WO2010058426A2 - Inhibition de la sécrétion de vegf-a, l’angiogenèse et/ou la néo-angiogenèse par inactivation véhiculée par ansi de vegf-c et rhoa - Google Patents

Inhibition de la sécrétion de vegf-a, l’angiogenèse et/ou la néo-angiogenèse par inactivation véhiculée par ansi de vegf-c et rhoa Download PDF

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WO2010058426A2
WO2010058426A2 PCT/IN2009/000671 IN2009000671W WO2010058426A2 WO 2010058426 A2 WO2010058426 A2 WO 2010058426A2 IN 2009000671 W IN2009000671 W IN 2009000671W WO 2010058426 A2 WO2010058426 A2 WO 2010058426A2
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vegf
nucleic acid
sequence
nucleotides
acid molecule
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PCT/IN2009/000671
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WO2010058426A3 (fr
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Krishna Addepalli Murali
Kumar Bharat
Chile Shailaja
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Reliance Life Sciences Pvt. Ltd.
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Priority to CN2009801540536A priority Critical patent/CN102272307A/zh
Priority to US13/130,699 priority patent/US20110293625A1/en
Priority to EP09810851A priority patent/EP2358877A2/fr
Publication of WO2010058426A2 publication Critical patent/WO2010058426A2/fr
Publication of WO2010058426A3 publication Critical patent/WO2010058426A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to use of short nucleic acid molecules, such as short interfering nucleic acid (siNA) molecules, for modulating gene and protein expression, including compounds, compositions and synergistic combination of small nucleic acid molecules that modulate RhoA and/or VEGF-C gene expression.
  • short nucleic acid molecules such as short interfering nucleic acid (siNA) molecules
  • siNA short interfering nucleic acid
  • the compounds and methods of the present invention have applications in modulating Rho-A, VEGF-C and VEGF-A expression and secretion, angiogenesis and/or neoangiogenesis, either alone or in combination with other therapies.
  • Angiogenesis is the formation of new blood vessels or enlargement of existing vessels, while lymphangiogenesis is the equivalent process in lymphatic vessels.
  • the processes of blood and lymphatic angiogenesis are tightly regulated by several key angiogenic factors.
  • Anti-angiogenic factors include thrombospondin,platelet factor IV, TNF-alpha (in vitro), TGF-beta, interferons, angiostatin, integrin inhibitors, 16kD prolactin, endostatin, and ANG-2. Certain steroids may inhibit angiogenesis.
  • Proangiogenic factors for blood and lymph vessels include FGF (fibroblast growth factor), VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor), and angiopoietin families.
  • FGF fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived growth factor
  • angiopoietin families include FGF (fibroblast growth factor), VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor), and angiopoietin families.
  • FGF-I and FGF-2 are potent angiogenic factors in vivo, although the physiological and pathological relevance of these factors in regulation of angiogenesis is unclear.
  • the VEGF family includes at least five structurally related proteins, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PlGF). These molecules interact with a set of cell surface receptors, VEGFR-I, VEGFR-2, and VEGFR-3, that show varying specificity and function. VEGF-C and VEGF-D bind to both VEGFR-2 and VEGFR-3 and promote formation of blood and lymph vessels. VEGF-B and PlGF bind to VEGFR-I and modulate the effects of VEGF-A, but their roles in stimulation of angiogenesis remain controversial.
  • VEGF-A acts as a potent vascular permeability factor (VPF).
  • VEGFR-2 is the receptor that mediates VEGF-A-induced angiogenic and permeability effects.
  • VEGF-B and PlGF which only interact with VEGFR-I, lack angiogenic and vascular permeability activity.
  • VEGF-A, VEGF-C, and FGF-2 determine the thickness of the endothelial cell layer in capillaries (in order of importance, VEGF- A ⁇ VEGF-C ⁇ FGF-2). In contrast to blood capillaries, lymph capillaries generated by VEGF-C completely lack fenestrations.
  • VEGF-C vascular endothelial apoptosis
  • angiogenesis is critical for numerous normal and pathological processes, including embryonic development, wound healing, tumor growth and metastasis, rheumatoid arthritis, diabetic retinopathy, atherosclerosis, and revascularization of ischemic myocardium, hind limb muscles, and brain. Lymphangiogenesis is critically important for tumor spread via the lymphatic system.
  • Neovascularization has been shown to cause or exacerbate ocular diseases including, but not limited to, macular degeneration, neovascular glaucoma, diabetic retinopathy, myopic degeneration, and trachoma. Norrby APMIS 105, 417-437(1997). It has been reported that the ocular fluid of a majority of patients suffering from diabetic retinopathy and other retinal disorders contains a high concentration of VEGF-A. Aiello et al. New Engl. J. Med. 331, 1480 (1994). Elevated levels of VEGF mRNA in patients suffering from retinal ischemia have also been reported. Miller et al., Am. J. Pathol. 145, 574 (1994). Thus, VEGF-A may have a direct role in ocular diseases. Other factors, including those that stimulate VEGF synthesis, may also contribute to these indications.
  • Rho proteins are involved in the regulation and timing of cell division, and include Rho.
  • the Rho family of proteins includes RhoA, RhoB, RhoC, Racl, Rac2 and Cdc42, which share more than 50% sequence identity with each other.
  • Rho proteins are involved in inducing the formation of stress fibers and focal contacts in response to extracellular signals such as lysophosphatidic acid (LPA) and growth factors.
  • LPA lysophosphatidic acid
  • the Rho family is also considered to be implicated in physiological functions associated with cytoskeletal rearrangements, such as cell morphological change (Parterson et al., J.
  • Rho proteins are involved in the regulation of smooth muscle contraction (Hirata et al., J. Biol. Chem., 267, 8719-8722 (1992); Noda et al., FEBS Lett., 367, 246-250 (1995); Gong et al., Proc. Natl. Acad. Sci. USA, 93, 1340-1345 (1996)), and the expression of phosphatidylinositol 3-kinase (PI3 kinase) (Zhang et al., J. Biol.
  • RhoA is a small GTPase protein known to regulate the actin cytoskeleton in the formation of stress fibers.
  • Rho-kinase (ROCK) proteins are downstream effectors of RhoA and are activated through phosphorylation by activated Rho.
  • the mammalian ROCK family includes ROCK-I and ROCK-2, which are serine/threonine kinases of about 160 kDa, encoded by two different genes.
  • An object of the present invention is to provide a combination of short interfering nucleic acid (siNA) molecules for modulation of VEGF-C and/or RhoA gene expression that display better specificity and/or effectiveness than prior art short interfering nucleic acids directed against either protein.
  • siNA short interfering nucleic acid
  • RhoA gene expression so that a VEGF-A gene or protein, or a gene or protein involved in a VEGF-A mediated signaling pathway, is inhibited or reduced in amount, thereby regulating neoangiogenesis.
  • VEGF-C gene expression so that a VEGF-A gene or protein, or a gene or protein involved in a VEGF-A mediated signaling pathway, is inhibited or reduced in amount, thereby regulating neoangiogenesis.
  • RhoA and VEGF-C gene expression so that a VEGF-A gene or protein, or a gene or protein involved in a VEGF-A mediated signaling pathway, is inhibited or reduced in amount, for example, synergistically, thereby regulating neoangiogenesis.
  • RhoA and VEGF-C siNAs in combination to a cell, vessel, tissue or organism, whereby VEGF-A expression and secretion, angiogenesis and/or neoangiogenesis is inhibited in a synergistic manner, as compared to what is observed when adding the effects of using each of RhoA and/or VEGF-C siNA individually.
  • siNAs comprising about 19 to about 30 nucleotides that inhibit gene expression of RhoA and/or VEGF-C
  • siNA molecules that are site directed to a target gene.
  • siNA molecules that can be used for inhibiting angiogenesis.
  • AMD age related macular degeneration
  • neo-vascular glaucoma rubeosis
  • uveitis uveitis
  • choroidal neovascularization eye infections such as conjunctivitis, keratitis, blepharitis, sty, chalazion, ulceris, and stromal keratitis.
  • cancers such as breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer.
  • the present invention designs and presents nucleic acid molecules targeting RhoA and/or VEGF-C genes, which can specifically and effectively direct homology-specific post transcriptional gene silencing, and therefore are useful as highly effective, selective and potent therapeutics, with minimal side effects.
  • the present invention includes double stranded short nucleic acid (siNA) molecules that are specifically targeted.
  • the short nucleic acid molecules are RNA, including siRNA targeting RhoA and/or VEGF-C genes.
  • the invention presents a siNA molecule that down-regulates expression of a VEGF-C gene, wherein the siNA molecule comprises about 19 to about 30 base pairs.
  • the siNA is an siRNA that is 19, 20, 21, 22, 23,
  • the invention presents a siNA molecule that down-regulates expression of a RhoA gene, wherein the siNA molecule comprises about 19 to about 30 base pairs.
  • the siNA is an siRNA that is 19, 20, 21, 22, 23, 24,
  • the invention presents a siNA molecule comprising nucleotide sequence, for example, nucleotide sequence in the antisense region of the siNA molecule that is complementary to a nucleotide sequence or portion of sequence of a VEGF-C and/or RhoA gene.
  • the invention presents a siNA molecule comprising a region, for example, the antisense region of the siNA construct, complementary to a sequence comprising a VEGF-C and/or RhoA gene sequence or a portion thereof.
  • a siNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary to a nucleotide sequence or a portion thereof encoding a VEGF-C and/or a RhoA protein.
  • the siNA further comprises a sense strand, wherein said sense strand comprises a nucleotide sequence of a VEGF-C and/or a RhoA gene or a portion thereof.
  • a siNA molecule of the invention has RNA interference (RNAi) activity that modulates expression of an RNA encoded by a VEGF-C and/or a RhoA gene.
  • RNAi RNA interference
  • the invention presents a double-stranded short interfering nucleic acid (siNA) molecule that down-regulates expression of a VEGF-C and/or RhoA gene, wherein the siNA comprises an antisense region, complementary to a nucleotide sequence of the VEGF-C and/or RhoA gene or a portion thereof, and a sense region substantially similar to the nucleotide sequence of the VEGF-C and/or RhoA gene or a portion thereof.
  • siNA double-stranded short interfering nucleic acid
  • the antisense region and the sense region each comprise about 19 to about 30 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the antisense region comprises at least 19 nucleotides that are complementary to nucleotides of the sense region.
  • a siNA molecule of the invention comprises any of one of: RINA 6 comprising sense strand SEQ ID NO: 7 and antisense strand SEQ ID NO: 8; RINA 17 comprising sense strand SEQ ID NO: 9 and antisense strand SEQ ID NO: 10; RINA 30 comprising sense strand SEQ ID NO: 11 and antisense strand SEQ ID NO: 12; RINA 50 comprising sense strand SEQ ID NO: 13 and antisense strand SEQ ID NO: 14; RINA 51 comprising sense strand SEQ ID NO: 15 and antisense strand SEQ ID NO: 16; and RINA 52 comprising sense strand SEQ ID NO: 17 and antisense strand SEQ ID NO: 18.
  • the knockdown of VEGF-C expression using siNA leads to decreased activation of VEGFR-3, which is required for the initiation and sustaining of angiogenesis. Further, the present invention demonstrates for the first time that knockdown of VEGF-C also leads to decrease in expression of VEGF-A. Thus, the present invention establishes a direct relationship exists between expression levels of both VEGF-C and VEGF-A and thus provides a means to control or reverse angiogenesis.
  • the present disclosure provides short nucleic acid molecules for modulation of RhoA and/or VEGF-C gene expression.
  • the present invention provides RhoA and/or VEGF-C targeting short nucleic acid molecules for the inhibition of angiogenesis.
  • Such molecules may be used alone (RhoA and/or VEGF-C targeting molecules), or in combination with other therapies, for the management and treatment of various disorders associated with excessive angiogenesis, such as age related macular degeneration and diabetic retinopathy, hypertension, cancer, inflammation and autoimmune diseases.
  • the present invention provides siNAs having between 19 to 30 nucleotides, between 25 and 29 nucleotides, or having 27 nucleotides, where the sequence is designed for better stability and efficacy in knockdown (i.e., reduction) of RhoA and/or VEGF-C gene expression.
  • siNAs can be used alone or in combination with other therapies.
  • the nucleic acid molecule of the present invention comprises 19-30 nucleotides complementary to RNA corresponding to a RhoA and/or VEGF-C nucleic acid sequence.
  • the invention encompasses compounds, compositions and uses of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30-mers, including 27-mer siNA molecules, for modulation of RhoA and/or VEGF-C gene expression.
  • the present invention provides stable compositions of siNA with or without conjugation to cholesterol.
  • the compounds of the present invention are useful in therapy of angiogenesis either alone or in combination with other treatments or therapies.
  • the siNA of the present invention includes short interfering RNA (siRNA), a double stranded RNA (dsRNA), a micro RNA ( ⁇ RNA), and/or a short hairpin RNA (shRNA) molecule.
  • siRNA short interfering RNA
  • dsRNA double stranded RNA
  • ⁇ RNA micro RNA
  • shRNA short hairpin RNA
  • the siNA molecules can be unmodified or modified chemically.
  • the present invention relates to a siRNA having 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides on one strand.
  • Nucleotides of the present invention can be chemically synthesized, expressed from a vector, or enzymatically synthesized.
  • an siRNA composition of the invention comprises combination of two double stranded siRNAs, wherein one strand of the first double stranded siRNA molecule is complimentary to a portion of an RNA encoding RhoA, and one strand of the second double stranded siRNA molecule is complimentary to a portion of an RNA encoding VEGF-C.
  • one siRNA molecule of the invention comprises a double stranded RNA, wherein one strand of the RNA comprises a portion of a sequence of RNA encoding RhoA, and another siRNA molecule comprises a double stranded RNA wherein one strand of the RNA comprises a portion of a sequence of RNA encoding VEGF-C.
  • the invention targets RhoA and/or VEGF-C as set forth in GenBank Accession Numbers NM_001664 and NM_ 005429, respectively.
  • the present invention is not limited, however, to nucleotides targeting one variant of RhoA, but also includes nucleotides that target RhoA-related molecules including single nucleotide polymorphisms of RhoA, RhoA homologs, and RhoA splice and transcript variants.
  • the present invention also contemplates nucleotides that target genes involved in RhoA regulatory pathway as a means of regulating RhoA.
  • nucleotides targeting VEGF-C are also not limited to nucleotides targeting one variant of VEGF-C, but also include nucleotides that target VEGF-C-related molecules including single nucleotide polymorphisms of VEGF-C, VEGF-C homologs, and VEGF-C splice and transcript variants.
  • the present invention also contemplates nucleotides that target genes involved in VEGF-C regulatory pathway as a means of regulating VEGF-C.
  • the present invention provides compositions and methods used to regulate RhoA and/or VEGF-C expression or activity.
  • RhoA expression or activity may be regulated by a small nucleic acid molecule that targets RhoA directly, or by targeting molecules that regulate the RhoA pathway.
  • VEGF-C expression or activity may be regulated by a small nucleic acid molecule that targets VEGF-C directly, or by targeting molecules which regulate the VEGF-C pathway.
  • Small nucleic acid molecules that target RhoA and/or VEGF-C may be used in combination with other small nucleic acid molecules or small chemical molecules.
  • the targeting of RhoA and/or VEGF-C is used to regulate neovascular disease states that respond to modulation of RhoA and/or VEGF-C expression levels in the cell, such as age related macular degeneration, diabetic retinopathy and glaucoma, cancer, inflammation and autoimmune diseases.
  • neovascular disease states that respond to modulation of RhoA and/or VEGF-C expression levels in the cell, such as age related macular degeneration, diabetic retinopathy and glaucoma, cancer, inflammation and autoimmune diseases.
  • siNAs of 27 nucleotides in length are used to reduce expression levels of RhoA and VEGF-C, alone or in combination with other small nucleic acid molecules directed against genes that are involved in treatment of the same disease.
  • the siNAs are of 19, 20, 21, 22, 23, 24, 25, 26, 28, 29 or 30 nucleotides.
  • the present invention provides techniques for validating the efficacy of siNA using biomarkers for angiogenesis, such as expression levels of VEGF- A, and phosphorylation status of ROCK-I and ROCK-2.
  • the invention presents a mammalian cell, for example a human cell, comprising a small nucleic acid molecule of the invention.
  • the present invention presents a method of down-regulating (also called “knocking down") RhoA and/or VEGF-C activity in a cell, comprising contacting the cell with a nucleic acid molecule of the invention, under conditions suitable for down-regulating RhoA and VEGF-C activity.
  • the present invention also presents a method for treating a subject having a condition associated with elevated levels of RhoA and/or VEGF-C, comprising contacting cells of the subject with a nucleic acid molecule of the invention, under conditions suitable for the treatment.
  • the method is supplemented with a drug therapy for the same condition.
  • the present invention also presents a method using a therapeutic agent for the treatment of ocular diseases or disorders such as anterior and posterior ocular diseases.
  • the present invention provides a combination of short nucleic acid molecules which inhibit the expression of at least one gene associated with neovascularization and angiogenesis.
  • the present invention provides short nucleic acid molecules for treatment of diabetic retinopathy, age related macular degeneration (AMD), wet AMD, inflammatory conditions of the eye such as uveitis, rubeosis, conjunctivits, keratitis, blepharitis, sty, chalazion, ulceris, stromal keratitis, and cancers.
  • the present invention provides short nucleic acid molecules for the treatment of cancer, such as breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer, comprising administering to a subject a nucleic acid molecule of the invention under conditions suitable for said treatment.
  • cancer such as breast cancer, lung cancer, prostate cancer, colorectal cancer, brain cancer, esophageal cancer, stomach cancer, bladder cancer, pancreatic cancer, cervical cancer, head and neck cancer, ovarian cancer, melanoma, lymphoma, glioma, or multidrug resistant cancer
  • the invention provides a method to prevent activation of VEGFR-3 nullified by inhibiting its interacting ligand, VEGF-C.
  • the present invention establishes that a direct correlation exists between expression levels of VEGF-C and VEGF-A. Likewise, the present invention establishes that a homeostasis exists between expression levels of VEGF-C and VEGF-A, which is required for the initiation and containment of neoangiogenesis.
  • other drug therapies contemplated by the invention include chemical inhibitiors, monoclonal antibodies or a combination thereof.
  • the present invention presents compositions comprising the nucleic acid molecules of the invention in a pharmaceutically acceptable carrier.
  • the invention also presents a method of administering to a cell, such as mammalian cell (e.g., a human cell), a nucleic acid of the invention.
  • a cell such as mammalian cell (e.g., a human cell)
  • the method of administering comprises contacting the cell with a nucleic acid molecule of the invention under conditions suitable for such administration.
  • the method of administration may be in the presence of a delivery reagent, for example a lipid, cationic lipid, phospholipid, or liposome.
  • the site of administration may be selected depending on the disease, e.g., ocular diseases, and the method of delivery may be, for example, subconjunctival, intravenous, subcutaneous, eye drops and/or topical.
  • the present invention provides compositions comprising: (1) a first short nucleic acid molecule that modulates VEGF-C expression, wherein the first short nucleic acid molecule comprises a first nucleotide sequence, wherein a sequence of at least 19 contiguous nucleotides in the first nucleotide sequence is at least 95% complementary to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and/or (2) a second short nucleic acid molecule that modulates RhoA expression, wherein the second short nucleic acid molecule comprises a second nucleotide sequence, wherein a sequence of at least 19 contiguous nucleotides in the second nucleotide sequence is at least 95% complementary to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. See Table 1 below.
  • a sequence of at least 19 contiguous nucleotides in the first nucleotide sequence is completely (100%) complementary to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and a sequence of at least 19 contiguous nucleotides in the second nucleotide sequence is completely (100%) complementary to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • a first short nucleic acid molecule comprises a siNA selected from the group consisting of: RJNA 6 comprising sense strand SEQ ID NO: 7 and antisense strand SEQ ID NO: 8; RINA 17 comprising sense strand SEQ ID NO: 9 and antisense strand SEQ ID NO: 10; and RINA 30 comprising sense strand SEQ ID NO: 1 1 and antisense strand SEQ ID NO: 12. See Table 2 below.
  • a second short nucleic acid molecule comprises a siNA selected from the group consisting of: RINA 50 comprising sense strand SEQ ID NO: 13 and antisense strand SEQ ID NO: 14; RTNA 51 comprising sense strand SEQ ID NO: 15 and antisense strand SEQ ID NO: 16; and RINA 52 comprising sense strand SEQ ID NO: 17 and antisense strand SEQ ID NO: 18. See Table 2 below.
  • a first short nucleic acid molecule comprises RlNA 30 comprising sense strand SEQ ID NO: 11 and antisense strand SEQ ID NO: 12
  • the second short nucleic acid molecule comprises RINA 52 comprising sense strand SEQ ID NO: 17 and antisense strand SEQ ID NO: 18.
  • the present invention also provides compositions for the treatment of an ocular disorder comprising one of the compositions described herein, and a pharmaceutically acceptable carrier.
  • the ocular disorder is retinopathy or glaucoma.
  • the present invention relates to compositions for the treatment of a neovascular disease comprising one of the compositions described herein, and a pharmaceutically acceptable carrier.
  • compositions of the invention comprise a lipid, polymer and/or a monoclonal antibody.
  • at least one short nucleic acid molecule is conjugated to cholesterol.
  • the invention comprises methods of inhibiting VEGF-C, VEGF-A, and/or RhoA by administering a siRNA, including the siRNA's of the invention, and related methods of use and methods of treatment.
  • the method comprises comprising contacting the cell with a short nucleic acid molecule comprising a sequence of at least 19 contiguous nucleotides that is at least 95% complementary to a sequence of at least 19 contiguous nucleotides within a full length VEGF-C or RhoA gene.
  • the sequence is 100% complementary.
  • the nucleic acid is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides on one strand.
  • the short nucleic acid molecule comprises a antisense strand consisting of 27 nucleotides, wherein the 27 nucleotide strand sequence is at least 95% complementary to 27 contiguous nucleotides within a full length VEGF-C or RhoA gene.
  • the present invention provides methods for reducing or down- regulating VEGF-A expression, or VEGF-A secretion from a cell, comprising contacting the cell with a short nucleic acid molecule comprising a sequence of at least 19 contiguous nucleotides that is at least 95% complementary to a sequence of at least 19 contiguous nucleotides within a full length VEGF-C or RhoA gene.
  • the sequence is 100% complementary.
  • the nucleic acid is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides on one strand.
  • the short nucleic acid molecule comprises a antisense strand consisting of 27 nucleotides, wherein the 27 nucleotide strand sequence is at least 95% complementary to 27 contiguous nucleotides within a full length VEGF-C or RhoA gene.
  • VEGF-A secretion is reduced or down-regulated by about 50% in a cell after contacting the cell with a short nucleic acid molecule at a concentration of about 0.6 nM.
  • the short nucleic acid molecule (1) directly modulates expression of VEGF-C or RhoA; (2) inhibits VEGF-C and/or RhoA expression; (3) inhibits VEGF- A expression or secretion; and/or (4) binds to at least one molecule that modulates expression or secretion of VEGF-A.
  • the first short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is at least 95% to a sequence of at least 19 contiguous nucleotides within a full length VEGF-C gene
  • the second short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is at least 95% to a sequence of at least 19 contiguous nucleotides within a full length RhoA gene.
  • the nucleic acid is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • the sequence is completely (100%) complementary.
  • the siRNA molecules are selected from:
  • RINA 6 comprising sense strand SEQ ID NO: 7 and antisense strand SEQ ID NO: 8
  • RINA 17 comprising sense strand SEQ ID NO: 9 and antisense strand SEQ ID NO: 10
  • RINA 30 comprising sense strand SEQ ID NO: 11 and antisense strand SEQ ID NO: 12
  • RINA 50 comprising sense strand SEQ ID NO: 13 and antisense strand SEQ ID NO: 14
  • RINA 51 comprising sense strand SEQ ID NO: 15 and antisense strand SEQ ID NO: 16
  • RINA 52 comprising sense strand SEQ ID NO: 17 and antisense strand SEQ ID NO: 18.
  • the present invention provides methods for reducing or down- regulating VEGF-A secretion from a cell comprising contacting the cell with a short nucleic acid molecule comprising a sequence of at least 19 contiguous nucleotides that is completely (100%) complementary to a sequence of at least 19 contiguous nucleotides within a full length VEGF-C or RhoA gene.
  • the nucleic acid is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.
  • the short nucleic acid molecule comprises a antisense strand consisting of 27 nucleotides, wherein the 27 nucleotide strand sequence is completely (100%) complementary to 27 contiguous nucleotides within a full length VEGF-C or RhoA gene.
  • the present invention provides methods for reducing or down- regulating VEGF-A secretion from a cell comprising contacting the cell with a short nucleic acid molecule comprising a nucleotide sequence that is at least 95% complementary to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
  • the short nucleic acid molecule comprises a nucleotide sequence that is completely (100%) complementary to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.
  • the present invention provides methods for reducing or down- regulating VEGF-A secretion from a cell comprising contacting the cell with a short nucleic acid molecule comprising a nucleotide sequence that is at least 95% complementary to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • the short nucleic acid molecule comprises a nucleotide sequence that is completely (100%) complementary to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • the present invention provides methods for reducing or down- regulating VEGF-A secretion from a cell comprising contacting the cell with a first short nucleic acid molecule and second short nucleic acid molecule, wherein the first short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is at least 95% complementary to a sequence of at least 19 contiguous nucleotides within a full length VEGF-C gene, and wherein the second short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is at least 95% complementary to at least 19 contiguous nucleotides within a full length RhoA gene.
  • the first short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is completely (100%) complementary to a sequence of at least 19 contiguous nucleotides within a full length VEGF-C gene
  • the second short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is completely (100%) complementary to a sequence of at least 19 contiguous nucleotides within a full length RhoA gene.
  • the present invention also provides methods for treating an ocular disorder, neovascular disease, and/or cancer comprising administering at least one of the compositions described herein.
  • the present invention provides methods for reducing or inhibiting angiogenesis or neoangiogenesis in a tissue comprising contacting the tissue with a first short nucleic acid molecule and second short nucleic acid molecule, wherein the first short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is at least 95% complementary to at least 19 contiguous nucleotides within a full length VEGF-C gene, and wherein the second short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is at least 95% complementary to a sequence of at least 19 contiguous nucleotides within a full length RhoA gene.
  • the first short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is completely (100%) complementary to a sequence of at least 19 contiguous nucleotides within a full length VEGF-C gene
  • the second short nucleic acid molecule comprises a sequence of at least 19 contiguous nucleotides that is completely (100%) complementary to a sequence of at least 19 contiguous nucleotides within a full length RhoA gene.
  • the nucleic acid is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length
  • the present invention presents methods for reducing or inhibiting angiogenesis or neoangiogenesis in a tissue comprising contacting the tissue with at least one of the compositions described herein.
  • the present invention provides methods for reducing or inhibiting ROCK phosphorylation, such as ROCK-2 phosphorylation, in a cell comprising contacting the cell with a short nucleic acid molecule comprising a sequence of at least 19 contiguous nucleotides that is at least 95% complementary to a sequence of at least 19 contiguous nucleotides within a full length RhoA gene, wherein phosphorylation of ROCK-2 is reduced or inhibited, but ROCK expression is not knocked down.
  • ROCK phosphorylation such as ROCK-2 phosphorylation
  • the short nucleic acid molecule modulates RhoA expression and comprises a nucleotide sequence of at least 19 contiguous nucleotides at least 95% complementary to a sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.
  • the short nucleic acid molecule comprises a siNA selected from the group consisting of: RINA 50 comprising sense strand SEQ ID NO: 13 and antisense strand SEQ ID NO: 14; RINA 51 comprising sense strand SEQ ID NO: 15 and antisense strand SEQ ID NO: 16; and RINA 52 comprising sense strand SEQ ID NO: 17 and antisense strand SEQ ID NO: 18.
  • Figure 1 Reverse transcriptase PCR products amplified by gene specific primers were resolved over 2% agarose gel electrophoresis and stained with ethidium bromide to visualize the amplicons of interest. Arrowheads indicate amplicons of specific gene products, showing that two cell lines, HUVEC (Human Umblical Vascular Endothelial Cells, ATCC) and PC3 (Prostate Cancer cells, ATCC), express both VEGF-C and RhoA.
  • HUVEC Human Umblical Vascular Endothelial Cells, ATCC
  • PC3 Prostate Cancer cells, ATCC
  • Lane 2 PC3 VEGF-C no template (c-DNA) added;
  • Lane 5 lOObp ladder
  • Lane 6 HUVEC VEGF-C
  • Lane 7 HUVEC VEGF-C no template (c-DNA) added:
  • Lane 8 HUVEC RhoA
  • Lane 9 HUVEC RhoA no template (c-DNA) added
  • Lane 10 lOObp ladder.
  • Figure 2 Standard curve for VEGF-A concentration determination, as obtained from a sandwich ELISA kit and using standards for VEGF-A provided in the kit.
  • Figure 3 Figure 3A. Knockdown of VEGF-C at various concentrations of RINA 30 ranging from 0.01 to 100 nM, at 10-fold increments, where results reach a plateau at 70% inhibition. The IC50 value is 0.5 nM for RINA 30.
  • Figure 3B Knockdown of VEGF-A at various concentrations of RINA 30 ranging from 0.01 to 100 nM, at 10-fold increments, where results reach a plateau at 48% inhibition. The IC50 value is 0.8 nM for RINA 30.
  • Figure 4 Figure 4A. Retinal pigmented cells were transfected with RhoA RINAs 50, 51 and 52. Protein from lysed transfected cells were blotted onto pre-wet nitrocellulose membrane. Phosphorylated ROCK-2 was detected on the nitrocellulose using an anti- phospho-Rho-associated kinase-alpha antibody ABCAM ab 24843 (ABCAM PIc, Cambridge MA, USA) and an ALP-conjugated secondary antibody, anti-mouse gamma chain specific ALP conjugate (Sigma). Arrowheads indicate the 160 KDa phosphorylated ROCK-2 protein band, and the 51KDa tubulin protein band as an internal control, used to ensure that equal quantities of protein were loaded in all wells.
  • ABCAM ab 24843 ABCAM PIc, Cambridge MA, USA
  • ALP-conjugated secondary antibody anti-mouse gamma chain specific ALP conjugate
  • Lane 1 RINA 50 transfected cells
  • Lane 2 RINA 51 transfected cells
  • Lane 3 RINA 52 transfected cells
  • Lane 4 Negative RINA tranfected cells
  • Lane 5 Untreated RPE- 19 cells
  • M represents molecular weight markers.
  • FIG 4B Retinal pigmented cells were transfected with RINA 52 and protein from lysed transfected cells was blotted onto pre-wet nitrocellulose membrane.
  • ROCK-I was detected on the nitrocellulose using an anti-ROCK-1 mouse monoclonal IgGl (Santa Cruz) and ALP-conjugated secondary antibody, anti-mouse gamma chain specific ALP conjugate (Sigma).
  • Arrowheads indicate the 160 KDa ROCK-I protein band, and the 51 KDa tubulin protein band (internal control, used to ensure that equal quantities of protein were loaded in all wells).
  • FIG. 5 Untreated HUVEC cells, negative RINA treated and RINA 30 treated HUVEC cells were allowed to form vessels on extracellular matrix (ECM) coated wells of a 96- well plate. After 8 h of incubation on ECM, light microscopic observations were made and photographs were taken at 1 Ox magnification.
  • Figure 5A shows untreated HUVEC cells initiating pentagonal network of vessels. Arrowhead indicates the branching of vessels.
  • Figure 5B shows negative RINA treated cells forming vessels. Arrowhead indicates vessels formed.
  • Figure 5C shows RINA 30 treated cells. Cells separated from each other, and exhibited no signs of cell migration or initiation of vessel formation.
  • FIG. 6 VEGF after knockdown. Decreased VEGF-A production with the increase in concentration of siRNA462 in RPE19 (Figure 6A) as well as MCF7 cell lines ( Figure 6B) However, S ⁇ RNA462 in both cell lines caused an increase in secreted VEGF-C with concentration. Figure 6C shows MCF7.
  • FIG. 7A-D RPE 19 cells were knocked down with siRNA462, RINA 30 or both under hyperglycemic condition or normal conditions and examined for their affect on VEGF-A and VEGF-C secretion.
  • Figure 7A Effect of siRNAs on VEGF-A secretion under hyperglycemia or normoglycemia.
  • Figure 7B Effect of siRNAs on VEGF-C secretion under hyperglycemia or normoglycemia.
  • Figure 7C Effect of siRNAs on VEGF-A secretion under hypoxia or normaxia.
  • Figure 7D Effect of siRNAs on VEGF-C secretion under hypoxia or normaxia.
  • FIG. 8 RPE 19 and MCF7 cells were transfected with RINA 30 at various concentrations (ranging from 0.01 -100 nM with one log increment each) and analyzed cell culture supernatants at the end of 72 h post transfection. Analysis of cell culture supernatants for VEGF-C ELISA have shown decrease in secretary levels of VEGF-C with the increase in concentration of RINA30 in RPE19 ( Figure 8A) as well as MCF7
  • Figure 8B cells. Similarly the secretary levels of VEGF-A were also found to be decreased with the increase in concentration of RINA30 in both cell lines.
  • Figure 8C shows RPE 19; and Figure 8D shows MCF7.
  • Figure 9 HUVEC were transfected with RINA52 and analyzed for their ability to reduce protein levels and its effect on angiogenesis.
  • Figure 9A shows protein blot analysis of HUVEC cells for RhoA protein knockdown after transfection with RINA52
  • Figure 9B-C shows proliferation after knockdown by RINA52, with or without supplementation with external VEGF-A or VEGF-C.
  • UT untreated with siRNA.
  • Figure 9B shows HUVEC cells
  • Figure 9C shows HMVEC cells.
  • Figure 9D shows the % inhibition of angiogenesis by HUVEC cells associated with treatment of cells with siRNA (RINA52), and that this affect cannot be reversed by external supplementation with VEGF-A and/or VEGF-C.
  • Figure 10 HUVEC cells transfected.
  • Figure 1OA shows cells knocked down for RhoA where the arrowhead indicates absence of F-actin filaments.
  • Figure 1OB shows cells transfected with negative RINA where arrowhead indicates intact F-actin filaments.
  • Figure 1OC shows untransfected cells where F-actin filaments were prominent.
  • FIG. 1 Fold change in secreted VEGF-A (Figure HA) and VEGF-C ( Figure HB) at different glucose concentrations in cell culture media.
  • FIG. 12 HUVEC cell proliferation in response to addition of VEGF-A or VEGF-C to culture supernatant.
  • VEGF-C A
  • VEGF-A ⁇
  • FIG. 13 Knockdown of VEGF-C or VEGF-A expression following transfection with different siRNA targeting VEGF-C.
  • short nucleic acid molecule refers to any nucleic acid molecule capable of modulating gene expression.
  • short interfering nucleic acid refers to any nucleic acid molecule capable of modulating gene expression.
  • short interfering nucleic acid refers to any nucleic acid molecule capable of inhibiting, down- regulating or knocking down gene expression.
  • RNA as used herein means a molecule comprising at least one ribonucleotide residue and includes double stranded RNA, single stranded RNA, isolated RNA, partially purified, pure or synthetic RNA, recombinantly produced RNA, as well as altered RNA such as analogs or analogs of naturally occurring RNA.
  • RINA refers to siRNA duplexes, having sense and antisense strands.
  • RINA 6, RINA 17, RINA 30, RINA 50, RINA 51 and RINA 52 refers to specific siRNA duplexes having certain nucleotide sequences, as presented in Table 2 below.
  • negative RINA is a commercially available negative control comprises of a scrambled sequence, obtained from Ambion Inc.
  • modulate means that gene expression or level of RNA molecule or equivalent RNA molecules encoding one or more protein or protein subunits or peptides, or activity of one or more protein subunits or peptides, is up-regulated or down-regulated such that the expression, level, or activity is greater than or less than that observed in the absence of the modulator.
  • modulate includes “inhibit.”
  • inhibitors or “inhibit”
  • down-regulation or “down-regulate”
  • knockdown of a gene means that expression of a gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of an inhibitory nucleic acid molecule (e.g., siNA) of the present invention.
  • inhibition or down-regulation observed in the presence of one or more siNA molecules is greater than inhibition or down-regulation observed in the absence of the siNA(s) or in the presence of, for example, a siNA molecule with a scrambled sequence or with mismatches.
  • expression of a gene or protein is "knocked down" in the presence of one or more siNA molecules, as compared to gene or protein expression observed in absence of the siNA molecule(s), or in the presence of, for example, a siNA molecule with a scrambled sequence or with mismatches.
  • RNA refers to a nucleic acid that encodes an RNA, for example, nucleic acid sequences including, but not limited to, a structural gene encoding a polypeptide.
  • RhoA refers to any RhoA protein, peptide, or polypeptide or a derivative thereof, such as encoded by Genbank Accession number NM_001664, having RhoA activity.
  • RhoA also refers to nucleic acid sequences encoding any RhoA protein, peptide, polypeptide, or polypeptide having isoforms, mutant genes, splice variants or polymorphisms, having RhoA activity.
  • ROCK refers to any Rho kinase (e.g., ROCK-I, ROCK-2) protein, peptide, or polypeptide or a derivative thereof, such as encoded by Genbank Accession numbers NM_005406 (ROCK-I) and NM_004850 (ROCK-2) having Rho kinase activity.
  • ROCK also refers to nucleic acid sequences encoding any RhoA kinase protein, peptide, polypeptide, or polypeptide having isoforms, mutant genes, splice variants or polymorphisms, having RhoA kinase activity.
  • VEGF refers to any vascular endothelial growth factor (e.g., VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D) protein, peptide, or polypeptide having vascular endothelial growth factor activity.
  • VEGF also refers to nucleic acid sequences encoding any vascular endothelial growth factor protein, peptide, polypeptide, or polypeptide having isoforms, mutant genes, splice variants or polymorphisms, having vascular endothelial growth factor activity.
  • VEGF-C refers to any protein, peptide, or polypeptide or a derivative thereof, such as encoded by Genbank Accession number NM 005429, having vascular endothelial growth factor type C activity.
  • the term VEGF-C also refers to nucleic acid sequences encoding any VEGF-C protein, peptide, polypeptide, or polypeptide having isoforms, mutant genes, splice variants or polymorphisms, having VEGF-C activity.
  • VEGF-A refers to any protein, peptide, or polypeptide or a derivative thereof, such as encoded by Genbank Accession number NM OO 1025366, having vascular endothelial growth factor type A activity.
  • the term VEGF-A also refers to nucleic acid sequences encoding any VEGF-A protein, peptide, polypeptide, or polypeptide having isoforms, mutant genes, splice variants or polymorphisms, having VEGF-A activity.
  • the target nucleic acid can be DNA or RNA.
  • siNA region refers to a nucleotide sequence of a siNA molecule that is complementary to an antisense region of the siNA molecule.
  • the sense region of a small nucleic acid molecule can comprise a nucleic acid sequence having homology with a target nucleic acid sequence.
  • antisense region means a nucleotide sequence of a siNA molecule that is complementary to a target nucleic acid sequence. It can also comprise a nucleic acid sequence that is complementary to a sense region of a siNA molecule.
  • nucleic acid sequence can form hydrogen bond(s) with another nucleic acid sequence.
  • a nucleic acid molecule comprising two or more nucleic acids may be partially or completely (100%) complementary to another nucleic acid molecule, for example, with regard to corresponding nucleic acids that are capable of forming a double stranded molecule.
  • a percent “complementarity” or “complementary” indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds with a second nucleic acid sequence.
  • a first sequence is 95% complementary to a second sequence if 19 out of 20 contiguous nucleotides in the first sequence form hydrogen bonds with 19 out of 20 contiguous nucleotides in the second sequence.
  • “Completely complementary” means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • a 23-mer nucleic acid may be completely (100%) complementary to a 27-mer nucleic acid with regard to 23 contiguous nucleic acids.
  • ocular disorder refers to any disease or disorder that affects the eye, such as anterior and posterior ocular diseases, including retinopathy, age related macular degeneration (AMD), diabetic retinopathy, glaucoma, inflammatory conditions of the eye, such as uveitis, rubeosis, conjunctivits, keratitis, blepharitis, sty, chalazion, ulceris, stromal keratitis, and cancers
  • anterior and posterior ocular diseases including retinopathy, age related macular degeneration (AMD), diabetic retinopathy, glaucoma, inflammatory conditions of the eye, such as uveitis, rubeosis, conjunctivits, keratitis, blepharitis, sty, chalazion, ulceris, stromal keratitis, and cancers
  • neovascular disease refers to any disease or disorder involving abnormal neovascularization or neoangiogenesis, including cancers and ocular neovascular diseases such as retinal neovascularization and choroidal neovascularization.
  • retinopathy refers to any type of retinopathy such as angiopathic retinopathy, arteriosclerosis retinopathy, central angiospastic retinopathy, central serous retinopathy, circinate retinopathy, diabetic retinopathy, dysproteinemic retinopathy, hypertensive retinopathy, leukemic retinopathy, lipemic retinopathy, proliferative retinopathy, renal retinopathy, sickle cell disease , retinopathy, toxemic retinopathy of pregnancy and the like.
  • angiopathic retinopathy such as arteriosclerosis retinopathy, central angiospastic retinopathy, central serous retinopathy, circinate retinopathy, diabetic retinopathy, dysproteinemic retinopathy, hypertensive retinopathy, leukemic retinopathy, lipemic retinopathy, proliferative retinopathy, renal retinopathy, sickle cell disease ,
  • glaucoma includes primary open angle glaucoma, normal pressure glaucoma, hypersecretion glaucoma, ocular hypertension, acute angle closure glaucoma, chronic angle closer glaucoma, plateau iris syndrome, combined-mechanism glaucoma, steroid glaucoma, capsular glaucoma, pigmentary glaucoma, secondary glaucoma associated with amyloidosis, neovascular glaucoma, malignant glaucoma and the like.
  • Methods and compositions of the present invention for prophylaxis and treatment of glaucoma may have intraocular pressure lowering action, optic disc blood flow improving action and/or aqueous humor outflow promoting action.
  • angiogenesis refers to the generation of new blood supply, e.g., blood capillaries, vessels, and veins, e.g., from existing blood vessel tissue (e.g., vasculature).
  • the process of angiogenesis can involve a number of tissue cell types including, for example, endothelial cells that form a single cell layer.
  • neoangiogenesis refers to the proliferation of new blood capillaries, vessels, and veins in tissue. It also refers to the abnormal or excessive formation of new blood capillaries, vessels, and veins in tissue.
  • Neoangiogenesis differs from angiogenesis in that angiogenesis usually involves the protrusion and outgrowth of capillary buds and sprouts from pre-existing blood vessels.
  • cancer or “proliferative disease” as used herein means any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as is known in the art. It can include all types of cancer, tumors, lymphomas, carcinomas that can respond to the modulation of disease-related VEGF-C and/or RhoA gene expression in a cell or tissue alone or in combination with other therapies. In one embodiment, cancers associated with the eye are contemplated.
  • Rho kinases such as ROCK-I and ROCK- 2.
  • Other separate work has examined the inhibition of VEGF-C.
  • the present invention discloses the combination/synergistic approach of inhibiting both Rho kinases (via RhoA) and VEGF-C via small nucleic acids.
  • the present invention knocks down VEGF-C gene expression, by which one can reduce VEGF-C protein levels and thus reverse anti-apoptosis mechanisms activated by this factor.
  • the invention establishes for the first time that knockdown (i.e., reduction) of VEGF-C expression decreases levels of VEGF-A, which is primarily required for angiogenesis.
  • the present invention establishes that both VEGF-C and VEGF-A secretions are interdependent and remain in direct proportion to each other quantitatively. Because the knockdown of VEGF-C inhibits the secretion of VEGF-A, such knockdown help arrest angiogenesis.
  • RhoA plays an important role in regulation of VEGF-induced endothelial cell migration by phosphorylating the RhoA effector molecules ROCK-I and ROCK-2.
  • the present invention knocks down RhoA, thereby inhibiting (i.e., reducing) the signaling mechanisms of RhoA, which are responsible for endothelial cell migration and cell adhesion.
  • the present invention is directed to short nucleic acids targeting RhoA and/or VEGF-C genes that can specifically and effectively direct homology-specific post transcript gene silencing, and therefore act as highly effective, selective and potent therapeutics, with minimal side effects.
  • the present invention provides compounds, compositions and methods for the treatment of angiogenesis.
  • the invention uses siNA-mediated inhibition of RhoA and VEGF-C.
  • the present invention provides a combination of nucleic acids targeting RhoA gene and/or VEFG-C gene.
  • the present invention targets RhoA and therefore inhibits the activation of its effector molecules, e.g., ROCK-I and ROCK-2.
  • the present invention knocks down RhoA protein levels and thus limits its ability to phosphorylate ROCK-I and ROCK-2, but does not inhibit protein expression of ROCK-I and ROCK-2.
  • the present invention down regulates the protein levels of RhoA and thus inhibits VEGF-driven endothelial cell migration. In another embodiment, the present invention knocks down RhoA in the trabecular meshwork, and thus inhibits the intraocular pressure by regulating stress fiber formation.
  • the present invention knocks down VEGF-C expression and secretion, which causes a reduction in the secretion of VEGF-A from cells.
  • the present invention provides short interfering nucleic acid (siNA) molecules and their uses in modulation of RhoA and VEGF-C gene expression.
  • siNA short interfering nucleic acid
  • design of suitable siNA involved the design of the siRNA with 21, 23, and 27 nucleotides for modulation of RhoA and VEGF-C, without chemical modification.
  • Target genes such as RhoA and VEGF-C, were screened for accessible sites and siRNA were synthesized considering the open reading frame (ORF) sequences of RhoA and VEGF-C.
  • the first 200 bases were omitted from the start codon to avoid binding to regulatory element, iv.
  • Each siNA duplex was checked in silico to avoid silencing of off-target effects made on BLAST search considering the following parameters:
  • Expected value threshold was set at 1000 to avoid the probability of short sequence occurrence.
  • siNA was done by commercially available methods (e.g., Qiagen) using chemically-protected phosphoramidite monomers. Resultant oligomers were purified by PAGE, desalting, or IE-HPLC. The quality of each siNA was analyzed by MALDI-TOF and yields were determined by an integrated spectrophotometer.
  • siRNAs to reduce the secretary levels of VEGF-C and VEGF-A were determined by sandwich ELISA in various cell lines including apical retinal pigmented epithelial cells.
  • IC50 values for RINA 30 transfected cells may be determined by estimating quantities of VEGF-C and VEGF-A using sandwich ELISA in breast cancer cell lines VEGF-C amounts are calculated on the basis of standard graph for VEGF-C (graph not provided). Percentage VEGF-C knockdown was calculated as the proportion of VEGF-C secreted in siRNA treated sample against control.
  • the cytotoxicity of siRNAs on cells may be analyzed by measuring amount of LDH released into the medium due to cell necrosis.
  • the effect of siRNAs on various cytokines being released from the cells may be estimated using a cytokine array kit.
  • the angiogenic or vessel formation ability of endothelial cells were analyzed upon knocking down of VEGF-C and RhoA with the corresponding siRNA under in-vitro conditions either alone or in combination.
  • the ability of VEGF-A alone to induce vessel formation, after VEGF-C and RhoA knockdown was analyzed using an in vitro angiogenesis assay using Human Umbilical Vein Endothelial Cells (HUVEC) cells (ATCC).
  • HUVEC Human Umbilical Vein Endothelial Cells
  • VEGF-C The ability of VEGF-C alone to induce angiogenesis in absence of VEGF-A and RhoA was also analyzed under in vitro conditions employing HUVEC cells. For example, RhoA expression was knocked down using siNAs and VEGF-A was not added. In addition, RhoA expression was knocked down using siNAs and VEGF-A is neutralized using VEGF-A receptors.
  • RhoA expression was knocked down using siNAs and VEGF-A is neutralized using VEGF-A receptors
  • the ability of VEGF-C and VEGF-A together to induce angiogenesis in the absence of RhoA can be analyzed under in vitro conditions.
  • RhoA knockdown using siNA was determined by observing the formation of stress fibers using FITC labeled phalloidon.
  • siRNA treated cells may also be examined for the expression of CD31 antigen, a marker for neoangiogenesis by ELISA, western blot, as well as immunoflourescence assays.
  • the siRNA treated cells may also be analyzed for their ability to prevent wound healing using dermal endothelial cells in wound healing assays.
  • Cells transfected with the siRNA may also be analyzed for reduction in levels of specific mRNAs using real time quantitative PCR analysis or Northern blot analysis.
  • real-time PCR the preparation of the first strand cDNA is carried out using kits from Qiagen.
  • the first strand cDNA from siRNA, negative RINA ⁇ i.e., scrambled siRNA, such as purchased from Ambion Inc.) and untreated samples is used as templates and mRNA quantity is detected by normalizing against internal control.
  • the fold change in mRNA levels is determined by the protocol of Kenneth and Thomas ("Analysis of relative gene expression data using real-time quantitative PCR and 2 " ⁇ ° method," Methods 2001; 25: 402-408). See Table 6 below.
  • the siRNA treated cells are analyzed for the gene expression levels of CD31 antigen by quantitative Real time PCR. Protein
  • VEGF-C vascular endothelial growth factor-C
  • VEGF-A vascular endothelial growth factor-A
  • RhoA vascular endothelial growth factor-A
  • siRNA transfections exhibited reduced mRNA levels and reflect gene expression levels, the determination of protein levels established that the RNA acted as an siRNA rather than an miRNA.
  • miRNA are short RNA molecules (e.g., 23 mer in length), where contiguous sequence do not exactly match the mRNA sequence of a target protein. miRNA bind to mRNA and prevent translation but do not cause mRNA degradation.
  • siRNA the siRNA complementary binding to a target causes degradation of mRNA and thus causes translation repression.
  • siRNA levels also go down.
  • the effect of thrombin on induction of RhoA levels and efficiency of siRNA in its inhibition may be determined by GST-RBD pull down assays.
  • the present siNA may be used with or without additional factors.
  • the present invention also provides cholesterol conjugated siNA of VEGF-C and RhoA complexed with PEI.
  • the siNA molecules can also be chemically modified by introduction of a 2'-O-Methoxy modification and thus made nuclease resistant. The serum stability of these molecules may also be studied.
  • mice, rat or rabbit may be subjected to laser degeneration of Brunches membrane and therefore angiogenesis is induced.
  • siRNA is applied either by intravitrial, periocular or intravenous routes, and the efficacy of these is tested for inhibition of neoangiogenesis.
  • Nuclease resistant or unmodified siNA is administered by intravitrial, periocular, intravenous, or retroorbital routes to derive the benefits of anti-angiogenic properties in treating various ocular disorders.
  • the siRNA is complexed or encapsulated in liposomes, and is applied to treat neoangiogenic, glaucoma and other ocular disorders which include age related macular degeneration, diabetic retinopathy and glaucoma apart from other neoangiogenic diseases.
  • neoangiogenic, glaucoma and other ocular disorders which include age related macular degeneration, diabetic retinopathy and glaucoma apart from other neoangiogenic diseases.
  • EXAMPLE 1 Design of 21, 22, 23, and 27 mer siIRNAs for modulation of RhoA and VEGF-C gene expression
  • AU siR ⁇ A has GC content between 30-50%.
  • each siR ⁇ A has a over hang of dTdT.
  • AU siR ⁇ As start at 5'- either with G/C.
  • GC content of duplex is between 40 -55%.
  • Sense strand is 25 nucleotides, where as antisense strand is always 27 nucleotides resulting in overhang at 3'- of antisense strand.
  • Last 2 nucleotides of 3'-sense strand contains deoxy sugar instead of ribosugar backbone.
  • VEGF-C and RhoA genes were screened for accessible sites that could meet the above-mentioned criteria using various online available algorithms, as well as manually. Based on these and other criteria, the following target sequences were chosed to create siRNA to VEGF-C and RhoA.
  • Table 1 Target ORF sequences of VEGF-C and RhoA for siRNA synthesis
  • siNA molecules were synthesized by chemical means, employing commercially available machinery. Chemical methods were classified based on the type of protecting group incorporated at 2'-carbon position of the ribose sugar:
  • the cycle began with the 3'-most nucleoside attached to solid support material or bead.
  • the second nucleotide was coupled to the 5-hydroxyl of the first nucleoside. Capping prevented effectively the propagation of failed or short nucleosides.
  • the internucleotidic phosphate bond was then oxidized to the final P(V) state.
  • the 5'-protecting group on the new nucleotide was removed and the growing oligonucleotide was ready for addition of the next nucleotide. Once the nucleic acid molecule reached the desired length, it was further deprotected, cleaved from the solid support, and analyzed for purity and yield.
  • siRNAs were purified by desalting, followed by PAGE (polyacrylamide gel electrophoresis), or by IE-HPLC (Ion Exchange - High Performance Liquid Chromatography).
  • the quality of each RNA strand was analyzed by MALDI-TOF and the yield was determined by integrated spectrophotometer absorbance at 30nm. During quality control by MALDI-TOF, a difference of 4 atomic mass units is the maximum allowed difference from that predicted. After obtaining comparable yields for each strand as determined by absorbance at 30 nm, sense and antisense strands were annealed, and vacuum lyophilized.
  • the lyophilized powders were suspended in RNA suspension buffer consisting of 100 mM KCl, 30 mM HEPES buffer (pH 7.5), and 1 mM MgCl 2 , and heated for 1 min at 90 0 C and the incubated at 37°C for 1 h to dissolve the lyophilized powder.
  • RNA suspension buffer consisting of 100 mM KCl, 30 mM HEPES buffer (pH 7.5), and 1 mM MgCl 2
  • Table 2 siRNA synthesized with end modifications for VEGF-C and RhoA genes.
  • EXAMPLE 3 VEGF-C and RhoA expression analysis by reverse transcriptase PCR
  • HUVEC Human Umblical Vascular Endothelial Cells, ATCC
  • PC3 Prostate cancer cells, ATCC
  • HUVEC Human Umblical Vascular Endothelial Cells, ATCC
  • PC3 Prostate cancer cells, ATCC
  • Cells at 60-70% confluence were subjected to total RNA isolation followed by first strand cDNA preparation using the Qiagen Fast lane cell cDNA kit with minor modifications. Briefly, 20,000 cells were pelleted and washed once with buffer FCW (Qiagen). Cells were lysed for 15 min. at room temperature using buffer FCP (Qiagen). Genomic DNA contamination was eliminated by the addition of gDNA wipeout buffer (Qiagen) by incubating at 42.5°C for 30 min.
  • First strand cDNA was synthesized by the addition of Quantiscript reverse transcriptase at 42.5°C for 45 min. followed by incubation at 95°C for 3 min. The first strand cDNA prepared was either used immediately for reverse transcriptase PCR or stored until further use at -20 0 C. First strand cDNA were amplified by PCR using the following primer sequences:
  • VEGF-C Forward Primer 5'-AAAGAACCTGCCCCAGAAAT-S' (SEQ ID NO: 19)
  • VEGF-C Reverse Primer 5'-TGGTGGTGGAACTTCTTTCC-S' (SEQ ID NO:20)
  • VEGF-C Probe 5'- ⁇ -FAM-AATCCTGGAAAATGTGCCTG-S' (SEQ ID NO:21)
  • RhoA Forward Primer 5'-TATCGAGGTGGATGGAAAGC-S' (SEQ ID NO:22)
  • RhoA Reverse Primer 5'-TTCTGGGGTCCACTTTTCTG-S' (SEQ ID NO:23)
  • RhoA Probe 5'- ⁇ -FAM-CCATCGACAGCCCTGATAGT-S' (SEQ ID NO:24)
  • HUVEC cells human umbilical vascular endothelial cells
  • HeLa cervical cancer
  • PC-3 prostate cancer
  • HTB-38 colonal carcinoma
  • ARPE- 19 normal diploid retinal pigmented epithelial cells
  • siRNA and transfection mixes were performed as master mixes from which appropriate volumes were added to the wells seeded with cells. At the end of incubation, siRNA-liposome complexes were mixed thoroughly and gently added dropwise to each ' well. The wells of a 24-weIl plate were mixed to uniformity and the plates incubated for appropriate incubation times in 37°C CO 2 incubators for further analysis of cells. Transfection efficiencies are obtained for each cell line by counting number of cells showing Cy3-labeled siRNA (using negative negative RINA from Ambion) after 16 h of transfection. After 16 h of transfection, cells were trypsinised and washed once in PBS and suspended in the same.
  • EXAMPLE 5 VEGF-C and RhoA siRNAs inhibit VEGF-A secretion
  • PC3 prostate cancer
  • ARPE- 19 retina pigmented epithelial cells
  • HeLa cervical cancer
  • HCC-38 breast cancer
  • 10 nM siRNA i.e., one of six siRNAs (RINA 6, 17, 30 against VEGF-C, and RINA 50, 51, 52 against RhoA)
  • HiPerFect Transfection Reagent following protocol of manufacturer (Qiagen).
  • supernatants were analyzed by Sandwich ELISA as per the instructions of VEGF-A ELISA kit (Calbiochem).
  • a standard curve was obtained for concentrations (15.6 pg/mL to 1000 pg/mL) of VEGF-A, as shown in Figure 2.
  • VEGF-A The quantity of secreted VEGF-A in supernatants was estimated from the standard curve for VEGF-A ( Figure 2). All tested VEGF-C siRNAs (RINA 6, 17 and RINA 30) caused inhibition of VEGF-A secretion in two or more cell lines (PC3, ARPE- 19 and/or HeLa) upon transfection. See Table 4 below. Of the three VEGF-C siRNAs tested, RINA 30 transfection of HeLa cells showed the greatest effect. RINA 30 inhibited secretion of VEGF-A by 68% in HeLa, as compared to mock (negative RINA) treated cells.
  • RhoA siRNAs caused inhibition of VEGF-A secretion in at least one cell line (ARPE-19, HeLa and/or HCC-38) upon transfection.
  • RINA 50 showed the greatest effect.
  • RINA 50 inhibited secretion of VEGF-A by 40% in HeLa cells, as compared to mock treated cells.
  • RINA 52 inhibited secretion of VEGF-A by 27% in ARPE-19 cells. Table 5. Knockdown of RhoA inhibits VEGF-A secretion
  • results presented herein indicate that siRNAs directed to either VEGF-C or RhoA unexpectedly inhibit expression and secretion of VEGF-A.
  • the results likewise indicate for the first time that inhibition of either VEGF-C or RhoA expression leads to an inhibition of VEGF-A expression, secretion and VEGF-A-specific cell signaling.
  • Results will indicate that use of a VEGF-C siRNA and a Rho siRNA together inhibit VEGF-A expression and secretion in a synergistic manner, as compared to additive effects of using VEGF-C siRNA or RhoA siRNA alone, and more than direct inhibition of VEGF-A
  • VEGF-C and RhoA genes were determined by Real time PCR.
  • the cell lines used in this study included PC3 (prostate cancer) HeLa (cervical cancer), ARPE-19 (retinal pigmented epithelial cells) and HTB-38 (colorectal cancer) obtained from ATCC. Cells were transfected either with 10 nM of RINA 52 or RINA 30 and negative negative RINA. At the end of 72 h of post transfection, the first strand cDNA preparation was carried-out using Qiagen Fast lane cell cDNA kit with minor modifications. Briefly 20,000 cells were pelleted and washed once with buffer FCW (Qiagen). Cells were lysed for 15 min at room temperature using buffer FCP (Qiagen).
  • Genomic DNA contamination was eliminated by the addition of gDNA wipeout buffer (Qiagen) by incubating at 42.5 0 C for 30min.
  • First strand cDNA was synthesized by the addition of Quantiscript reverse transcriptase at 42.5 0 C for 45 min followed by incubation at 95 0 C for 3 min.
  • the first strand cDNA prepared was either used immediately for quantitative Real time PCR or stored till further use at -20 0 C. Real time quantitative PCR can be accomplished following standard protocols and using commercially available machines.
  • First strand cDNA from antisense, negative RINA and untreated samples were used as template, and the levels of mRNA was quantified by normalizing against the internal control ⁇ -actin.
  • the expression of RhoA and VEGF-C was determined as a percent decrease in expression level over untreated cells as indicated in Table 6.
  • Quantitative real time PCR analysis of RINA 52 transfected cells shows a 96% decrease in mRNA levels of RhoA gene in HeLa and HTB-38 cells, as compared to mock transfected cells.
  • RINA 30 transfection resulted in a decrease in mRNA levels of gene VEGF-C by 87% and 85% respectively in the case of PC3 and ARPE- 19 cells.
  • VEGF-C RINA 30 0.01, 0.1, 1.0, lO.O, and 100.0 nM
  • negative RINA 30 0.01, 0.1, 1.0, lO.O, and 100.0 nM
  • cell culture supernatants were clarified from respective wells and subjected to Sandwich ELISA to determine the level inhibitory levels for VEGF-C cell expression, as well as VEGF-A expression.
  • VEGF ELISA kit Human,Cat.no QIA51, CALBIOCHEM
  • Quantikine VEGF-C Human, kit R&D systems, Cat.no. DVECOO
  • VEGF-C expression (as compared negative RINA transfected cells), RINA 30 reached a plateau at 70% knockdown of VEGF-C expression at 10 nM concentration, and exhibited 50% knockdown at 0.8 nM, as shown in Figure 3A.
  • VEGF-A reached a plateau of 48% knockdown of VEGF-A at 10 nM concentration of RINA 30, while its IC50 value remained at 0.8 nM, as shown in the Figure 3B.
  • the IC50 values and Real time PCR data show that RINA 30 is highly potent and works to inhibit VEGF-C and VEGF-A expressions at 0.8 nM.
  • the results presented herein show that expression of VEGF-C regulates expression levels of VEGF-A, indicating that there exists a homeostasis and that both VEGF-C and VEGF-A are required for neoangiogenesis.
  • Cells (HeIa, PC3, MCF-7, HTb-38, ARPE-19 and HUVEC) are transfected with RINA 6, 17 and 30.
  • protein lysates are obtained employing Mammalian protein extraction reagent, MPER (Pierce) and are subjected to Western blot analysis.
  • MPER Mammalian protein extraction reagent
  • the protein knockdown levels are detected from the analysis of protein blots.
  • VEGF-C expression levels are inhibited in the cells upon transfection with RINA 6, 17 and/or 30, as compared to negative RINA transfected cells.
  • Cells (HeIa, PC3, MCF-7, HTb-38, ARPE-19 and HUVEC) are transfected with RINA 50, 51 and 52.
  • RINA 50, 51 and 52 At the end of 72 h of transfection, protein lysates are obtained employing Mammalian protein extraction reagent, MPER (Pierce) and are subjected to Western blot analysis. The protein knockdown levels are detected from the analysis of protein blots. RhoA expression levels are inhibited in the cells upon transfection with RINA 50, 51 and/or 52, as compared to negative RINA transfected cells.
  • Rho Kinase 1 (ROCK-I) and Rho kinase 2 (ROCK-2) are Ser/Thr kinases that are activated by RhoA and act as effector molecules of RhoA, resulting in cytoskeletal reorganization. This reorganization results in endothelial cell morphogenesis leading to formation of new blood vessels.
  • RhoA Knockdown of RhoA and its effect on activation of Rho kinases was determined.
  • Cells (ARPE-19) were transfected with RINA 50, 51 or 52 and analyzed for protein expression or phosphorylation status of ROCK-I or ROCK-2 at the end of 72 h of transfection ( Figure 4A and 4B).
  • Protein lysates were made using mammalian protein extraction reagent (MPER, Calbiochem) following manufactures protocol. Based on Bradford total protein estimations, equal quantities of proteins were resolved over 10% SDS PAGE. Proteins resolved over the SDS-PAGE were subjected to Western blot transfer at 1 10 V for 70 min. on to a pre-wet nitrocellulose membrane along with pre-stained rainbow molecular weight markers (Amersham Biosciences).
  • the transfer of proteins by electro- blotting was confirmed by Ponceau S staining (Sigma).
  • the blot was incubated in blocking solution (5% skim milk powder) for 1 h at room temperature on a rocking platform.
  • blocking solution 5% skim milk powder
  • the blot was washed over an orbital shaker for 5 min each with change of PBST (phosphate buffered saline containing 0.1% Tween 20).
  • PBST phosphate buffered saline containing 0.1% Tween 20
  • the blot was incubated with primary antibody overnight at 4°C.
  • blots were incubated with secondary antibodies conjugated with alkaline phosphatase for two hours at room temperature over an orbital shaker.
  • Secondary antibodies included rabbit anti-mouse antibody conjugated with alkaline phosphatase (SIGMA) to detect tubulin and Rock-1, and goat anti-rabbit antibody (SIGMA) to detect ROCK-2 phospho-antibody.
  • SIGMA rabbit anti-mouse antibody conjugated with alkaline phosphatase
  • SIGMA goat anti-rabbit antibody
  • primary antibodies were Rabbit anti phospho Rho kinase alpha(Rock-2) antibody (Abeam, Cat. No. ab24843.2.), Mouse Anti alpha Tubulin Antibody (SIGMA Cat. No. T6199).
  • Secondary antibodies were rabbit anti-mouse antibody(gamma chain specific) conjugated with alkaline phosphatase (SIGMA Cat. No. A3438); Goat anti Rabbit antibody (Whole molecule) conjugated with alkaline phosphatase (SIGMA Cat no A3687).
  • the primary antibodies are Anti ROCK-I mouse monoclonal IgGl (Santa Cruz Cat. No. SC 17794) and Mouse Anti alpha Tubulin Antibody, (SIGMA Cat. No. T6199); while the secondary antibodies was rabbit anti-mouse antibody (gamma chain specific) conjugated with alkaline phosphatase (SIGMA Cat. No. A3438). Blots were washed three times with PBST for 10 min each before being developed with BCIP/NAT substrate solution (SIGMA). Protein bands corresponding to phosphorylated ROCK-2 (160 KDa) and tubulin (51 Kda) as endogenous control were detected, as shown in the Figure 4A.
  • siRNAs directed to RhoA inhibit ROCK phosphorylation (as demonstrated for ROCK-2 here) in relevant cells, such as retinal pigmented epithelial cells.
  • siRNAs of the present invention inhibit the ROCK signaling pathway, without affecting expression of ROCK itself as seen in regard ROCK 1 in Figure 4B. This is in contrast to results expected when using a siRNA directed to ROCK itself, which presumably affects gene expression of ROCK.
  • Retinal pigmented cells were transfected with 10 nM VEGF-C siRNA (RINA 6 and 30) and RhoA siRNA (RINA 50, 51 and 52). At the end of 20 h of transfection, cells were lysed and the otal RNA was prepared.
  • Interferon response pathway specific gene expression was determined for the following genes following the manufacturers instructions from the Interferon Response Detection Kit for validation of siRNA experiments (SBI): Interferon response genes OAS1(NM_016816) and OAS2 (NM 016817.1) represents 2',5'-otigoadenylate synthetase (OAS); MXl (NM 002462.2) represents Myxovirus (Influenza virus) resistance protein family; and IFITMl (NM 003641.2) represents interferon inducible trans-membrane proteins.
  • RINA 30 and 52 caused only a low level enhancement of expression with regard to genes MXl or ISGF3 ⁇ , respectively, and caused little to no measurable enhancement of expression of other genes involved in eliciting an interferon response. See Table 7. Thus, results presented herein demonstrate that cellular responses observed upon transfection with siRNAs of the present invention, such as VEGF-C RINA 30 and RhoA RINA 52, are not due to an activation of an interferon response, but rather are due to inhibition of expression of the specific gene(s) of interest.
  • HUVEC Human umbilical vein endothelial cells obtained from ATCC were cultured in endothelial cell culture medium following directions from ATCC.
  • HUVEC cells were transfected individually with 5 nM of RINA 6, 30, 50, 52 and negative RINA.
  • ECM extracellular matrix coated wells of a 96-well plate in triplicate, each at a concentration of 5000 cells per well.
  • Cells seeded onto ECM coated 96-well plates were cultured and observations were made under light microscope for the following parameters to be quantified at the end of 8 h of incubation on the ECM:
  • FIG. 5 shows angiogenesis in untreated HUVEC cells (Figure 5A) or cells treated with negative RINA ( Figure 5B), with arrows showing vessel branching.
  • Figure 5C by contrast, HUVEC cells treated with RINA30 show cells separated from each other, and exhibited no signs of cell migration or initiation of vessel formation. Results herein show that transfection of HUVEC cells with VEGF-C or RhoA siRNA caused an inhibition of the formation of vessels, as compared to that seen in mock or untreated cells.
  • RJNA 30 and 52 exhibited maximum inhibition of the vessels formation. In addition, no vessel formation was noted even after 16 h after plating on ECM in cells transfected with both VEGF-C RINA 30 and RhoA RINA 52. These results indicate that knockdown of either RhoA or VEGF-C inhibits vessel formation and that this inhibition is durable, as shown in Table 8.
  • EXAMPLE 9 Effect of knockdown of VEGF-C and RhoA on cytokine profile in ARPE-19 cells
  • Retinal pigmented epithelial cells are transfected with RINA 30, 52 or their combination. At the end of 72 h of transfection, cell supernatants are obtained from the RINA treated, negative RINA treated and untreated cells. The supernatants are analyzed for 28 different cytokines following the protocol of Human Cytokine Array Panel A Array kit from R & D systems. The implications of change in expression profiles of various cytokines in relation to angiogenesis is obtained. Most of the pro-angiogeneic cytokines are inhibited to various degrees, while anti-angiogenic cytokines are over-expressed. EXAMPLE 10: Transcriptome analysis of VEGF-C and RhoA knockdown in retinal pigmented epithelial cells
  • retinal pigmented epithelial cells are transfected with RINA 30, 52 or their combination as described earlier.
  • total RNA is prepared following the protocol of Qiagen total RNA isolation kit (RNeasy Mini kit).
  • Total RNA of 2 ⁇ g is suspended in lO ⁇ L of water.
  • the quality of RNA is checked on denaturing formaldehyde gels and the OD ratio is determined using a Perkin Elmer Spectrophotometer.
  • One ⁇ g of total RNA is converted into DIG labeled cRNA following the protocol of Nano In-vitro Transcription amplification kit from Applied Biosystems.
  • the human cell lines RPE 19 and MCF7 (American Type Culture Collection, Manassas,
  • HUVEC and HMVEC Longza, Walkersville, MD
  • VEGF-C Human VEGF-C
  • human RhoA Human RhoA
  • NM OO 1664 Three double-stranded siRNAs for each targeting genes VEGF-C and RhoA were custom synthesized through Qiagen (Hilden,
  • Pre-validated siRNA 462 targeting VEGF-A was obtained from Ambion cat no (siRNA i.d s462) (Austin, TX, USA).
  • Hiperfect transfection reagent Qiagen as per the manufacturer's instructions.
  • the transfection efficiency in cell lines was determined using Cy3 labeled control - siRNA (negative RINA was labeled with Cy3 using Silencer siRNA labeling kit-Cy3 from Ambion following manufacturer's instructions).
  • VEGF-A and VEGF-C ELISA was performed according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Data was obtained from triplicate wells and statistical significance was determined at P ⁇ 0.05 by student's t test.
  • Protein lysates were prepared from siRNA-transfected cells (after 72 h) with Mammalian cells
  • Protein Extraction Reagent (Pierce, Rockford, IL, USA), and the protein content of the lysate was estimated using the Bradford reagent (Biorad, Hercules, CA, USA). Equal amounts of protein were loaded onto SDS-PAGE and western blot analysis was carried out using monoclonal antibody to RhoA (sc-418, Santa Cruz Biotech., Santa Cruz, CA, USA), as previous described (Suarez et al. "The role of VEGF receptors in angiogenesis; complex partnerships," Cell MoI Life Sci 63: 601-6125 (2006)). Monoclonal antibody against ⁇ -tubulin (clone DMlA, T6199, Sigma) was used to detect ⁇ -tubulin, as an internal control.
  • Goat anti-mouse IgG ( ⁇ -chain specific)-Alkaline phosphatase-(A3438, Sigma, St. Louis, MO, USA) was the secondary antibody. All protein blots were repeated three times from independent transfections and their standard deviations (S. D) were determined from densitometric analysis. Quantitative Real Time PCR
  • First strand cDNA was prepared from siRNA transfected RPE 19 and MCF7 cells using Fast cell cDNA kit (Qiagen) as per the manufacturer's instructions. Quantitative Real Time PCR was performed using the Applied Biosystems 7500 system (ABI, Foster city, CA, USA) and data analyzed as per manufacturer's instructions. mRNA abundance was measured by real-time quantitative PCR at 72h post-transfection for screening siRNAs designed. The efficacy of siRNA was determined at 72 h post transfection. ⁇ -actin was used as the internal reference control. All the experiments were repeated twice from ' independent transfections, each in triplicate. Dosage studies
  • the oxygen content was set to 10% with the loss of partial oxygen pressure replaced with nitrogen and CO 2 .
  • Cell lines were maintained under hypoxic conditions for 24 h prior to transfection, and 72 h post-transfection. Hyperglycemic experiments
  • HUVEC cells were transfected with RINA52 or negative RINA at a concentration of 10OnM. After 24h, the cells were trypsinised and seeded at a cell density of 3000 cells/well in a 96-well plate. This was followed by the addition of VEGF-A (Sigma), VEGF-C (Prospec), or their combination, at 5 ng.mL "1 . Cells with and without bovine brain extract was included as controls. After 48h, proliferation was measured using the Cell Titer aqueous one solution cell proliferation assay (Promega Corp., Madison, WI, USA) as per manufacturer's instructions.
  • In vitro tube formation was assessed using the in vitro angiogenesis assay kit of Chemicon (Temecula, CA, USA) as per the manufacturer's instructions. Briefly, 72 h after transfection with RINA52, HUVEC or HMVEC cells were seeded on extracellular matrix and allowed to form a capillary tube. The culture medium was supplemented with growth factors such as VEGF-C or VEGF-A as, and where, applicable. Capillaries were observed at the end of 18 h from the time of seeding. Capillary tube widths, total lengths, and number of branch points was quantitated in three random fields, scores assigned, and the values averaged.
  • HUVEC cells were transfected with RINA52 on coverslips, and after 72h; the cells were fixed with 4% paraformaldehyde at room temperature for 20 min and then incubated with ice-cold 100% acetone at -2O 0 C for an additional 20 min.
  • the fixed cells were incubated with a 1 : 200 dilution of Texas Red -X phalloidin (Molecular probes, Eugene, Oregon, USA) for 1 h at room temperature.
  • the cover slips were mounted using Vectashield with DAPI (Vector Laboratories) and the cells were examined under Nikon Eclipse TE300 fluorescent microscope.
  • Cy-3 labeled Negative RINA at 10 nM was introduced into cells using Hiperfect transfection reagent. Delivery of siRNA by Hiperfect transfection reagent resulted in transfection efficiencies ranging from 87-98 % (Table 9).
  • VEGF-C compensates expression levels of VEGF-A in dosage dependent manner
  • RINA30 and RINA52 showed highest efficacy in knocking down VEGF-C and RhoA, respectively, and did not elicit an interferon response.
  • VEGF-A knockdown cells increased VEGF-C expression over negative RINA treated or untransfected cells in a dose-dependent manner. ( Figures 6C).
  • VEGF-C knockdown decreased VEGF-A expression ( Figures 8C-D).
  • Similar results were observed in both hyperglycemic (for RPEl 9 cells) and hypoxic (for MCF7 cells) conditions respectively ⁇ see Figure 7).
  • Quantitative real time PCR also indicated a decrease in mRNA levels of both VEGF-C and A at the end of 72 h post transfection (Table 5).
  • RhoA The cells knocked down for RhoA showed decreased proliferation over negative RFNA treated (HUVEC as well as in HMVEC cells) at the end of 72 h post transfection ( Figures 9B and 9C), indicating that RhoA plays a crucial role in neo-angiogenesis. Supplementation of media with VEGF-A, VEGF-C, or both together ( Figures 9B and 9C), did not reverse the inhibition of proliferation caused by RhoA knockdown.
  • RhoA knockdown decreases stress fiber formation
  • RhoA inhibits angiogenesis was by regulating cytoskeletal proteins such as F- actin to form stress fibers and thus effects the migration, proliferation and contractility of endothelial cells.
  • VEGF-A causes increases VEGF-C expression in both RPE 19 and MCF7 cells. Similar results were obtained in hypoxic as well as hyperglycemic conditions (Hyperglycemic conditions have induced expression levels of both VEGF-A and C as shown in Figure 1 1), indicating the possibility of similar mechanisms being operated in proliferative retinopathies as well as tumor growth. The increase in expression was observed at the level of both secreted protein and mRNA, indicating that VEGF-A acts at a transcriptional level on VEGF-C.
  • VEGF-C significantly decreased VEGF-A and VEGF-C expression in both RPEl 9 as well as MCF7 cells, and acted at a transcriptional level on VEGF-A.
  • the decrease in expression levels of VEGF-A was observed not only with RINA30 but also with R.INA6 and 17, which target different regions of VEGF-C ( Figure 13).
  • the results obtained in the present study signify that knocking down VEGF-C also regulates neo- angiogenesis being stimulated by VEGF-A by lowering its expression levels.
  • VEGF-C is a superior target than VEGF-A to inhibit not only lymphangiogenesis but also neo- angiogenesis.
  • the inhibition of VEGF-A, or its receptor (VEGFR2) have been shown to have therapeutic advantages in controlling neo-angiogenesis in various indications such as cancer and proliferative retinopathies.
  • a number of anti- VEGF small molecule inhibitors eg: sunitinib
  • anti-VEGF monoclonal antibodies e.g., Avastin
  • Their therapeutic efficacy was found to be modest and transitory followed by restoration of neo-angiogenesis and disease progression, however.
  • the present inventors have shown that the knockdown of VEGF-A leads to enhanced expression of VEGF-C and counteracts the effect of VEGF-A inhibition.
  • the present inventors have also shown that VEGF-C acts through RhoA to mediate angiogenesis, similar to that of VEGF-A.
  • knockdown of VEGF-C along with RhoA has synergistic effects on inhibiting VEGF-A, and inhibiting angiogenesis.
  • the present inventors demonstrate that VEGF-C knockdown results in simultaneous decrease in expression levels of VEGF-A under normaxic, hypoxic, normal and hyperglycemic conditions, and therefore the present invention is applicable under a variety of conditions.
  • VEGF vascular endothelial growth factor
  • VEGF receptor system The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin Sci (Lond). 2005, 109: 227-241. 13. Suarez SC, Fjallman Z, Hofer KB. The role of VEGF receptors in angiogenesis; complex partnerships. Cell MoI Life Sci 2006; 63: 601-6125.
  • Jain RK Antiangiogenic therapy for cancer : Current and emerging tconcepts. Oncology (Williston park) 2005, 19: 7-16.
  • Rho activity critically and selectively regulates endothelial cell organization during angiogenesis. Proc. Natl Acad. Sci 2004; 101: 1874- 1879.
  • VEGF-C induced angiogenesis preferentially occurs at a distance from lypmphangiogenesis. Cardiovascular Res. 2008; 78:315-323.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

La présente invention concerne l’utilisation de molécules d’acide nucléique court interférant (ANsi, tels que des ARNsi) qui modulent l’expression de VEGF-C et/ou RhoA impliqués dans l’angiogenèse néovasculaire. Dans la présente invention, l’inhibition de l’expression génique de VEGF-C et/ou RhoA conduit à une expression diminuée de VEGF-A, qui est requise pour l’initiation et le maintien de l’angiogenèse. De plus, l’invention concerne en outre l’inhibition des taux d’expression de RhoA ainsi que VEGF-C, de manière à produire les bénéfices de la régulation à la baisse de deux différentes cibles requises pour l’angiogenèse. La présente invention décrit des composés, des compositions et des procédés utiles pour l’inhibition de la néo-angiogenèse. Dans certain modes de réalisation, l’invention concerne des procédés pour inhiber la néovascularisation, ainsi que des composés, tels que des ARNsi de VEGF-C et RhoA, utiles dans le traitement de troubles oculaires tels que la dégénérescence maculaire liée à l’âge (DMLA), la rétinopathie diabétique, le glaucome et d’autres troubles néovasculaires.
PCT/IN2009/000671 2008-11-21 2009-11-20 Inhibition de la sécrétion de vegf-a, l’angiogenèse et/ou la néo-angiogenèse par inactivation véhiculée par ansi de vegf-c et rhoa WO2010058426A2 (fr)

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US13/130,699 US20110293625A1 (en) 2008-11-21 2009-11-20 Inhibition of vegf-a secretion, angiogenesis and/or neoangiogenesis by sina mediated knockdown of vegf-c and rhoa
EP09810851A EP2358877A2 (fr) 2008-11-21 2009-11-20 Inhibition de la sécrétion de vegf-a, l angiogenèse et/ou la néo-angiogenèse par inactivation véhiculée par ansi de vegf-c et rhoa

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WO2011163436A1 (fr) * 2010-06-24 2011-12-29 Quark Pharmaceuticals, Inc. Composés à base d'arn double brin pour le gène rhoa et leur utilisation
AU2011270896B2 (en) * 2010-06-24 2015-04-09 Quark Pharmaceuticals, Inc. Double stranded RNA compounds to RhoA and use thereof
US9045755B2 (en) 2010-06-24 2015-06-02 Quark Pharmaceuticals, Inc. Double stranded RNA compounds to RhoA and use thereof

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