 42G U 2 U C200 00G 4 0 0 0 0 <C 2 2 < 0 0 0 0 0 0 0 0 mC 2 m 0 0 0 0 0 0 <C 2 0 0 0 0 0 0 0 0 0 0 0 0 2 2 m 2 <C 0 0 0 0 0 0 2 <C 2 < 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0 <C 2 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A 4 2 4 2 4 2 <2 2 U Am U Am U Am U Am <2 2 <2 2 2 < 2 <2 Am U Am U U Am U Am U Am U Am <2 2 m 2 m m m m m 2 o o o U o o o m q m UM N W O H iN N M C n I I I I I I I I I I I E B E I 0. . . 0 0 0 0. 0. . 0 0. 0 0 u 0 0 0 Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Am U 2 Am U< <2 U U <2~~~ Am Am U A<U< 2 U U U <2A A L cu Lm 2 Am Am L <2~~ Am2A m2U Am U U<<2A2Am 2~ Am U 2<2A <2~ U U 22AU U ~~ <2UU<22A

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U 2 U K U K 0 0 2 0 0 2 U U 2 U K K U 2 U 0 0 0 U 2 U U 2 U K U K K U K 0 K U 2 U U 2 U 2 2 0 K 0 K K 4 U 2 U U 2 K 0 U 0 U 0 U K K U 2 U U 0 2 0 U 2 U 0 U K U 2 U 2 U U 0 u u 4 < m 0 2 K 0 U K K U 2 2 2 K U U U 2 0 K 0 U 4 K K U 2 0 K U K 2 0 K 0 U K K U K 2 2 0 K 2 0 K 2 0 U 2 U 2 2 U 4 K U Gv K 2 0 K K U K LU m > m 02 Am ki< mi m> < a 2 0 m H Am Am L Am L Am L U A A LO A LO A U A U A -L A L L -L <2 2 A -L A U Am H H H A U H Am U m 2 H H H H U A H <C 2 o Am 2 4 42 2 U Am 2 4 2 4 Am U 2 <2 Am U 42 2 U Am U Am U Am U Am U Am U Am Am U U Am 2 42 42 2 U Am U Am 2 42 42 2 U Am Am U 42 2 U Am U Am 2 42 2 42 U Am 42 2 Am U U Am 2 42 42 2 42 2 U Am 42 2 2 42 42 2 Am U 42 2 U Am 2 42 2 42 2 SAm 42 2 Am U U Am 2 42 2 42 42 2 U Am 42 2 Am U 42 2 Am U 42 2 U Am U Am 2 Am U U Am 42 2 Am U U Am U Am 2 42 U Am 42 2 U Am Am U 42 2 Am U 2 42 2 42 U SAm Am U U Am 42 2 Am U Am U U Am 2 <2 U Am 2 42 U Am Am U 42 2 42 2 42 2 2 42 Am U U Am Am U U Am 42 2 42 2 Am U 42 2 U Am 2 2 <2 U Am Am U U Am U Am <2 2 42 2 Am U U Am Am U U Am U Am <2 2 U Am 2 <2 4 2 <2 2 2 <2 U Am <2 2 <2 2 U Am S Am <2 2 Am U U Am Am U Am U U Am U Am <2 2 U Am <2 2 <2 2 2 <2 Am U Am U <2 2 2 <2 U Am <2 2 Am U U Am U Am Am U Am U U Am U Am U Am <2 2 <2 2 Am U Am U Am U <2 2 2 <2 U Am <2 2 Am U Am U U Am 2 <2 U Am 2 <2 U Am U Am <2 2 2 <2 2 <2 Am U 2 <2 <2 2 2 <2 U Am <2 2 <2 2 Am U U Am Am U <2 2 2 <2 U Am U Am Am U Am U 2 <2 S Am 2 <2 <2 2 2 <2 U Am U Am <2 2 <2 2 2 <2 U Am <2 2 2 <2 U Am Am U Am U Am U 2 <2 U Am 2 <2 <2 2 2 <2 2 <2 U Am 2 <2 U Am U Am U Am <2 2 2 <2 2 <2 2 <2 Am U Am U 2 <2 U Am 2 <2 <2 2 <2 2 2 <2 U Am <2 2 Am U U Am U Am <2 2 U Am U Am 2 <2 2 <2 Am U 2 <2 U Am 2 <2 2 <2 <2 2 Am U 2 <2 2 <2 Am U U Am U Am U Am U Am U Am <2 2 2 <2 Am U 2 <2 U Am U Am 2 <2 <2 2 U Am U Am 2 <2 Am U U Am U Am U Am U Am Am U <2 2 2 <2 Am U 2 <2 2 <2 U Am U Am Am U <2 2 U Am 2 <2 Am U <2 2 <2 2 U Am Am U Am U <2 2 2 <2 Am U Am U 2 <2 Am U <2 2 2 <2 <2 2 U Am 2 <2 Am U Am U <2 2 m m m m 4 Am m m A A m m m 2 U LO Am LO Am LO Am LO Am -LO 2 -L Am LO Am -L U -LO U LO Am LO Am LO Am LO Am -LO U LO Am -LO 2 N O N L ~ C N H m m O N N N N N M M O L O O O H C C C C C C C C C C C C C 0. 0 0 . 0 0. 0. 0. 0 0. . 0. 0. 0 0 A A A A A A A A A A A A A A A I I I I I I I I I I I I I I I I o 2 Am U U Am Am U U 2 Am <2 Am <2 U 2 Am U U <2 U U U 2 U <2 2 <2 U 2 A 2 A <2 U U U U o 2 <2 U 2 U A <2 U U 2 Am U 2 <2 U <2 2 <2 Am <2 U 2 <2 A U 2 <2 U A <2 A <2 U o <2 Am U 2 U 4 U A <2 A m U <2 Am U 2 U 2 U A <2 < S Am U <2 A <2 U <2 2 U A U <2 Am U Am U <2 U 2 <2 2 U <2C <2 A U A U U <2 U < <2 2 A <2 o <2 Am U Am Am U U U <2 Am Am 2 U <2 Am U 2 U 2 U U <2 2 Am <2 2 U <2 A U A <2 2 U U A 2 <2 2 U <2 <2 2 U <2 2 U Am Am o 2 <2 2 U 2 U U U <2 2 2 Am 2 U 2 <2 2 U <2 Am U U <2 U2 Am 2 U 2 <2 Am 2 2 Am U U UU 2 Am 2 U 2 <2 U U 2 Am U UU <2 2 Am 2 U U Am <2 U 2 Am <2C Am <2 2 Am 2 Am <2 2 <2 U 2 Am <C Am Am <2 2 Am U U U 2 <2 U <2 Am L L L L L L Am Am Am 0pm A 2 2 A m 0 Am p ~ 2 p2 Am pm <2 2 A u0 0 LD Am p m A ~ 2 Am m 0 Am m 0 Am < 2 m m u m 2 L Am Lm 0 LD m m L2 o mD < 2 < 2 D 0 LD 0 D 0 LD 0 < m < 2 LD 0 D 0 0 LD 0 D 0 L 0 D L 0 o mD 0 mD 0 m < 2 < 2 D 0 LD 0 D u L 0 < 2 < 2 < 2 < 2 < 2 < 2 2 < H OOLD OLD OLD O LD <22<22L LDQ HLO LDO HLO 2<22<22<2L2< L OLD m U 2 <2 2 <2 2 2 OL OL OL OLD u 0u0 <22 <22 <2 <2 <2 <2 0 A 2<2 OD4 OLD 2<2 24G 2<2 2<2 OLD OLD OLD OLD LDO LDO LDO LDO <22 m2 2 2 <2 2 <2 0 u < 0 Am 0 Am 2 <2 2 <2 <2 0 u u m m 4 m 4 m u 0 u 0 u 0 u 0 < m 4 m 4 m 4 m 4 m 4 m 4 0 OD4 <22<222<22<2D OD4 OLD 2 3 2 G 2<22<2<22<22<22<22 G 4 4 2 0 Am 0 4 2 m 0 Am 0 A 2 4 0 A m 0 A 0 Am 0 A < 2 Am 0 <2 2 < 2 0 Am 0 Am m2 2 <2 2 2 <2 2 0 Am 0 A 0 Am 0 Am 0 Am 0 Am Am 0 A m 0 Am 0 Am 0 < 2 < 2 0 Am 0 A m m2 2 <2 2 2 m2 2 m2 2 m 0 2 m L 0 L 0 4 0 4 0 m 2 m 2 4 0 LD < 2 m 2 < 2 4 2 4 2 <2 < 2 L 0 2 m 2 m A 0 A 0 Am 0 m 0 < 2 < 2 0 A 0 A 0 A 0 A 0 A 0 0 A m u Am 0 m 0 2 m 2 m 0 A 0 Am < m 0 < 2 < 2 2 m 2 m 2 m 2 0 A 2 < A u A u A 0 0 2 < 2 < Am U A 0 Am 0 m 0 m 2 m 2 m 2 < 2 2 m A 2 < < A u A u A u A u < 2 4 Am 0 A 0 0 A 0 A 0 A 0 A 2 SA 0 A 0 A 2 < 2 < A u A u U A u 2 4 2 4 4 2 4 2 4 2 4 2 u A m A 0 A 0 A 0 A 0 2 < 2 < U A 0 A u A 0 m 0 m 0 m 0 m 0 2 4 4 2 0 A 0 A 0 A 0 A 0 A 0 A 2 < 2 < A u A u 0 A 0 A 0 A 0 4 2 A m 2 A <A Am Am Am Am Am 0 < Am A < m < m Amm u m Am m 0 A Am A m 2 A Am m Am m Am m Am m Am m Am m Am m Am m Am m Am m Am -m 2 H1 C H m H H H m H 4 H m H HL L L L H L LO L LO L H H N L O 0. H. 0. 0 H. co 0 0. 0n 0. 0 H. H. 0 0. L L L Q Q QO HQ Q Am~~~ <2A < O <LOAm Am <LO<L 2 O 0O< A O AmL L 00 N O O 2 2 0 0 O AO <2O M 2 0 2 A O~ <2 2m 0< Am~~ <20<<22A 2 ~ ~~ AmA 2 2 AmAA AA 2 O 2 Am Am2m O 0 2 Am AmA< 0 0 0 2 m Am< <2 0 0 0 2 2 0 Am <2 0 0 04m< <2 <2 2 <2 2 <2 2 <2 2 <2 2 <2 2 <2 2 <2 Am~ ~~~~ Am m2 2 F- -f m -l 2 < m C 2 2m 2 <2 Am Am Am C Am A ) Am p) Am Am pm m m m Am m o m < o m < 2 m A m m m 2m < m H LA 4 0 A LO A LO u 4 u 0 0 u 0 4 H LO 0 A H LO u 0 A 0 H LO L A C) 2 2 2 < ) Am A C) 2 2 2 < ) Am A2 2 A) Am Am A Am C 2 A m C) A < 2 <2 2 A C A C) C A < 2 Am A A C) C) Am <2 2 <2 2 2 <2 Am C) Am Am ) <2 2 2 2 2 <2 Am C) 2 <2 <2 2 2 <2 C A ) Am <2 2 C Am C A Am 2 Am 2 < C) Am Am ) Am C <2 A C A C) Am A C) C A 2 <2 2 A A 2 <2 <2 2 ) Am A 2 2 C A C A Am 2 <2 <2 2 A ) <2 2 2 <2 <2 2 2 <2 2 <2 <2 2 C Am <A <2 2 2 <2 2 <2 C Am C Am Am <2 2 Am A ) A) Am <2 2 <2 2 Am C 2 <2m 2 A <2 2 <2 2 A C ) Am Am A Am A A <2 2 <2 2 A 2 <2 Am <2 2 <2 2 <2 m <2 2 A C A C) 2 AmC 2< 2< <22 u 0u AmC <2 <2 2< C)Am AmC C)A <2 AmC <2 <2 <22 AmC) u C)A C)A AmC CA C) A< A m ) Am ) 2 <2 ) AmA 2 <2 A C) A C) C) A Am C) C) Am <2 2 2 <2 <2 2 C) Am A C) Am C) 2 <2 C) A <2 2 2 < Am C) <2 2 <2 2 2 <2 A C) A C) C) Am <2 2 2 <2 Am C) <2 2 C) A 2 2 <2 Am C) Am C) Am C) Am C) Am <2 2 Am C) Am C) 2 <2 C) Am C) Am Am C) <2 2 <2 2 Am C) 2 <2 2 <2 Am C <2 2 C) Am C) Am <2 2 <2 2 <2 2 2 <2 2 <2 C) Am A C) 2 <2 C) Am C) Am A C) 2 <2 <2 2 m Am m C) m Am Am A A C) A 2 Am C) Am ) Am C) A Am A 2 m Am m 2 A C) A A Am N N m H q uQ 4 w w C U H C m H N 4q H 0.~~ . 0. . 0. . 0. 0. 0.A 0 A 0 0 0.A 0 A 0 Q 4 Q Q Q4 Q4 U U 4 Q Q Q Q Q Q 4 Q Q p I 4 4 I I 4 4 4 p I p p I I 4 4) <2 2 2 Am <2 C) 2 2 Am 4 ) C) C) C) <2 <2 C) Am4 4 Am 2 4 C4 < <2 <2 Am4 2 C) 2 4 2 <2 2 2 Am C) <2 C) C) 4 ) <2 <2 4 ) <2 U m 4 2 C A 4 2 U <2 Am 4C4 <2 Am Am4 C Am <24 2 AmU 2 <2G 2 C)4 C)4 <2Q C) U 4 Q s 4 Q U U D U 4 Q4 4Q QU 4Q 4Q Q U G U 4 C) Am4 <24Q AmG Am AmU AmG C)G 2 2 Am U GU Q 2 4 C) C)G U 2 <2 Am4 <2Q 2 <2U 2 2 <2 4 C) C ) <2 2 4C)4 4 C4 AmQ <2 AmU Am C) 4 <2 4 <4 Am4 2 4 <24 Am <24 Am4 4 C) AmG Am Am4 <2Q 4 Am4 2 AmD <2 4 3 U <2 4 Am <2G U AmU 2 2 <2 Am <24 <2U 2 C)Q Am Q U Am <2D 4<24 4 A4 C)4 C)Q Am Am4 C)Q C) <2 4 A4 AmQ 2 C) 2 AmU C)U G AmQ C)G <2Q 2 <2 C) Am4 Am4 2 C)Q <2D 2 <2 2 2 <2 4 Am C)G C)G Am 2 2 <2 <24 Am4 Am4 <2 Am <2U 2 Am4 Am4 C)Q 4 <2 2 AmG <2 4 C) 2 2 Am 4 A4m 2 4 A4 C)4 4 C4 AmQ C)G C) C) <2 4 C4 Am4 2 <24 C) 4 Am H) 2 2 <2 <2 Am <) <2 Am C) 2 <2 2 Am H) L) C)L <2O L AmL AmL 2 L C)L C)L AmL <2L <2L AmL 2 L 2 L Am C) C C) COA ALC <2 L) C 2 2 < L<2 L L) L2 < ) A ) C m 2< L2 L <2L 2 L <L L AL L AL L AL L AL 2 L <L L <L L AL L <L 2 2 <2 A A <A A <2 A A A <2 <2 2 <2 A A <2 j- u p u C) p) -n m2& 4 < & & H L 2 <2 L 2 L 2 C OD H LI C) IL C) O) C) LO L2 H L H 0 0 A 0 A R H 0 o o u o 4 <2 7 <2 <2 4 ) 0 4 0 4 2 40 <2 C) C) < <2 0 <2m< C) G U G U G C 42 <24 <2Q <2U <2 <2 42 <24 u 0 4 H A H B u 0 0 0 0 u 0 4 <2 <2 2 <2 <4 4 2 C) C) C) C) C) 4 2 C C) U < <2 C) C) C) C) <2 <2 42 C)Q C)U C)U CU CU C C) C 42 <24 C)Q C)U C)U CU C C)U H 2 F] ) H H N L 0 m L L N N O H H H H N O LO O OC H N H C O H C H C C H C H C H H H C H I I I I I I I I I I I I I I I I 4 C m m < 4C) 4Cm C 42 <2 4C) 4) < 4<2 4 < C) 4<2 <2< <2 <2< 42 C < <2 4 4C 4 <2 ) 4< C) 4< C) C 42 C) 4 42 C) 42 C C) 4 C)3 4 42 4) 42 C) C) 4) 4 <2 C4 C) <2 2 L F- - LK 2 K 2 0 2 L p - - 2 0 L p A 2 F o 2 0 2 m 0 2 0 2 0 K 2 m 2 m 2 m 2 m 0 2 0 2 < 2 < 2 m 2 < m 2 m 2 2 0 m m m 2m 0 2 2 0 <2 20 <2 u2 K2 E2 K2 K2 E2 E, 2 0 2 0 2 20 2m 02 m2 2 2 2 m2 20 2 0 K24 K24 24 2K4 42 402 02 42 42Q 42D K2Q 42 42 2 0 2 0 0 2 2 0 < 2 < 2 0 2 0 2 0 2 0 2 0 2 K m 2 4 0 2 m 2 0 2 0 < 2 u 2 0 2 0 2 0 K 2 K 2 K ~~ 2m 0 K 2 m 2 m 2 < 2 0 2 0 m 0 m 0 < 0 4 2 m 0 0 2 K 2 4 0 4 0 2 0 2 0 0 2 0 r m 2 m 2 2 K K 2 2 0 4 0 0 2 m 2 m 2 m 2 m 2 K 2 K 2 m 4 m 4 m < m 4 m m < 0 4 m m m m m < m 4 0 4 0 K 2 0 < 4 2 u m < 0 0 0 0 4 0 4 0 4 0 4 0 4 0 0 m 0 m 4 0 0 u 0 u u 0 4 0 4 m 4 u 41 11 El 1 1 4 u 1 1eo e o e K 2 K 2 K 2 K 2 K 2 2 K 2 0 4 2 4 2 4 2 4 2 2 K 2 K H 4 cl 0) H- r li V r H 4 Hn Ln Lw H L - m Lw o) LHU L O H L M L H cLO M o Ln L0 200 K 2 K K 2 2N 0 2 L0 2 u 4 u Am 2 mm F' Au2 A ~ 0 A ' A ' A 2 A m A ' A m A m F u m u u U; U; U; U ; U; U; 4; U U U U U ; U ; U m N 4H H Um a < C C H 4 Q 4 Q 4 Q Q 4 04 4 4 2 2 2 2 K L 2 0 K K K 0u 0 Km 2u 2m Km 2 2 0 2 < 2 2 2 2 2m + + + + + + 9z I- F-O Hn -L )< H - I F-Io H)< ~ ' 0 9z-'- 4*:c CA CA CA Table 4. In vitro efficacy of Modified AL-DP-4094 series 5'-sense strand-3' SiRNA Efficacy 3' -antisense strand-5' AL-DP-4198 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA-'5 AL-DP-4165 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4558 '3-GSUOMeCMeGUGUAUCCUCUCUACUGAsA-'5 AL-DP-4166 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4559 '3-GSUOMeCMeGUGUAUOMeCCUCUCUACUCGASA- '5 AL-DP-4167 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4560 '3-GSUOMeCGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA- '5 AL-DP-4168 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4561 '3-GSUOMeCOMeGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA- '5 AL-DP-4169 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4562 '3-GSUOMedCGUOMeGUOMeAUOMeCCUCUCUAdCUCGAA- '5 AL-DP-4170 AL4555 +++ 5'- GsCACAU 2 'OMeAGGAGAGAUGAGCUsU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA -'5 AL-DP-4171 AL4555 +++ 5'- GsCACAU 2 'OMeAGGAGAGAUGAGCUsU-3' AL4558 '3-GSUOMeCOMeGUGUAUCCUCUCUACUGAsA- '5 AL-DP-4172 AL4555 +++ 5'- GsCACAU 2 'OMeAGGAGAGAUGAGCUsU-3' AL4559 '3-GsUOMeCOMeGUGUAUOMeCCUCUCUACUGASA- '5 AL-DP-4173 AL4555 +++ 5'- GsCACAU 2 'OMeAGGAGAGAUGAGCUsU-3' AL4560 '3-GSUOMeCGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA- '5 AL-DP-4174 AL4555 +++ 5'- GsCACAU 2 'OMeAGGAGAGAUGAGCUsU-3' AL4561 '3-GSUOMeCOMeGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA-'5 AL-DP-4175 AL4555 +++ 5'- GsCACAU 2 'OMeAGGAGAGAUGAGCUsU-3' AL4562 '3-GSUOMedCGUOMeGUOMeAUOMeCCUCUCUAdCUCGAA-'5 AL-DP-4176 AL4556 +++ 5 '-GCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA -'5 AL-DP-4177 AL4556 +++ 5 '-GCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4558 '3-GSUOMeCOMeGUGUAUCCUCUCUACUGAsA-'5 AL-DP-4178 AL4556 +++ 5 '-GCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4559 '3-GSUOMeCOMeGUGUAUOMeCCUCUCUACUGASA- '5 AL-DP-4179 AL4556 +++ 5'-GCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4560 '3-GSUOMeCGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA-'5 AL-DP-4180 AL4556 +++ 5'-GCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4561 '3-GSUOMeCOMeGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA-'5 110 AL-DP-4181 AL4556 ++ 5 '-GCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4562 '3-GsUOMedCGUOMeGUOMeAUOMeCCUCUCUAdCUCGAA- '5 AL-DP-4220 AL2780 +++ 5'-GsCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL2781 '3-GsUOMeC OMeGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAsA-'5 AL-DP-4182 AL4563 +++ 5 '-G dC A dC AUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA -'5 AL-DP-4183 AL4563 +++ 5 '-G dC A dC AUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4558 '3-GsUOMe COMeGUGUAUCCUCUCUACUGAsA-'5 AL-DP-4184 AL4563 +++ 5 '-G dC A dC AUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4559 '3-GsUOMe COMeGUGUAUOMeCCUCUCUACUGAsA- '5 AL-DP-4185 AL4563 +++ 5 '-G dC A dC AUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4560 '3-GsUOMeCGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA- '5 AL-DP-4186 AL4563 +++ 5 '-G dC A dC AUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4561 '3-GsUOMeC OMeGUOMeGUOMeAUOMeCCUCUCUACOMeUCGAA-'5 AL-DP-4187 AL4563 +++ 5 '-G dC A dC AUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4562 '3-GsUOMedCGUOMeGUOMeAUOMeCCUCUCUAdCUCGAA- '5 AL-DP-4188 AL4564 +++ 5'-GsCACAUFAGGAGAGAUGAGCUsU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA -'5 AL-DP-4189 AL4565 +++ 5'-GCFACFAUFAGGAGAGAUFGAGCUFsU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA -'5 AL-DP-4190 AL4566 +++ 5 '-GCFACFAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA -'5 AL-DP-4191 AL4567 +++ 5 '-GCOMeACOMeAUFAGGAGAGAUFGAGCUFsU-3' AL4557 '3-GsUCGUGUAUCCUCUCUACUCGAsA -'5 AL-DP-4192 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4568 '3-GsUFCGUFGUFAUFCCUCUCUACFUCGAA-'5 AL-DP-4193 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4569 '3-GsUFCGUFGUFAUFCCUCUCUACOMeUCGAA- '5 AL-DP-4194 AL4554 +++ 5'- GsCACAUAGGAGAGAUGAGCUsU-3' AL4570 '3-GsUOMeCGUOMeGUOMeAUOMeCCUCUCUACFUCGAA- '5 AL-DP-4197 AL4556 ND 5'- GCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL4568 '3-GsUFCGUFGUFAUFCCUCUCUACFUCGAA-'5 AL-DP-4221 AL2780 +++ 5'- GsCOMeACOMeAUOMeAGGAGAGAUOMeGAGCUOMesU-3' AL2782 '3-GsUFCGUFGUFAUFCCUCUCUACFUCGAsA-'5 "Atugen Design" based on single overhang AL-DP-4195 AL4571 + 5'-GcAcAuAgGaGaGaUgAgCusU-3' 111 AL4572 '3-gsUcGuGuAuCcUcUcUaCuCgAa-'5 d deoxynucleotide OMe 2'0-Methyl F 2'Flouro s phosphorothioate linkage 5 N Mismatches in scrambled controls 112 Table 5. In vitro efficacy of siRNAs in HeLa cells siRNA Unmodified Strand # Efficacy 5'-sense strand- 3' parent 3'- antisense strand-5' AL-DP-4374 AL-DP-4055 AL2732 +++ 5' CsAAGUGGUCCCAGGCUGCATsT 3' AL2740 3' TsTGUUCACCAGGGUCCGACGsU 5' AL-DP-4375 AL-DP-4015 AL2728 +++ 5' GsGACAUCUUCCAGGAGUACTsT 3' AL2730 3' TsTCCUGUAGAAGGUCCUCAUsG 5' AL-DP-4379 AL-DP-4088 AL2963 +++ 5' COMeCOMeAAGUOMeGGUOMeCOMeCOMeCOMeAGGCOMeUOMeGCOMeTST3 AL2964 3' TsTGGUFUFCFACFCFAGGGUFCFCFGACFG 5' AL-DP-4380 AL-DP-4014 AL2966 +++ 5' GCOMeGGAUOMeCOMeAAACOMeCOMeUOMeCOMeACOMeCOMeAATsT 3 AL2967 3' 'TsTCFGCFCFUFAGUFUFUFGGAGUFGGUFUF,5' AL-DP-4219 AL-DP-4004 AL2712 +++ 5' GsUGAAUGCAGACCAAAGAAAsG 3' AL2720 13' UsACACUUACGUCUGGUUUCUUUsC 5' AL-DP-4140 AL-DP-4014 AL2281 - 5' GsCsGGAACAAUCCUGACCAsAsTsT 3' AL2282 3' TsTCGCCUUGUUAGGACUGGsUsU 3' OMe 2'0-Methyl F 2'Flouro 5 s phosphorothioate linkage N Mismatches in scrambled controls 113 c r- C) LO (0 M C) M (0 M .N N N N N N N N N cli m o o m co m co m - co - co - o- - (0 (0 (0 (0 (0 (0 (0 (0 (0 (0 (0 (0 (0 (0 (0 (0 tu + + + + + + + w - o - o- LOI-o 0 0- -o o o -o o clc 0 0 0 _0 _0_ 0- U) 0 U 0 )U - o - o U o - 00 -0 0 U . - 2 2 o 0 _o0 S0o 2 ( 0 D D 6D D24 0 L ... 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Claims (20)

1. An isolated iRNA agent, comprising a sense sequence and an antisense sequence, wherein the sense and the antisense sequences form an RNA duplex, and wherein the antisense sequence comprises a nucleotide sequence sufficiently complementary to a target sequence of about 19 to 23 nucleotides of a VEGF nucleotide sequence and wherein said target sequence differs by no more than 1, 2, or 3 nucleotides from a sequence selected from the group consisting of SEQ ID NOs: 1-401.

2. The iRNA agent of claim 1, wherein the sense sequence comprises a sequence that differs by no more than 1, 2, or 3 nucleotides from a sequence selected from the group consisting of SEQ ID NOs: 2-401.

3. The iRNA agent of claim 1, wherein the sense sequence comprises a sequence that differs by no more than 1, 2, or 3 nucleotides from a sequence selected from the group consisting of SEQ ID NO:456, SEQ ID NO:546, SEQ ID NO:548, SEQ ID NO:550, SEQ ID NO:552, SEQ ID NO:590, SEQ ID NO:592, SEQ ID NO:594, SEQ ID NO:596, SEQ ID NO:608, SEQ ID NO:610, SEQ ID NO:612, SEQ ID NO:614, SEQ ID NO:634, SEQ ID NO:636, SEQ ID NO:638, SEQ ID NO:640, SEQ ID NO:646, SEQ ID NO: 648, and SEQ ID NO:650.

4. The sense sequence of claim 3, wherein the sequence differs by no more than 1, 2, or 3 nucleotides from SEQ ID NO: 608.

5. The iRNA agent of claim 1, wherein the antisense sequence comprises a sequence that differs by no more than 1, 2, or 3 nucleotides from a sequence selected from the group consisting of SEQ ID NO:457, SEQ ID NO:547, SEQ ID NO:549, SEQ ID NO:551, SEQ ID NO:553, SEQ ID NO:591, SEQ ID NO:593, SEQ ID NO:595, SEQ ID NO:597, SEQ ID NO:609, SEQ ID NO:611, SEQ ID NO:613, SEQ ID NO:615, SEQ ID NO:635, SEQ ID NO:637, SEQ ID NO:639, SEQ ID NO:641, SEQ ID NO:647, SEQ ID NO: 649, and SEQ ID NO:651. 146

6. The antisense sequence of claim 5, wherein the sequence differs by no more than 1, 2, or 3 nucleotides from SEQ ID NO: 609.

7. The iRNA agent of claim 1, wherein the iRNA agent further comprises a non nucleotide moiety.

8. The iRNA of claim 7, wherein the sense and antisense sequences are stabilized against nucleolytic degradation.

9. The iRNA agent of claim 1, further comprising one 3'-overhang wherein said 3'-overhang comprises from 1 to 6 nucleotides.

10. The iRNA of claim 9, further comprising a second 3'-overhang wherein said second 3'-overhang comprises from 1 to 6 nucleotides.

11. The iRNA agent of claim 1, further comprising a phosphorothioate at the first internucleotide linkage at the 5' end of the antisense and sense sequences.

12. The iRNA agent of claim 1, further comprising a phosphorothioate at the first internucleotide linkage at the 3' end of the antisense and sense sequences.

13. The iRNA agent of claim 1, further comprising a phosphorothioate at the first internucleotide linkage at the 5' end of the antisense and sense sequences, and a phosphorothioate at the first internucleotide linkage at the 3' end of the antisense and sense sequences.

14. The iRNA agent of claim 1, further comprising a 2'-modified nucleotide.

15. The iRNA agent of claim 14, wherein the 2'-modified nucleotide comprises a modification selected from the group consisting of: 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-0 methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-0 147 dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-0 dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-O-N-methylacetamido (2'-0 NMA).

16. A method of reducing the amount of VEGF RNA in a cell of a subject, comprising contacting the cell with an iRNA agent of any one of the claims 1-15.

17. A method of making an iRNA agent of any of claims 1-15, the method comprising the synthesis of the iRNA agent, wherein the sense and antisense strands comprise at least one modification that stabilizes the iRNA agent against nucleolytic degradation.

18. A pharmaceutical composition comprising an iRNA agent of claim 1 and a pharmaceutically acceptable carrier.

19. A method of inhibiting VEGF expression comprising administering an effective amount of an iRNA agent of claim 1.

20. A method of treating a human diagnosed as having or at risk for having adult macular degeneration (AMD), comprising administering to a subject in need of such treatment a therapeutically effective amount of an iRNA agent of claim 1. DATED this NINTH day of AUGUST, 2013 Alnylam Pharmaceuticals, Inc. By Patent Attorneys for the Applicant: FB Rice 148