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Definitions

• the present invention is in the filed of iRNA agents that can inhibit expression of vascular endothelial growth factor (VEGF).
• VEGF vascular endothelial growth factor
• 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 VEGF also known as vascular permeability factor, VPF
• VPF vascular permeability factor
• 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.
• 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 ofthe retina malfunction or loose function and which can result in debilitating loss of vital central or detail vision.
• AMD which is the most common form of macular degeneration, occurs in two main forms. Ninety percent of people with AMD have the form described as "dry" macular degeneration.
• ⁇ AMD ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
• RNA interference or "RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al, Nature 391:806-811, 1998). Short dsRNA directs gene- specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi 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.
• 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.
• RNAi RNA interference
• small nucleic acid molecules such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA) and short hai ⁇ in RNA (shRNA) molecules, which collectively fall under 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.
• 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 sequences such as may exist 5 ' or 3 ' of a VEGF open reading frame (ORF) .
• 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.
• the VEGF sequence that is targeted includes the sequence of any one of SEQ ID NOs:2-401 (see Table 1).
• the sense sequence ofthe iRNA agent includes a sequence identical or substantially identical to any ofthe VEGF target sequences, e.g., substantially identical to any of sense sequences provided in Table 1, SEQ ID NOs:2-401.
• the antisense sequence ofthe iRNA agent can include a sequence complementary to or substantially complementary to, any ofthe target sequences, e.g., complementary to any of SEQ ID NOs: 2- 401.
• substantially identical is meant that the mismatch between the nucleotide sequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%.
• no more than 1, 2, 3, 4, or 5 nucleotides differ between the target sequence and sense sequence.
• sequences that are "complementary" to each other can be fully complementary, or can have no more than 1, 2, 3, 4, or 5 nucleotides that lack full complementarity.
• the sense and antisense pairs of sequences of an iRNA agent includes any one ofthe agents provided in Table 2, or a sequence which differs in the sense strand from 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.
• 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 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.
• the antisense sequence ofthe iRNA agent includes a sequence fully complementary or substantially complementary to any ofthe VEGF target sequences, e.g., complementary or substantially complementary to any of SEQ ID NOs:2-401.
• the antisense sequence of an iRNA agent includes a sequence selected from the group consisting any ofthe 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.
• this antisense sequence is fully complementary to a sense sequence or has no more than 1, 2, 3, 4, or 5 nucleotide mismatches with the sense sequence.
• 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.
• the iRNA agent is chemically modified.
• 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.
• conjugates can be used to increase uptake and target uptake ofthe iRNA agent to particular cell types.
• the iRNA agent includes a 3 '-overhang that ranges from 1 to about 6 nucleotides.
• 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' 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.
• the iRNA agent ofthe invention includes an antisense sequence having 23 nucleotides complementary to the target VEGF sequence and a sense 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.
• both the sense and antisense sequences ofthe iRNA agent include a 3' overhang, the length of which can be the same or different for each sequence.
• the 3' overhang on each sequence ranges from 1 to about 6 (e.g., from 1 to about 3) nucleotides in length.
• the 3' overhang is on both sequences ofthe iRNA agent and is two nucleotides in length.
• the 3' overhang is on both sequences ofthe iRNA agent and the 3 ' overhangs include two thymidylic acid residues ("TT").
• an iRNA agent includes an antisense sequence having about 19 to 25
• 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 complementary nucleotides.
• an iRNA agent ofthe 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 about 19 complementary nucleotides.
• the sense sequence can include a nucleotide sequence that is substantially identical to a nucleotide sequence of VEGF.
• 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- nucleotide moiety.
• the sense or antisense sequence ofthe 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'- O-aminopropyl (2'-O-AP), 2'-O-N-methylacetamido (2'-O-NMA), 2'-O- dimethylaminoethlyoxyethyl (2'-DMAEOE), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-AP), 2'-hydroxy nucleotide, or a 2 -ara-fluoro nucleotide, or a locked nucleot
• a 2' modification is preferably a 2'-OMe modification, and more preferably, a 2'- fluoro modification.
• one or more 2' modified nucleotides are on the sense strand ofthe iRNA agent.
• 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 ofthe terminal nucleotides ofthe iRNA agent.
• An alkylamino-dT conjugate is preferably attached to the 3' end ofthe sense or antisense strand of an iRNA agent.
• a pyrrolidine linker is preferably attached to the 3' or 5' end ofthe sense strand, or the 3' end ofthe antisense strand.
• an allyl amine uridine is preferably on the 3' or 5' end ofthe sense strand, and not on the 5' end ofthe antisense strand.
• An aminooxy conjugate can be attached to a hydroxyl prolinol and at the 3' or 5 ' end of either the sense or antisense strands.
• 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.
• the conjugate can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, retinol or a C5 pyrimidine linker.
• the conjugates are glyceride lipid conjugates (e.g. a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols.
• conjugates are on the 3' end ofthe antisense strand, or on the 5' or 3' end ofthe sense strand, and preferably the conjugates are not on the 3' end ofthe antisense strand and on the 3' end ofthe sense strand.
• the conjugate is naproxen, and the conjugate is preferably on the 5' or 3' end ofthe sense or antisense strands.
• the conjugate is cholesterol or thiocholesterol, and the conjugate is preferably on the 5 ' or 3' end ofthe sense strand and preferably not present on the antisense strand.
• the cholesterol is conjugated to the iRNA agent by a pyrrolidine linker, or serinol linker, or hydroxyprolinol linker.
• the conjugate is cholanic acid, and the cholanic acid is attached to the 5' or 3' end ofthe sense strand, or the 3' end ofthe antisense strand.
• the cholanic acid is attached to the 3' end ofthe sense strand and the 3' end ofthe antisense strand.
• the conjugate is retinol acid, and the retinol acid is attached to the 5' or 3' end ofthe sense strand, or the 3' end ofthe antisense strand.
• the retinol acid is attached to the 3' end ofthe sense strand and the 3' end ofthe antisense strand.
• an iRNA agent ofthe 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 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. In one embodiment, an iRNA agent is chemically modified.
• the iRNA agent includes a duplex molecule wherein one or more sequences ofthe duplex molecule is chemically modified.
• chemical modifications include phosphorothioate internucleotide linkages, 2 '-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2 '-deoxy-2' -fluoro ribonucleotides, "universal base” nucleotides, "acyclic" nucleotides, 5'-C-methyl nucleotides, and terminal glyceryl and/or inverted deoxy abasic residue inco ⁇ oration.
• an iRNA agent when used in iRNA agents, can help to preserve RNAi activity ofthe agents in cells and can increase the serum stability ofthe iRNA agents.
• an iRNA agent includes one or more chemical modifications and the sense and antisense sequences ofthe double-stranded RNA is about 21 nucleotides long.
• the first and preferably the first two internucleotide linkages at " the 5' end ofthe antisense and/or sense sequences are modified, preferably by a phosphorothioate.
• 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 sequences, and the 3' end of both the sense and antisense sequences are modified as described.
• 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 ofthe iRNA construct.
• the invention features an iRNA agent that includes one or more chemical modifications that can modulate the cellular uptake ofthe iRNA agent.
• the invention features an iRNA agent that includes one or more chemical modifications that improve the pharmacokinetics ofthe iRNA agent.
• 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; 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.
• conjugates such as ligands for cellular receptors, e.g., peptides derived from naturally occurring protein ligands; protein localization sequences; antibodies; nucleic acid aptamers; vitamins and other co-factors, such as folate, retinoids and N- acetylgal
• 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- 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).
• AL-DP-4003 AL-DP-4116, AL-DP-4015, AL-DP-4120, AL-DP-4002, AL-DP- 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-4
• the iRNA agent includes a duplex described as AL-DP- 4094, which includes the antisense sequence 5'AAGCUCAUCUCUCCUAUGUGCUG 3' (SEQ ID NO:609) and the sense sequence 5' GCACAUAGGAGAGAUGAGCUU 3' (SEQ ID NO:608).
• 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 NO:634).
• 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).
• 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).
• the antisense sequence of an iRNA agent described herein does not hybridize to an off-target sequence.
• the antisense sequence can have less than 5, 4, 3, 2, or 1 nucleotides complementary to an off-target sequence.
• off-target is meant a sequence other than a VEGF nucleotide sequence.
• 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 a pyrrolidine linker.
• 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.
• the invention provides a method of delivering an iRNA agent, e.g., an 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.
• a mammalian subject such as a mouse, a rat, a monkey or a human.
• 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.
• the iRNA agent can be delivered to a cell or cells in a choroid region ofthe eye.
• the iRNA agent down-regulates expression ofthe VEGF gene at a target site within the eye.
• An iRNA agent delivered to the eye e.g., choroid cells ofthe eye, can be an unmodified iRNA agent.
• the iRNA agent can be stabilized with phosphorothioate linkages.
• the 3' end ofthe sense or antisense sequences, or both, ofthe iRNA agent 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 ofthe terminal nucleotides ofthe iRNA agent.
• the modification can be an external or terminal cationic residue.
• a pyrrolidine cap is attached to the 3 ' or 5' end ofthe sense strand, or the 3' end ofthe antisense strand.
• the sense or antisense sequence, or both, ofthe 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.
• the iRNA agent can be conjugated to an enzyme substrate, e.g., a substrate for which the relative enzyme is present in a higher amount, as compared to the enzyme level in other tissues ofthe body, e.g., in tissues other than the eye. In one embodiment, at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more ofthe iRNA agent administered to the subject reaches the eye.
• the invention features a composition, e.g., a pharmaceutical composition that includes an iRNA agent ofthe present invention in a pharmaceutically acceptable carrier or diluent.
• the iRNA agent can be any agent described herein.
• 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.
• 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 ofthe invention under conditions suitable for the treatment or prevention ofthe disease or condition in the subject, alone or in conjunction with one or more other therapeutic compounds.
• the iRNA agent is administered at or near the site of unwanted VEGF 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).
• the iRNA agent is administered via an intraocular implant, which can be inserted, for example, into an anterior or posterior chamber ofthe eye; or into the sclera, transchoroidal space, or an avascularized region exterior to the vitreous.
• the implant is positioned over an avascular region, such as on the sclera, so as to allow for transcleral diffusion ofthe drug to the desired site of treatment, e.g., to the intraocular space and macula ofthe eye.
• the site of transcleral diffusion is preferably in proximity to the macula.
• an iRNA agent is administered to the eye by injection, e.g., by intraocular, retinal, or subretinal injection.
• 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.
• an iRNA is delivered at or near a site of neovascularization.
• 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 period of time, or until the patient no longer requires therapy.
• sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the area ofthe target VEGF nucleotide sequences.
• an iRNA agent is delivered to the eye at a dosage on the order of 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.
• an iRNA agent is administered prophylactically such as to prevent or slow the onset of a disorder or condition that affects the eye.
• an iRNA can be administered to a patient who is susceptible to or otherwise at risk for a neovascular disorder.
• one eye of a human is treated with an iRNA agent described herein, and in another embodiment, both eyes of a human are treated.
• a method of inhibiting VEGF expression is provided.
• One such method includes administering an effective amount of an iRNA agent ofthe present invention.
• a method of treating adult onset macular degeneration is provided. The method includes administering a therapeutically effective amount of an iRNA agent ofthe present invention.
• a human has been diagnosed with dry adult macular degeneration
• a human treated with an iRNA agent described herein is over the age of 50, e.g., between the ages of 75 and 80, and the human has been diagnosed with adult onset macular degeneration.
• 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.
• 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 degeneration.
• 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.
• methods of treating any disease or disorder characterized by unwanted VEGF expression are provided.
• Particularly preferred embodiments include the treatment of disorders ofthe 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.
• the invention features a kit containing an iRNA agent ofthe invention.
• the iRNA agent ofthe kit can be chemically modified and can be useful for modulating the expression of a VEGF target gene in a cell, tissue or organism.
• the kit contains more than one iRNA agent ofthe invention.
• FIGURE 1 is the nucleotide sequence ofthe mRNA of the 121 amino acid form of vascular endothelial growth factor, VEGF121.
• the first nucleotide ofthe initiator codon is nucleotide 1.
• the signal peptide is from nucleotide 1 through 78.
• FIGURE 2 is a graphical representation of a comparative analysis ofthe 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.
• FIGURE 3 is a graphical representation of a comparative analysis ofthe 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 siRNAs; dashed lines represent the control siRNAs.
• the control siRNA hVEGF is described in Reich et al. (Mol. Vis.
• FIGURE 4 is a graphical representation of a comparative analysis ofthe siRNAs activities 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 hrm VEGF is described in Filleur et al. (supra).
• L2000 refers to Lipofectamine 2000 reagent.
• hVEGF expression (y-axis) refers to endogenous VEGF expression.
• FIGURE 5 is a graphical representation of a comparative analysis ofthe 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 hrm VEGF is described in Filleur et al. (supra).
• L2000 refers to Lipofectamine 2000 reagent.
• hVEGF expression (y-axis) refers to endogenous VEGF expression.
• FIGURE 6A is a graphical representation ofthe 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).
• FIGURE 6B is a graphical representation ofthe 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 ofthe 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 (130 ⁇ M defoxamine).
• FIGURE 7 is a graphical representation ofthe comparative activities of double-overhang
• siRNAs targeting ORF 319 SEQ ID NO:320
• 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 8 A is a graphical representation ofthe 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 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.
• FIGURE 8B is a graphical representation ofthe 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 ofthe activities of siRNAs having the sequence of AL-DP-4094 but differing in the inclusion of nucleotide modifications (see Table 4).
• 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 ofthe hrmVEGF control siRNA of FIGURE. 7.
• FIGURE 10 is a graphical representation of siRNA silencing activity in vitro in HeLa 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.
• 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 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).
• 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 Tables 6 and 7.
• FIGURE 40 Cholesterol and 5/3-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 5Me C and 5Me U RNA building blocks for oligonucleotide synthesis. These building blocks were used for syntheses of selected sequences listed in Table 8.
• FIGURE 42 Naproxen - trans A- 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 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. 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.
• FIGURE 48 P-methylphosphonamidite of ribo 5Me U 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.
• FIGURE 50 Thiocholesterol conjugate building blocks.
• Table 1 provides the sequences in the VEGF gene that are targeted by the agents ofthe present invention. These sequence can also be the sense strand of some ofthe iRNA agents of 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.
• 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 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.
• 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.
• 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 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.
• Table 17 provides iRNA agents containing a variety and combinations ofthe modifications as noted in the Table.
• Table 18 provides physical characterization of iRNA agents containing a variety and combinations ofthe modifications as noted in the Table.
• Double-stranded directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi).
• RNAi RNA interference
• 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound by theory, it may be that a molecular signal, which may be merely the specific length ofthe fragments, present in these 21 -23 nt fragments recruits cellular factors that mediate RNAi.
• iRNA agents or recombinantly produced or chemically synthesized oligonucleotides ofthe same or similar nature
• dsRNA agent fragments can also be used, e.g., as described below.
• the length ofthe sense and antisense 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.
• a composition of iRNA agents e.g., formulated as described herein
• use of a 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.
• iRNA agent such as an iRNA duplex
• 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'- phosphate have been shown to be active when introduced exogenously, suggesting that 5'- phosphorylation of siRNA constructs may occur in vivo.
• VEGF isoforms are expressed in endothelial cells.
• the 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, 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.
• VEGF145 induces angiogenesis and like VEGF189 (but unlike VEGF165)
• VEGF145 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:l) 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 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 occurs during asthmatic episodes.
• RNA agent is an unmodified RNA, modified RNA, or nucleoside surrogate. Preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs.
• RNA agent is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA.
• an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms.
• An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5' modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
• the iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation ofthe target gene.
• nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage ofthe term "ribonucleotide" or
• nucleotide herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.
• the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage ofthe target RNA, e.g., mRNA.
• Complementarity, or degree of homology with the target strand is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides ofthe 5' and/or 3' terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double strand character ofthe 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 hai ⁇ in structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3'- or 5'-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored.
• Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.
• iRNA agents include: molecules that are long enough to trigger the interferon response
• RISC RNAi-induced silencing complex
• molecules that are sufficiently short that they do not trigger the interferon response which molecules can also be cleaved by Dicer and/or enter a RISC
• 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 refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs.
• the sRNA agent, or a cleavage product thereof can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.
• 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.
• 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.
• a "single strand iRNA agent" as used herein, is an iRNA agent which is made up of a single molecule.
• Single strand iRNA agents are preferably antisense with regard to the target molecule.
• single strand iRNA agents are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus.
• 5'- phosphate modifications include those which are compatible with RISC mediated gene silencing.
• Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O- P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5'
• a single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA.
• a single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length. Hai ⁇ in 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, 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 hai ⁇ in will preferably have a single strand overhang or terminal unpaired region, preferably the 3', and preferably ofthe antisense side ofthe hai ⁇ in. Preferred overhangs are 2-3 nucleotides in length.
• a "double stranded (ds) iRNA agent" as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.
• An iRNA agent can include a non-naturally occurring base, such as the bases described in co-owned 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 linkage) useful for increasing nuclease resistance.
• 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 Application No. PCT/US2004/07070 filed on March 8, 2004.
• An iRNA agent can be complexed with an amphipathic moiety.
• iRNA agents for use with iRNA agents are described in co-owned PCT Application No. PCT US2004/07070 filed on March 8, 2004.
• the iRNA agent can be complexed to a delivery agent that features a modular complex.
• the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type.
• a condensing agent e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions
• a fusogenic agent e.g., an agent capable of fusing and/or being transported
• 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 ofthe iRNA duplex.
• Exemplary features of non-canonical iRNA agents are described 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.
• 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 ofthe 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 pu ⁇ oses in humans.
• 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 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.
• 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 ofthe modifications provided in Tables 3-18.
• Candidate iRNA agents can be designed by performing, as done herein, a gene walk analysis ofthe VEGF gene that will serve as the iRNA target. Overlapping, adjacent, or closely spaced candidate agents corresponding to all or some ofthe transcribed region can be generated and tested. Each ofthe iRNA agents can be tested and evaluated for the ability to down regulate the target gene expression (see below, "Evaluation of Candidate iRNA agents").
• the iRNA agents ofthe present invention are based on and comprise at least 15 or more contiguous nucleotides from one ofthe iRNA agents shown to be active in Table 2, or the modified sequences provided in Tables 3-18.
• the agent can comprise, consist of or consist essentially ofthe entire sequence provided in the Table or can comprise 15 or more contiguous residues along with additional nucleotides from contiguous regions ofthe target gene.
• An iRNA agent can be rationally designed based on sequence information and desired characteristics and the information ofthe target sequence provided in Table 1. For example, an iRNA agent can be designed according to the relative melting temperature ofthe candidate duplex. Generally, the duplex should have a lower melting temperature at the 5' end ofthe antisense strand than at the 3 ' end ofthe antisense strand.
• 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 ofthe 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, 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 ofthe agents in Table 2 (or are complementary to the target sequence provided in Table 1) but are not longer than 25 nucleotides in length.
• the sense strand of an iRNA agent should be equal to or at least 15, 16 17, 18, 19, 25, 29,
• Exemplified iRNA agents include those that comprise 15 or more nucleotides from one ofthe agents in Table 2 (or the target sequence in Table 2) but are not longer than 25 nucleotides in 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.
• 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 ofthe 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 modification and agents provided in Tables 3-18.
• the iRNA agents ofthe 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 ofthe VEGF gene.
• the antisense strands of the iRNA agents of the present invention are preferably fully complementary to the mRNA sequences of VEGF gene.
• the iRNA agents of the instant invention include agents comprising a sense 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 ofthe sequences ofthe 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.
• agents comprising a sense 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 ofthe sequences ofthe 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
• These agents will therefore possess 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 within 6, 5, 4, or 3 nucleotides ofthe 5'-terminus ofthe sense strand or the 3'-terminus ofthe antisense strand.
• the sense strand need only be sufficiently complementary with the antisense strand to maintain the overall double stranded character ofthe 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 ofthe molecule, such as those exemplified in Table 2 (as well as Tables 3-18).
• 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 ofthe iRNA agent.
• the overhangs can be the result of one strand being longer than the other, or the result of two strands ofthe same length being staggered.
• 5'-ends are preferably phosphorylated.
• 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 ofthe siRNA agent are linked, e.g., covalently linked are also included. Hai ⁇ in, or other single strand structures which provide the required double stranded region, and preferably a 3' overhang are also within the invention.
• Oligonucleotides e.g., certain modified oligonucleotides or portions of oligonucleotides lacking ribonucleotides
• Oligonucleotides can be synthesized using protocols known in the art, for example as described in Caruthers et al., Methods in Enzymology 21_1 :3, 1992;
• RNA including certain iRNA agents ofthe 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
• nucleic acid molecules ofthe present invention can be synthesized separately and joined together post-synthetically, for example, by ligation (Moore et ah, 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.
• 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 ofthe 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- 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 inco ⁇ orated herein by reference) and re-suspended in water.
• 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.
• viral vectors can be used that provide for transient expression of iRNA agents.
• An iRNA agent may be susceptible to cleavage by an endonuclease or exonuclease, such as when the iRNA agent is introduced into the body of a subject. Methods can be used to 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
• cleavage e.g., cleavage by a component found in the body of a subject.
• the component can be specific for a particular area ofthe 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 ofthe body may be resistant to cleavage in other areas ofthe body.
• a method for evaluating an iRNA agent can include: (1) determining the point or points at which a substance present in the body of a subject, and preferably a component present in a compartment ofthe 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) identifying one or more points of cleavage, e.g., endonucleolytic, exonucleolytic, or both, in the dsRNA.
• the method further includes providing an RNA (e.g., an iRNA agent) modified to inhibit cleavage at such sites.
• an RNA e.g., an iRNA agent
• the steps described above can be accomplished by using one or more ofthe following assays: (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.
• a time course is taken and a number of samples incubated for different times are applied to the size-based assay.
• the 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.
• a size-based assay e.g., gel electrophoresis to determine if the iRNA agent is cleaved.
• a time course is taken where a number of samples are incubated for different times and applied to the size-based assay.
• the determination is made under conditions that allow determination ofthe number of nucleotides present in a fragment.
• an incubated sample is run on a gel having markers that allow assignment ofthe 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 one strand is labeled in a particular experiment.
• the label can be inco ⁇ orated 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 ofthe fragment based on the ladder and mapping using a site-specific endonuclease such as RNAse TI.
• Fragments produced by any method can be analyzed by mass spectrometry.
• 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.
• 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.
• modifications include modifications that inhibit endonucleolytic degradation, including the modifications described herein.
• Particularly favored modifications include: 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'-O- 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).
• 2' modification e.g., a 2'-O-methylated nucleotide or 2'-deoxy nucleotide (e.g., 2'deoxy- cytodine), or a 2 '-fluoro, difluoroto
• the 2' modification is on the uridine of at least one 5'-uridine-adenine-3' (SKUAS') 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 5 ) 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.
• SKUAS' 5'-uridine-adenine-3'
• the 2' modification can also be applied to all the pyrimidines in an iRNA agent.
• the 2' modification is a 2'OMe modification on the sense strand of an iRNA agent.
• the 2' modification is a 2' fluoro modification, and the 2' fluoro is on the sense or antisense strand or on both strands.
• Modification ofthe backbone e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification can be used to inhibit endonuclease activity.
• an iRNA agent has been modified by replacing one or more ribonucleotides with deoxyribonucleotides.
• 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 ofthe U with a C5 amino linker; replacement of an A with a G (sequence changes 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 ofthe iRNA agent.
• an iRNA agent includes a phosphorothioate linkage or P-alkyl modification in the linkages between one or more ofthe terminal nucleotides of an iRNA agent.
• one or more terminal nucleotides of an iRNA agent include a sugar modification, e.g., a 2' or 3' sugar modification.
• Exemplary sugar modifications include, for example, a 2'-O- 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'-O- dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-AP), 2'-hydroxy nucleotide, or a 2'-ara-fluoro nucleotide, or a locked nucleic acid (LNA), extended nucleic acid (ENA), hex
• 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.
• at least one terminal nucleotide of an iRNA agent has a phosphorothioate linkage and a 2' sugar modification, e.g., a 2'F or 2'OMe modification.
• 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.
• an iRNA agent can include 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 or a hydroxyprolinol conjugate, on one or more ofthe terminal nucleotides ofthe iRNA agent.
• An alkylamino-dT conjugate is preferably attached to the 3' end ofthe sense or antisense strand of an iRNA agent.
• a pyrrolidine linker is preferably attached to the 3 ' or 5' end ofthe sense strand, or the 3' end ofthe antisense strand.
• An allyl amine uridine is preferably on the 3' or 5' end ofthe sense strand, and not on the 5' end ofthe antisense strand.
• the iRNA agent includes a conjugate on one or more ofthe terminal nucleotides ofthe iRNA agent.
• the conjugate can be, for example, a lipophile, a te ⁇ ene, a protein binding agent, a vitamin, a carbohydrate, a retiniod, or a peptide.
• the conjugate can be naproxen, nitroindole (or another conjugate that contributes to stacking interactions), folate, ibuprofen, cholesterol, retinoids, PEG, or a C5 pyrimidine linker.
• the conjugates are glyceride lipid conjugates (e.g. a dialkyl glyceride derivatives), vitamin E conjugates, or thio-cholesterols.
• conjugates are on the 3' end ofthe antisense strand, or on the 5' or 3' end ofthe sense strand, and preferably the conjugates are not on the 3 ' end ofthe antisense strand and on the 3 ' end ofthe sense strand.
• the conjugate is naproxen, and the conjugate is preferably on the 5' or 3' end ofthe sense or antisense strands.
• the conjugate is cholesterol, and the conjugate is preferably on the 5' or 3 ' end ofthe sense strand and preferably not present on the antisense strand.
• the cholesterol is conjugated to the iRNA agent by a pyrrolidine linker, or serinol linker, aminooxy, or hydroxyprolinol linker.
• the conjugate is a dU-cholesterol, or cholesterol is conjugated to the iRNA agent by a disulfide linkage.
• the conjugate is cholanic acid, and the cholanic acid is attached to the 5' or 3' end ofthe sense strand, or the 3' end ofthe antisense strand. In one embodiment, the cholanic acid is attached to the 3' end ofthe sense strand and the 3' end ofthe antisense 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 ofthe sense strand.
• the iRNA agent includes an L-sugar, preferably at the 5' or 3' end of the sense strand. 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 ofthe sense or antisense strands ofthe iRNA agent. In one embodiment, an iRNA agent has been modified by replacing one or more ribonucleotides with deoxyribonucleotides.
• 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.
• DFT difluorotoluyl
• the methods described can be used to select and/or optimize a therapeutic dsRNA, e.g., iRNA agent.
• dsRNAs e.g., iRNA agents
• 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 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.
• modify it e.g., as described herein, e.g., to inhibit 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 ofthe nucleic acid molecules ofthe invention Chemically synthesizing nucleic acid molecules with modifications (base, sugar and/or phosphate) can 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.
• oligonucleotides are modified to enhance stability and/or enhance biological activity by modification with nuclease resistant groups, for example, 2'- amino, 2'-C-allyl, 2'- fluoro, 2'-O-methyl, 2'-O- allyl, 2'-H, nucleotide base modifications (for a review see Usman and Cedergren, Trends in Biochem. Sci.
• 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 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 ofthe molecule. They can include modification or replacement of an entire terminal phosphate or of one or more ofthe atoms ofthe phosphate group.
• an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
• labeling moieties e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
• the functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer.
• the terminal atom ofthe spacer can connect to or replace the linking atom ofthe phosphate group or the C-3' or C-5' O, N, S or C group ofthe sugar.
• the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs).
• PNAs nucleotide surrogate
• the array can substitute for a hai ⁇ in RNA loop in a hai ⁇ in-t pe 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.
• terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), po ⁇ hyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, l,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)
• 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.
• 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 ofthe 5' end with phosphate or phosphate analogs.
• iRNA agents, especially antisense sequences are 5' phosphorylated or include a phosphoryl analog at the 5' prime terminus.
• 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'- monophosphate ((HO)2(O)P-O-5'); 5'-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5*); 5'- tiiphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5*-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(0)P-0-(HO)(O)P-O-P(HO)(O)-O-5'); 5 * -adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O- (HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (
• the invention features conjugates and/or complexes of iRNA agents ofthe invention.
• conjugates and/or complexes can be used to facilitate delivery of 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 ofthe invention.
• the present invention encompasses the design and synthesis of novel conjugates and complexes for the delivery of molecules, including, but not 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.
• molecules including, but not 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.
• the transporters described are designed to be used either individually or as part of a multi-component system, with or without degradable linkers.
• Conjugates ofthe molecules described herein can be attached to biologically active molecules via linkers that are biodegradable, such as biodegradable nucleic acid linker molecules.
• a patient who has been diagnosed with a disorder characterized by unwanted VEGF expression can be treated by admimstration of an iRNA agent described herein to block the negative effects of VEGF, thereby alleviating the symptoms associated with unwanted VEGF gene expression.
• the iRNA agent can alleviate symptoms associated with a disease ofthe eye, such as a neovascular disorder.
• 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 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.
• Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by ionophoresis, or by inco ⁇ oration 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 andNormand, International PCT Publication No. WO 00/53722).
• 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.
• the iRNA agent is administered as naked iRNA.
• the iRNA agent ofthe invention can be administered to the subject by any means suitable for delivering the iRNA agent to the cells ofthe tissue at or near the area of unwanted VEGF expression, such as at or near an area of neovascularization.
• 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., 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 (e.g., a retinal pellet or an implant comprising a porous, non-porous, or gelatinous material).
• intravascular administration e.g., 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
• the iRNA agent ofthe invention can be delivered using an intraocular implant.
• Such implants can be biodegradable and/or biocompatible implants, or may be non-biodegradable implants.
• the implants may be permeable or impermeable to the active agent, and may be inserted into a chamber ofthe 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.
• the implant may be positioned over an avascular region, such as on the sclera, so as to allow for transcleral diffusion ofthe drug to the desired site of treatment, e.g., the intraocular space and macula ofthe eye.
• the site of transcleral diffusion is preferably in proximity to the macula.
• the iRNA agent ofthe 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 eyedroppers.
• the compositions can be administered directly to the surface ofthe eye or to the interior ofthe eyelid.
• 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.
• the iRNA agent ofthe 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 ofthe 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.
• a sustained release composition may be appropriate.
• An iRNA agent can be injected into the interior ofthe eye, such as with a needle or other delivery device.
• the iRNA agent ofthe invention can be administered in a single dose or in multiple doses. Where the administration ofthe iRNA agent ofthe invention is by infusion, the infusion can be a single sustained dose or can be delivered by multiple infusions. Injection ofthe agent directly into the tissue is at or near the site of neovascularization is preferred. Multiple injections ofthe agent into the tissue at or near the site of neovascularization are also preferred.
• Dosage levels on the order of about 1 ⁇ g/kg to 100 mg/kg of body weight per administration are useful in the treatment ofthe neovascular diseases.
• 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 ofthe invention to a given subject.
• the iRNA agent can be administered to the subject once, e.g., as a single injection or deposition at or near the neovascularization site.
• 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.
• 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.
• a dosage regimen comprises multiple 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.
• 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 ofthe formulation, the rate of excretion, the particular disorder being treated, the severity ofthe disorder, the pharmacodynamics ofthe iRNA agent, and the age, sex, weight, and general health ofthe patient. Wide variations in the necessary dosage level are to be expected in view ofthe differing efficiencies ofthe various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous or intravitreal injection. Variations in these dosage levels can be adjusted using standard empirical routines of optimization, which are well-known in the art.
• iRNA agents ofthe invention can be administered prophylactically in order to prevent or slow the onset of these and related disorders.
• an iRNA ofthe 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.
• Pharmaceutical compositions ofthe present invention are characterized as being at least sterile and pyrogen-free.
• pharmaceutical formulations include formulations for human and veterinary use.
• compositions ofthe 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 Pharmacy, 2003, Gennaro et al, the entire disclosures of which are herein inco ⁇ orated by reference.
• the present pharmaceutical formulations comprise an iRNA agent ofthe 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, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.
• compositions ofthe 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), 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 ofthe invention can be packaged for use in liquid form, or can be lyophilized.
• a solid pharmaceutical composition for oral administration can comprise any ofthe carriers and excipients listed above and 10-95%, preferably 25%-75%, of one or more iRNA agents ofthe invention.
• pharmaceutically acceptable formulation is meant a composition or formulation that allows for the effective distribution ofthe nucleic acid molecules ofthe instant invention in the physical location most suitable for their desired activity.
• Non-limiting examples of agents suitable for formulation with the nucleic acid molecules ofthe 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.
• P- glycoprotein inhibitors such as PluronicP85
• 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 ofthe instant invention 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.
• the invention also features the use ofthe composition comprising surface-modified 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; 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. Ada 1238:86, 1995).
• the long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues ofthe 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).
• compositions prepared for storage or administration that include a pharmaceutically effective amount ofthe 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 Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985), hereby inco ⁇ orated by reference herein.
• preservatives, stabilizers, dyes and flavoring agents can be provided.
• nucleic acid molecules ofthe present invention can also be administered to a subject 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.
• certain iRNA agents ofthe instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, Science 229:345, 1985; McGarry and Lindquist, Proc. Natl. Acad. Sci. USA 83:399, 1986; Scanlon et al, Proc.Natl. Acad. Sci.
• 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 Acids Res. 21:3249, 1993; Chowrira et al, J. Biol. Chem.
• RNA molecules ofthe present invention can be expressed from transcription units (see for example Couture et al, Trends in Genetics 12:510, 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 to, adeno-associated virus, retrovirus, adenovirus, or alphavirus.
• pol III based constructs are used to express nucleic acid molecules ofthe invention (see for example Thompson, U. S. Pats. Nos. 5,902,880 and 6,146,886).
• the recomb ⁇ nant vectors capable of expressing the iRNA agents can be delivered as described above, a. ⁇ d persist in target cells.
• viral vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary.
• the iRNA agent interacts with the target mRNA and generates an RNA-_i 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 desired target cell (for a review see Couture et al, Trends in Genetics 12:510, 1996).
• Additional ophthalmic indications for the iRNA agents of tlie invention include proliferative diabetic retinopathy (the most severe stage of diabetic; retinopathy), uveitis (an inflammatory condition ofthe eye that often leads to macular edema), cystoid macular edema following cataract surgery, myopic degeneration (a condition in which a patient with a high 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 ofthe iris).
• proliferative diabetic retinopathy the most severe stage of diabetic; retinopathy
• uveitis an inflammatory condition ofthe eye that often leads to macular edema
• 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.
• siRNA Design Four hundred target sequences were identified within exons 1-5 ofthe VEGF-A121 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 ofthe BLAST algorithm is described in 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 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).
• RNA was synthesized on Expedite 8909, ABI 392 and AB 1394 Synthesizers (Applied Biosystems, Applera Kunststoff GmbH, Frankfurter Str. 129b, 64293 Damistadt, Germany) at 1 ⁇ 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 Leuschne ⁇ ark 4:64347 Griesheim, Germany).
• Phosphorothioate linkages were introduced by replacement ofthe iodine oxidizer solution with a solution ofthe Beaucage reagent in acetonitrile (5% weight per volume). Cleavage ofthe 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 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 Biosciences Europe GmbH, Munzinger Strasse 9, 79111 Freiburg, Germany, ESI-MS: LCQ, Deca XP, Thermo Finnigan, Im Steinground 4-6, 63303 Dreieich, Germany) and capillary electrophoresis (P/ACE MDQ Capillary Electrophoresis System, Beckman Coulter GmbH, 85702 UnterschleiBheim, Germany). Purity ofthe isolated oligoribonucleotides was at least 85%. Yields and concentrations were determined by UV abso ⁇ tion of a solution ofthe 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.
• 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 ofthe siRNAs in their ability to inhibit expression of an endogenous VEGF gene. In this process the number of candidate 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 ⁇ l 10% fetal bovine serum in Dulbecco's Modified Eagle Medium (DMEM).
• the cells 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 ⁇ l of transfection reagent LipofectamineTM 2000 (Invitrogen Co ⁇ oration, Carlsbad, CA) was 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 well of a 96 well plate, 0.4 ⁇ l was diluted in 25 ⁇ l 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 ⁇ l ofthe diluted siRNA was combined with the diluted LipofectamineTM 2000 (total volume is 26.4 ⁇ 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 ⁇ l of 10% fetal bovine serum in DMEM was added to each of the siRNA:LipofectamineTM 2000 complexes and mixed gently by rocking the plate back and forth.
• 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 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.
• 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 ofthe siRNAs were compared to the activity ofthe control human VEGF siRNA of Reich et al. (supra) with "+” representing a lower activity, "++” representing similar 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 ofthe siRNAs are more active than the control siRNAs and may inhibit expression of VEGF by approximately 80%.
• the siRNA from Reich et al. reduced the level of endogenous hVEGF by approximately 20% 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.
• 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 ofthe siRNAs are more active than the control siRNAs and may inhibit expression of VEGF by approximately 90%.
• the siRNA from Reich et al. reduced the level of hVEGF by approximately 35% under the same experimental conditions.
• the siRNA based on consensus sequence hrmVEGF (Filleur et al, supra) reduced the expression level by approximately 70%.
• 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 ⁇ l 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 starting at 30 nM. 0.2 ⁇ l of LipofectamineTM 2000 transfection reagent (Invitrogen Co ⁇ oration, 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.
• siRNAs both single overhang siRNAs and double overhangs siRNAs directed against ORF regions having the first nucleotides corresponding to 319 and 343 respectively, together with the control siRNAs.
• FIG. 6B 1% oxygen
• FIG. 6C three ofthe experimental siRNAs achieved almost 95% inhibition of expression of VEGF, namely AL-DP-4094 (single-overhang) directed at ORF 343, and both ofthe siRNAs (single and double-overhangs) directed at ORF 319.
• AL-DP-4094 single-overhang
• siRNAs single and double-overhangs
• FIGs. 8 A 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).
• the unmodified (AL-DP-4014) and the phosphorothioate-modified siRNA (AL-DP-4127) reduced endogenous VEGF expression to less than 20% of its original expression level.
• the phosphorothioate-modified siRNA essentially abolished VEGF expression.
• Example 5 Modified VEGF siRNA molecules retain full activity and show enhanced 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.
• a panel of siRNAs were synthesized that retained the sequence ofthe AL-DP-4094 siRNA (Table 1) but included different modifications including phosphorothioate linkages, O- methyl-modified nucleotides, and 2'-fiuoro-modif ⁇ ed nucleotides (Table 4). The panel of siRNAs was tested in HeLa cells, and FIGs.
• 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).
• the Stains- All technique (Sigma, St. Louis, MO) was performed to examine the stability ofthe modified siRNAs.
• an siRNA duplex was incubated in 90% human serum at 37°C. Samples ofthe 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 ofthe RNA over the time course provided information regarding the susceptibility of the siRNA duplex to serum nuclease degradation.
• O-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.
• modified versions ofthe AL-DP-4094 siRNA included a phosphorothioate-modified siRNA (AL-DP-4198), a phosphorothioate plus O-methyl modified siRNAs (e.g., AL-DP-4180, AL-DP-4175, and AL-DP-4220), and phosphorothioate plus O-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 O-methyl and 2'- fluoro modifications stabilized the siRNAs against endonucleolytic degradation.
• 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 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 aCL, lmMNa2EDTA, 0.5% Na-deoxycholate deoxycholic acid, 1% IGEPAL CA-630, 0.05% SDS
• RIPA buffer 50 mM Tris-HCl, pH 8.0, 150 mM aCL, lmMNa2EDTA, 0.5% Na-deoxycholate deoxycholic acid, 1% IGEPAL CA-630, 0.05% SDS
• PNK Polynucleo
• siRNA duplex was added to 18 ⁇ l serum or tissue lysate or buffer control in PCR tube (0.2ml).
• a zero time point sample was removed immediately following the addition ofthe siRNA duplex by removing 2 ⁇ l and adding it to 18 ⁇ l 90%) formamide, 50 mM EDTA, 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, IX working PBS contains 0.14 M Sodium Chloride, 0.003 M Potassium Chloride, 0.002 M Potassium phosphate, 0.01 M Sodium phosphate) were taken at zero and the last time point ofthe experiment.
• 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 O-methyl modifications, as in AL-DP-4180 and AL-DP-4220, stabilized the siRNAs in rat whole eye extracts.
• 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 the siRNA following incubation in serum FIG. 11. Following incubation ofthe -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.
• 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'-0-methyl (2'OMe) or 2'-fluoro (2'F). Both pyrimidines (Py) and purines (Pu) could be modified as provided in Table 6. The first four pattems(A-D) inco ⁇ orated 2'OMe on both strands at every other position.
• 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 ofthe antisense strand and 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).
• 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) 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 1.
• siRNA duplexes 2 ⁇ M 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 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.
• 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 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.
• siRNAs were screened including the unmodified parent duplexes plus the four modular chemistries. Ofthe 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 both strands or 2'OMe pyrimidines in the sense strand and 2'F in the antisense strand (chemistries 1 and 3 above). Ofthe 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.
• RNA Synthesis ofthe iRNA agents RNA Synthesis using "fast" deprotection monomers 1.
• RNA synthesis Oligoribonucleotides were synthesized using phosphoramidite technology on solid phase employing an AKTA 10 synthesizer (Amersham Biosciences) at scales ranging from 35 to 60 ⁇ mol. Synthesis was performed on solid supports made of controlled pore glass (CPG, 520A, with a loading of 70 ⁇ mol/g) or polystyrene (with a loading of 71 ⁇ mol/g). All amidites were dissolved in anhydrous acetonitrile (70 mM) and molecular sieves (3A) were added.
• ETT 5-Ethyl thiotefrazole
• ETT 600 mM in acetonitrile
• 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-l,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'- end ofthe 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.
• the Cleavage and deprotection of support bound oligonucleotide After solid-phase synthesis, the RNA was cleaved from the support by passing 14 mL of 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.
• RNA 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 ⁇ L N-methylpyrolidin-2-one ( ⁇ MP), and 120 ⁇ L triethylamine (TEA) and 160 ⁇ L TEA-3HF were added. This mixture was brought to 65°C for 2h. After cooling to ambient temperature, 1.5 mL ⁇ MP and 1 mL of ethoxytrimethylsilane were added.
• ⁇ MP N-methylpyrolidin-2-one
• TEA triethylamine
• 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 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 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.
• DMT-on oligoribonucleotides a gradient of 7% B to 45% B within 10 column volumes (CN) was used and for cholesterol-conjugated R ⁇ A 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 with one-third volume IM ⁇ aOAc, 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.
• oligonucleotide solutions were concentrated to a volume ⁇ 15 mL. The concentrations ofthe solutions were determined by measurement ofthe absorbance at 260 nm in a UV spectrophotometer. Until annealing the individual strands were 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 (BeckmanCoulter) with an effective length of 20 cm was used.
• RNA samples were analyzed under denaturing conditions containing 6 M urea (eCap ssDNAlOO 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 wavelength detector (260 and 280 nm), column oven, autosampler, and internal degasser. A Dionex DNAPac PI 00 column (4*250mm) was used as at a flow rate of 1.0 mL/min and 30°C. Unconjugated RNA (20 ⁇ L, 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 80% B over 20 min was employed.
• LC-MS analysis was performed on an Ettan ⁇ LC-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 ⁇ L sample, 1 OD/mL concentration in water for unconjugated RNA and 40 ⁇ L for cholesterol-conjugated RNA) were injected onto a Waters Xterra C8 MS column (2.1 x 50 mm; 2.5 ⁇ m particle size) with a flow rate of 200 ⁇ L/min at 60°C.
• Composition of eluent A was 400 mM hexafluoroisopropanol (HFIP), 16.3 mM TEA in H 2 O, pH 7.9 and eluent B was methanol.
• HFIP hexafluoroisopropanol
• eluent B was methanol.
• oligoribonucleotides 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 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).
• PAGE polyacrylamide gel electrophoresis
• RNA Synthesis using "standard” deprotection monomers including ribo and 2 '-O-methyl 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 2' OMe RNA phosphoramidites with standard protecting groups and used at concentrations of 0.15 M in acetonitrile (CH CN) unless otherwise stated.
• CH CN acetonitrile
• RNA phosphoramidites were 5 '-O-Dimethoxytrityl-N -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-((8-cyanoethyl-N,N'-diisopropyl)phosphoramidite, 5'-O- Dimethoxytrityl-N 4 -acetyl-2'-O-tbutyldimethylsilyl-cytidine-3'-O-(i8-cyanoethyl-N,N'- diisopropyl)phosphoramidite and 5'-O-Dimethoxytrityl
• the controlled pore glass (CPG) was transferred to a screw 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 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 uL of NMP/Et 3 N/Et 3 N-HF and heated at 65°C 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 ofthe tube. Residual ether was removed in the 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 -20°C.
• 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 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 ⁇ L 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% 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
• 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 CPG (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.
• RNA phosphoramidites were 5'-O-Dimethoxytrityl-N 6 -benzoyl-2'-O- tbutyldimethylsilyl-adenosine-3 '-O-(j8-cyanoethyl-N,N'-diisopropyl) phosphoramidite , 5 '-O- Dimethoxytrityl-N 2 -isobutyryl-2'-O-tbutyldimethylsilyl-guanosine-3'-0-(/3-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-D
• Activator 5-ethyl thiotefrazole (0.25 M); Cap A: 5% acetic anhydride/THF/pyridine; Cap B: 10% N-methylimidazole/THF; 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 ofthe 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 deprotected with 1.0 mL of a mixture of ethanolic ammonia (1:3) for 7 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 uL of NMP/Et 3 N/Et 3 N-HF and heated at 50°C for 16 h to remove the TBDMS groups at the 2'position.
• 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 ofthe 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 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.
• RNA oligoribonucleotides 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 ⁇ mole 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.
• RNA phosphoramidites were 5'-O-Dimethoxytrityl- N 6 -benzoyl-2'-O-tbutyldimethylsilyl- adenosine- 3'-O-((8-cyanoethyl-N,N'-diisopropyl) phosphoramidite , 5'-O-Dimethoxytrityl- N -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-Dimethoxytrity
• the coupling times were 10 min. Details of the other reagents are as follows: activator: 5-ethyl thiotefrazole (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 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.
• the white residue obtained was resuspended in 200 ⁇ L of TEA3HF 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 ⁇ L 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 (Table 7)
• A Synthesis of CPGs for 2'F.
• CPGs of 5'-O-DMTr-2'-deoxy-2'-fluororibonucleosides with appropriate base protection were synthesized as shown in Scheme A.
• 5'-O-DMTr-2'-Deoxy-2'-fluoro-N Bz -A and 5'-O- DMTr-2'-Deoxy-2'-fluoro-N' Bu -G were synthesized as reported (Kawasaki et al, J. Med. Chem., 1993, 36, 831).
• 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 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.
• RNA phosphoramidites were 5'-O-Dimethoxytrityl-N 6 -benzoyl-2'-O- methyl-adenosine-3 '-O-(/3-cyanoethyl-N,N'-diisopropyl ⁇ phosphoramidite, 5 '-O- Dimethoxytrityl-N 2 -isobutyryl-2'-O-methyl-guanosine-3'-O-(j8-cyanoethyl-N,N'- diisopropyl)phosphoramidite, 5 ' -O-Dimethoxytrityl-N 4 - acetyl-2 ' -O-methyl-cytidine-3 ' -O-(
• the 2'F RNA phosphoramidites 5'-O- Dimethoxytrityl-N 4 -acetyl-2'-fluoro-2'-deoxy-cytidine-3'-O-(i8-cyanoethyl-N,N'- diisopropyl)phosphoramidite, 5 ' -O-Dimethoxytrityl-2 '-:fluoro-2'-deoxy-uridine-3 '-O-(/3- cyanoethyl-N,N'-diisopropyl)phosphoramidite.
• Activator 5-ethyl thiotefrazole (0.25M); Cap A: 5% acetic anhydride/THF/pyridine; Cap B: 10% N-methylimidazole/THF; phosphate oxidation involved 0.02M I 2 /THF/H 2 O, 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 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 55°C.
• the tube was cooled briefly on ice and then the solution was transfened 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 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°C.
• 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 the monomers were RNA phosphoramidites with standard protecting groups (5 ' -O- 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-cyanoethy
• the reaction was then quenched with isopropoxytrimethylsilane (iPrOMe Si, 400 ul) and further incubated on the heating block leaving the caps open for lOmin; This causes the volatile isopropoxytrimethylsilylfluoride 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.
• 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.
• the oligonucleotides were purified by vertical slab polyacrylamide gel electrophoresis (PAGE) using an Owl's Separation Systems (Portsmouth, NH). Electrophoresis grade 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 ethylenediaminetefraacetate) were from National Diagnostics (Atlanta, GA).
• the 12 % denaturing gel was prepared for purification of unmodified and modified oligoribonucleotides.
• the thickness ofthe preparative gels was 1.5 mm.
• Loading buffer was 80% formamide in lOx TBE.
• 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 was removed by drying in a speed vac.
• 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) a.nd the monomers were RNA phosphoramidites with standard protecting groups (5 '-O- dimethoxytrityl N6-Benzoyl-2'O-t-butyldimethylsilyl- adenosine-3'-O-N,N'-diisopropyl-2- cyanoethylphosphoramidite, 5 '-O-dimethoxytrityl-N4-acetyl-2 '-O-t-butyldimethylsilyl-cytidirie- 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-lH-tetrazole (0.25M) was used as an 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).
• MS analysis Samples ofthe RNA ( 0.1 OD) were analyzed using MS.
• the oligonucleotides were purified by vertical slab polyacrylamide gel electrophoresis (PAGE) using an Owl's Separation Systems (Portsmouth, NH). Electrophoresis grade acrylamide (40%), N,N'-methylene-bis(acrylamide) (BIS), ammonium persulfate (APS,
• N,N,NTSf '-teframethyl enediamine (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 ofthe preparative gel was 1.5 mm. Loading buffer was 80% formamide in lOx TBE.
• RNAse free water was applied to 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
• 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 conjugation - oxime approach 1 for 5 '-conjugation.
• 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 with diethyl ether and filtered. Residue was washed with ether, filtered and combined the filtrate. Hexane was added dropwise into the filfrate 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).
• 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 through a celite bed over a sintered funnel. The residue was subsequently passed through a 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, CDC1 3 , 25 °C) 7.83-7.81 (bm.
• Step 3 Compoimd 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 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.
• Step 4 Conjugation of all-traws-retinal to Oligonucleotide: All-tr ⁇ Tz.s-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 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).
• RNA oligonucleotide deprotection and purification under dark yielded the desired oligonucleotide-retinal conjugate 109.
• Compound 109 was also obtained from compound 110 as shown in Scheme C. Complete deprotection and purification of compound 106 yielded an unbound free oligonucleotide 110 which was subsequently reacted with all-tr ⁇ z.y-retinal to afford the desired compound 109.
• Phosphoramidite 104 (standard oligonucleotide synthesis cycle); (ii) Hydrazinium hydrate/Py/AcOH (0.124/4/7); (iii) all-traws-retinal in DMF or MeCN; (iv) Oligonucleotide (RNA) deprotection (MeNH 2 , TEA.3HF) and purification; (v) Oligonucleotide (RNA) deprotection (MeNH 2 , TEA.3HF) and purification; (vi) all-tr ⁇ «.s*-retinal in DMSO-H 2 O
• 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
• RNA phosphoramidites with standard protecting groups 5'-O-dimethoxytrityl-N6-benzoyl-2'-t- buxyldimethylsilyl-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-
• 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). Coupling/recycling time of 16 minutes was used.
• the activator was 5-ethyl thiotefrazole
• the aminooxy-linker phosphoramidite was synthesized as described above and used at a concentration of 0.15M in acetonitrile. Coupling time for the aminooxy-linker phosphoramidite was 15 minutes. For all sequences, coupling ofthe aminooxy-linker phosphoramidite was carried out on the ABI 390 DNA synthesizer.
• Step 4.2 Cleavage ofthe phthalimido-protecting group from the aminoxy-linker oligonucleotides
• 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.
• the CPG was washed with 2x5 ml of pyridine followed by 3x5ml of acetonitrile.
• Step 4.3 On support conjugation with the aldehydes
• the 1-pyrene-carboxaldehyde and the all-tr ⁇ ns-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 DMF.
• the CPG from above was added to the aldehyde solutions. Conjugation was carried out 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.
• sequence AL-3213 the conjugation with both all-tr ⁇ ras-retinal and 1-pyrene-carboxaldehyde was also carried out in acetonitrile.
• 1-pyrene-carboxaldehyde the aldehyde did not fully dissolved at 0.5M and the solution was used as is without filfration to get rid ofthe undissolved aldehyde.
• Step 4.4 Deprotection- 1 fNucleobase Deprotection of on support conjugated oligonucleotides
• the support was fransfened to a 5 ml 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. Step 4.5.
• Deprotection-II Removal of 2' TBDMS group
• TREAT-HF triethylamine trihydrofluoride
• TDMS tert-butyldimethylsilyl
• Step 4.6 After deprotection conjugation with aldehydes Conjugation with the aldehydes (1-pyrene-carboxaldehyde and all-trara-retinai) after deprotection ofthe aminooxy-linker oligonucleotides was also carried out as an alternative 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 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 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.
• Step 4.8 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
• Deprotection- II Removal of 2' TBDMS group
• TBDMS tert-butyldimethylsilyl
• Step 4.9 Ouantitation of Crude Oligomer or Raw Analysis
• a l ⁇ l, a lO ⁇ l or 30 ⁇ l aliqoute was diluted with 999 ⁇ l, 990 ⁇ l or 970 ⁇ l of 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 abundance ofthe expected final product.
• Step 4.11 Desalting of purified oligonucleotides
• 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.
• Step 4.12. Capillary Gel Electrophoresis (CGE). Ion-Exchange HPLC (IEX) and
• 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 D a .
• Step 5.2 Compound 13: Compound 112 (9.4 g, 14.54 mmol) was suspended in 15 mL 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 ofthe 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- hexane (2:1) followed by neat dichloromethane.
• the protected RNA was assembled on an AKTA Oligo Pilot 100 on a 100-150 ⁇ mol 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.
• the support-bound RNA was treated with aqueous CH 3 NH 2 (40%)for 90 minutes at 45°C, cooled, filtered and washed with DMSO (3x40mL).
• 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.
• Oigonucleotide - PEG Conjugate a (i) Solid phase Oligonucleotide synthesis; (ii) Deprotection and purification; (iii) PEG- NHS ester, NaHCO 3 , pH 8.1, 1 h. Step 2. Deprotection- 1 (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 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 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 For all samples, a lO ⁇ l aliqoute was diluted with 990 ⁇ l of deionised nuclease free water (1.0 mL) and absorbance reading obtained at 260 nm.
• TREAT-HF triethylamine trihydrofluoride
• TDMS tert-butyldimethylsilyl
• Step 5 Purification of Oligomers fa) HPLC Purification
• the crude oligomers were first analyzed by HPLC (Dionex PA 100).
• 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 ⁇ l 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 fractions containing the fulllength oligonucleotides were then pooled together, evaporated and reconstituted to ⁇ 100 ml with deionised water.
• 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 fractions were combined together, dried down and reconstituted in 50 ml of RNase free water.
• Step 7 Capillary Gel Electrophoresis (CGE) and Electrosprav LC/Ms Approximately 0.15 OD of desalted oligonucleotides were diluted in water to 150 ⁇ l and then pipetted in special vials for CGE and LC/MS analysis.
• CGE Capillary Gel Electrophoresis
• Electrosprav LC/Ms Approximately 0.15 OD of desalted oligonucleotides were diluted in water to 150 ⁇ l and then pipetted in special vials for CGE and LC/MS analysis.
• Step 8 PEG conjugation.
• A) Initial reaction conditions. The purified and desalted RNA was lyophilized. RNA (lmg) was dissolved in aq.NaHCO 3 (0.1M, 200 ⁇ L, pH 8.1) and DMF (200 ⁇ L each). 5 K (13 equivalents, lOmg) or 20KPEG (3.4 equivalents, lOmg) was added directly to 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. NaOAc (0.05M, pH 5.5) until the pH was ⁇ 7. B) Borate buffer conjugation.
• RNA was lyophilized.
• a sample of RNA (lmg) was dissolved in sodium borate buffer (200 ⁇ L, 0.05M,pH10).
• 5KPEG (3mg, 4.5 equivalents Sunbright ME-50HS, NOF Corp.) was dissolved in CH 3 CN (200 ⁇ L).
• 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.
• RNA (lmg) was dissolved in aq, NaHCO 3 (0.1M, 200 ⁇ L, pH 8.1) and DMF (200 ⁇ L).
• 5KPEG (13.5 eq, lOmg, 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. NaOAc (0.05M, pH 5.5) until the pH was ⁇ 7.
• RNA 50mg was dissolved in aq. NaHC0 3 ( 0.1M, 2mL pH 8.1) and DMF (lmL).
• 20KPEG approximately 2.7 eq, 400-520mg Sunbright ME-200HS, different amounts for different sequences within this range
• CH 3 CN 2mL
• the RNA solution was added to the PEG solution and vortexed thoroughly.
• H 2 O 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.
• 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 ofthe activities in HeLa cells for each ofthe modifications described above.
• RNA molecules were synthesized on a 3 94 ABI machine using the standard cycle written by the manufacturer with modifications to a few wait steps.
• the solid support was 500 A dT CPG (2 umole).
• the monomers were either RNA-. phosphoramidites or the ribo-difluorotoluyl amidite. All had standard protecting groups and were used at concentrations of 0.15 M in acetonitrile (CH 3 CN) unless otherwise stated.
• the phosphoramidites were 5'-O- Dimethoxytrityl-N 6 -benzoyl-2'-O-tbutyldimethylsilyl-adenosine-3'-O-(jS-cyanoethyl-N,N'- diisopropyl) phosphoramidite, 5'-O-Dimethoxytrityl -N 2 -isobutyryl-2'-O-tbutyldimethylsilyl- guanosine-3 '-O-(j8-cyanoethyl-N,N'-diisopropyl)phosphoramidite, 5 ' -O-Dimethoxytrityl-N 4 - acetyl-2'-O-tbutyldimethylsilyl-cytidine-3'-O-(/3-cyanoethyl-N,N'-diisopropyl)phosphoramidite, and 5 '-O-Dimeth
• RNA monomers were 7 min for all RNA monomers and 10 min for the DFT monomer.
• Activator 5-ethylthio-lH-tetrazole (0.25M)
• Cap A 5% acetic anhydride/THF/pyridine
• Cap B 10% N-methylimidazole/THF
• phosphate oxidation involved 0.02M I 2 /THF/H 2 O.
• Detritylation was achieved with 3% TCA/dichloromethane.
• the DMT protecting group was removed after the last step ofthe 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 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.
• the white residue obtained was resuspended in 200 uL of triethylamine trihydro fluoride and heated at 65°C for 1.5 h to remove the TBDMS groups at the 2'-position.
• oligonucleotides were then precipitated in dry methanol (400 uL). The liquid was removed carefully to yield a pellet at the bottom ofthe 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. 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 ofthe activities in HeLa cells for each ofthe modifications described above.
• 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 500 A dT CPG (2 ⁇ mole).
• the monomers were either RNA phosphoramidites, or 2'- r fluro-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.
• RNA phosphoramidites were 5'-O-Dimethoxytrityl-N 6 -benzoyl-2'-O-tbutyldimethylsilyl- adenosine-3 '-O-(c ⁇ -cyanoethyl-N,N'-diisopropyl) phosphoramidite, 5 '-O-Dimethoxytrityl-N 2 - isobutyryl-2'-O-tbutyldimethylsilyl-guanosine-3'-O-(jS-cyanoethyl-N,N'- diisopropyl)phosphoramidite, 5'-O-Dimethoxytrityl-N 4 -acetyl-2'-O-tbutyldimethylsilyl-cytidine- 3 '-O-(/3-cyanoethyl-N,N'-diisopropyl)phosphoramidite, and 5 ' -O-D
• the oligonucleotide was cleaved and the base and phosphate groups were simultaneously deprotected with 1.0 mL of a mixture of ethanolic ammonia cone (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 ⁇ L of triethylamine trihydrofluoride and heated at 65°C for 1.5h to remove the TBDMS groups at the 2'-OH position.
• oligonucleotides were then precipitated in dry methanol (400 ⁇ L). The liquid was removed carefully to yield a pellet at the bottom ofthe 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. 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 ofthe activities in HeLa cells for each ofthe modifications described above.
• Example 15 Deprotection of Methylphosphonate Modified siRNAs (Table 15) Deprotection step 1 : After completion ofthe synthesis, the controlled pore glass (CPG) w ⁇ as transferred to a screw cap vial. A solution (0.5 ml) consisting of Acetonifrile/Ethanol NH OH (45:45:10) was 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).
• CPG controlled pore glass
• the combined supernatant was diluted with water (15 mL).
• the pH was adjusted to 7.0 with 6 M HCl in AcCN/H 2 O (1:9).
• the sample was desalted using a Sep- pak C ⁇ 8 cartridge and then dried in a speed vac.
• Deprotection step 2 Removal of 2'-O- TBDMS group
• the white residue obtained was resuspended in a mixture of triethylamine, triethylamine trihydrofluoride (TEA.3HF ca, 24% HF) and l-Methyl-2-Pyrrolidinone (NMP) (4:3:7) (400 ul) and heated at 65°C for 90 min to remove the tert-butyldimethylsilyl (TBDM S) groups at the 2'- position.
• TEA.3HF ca triethylamine trihydrofluoride
• NMP l-Methyl-2-Pyrrolidinone
• Duplexes are shown with the sense strand written 5 ' to 3 ' .
• the complementary antisense strand is written below the sense strand in the 3 ' to 5 ' direction.
• Lower case “d” indicates a deoxy nucleotide; all other positions are ribo.
• Lower case “s” indicates a phosphorothioate linkage.
• Subscript “OMe” indicates a 2'-0-methyl sugar and subscript “F” indicates a 2'-fluoro modified sugar.
• the extinction coefficient is the value at 260 nm (*10 "3 ).
• Duplexes are shown with the sense strand written 5' to 3'.
• the complementary antisense strand is written below the sense strand in the 3' to 5' direction.
• Lower case “s” indicates a phosphorothioate linkage.
• Subscript "OMe” indicates a 2'-0-methyl sugar and subscript “F” indicates a 2'-fluoro modified sugar.
• the parent duplexes had unpaired nucleotides at one or both ends ofthe duplex. These duplexes have blunt ends.
• the extinction coefficient is the value at 260 nm(*10 "3 ).
• Table 8A-B Cholesterol and cholanic acid conjugates of active VEGF sequences (single strands).
• the antisense strand ofthe duplex is shown written 5' to 3'.
• Lower case “s” indicates a phosphorothioate linkage.
• Lower case “d” indicates a deoxy.
• Subscript “F” indicates a 2'-fluor sugar.
• 5 e U indicates a 5-methyl-uridine.
• Naproxen indicates a naproxen conjugated to the oligonucleotide through a serinol linker.
• oligonucleotides are written 5' to 3'.
• Lower case “s” indicates a phosphorothioate linkage.
• Lower case “d” indicates a deoxy.
• Subscript “OMe” indicates a 2'-0-methyl sugar and subscript “F” indicates a 2'-fluoro modified sugar.
• 5Me u indicates a 5-methyl uridine.
• Table 1 la-b Conjugation of aldehydes. Retinal and other Retinoids to VEGF siRNAs and model oligonucleotides.
• NH2 indicates a hydroxyprolinol amine conjugate used as a control.
• HP-NH2-20KPEG or “20KPEG-NH2-HP” indicates conjugation to polyethylene glycol (20K) through the hydroxyprolinol linker.
• HP-NH2-5KPEG or 5KPEG-NH2-HP” indicates conjugation to polyethylene glycol (20K) through the hydroxyprolinol linker.
• the control in this column indicates that the oligonucleotide is not complementary to VEGF.
• Oligonucleotides 3164, 3170, and 3171 target ApoB and oligonucleotides 2936, 3187, 3188, 2937, 3172, and 3173 target luciferase.
• the range in observed molecular weight is due to the polydispersity of PEG starting material.
• Duplexes are shown with the sense strand written 5' to 3'.
• the complementary antisense strand is written 3' to 5'.
• Lower case “d” indicates a deoxy nucleotide; all other positions are ribo.
• Lower case “s” indicates a phosphorothioate linkage.
• “F” indicates a ribo-difluorotoluyl modification. Positions altered relative to the control duplex are indicated in bold face type.
• oligonucleotides are shown written 5' to 3'. Lower case “s” indicates a phosphorothioate linkage. Subscript “mp” indicates a methyl phosphonate linkage. Lower case “d” indicates a deoxy nucleotide.
• oligonucleotides are shown written 5' to 3'.
• Lower case “s” indicates a phosphorothioate linkage.
• Subscript “aa” indicates an allyamino modification.
• Lower case “d” indicates a deoxy nucleotide.

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