CA2846756A1 - Short interfering ribonucleic acid (sirna) for oral administration - Google Patents

Abstract

Short interfering ribonucleic acid (siRNA) for oral administration, said siRNA

comprising two separate RNA strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein at least one of which strands contains at least one chemical modification.

Description

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This is a divisional application of Canadian application serial No. 2,649,876 Short interfering ribonucleic acid (siRNA) for oral administration Background:
RNA interference initially discovered in plants as Post-Transcriptional Gene Silencing (PTGS), is a highly conserved mechanism triggered by double-stranded RNA
(dsRNA) and able to down regulate transcript of genes homologous to the dsRNA.I. The dsRNA
is first processed by Dicer into short duplexes of 21-23 nt, called short interfering RNAs (siRNAs)2.
Incorporated in RNA-induced silencing complex (RISC) they are able to mediate gene silencing through cleavage of the target rnRNA in the center of the region of homology by Argonaute 2, a component of RISC3. In 2001, Elbashir et a14 demonstrated that the direct introduction of synthetic siRNAs would mediate RNA interference gene silencing in drosophila but also in mammalian cells. Since then, siRNA-mediated gene silencing has become a powerful and widely-used molecular biology tool in both target identification target validation studies. Use of siRNAs for gene silencing in animal studies has been described in a limited amount of animal models. Unmodified siRNAs were delivered locally in the eyes, intrathecally or intracerebellarly in the central nervous systems, and intranasally for the inhibition of respiratory viruses?. Intravenous hydrodynamic tail vein injection of unmodified siRNAs has also been studied. This approach allows a rapid delivery, mainly to the livers. A
very limited number of studies have been reported on the systemic administration of unmodified siRNAs. Duxbury et als administered intravenously unmodified siRNAs targeting Focal Adhesion ICinase to an orthotopic tumor xenograft mice model, and observed a tumor growth inhibition as well as a chemosensitization to gemcitabine. Soutscheck et al reported the systemic use of highly chemically modified siRNAs for the endogeneous silencing Apolipoprotein B. Intraperitoneal administration of most anti-ApoB siRNA at the high dose of 50 mg/kg reduced ApoB protein level and Lipoprotein concentration's.
Despite these examples, in vivo use of siRNAs upon systemic delivery requires improvements in order to make this technology widely applicable for target validation or therapeutic applications.
Indeed, unmodified siRNAs are subject to enzymatic digestion, mainly by nucleases abundant in the blood stream. In order to improve pharmacological properties of siRNAs several groups investigated chemical modification of these reagents. While the approaches described are very different among themselves and that no systematic study was yet performed, an overview of the results allows to determine the tolerance of siRNAs to chemical modifications. Several chemistries such as phosphorothioatesu or boranophosphatesu, 2'-0-Methy113, 2'-0-ally114, 2'-methoxyethyl (MOE) and 2'-deoxyfluoronucleotidesi5 or Locked Nucleic Acids (LNA)I6 have been investigated. These studies highlighted that tolerance for modification is not only chemistry-dependent, but also position-dependent.
The present invention provides a minimally modified siRNA with improved pharmacological properties_ The minimally modified siRNAs are 19bp double-stranded RNA
modified on the 3'-end of each strand in order to prevent 3'-exonuelease digestion: the 3'-dideoxynucleotide overhang of 21-nt siRNA has been replaced by a universal 3'-hydroxypropyl phosphodiester moiety and the modification of the two first base-pairing nucleotides on 3'-end of each strand further enhances serum stability. Applied intraperitoneally or orally to adult mice, the modified siRNAs displayed higher potency in a growth factor induce angiogenesis model which correlates with their increased serum stability.
Summary:
In one aspect, the present invention provides a short interfering ribonucleic acid (siRNA) for oral administration, said siRNA comprising two separate RNA
strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein at least one of which strands contains at least one chemical modification.
In one embodiment, the siRNA comprises at least one modified nucleotide.
In another embodiment, the siRNA comprises at least one 3' end cap.
In another embodiment, said modified nucleotide is selected from among 2' alkoxyribonucleotide, 2' alkoxyalkoxy ribonucleotide, a locked nucleic acid ribonucleotide (LNA), 2'-fluoro ribonucleotide, morpholino nucleotide.
In another embodiment, said modified nucleotide is selected from among nucleotides having a modified internucleoside linkage selected from among phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, and amide linkages.
In another embodiment, said two RNA strands are fully complementary to each other_ In another embodiment, said siRNA comprises a 1 to 6 nucleotide overhang on at least one of the 5' end or 3' end.

In another embodiment, the siRNA contains at least one 3' cap, which is chemical moiety conjugated to the 3' end via the 3' carbon and is selected from among compounds of Formula I:
R, 3'01¨R2 [Formula I]
wherein X is 0 or S
Ri and R2 are independently OH, NH2, SH, alkyl, aryl, alkyl-atyl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms and functional groups, preferably a heteroatom selected from the group of N, 0, or S or a functional group selected from the group OH, NH2, SH, carboxylic acid or ester;
or R1 and R2 may be of formula Y-Z where Y is 0, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, 0, or S.
In another embodiment, the siRNA contains at least one strand which is complementary over at least 15 nucleotides to the mRNA or pre-mRNA of VEGFR-1, VEGFR-2, VEGFR3, Tie2, bFGFR, IL8RA, IL8RB, Fas, or IGF2R.
In another embodiment, the siRNA contains at least one strand which comprises a sequence selected from SEQ ID NO I - 900.
In another embodiment, the siRNA is chosen from the group consisting of SEQ ID
NO
901-930.
In another embodiment, the siRNA has a stability in a standard gastric acid assay that is greater than an unmodified siRNA with the same nucleotide sequence_ In another embodiment, the siRNA has a stability in a standard gastric acid assay that is greater than or equal to 50% after 30 minutes exposure.
In another embodiment, the siRNA has a stability in a standard serum assay greater than unmodified siRNA.

In another embodiment, the siRNA has a stability in a standard serum assay that is greater than or equal to 50% after 30 minutes exposure.
In another embodiment, the siRNA has a stability in a standard intestinal lavage assay that is greater than unmodified siRNA.
In another embodiment, the siRNA has an enhanced oral bioavailability compared to an unmodified siRNA of the same nucleotide sequence.
In one aspect, the invention provides a pharmaceutical composition comprising an siRNA with any one or more of the above properties.
In another aspect, the invention provides an siRNA with any one or more of the above properties for use as a medicament.
In another aspect, the invention provides the use of an siRNA with any one or more of the above properties in the preparation of a medicament for treating an angiogenic disorder.
In another aspect, the invention provides the use of an siRNA with any one or more of the above properties to inhibit an angiogenic process in vitro.
In another embodiment, the present invention provides a short interfering ribonucleic acid (siRNA), said siRNA comprising two RNA strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein the 3'-terminus of at least one strand comprises a modification at the 3' carbon, wherein the modification is:

, 21489-11000D3 01=0 ===
HO" SO
In another embodiment, the present invention provides a short interfering ribonucleic acid (siRNA), said siRNA comprising two RNA strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein the 3'-terminus of each strand comprises a modification at the 3' carbon, wherein the modification is:

0-1=0 HO''l 4a Brief Description of the Drawinas:
Figure la, lb, lc, id and le: Metabolic degradation of unmodified siRNA
pG13-siRNA (wild-type siRNA in mouse serum); a-c) Ion Exchange-HPLC analysis of unmodified siRNAs after incubation in mouse serum for 0', 30' and 180'; After 30' of incubation at 37 C, major peak in the Ion Exchange HPLC was isolated and re-injected in LC-MS, d) table of detected molecular weights and their assignments; e) ESI-MS
spectrum.
Figure 2: illustration of four double-stranded RNA formats: wild-type (or unmodified) siRNA. MOE o/h siRNA, C3-siRNA and C3-MOE siRNA.
Figure 3: Stability of siRNA in 3 different formats in mouse gastric acid.
Samples were incubated at 37 C in mouse gastric acid at a 2 micromolar concentration.
Disappearance of parent compound was followed over a 2-6 hours period by quantifying the parent compound band.
Lane 1-7: wild-type siRNA in gastric acid at t=0, 5, 10, 15, 30, 60 and 120 min 4b Lane 8: ds RNA ladder (30, 21, 19, 16, 13, 10 bp) Lane 9-15: C3 siRNA in gastric acid at t=0, 5, 10, 15, 30, 60 and 120 min Lane 16: ds RNA ladder (30, 21, 19, 16, 13, 10 bp) Lane 17-24: C3-MOE siRNA in gastric acid at t:), 5, 10, 15, 30, 60 and 120 min Figure 4: Stability of siRNA in 4 different formats in intestinal lavage.
Samples were incubated at 37 C in liver microsomes at a 5 micromolar concentration.
(From left to right) Lane 1: ds RNA ladder (30, 21, 19, 16, 13, 10 bp) Lane 2-7: wild-type siRNA in intestinal lavage at t=0, 15, 30, 60, 180 and 360 min Lane 8-13: moe o/h siRNA in intestinal lavage at t=0, 15, 30, 60, 180 and 360 min Lane 14-19: C3 siRNA in intestinal lavage at t=0, 15, 30, 60, 180 and 360 min Lane 20-25: C3-MOE siRNA in intestinal lavage at t-A), 15, 30, 60, 180 and 360 min Figure 5: Stability of siRNA in 4 different formats in liver microsomes.
Samples were incubated at 37 C in intestinal fluid from rat intestinal lavage at'a 2 micromolar concentration.
(From left to right) Lane 1: cis Figure 6: Stability of siRNA in 4 different formats in mouse serum. Samples were incubated at 37 C in mouse serum at a 2 micromolar concentration.
Disappearance of parent compound was followed over a 6 hours period by quantifying the parent compound band.
(From left to right) Lane 1: ds RNA ladder (30, 21, 19, 16, 13, 10 bp) RNA ladder (30,21, 19, 16, 13, 10 bp) Lane 2: wild-type siRNA untreated Lane 3: moe o/h siRNA untreated Lane 4: Ci siRNA untreated Lane 5: C3-MOE siRNA untreated Lane 6-9: same as 2-5 in liver microsomes -t=0 Lane 10-13: same as 2-5 in liver microsomes t=60' Lane 14-17: same as 2-5 in supernatant S12 t=0 Lane 18-21: same as 2-5 in supernatant S12 t=60' Lane 2-7: wild-type siRNA in mouse serum at trJ, IS, 30, 60, 180 and 360 min =

Lane 8-13: moe o/h siRNA in mouse serum at t=0, 15, 30, 60, 180 and 360 ruin Lane 14-19: C3 siRNA in mouse serum at r=0, 15, 30, 60, 180 and 360 min Lane 20-25: C3-MOE siRNA mouse serum at t=0, 15, 30, 60, 180 and 360 min Figure 7: Characterization in cellulo of 3 formats of anti-VEGFR2 siRNA (2 independent sequences). Wild-type siRNA, C3-siRNA and C3-MOE siRNA were transfected into MS1 cells at three concentrations (I, 5, 10 nM). Silencing potency was assessed by measuring VEGFR2 cell surface level by FACS.
Figure 8a and 8b: In vivo testing of wild-type siRNA, C3-siRNA and C3-Moe siRNA in a growth factor induced angiogenesis "Agar Chamber" mouse model.
Figure 8a shows the results of controls, unmodified VEGFR2 siRNA and C3 modified VEGFR2 siRNA
at 1, 5 and 25 micrograms per mouse per day. Figure 8b shows controls, C3 modified VEGFR2 siRNA and of C3-MOE VEGFR2 siRNA at 0.2, 1 and 5 micrograms per mouse per day. In each case pools of 2 anti-VEGFR2 siRNAs were given daily intraperitoneally for three days.
Figure 9: In vivo testing of anti-VEGFR2 C3-MOE siRNA given intraperitoneally (i.p.) in a B16 homograft melanoma tumor mouse model at 5 and 20 micrograms per mouse per day. Figure 9a shows that i.p. treatment with modified VEGFR2 siRNA
significantly reduces tumour development. Figure 9b also shows that i.p. injection of VEGFR2 siRNA at 20 ug per mouse results in significant inhibition of tumour growth.
Figure 10: In vivo testing of C3-MOE siRNA in a growth factor induced angiogenesis mouse model anti-VEGFR2 siRNAs were given daily orally for three days at 20 micrograms per mouse per day.
Figure 11: In vivo testing of C3-MOE siRNA in a growth factor induced angiogenesis mouse model. anti-Tie2 siRNAs were given daily intraperitoneally (1 and 0.2 micrograms per mouse per day) or orally (20 and 5 micrograms per mouse per day) for three days. Figure 11 a: weight of excised tissue; Figure 1 lb: Tie2 protein knock-down Detailed Disclosure of the Invention:
The present invention relates to compositions and methods for treating angiogenic disorders in a mammal. Specifically, the invention relates to small-interfering RNA
("siRNA") which may be used to treat angiogenic disorders upon oral administration to a mammal.
Angiogenesis targets in vascular endothelial cells include the following targets/genes:
VEGFR-1 (GenBank Accession # AF06365); VEGFR-2 (GenBank Accession # AF063658);

VEGFR-3 (GenBank Accession # (NM 002020); Tie2 (TEK) (GenBank Accession #
NM_000459); bFGFR (GenBank Accession # M60485); IL8RA (GenBank Accession #
L19591); IL8RB (GenBank Accession # L19593); Fas (GenBank Accession # X89101);

IGF2R (GenBank Accession # NM_000876).
The siRNA molecules according to the present invention mediate RNA
interference ("RNAi"). The term "RNAi" is well known in the art and is commonly understood to mean the inhibition of one or more target genes in a cell by siRNA with a region which is complementary to the target gene. Various assays are known in the art to test siRNA for its ability to mediate RNAi (see for instance Elbashir et al., Methods 26 (2002), 199-213). The effect of the siRNA according to the present invention on gene expression will typically result in expression of the target gene being inhibited by at least 10%, 33%, 50%, 90%, 95% or 99%
when compared to a cell not treated with the RNA molecules according to the present invention "siRNA" or "small-interfering ribonucleic acid" according to the invention has the meanings known in the art, including the following aspects. The siRNA consists of two strands of ribonucleotides which hybridize along a complementary region under physiological conditions. The strands are separate but they may be joined by a molecular linker in certain embodiments_ The individual ribonucleotides may be unmodified naturally occurring ribonucleotides, unmodified naturally occurring deoxyribonucleotides or they may be chemically modified or synthetic as described elsewhere herein.
The siRNA molecules in accordance with the present invention comprise a double-stranded region which is substantially identical to a region of the mRNA of the target gene. A
region with 100% identity to the corresponding sequence of the target gene is suitable. This state is referred to as "fully complementary". However, the region may also contain one, two or three mismatches as compared to the corresponding region of the target gene, depending on the length of the region of the mRNA that is targeted, and as such may be not fully complementary. In an embodiment, the RNA molecules of the present invention specifically target one given gene. In order to only target the desired mRNA, the siRNA
reagent may have 100% homology to the target mRNA and at least 2 mismatched nucleotides to all other genes present in the cell or organism. Methods to analyze and identify siRNAs with sufficient sequence identity in order to effectively inhibit expression of a specific target sequence are known in the art. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group).
Another factor affecting the efficiency of the RNAi reagent is the target region of the target gene. The region of a target gene effective for inhibition by the RNAi reagent may be determined by experimentation. A suitable mRNA target region would be the coding region.
Also suitable are untranslated regions, such as the 5'-UTR, the 3'-UTR, and splice junctions.
For instance, transfection assays as described in Elbashir S.M. et at, 2001 EMBO J., 20,6877-6888 may be performed for this purpose_ A number of other suitable assays and methods exist in the art which are well known to the skilled person.
The length of the region of the siRNA complementary to the target, in accordance with the present invention, may befrom 10 to 100 nucleotides, 12 to 25 nucleotides, 14 to 22 nucleotides or 15, 16, 17 or 18 nucleotides. Where there are mismatches to the corresponding target region, the length of the complementary region is generally required to be somewhat longer.
Because the siRNA may carry overhanging ends (which may or may not be complementary to the target), or additional nucleotides complementary to itself but not the target gene, the total length of each separate strand of siRNA may be 10 to 100 nucleotides, 15 to 49 nucleotides, 17 to 30 nucleotides or 19 to 25 nucleotides.
The phrase "each strand is 49 nucleotides or less" means the total number of consecutive nucleotides in the strand, including all modified or unmodified nucleotides, but =

not including any chemical moieties which may be added to the 3' or 5' end of the strand.
Short chemical moieties inserted into the strand are not counted, but a chemical linker designed to join two separate strands is not considered to create consecutive nucleotides.
The phrase "a 1 to 6 nucleotide overhang on at least one of the 5' end or 3' end"
refers to the architecture of the complementary siRNA that forms from two separate strands under physiological conditions. If the terminal nucleotides are part of the double-stranded region of the siRNA, the siRNA is considered blunt ended. If one or more nucleotides are unpaired on an end, an overhang is created. The overhang length is measured by the number of overhanging nucleotides. The overhanging nucleotides can be either on the 5' end or 3' end of either strand.
The siRNA according to the present invention confer a high in vivo stability suitable for oral delivery by including at least one modified nucleotide in at least one of the strands.
Thus the siRNA according to the present invention contains at least one modified or non-natural ribonucleotide. A lengthy description of many known chemical modifications are set out in published PCT patent application WO 200370918 and will not be repeated here.
Suitable modifications for oral delivery are more specifically set out in the Examples and description herein. Suitable modifications include, but are not limited to modifications to the sugar moiety (i.e. the 2' position of the sugar moiety, such as for instance 2'4)42-methoxyethyl) or 2'-M0E) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group) or the base moiety (i.e. a non-natural or modified base which maintains ability to pair with another specific base in an alternate nucleotide chain).
Other modifications include so-called 'backbone' modifications including, but not limited to, replacing the phosphoester group (connecting adjacent ribonuclotides with for instance phosphorothioates, chiral phosphorothioates or phosphorodithioates). Finally, end modifications sometimes referred to herein as 3' caps or 5' caps may be of significance_ As illustrated in Table 1, caps may consist of simply adding additional nucleotides, such as "T-T"
which has been found to confer stability on an siRNA. Caps may consist of more complex chemistries which are known to those skilled in the art.
In an embodiment used in the Examples below, the 3' cap is a chemical moiety conjugated to the 3' end via the 3' carbon and is selected from among compounds of Formula CA 02846756 2014-03-17 i = -! :=

R, 3' X [Formula I]
wherein X is 0 or S
RI and R2 are independently OH, NH2, SH, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms and functional groups, preferably a heteroatom selected from the group of N, 0, or S or a functional group selected from the group OH, NH2, SH, carboxylic acid or ester;
or R1 and R2 may be of formula Y-Z where Y is 0, N, S and Z is H, alkyl, aryl, alkyl-aryl, aryl-alkyl, where alkyl, aryl, alkyl-aryl, aryl-alkyl can be substituted by additional heteroatoms, preferably a heteroatom selected from the group of N, 0, or S.
Examples of modifications on the sugar moiety include 2' alkoxyribonucleotide, T
alkoxyalkoxy ribonucleotide, locked nucleic acid ribonucleotide (LNA), 2'-fluoro ribonucleotide, morplaolino nucleotide.
The intemucleoside linkage may also be modified. Examples of internucleoside linkage include phosphorothioate, phosphorodithioate, phosphoramidate, and amide linkages.
R1 may be OH.
R1 and R2 together may comprise from 1 to 24 C-atoms, from 1 to 12 C-atoms, from 2 to 10 C-atoms, from 1 to 8 or from 2 to 6 C-atoms. In another embodiment, R1 and R2 are independently OH, lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl, where lower alkyl, lower aryl, lower alkyl-aryl, lower aryl-alkyl can be substituted by additional heteroatoms and functional groups As defined above. In another embodiment, R1 and R2 are not both OH.
The term "lower" in connection with organic radicals or compounds means a compound or radical which may be branched or unbranched with up to and including 7 carbon 21489-11000 =
atoms, preferably 1-4 'carbon atoms. Lower alkyl represents, for example, methyl, ethyl, n-*
propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and branched pentyl, n-hexyl and branched hexyl.
Examples of allcoxys include 0-Met, 0-Eth, 0-prop, 0-but, 0-pent, 0-hex.
=
Methods for the synthesis of siRNA, including siRNA containing at least one modified . or non-natural ribonucleotides are well known and readily available to those of skill in the art.
= For example, a variety of synthetic chemistries are set out in published PCT patent applications W02005021749 and W0200370948.. The reaction may he carried out, in solution or, preferably, on solid phase or by using polymer supported'reagents, followed by combining the synthesized RNA strands under conditions, wherein a siRNA molecule is formed, which is papable of mediating RNAi.
The present invention provides an siRNA containing at least one' modified nucleotide -which is suitable for oral delivery. In functional terms this means siRNA will have suitable pharmacokinetics and biodistribution upon oral administration to achieve delivery to the target tissue of concern. In particular this requires serum stability, lack of immune response, = and drug like behaviour.. Many of these features of siRNA can be anticipated based on the standard gastric acid assays and standard serum assays disclosed elsewhere herein.
,==
In another aspect, the present invention provides methods for the inhibition of a target gene comprising introducing into a cell and siRNA according to the present invention, which is capable of inhibiting at least one target gene by RNAi. Also, more than one species of siRNA, which are each specific for another target region, may be introduced into a cell at the = same time or sequentially.
=
The present invention is not limited to any type of target gene or nucleotide sequence.
For example, the target gene can be a cellular gene, an endogenous gene, .a pathogen- -associated gene, a viral gene or an oncogene. Angiogenic genes are of particular importance to the invention because some of the Examples highlight that the orally delivered siRNA of .the invention may accumulate at sites of vasculogenesis, neovascularization or angiogenesis.
An updated fisting of angiogenic genes at these sites of particular interest for the invention are listed in AngioDB: database of angiogenesis and angiogenesis-related molecules Tae-Kwon .c Sohn, Eun-Joung Moonl, Seok-ICi Lee l, Hwan-Gue Cho2 and 1CSru-Won IChn.3, Nucleic = Acids Research, 2002, Vol. 30, No. 1 369-371 . Genes _ .
of particular significance have been analyzed in detail and are set out elsewhere herein.
In another aspect, the invention also provides a kit comprising reagents for inhibiting= .
expression of a target gene in a cell, wherein said kit comprises dsRNA
according to the present invention. The kit Comprises at least one of the reagents necessary to carry out the in vitro or in vivo introduction of the dsRNA according to the present invention to test samples .
or subjects. In a preferred embodiment, such kits also comprise instructions detailing the =
procedures by which the kit components are to be used.
"Treatment of an angiogenic disorder" as used in this disclosure means use of a modified siRNA of the invention in a pharmaceutical composition for the treatment of = diseases involving the physiological and pathological processes of neovascularization, vasculogenesis and/or angiogenesis. As such, these pharmaceutical compositions are useful for treating diseases, conditions and disorders that require inhibition of neovascularization, vasculogenesis or angiogenesis, including but not limited to cancer tumour growth and metastasis, neoplasm, ocular neovascularization (including macular degeneration, diabetic retinopathy, ischemic retinopathy, retinopathy of prematurity, choroidal neovascularization), rheumatoid arthritis, osteoarthritis, chronic asthma, spectic shock, inflammatory diseases, -synovitis, bone and cartilage destruction, pannirs growth, osteophyte formation, osteomyelitis, psoriasis, obesity, haemangioma, Kaposi's sarcoma, atherosclerosis (including atherosclerotic plaque rupture), endometriosis, warts, excess hair growth, scar keloids, allergic oedema, dysfunctional uterine bleeding, follicular cysts, ovarian hyperstimulation, endometriosis, osteomyelitis, inflammatory and infectious processes (hepatitis, pneumonia, glumerulonephtritis), asthma, nasal polyps, transplantation, liver regeneration, leukomalacia, thyroiditis, thyroid enlargement, lymphoproliferative disorders, haematologie malignancies, .
vascular malformations, and pre-eclampsia.
As used herein, "treatment" means an action taken to inhibit or reduce a process of a disease, disorder or condition, to inhibit or reduce a symptom of a disease, disorder or-condition, or to prophylactically prevent the onset or further development of a disease, disorder or condition. "Treat" is the cognitive verb thereof.
An effective dose of the therapeutic agent of the invention is that dose required to treat = a disease state. The effective dose depends on the type of disease, the composition used, the =

f CA 02846756 2014-03-17 route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of siRNA is administered dependent upon potency. The nucleic acid molecules of the invention and formulations thereof can be administered orally, topically, parenterally, by inhalation or spray, or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and/or vehicles. The term parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, intraperitoneal, or intrathecal injection, or infusion techniques and the like. In addition, there is provided a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier. One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients. The pharmaceutical compositions containing nucleic acid molecules of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets. These excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearie acid or talc. The tablets can be uncoated or they can be coated by known techniques. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions.

Oral administration of the compositions of the invention include all standard techniques for administering substances directly to the stomach or gut, most importantly by patient controlled swallowing of the dosage form, but also by other mechanical and assisted means of such delivery.
Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above- indicated conditions (about 0.5 mg to about 7 g per subject per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration. Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. It is understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Therapeutic effect of the therapeutic agents of the invention may be enhanced by combination with other agents. Typically such other agents will include agents known for use in treating similar diseases, such as angiogenic disorders. Alternatively, such agents may be used to reduce side-effects or unwanted effects caused by the therapeutic agents of the invention.
The siRNA of the invention also have important research uses. One such study includes research into an angiogenic process in vitro. By "angiogenic process in vitro" is meant any process for studying angiogenesis or vasculogenesis which does not employ a whole animal. As such, in vitro or ex vivo methods and assays which study the steps of the angiogenic process using markers or indicators of angiogenesis are included hereby.
RNA Strand Nucleotide Sequences The siRNA strand sequences identified in Table I have been identified as suitable siRNA
sequences against the following targets: VEGFR-1 (GenBank Accession #
AF06365);
VEGFR-2 (GenBank Accession # AF063658); VEGFR-3 (GenBank Accession #
(NM_002020); Tie2 (TEK) (GenBank Accession # NM 000459); bFGFR (GenBank Accession # M60485); IL8RA (GenBank Accession # L19591); IL8RB (GenBank Accession # L19593); Fas (GenBank Accession # X89101); IGF2R (GenBank Accession #
NM 000876).
Table 1: siRNAs against human VEGFR-1, VEGFR-2, VEGFR-3, Tie2, bFGFR, IL8RA, IL8RB, Fas, IGF2R
SEQ SEQ
pos ID ID
Target Name siRNA guide sequence NO siRNA complement NO
VEGFR-CAGUUAACAAGUUCUUAUATT _ 451 VEGFR-VEGFR-1 1209 UUUAUGCUCAGCAAGAUUGTA 3 , CAAUCUUGCUGAGCAUAAATT _ 453 VEGFR-VEGFR-VEGFR-VEGFR-1 1091 UUAACCAUACAACUUCCGGCG _ 7 CCGGAAGUUGUAUGGUUAATT 457 VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-1 2971 , UUCCAUAGUGAUGGGCUCCTT 14 VEGFR-1 1774 UCUGUUAUUAACUGUCCGCAG 15 GCGGACAGUUAAUAACAGATT _ 465 VEGFR-VEGFR-1 2269 UGUUAGAGUGAUCAGCUCCAG 17 , GGAGCUGAUCACUCUAACATT 467 , VEGFR-VEGFR-VEGFR-1 2246 UAGACUUGUCCGAGGUUCCTT 20_ GGAACCUCGGACAAGUCUATT 470 VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-CAGUCAAUGGGCAUUUGUATT , 487 VEGFR-VEGFR-VEGFR-CAACUACCUCAAGAGCAAATT 490 _ VEGFR-VEGFR-VEGFR-VEGFR-1 1370 UUGUAGGUUGAGGGAUACCAT 44 , VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-2 617 UA.AUAGACUGGUAACUUUCAT 51 - = CA 02846756 2014-03-17 2 561 _UAGCUGAUCAUGUAGCUGGGA

GUCUCAUGGAAUUGAACUATT 514 .

VEGFR-2 _ 324 UAAAUGACCGAGGCCAAGUCA 80 VEGFR-VEGFR-2 56 UAGGCAAACCCACAGAGGCGG 82 GCCUCUGUGGGUUUGCCUATT 532 .
VEGFR-2 _ 2453 UGGCAUCAUAAGGCAGUCGTT 83 VEGFR-VEGFR-2 _ 1813 UUUCCAAAGAGUAUCCAAGTT 85 VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEG FR-VEGFR-VEGFR-VEGFR-VEGFR-VEG FR-VEGFR-VEGFR-UUCCUGUUGACCAAGAGCGTG 101 CGCUCUUGGUCAACAGGAATT 551_ VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-_ 3 1954 AUAGUGGCCCUCGUGCUCGGG _ CGAGCACGAGGGCCACUAUTT 565 , VEGFR-GCCUUGCCCGGGACAUCUATT 578 _ =

VEGFR-VEGFR-3 _1684 VEGFR-VEGFR-VEGFR-UUGUCGAAGAUGCUUUCAGGG 138 _CUGAAAGCAUCUUCGACAATT 589 VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-VEGFR-Tie-2 (TEK) 1223 UAAGCUUACAAUCUGGCCCGT 151 GGGCCAGAUUGUAAGCUUATT 601 Tie-2 (TEK) 2350 UAUCUUCACAUCAACGUGCTG 152 GCACGUUGAUGUGAAGAUATT 602 Tie-2 (TEK) 706 Tie-2 (TEK) 3561 UUUAAGGACACCAAUAUCUGG 154 AGAUAUUGGUGUCCUUAAATT 604 Tie-2 (TEK) 2763 UGAAAUUUGAUGUCAUUCCAG 155 GGAAUGACAUCAAAUUUCATT 605 , Tie-2 (TEK) 174 Tie-2 (TEK) 1183 UUCAUUGCACUGCAGACCCTT 157 GGGUCUGCAGUGCAAUGAATT 607 Tie-2 (TEK) 805 Tie-2 (TEK) 2601 UUCAAUUGCAAUAUGAUCAGA 159 UGAUCAUAUUGCAAUUGAATT 609 Tie-2 (TEK) 2277 UAGCCAUCCAAUAUUGUCCAA 160 GGACAAUAUUGGAUGGCUATT 610 Tie-2 (TEK) 1366 UACUUCUAUAUGAUCUGG CAA 161 GCCAGAUCAUAUAGAAGUATT 611 , Tie-2 (TEK) 32 Tie-2 163 (TEK) 4085 UGUACUAUCAGGGUCAUUGTT

Tie-2 164 (TEK) 3881 UUCUGAUUUCAGCCCAUUCTT

lie-2 165 (TEK) 646 UUGUUGACGCAUCUUCAUGGT

Tie-2 166 (TEK) 4021 AUAGCAUUCAACAUAAAGGTA

Tie-2 167 Tie-2 168 (TEK) 4223 UAAAUGAAACGGGACUGGCTG

Tie-2 169 (TEK) 3961_ UACUAAUUGUACUCACGCCTT

Tie-2 170 (TEK) 1771 UUGAAUAUGUUGCCAAGCCTC

Tie-2 171 (TEK) 3909 UUAUUGCAUAUGAAACCACAA

Tie-2 172 (TEK) 3606 UAAAGCGUGGUAUUCACGUAG

Tie-2 173 (TEK) 477 AUUAAGGCUUCAAAGUCCCTT

Tie-2 174 (TEK) 3421 UUCUGCACAAGUCAUCCCGCA

Tie-2 175 (TEK) 2730 UAAAUUGUAGGAUCUGGGUTG

Tie-2 176 (TEK) 1800 UAGUUGAGUGUAACAAUCUCA

Tie-2 (TEK) 3385 UAAGCUAACAAUCUCCCAUAG 177 AUGGGAGAUUGUUAGCUUATT 627 Tie-2 (TEK) 1692 UAAGGCUCAGAGCUGAUGUTG 178 ACAUCAGCUCUGAGCCUUATT 628 , Tie-2 (TEK) 1657 AU GUCCAG UGUCAAUCACGTT 179 CGUGAUUGACACUGGACAUTT 629 Tie-2 (TEK) 3665 UUCUGUCCUAGGCCGCUUCTT 180 GAAGCGGCCUAGGACAGAATT 630 Tie-2 (TEK) 2091 UUAAGUAGCACCGAAGUCAAG 181 UGACUUCGGUGCUACUUAATT 631 Tie-2 (TEK) 2827 UAACCCAUCCUUCUUGAUGCG 182 CAUCAAGAAGGAUGGGUUATT 632 Tie-2 (TEK) 1979 UUGGUUGCCAGGUCAAAUUTA 183 AAUUUGACCUGGCAACCAATT 633 Tie-2 (TEK) 67 Tie-2 (TEK) 3459 UUCUCCAGUCUGUAGCCCUGG 185 AGGGCUACAGACUGGAGAATT 635 Tie-2 (TEK) 2764 UUGAAAUUUGAUGUCAUUCCA 186 GAAUGACAUCAAAUUUCAATT 636 lie-2 (TEK) 3560 UUAAGGACACCAAUAUCUGGG 187 CAGAUAUUGGUGUCCUUAATT 637 Tie-2 (TEK) 715 Tie-2 (TEK) 1368 UUUACUUCUAUAUGAUCUGGC 189 CAGAUCAUAUAGAAGUAAATT 639 Tie-2 (TEK) 2351 U UAU CU UCACAUCAACGUG CT _ 190 CACGUUGAUGUGAAGAUAATT 640 Tie-2 (TEK) 205 UGACUUUCCAUUAGCAUCGTC 191 CGAUGCUAAUGGAAAGUCATT 641 Tie-2 _(TEK) 3957 AAUUGUACUCACGCCUUCCTA 192_ GGAAGGCGUGAGUACAAUUTT 642 Tie-2 (TEK) 3962 AUACUAAUUGUACUCACGCCT 193 GCGUGAGUACAAUUAGUAUTT 643 Tie-2 (TEK) 2352 UUUAUCUUCACAUCAACGUGC 194 ACGUUGAUGUGAAGAUAAATT 644 Tie-2 (TEK) 3963 UAUACUAAUUGUACUCACGCC 195_ CGUGAGUACAAUUAGUAUATT 645 Tie-2 (TEK) 1777 UGUCACUUGAAUAUGUUGCCA 196 GCAACAUAU(JCAAGUGACATT _ 646 Tie-2 (TEK) 3388 UCCUAAGCUAACAAUCUCCCA 197 GGAGAUUGUUAGCUUAGGATT _ 647 Tie-2 (TEK) 636 AUCUUCAUGGUUCGUAUCCTG 198 GGAUACGAACCAUGAAGAUTT 648 Tie-2 (TEK) 74 UCCUUUGUAGAUUAGGAUGGG 199 CAUCCUAAUCUACAAAGGATT 649 Tie-2 (TEK) 707 AUAUGUUCACGUUAUCUCCCT 200 GGAGAUAACGUGAACAUAUTT 650 bFGFR 3814 UAAAUCUCUGGUAACGACCCT 201 GGUCGUUACCAGAGAUUUATT 651 bFGFR 1478 _UUACACAUGAACUCCACGUTG 202 ACGUGGAGUUCAUGUGUAATT 652 bFGFR 3773 UAUACUCAGAUUUAUCAACTT 203 GUUGAUAAAUCUGAGUAUATT 653 bFGFR 715 UAGCGGUGCAGAGUGUGGCTG 204 GCCACACUCUGCACCGCUATT 654 bFGFR 575 UUCAAACUGACCCUCGCUCGG 205 GAGCGAGGGUCAGUUUGAATT 655 bFGFR 646 UUCUGCAGUUAGAGGUUGGTG 206 CCAACCUCUAACUGCAGAATT 656 bFGFR 3625 AUCGGAAUUAAUAAGCCACTG 207 GUGGCUUAUUAAUUCCGAUTT 657 bFGFR 2318 UACAAGGGACCAUCCUGCGTG 208 CGCAGGAUGGUCCCUUGUATT 658 bFGFR 1439 UUGUUGGCGGGCAACCCUGCT 209 CAGGGUUGCCCGCCAACAATT 659 bFGFR 3860 AUAGCAACUGAUGCCUCCCAG 210 GGGAGGCAUCAGUUGCUAUTT 660 bFGFR 3163 UGAGGGUUACAGCUGACGGTG 211 CCGUCAGCUGUAACCCUCATT 661 bFGFR 2600 UCGAUGUGGUGAAUGUCCCGT 212 GGGACAUUCACCACAUCGATT 662 bFGFR 2513 UCUCGGUGUAUGCACUUCUTG 213 AGAAGUGCAUACACCGAGATT 663 bFGFR 2214 UUUCUCUGUUGCGUCCGACTT 214 GUCGGACGCAACAGAGAAATT 664 bFGFR 1346 UUCUCCACAAUGCAGGUGUAG 215 ACACCUGCAUUGUGGAGAATT 665 bFGFR 1556 UUGUCUGGGCCAAUCUUGCTC 216 GCAAGAUUGGCCCAGACAATT 666 bFGFR 2671 UCCGGUCAAAUAAUGCCUCGG 217 GAGGCAUUAUUUGACCGGATT 667 bFGFR 3105 UUUGAGUCCGCCAUUGGCAAG 218 UGCCAAUGGCGGACUCAAATT 668 bFGFR 2091 UUUGCCUAAGACCAGUCUGTC 219 CAGACUGGUCUUAGGCAAATT 669 bFGFR 1590 UCCAGCAGUCUUCAAGAUCTG 220 GAUCUUGAAGACUGCUGGATT 670 bFGFR 1689 UCCGAUAGAGUUACCCGCCAA 221 GGCGGGUAACUCUAUCGGATF 671 bFGFR 1319 UUGUCAGAGGGCACCACAGAG 222 CUGUGGUGCCCUCUGACAATT 672 bFGFR 2342 UUGGAGGCAUACUCCACGATG 223 UCGUGGAGUAUGCCUCCAATT 673 bFGFR 107 UCUCGGUCCCGACCGGACGTG 224 CGUCCGGUCGGGACCGAGATT 674 bFGFR _3662 UCUGGUACCAGGCAUUUGGTC _ 225 CCAAAUGCCUGGUACCAGATI 675 bFGFR 2150 UUGUCCAGCCCGAUAGCCUCT 226 AGGCUAUCGGGCUGGACAATT 676 bFGFR 1517 UUUAGCCACUGGAUGUGCGGC 227 CGCACAUCCAGUGGCUAAATT 677 bFGFR 1264 UGUAGCCUCCAAUUCUGUGGT 228 CACAGAAUUGGAGGCUACATT 678 bFGFR 3576 UUCAAUCGUGGCUCGAAGCAC 229 GCUUCGAGCCACGAUUGAATT 679 bFGFR 613 AUCUCCAUGGAUACUCCACAG 230 GUGGAGUAUCCAUGGAGAUTT 680 bFGFR 1221 UUUCAACCAGCGCAGUGUGGG 231 CACACUGCGCUGGUUGAAATT 681 bFGFR 3004 UAGAGCUCCGGGUGUCGGGAA 232 CCCGACACCCGGAGCUCUATT 682 bFGFR 3825 UUACCGAUGGGUAAAUCUCTG 233 GAGAUUUACCCAUCGGUAATT 683 bFGFR 3813 AAAUCUCUGGUAACGACCCTT 234 GGGUCGUUACCAGAGAUUUTT 684 bFGFR 3861 UAUAGCAACUGAUGCCUCCCA 235 GGAGGCAUCAGUUGCUAUATT 685 bFGFR 576 UUUCAAACUGACCCUCGCUCG 236 AGCGAGGGUCAGUUUGAAATT 686 bFGFR 3772 AUACUCAGAUUUAUCAACUTT 237 bFGFR 3824 UACCGAUGGGUAAAUCUCUGG 238 AGAGAUUUACCCAUCGGUA'TT 688 bFGFR 2319 AUACAAGGGACCAUCCUGCGT 239 GCAGGAUGGUCCCUUGUAUTT 689 bFGFR 3771 UACUCAGAUUUAUCAACUUTG 240 AAGUUGAUAAAUCUGAGUATT 690 bFGFR 2511 UCGGUGUAUGCACUUCUUGGA 241 CAAGAAGUGCAUACACCGATT 691 bFGFR 2333 UACUCCACGAUGACAUACAAG 242 UGUAUGUCAUCGUGGAGUATT 692 bFGFR 3624 UCGGAAUUAAUAAGCCACUGG 243 AGUGGCUUAUUAAUUCCGATT 693 bFGFR 1304 ACAGAGUCCAUUAUGAUGCTC 244 GCAUCAUAAUGGACUCUGUTT 694 bFGFR 1608 UUUGUCGGUGGUAUUAACUCC 245 AGUUAAUACCACCGACAAATT 695 bFGFR 1301 GAGUCCAUUAUGAUGCUCCAG 246 GGAGCAUCAUAAUGGACUCTT 696 bFGFR 3626 UAUCGGAAUUAAUAAGCCACT _ 247 UGGCUUAUUAAUUCCGAUATT 697 bFGFR 2672 AUCCGGUCAAAUAAUGCCUCG 248 AGGCAUUAUUUGACCGGAUTT 698 bFGFR 2213 UUCUCUGUUGCGUCCGACUTC 249 AGUCGGACGCAACAGAGAATT 699 CA 02846756 2014-03-17 =

bFGFR 2597 AUGUGGUGAAUGUCCCGUGCG 250 CACGGGACAUUCACCACAUTT 700 IL8RA 1432 _ CUAAUUAGCCAGUUAGUGGGT 284 CCACUAACUGGCUAAUUAGTT 734 =

1L8RA 1082_ UUGCUGACCAGGCCAUGCATA 288 UGCAUGGCCUGGUCAGCAATT 738 1L8FtA 81 1L8RA _1372 UCCAUGGAGGUGCAAAGGCCG 291 GCCUUUGCACCUCCAUGGATT 741 1L8RA 1524 AGGGUCAACGAGAGCAUCCAG 295_ GGAUGCUCUCGUUGACCCUTT 745 1L8RA 237 AUAGGCGAUGAUCACAACATA r 296 UGUUGUGAUCAUCGCCUAUTT 746 !URA 1972 CUUUAUUAGGAACAUCUGCCT 299 GCAGAUGUUCCUAAUAAAGTT 749 1L8RB 2648 UUAAGUGUCAAUUUAGUGGCA 301 CCACUAAAUUGACACUUAATT. 751 , 1L8RB 2184 UUUCUUGUGGGUCAAUUCCTA 302 _ GGAAUUGACCCACAAGAAATT 752 1L8RB _ 960 UUGGAUGAGUAGACGGUCCTT 305 GGACCGUCUACUCAUCCAATT 755 iL8RB 2750 UUGGUUUAAUCAGCCUUGGTG 307 CCAAGGCUGAUUAAACCAATT 757 1L8RB 1149 AAGAUGACCCGCAUGGCCCGG 311 GGGCCAUGCGGGUCAUCUUTT_ 761 11_812B 2464 UCUCAGUACCUCAUGUAGGTG 312 CCUACAUGAGGUACUGAGATT 762 11_813B 2360 AUGAGUACUUCAUUCCUCUTT 315 AGAGGAAUGAAGUACUCAUTT 765 ILBRB 265 UUGGGUGGUAGUCAGAGCUGT 316 AGCUCUGACUACCACCCAATI- 766_ 1L8RB 2627 UUAAGUCACAUUGCGGUACAA 319 _GUACCGCAAUGUGACUUAATT 769 1L8RB _1000 UGUAUUGUUGCCCAUGUCCTC 320 GGACAUGGGCAACAAUACATT _ 770 1L8RB 315 UGACCUGCUGUUAUUGGAGTG 321 CUCCAAUAACAGCAGGUCATT 771 .

IL8RB 2389_ UUUCAAGGUUCGUCCGUGUTG 324 ACACGGACGAACCUUGAAATT 774 IL8RB 385_ UGAGGUAAACUUAAAUCCUGA 325 _AGGAUUUAAGUUUACCUCATT 775 ILBRB 2755 _ UAGCCUUGGUUUAAUCAGCCT 333 GCUGAUUAAACCAAGGCUATT 783 IL8RB 2183, UUCUUGUGGGUCAAUUCCUAT 334 AGGAAUUGACCCACAAGAATT 784 IL8RB 2143 UGUGUUAAUUCUAUGUCUGAA 337 , CAGACAUAGAAUUAACACATT 787 IL8RB 956_ AUGAGUAGACGGUCCUUCGGA 344 CGAAGGACCGUCUACUCAUTT 794 IL8RB 456_ UAAUUACUAAGAUCUUCACCT 345 IL8RB 226 UGAAACAACCUUGACGAUGAA 346 , CAUCGUCAAGGUUGUUUCATT 796 IL8RB 1394 UGAUCAAGCCAUGUAUAGCTA _ 347 GCUAUACAUGGCUUGAUCATT 797 IL8RB _ 881 UGAAUUUGACCAAGUAGCGCT _ 348 CGCUACUUGGUCAAAUUCATT 799 IL8RB 2327 , UACUUCGUUAGGUACAUAUCA 350 AUAUGUACCUAACGAAGUATT 800 Fas 109 UGUAGUAACAGUCUUCCUCAA 351 GAGGAAGACUGUUACUACATT 801 Fas 41 Fas 161_ UAUGGCAGAAUUGGCCAUCAT 353 GAUGGCCAAUUCUGCCAUATT 803 Fas 182 UUUCACCUGGAGGACAGGGCT 354 CCCUGUCCUCCAGGUGAAATT 804 Fas 62 Fas 377 ACUUCCUCUUUGCACUUGGTG 358 CCAAGUGCAAAGAGGAAGUTT 806 Fas 349 UGAGUGUGCAUUCCUUGAUGA 357 AUCAAGGAAUGCACACUCATT 807 Fas 245 UCCCUUCUUGGCAGGGCACGC 358 GUGCCCUGCCAAGAAGGGATT _ 808 Fas 205 GACUGUGCAGUCCCUAGCUTT 359 AGCUAGGGACUGCACAGUCTT 809 Fas 145 AUCAUGAUGCAGGCCUUCCAA 360 GGAAGGCCUGCAUCAUGAUTT 810 Fas 123 UUCUGAGUCUCAACUGUAGTA 361 CUACAGUUGAGACUCAGAATT 811 Fas 34 UAAUCUAGCAACAGACGUAAG 362 UACGUCUGUUGCUAGAUUATT 812 Fas 114 UCAACUGUAGUAACAGUCUTC 363 AGACUGUUACUACAGUUGATT 813 Fas 115 CUCAACUGUAGUAACAGUCTT 364 GACUGUUACUACAGUUGAGTT 814 Fas 28 AGCAACAGACGUAAGAACCAG 365 GGUUCUUACGUCUGUUGCUTT 815 _ Fas 122 UCUGAGUCUCAACUGUAGUAA 366 ACUACAGUUGAGACUCAGATT 816 _ Fas 186 UUCCUUUCACCUGGAGGACAG 367_ GUCCUCCAGGUGAAAGGAATT 817 Fas 42 UUGGACGAUAAUCUAGCAACA 368 UUGCUAGAUUAUCGUCCAATT 818 Fas 111 ACUGUAGUAACAGUCUUCCTC 369_ GGAAGACUGUUACUACAGUTT 819 Fas 144 UCAUGAUGCAGGCCUUCCAAG 370_ UGGAAGGCCUGCAUCAUGATT 820 Fas 92 UCAAUUCCAAUCCCUUGGAGT 371 UCCAAGGGAUUGGAAUUGATT 821 Fas 201 GUGCAGUCCCUAGCUUUCCTT 372 GGAAAGCUAGGGACUGCACTT 822 Fas 128 CCAAGUUCUGAGUCUCAACTG 373 _ GUUGAGACUCAGAACUUGGTT 823 Fas 36 GAUAAUCUAGCAACAGACGTA 374 CGUCUGUUGCUAGAUUAUCTT 824 Fas 162 UUAUGGCAGAAUUGGCCAUCA 375 AUGGCCAAUUCUGCCAUANIT 825 Fas 127 CAAGUUCUGAGUCUCAACUGT

Fas 202 UGUGCAGUCCCUAGCUUUCCT 377 GAAAGCUAGGGACUGCACATT 827 Fas 82 UCCCUUGGAGUUGAUGUCAGT 378 UGACAUCAACUCCAAGG GATT 828 Fas 160 AUGGCAGAAUUGGCCAUCATG 379 _ UGAUGGCCAAUUCUGCCAUTT 829 Fas 150 UGGCCAUCAUGAUGCAGGCCT 360 GCCUGCAUCAUGAUGGCCATT 830 . Fas 63 GUCACUUGGGCAUUAACACTT 381 GUGUUAAUGCCCAAGUGACTT 831 Fas 164 GCUUAUGGCAGAAUUGGCCAT 382 GGCCAAUUCUGCCAUAAGCTT 832 Fas 37 CGAUAAUCUAGCAACAGACGT 388 GUCUGUUGCUAGAUUAUCGTT 833 Fas 116 UCUCAACUGUAGUAACAGUCT 384 ACUGUUACUACAGUUGAGATT 834 Fas 32 AUCUAGCAACAGACGUAAGAA 385 CUUACGUCUGUUGCUAGAUTT 835 Fas 64 AGUCACUUGGGCAUUAACACT 386 UGUUAAUGCCCAAGUGACUTT 836 Fas 167 AGGGCUUAUGGCAGAAUUGGC 387 CAAUUCUGCCAUAAGCCCUTT 837 Fas 120 UGAGUCUCAACUGUAGUAACA 388 UUACUACAGUUGAGACUCATT 838 Fas 125 AGUUCUGAGUCUCAACUGUAG 389 ACAGUUGAGACUCAGAACUTT 839 Fas 43 UUUGGACGAUAAUCUAGCAAC 300 UGCUAGAUUAUCGUCCAAATT 840 Fas 94 CCUCAAUUCCAAUCCCUUGGA 391 CAAGGGAUUGGAAUUGAGGTT 841 Fas 159 UGGCAGAAUUGGCCAUCAUGA 392 AUGAUGGCCAAUUCUGCCATT 842 Fas 110 CUGUAGUAACAGUCUUCCUCA 393 AGGAAGACUGUUACUACAGTT 843 Fas 31 UCUAGCAACAGACGUAAGAAC 394 UCUUACGUCUGUUGCUAGATT 844 Fas 38 ACGAUAAUCUAGCAACAGACG 395 UCUGUUGCUAGAUUAUCGUTT 845_ Fas Fas Fas Fas Fas IGF2R _6340 UUUGUCACCUAUGACACCCAG 401 GGGUGUCAUAGGUGACAAATT 851 IGF2R 4572 UUCACUUGGCUCUCGCUGCAG 425 GCAGCGAGAGCCAAGUGAATT 875.

= IGF2R 3153 UUCUCAAUUCCGACUGGCCTT 427 GGCCAGUCGGAAUUGAGAATT 877 IGF2R 9029 UAUUACAGUAAAGUUGAUUGA 428 AAUCAACUUUACUGUAAUATT _ 878 IGF2R 6702 UUGGCUCCAGAGCACGCCGGG _ 432 CGGCGUGCUCUGGAGCCAATT 882 IGF2R 6203 UAUAGUACGAGACUCCGUUGT , 436 AACGGAGUCUCGUACUAUATT 886 , IGF2R 753 AUGAAUAGAGAAGUGUCCGGA 437 CGGACACUUCUCUAUUCAUTI" 887 IGF2R 1187 A.AAUAUAGGAUGAACCUCCGC 445 GGAGGUUCAUCCUAUAUUUTT 895 IGF2R , 7190 UUGAGUAUUUGUAGGACACGT 447 GUGUCCUACAAAUACUCAATT 897 IGF2R 2941 AUCCCUUAUAGAGCAAGCCTd 449 GGCUUGCUCUAUAAGGGAUTT 899 Chemical Modification of RNA Strand Nucleotides The siRNA according to the invention may comprise at least one modified nucleotide in at least one of the RNA strands. A range of potential modified nucleotides are disclosed elsewhere herein. Useful modifications and combinations of modifications for use according to the invention are shown in Table 2:
Table 2: Chemical Modifications and Sequence Architecture ii Modification Format Name I PS DNA o/h NNNNNNNNNNNNNNNNNNNsnsn nsnsNNNNNNNNNNNNNNNNNNN
2 Full PS NsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsN
NsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsNsN

NNNNNNNNNNNNNNNNNNNN

4 Blunt-ended 2'-0Me o/h PNP
_ _ NPN ' 6 2' -0Me/2`F PNP
NN' 7 LNA (3-7 snsn incorporations nsnsNNNNNNNNNNNNNNNNNNN
in ds region) N = any unmodified RNA nucleotide n = unmodified DNA nucleotide = modified RNA nucleotide s = identifies phosphorthioate intemucleoside linkage o/h = overhang The following modifications added to the 3' position of the 3 '-terminus of the siRNA
strands , sometimes referred to as a '3' end cap' are also recognized as useful embodiments of the invention and may be used with any of the siRNA according to the invention:

5 c%0H
.Base BASE
$ s...
OH 1:1F4 0 OH
_ 04=0 0-P=0 0-P=0 OH
C3 C6 C12 Triethylene glycol LO.BASE0 gASE Lc_O_TBASE 1..s.cijo õBASE LclyBASE
d 0 OH $
0 OH e 0 bH
_ = _ = _ = _ = _ =
0-P=0 0-P=0 0-P=0 41) 0*
O
HO' a OH
Cyclohexyl Phenyl Biphenyl Adamantane Lithocholic acid Specific compounds with activity according to the invention include the following, shown in Table 3:

f wo 2007/128477 Table 3: Sequences and Chemistries of siRNA used in Examples Name strand Sequence (N: RNA; dN: DNA; n: 2'-moe RNA; s: phosphorothioate) SEQ ID NO
guide strand 901 pGI3-s1RNA UCG MG UAC UCA GCG UAA
GdTdT
complement 902 strand CUU ACG COG AGU ACU UCG
AdTdT
guide strand 903 pGL3 MOE o/h CUU ACG COG AGU ACU UCG Atst siRNA
complement 904 strand UCG AAG UAC UCAOCG UAA Gtst guide strand 905 pG13-C3-siRNA UCG AAG UAC UCA GCG UM G-C3 complement 906 strand CUU ACG CUG AGU ACU UCG A-C3 guide strand 907 - pG13-C3-M0E- UCG AAG UAC UCA GCG UAa g-C3 siRNA
complement 908 strand CUU ACG CUG AGU ACU UCg a-C3 guide strand 909 VEGFR2-siRNA1 UUG AGG UUU GM AUC GAC
CdCdT
complement 910 strand GGU CGA UUU CAA ACC UCA
AdTdT
guide strand 911 VEGFR2-siRNA2 UAA UUU GUU CCU GUC UUC
CdAdG
complement 912 strand GGA AGA CAG GM CM AUU
AdTdT
guide strand 913 siRNA control ACG UGA CAC GUU CGG AGA
AdTdT
complement 914 strand UUC UCC GM CGU GUC ACG
UdTdT
guide strand 915 VEGFR2-C3- UUG AGG UUU GM. AUC GAC C-siRNA1 C3 complement 916 strand GGU CGA UUU CM ACC UCA A-guide strand 917 siRNA2 C3 complement 918 strand GGA AGA CAC GM CM AUU A-CA 02846756 2014-03-17 v guide strand 919 C3-siRNA control ACG UGA CAC GUU CGG AGA A-complement 920 strand UUC UCC GM CGU GUC ACG U-guide strand 921 VEGFR2-C3- UUG AGG UUU GM AUG GAc c-MOE-s1RNA1 C3 complement 922 strand GGU CGA UUU CM ACC UCa a-guide strand 923 VEGFR2-C3- UAA UUU GUU CCU GUC UUc c-MOE-siRNA2 C3 complement 924 strand GGA AGA CAG GM CAA AUu a-C3 guide strand 925 Tie2-C3-M0E- UUC UUC UUU MU UAA CAc c-C3 siRNA1 complement 926 strand GGU GUU MU UAA AGA AGa a-guide strand 927 Tie2-C3-M0E- UCU GAG UUU GUA MU AUc g-C3 siRNA2 complement 928 strand CGA UAU UUA CM ACU CAg a-C3 guide strand 929 C3-M0E-siRNA ACG UGA CAC GUU CGG AGa a-control C3 complement 930 strand UUC UCC GM CGU GUC ACg t-C3 Examples:
The following Examples illustrate aspects of the invention, and are not intended to limit the embodiments included in the claims recited below. The results and discussion section farther below refers to experiments conducted according to the following protocols and employing the following materials. Materials and protocols that are not specifically described are considered to be routinely available to those skilled in the art.
Example 1 Preparation of siRNAs.
Single strand siRNA derivatives were synthesized by standard 2'-0-TOM
phosphoamidite technology and purified by Oasis HLB Extraction Plates (Waters). Sense- and antisense = CA 02846756 2014-03-17 stranded siRNA were mixed in hybridization buffer (100 mM potassium acetate, 2 mM
magnesium acetate, 30 mM Hepes, pH 7.6) heat-denatured at 900 C for 3 min and annealed at 37 C for 60 min. 100 AM stock solutions of siRNA duplexes were stored at -20 C.
Example 2 Incubation in Serum and analysis by IE-HPLC (LC-MS).
In a standard serum assay, 6 AL 20 AM of each siRNA were mixed with 54 AL
serum or CSF
and heated at 37 C in an incubator. 50 AL of the cooled mixture was loaded on an analytical DNA-pac PA-100 Column (Dionex) and analyzed with a NaC1 gradient (0 ¨ 0.6 M in 30 min) in a 1:10 Acetonitrile:Buffer (20 mM sodium acetate, 1 mM magnesium acetate, pH 6.5) solution.
For LC-MS analysis 100 AL (20 AM or 50 AM) each siRNA was mixed with 900 1., sterile fetal bovine serum (GIBCO) incubated at 37 C and separated by HPLC as indicated previously (except of the NaC1 gradient: OM - 0.36M in 9' / 0.36M - 0.6M in 12'). Degradation products were desalted on NAP columns and analyzed by LC-EST-MS.
Example 3 Incubation in gastric acid To prepare a standard gastric acid assay, FVB and C57BL6 mice, weighing 18 to 20 g (6 to 8 weeks old), were obtained from Charles River Laboratories (Les Oncins, France). Animals were sacrificed using CO2, and then stomachs were quickly recovered. Gastric fluid as well as stomach contents were collected and pooled, then loaded on centrifugal filter devices (Ultrafree MC, Millipores). Filter units were spun for 10 minutes according to manufacturer's recommendations. The filtrate, corresponding to mouse gastric fluid, was recovered, aliquoted and frozen prior further experiments.
For each assay, 20 AM of siRNA solutions were diluted in 9x volume of gastric acid as above described and incubated at 37 C for 0, 5, 10, 15, 30, 60 and 120 min.
Example 4 Incubation in intestinal lavage To prepare a standard intestinal lavage assay, Male Wistar rat were fasted, anesthetized with isoflurane. Intestinal lavage was obtained by in situ perfusion of the small intestine (duodenum, jejunum, ileum) with 10 mL saline (0.5 mL/min) followed by 20 inL
water (1 =

wo 2007/128477 mL/min). Outlet collected was centrifuged (3000 x g, 15 min, 22 C), and supernatant passed through a 1.2-pm filter and stored at -20 C.
For each assay, 20 pM siRNA solutions were diluted in 9x volume of intestinal lavage and incubated at 37 C for 0, 15, 30,60, 180 and 360 min.
Example 5 Incubation in mouse liver microsomes In a standard liver microsome assay, to 10p.1 of a 250 AM solution of siRNA
were added 25 pl of mouse liver microsomes (GEntest 452701 Charge 11) at 20 mg protein /ml, 365 Al of 100 mM phosphate buffer (pH 7.4), 50 p.1 of UDPGA cofactor (24 misil in water), 50 p.1 of NADPH. Incubation was quenched by freezing at t=0 min and t= 60 min.
Example 6 Incubation in rat SI2 supernatant For a standard rat S12 supernatant assay, 10 pl of a 250 pM solution of siRNA
were added to 17 pi of rat liver S12 at 29.9 mg protein /ml, 373 p.I of 100 mM phosphate buffer (pH 7.4), 50 pl of UDPGA cofactor (24 mM in water), 50 AI of NADPH. Incubation was quenched by freezing at t=0 min and t= 60 min.
Example 7 Incubation in mouse serum For a standard incubation in mouse serum, 20 pM siRNA solutions were diluted in 9x volume of murine serum (Harlan nude mouse) and incubated at 37 C for 0, 15, 30,60, 180 and 360 min.
Example 8 Gel electrophoresis stability assay.
A 10 AL aliquot of incubation solution was taken immediately after shaking and shock-frozen on dry ice, the mixtures were incubated at 37 C and aliquots were shock frozen at various time points. Aliquots were thawed in 30 jiL (15 pL respectively) Loading Buffer (Elchrom Sc., Cham, Switzerland) and separated on a SF50 gels (Elchrom Sc., Cham, Switzerland) at 120 V, 8 C for 240 min. Bands were stained with SYBR Gold (Molecular Probes) and picture were taken with a BIORAD ChemiDocTm XRS system.

Example 9 Cell culture The mouse immortalized endothelial cell line MS1 (ATCC CRL-2279) was grown in DMEM
high glucose (4.5g/1) supplemented with L-Glutamine and 10% heat-inactivated FCS
(AMIMED, Switzerland) on 1.5% Gelatine-coated culture dishes. MS1 cells were transfected in 24 well-format with siRNA using HiPerfect (QIAGEN) according to manufacturer procedure (tetraplicate, final siRNA concentration was lOnM or as indicated).
Example 10 FACS analysis Non-transfected and siRNA transfected MS1 cells were analyzed by FACS for levels. Briefly, cells were trypsinized from duplicate or triplicate wells, pooled for each conditions, then washed twice with PBS+10% FCS and incubated 10 minutes on ice prior addition of RPE-conjugated anti-VEGFR2 Ab (11.18/106 cells; Avas 12al, BD
Phanningen).
RPE-labeled isotype IgG2a were used as FACS control (BD Phanningen). FACS
acquisition and analysis were performed on a FACScalibur using Cell Quest Software (Becton-Dickinson).
Example 11 Animal studies Female FVB mice (6 to 8 weeks old), were obtained from Charles River Laboratories (Les Oncins, France). Mice were identified via ear markings and kept in groups (6 animals per cage) under normal conditions and observed daily. Six mice were used per treatment group and all animal experiments were performed in strict adherence to the Swiss law for animal protection.
The reference chamber model has been described in publications (e.g. Wood J, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2000;60:2178-89) In brief, porous tissue chambers made of perfluoro-alkoxy-Teflon (Teflon -PFA, 21 mm x 8 mm diameter, 550 pl volume) were filled with 0.8% agar (BBL Nr. 11849, Becton Dickinson, Meylan, France) and 20U/m1 heparin, (Novo Nordisk AJS, Bagsvaerd, Denmark) supplemented with or without 3 1.1.g/m1 recombinant human VEGF and siRNAs as indicated.
=

' CA 02846756 2014-03-17 Solutions were maintained at 42 C prior the filling procedure. Mice were anesthetized using 3% Isoflurane (Forene0, Abbott AG, Cham, Switzerland) inhalation. For subcutaneous implantation, a small skin incision was made at the base of the tail to allow the insertion of an implant trocar. The chamber was implanted under aseptic conditions through the small incision onto the back of the animal. The skin incision was closed by wound clips (Autoclip 9 mm Clay Adams). Depending on the required dose, siRNAs were diluted in "injectable quality grade" 0.9% saline solution then delivered to animals either i.p. (200 L /dose) or p.o.
by gavage (1001AL /dose). The mice were receiving the first dose 2 to 4 hours before implanting chambers; then treated daily for 2 days. If not otherwise indicated, mice were sacrificed three days after implantation, chambers excised and the vascularized fibrous tissue formed around each implant carefully removed. Body weight was used to monitor the general condition of the mice. Statistical analysis was done using one-way ANOVA
followed by Dunnett test.
Example 12 B16 melanoma xenograft model The syngeneic B16/131-6 murine melanoma model, previously identified to be responsive to antiangiogenic therapy (e.g. LaMontagne K, Littlewood-Evans A, Schnell C, O'Reilly T, Wyder L, Sanchez T, Probst B, Butler J, Wood A, Liau G, Billy E, Theuer A, Hla T, Wood J.
Antagonism of sphingosine- 1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization. Cancer Res. 2006 Jan 1;66(1):221-31), was used to evaluate the antitumor activity of standard or modified siRNAs. Tumor cells (1 p.L, 5 x 1044L) were injected intradermally into the dorsal pinna of both ears of syngeneic female C57BL/6 mice.
Measurements of primary tumor area (mm2) were carried out on days 7, 14, and 21 after tumor cell inoculation using computer-assisted image analysis software (KS-400 3.0 imaging system, Zeiss) and a specifically designed macro. From days 7 to 21, mice were receiving siRNAs diluted in" injectable quality grade" 0.9% saline solution either i.p.
(200 L /dose) or p.o. by gavage (1001iL /dose) once a day. Mice were sacrificed on day 21, and cranial lymph node metastases were weighed and then frozen.
=
In these results, actual siRNA sequences and chemistries employed may be determined by reference to Table 3.

Wild-type siRNAs are degraded in mouse serum from both 3'-ends Oligonucleotide degradation by nucleases is predominantly 3'-exonucIeolytic.
Modification of antisense oligonueleotides at their termini by the introduction of aromatic or lipophilic residues delays their nueleolytic degradation". To verify whether this metabolic pathway would also be dominant for siRNA, we incubated at 37 C a unmodified siRNA
(wild-type siRNA) in mouse serum for up to 3 hours.
The unmodified siRNA sequence employed was pG13-siRNA (see Table 3) The mixtures were analyzed with Strong Anion Exchange HPLC at t0 min., t=30 min, t= 180 min.
As shown in Figure la, lb and lc, at t 30 min, a well defined peak corresponding to blunt ended siRNA was observed. By 1=312 substantial degradation is observed.
Figure Id and le illustrate the metabolites identified by HPLC-ESI-MS analysis. This analysis revealed the presence of several metabolites corresponding to the loss of the 3' overhangs and of the 3'- terminal first base pairing ribonucleotide on both strands. Digestion of the 5'-terminal ribonucleotide of the guide strand was also observed.
Figure 1 suggests the degradation pathway of unmodified siRNAs in serum. DNA
overhangs are first digested, possibly by 3'-exonueleases. In the LC-MS, additional metabolites were also detected which correspond to the loss of the first base-pairing 3'-ribonucleotide of both strands and also the first 5'-base-pairing ribonucleotide of the guide strand.
3'-modified siRNAs are stable through the GI tract siRNAs with 2'-methoxyethyl ribonucleotides overhangs (MOE o/h siRNA), blunt-ended siRNAs 3'-capped with a hydroxypropoxy phosphodiester moiety (C3-siRNA), and hydroxypropoxy phosphodiester 3'-capped siRNAs where the two first base paring nucleotide at 3'-end of each strand were modified by 2'-methoxyethyl ribonucleotides residues (C3-MOE siRNA) were synthesized. These compounds are illustrated schematically in Figure 2.

CA 02846756 2014-03-17 ,` =

First siRNAs were incubated in mouse gastric acid for 2h (Figure 3). No degradation was observed in the cases of C3 siRNA and C3-MOE sRNA, while degradation of wild-type siRNA was observed after 30 minutes.
Stability in intestinal fluid obtained from intestinal lavage of rats revealed almost complete degradation of wild-type siRNA after 15 minutes whereas parent compound in the MOE o/h siRNA, C3-siRNA and C3-Moe siRNA were observed for 60 minutes. (Figure 4) Stability in liver was evaluated using a liver microsome assay and a S12 assay (representative of liver cytosolic enzymatic activity). Results are shown in Figure 5. In both cases, no degradation was observed after 60 minutes of incubation.
Finally, siRNAs were tested in mouse serum by incubation at 2 micromolar for up to 6 hours at 37 C (results in Figure 6). Parent compound stability was followed by gel electrophoresis. In the cases of modified siRNAs (C3 siRNA, C3-MOE siRNA of MOE o/h siRNA), no significant degradation was observed while the wild-type siRNA
This study indicate that wild type (unmodified) siRNAs are metabolized in mouse gastric acid and in mouse serum, hi case of 3'-ends modified siRNAs, no degradation was observed in the GI tract. Therefore it is likely that 3'-modified siRNAs will have a higher oral bioavaiiability than wild-type siRNAs Systemically delivered 3'-modified siRNAs are more active in an in vivo growth factor induced angiogenesis model's.
Firstly, the ability of modified siRNAs (C3-siRNA and CE-MOE siRNA) to down regulate a target gene was checked in cellulo by measuring VEGFR2 surface level of MS) cells transfected with anti-VEGFR2 siRNAs.
Pools of 2 anti VEGFR2 siRNAs as wild-type siRNAs, C3-siRNAs and C3-MOE
siRNAs were administered intraperitoneally. Results are shown in Figure 7.
Pooled Wild type siRNAs reduced significantly the VEGF induced vascularization at the higher dose of 25 micrograms per mice per day. The same level of inhibition was observed at a 5-fold lower dose with C3-siRNA. In the case of the C3-MOE siRNAs pool, significant reduction of vascularized tissue weight was observed at all tested doses including the lowest 0.2 microgram per mouse per day.

CA 02846756 2014-03-17 , Figure 8a and 8b show that, when given intraperitoneally, both VEGFR2 - C3 and C3-MOE siRNAs were active at below 1 microgram per mouse per day dose.
In vivo testing of anti-VEGFR2 C3-MOE siRNA given intraperitoneally (i.p.) in a B16 homog,raft melanoma tumor mouse model. Figure 9a shows that i.p. treatment with modified VEGFR2-C3-M0E-siRNA significantly reduces tumour development. Figure 9b also shows that i.p. injection of VEGFR2-C3-M0E-siRNA at 20 ug per mouse results in significant inhibition of tumour growth.
Oral Delivery of siRNA for Treatment of Angiogenic Disorders Figure 10 shows that given orally, at a dose of 20 micrograms per mouse per day, the VEGFR2-C3-M0E-siRNA1 reduced vascularization weight down to basal level (e.g.
weight without growth factor induction). Actual siRNA sequences used are referred to in Table 3.
Anti Tie2 C3-MOE siRNAs were also tested in the growth factor induced angiogenesis model under both intraperitoneal and oral deliveries. Figure lla and llb show that given orally, both C3-MOE siRNAs directed at Tie2 were active at 20 microgram per mouse per day. Actual siRNA sequences used may be determined by reference to Table 3.
The data shows that 3'-end modified siRNAs with or without additional internal modifications are able to demonstrate therapeutic effect at reasonable doses upon oral administration.

= CA 02846756 2014-03-17 REFERENCES
1. a) Y. Tomari et al. Genes and Development 19 (2005), 517; b) P. Shankar et al. JAMA
11 (2005), 1367; c)Y. Dorsett et at. Nature Reviews 3 (2004), 318 2. a) P.D. Zamore et at. Cell101, (2000), 25; b) S.M. Hammond et al. Nature (2000), 293 3. a) G. Meister et at. Molecular Cell 15 (2004), 185.
4. S.M. Elbashir et at. Genes Dev. 15 (2001), 188.
5. S.J. Reich et al. Molecular Vision 9 (2003), 210.
6. a) Dorn et al. Nucleic Acids Research 32 (2004), e49; b) D. R.Thakker et al. PNAS 101 (2004), 17270; c) D.R. Thakker etal. Molecular Psychiatry 10 (2005), 714 7. V. Bitko et at. Nature Medicine 11 (2005), 50.

8. E. Song et al. Nature Medicine 9(2003), 347.

9. D.A.Braasch et at. Biochemistry 42 (2003), 7967.

10. Harborth, Antisense Nucleic Acid Drug Devt, 2003 11. A.H.S. Hall et al. Nucleic Acids Research 32 (2004), 5991.

12. M. Amarzguioui et al. Nucleic Acids Research 31 (2003), 589.

13. F. Czaudema et aL Nucleic Acids Research 31 (2003), 2705.

14. T. Prakash et al. Journal of Medicinal Chemistry 48 (2005), 4247.

15. J. Etmen et al. Nucleic Acids Research 33 (2005), 439.

16. A.S. Boutorin, L.V. Guskova, E.M. Ivanova, N.D. Kobetz, V.F. Zafytova, A.S. Ryte, L.V. Yurchenko and V.V. Vlassov FEBS Lett. 254 (1989), p. 129 17. J. Wood et al. Cancer Research 60 (2000), 2178.

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Claims (24)

1. A short interfering ribonucleic acid (siRNA), said siRNA comprising two RNA
strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein the 3'-terminus of at least one strand comprises a modification at the 3' carbon, wherein the modification is:

2. A short interfering ribonucleic acid (siRNA), said siRNA comprising two RNA
strands that are complementary to each other over at least 15 nucleotides, wherein each strand is 49 nucleotides or less, and wherein the 3'-terminus of each strand comprises a modification at the 3' carbon, wherein the modification is:

3. The siRNA according to claim 1, wherein the first two base-pairing nucleotides at the 3' end of each strand are modified.

4. The siRNA according to claim 1, wherein the first two base-pairing nucleotides at the 3' end of each strand are 2'-methoxyethyl ribonucleotides residues.

5. The siRNA according to claim 1, wherein the two strands are complementary to each other over at least 19 nucleotides.

6. The siRNA according to claim 5, wherein each strand is 19 nucleotides.

7. The siRNA according to claim 1, wherein both ends of the siRNA are blunt-ended.

8. The siRNA according to claim 1, wherein each strand is 19 nucleotides.

9. The siRNA according to claim 1, wherein the two strands are fully complementary to each other over 19 nucleotides and wherein the siRNA is blunt-ended.

10. The siRNA according to claim 3, wherein at least one additional nucleotide is modified.

11. The siRNA according to claim 1, having stability in a standard gastric acid assay that is greater than an unmodified siRNA with the same nucleotide sequence.

12. The siRNA according to claim 1, having stability in a standard gastric acid assay that is greater than or equal to 50% after 30 minutes exposure.

13. The siRNA according to claim 1, having stability in a standard serum assay is greater than an unmodified siRNA with the same nucleotide sequence.

14. The siRNA according to claim 1, having stability in a standard serum assay is greater than or equal to 50% after 30 minutes exposure.

15. The siRNA according to claim 1, having stability in a standard intestinal lavage assay that is greater than an unmodified siRNA with the same nucleotide sequence.

16. The siRNA according to claim 1, having an enhanced bioavailability compared to an unmodified siRNA of the same nucleotide sequence.

17. Pharmaceutical composition comprising the siRNA according to claim 1, and a pharmaceutically acceptable carrier.

18. The siRNA according to claim 1, for use as a medicament.

19. The siRNA according to claim 1, wherein the first two base-pairing nucleotides at the 3' end of each strand are modified, wherein each modified nucleotide has an internucleoside linkage which is an amide linkage.

20. The siRNA according to claim 1, for use as a medicament which is administered parenterally.

21. The siRNA according to claim 1, wherein the first two base-pairing nucleotides at the 3' end of each strand are modified, wherein each modified nucleotide is selected from among nucleotides having a modified internucleoside linkage selected from among phosphorothioate, phosphorodithioate, phosphoramidate, boranophosphonoate, and amide linkages.

22. The siRNA according to claim 5, wherein one end of the siRNA is blunt-ended.

23. The siRNA according to claim 1, comprising a 1 to 6 nucleotide overhang on at least one of the 5' end or 3' end.

24. The siRNA according to claim 1, for use as a medicament which is administered orally, topically, parenterally, by inhalation or spray, or rectally, or by percutaneous, subcutaneous, intravascular, intravenous, intramuscular, intraperitoneal, intrathecal or infusion technique.