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WO2008154482A2 - Compositions d'arnsi et procédés d'utilisation dans le traitement de maladies oculaires - Google Patents

Compositions d'arnsi et procédés d'utilisation dans le traitement de maladies oculaires Download PDF

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WO2008154482A2
WO2008154482A2 PCT/US2008/066298 US2008066298W WO2008154482A2 WO 2008154482 A2 WO2008154482 A2 WO 2008154482A2 US 2008066298 W US2008066298 W US 2008066298W WO 2008154482 A2 WO2008154482 A2 WO 2008154482A2
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sirna
sirna molecule
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sense strand
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WO2008154482A3 (fr
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Patrick Y. Lu
Xiao-Ling Liang
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Sirnaomics, Inc.
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    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
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    • 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
    • C12N15/1136—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 against growth factors, growth regulators, cytokines, lymphokines or hormones
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2320/00—Applications; Uses
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    • C12N2320/31—Combination therapy
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention provides compositions and methods for treatment of various ocular diseases using small interfering RNA (siRNA) mixtures containing a plurality of sequences targeting multiple disease causing genes.
  • siRNA small interfering RNA
  • the invention provides siRNA sequences targeting 1) viral genes expressed in ocular infections; 2) genes whose expression causes inflammation; and 3) genes whose expression promotes pro-angiogenesis activity.
  • ocular diseases There are variety of ocular diseases having unmet medical needs. Those diseases are ranging from infections, allergenic disorder, cardiovascular disorder and age related conditions. Many diverse ocular diseases are the result of excessive neovascularization (NV), an abnormal proliferation and growth of blood vessels within the eye.
  • NV neovascularization
  • the development of ocular NV itself has adverse consequences for vision but also is an early pathological step in many serious eye diseases; despite introduction of new therapeutic agents it remains the most common cause of permanent blindness in United States and Europe.
  • Several major eye diseases promote an abnormal neovascularization, which leads to further damage to the eyes causing loss of vision. Unfortunately, few treatment options exist for patients with any of these ocular NV diseases.
  • the most commonly used approved therapy is a photodynamic treatment, Visudyne, that uses light to activate a photosensitizer in the vicinity of the neovascularization to destroy unwanted blood vessels. It is not effective in many patients and cannot prevent recurrence even when it is effective.
  • a recently approved agent, Macugen provides some benefit but also is ineffective in most patients.
  • the intraocular administration of Macugen leads to irritation and risk of infection, both of which are adverse since they exacerbate the neovascularization pathology.
  • the ocular neovascularization diseases can be divided into diseases affecting the anterior, or front, of the eye and those affecting the posterior, or retinal, part of the eye. Development of NV at these different regions may have different origins, but the biochemical and physiological nature of the NV process appears to be virtually identical, regardless of eye region. Consequently, an effective means to intervene in the biochemical nature of ocular NV offers the prospect for providing an effective treatment for any ocular disease that involves ocular NV as the major pathology or as the underlying pathology, regardless of whether the disease afflicts the anterior or posterior of the eye. Nonetheless, the anterior and posterior ocular tissues differ considerably and these differences can have a dramatic influence on the most effective means to administer therapeutic treatments so that the tissue and cells are reached by the therapeutic agent.
  • ROP Retinopathy of prematurity
  • ROP is a potentially blinding eye disorder that primarily affects premature infants weighing about 2% pounds (1250 grams) or less that are born before 31 weeks of gestation (a full-term pregnancy has a gestation of 38-42 weeks).
  • This disorder which usually develops in both eyes — is one of the most common causes of visual loss in childhood and can lead to lifelong vision impairment and blindness.
  • ROP The most effective proven treatments for ROP are laser therapy or cryotherapy.
  • Laser therapy "burns away" the periphery of the retina, which has no normal blood vessels.
  • cryotherapy physicians use an instrument that generates freezing temperatures to briefly touch spots on the surface of the eye that overlie the periphery of the retina.
  • Both laser treatment and cryotherapy destroy the peripheral areas of the retina, slowing or reversing the abnormal growth of blood vessels.
  • the treatments also destroy some side vision. This is done to save the most important part of our sight — the sharp, central vision we need for "straight ahead" activities such as reading, sewing, and driving.
  • Both laser treatments and cryotherapy are performed only on infants with advanced ROP.
  • ROP treatment decreases the chances for vision loss, it does not always prevent it. Not all babies respond to ROP treatment, and the disease may get worse. If treatment for ROP does not work, a retinal detachment may develop. If the center of the retina or the entire retina detaches, central vision is threatened, and surgery may be recommended to reattach the retina.
  • the current methods of treatment of ROP are invasive surgeries, not able to prevent the disease getting worse and have potential long-term side effects.
  • PDR Proliferate Diabetic Retinopathy
  • PDR occurs when the tiny blood vessels providing oxygen to the retina become damaged. The damage allows blood and fluid to escape into the retina, and also results in new blood vessel growth. These new vessels are more fragile and frequently bleed into the vitreous region of the eye, interfering in vision. Patients with the most serious form of DR are at a substantial risk for severe visual loss without treatment.
  • neovascularization is a central pathology of the disease.
  • Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness in American adults. In some people with diabetic retinopathy, blood vessels may swell and leak fluid. In other people, abnormal new blood vessels grow on the surface of the retina. The retina is the light-sensitive tissue at the back of the eye.
  • Scatter laser treatment helps to shrink the abnormal blood vessels. Physician places 1,000 to 2,000 laser burns in the areas of the retina away from the macula, causing the abnormal blood vessels to shrink. Because a high number of laser burns are necessary, two or more sessions usually are required to complete treatment. This treatment may cause some loss of the side vision, but can save the rest of sight. Scatter laser treatment may slightly reduce color vision and night vision. As we can see, the scatter laser treatment is still not ideal.
  • Age related macular degeneration (AMD):
  • AMD is the leading cause of blindness in people over 60 years and each year the problem becomes more acute. In AMD central vision is lost making it impossible to appreciate fine detail. Given the magnitude of the burden of AMD on individuals and society as a whole, it is perhaps surprising that more is not known of the causes of the disease and how it develops. It is clear, however, that the retinal pigment epithelium (RPE) plays a pivotal role. Abnormal waste material builds up beneath and within the RPE and RPE cells eventually die. The rods and cones in the retina depend for their survival upon normal functioning RPE and this RPE failure leads to progressive loss of vision. The disease provokes a scarring process at the back of the eye inducing formation of new blood vessels, neovascularization.
  • RPE retinal pigment epithelium
  • Rubeosis is a term that describes abnormal blood vessel growth on the iris and the structures in the front of the eye. Normally there are no visible blood vessels in these areas. When the retina has been deprived of oxygen, or is ischemic, as with diabetic retinopathy or vein occlusion, abnormal vessels form to supply oxygen to the eye. Unfortunately, the formation of these vessels obstructs the drainage of aqueous fluid from the front of the eye, causing the eye pressure to become elevated. This usually leads to neovascular glaucoma.
  • Uveitis is a broad group of diseases originating from inflammation of tissues on the inside of the eye. This disease is most commonly classified anatomically as anterior, intermediate, posterior or diffuse. Ocular complications of uveitis may produce profound and irreversible loss of vision, especially when unrecognized or treated improperly. The most frequent complications include cataract; glaucoma; retinal detachment; neovascularization of the retina, optic nerve, or iris and the like.
  • a related group of ocular diseases are the consequence of eye infections, including Conjunctivitis, Keratitis, Blepharitis, Sty, Chalazion and Iritis, again all major causes of ocular neovascularization that leads to vision loss.
  • Recurrent HSV infection is the most common infectious cause of corneal blindness in the U.S. This viral infection causes blinding lesions called stromal keratitis (SK). Corneal NV is an early step in vision loss from herpetic SK.
  • ocular tissues are in a continuous state of maintenance which often entails neovascularization.
  • ocular neovascularization becomes the major symptom and this abnormal physiological change is the key pathology required treatment.
  • This ocular neovascularization results in excessive growth of damaging new blood vessels, and it appears to be virtually identical regardless of the region of the eye and disease, although the originating cause of the pathology as well as the role in vision loss differs widely.
  • the commonality of the pathological process offers means to provide therapeutic interventions that are effective in these diverse diseases of the eye.
  • Herpes Simplex Virus type-1 infection can cause Herpetic Stromal Keratitis (HSK) which induces corneal neovascularization.
  • HSV Herpes Simplex Virus type-1 infection
  • the angiogenic factor production occurs initially from virus-infected corneal epithelial, non-inflammatory, cells followed by expression in a clinical phase from inflammatory cells (PMNs and macrophages) in the stroma.
  • PMNs and macrophages inflammatory cells
  • a mouse model of HSV induced corneal NV was developed by implantation of purified HSV viral DNA fragments (HSV DNA, rich in CpG motifs) or synthetic CpG oligonucleotides (CpG ODN).
  • HSV-I Hypoxia inducible factor- 1
  • HIF-I Hypoxia inducible factor- 1
  • HIF-I transactivates multiple genes whose products play key roles in oxygen homeostasis, including vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • VEGF-mediated neovascularization and angiogenesis is one of the common pathological pathways of many ocular neovascularization diseases.
  • the VEGF-mediated angiogenesis pathway plays a central role in angiogenesis of all these NV-related eye diseases.
  • the VEGF family is composed of five structurally related growth factors: VEGF-A, Placenta Growth factor (PIGF), VEGF-B, VEGF-C, and VEGF-D.
  • VEGFR-I FIt-I
  • VEGFR-2 KDR or FIk-I
  • VEGFR-3 VEGFR-3
  • VEGF-A binds VEGFR-I and VEGFR-2 and is known to induce neovascularization and angiogenesis, as well as vascular permeability.
  • VEGFR- 1 and VEGFR-2 are both up-regulated in proliferating endothelium that may be a direct response to VEGF-A or hypoxia.
  • VEGFR-I has higher affinity to VEGF-A than VEGFR-2.
  • VEGFR-2 is responsible for angiogenic signals for blood vessel growth, but the function of VEGFR-I is poorly understood. Some studies suggested a direct role in transducing angiogenic signals, and roles in motility and permeability. This understanding of key players in the VEGF pathway of angiogenesis has led to studies with inhibitors of VEGF-A as candidate therapeutic agents, including Macugen® (an aptamer oligonucleotide inhibiting VEGF binding to its receptor) and Lucentis® (a monoclonal antibody against VEGF). While these studies in ocular angiogenesis, as well as in other angiogenesis diseases such as tumor growth, have validated the value of the VEGF pathway for clinical effect, the experimental agents are far from effective for many patients. It is clear that better inhibitors of the VEGF pathway are needed if we are to develop treatments for these major eye diseases.
  • Matrix metalloproteinases are a family of extracellular matrix-degrading enzymes associated with neovascularization. Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal neovascularization (CNV).
  • CNV choroidal neovascularization
  • RPE Retinal pigment epithelial cells
  • MMP-2 secretion was also increased by stimulation with VEGF, but not bFGF.
  • MMP-2 activity compromises retinal pericyte survival possibly through MMP-2 action on ECM proteins and/or direct association of MMP-2 with integrins, which promotes apoptosis/anoikis by loss of cell contact with an appropriate ECM.
  • pshRNA shRNA
  • two plasmids were generated encoding shRNA (pshRNA) targeted against two distinct MMP-9 gene sequences. Transfection of these pshMMP-9s have shown specific inhibition of MMP-9 expression both in vivo and in vitro. In vivo delivery of pshMMP- 9 subconjunctivally was also effective at inhibiting MMP-9 protein expression in the mouse cornea. Delivery of the pshMMP-9 stopped angiogenesis and decreased the severity of herpetic stromal keratitis. Therefore, we strongly believe that MMP-2 and MMP-9 should be another set of targets for anti-angiogenesis treatment.
  • alpha v beta 3 and alpha v beta 5 in the ocular angiogenic process.
  • PDR proliferative diabetic retinopathy
  • Fibroblast growth factors such as FGF-2 are potent mitogens for endothelial cells and induce their assembly into vascular-like structures in culture and in in vivo assays.
  • a study on FGF-2 functions during physiological vascularization are poorly documented has indicated that the major functions of FGF-2 at different early stages of physiological vascularization. Both the failure in hyaloid regression and the intense angiogenic invasion of endothelial cells into the retina may serve as a model for some related human ocular pathologies.
  • Expression of FGF-2 interestingly, resulted in beneficial therapeutic effect on herpetic SK progression via its role in wound healing.
  • EGF epithelial growth factor
  • PDGFR platelet-derived growth factor receptor alpha and beta
  • PIGF placenta growth factor
  • Inflammation is a process that involves many cells and biochemical factors, but despite its complexity the process is highly conserved across tissues.
  • One of the early events in inflammation is secretion of activating factors as a result of tissue hypoxia, damage, or other insults. These factors activate cells and induce recruitment of inflammatory cells into the tissue, which secrete additional activating factors.
  • One common biochemical pathway for induction of inflammation is secretion of TNF alpha and IL-lbeta, as well as up regulation of cyclooxygenase (COX)-2.
  • COX cyclooxygenase
  • RNA interference RNA interference
  • siRNA Small Interfering RNA
  • RNA interference is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene (11).
  • RNA interference a double stranded RNA is cleaved by an RNase IIMielicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-nt overhangs at the 3' ends.
  • siRNA small interfering RNA
  • RISC RNA-induced-silencing-complex
  • siRNA-directed endonuclease digests the RNA, thereby inactivating it.
  • Recent studies have revealed that the use of chemically synthesized 21-25-nt siRNAs exhibit RNAi effects in mammalian cells 20, and the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function.
  • target designate an siRNA molecule which is specifically complementary to a sequence on a target RNA molecule, and inactivates the target RNA molecule. While not wishing to bound by theory, the inactivation is thought to proceed through the RNAi mechanism described in this paragraph.
  • RNAi in mammalian cells in laboratory or potentially, in therapeutic applications, uses either chemically synthesized siRNAs or endogenously expressed molecules (24).
  • the endogenous siRNA is first expressed as a small hairpin RNAs (shRNAs) by an expression vector (plasmid or virus vector), and then processed by Dicer into siRNAs. It is thought that siRNAs hold great promise to be therapeutics for human diseases especially that caused by viral infections (27-30).
  • siRNA polynucleotide that is, with certainty, capable of specifically altering the expression of a given mRNA.
  • the present invention provides siRNA compositions directed toward RNA targets implicated in various diseases and pathologies of the eye. These include both virally induced diseases and a variety of spontaneous afflictions that affect mammalian eyes, especially the human eye.
  • the invention is also directed to methods of treatment of such diseases and pathologies using the compositions disclosed herein.
  • a mixture in a first aspect includes a plurality of small interfering RNA (siRNA) oligonucleotides and a pharmaceutical carrier, wherein each of the siRNA molecules targets an RNA molecule encoding a gene product whose activity promotes at least one of inflammation, neovascularization and angiogenesis in the eye arising in an ocular disease.
  • siRNA small interfering RNA
  • the ocular disease is selected from the group consisting of herpetic stromal keratitis, uveitis, rubeosis, conjunctivitis, keratitis, blepharitis, sty, chalazion, crizis, age-related macular degeneration, proliferate diabetic retinopathy and retinopathy of prematurity.
  • a targeted RNA molecule encodes a gene selected from the group consisting of herpesvirus essential genes, pro-inflammatory pathway genes, pro-angiogenesis pathway genes, pro-cell proliferation pathway genes, and viral infectious agent genome RNA, and viral infectious agent genes.
  • a component siRNA molecule targets an mRNA molecule, whereas in alternative embodiments a component siRNA molecule targets a viral RNA molecule.
  • the same siRNA targets mRNA molecules that encode orthologous human and mouse genes.
  • the mixture includes at least three siRNA molecules targeting a herpes virus UL5 gene, a herpes virus UL29 gene, and human and mouse matrix metalloproteinase (MMP) 9 gene (Table 23).
  • a mixture includes at least three siRNA molecules targeting human and mouse tumor necrosis factor alpha, interleukin- 1 beta, and cyclooxygenase-2 genes (Table 18).
  • a mixture includes at least three siRNA molecules targeting human and mouse vascular endothelial growth factor (VEGF)-A, VEGF receptor 1 and VEGF receptor 2 genes (Table 19).
  • VEGF vascular endothelial growth factor
  • a mixture includes at least three siRNA molecules targeting human and mouse VEGF receptor 2, VEGF A, and VEGF receptor 1 genes (Table 20). In yet an additional embodiment a mixture includes at least three siRNA molecules targeting human and mouse MMP-2, VEGF A, and VEGF receptor 1 genes (Table 22).
  • various mixtures include combinations of alternative choices of at least three siRNA molecules targeting human and mouse placenta growth factor (PIGF), VEGF A, and VEGF receptor 1 genes (Table 25).
  • PIGF human and mouse placenta growth factor
  • VEGF A vascular endothelial growth factor
  • VEGF receptor 1 genes Table 25.
  • a mixture includes at least four siRNA molecules targeting human and mouse PIGF, VEGF A, VEGF receptor 1, and basic fibroblast growth factor (b- FGF) genes (Table 25).
  • a mixture includes at least four siRNA molecules targeting human and mouse A-RAF, mTOR, hypoxia inducible factor- 1 (HIF-I) alpha, and integrin alpha V genes (Table 24).
  • a mixture includes at least three siRNA molecules targeting human and mouse A-RAF, mTOR, and HIF-I alpha genes (Table 24).
  • a mixture includes at least three siRNA molecules targeting human and mouse A-RAF, m TOR, and integrin receptor alpha V genes (Table 24).
  • a mixture includes at least four siRNA molecules targeting human and mouse PIGF 5 VEGF A, VEGF receptor 1, and VEGF receptor 2 genes (Table 25).
  • a mixture includes at least four siRNA molecules targeting human and mouse PIGF, VEGF A, VEGF B, and b-FGF genes (Table 25).
  • a mixture includes at least four siRNA molecules targeting human and mouse MMP-9, VEGF A, herpes virus UL5, and herpes virus UL29 genes (Table 23).
  • a targeted mRNA molecule includes a VEGF pathway gene, an FGF pathway gene, a protein kinase gene, a pro-angiogenesis gene, a proinflammatory gene, an endothelial cell proliferation gene, or a herpes simplex virus gene.
  • the pharmaceutical carrier of the mixture is selected from the group of a saline solution, sugars, polymer, lipid, or micelle solutions, and in more particular embodiments the carrier is selected from among a polycationic binding agent, a cationic lipid, a cationic micelle, a cationic polypeptide, a hydrophilic polymer grafted polymer, a non-natural cationic polymer, a cationic polyacetal, a hydrophilic polymer grafted polyacetal, a ligand functionalized cationic polymer, and a ligand functionalized-hydrophilic polymer grafted polymer.
  • the carrier includes a histidine-lysine copolymer which forms a nanoparticle with an siRNA molecule.
  • an siRNA molecule contains naturally occurring nucleotides, and in other embodiments an siRNA molecule includes one or more chemically modified nucleotides.
  • a method for treating ocular disease in a subject, wherein the disease is characterized at least in part by inflammation, neovascularization, and/or angiogenesis.
  • the method includes administering to the subject a mixture that includes a plurality of small interfering RNA (siRNA) oligonucleotides and a pharmaceutical carrier, wherein each of the siRNA molecules targets an RNA molecule encoding a gene product whose activity promotes at least one of inflammation, neovascularization and angiogenesis in the eye arising in an ocular disease of said subject.
  • siRNA small interfering RNA
  • the mixture is administered at a site distal to the eye wherein said site is selected from the group consisting of a subconjunctival site, an intravenous site, an intraocular site, and a subcutaneous site, and in certain other embodiments the mixture is administered topically to the eye.
  • the method is directed against an ocular disease selected from the group consisting of herpetic stromal keratitis, uveitis, rubeosis, conjunctivitis, keratitis, blepharitis, sty, chalazion, crizis, age-related macular degeneration, proliferate diabetic retinopathy and retinopathy of prematurity.
  • the mixture inhibits expression of at least one gene selected from among herpesvirus essential genes, pro-inflammatory pathway genes, pro- angiogenesis pathway genes, pro-cell proliferation pathway genes, and viral infectious agent genome RNA, and viral infectious agent genes.
  • the mixture includes siRNA molecules that target sequences selected from among the following sets of sequences: SEQ ID NOS: 61-158; or SEQ ID NOS:1-60 and SEQ ID NOS:486-536; or SEQ ID NOS:189-218 and SEQ ID NOS:279-365; or SEQ ID NOS: 159-218 and SEQ ID NOS:279-335; or SEQ ID NOS: 189- 218, SEQ ID NOS:279-335 and SEQ ID NOS:456-485; or SEQ ID NOS: 189-218, and SEQ ID NOS:249-335; or SEQ ID NOS: 189-218, SEQ ID NOS:279-335, and SEQ ID NOS:426- 455; or SEQ ID NOS:159-188 and SEQ ID NOS:396-455; or SEQ ID NOS:159-188, SEQ ID NOS:396-455, and SEQ ID NOS:537-566; or a mixture that inhibit
  • the carrier is selected from among a polycationic binding agent, a cationic lipid, a cationic micelle, a cationic polypeptide, a hydrophilic polymer grafted polymer, a non-natural cationic polymer, a cationic polyacetal, a hydrophilic polymer grafted polyacetal, a ligand functionalized cationic polymer, and a ligand functionalized-hydrophilic polymer grafted polymer.
  • composition in an additional aspect includes one or more siRNA oligonucleotides and a pharmaceutical carrier, wherein the one or more siRNA molecules targets an RNA molecule encoding a gene product whose activity promotes at least one of inflammation, neovascularization and angiogenesis in the eye arising in an ocular disease.
  • HKP-siRNA nanoparticle enhances siRNA delivery into the rabbit ocular tissue.
  • Each of the 250 ⁇ g of siRNA labeled with detection signal was intravitreously injected into the rabbit eye balls. Four eyes were measured from each test group.
  • Fig. 2 Polymer-siRNA nanoparticles.
  • siRNA designs targeting mVEGF (Accession: M95200), mVEGRl (Accession: D88689) and mVEGFR2 (Accession: X70842) with two siRNA oligos for each gene
  • mVEGF mVEGF
  • mVEGFRl mVEGFR2
  • mVEGFR2 mVEGFR2
  • the total RNA samples were collected from the cell culture experiments after transfection of simVEab, simRlab and simR2ab (Table 26). Two dosages of the siRNA oligos were used against the same dosage of control siRNA oligos.
  • HKP-siRNA particles can be formed when HKP aqueous solution is mixed with siRNA solution, and likewise for RPP-siRNA particles, (d) siRNA oligos were delivered through both local and systemic administration routes.
  • siRNA oligos were delivered through both local and systemic administration routes.
  • HKP-siRNA particles were mainly for local delivery through either subconjunctival (SCJ) or intravitreous (IVT) routes.
  • RPP- siRNA particles were applied for systemic deliveries through either intraperitoneal (IP) or intravenous (IV) routes, respectively, reaching to the blood stream first and then to ocular neo vasculature,
  • IP intraperitoneal
  • IV intravenous
  • H3K4b ocular neo vasculature
  • siRNA oligos were locally and systemically delivered into the ocular NV tissues, (al) FITC-labeled siClab was observed in angiogenic corneal cryosection 24 hr after SCJ administration, compared to the cryosection from the group (a2) treated with naked FITC-labeled siClab through the same route of delivery, (bl) FITC-labeled siClab was observed in angiogenic corneal cryosection 24 hr after IV injection of RPP-siClab, compared to the cryosection from the group (b2) treated with naked FITC-labeled siClab through the same systemic delivery, (c) ELISA analysis identified mVEGF protein down regulation.
  • Fig. 4 Anti-angiogenesis efficacy on CpG induced ocular NV.
  • (b) Images from mouse eyes representing each treated group marked with the same pattern as (a)
  • SCJ locally
  • HSK-simVmix and systemically
  • IV delivered RPP-simVmix demonstrated potent anti-angiogenesis efficacy.
  • (d) Images of mouse eyes from both treated and control groups, indicated by the same marks as (c).
  • Fig. 5 Inhibition of Corneal NV with locally administration of siRNA duplexes in HSK model.
  • the siRNA duplexes targeting only one gene were able to inhibit corneal NV compared to control siRNA (a) in the eyes of HSK models.
  • Locally delivered simVmix demonstrated stronger inhibition for corneal NV (b) compared to the siRNA targeting only a single gene. The pictures were taken at P4.
  • Fig. 6 Pharmacodynamics of the simVmix in mouse HSK model, (a) the HSK disease scores were measured at P6, 10, 14 and 22, after either local (SCJ) or systemic (IV) administrations of HKP-simVmix (see page 51) and RPP-simVmix at Pl and P3. Significant inhibition was observed for systemic (IV) delivered RPP-simVmix compared to RPP-siClab (* P ⁇ 0.05), at P14 and P22. The potency is comparable to the locally delivered HKP- simVmix . (b) Therapeutic efficacy of simVmix in the HSK model.
  • the HKP- simVmix (stripe) and the RPP-simVmix (dot) treated groups exhibit healthy eyes compared to HKP-siClab (black) and RPP-siClab (grey) treated groups, which show severer HSK disease symptom, deformed and bloody corneal surfaces.
  • Fig. 7 Inhibition of Corneal NV with systemic administration of siRNA duplexes in HSK model.
  • the siRNA duplexes targeting only one gene were able to inhibit corneal NV compared to control siRNA (a) in the eyes of HSK models.
  • Systemically delivered simVmix demonstrated stronger inhibition for corneal NV (b) compared to the siRNA targeting only a single gene. The pictures were taken at P4.
  • Fig. 9 Fluorescein-labeled retinal whole mount changes. The changes were examined in ischemic retinopathy mice with siRNA duplexes treated though subconjuctival, intravitreal or intraperitoneal injection, (al, 2) Normal retinal vasculature in a normal (control) mouse at P 17.
  • Fig. 10 Histological changes in ischemic retinopathy mice. The histological changes were examined with siRNA treated though subconjuctival, intravitreal or intraperitoneal injection, (al, 2) Normal retinal vasculature in a normal (control) mouse at P17.
  • Fig. 11 SiRNA cocktail significantly silenced expression of VEGF, VEGFRl and VEGFR2.
  • VEGF vascular endothelial growth factor
  • VEGFRl vascular endothelial growth factor
  • a Q-RT-PCR analyses of mRNA levels.
  • Fig. 12 RT-PCR analysis of VEGF, VEGFRl, VEGFR2 mRNA levels.
  • the mRNA levels in the retina tissues at P14 and P17 were examined after intravitreal and intraperitoneal injection of simVmix (see page 51).
  • IPN and IVT represent intraperitoneal or intravitreal administration respectively
  • Fig. 13 Suppression of expression of human VEGF in human cells.
  • Fig. 14 Comparison of siRNA effectiveness in suppressing VEGF in human and mouse cells.
  • Fig. 15. Comparison of effectiveness of five 25 mer VEGF-Rl specific siRNA duplexes in suppressing gene expression.
  • Fig. 16 Comparison of effectiveness of three 25 mer MMP-9 specific siRNA duplexes in suppressing gene expression.
  • Fig. 17 Comparison of effectiveness of three 25 mer PDGF specific siRNA duplexes in suppressing gene expression.
  • the present invention provides novel siRNA targeting sequences. There are three important characteristics that distinguish the instant siRNA molecules and their targets:
  • siRNA duplexes are identical in the human and mouse sequences of the same, or orthologous, gene. That means each of the siRNA duplexes will be able to suppress the expression of the same gene target in both human and mouse cells, which is advantageous in drug development. For example, a potent siRNA specific to VEGF gene will be able to knockdown both human VEGF in human cells and mouse VEGF gene expression in mouse cells.
  • the sequences generally have three different lengths: 21 nucleotides (nt), 23 nt and 25 nt.
  • 21 nucleotides (nt) 21 nucleotides (nt)
  • 23 nt and 25 nt 25 nucleotides
  • siRNAs that are 23 nt or 25 nt in length are usually more potent than 21 nt siRNAs, but on the other hand 25 nt siRNAs may induce unwanted interferon response more than shorter length oligonucleotides. Therefore, siRNA duplexes at various lengths will provide the best chance to identify potent inhibitors with minimal interferon response.
  • the siRNA oligos are provided in either blunt end or sticky end form,.
  • a "blunt” end designates a duplex in which the terminal nucleotide on each strand is paired with a nucleotide on the opposite strand.
  • a "sticky" end or staggered end designates a duplex having a terminus in which one strand extends additional unpaired nucleotides beyond a paired nucleotide on the opposing strand.
  • the additional unpaired nucleotides at a staggered end are termed an "overhang" herein, and such nucleotides are termed overhanging nucleotides.
  • oligonucleotides may be sensitive to degradation while the blunt end may activate the cellular interferon response.
  • oligonucleotides "oligos", and similar terms based on this relate to short oligonucleotides composed of naturally occurring nucleotides as well as to oligonucleotides composed of synthetic or modified nucleotides.
  • siRNAs provided herein may be constituted purely of ribonucleotides, or they may have certain designated positions occupied by deoxynucleotides.
  • oligonucleotides provided in this invention may be constituted entirely of deoxynucleotides which provide siRNA molecules by transcription processes.
  • an oligonucleotide that is an siRNA may have any number of nucleotides between 19 and 35 nucleotides. In many embodiments an siRNA may have any number of nucleotides between 19 and 27 nucleotides. Oligonucleotides may be 19 or more nucleotides in length, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or more nucleotides in length, including any integral number of nucleotides up to 35 or more in length. siRNA oligonucleotides are generally double stranded and include a sense strand and an antisense strand.
  • siRNA may be single stranded at a certain point in its preparation prior to being paired with a complementary strand to form a duplex. Either of such single stranded oligonucleotides is also included within the use of the term siRNA herein.
  • siRNA molecules are termed "targeting" oligonucleotides or "directed” oligonucleotides herein.
  • the base sequence of the siRNA directs or targets the siRNA to a particular target sequence within an mRNA or viral RNA whose expression is intended to be suppressed.
  • the base sequence within the mRNA so targeted by the siRNA is the "target" sequence as used herein.
  • an siRNA may have two blunt ends, or two sticky ends, or it may have one blunt end and one sticky end.
  • the overhang nucleotides of a sticky end can range from one to four or more.
  • the invention provides siRNA compositions of 21, 23 and 25 base pairs with blunt ends.
  • polynucleotide and “oligonucleotide” are used synonymously herein.
  • a "mixture”, and related words and phrases relates to a composition that contains a plurality of siRNA molecules.
  • a mixture of siRNA molecules may have improved beneficial effects, when used to treat a disease or pathology, than a composition that contains only a single species of siRNA.
  • a mixture may contain two siRNA species, or three siRNA species, or four siRNA species, or even more.
  • a "cocktail" of siRNA molecules and a mixture thereof are synonymous.
  • a composition of the invention may include a single siRNA oligonucleotide, or it may contain a plurality of targeting siRNA molecules.
  • a composition or mixture may further include, in addition to the at least one siRNA molecule, a polymeric and/or a liposome carrier.
  • a polymeric carrier may comprise a cationic polymer that binds to the RNA molecule to form a nanoparticle (between 50-500 nm in diameter).
  • the cationic polymer may be an amino acid copolymer (such as a copolypeptide), containing, for example, histidine and lysine residues.
  • the polymer may additionally comprise a branched polymer.
  • the composition may comprise a targeting synthetic carrier.
  • the synthetic carrier may comprise a cationic polymer, a hydrophilic polymer, and a targeting ligand.
  • the polymer may comprise a polyethyleneimine
  • the hydrophilic polymer may comprise a polyethylene glycol or a polyacetal
  • the targeting ligand may comprise a peptide comprising an RGD sequence, a transferrin targeting ligand, a protamine or a single chain antibody, etc.
  • an electric field may be applied to a tissue substantially contemporaneously with the composition.
  • the composition and method of the invention comprises dsRNA oligonucleotides with a sequence matching an endogenous human gene or a mutated endogenous gene, and at least one mutation in the mutated gene may be in a coding or regulatory region of the gene.
  • the endogenous gene may be selected from the group consisting of angiogenesis related genes including growth factor genes, protein serine/threonine kinase genes, protein tyrosine kinase genes, protein serine/threonine phosphatase genes, protein tyrosine phosphatase genes, receptor genes, and transcription factor genes.
  • RNAi agents utilize an endogenous process active against dsRNA viral infections but can be used to inhibit expression from virtually any mRNA, and with a high degree of selectivity.
  • the invention provides for RNAi agents for inhibiting ocular viral infections as a means to intervene in ocular neovascularization.
  • the RNAi agents of the invention include short dsRNA oligonucleotides, siRNA, with a sequence matching viral gene sequences and lacking sequence specificity for human genes.
  • RNAi agents of the invention inhibit mRNA expressed by either DNA or RNA viral infections and they degrade the genome of dsRNA viral infections.
  • One DNA viral infection inhibited by the RNAi agents of the invention is HSV, which causes herpetic stromal keratitis. This virus has a relative large genome that remains episomal and where expression levels of viral mRNA rise and fall over time. The continuous low levels of HSV viral mRNA expression result in a persistent, albeit quiescent, infection that flairs up from time to time.
  • the RNAi agents of the invention are useful to inhibit rising HSV mRNA expression associated with recurrence of infection. By reducing the ability of the infection to flair up, the RNAi agents protect from induction of ocular neovascularization disease.
  • RNAi agents also are useful to diminish the continuous, low level HSV mRNA expression to even lower levels, which diminishes the ability of the HSV infection to flair up.
  • the RNAi agents are effective to inhibit the DNA and RNA viral infections of ocular tissues that lead to ocular neovascularization.
  • the siRNA sequences were designed targeting respectively to HSV-I and HSV-2 viral UL5 and UL29 genes, with ten 21-mer, ten 23-mer and ten 25-mer in length.
  • the inhibitory siRNA duplexes can be synthesized according to those targeted sequences as one sense single-stranded RNA oligo and one antisense single- stranded RNA oligo first.
  • the double- stranded siRNA duplexes can be annealed as either blunt-ended or overhang (3' end).
  • the sequences of UL5 gene of both HSV- 1 and HSV-2 targeted by designed siRNAs are listed in Table 1.
  • the sequences of UL29 gene of both HSV-I and HSV-2 targeted by designed siRNAs are listed in Table 2.
  • HSV UL5 and UL29 genes are included in the group of genes considered to be herpesvirus essential genes. (Note: the lists of the sequences in this application are the target sequences from cDNA.
  • sequences can easily be transcribed into RNA sequences for preparation of siRNA' s by changing thymidine (t) to uridine (u). Only sense strand sequences are listed in the tables. The antisense sequences can be easily added by the base pairing rules).
  • Table 1 The targeted sequences ofUL5 gene of both HSV-I and HSV- ⁇ >
  • Inflammation is a process that involves many cells and biochemical factors, but despite its complexity the process is highly conserved across tissues.
  • One of the early events in inflammation is secretion of activating factors as a result of tissue hypoxia, damage, or other insults. These factors activate cells and induce recruitment of inflammatory cells into the tissue, which secrete additional activating factors.
  • One common biochemical pathway for induction of inflammation is secretion of TNF and IL-I. These factors act in a largely parallel manner so that strong inhibition of their activation of an inflammation cascade requires intervening in both simultaneously. Downstream of this point, the inflammatory cascade results in secretion of factors to induce neovascularization.
  • the inflammatory process offers many points for intervention: upstream at secreted factors initiating the cascade; and downstream at factors responsible for activating specific cells in the cascade, such as endothelial cell recruitment of neutrophils from the blood and endothelial cell induction of neovascularization.
  • the invention provides RNAi agents effective for inhibiting factors whose upregulation and role in inflammation depends on gene expression of the factor. While many secreted factors are present in cells and released to initiate inflammation, up regulation of expression of those same factors is important for continued expansion of the inflammation and for persistent of the inflammation.
  • the RNAi agents of the invention provide for inhibition of persistent inflammation, which is a greater contributor to the ocular neovascularization disease.
  • siRNA sequences target Interleukin-1 beta (IL- l ⁇ ) and Tumor Necroses Factor alpha (TNF ⁇ ) genes of both human and mouse.
  • IL- l ⁇ Interleukin-1 beta
  • TNF ⁇ Tumor Necroses Factor alpha
  • the siRNA targeted sequences are 21-mer, 23-mer and 25- mer in length.
  • the inhibitory siRNA duplexes can be synthesized according to those targeted sequences as one sense single- stranded RNA oligo and one antisense single- stranded RNA oligo first.
  • the double-stranded siRNA duplexes can be annealed as either blunt-ended or overhang (3' end).
  • the targeted sequences of IL- l ⁇ gene of both human and mouse are listed in Table 3.
  • the targeted sequences of TNF ⁇ gene of both human and mouse are listed in Table 4.
  • the targeted sequences of cyclooxygenase (COX)-2 gene of both human and mouse are listed in Table 5.
  • hypoxia inducible factor-1 is a transcription factor composed of HIF-lalpha and HIF-lbeta subunits. HIF-I transactivates multiple genes whose products play key roles in oxygen homeostasis, including vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • An early step is driven by the VEGF pathway that involves secretion of VEGF growth factors, which bind and activate cells bearing different members of the VEGF family of receptors.
  • the VEGF family is composed of five structurally related growth factors: VEGF-A, Placenta Growth factor Table 3: The targeted sequences of IL-Ip gene ofboth human and mouse.
  • PIGF vascular endothelial growth factor
  • VEGF-B vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor-B
  • VEGF-C vascular endothelial growth factor-D
  • VEGF-D vascular endothelial growth factor-B
  • VEGF-C vascular endothelial growth factor-B
  • VEGF-C vascular endothelial growth factor receptor
  • VEGF-D vascular endothelial growth factor receptors
  • Tables 4, 10 16, and 35 include sequences that target only the human ortholog of the gene, some of which include a -dtdt overhang at the 3' end.
  • Table 4 The targeted sequences of TNF ⁇ gene of both human and mouse.
  • s Sense strand
  • a Antisense strand Table 5.
  • COX cyclooxygenase
  • VEGF A gene of both human and mouse are listed in Table 7.
  • Table 7 The targeted sequences of VEGF A gene of both human and mouse.
  • the targeted sequences of VEGF B gene of both human and mouse are listed in Table 8.
  • the targeted sequences of PIGF gene of both human and mouse are listed in Table 9.
  • the targeted sequences of VEGFR-I (FIt-I) gene of both human and mouse are listed in Table 10 and corresponding siRNA sequences are in Table 35.
  • the targeted sequences of VEGFR- 2 gene of both human and mouse are listed in Table 11.
  • Table 8 The targeted sequences of VEGF B gene of both human and mouse.
  • bFGF Another major secreted angiogenic growth factor is bFGF, which actives a separate set of receptors. Both of these pathways activate endothelial cells in nearby vasculature, and stimulate their proliferation and migration to form new vasculature into the region secreting the growth factor stimulants.
  • the VEGF and bFGF pathways act somewhat in parallel up to a point where they become the same.
  • These secreted growth factor pathways of neovascularization represent a very useful point for therapeutic intervention, as provided by the invention, either by inhibiting the growth factors or their Table 9: The targeted sequences of PIGF gene of both human and mouse.
  • the invention also provides for inhibiting both pathways simultaneously, as well as for inhibition of intracellular signaling induced by these pathways such as the induced signal transduction kinases, or in a preferred embodiment the transcription factors.
  • the transcription factors have been established as useful points for therapeutic intervention but have been intractable to conventional therapeutic modalities.
  • the bFGF pathway is one of several FGF pathways.
  • the bFGF factor is a strong stimulator of angiogenesis and thus it and its receptors are an important point for therapeutic intervention.
  • the invention provides siRNA agents specific for inhibition of bFGF and its receptors, and intracellular signal transduction pathway. Table 10: The targeted sequences of VEGFR-I (FIt-I) gene of both human and mouse.
  • VEGFR1 VEGFR-I
  • Table 11 The targeted sequences of VEGFR- 2 gene of both human and mouse.
  • the targeted sequences of bFGF gene of both human and mouse are listed in Table 12.
  • the targeted sequences of A-RAF gene of both human and mouse are listed in Table 13.
  • the targeted sequences of mTOR gene of both human and mouse are listed in Table 14.
  • Table 12 The targeted sequences of bFGF gene of both human and mouse.
  • Table 13 The targeted sequences of A-RAF gene of both human and mouse.
  • gaggtgatccgtatgcaggacccga 398 Table 14 The targeted sequences of mTOR gene of both human and mouse.
  • Matrix metalloproteinases are a family of extracellular matrix-degrading enzymes associated with neovascularization. Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) are an important source of MMPs in the outer retinal environment, however little is known about the local factors that modulate MMP secretion in these cells. There are studies showed resting RPE cells secreted MMP-2 but not MMP-9, and stimulation with TNF-alpha induced secretion of MMP-9 and increased the secretion of MMP-2.
  • RPE Retinal pigment epithelial cells
  • MMP- 2 secretion was also increased by stimulation with VEGF, but not bFGF. Therefore, we strongly believe that MMP-2 and MMP-9 should be another set of targets for anti- angiogenesis treatment. There is evidence implicating the integrins alpha v beta 3 and alpha v beta 5 in the ocular angiogenic process.
  • alpha v beta 3 and alpha v beta 5 were present on vascular cells in tissues from patients with PDR.
  • the targeted sequences of MMP-2 gene of both human and mouse are listed in Table 15.
  • the targeted sequences of MMP-9 gene of both human and mouse are listed in Table 16.
  • the targeted sequences of integrins alpha v beta 3 (Integrin ⁇ V ⁇ 3) gene of both human and mouse are listed in Table 17.
  • Table 15 The targeted sequences of MMP-2 gene of both human and mouse.
  • Table 16 The targeted sequences of MMP-9 gene of both human and mouse.
  • Human 25-mer is UGGCACCACCACAACAUCACCUAUU 490 la AAUAGGUGAUGUUGUGGUGGUGCCA 491
  • Table 17 The targeted sequences of Integrin ⁇ V ⁇ 3 gene of both human and mouse.
  • the invention provides the therapeutic siRNA cocktail targeting multiple disease controlling genes in the same treatment.
  • "cocktail" and similar terms and phrases relates to a pharmaceutical composition that includes more than one siRNA molecule.
  • the invention provides for RNAi agents, such as siRNA oligonucleotides, that are chemically similar to the same source of supply and the same manufacturing process, and they are comprised of four types of nucleotides with different sequences.
  • the invention provides the siRNA cocktail drug for treatments of several types of ocular disease, including Uveitis, AMD, PDR and HSK, etc, acting on multiple aspects of the diseases and reducing potential toxicity.
  • the siRNA cocktail should contain at least two siRNA duplexes targeting at least two genes (not two sequences of the same gene) at a ratio of therapeutic requirement.
  • siRNA cocktail design for each combination must follow the understanding of the role of each gene in a background of the system biology network, such as these genes are functioning either in the same pathway or in a different one.
  • the chemical property of each siRNA duplexes in the cocktail must be the same in terms of source of supply, manufacturing process, chemical modification, storage conditions and formulation procedures.
  • Each individual siRNA duplex in the cocktail can be different in their lengths, with either blunt or sticky end, as long as their potencies have been defined.
  • siRNA cocktail Since siRNA cocktail is targeting multiple genes and a single cell type usually dose not express all those factors, the efficacy of a siRNA cocktail must be tested in a relevant disease model, either a multiple cell model, a tissue model or a animal model, after the confirmation of the potency of each individual siRNA duplex in the cell culture.
  • Each validated siRNA cocktail can be used for addressing one or more pathological conditions, for treatment of one or multiple types of diseases, such as, siRNA cocktail for suppressing inflammation, siRNA cocktail for antiangiogenesis activity and siRNA cocktail for autoimmune conditions.
  • siRNA cocktail must be administrated through the same route of delivery in the same formulation, although the regimen of dosing for each cocktail will be defined based on either the experimental design or therapeutic requirement.
  • Each siRNA cocktail can be applied either independently, or in combination with other drug modalities such as small molecule inhibitors, monoclonal antibodies, protein and peptides, and other siRNA cocktail drug(s).
  • Step 1 Defining the pathological condition for the siRNA cocktail design.
  • Inflammation is a process that involves many cells and biochemical factors, but despite its complexity the process is highly conserved across tissues.
  • One of the early events in inflammation is secretion of activating factors as a result of tissue hypoxia, damage, or other insults.
  • One common biochemical pathway for induction of inflammation is secretion of TNF alpha and IL- 1 beta.
  • Cyclooxygenases (COX) are rate-limiting enzymes involved in the conversion of PLA(2)-mobilized arachidonic acid into prostaglandins and thromboxanes.
  • COX-2 is a key mediator of inflammation during both physiologic and pathologic responses to endogenous stimuli and infectious agents.
  • siRNA cocktail may provide a potent anti-inflammatory agent for treatment of the ocular inflammatory disease such as Uveitis.
  • Step 2 Selection of potent siRNA duplexes targeting the selected genes in both mouse and human cell cultures.
  • Nonlimiting examples of highly potent siRNA duplexes are selected to be composed of the siRNA cocktail(s) as shown in Table 18.
  • the lists of the sequences in this application are generally the target sequences from cDNA.
  • the sequences can easily be transcribed into RNA sequences for preparation of siRNA' s by changing thymidine (t) to uridine (u).
  • t thymidine
  • u uridine
  • the combinations listed below are only examples. Therefore by following the same steps, various siRNA duplexes can be combined in various compositions to form a highly potent inhibitory cocktail for down regulation of a particular pathological process, as result, to treat various diseases).
  • siRNA cocktail Three combinations are selected to be tested in the Equine recurrent uveitis (ERU) model for autoimmune diseases, since it develops frequently and occurs spontaneously and has a similar pathogenic mechanism was assumed to exist in Uveitis.
  • ERU Equine recurrent uveitis
  • Step 4 When the potency of the siRNA cocktail is confirmed, it can be repeatedly tested in other autoimmune and inflammatory disease models in mouse. Since the sequences have homology to the same gene from both mouse and human, the siRNA cocktail can be directly developed for human therapeutic application. Using these procedures, many siRNA cocktail combinations deemed to be highly potent for various preclinical test and future therapeutics use have been identified, as described following: Table 19. siRNA cocktails targeting VEGF pathway. siRNA Cocktail Combinations (targeted sequences)
  • siRNA cocktails targeting VEGF pathway Table 20. siRNA Cocktail Combinations (targeted sequences)
  • VEGF VEGFR1 Human and Mouse homologues HIF-1 alpha SEQ ID NO:
  • siRNA cocktails targeting VEGF pathway Table 22. siRNA Cocktail Combinations (targeted sequences)
  • siRNA cocktail can be applied as an independent agent for treatment of ocular diseases, or applied with other drug modalities. Since siRNA therapeutics predominantly inhibit the target gene expression which is significantly different from the antagonist drugs such as the
  • siRNA cocktail and antagonist drug small molecule inhibitors and monoclonal antibodies. Therefore, use of the both siRNA cocktail and antagonist drug in the same therapeutic regimen may achieve better therapeutic benefit due to the too inhibitory mechanisms are in action.
  • siRNA cocktails can also be administrated in the same regimen for better clinical outcome.
  • the siRNA duplexes can be made as either the blunt end and sticky end at two ends, or one end is blunt and another is sticky. The ratios of each individual siRNA duplex in the cocktail can be even or different depending the target gene requirement for effective knockdown, to achieve therapeutic effect).
  • the invention provides compositions and methods for administering the therapeutic agents to treat ocular neovascularization diseases, and in particular to treat diseases in the anterior of the eye.
  • the invention also provides compositions and methods for administering the therapeutic agents to treat ocular neovascularization diseases anywhere in the eye including the posterior of the eye.
  • the tissues anywhere in the eye can be treated with neovasculature-targeted delivery of therapeutic agents, according to the invention, by local administration, by topical administration to the eye, and by intravenous administration at a distal site.
  • the tissues in the anterior of the eye can be treated, according to the invention, by local administration into the subconjunctival tissue, by topical administration to the eye, by periocular injection, by intraocular injection, and by intravenous administration at a distal site.
  • the compositions provided by the invention include 1) cationic agents that bind nucleic acids by an electrostatic interaction, including non-natural synthetic polymers, grafted polymers, block copolymers, peptides, lipids and micelles, 2) hydrophilic agents that reduce non-specific binding to tissues and cells, including non-natural synthetic polymers, peptides, and carbohydrates, 3) tissue and cell penetrating agents, including surfactants, peptides, non- natural synthetic polymers, and carbohydrates.
  • a preferred class of peptide is the histidine-lysine copolymer (HKP) that is a basic, cationic, broad class of peptides.
  • HTP histidine-lysine copolymer
  • Another preferred class of peptide is linear polylysine with histidine or imidazole monomers coupled to the epsilon amino moiety of the lysine monomers.
  • Another preferred class of peptide is branched polylysine and branched polylysine with histidine or imidazole monomers coupled to the epsilon amino moiety of the lysine monomers.
  • a preferred composition has a self-assembled complex of negatively charged therapeutic agent such as a nucleic acid with a cationic peptide with an excess of cationic charge of 2 fold to 10 fold and a more preferred cationic charge of 2 fold to 6 fold.
  • a preferred class of polylysine coupled with histidine or imidazole monomers has 30 to 70% coupling to primary amines of the lysine monomers.
  • Another preferred class of peptide is a polymer with a monomer comprised of the tripeptide histidine-histidine-lysine or the tetrapeptide of histidine-histidine-lysine-lysine, where the polymer is either linear or branched, the branched polymer having monomers coupled to either the alpha or epsilon amino group of another monomer, or both.
  • a preferred molecular weight of the polylysine class of polymers is in the range of 5,000 to 100,000, and a more preferred molecular weight of 10,000 to 30,000.
  • Fig. 1 demonstrates the significant enhancement of siRNA delivery into retina tissue.
  • a preferred class of grafted polymers is a peptide grafted with a hydrophilic polymer, where the hydrophilic polymer includes PEG, polyoxazoline, polyacetal (referred to in some instances as Fleximer), HPMA, and polyglycerol.
  • a preferred composition has a self- assembled complex of negatively charged therapeutic agent such as a nucleic acid with a cationic grafted polymer with an excess of cationic charge of 2 fold to 10 fold and a more preferred cationic charge of 2 fold to 6 fold.
  • a preferred molecular weight of the hydrophilic polymer is in the range of 2,000 to 10,000.
  • grafted polymers is a peptide grafted with a hydrophilic polymer further comprised of a ligand grafted to the hydrophilic polymer, where the ligand includes peptides, carbohydrates, vitamins, nutrients, and antibodies or their fragments.
  • a preferred class of non-natural synthetic cationic polymer is a polymer with a backbone repeating unit of ethyl-nitrogen ( -C-C-N- ), including polyoxazoline and polyethyleneimine (PEI).
  • a preferred composition has a self-assembled complex of negatively charged therapeutic agent such as a nucleic acid with a cationic polymer with an excess of cationic charge of 2 fold to 10 fold and a more preferred cationic charge of 2 fold to 6 fold.
  • the invention provides linear polyoxazoline or PEI derivatized with histidine or imidazole monomers.
  • Another preferred class of polymer is branched polyoxazoline or PEI derivatized with histidine or imidazole monomers.
  • a preferred class of polymer coupled with histidine or imidazole monomers has 30 to 70% of the basic moieties being imidazole.
  • a preferred molecular weight of the polymers is in the range of 5,000 to 100,000, and a more preferred molecular weight of 10,000 to 30,000.
  • a preferred class of grafted polymers is a polymer grafted with a hydrophilic polymer, where the hydrophilic polymer includes PEG, polyoxazoline, polyacetal (referred to in some instances as Fleximer), HPMA, and polyglycerol.
  • a preferred composition has a self-assembled complex of negatively charged therapeutic agent such as a nucleic acid with a cationic grafted polymer with an excess of cationic charge of 2 fold to 10 fold and a more preferred cationic charge of 2 fold to 6 fold.
  • grafted polymers is a polymer grafted with a hydrophilic polymer further comprised of a ligand grafted to the hydrophilic polymer, where the ligand includes peptides, carbohydrates, vitamins, nutrients, and antibodies or their fragments.
  • Another preferred class of cationic polymer is a polymer with a polyacetal backbone.
  • a preferred composition has a self-assembled complex of negatively charged therapeutic agent such as a nucleic acid with a cationic polyacetal polymer with an excess of cationic charge of 2 fold to 10 fold and a more preferred cationic charge of 2 fold to 6 fold.
  • the invention provides linear polyacetal derivatized with a basic moiety, where the basic moiety class includes mixture of lysine, primary amine, histidine, and imidazole monomers.
  • Another preferred class of polymer is branched polyacetal derivatized with a basic moiety (again including the class of lysine, amine, histidine, and imidazole monomers).
  • a preferred class of polyacetal polymer coupled with lysine, amine, histidine, and imidazole monomers has 30 to 70% if the basic moieties being imidazole.
  • a preferred molecular weight of the polymers is in the range of 5,000 to 100,000, and a more preferred molecular weight of 10,000 to 30,000.
  • a preferred class of grafted polymers is a polymer grafted with a hydrophilic polymer, where the hydrophilic polymer includes PEG, polyoxazoline, polyacetal (referred to in some instances as FleximerTM), HPMA, and polyglycerol.
  • grafted polymers is a polyacetal polymer grafted with a hydrophilic polymer further comprised of a ligand grafted to the hydrophilic polymer, where the ligand includes peptides, carbohydrates, vitamins, nutrients, and antibodies or their fragments.
  • RNAi the double stranded RNA (dsRNA)- induced sequence-specific degradation of messenger RNA (mRNA), often called gene silencing, has been proven to be a powerful tool for gene discovery and it holds great potential in targeted therapeutics.
  • dsRNA double stranded RNA
  • mRNA messenger RNA
  • mVEGF-A the double stranded RNA
  • mVEGF-Rl the double stranded RNA
  • mVEGF-R2 messenger RNA
  • the siRNAs are 21 -nucleotide long double stranded RNAs with 2-nt overhangs at either 3' termini, with the negative strand complementary to the targeted mRNA sequences.
  • siRNA duplexes targeting mVEGF, mVEGFRl and mVEGFR2.
  • Six siRNA duplexes were designed to target each of mVEGF, mVEGFRl and mVEGFR2 using algorithms from the public domain and chemically synthesized by Qiagen (Germantown, MD).
  • siRNAs were 21-nt long double stranded RNA oligos with dTdT overhang at 3' end without further modification (Table 26). After screenings for gene silencing potency in cell cultures, two out of six duplexes were selected for targeting the specified gene and those two were used in an equal molar mixture.
  • siRNA duplexes were used in pairs, the new name for each pairs are as follows: simVEab for mVEGF-siRNAab, simRlab for mVEGFRl-siRNAab, simR2ab for mVEGFR2-siRNAab, siClab for Control 1-siRNAab and siC2ab for Control2-siRNAab.
  • simVEGFmix The siRNA cocktail containing simVEab, simRlab and simR2ab with equal molar ratio was named as simVEGFmix.
  • HKP Optimal branched histidine-lysine polymer
  • HKP was synthesized on a Ranin Voyager synthesizer (PTI, Arlington, AZ) and complexed with siRNA duplexes as described previously (70) for local administration.
  • the HKP was dissolved in aqueous solution and then mixed with siRNA aqueous solution at a ratio of 4:1 by mass, forming nanoparticles of average size of 150-200 nm in diameter.
  • the targeted polymeric siRNA nanoparticles for systemic administration were prepared by chemical synthesis of tripartite polymer conjugate RPP, a PEGylated form of branched polyethyleneimine (PEI) having an RGF peptide at its distal end ( RGD-PEG- PEI) (71).
  • PEI branched polyethyleneimine
  • RGD-PEG- PEI RGD-PEG- PEI
  • RPP- siRNA nanoparticles can be self-assembled in aqueous solution by simply mixing two solutions together with a 2:1 molar ratio.
  • the measurements of particle size (120-150 nm) and ⁇ -potential indicate that the RPP- siRNA particles exhibit colloidal surface properties indicative of an outer steric polymer layer and potentially exposed RGD ligand to mediate cell-binding selectivity.
  • the HKP-siRNA and RPP-siRNA aqueous solutions were semi- transparent without noticeable aggregation of precipitate, and can be stored at 4 C for at least three months.
  • NV corneal neovascularization
  • the HSK model 1 2 was established using BALB/C (H-2 d ) mice 6-8 weeks of age purchased from Harlan Sprague- Dawley (Indianapolis, IN).
  • the NV in corneal stromal in the HSK model were induced through either corneal implantation of CpG-ON or HSV-I infection 1 ' 2 .
  • the CpG-ON induced model requires micropocket being made around 1 mm from the limbus under a stereomicroscope with pellets containing 1 ⁇ g of CpG-ON.
  • Angiogenesis was evaluated at day 4 and 7 post the pellet implantation by comparison of NV areas and the VEGF protein levels 34 .
  • HSV-induced HSK model was established by introducing 2 ⁇ l drop containing 1 x 10 5 plaque-forming units (PFU) of HSV-I RE into the mouse corneas with a 30-gauge needle.
  • PFU plaque-forming units
  • the NV in HSK model was evaluated through comparison of the NV area, angiogenesis score and clinical score, through 22 day period post the infection.
  • the animal studies followed guidelines of the Committee on the Care of Laboratory Animals Resources, Commission of Life Sciences, National Research Council. The animal facilities of the University of Tennessee (Knoxville, TN) are fully accredited by the American Association of Laboratory Animal Care.
  • ROP Mouse Retinopathy of prematurity
  • ROP model was established with C57BL/6 mice purchased from Center of Experimental animal of Guangzhou Medical college, Guangzhou University of traditional Chinese Medicine.
  • the retinal NV in ROP model was induced by hyperoxia 3 ' 4 . Briefly, the pups with the nursing dams were maintained in hyperoxia environment (75% + 2 oxygen) from postnatal days P7 to P12, then returned to room air (normoxia), followed by treatment with Polymeric siRNA nanoparticles via different routes of delivery.
  • the ocular NV in ROP model was evaluated with fluorescein perfusion/ flatmounting, cryosection staining, RT-PCR for mRNA levels and ELISA for protein levels. All investigations followed guidelines of the Committee on the Care of Laboratory Animals Resources, Commission of Life Science, National Research Council, China.
  • HKP-siRNA solution (10 ⁇ g/10 ⁇ l/eye) was delivered subconjunctivally (SCJ) with a 32-gauge Hamilton syringe (Hamilton company, Reno, Nevada) at 6 and 24 h after CpG-ON pellets implantation or day 1 and 3 after virus infection under deep anesthesia induced by Avertin (Pittman Moore, Mundelein, IL).
  • the RPP-siRNA solution (40 ⁇ g siRNA/100 ⁇ l/mice) was delivered intravenously (IV) at 6 and 24 h after CpG-ON pellets implantation or day 1 and 3 after virus infection.
  • simVmix is a mixture of the siRNA duplexes mVEGF-siRNA(a), mVEGF-siRNA(b), mVEGFRl-siRNA(a), mVEGFRl-siRNA(b), mVEGFR2-siRNA(a), and mVEGFR2-siRNA(b) (SEQ ID NOS:568-579; see Table 26).
  • HKP- simVmix solution (4 ⁇ g/2 ⁇ l/eye) was delivered either subconjunctivally (SCJ) or intravitreously (IVT) in the left eye and HKP-siC2ab (4 ⁇ g/2 ⁇ l/eye) in the right eye.
  • the RPP-simVmix solution (15 ⁇ g siRNA/50 ⁇ l/mice) was given intraperitoneally (IP). Both local and systemic administrations were in two regimens: P12, P 14, and P 16; or only on P 12 and P13. Mice were sacrificed followed by evaluations of NV status and the expressions of the targeted genes at P 14 and P 17. Retinal angiography.
  • RNA samples were tested by mRNA-specific PCR (RS-PCR) as described previously.
  • the set of primers for each mRNA include a 47-mer mRNA-specific primer for reverse transcription reaction (RTP), a 5'-end gene specific primer (GP) and a 3'-end universal primer (see Table 27). The lower cases indicated the sequences specific to the targets for reverse transcriptions.
  • the RNA samples were also quantified with GAPDH and ⁇ -actin specific RT-PCR. All PCR products were subjected to the gel electrophoresis analysis and quantification. Table 27.
  • VEGF and VEGFl, VEGFR2 ELISA analyses for VEGF and VEGFl, VEGFR2.
  • Retinas were collected after mice were sacrificed at P 14 and P 17, and homogenized in cell lysis buffer (Mammalian cell lysis Kit, Biotechnology Department Bio Basid Inc, Canada). The supernatants were subjected to ELISA analysis using BCA protein quantitative analysis Kit (Shenery Biocolor Bioscience & Technology Company, China).
  • Levels of VEGF, VEGFRl, and VEGFR2 were determined using the Quantikine M Murine VEGF, sVEGFRl, and sVEGFR2 Immunoassay Kits respectively (R&D Systems Inc., Minneapolis, MN). Six to 12 tissue samples were analyzed for each group and each time point.
  • VEGF vascular endothelial growth factor
  • VEGFRl vascular endothelial growth factor
  • VEGFR2 vascular endothelial growth factor 2
  • X70842 X70842
  • the siRNA duplexes were chemically synthesized by Qiagen (Germantown, MD, USA). Potencies of the siRNA duplexes for silencing the targeted genes were evaluated by in vitro transfection of RAW264.7 gamma NO (-) cells and SVR cells, followed by RT-PCR and ELISA analyses. Two most potent siRNA duplexes for each gene target were selected from the eight candidates (Fig.
  • the simVEab is able to target all three isoforms 38 of murine VEGF including VEGFigs, VEGF 164 and VEGF 12 O • These three pairs of siRNA agents were selected as the payloads of the nanoparticles for in vivo studies with murine ocular NV models.
  • Herpetic stromal keratitis (HSK) model was generated with CpG-ON pellet implant and HSV infection as described previously 34 , which represents a clinically relevant model of corneal neovascularization with typical characteristics of inflammation induced angiogenesis and lymphangiogenesis 40 . Measurements of the angiogenesis areas, HSK disease scores and protein expression levels are the most effective ways to evaluate the siRNA-mediated anti- angiogenesis activities in the anterior section of the eyes.
  • the retinopathy of prematurity (ROP) model reflects the characters of retinal NV with typical pathogenesis of ischemic and degenerative diseases such as PDR and AMD 41 .
  • Measurements of the angiogenesis areas through perfusion/flatmount, cryosection , and mRNA and protein expression levels are useful tools to evaluate anti-angiogenesis activity in the posterior section of the eyes.
  • integrin receptor subtypes ⁇ v ⁇ 3 and ⁇ v ⁇ 5 are selectively expressed in the proliferative endothelium of corneal, retinal and choroidal neovascular membranes 14 ' 15> 16 , which have been indicated as the ideal targets for RGD ligand binding 17> 34> 42 .
  • HK polymers Two polymeric carriers were selected for in vivo delivery of siRNA duplexes through either local or systemic administrations.
  • Optimal branched histidine-lysine (HK) polymers have been applied for siRNA deliveries in vitro 43 and in vivo 44 .
  • a particular species of the HK polymer (PT73, named as HKP) has a lysine backbone with four branches containing multiple repeats of Histidine, Lysine and Asparagine.
  • PT73 histidine-lysine
  • Asparagine Asparagine
  • HKP-siRNA solutions were used for the subconjunctival (SCJ) or intravitreous (IVT) administrations in both HSK and ROP models (Fig. 2c-2d).
  • SCJ subconjunctival
  • IVT intravitreous
  • a more complicated tripartite polymer conjugate, PEGylated branched polyethyleneimine (PEI) having an RGF peptide at its distal end (RGD-PEG-PEI, in short as RPP) 45 was applied in two previous in vivo studies 33> 34 .
  • PEI PEGylated branched polyethyleneimine
  • RPP RGF peptide at its distal end
  • RPP-siRNA nanoparticles can also be self-assembled in aqueous solution with sizes about 80-120 nm in diameter by simply mixing two solutions together with a 2: 1 molar ratio.
  • the RGD ligand directed RPP- siRNA nanoparticles were able to preferentially reach to the CpG-ON induced ocular NV tissue through IV delivery and to knockdown the VEGF expression which is the key factor for activation of the local angiogenesis.
  • SiRNA cocktail exhibited stronger anti-angiogenesis activity
  • VEGF, VEGFRl and VEGFR2 are key players in ocular NV through activation of HA and LA processes activated by CpG-ON induced inflammation 1 ' 2 . Therefore, inhibiting expression of each of these genes with the specific siRNA duplexes should down regulate progression of the ocular NV 35> 36 .
  • Systemically delivered RPP-simVEab, -simRlab and - simR2ab nanoparticles (40 ⁇ g/100 ⁇ l/eye) significantly minimized angiogenesis areas compared to the no treatment and siClab treated groups (Fig. 4a).
  • siRNA cocktail nanoparticles HKP-simVmix (see page 51; 4 ⁇ g/2 ⁇ l/eye) through SJV injection and RPP-simVmix (40 ⁇ g/100 ⁇ l/eye) through IV injection, at 6 and 24 hours after CpG-ON induction of corneal NV in mice (HSK model) as described in the method section.
  • the angiogenesis areas of each treatment groups were measured on day 4 and 7 followed by comparisons between the control and treatment groups (Fig. 5).
  • the siRNA cocktail treatments resulted in stronger anti-angiogenesis activities in the HSK models (Fig. 4c-d) compared to siRNAs targeting single gene (Fig.
  • HSV infection causes severe ocular NV and corneal lesion that usually present between 14 - 22 days after the infection.
  • both locally and systemically delivered siRNA cocktail nanoparticles displayed similar anti-angiogenesis benefits in this HSV induced HSK model.
  • the pharmacodynamics of both locally delivered HKP-simVmix and systemically delivered RPP-simVmix were very similar during the entire course of disease progression.
  • the 40% of disease inhibition at p22 also demonstrated the durable anti- angiogenesis potencies of both formulations.
  • the slightly higher scores for HKP-siClab treated group at P6 and PlO may be due to the physical prying by local injections of the nanoparticles.
  • the siRNA cocktail treatment the HSV infected eyes became highly angiogenic and finally deformed (Fig. 6b).
  • the systemically administrated RPP-simVmix demonstrated stronger inhibition for corneal NV (Fig. 7b) compared to the siRNA targeting only a single gene. (Fig. 7, c-e).
  • SiRNA cocktail displayed a potent anti-angiogenesis activity in ROP model
  • FIG. 8c A selection of the images for perfusion/flatmount analysis (Fig. 9) is demonstrated in Fig. 8c.
  • the HKP-simVmix and RPP-simVmix show marked differences between treatment groups and control groups (Fig. 8c, panels 2, 6, 3,7; see Figure Legend).
  • the similar therapeutic benefit could also be observed in Fig. 8d, where the samples from the groups treated with the siRNA cocktail through either IVT or IP delivery display minimum NV (Fig. 8d, panels 6-7), but the samples from the control groups revealed large fronds of retinal and subretinal NV extending above the internal limiting membrane (Fig. 8d, panels 2- 3).
  • siRNA cocktail (simVmix) exhibited potent anti-angiogenesis efficacy in vivo, delivered via either local or systemic routes, packaged with either HKP or RPP, and examined in either HSK or ROP model, we determined whether those anti-angiogenesis activities really came from silencing all three key angiogenesis factors in the same temporal and spatial locations.
  • Q-RT-PCR quantitative RT-PCR
  • the siRNA cocktail approach divulge a new paradigm that multiple targeted therapeutics are more efficacious than the single targeted therapeutics (Fig. 1 Ic) for treatment of ocular NV diseases.
  • siRNA cocktail simVmix
  • This simVmix is able to simultaneously knockdown genes for both angiogenic cytokine (VEGF) and its receptors (VEGFRl and VEGFR2), by reaching both retinal pigment epithelium cells and retinal vascular membrane endothelium cells through either local or systemic delivery, providing therapeutic benefit demonstrated in the ROP murine model.
  • VEGF angiogenic cytokine
  • VEGFRl and VEGFR2 angiogenic cytokine
  • This indicates that potent anti- angiogenesis efficacy is more likely to accrue from a cocktail approach than from targeting single target such as can be done with monoclonal antibody 10> u or chimerical receptor 7 drugs.
  • HKP and RPP polymeric nanoparticles have clearly demonstrated their capabilities for local and systemic deliveries of siRNA active agents.
  • the cyclic RGD ligand used in this study possesses high affinity to the integrin receptors, ⁇ v ⁇ 3 and ⁇ v ⁇ 5, which are not expressed in the normal and proliferative vitreoretinopathic tissues, but selectively expressed in corneal neovascular membranes 42 , retinal and choroidal neovascular membranes 16 ⁇ 20 .
  • Our data affirm that RPP-simVmix activity is mediated through RGD binding to ⁇ v ⁇ 3 and ⁇ v ⁇ 5 integrins expressed on the cell membrane of neovascular endothelium.
  • the unequivocal identification and quantification of new vessels and measurement of angiogenesis areas employed in the Examples enable objective data collection.
  • the local delivery effect and systemic delivery effect can be clearly distinguished.
  • the HSK model with severe corneal NV and ROP model with serious retinal NV reflect characteristics of almost all kinds of ocular angiogenesis diseases.
  • HSK or RPP carriers with either local or systemic delivery, are applicable for (1) inflammatory corneal NV such as HSK, Uveitis, Scleritis and Iritis; (2) ischemic retinal NV such as PDR, ROP and retinal vein occlusion (RVO); and (3) degenerative choroidal NV, AMD, polypoidal choroidal vasculopathy (PCV) and pathologic myopia, etc.
  • inflammatory corneal NV such as HSK, Uveitis, Scleritis and Iritis
  • ischemic retinal NV such as PDR, ROP and retinal vein occlusion (RVO)
  • degenerative choroidal NV AMD
  • PCV polypoidal choroidal vasculopathy
  • pathologic myopia etc.
  • siRNA cocktail administrated with polymeric nanoparticle, requires us to carefully evaluate the potential toxicity in vivo throughout the entire observation on both HSK and ROP murine models. Except some minor injures in the ocular tissue due to the repeated deliveries of polymeric-siRNA nanoparticles intravitreously into young mice (Table 29), we did not fine any physical, behavioral and appearance change of the treated mice, after repeated systemic administrations of RPP- simVmix nanoparticles, even with less than two week old C57BL/6 mice. This observation indicates that the dosage and regimen we used in those treatments were safe. Therefore, this siRNA cocktail therapeutic approach may possess a wide therapeutic window for the treatment of ocular NV.
  • Murine VEGF Accession: M95200
  • siRNA sequences were selected (forward sequence): SEQ ID NO: simVEa: GCCGUCCUGUGUGCCGCUGdtdt; 541 simVEb: CGAUGAAGCCCUGGAGUGCdtdt; 543 simVE-1: GUGGUCCCAGGCUGCACCCdtdt; 568 simVE-2: GAUCCGCAGACGUGUAAAUdtdt; 569 simVE-3: ACACAGACUCGCGUUGCAAdtdt; 570 simVE-4: CACAGACUCGCGUUGCAAGdtdt; 571 simVE-5: GGCGAGGC AGCUUGAGUUAdtdt; 572 simVE-6: ACGAACGUACUUGCAGAUGdtdt. 573
  • siRNA sequences were selected (forward sequence): simRla: GUUAAAAGUGCCUGAACUGdtdt 545 simRlb: GCAGGCCAGACUCUCUUUCdtdt 547 simRl-1: GGAGAGGACCUGAAACUGUdtdt, 574 simRl-2: GCAAGGAGGGCCUCUGAUGdtdt, 575 simRl-3: GGAGGGCCUCUGAUGGUGAdtdt, 576 simRl-4: CUACCUC AAGAGCAAACGUdtdt, 577 simRl-5: GUGGCCAGAGGCAUGGAGUdtdt, 578 simRl-6: AGUGCAUUCAUCGGGACCUdtdt. 579
  • Murine VEGFR-2 (FLK-I), Accession: X70842
  • siRNA candidates were selected (forward sequence): simR2a: GCUCAGCACACAGAAAGACdtdt 549 simR2b: UGCGGCGGUGGUGACAGUAdtdt 551 simR2-l: CAGAAUUUCCUGGGACAGCdtdt, 580 simR2-2: CUGAAGACAGGCUACUUGUdtdt, 581 simR2-3: GGACUUCCUGACCUUGGAGdtdt, 582 simR2-4: GUGGCUAAGGGCAUGGAGUdtdt, 583 simR2-6: AUGUACCAGACCAUGCUGGdtdt, 584 simR2-8: CAGUAAGCGAAAGAGCCGGdtdt, 585 Table 29. Toxicity responses to the intravitreal administration of simVmix.
  • Human colorectal carcinoma cells, DLD-I and human breast carcinoma cells, MDA-MB-435 American Type Culture Collection (ATCC; Centerville VA) were generally cultured and rinsed twice with 0.25% trypsin, 0.53 mM EDTA solution at room temperature (or at 37C) until the cells detached. Fresh culture medium was added, the cells were aspirated and dispensed into new culture flasks. Cells were transfected with naked siRNA using Lipo2000 (Invitrogen, Carlsbad, CA) according to the protocol provided by the vendor. Either 0.3 and 2.0 ug blunt ended siRNA was transfected in a given experiment.
  • Lipo2000 Invitrogen, Carlsbad, CA
  • the 25 mer siRNA 5'-ccaugccaaguggucccaggcugca-3' targets both human and mouse VEGF.
  • the 21 mer siRNA 5'- aaucgagacccugguggacau-3' targets human VEGF.
  • the mock was treated with buffer alone.
  • the amounts of mRNA were determined by quantitative-RT-PCR analysis using TaqMan® Gene Expression system (ABI, Forest City, CA).
  • Fig. 13 show that the 25 mer siRNA (SEQ ID NO: 596) is more potent for silencing effects tested than the 21 mer siRNA (SEQ ID NO 624).
  • VEGF-A Human embryo kidney cells, 293, and mouse myeloma cells, F3, were cultured and transfected with 8 different siRNA sequences designated VEGF-A through VEGF-H targeting human VEGF.
  • the expression ratio is determined by the changes of mRNA.
  • the amounts of mRNA were determined by quantitative-RT-PCR analysis using TaqMan® Gene Expression system (ABI, Forest City, CA).
  • Fig. 14 The results shown in Fig. 14 indicate that differing targeted sequences within the VEGF have differing susceptibilities for suppression of gene expression.
  • the siRNA designated VEGF-C is most effective in both human and mouse cells.
  • VEGF-Rl specific siRNA duplexes Comparison of effectiveness of five 25 mer VEGF-Rl specific siRNA duplexes in suppressing gene expression.
  • Mouse SVR cells were cultured and transfected with five different siRNA molecules each of which targets orthologous mouse and human genes, or with siRNA targeting green fluorescent protein (GFP), or were transfected with buffer alone. Either 0.3 or 2.0 ug siRNA per experiment was used. Relative expression levels compared to untreated cells were determined. The siRNA sequences used were
  • duplex molecules for use in various mixtures disclosed herein are listed in Table 34. duplex molecules
  • RNAs diced small interfering RNAs (siRNAs) specific to vascular endothelial growth factor (VEGF). Curr Eye Res. 2006; 31(2):171-80.
  • VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest. 2004; 113(7): 1040-50.
  • VEGF-D is the strongest angiogenic and lymphangiogenic effector among VEGFs delivered into skeletal muscle via adenoviruses. Circ Res. 2003; 92(10):1098-106.
  • VEGF-TRAP(R 1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier. J Cell Physiol. 2003; 195(2):241- 8.
  • Campochiaro PA Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders. Gene Ther. 2006; 13(6):559-62.
  • VEGF isoforms and their expression after a single episode of hypoxia or repeated fluctuations between hyperoxia and hypoxia: relevance to clinical ROP. MoI Vis. 2004 JuI 21; 10:512-20.

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Abstract

La présente invention concerne des compositions et procédés d'utilisation de mélanges d'ARNsi avec des systèmes d'administration appropriés pour traiter diverses maladies oculaires. L'invention comprend des compositions de séquences ciblées ARNsi, de duplex d'ARNsi et des mélanges d'ARNsi pour inhiber l'expression de gènes provoquant une maladie oculaire, soit à partir d'un virus infectieux soit apparaissant spontanément chez le patient. En outre, les compositions comprennent des polymères porteurs pour des agents d'ARNsi qui délivrent les agents à l'œil pour un traitement thérapeutique efficace.
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