Patent Publication Number: US-8541568-B2

Title: Compositions and methods using siRNA molecules for treatment of gliomas

Description:
This application claims priority to U.S. Provisional Patent Application No. 61/055,966, filed May 24, 2008, which is incorporated herein by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     This application is a U.S. national phase application of, and claims priority to and the benefit of, International Patent Application No. PCT/US2009/003196, filed May 26, 2009, which claims priority to, and the benefit of, U.S. Provisional Patent Application No. 61/055,966, filed May 24, 2008. The disclosures of these applications are expressly incorporated herein by reference in their entireties. 
     BACKGROUND 
     Major advances in molecular biology, cellular biology, and genomics have substantially improved our understanding of cancer. Now, these advances are being translated into therapy. Targeted therapy directed at specific molecular alterations is already creating a shift in the treatment of cancer patients. Glioblastoma multiforme (GBM), the most common brain cancer of adults, is highly suited for this new approach. GBM is among the most aggressive and deadly of neoplasms. Despite decades of aggressive surgical treatment, chemotherapy, radiotherapy, and extensive basic science and clinical research focused on combating this disease, the prognosis remains virtually unchanged, with survival rates still measured in months. The current genetic understanding of GBM has led to the identification of crucial intracellular molecules and their associated signaling pathways as potential therapeutic targets. Multiple genes are being identified as critical to the development of GBM and other similar tumors, such as gliomas and other astrocytomas. 
     Targets Involved in GBM 
     GBM, like most malignant tumors, exhibits multiple genetic abnormalities, including aberrant activation of intracellular signal-transduction pathways that regulate processes such as proliferation, angiogenesis, and apoptosis. For example, vascular endothelial growth factor (VEGF) is needed to promote tumorigenesis and angiogenesis in GBM (7). In addition, over 40% of all GBMs exhibit amplification, mutation, or rearrangement of the epidermal growth factor receptor (EGFR) (2). As is common in malignant neoplasms, EGFR overexpression leads to aberrant activation of crucial downstream targets, including the PI3K/AKT signaling pathway, which mediates cell survival, and the mitogenic RAS/MAPK cascade. Recent studies implicate a mutant variant of EGFR, EGFRvIII, in the activation of other receptor tyrosine kinases (RTKs), such as MET, thereby providing multiple inputs to these downstream signaling pathways for cellular proliferation and evasion of apoptosis. This redundant signaling could partially account for the modest response of GBMs to RTK inhibitors, such as erlotinib and gefitinib. In addition, GBM is frequently associated with elevated levels of O 6 -methylguanine-DNA-methlytransferase (AGT), a DNA repair enzyme that enhances neoplastic resistance to chemotherapeutics such as Temozolomide (TMZ). 
     Matrix metalloproteinases (MMPs) enhance tumor cell invasion by degrading extracellular matrix proteins, by activating signal transduction cascades that promote motility, and by solubilizing extracellular matrix-bound growth factors. MMP-9 and MMP-2 promote GBM invasion in vitro and in xenograft models, and their inhibition dramatically reduces the invasive phenotype (1). The serine/threonine kinase Raf-1 is involved in the regulation of tumor cell survival, proliferation and metastasis formation, and has therefore emerged as a promising target for cancer therapy. Raf-1 silencing appears as a potential therapeutic strategy to inhibit brain tumor angiogenesis and thereby outgrowth of highly vascularized glioblastoma multiforme (28). RNA interference targeting TGF-beta 1, 2 results in a glioma cell phenotype that is more sensitive to immune cell lysis and less motile in vitro and nontumorigenic in nude mice (29). The mammalian target of rapamycin (mTOR) activity is required for the survival of some cells within these GBMs, and mTOR appears required for the maintenance of astrocytic character in the surviving cells (30). A small molecule inhibitor of Cox-2 enhanced glioblastoma radiosensitivity, reduced tumor cell viability and prolonged survival of implanted glioblastoma mice by inhibition of tumor angiogenesis and causing extensive tumor necrosis (31). 
     RNA Interference (RNAi) and Small Interfering RNA (siRNA) 
     RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knock down, or silence, theoretically any gene. In naturally occurring RNAi, a double-stranded RNA (dsRNA) is cleaved by an RNase III/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-27 nucleotides (nt) with 2-nt overhangs at the 3′ ends. These siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced silencing complex (RISC). One strand of siRNA remains associated with RISC and guides the complex toward a cognate RNA that has sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it. Recent studies have revealed that chemically synthesized 21-27-nt siRNAs exhibit RNAi effects in mammalian cells, and the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule&#39;s function. These and other characteristics of RISC, siRNA molecules, and RNAi have been described. 
     Applications of RNAi in mammalian cells in the laboratory or, potentially, in therapeutic settings, use either chemically synthesized siRNAs or endogenously expressed molecules. The endogenous siRNA is first expressed as small hairpin RNAs (shRNAs) by an expression vector (plasmid or virus vector) and is then processed by Dicer into siRNAs. It is thought that siRNAs hold great promise to be therapeutics for human diseases, especially those caused by viral infections. Certain siRNA therapeutics are described in PCT application PCT/US2005/003858 for “Compositions and Methods for Combination RNAi Therapeutics”, which is incorporated herein by reference in its entirety. 
     Importantly, it is presently not possible to predict with any degree of confidence which of many possible candidate siRNA sequences potentially targeting a genomic sequence (e.g., oligonucleotides of about 16-30 base pairs) will in fact exhibit effective siRNA activity. Instead, individual, specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested to determine whether the intended interference with expression of a targeted gene has occurred. Accordingly, no routine method exists for designing an siRNA polynucleotide that is, with certainty, capable of specifically altering the expression of a given mRNA. 
     SUMMARY OF THE INVENTION 
     The present invention provides an isolated small interfering RNA (siRNA) molecule that binds to a single-stranded RNA molecule, wherein the single-stranded RNA molecule comprises an mRNA that encodes at least part of a peptide or protein whose activity promotes tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal, or wherein the single stranded RNA molecule comprises an miRNA whose activity promotes tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal. 
     The invention also provides a composition comprising at least three of the siRNA molecules. In one embodiment, the molecules bind to multiple, different target sequences in the single-stranded RNA molecule or in different single stranded RNA molecules. 
     The invention further provides compositions comprising one or more of the siRNA molecules in a pharmaceutically acceptable carrier. These compositions may include, or be used with, a therapeutic agent that prevents or hinders tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal. 
     The invention also provides a method for treating a glioma in a subject comprising administering to the subject an effective amount of a composition of the invention. In one embodiment, the subject is a human. In another embodiment, the glioma is a glioblastoma multiforme (GBM). 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 . Identification of siRNA duplexes silencing MGMT expression. The total RNA and protein samples were collected after H80TR cells were transfected with corresponding siRNA duplexes, followed by RT-PCR, quantitative PCR and Western blot analyses. Selection of potent siRNA duplexes targeting both human and mouse MGMT genes. 
         FIG. 2 . Identification of siRNA duplexes silencing EGFR expression. The total RNA and protein samples were collected after H898 cells were transfected with corresponding siRNA duplexes, followed by RT-PCR, quantitative PCR and Western blot analyses. Selection of potent siRNA duplexes targeting both human and mouse EGFR. 
         FIG. 3 . Identification of potent siRNA duplexes silencing VEGF-A expression. The total RNA and protein samples were collected after 293 cells and F3 cells were transfected with corresponding siRNA duplexes, followed by quantitative PCR analyses. Selection of potent siRNA duplexes targeting both human and mouse VEGF. 
         FIG. 4 . Selection of potent siRNA for MMP-2 knockdown. On the day before transfection, 2.5×10 5  PC-3 cells were plated on the wells of 6-well plates in 2 ml of culture medium without antibiotics. On the next day, the cells were transfected with siRNA-hMMP-2 #1, #2, and #3 and Luc siRNA at different concentrations—0.5 μg/well, 1.0 μg/well, and 2.0 μg/well. For each transfection sample (in duplicate), oligomer-Lipofectamine 2000 complexes were prepared: 
       250 μl of Optimem I Reduced Serum Medium+1.4 μl siRNA (0.5 μg) (28 pmole/well) 
       250 μl of Optimem I Reduced Serum Medium+2.8 μl siRNA (1 μg) (56 pmole/well) 
       250 μl of Optimem I Reduced Serum Medium+5.6 μl (siRNA (2 μg) (112 pmole/well) 
       Small interfering ultimobranchial body cells were used for negative control (0.5 μg/well-28 pmole). After 6 hours, the medium was changed. mRNA was isolated by using an RNAqueous-4PCR kit. 
         FIG. 5 . Selection of potent MMP-9 siRNA. mRNA knockdown after transfection of the PC-3 cells with siRNA (hMMP-9). On the day before transfection, 2.5×10 5  PC-3 cells were plated on the wells of 6-well plates in 2 ml of culture medium without antibiotics. On the next day, cells were transfected with siRNA hMMP-9 #1, siRNA hMMP-9 #2, siRNA hMMP-9 #3, and siRNA Luc at different concentrations—0.9 μg/well (50 pmole), 1.8 μg/well (100 pmole), and 2.7 μg/well (150 pmole). After 6 hours, the medium was changed. Cells were incubated at 37° C. for 48 hours. 
         FIG. 6 . Selection of potent PDGF siRNA using the same procedures as above. 
         FIG. 7 . Comparison of silencing potencies between 25mer and 21mer siRNA duplexes. The most potent 25 mer and 21mer siRNA were selected first from each set of 6 duplexes. Then comparison was carried out with two tumor cell lines expressing human VEGF protein (DLD-1, colon carcinoma and MBA-MD-435, breast carcinoma) using in vitro transfection with Lipo2000 (Invitrogen, CA) followed by RT-PCR analyses. At either 0.3 μg or 2.0 μg doses, 25mer siRNA demonstrated stronger inhibitory activity than 21mer siRNA, especially at 2.0 μg. We found that 25mer duplexes with blunt ends are the most potent inhibitors, up to 60% either MBA-MD-435 or DLD-1 cells and in tumor bearing in animals. We have tested a 25mer siRNA duplex targeting human VEGF gene, hVEGF-25c (sense: 5′-CACAACAAAUGUGAAUGCAGACCAA-3′ (SEQ ID NO: 1); Antisense: 5′-UUGGUCUGCAUUCACAUUUGUUGUG-3′ (SEQ ID NO: 2)), comparing to a 21mer siRNA duplex which has been tested many times as one of the most potent VEGF specific inhibitory duplexes, hVEGF-21a (sense: 5′-UCGAGACCCUGGUGGACAUTT-3′ (SEQ ID NO: 3); antisense: 5′-AUGUCCACCAGGGUCUCGATT-3′ (SEQ ID NO: 4)), in the cell culture followed with Q-RT-PCR analysis.  FIG. 7  demonstrates that the 25 mer blunt end siRNA is more potent than the 21mer sticky end siRNA, which supports using 25mer siRNA duplexes in the proposed study of multi-targeted siRNA cocktail therapeutics for cancer treatment. 
         FIG. 8 . Selection of potent siRNA targeting RAF-1. (A) The lower panel illustrates selection of eight 25 mer siRNA duplexes with control siRNA were transfected into human MDA-MB-231 cells and mouse CT26 cells. 24 hr later, mRNA were collected and subject to Q-RT-PCR with the standard control gene target Rigs15. Based on the gene silencing activity observed, the most potent Raf1-siRNA was selected as a component of a future siRNA cocktail for both in vitro and in vivo study. RAF-1-siRNA: 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ (SEQ ID NO: 5). 
         FIG. 9 . Selection of potent siRNA targeting mTOR. (A) The lower panel illustrates selection of eight 25 mer siRNA duplexes with control siRNA were transfected into human MDA-MB-231 cells and mouse CT26 cells. 24 hr later, mRNA were collected and subject to Q-RT-PCR with the standard control gene target Rigs15. Based on the gene silencing activity observed, the most potent mTOR-siRNA was selected as a component of a future siRNA cocktail for both in vitro and in vivo study. mTOR-siRNA: 5′-GGUCUGGUGCCUGGUCUGAUGAUGU-3′ (SEQ ID NO: 6). 
         FIG. 10 . RT-PCR analysis for selection of potent siRNA targeting TGFβ1 The silencing activities of three siRNA oligos targeting the TGFβ-1 gene expression was demonstrated through gel electrophoresis analysis. A potent siRNA oligo was identified based on the silencing activities in both human and moue cells. hmTGFβ1: sense, 5′-CCCAAGGGCUACCAUGCCAACUUCU-3′ (SEQ ID NO: 7), antisense, 5′-AGAAGUUGGCAUGGUAGCCCUUGGG-3′ (SEQ ID NO: 8). 
         FIG. 11 . RT-PCR analysis for selection of potent siRNA targeting Cox-2 The silencing activities of three siRNA oligos targeting the Cox-2 gene expression was demonstrated through gel electrophoresis analysis. A potent siRNA oligo was identified based on the silencing activities in both human and moue cells. hmCox-2: sense, 5′-GGUCUGGUGCCUGGUCUGAUGAUGU-3′ (SEQ ID NO: 6), antisense, 5′-ACAUCAUCAGACCAGGCACCAGACC-3′ (SEQ ID NO: 9). 
         FIG. 12 . HKP for in vivo siRNA Delivery (A) Polymeric Nanoparticle-siRNA Systems Histidine-Lysine polymer (HKP) mixed with siRNA duplexes resulted in HKP-siRNA nanoparticles. Structures of two species of HKP, H3K4b and PT73, were showed with the scanning electron microscope (SEM) image of HKP-siRNA nanoparticle.  FIG. 12A  discloses SEQ ID NOS 255-256, respectively, in order of appearance. (B) Cocktail siRNAs are more potent than single siRNA for anti-angiogenesis efficacy of siRNA cocktail on ocular NV. Local delivery of HKP-siRNA cocktail (black bar) significantly minimized angiogenesis areas in mouse eyes at P4 (**P&lt;0.01, n=8) compared to single gene targeted HKP-siRNA: VEGF (grey), VEGFR1 and VEGFR2 (stripe and dot) at P4 ((*P&lt;0.05). At P7, VEGF and VEGFR1 specific siRNA lost the inhibitory activities, while the cocktail siRNA are still demonstrated its activity. 
         FIG. 13 . HKP for Systemic Tumor siRNA Delivery Accumulation of intravenously (IV) delivered HKP-encapsulated siRNA molecules in established head and neck squamous cancer 1483 xenograft tumors. A: Images of mouse tissues and tumors with Alexa Fluor 555 labeled fluorescent siRNA CT-2 deposits following tail vein injection of HKP-siRNA. The tissues were harvested at the indicated time, freshly frozen, sectioned, and analyzed by fluorescent microscopy. Magnification: 400×. B: Accumulation of fluorescent CT-2 (Red deposits indicated with arrows) in the tumors in proximity to blood vessels (Brown; CD31 immunostaining). Magnification: 400×. Inset: Detail showing labeled siRNA in tumor tissue. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention provides siRNA molecules, compositions containing the molecules, and methods of using the compositions to treat a glioma in a subject. As used herein, an “siRNA molecule” is a duplex oligonucleotide, that is a short, double-stranded oligonucleotide, that interferes with the expression of a gene in a cell that produces RNA, after the molecule is introduced into the cell. Such molecules are constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos. 5,898,031, 6,107,094, 6,506,559, 7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties. 
     The siRNA molecule of the invention is an isolated siRNA molecule that binds to a single stranded RNA molecule, which is a messenger RNA (mRNA) that encodes at least part of a peptide or protein whose activity promotes tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal, or which is a micro-RNA (miRNA) whose activity promotes tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal. In one embodiment, the molecule is an oligonucleotide with a length of about 19 to about 35 base pairs. In another embodiment, the molecule is an oligonucleotide with a length of about 19 to about 27 base pairs. In still another embodiment, the molecule is an oligonucleotide with a length of about 21 to about 25 base pairs. In all of these embodiments, the molecule may have blunt ends at both ends, or sticky ends at both ends, or a blunt end at one end and a sticky end at the other. 
     The siRNA molecule can be made of naturally occurring ribonucleotides, i.e., those found in living cells, or one or more of its nucleotides can be chemically modified by techniques known in the art. In addition to being modified at the level of one or more of its individual nucleotides, the backbone of the oligonucleotide also can be modified. Additional modifications include the use of small molecules (e.g. sugar molecules), amino acid molecules, peptides, cholesterol and other large molecules for conjugation onto the siRNA molecules. 
     In one embodiment, the siRNA molecule binds to an mRNA that encodes at least part of a peptide or protein whose activity promotes tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal. Such may be the case when the mRNA molecule encodes a protein in a pro-tumorigenic pathway, pro-angiogenesis pathway, pro-cell proliferation pathway, or anti-apoptotic pathway. For example, the protein can be a VEGF pathway protein, EGFR pathway protein, MGMT pathway protein, RAF pathway protein, MMP pathway protein, mTOR pathway protein, TGFβ pathway protein, or Cox-2 pathway protein. In one embodiment, the protein is one of the following: VEGF, EGFR, PI3K, AKT, AGT, RAF1, RAS, MAPK, ERK, MGMT, MMP-2, MMP-9, PDGF, PDGFR, IGF-1, HGF, mTOR, Cox-2, or TGFβ1. In another embodiment, the protein is VEGF, EGFR, MGMT, MMP-2, MMP-9, or PDGF. In still another embodiment, the protein is RAF1, mTOR, Cox-2, or TGFβ1. 
     In one embodiment, the siRNA molecule binds to both a human mRNA molecule and a homologous mouse mRNA molecule. That is, the human and mouse mRNA molecules encode proteins that are substantially the same in structure or function. Therefore, the efficacy and toxicity reactions observed in the mouse disease models (e.g. tumor models) will allow us to have a good understanding about what is going to happen in humans. More importantly, the siRNA molecules tested in the mouse model are good candidates for human pharmaceutical agents. The human/mouse homology design of an siRNA drug agent can eliminate the toxicity and adverse effect of those species specificities observed in monoclonal antibody drugs. 
     In another embodiment, the siRNA molecule binds to an miRNA whose activity promotes tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal. As used herein, an miRNA is a short, single-stranded RNA molecule that down-regulates gene expression through a loose homology binding to the 3′ end of the untranslated region of a particular gene target. Such molecules are transcribed from DNA, but are not translated into a polypeptide. It has been associated with certain diseases (e.g. glioblastoma). 
     Similarly, siRNA molecules can be designed to bind to other single-stranded RNA molecules that can regulate gene expression through mechanisms other than the RNAi effect. 
     The siRNA molecules of the invention are used to treat one or more gliomas in the brain or spinal cord of a subject. In practice, a plurality of the molecules are used. The siRNA molecules may bind to a peptide or protein that causes or promotes the growth of a glioma in the subject. In one embodiment, the subject is a mammal, such as a mouse, non-human primate, or human. In another embodiment, the subject is a human. In one embodiment, the glioma is an astrocytoma. In another embodiment, it is a type of astrocytoma known as a glioblastoma multiforme (GBM). The molecules are delivered to the subject in pharmaceutically acceptable carriers known to those skilled in the by techniques known to those skilled in the art. 
     The invention also includes compositions of a plurality of the siRNA molecules, where each one targets a different RNA nucleotide sequence, which can be on the same RNA target molecule, different RNA target molecules, or any combination thereof. These compositions, in combination with pharmaceutically acceptable carriers, such as those described herein, are sometimes called siRNA cocktails. Thus, the invention provides multi-targeted siRNA cocktails for the treatment of gliomas. 
     All possible combinations of types of molecules and targets are included in the invention. For example, the targeted RNA molecules may encode or regulate the expression of one or more proteins in the subject. The proteins can be in the same or different pathways. The pathways, categories of proteins, and specific proteins are the ones identified previously herein. In one embodiment, the composition comprises two or more different siRNA molecules, each binding to a different RNA target sequence. In another embodiment, the composition comprises three different siRNA molecules, each binding to a different RNA target sequence. In still another embodiment, the composition comprises more than three different siRNA molecules, each binding to a different RNA target sequence. In one embodiment, the siRNA molecules target one or more of the mRNA molecules that are transcribed from one or more of the gene sequences listed in Tables 1-6 herein. In another embodiment, the siRNA molecules are selected from those listed in Tables 7-12 herein. 
     In yet another embodiment, the composition comprises at least one siRNA (sense: 5′-CUGUAGACACACCCACCCACAUACA-3′ (SEQ ID NO: 10), antisense: 5′-UGUAUGUGGGUGGGUGUGUCUACAG-3′ (SEQ ID NO: 11)) or (sense 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ (SEQ ID NO: 12), antisense 5′-UACUCCUGGAAGAUGUCCACCAGGG-3′ (SEQ ID NO: 13)) that binds to an mRNA molecule that encodes both a human and a mouse VEGF protein, at least one siRNA molecule (sense: 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ (SEQ ID NO: 14), antisense: 5′-ACCAUGAUCAUGUAGACAUCGAUGG-3′ (SEQ ID NO: 15)) or (sense 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ (SEQ ID NO: 16), antisense 5′-UAUCAUCCAGCACUUGACCAUGAUC-3′ (SEQ ID NO: 17)) that binds to an mRNA molecule that encodes both a human and mouse EGFR protein, and at least one siRNA molecule (sense: 5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′ (SEQ ID NO: 18), antisense: 5′-UUCUCCGAAUUUCACAACCUUCAGC-3′ (SEQ ID NO: 19)) or (sense 5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′ (SEQ ID NO: 20), antisense 5′-UCCGAAUUUCACAACCUUCAGCAGC-3′ (SEQ ID NO: 21)) that binds to an mRNA molecule that encodes both a human and mouse MGMT protein. 
     As previously mentioned, the siRNA cocktails of the invention comprise two or more different siRNA molecules of the invention in a pharmaceutically acceptable carrier. Such carriers are generally known to those skilled in the art and include saline, sugars, polypeptides, polymers, lipids, creams, gels, micelle materials, and metal nanoparticles. In one embodiment, the carrier comprises at least one of the following: a glucose solution, 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, a ligand functionalized-hydrophilic polymer grafted polymer, and a ligand functionalized liposome. In another embodiment, the polymers comprise a biodegradable histidine-lysine polymer, a biodegradable polyester, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer, a cationic lipid (such as DOTAP), or a PEGylated PEI. In still another embodiment, the carrier is a histidine-lysine copolymer that forms a nanoparticle with the siRNA molecule, wherein the diameter of the nanoparticle is about 100 nm to about 500 nm. In a further embodiment, the ligand comprises one or more of an RGD peptide, such as H-ACRGDMFGCA-OH, an RVG peptide, such as H-YTIWMPENPRPGTPCDIFTNSRGKRASNG-OH, or a FROP peptide, such as H-EDYELMDLLAYL-OH. 
     The invention also provides a nanoparticle comprising the siRNA molecule of the invention, a carrier, such as one or more of those described herein, and a targeting ligand. Examples of targeting ligands include EGF receptor ligands, IL13 ligand, hepatocyte growth factor ligand, single chain monoclonal antibodies, RGD peptide ligands, and RVG peptide ligands. In one embodiment, the nanoparticle comprises an RGD peptide ligand. In another embodiment, it comprises an RVG peptide ligand. 
     These nanoparticles may be used to prepare the siRNA cocktails previously described herein. Thus, the invention also includes a composition comprising 3 or more of the nanoparticles described herein. 
     The invention also provides a method of treating a subject with a glioma by administering to the subject an effective amount of one or more of the siRNA molecules of the invention or the compositions of the invention. The subject is a mammal. In one embodiment, the mammal is a mouse or rat. In another embodiment, it is a non-human primate. In another embodiment, the subject is a human patient. The glioma may be characterized at least in part by neovascularization and inflammation in the subject&#39;s brain or spinal cord. In one embodiment, the glioma is an astrocytoma. In another embodiment, the astrocytoma is a glioblastome multiforme. 
     The compositions are administered by techniques known to those skilled in the art. In one embodiment, the composition comprises at least three siRNA molecules at a ratio determined by the potency of each siRNA molecule and the therapeutic needs of the subject. In another embodiment, the composition comprises three different siRNA molecules at a ratio of 1:1:1, 1:1.5:0.5, or 0.5:0.5:2. 
     The compositions of the invention can be used with an effective amount of other anti-cancer therapeutic agents. These include ones that impede or block tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of a mammal. In one embodiment, the agent impedes or blocks the activity of a peptide or protein whose activity promotes tumorigenesis, angiogenesis, or cell proliferation in the brain or spinal cord of the mammal. For example, it may impede or block the activity of a peptide or protein that causes or promotes the growth of a glioma. In one embodiment, it impedes or blocks the activity of a protein that is a pro-tumorigenic pathway protein, a pro-angiogenesis pathway protein, a pro-cell proliferation pathway protein, or an anti-apoptotic pathway protein. Such proteins include, but are not limited to, a VEGF pathway protein, EGFR pathway protein, MGMT pathway protein, RAF pathway protein, MMP pathway protein, mTOR pathway protein, TGFβ pathway protein, or Cox-2 pathway protein. Particular examples of proteins that may be targeted by the therapeutic agent are VEGF, EGFR, PI3K, AKT, RAF1, RAS, MAPK, ERK, MGMT, MMP-2, MMP-9, PDGF, PDGFR, IGF-1, HGF, mTOR, Cox-2, and TGFβ. 
     In one embodiment of the invention, the therapeutic agent is selected from the group consisting of bevacizumab (trade name Avastin), sunitinib (trade name Sutent), sorafenib (trade name Nexavar), temsirolimus (trade name Torisel), and temozolomide (trade name Temodar). In one embodiment, a composition of the invention comprises temozolomide and siRNA molecules that inhibit the expression of MGMT and two of the following: EGFR, VEGF, PDFG, MMP-2, and MMP-9. In another embodiment, a composition of the invention comprises bevacizumab and siRNA molecules that inhibit the expression of two of the following: EGFR, PDFG, MMP-2, and MMP-9. In still another embodiment, a composition of the invention comprises temozolomide and siRNA molecules that inhibit the expression of EGFR, VEGF, and MGMT. 
     EXAMPLES 
     The following examples illustrate certain aspects and embodiments of the invention and should not be construed as limiting the scope thereof. 
     Designing siRNA Inhibitors 
     Our experimental approach provides a novel approach to designing siRNA targeting sequences. It differs from other approaches in three important aspects:
         (1) The sequences designed to be targeted by siRNA duplexes have homology to both human and mouse sequences of the same gene. That means that each of the designed siRNA duplexes will be able to knock down the same gene target in either human or mouse cells. For example, a potent siRNA specific to the VEGF gene will be able to knock down both human VEGF and mouse VEGF gene expression.   (2) The sequences were designed in three different lengths: 21-mer, 23-mer, and 25-mer. One consideration is that 23-mer and 25-mer are usually more potent than 21-mer siRNA, but 25-mer may have a greater potential for inducing an unwanted interferon response. Therefore, siRNA duplexes at various lengths may provide the best chance to achieve potent inhibition with less interferon response.   (3) The siRNA oligos can be obtained in either blunt end or sticky end form, according to the synthesis design and annealing. One consideration is that the sticky end siRNA oligos may be sensitive to degradation, and the blunt end siRNA oligos may activate the cellular interferon response.       

     As used herein, “oligonucleotides” and similar terms relate to short oligos composed of naturally occurring nucleotides as well as to oligos composed of synthetic or modified nucleotides, as described in the preceding section on RNAi and siRNA. The terms “polynucleotide” and “oligonucleotide” are used synonymously. 
     Design of siRNA Sequences Against Targets 
     Tables 1-6 present the sequences of six genes targeted by specific siRNA sequences. To identify the most potent siRNA duplex targeting three of these genes, EGFR, VEGF, and MGMT, we generated EGFR, VEGF, and MGMT siRNAs and evaluated the effect of siRNA-mediated gene knockdown in vitro. They provide templates for the design of specific siRNAs. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 The targeted sequences of VEGF-A gene of both human and mouse 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Organism 
                 Gene 
                 homology 
                 Length 
                 No. 
                 Sense Sequences 
                 SEQ ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Human 
                 VEGF165 
                 Human 
                 21-mer 
                 1 
                 gtgtgcgcagacagtgctcca 
                 25 
               
               
                 Mouse 
                   
                 Mouse 
                   
                 2 
                 ccaccatgccaagtggtccca 
                 26 
               
               
                   
                   
                   
                   
                 3 
                 cctggtggacatcttccagga 
                 27 
               
               
                   
                   
                   
                   
                 4 
                 gcacataggagagatgagctt 
                 28 
               
               
                   
                   
                   
                   
                 5 
                 caagatccgcagacgtgtaaa 
                 29 
               
               
                   
                   
                   
                   
                 6 
                 ggcgaggcagcttgagttaaa 
                 30 
               
               
                   
                   
                   
                   
                 7 
                 cttgagttaaacgaacgtact 
                 31 
               
               
                   
                   
                   
                   
                 8 
                 ggaaggagcctccctcagggt 
                 32 
               
               
                   
                   
                   
                   
                 9 
                 cactttgggtccggagggcga 
                 33 
               
               
                   
                   
                   
                   
                 10 
                 cagtattcttggttaatattt 
                 34 
               
               
                   
                   
                   
                 23-mer 
                 1 
                 gcctccgaaaccatgaactttct 
                 35 
               
               
                   
                   
                   
                   
                 2 
                 ctccaccatgccaagtggtccca 
                 36 
               
               
                   
                   
                   
                   
                 3 
                 cctggtggacatcttccaggagt 
                 37 
               
               
                   
                   
                   
                   
                 4 
                 cagcacataggagagatgagctt 
                 38 
               
               
                   
                   
                   
                   
                 5 
                 gcttgagttaaacgaacgtactt 
                 39 
               
               
                   
                   
                   
                   
                 6 
                 gttaaacgaacgtacttgcagat 
                 40 
               
               
                   
                   
                   
                   
                 7 
                 ggaaggagcctccctcagggttt 
                 41 
               
               
                   
                   
                   
                   
                 8 
                 ctccctcagggtttcgggaacca 
                 42 
               
               
                   
                   
                   
                   
                 9 
                 ctaatgttattggtgtcttcact 
                 43 
               
               
                   
                   
                   
                   
                 10 
                 gagaaagtgttttatatacggta 
                 44 
               
               
                   
                   
                   
                 25-mer 
                 1 
                 cctccgaaaccatgaactttctgct 
                 45 
               
               
                   
                   
                   
                   
                 2 
                 ccaccatgccaagtggtcccaggct 
                 46 
               
               
                   
                   
                   
                   
                 3 
                 ccctggtggacatcttccaggagta 
                 47 
               
               
                   
                   
                   
                   
                 4 
                 gatccgcagacgtgtaaatgttcct 
                 48 
               
               
                   
                   
                   
                   
                 5 
                 cgcagacgtgtaaatgttcctgcaa 
                 49 
               
               
                   
                   
                   
                   
                 6 
                 gtaaatgttcctgcaaaaacacaga 
                 50 
               
               
                   
                   
                   
                   
                 7 
                 cagcttgagttaaacgaacgtactt 
                 51 
               
               
                   
                   
                   
                   
                 8 
                 gttaaacgaacgtacttgcagatgt 
                 52 
               
               
                   
                   
                   
                   
                 9 
                 ccatgccaagtggtcccaggctgca 
                 53 
               
               
                   
                   
                   
                   
                 10 
                 ccctggtggacatcttccaggagta 
                 47 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 The targeted sequences of EGFR gene of both human and mouse 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Organism 
                 Gene 
                 homology 
                 Length 
                 No. 
                 Sense Sequences 
                 SEQ ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Human 
                 EGFR 
                 Human 
                 21-mer 
                 1 
                 ccctgactaccagcaggactt 
                 54 
               
               
                 Mouse 
                   
                 Mouse 
                   
                 2 
                 ctgactaccagcaggacttct 
                 55 
               
               
                   
                   
                   
                   
                 3 
                 cagggggatgaaagaatgcat 
                 56 
               
               
                   
                   
                   
                   
                 4 
                 gggggatgaaagaatgcattt 
                 57 
               
               
                   
                   
                   
                   
                 5 
                 gaattctccaaaatggcccga 
                 58 
               
               
                   
                   
                   
                   
                 6 
                 ccatcgatgtctacatgatca 
                 59 
               
               
                   
                   
                   
                   
                 7 
                 gatcatggtcaagtgctggat 
                 60 
               
               
                   
                   
                   
                   
                 8 
                 cgatgtctacatgatcatggt 
                 61 
               
               
                   
                   
                   
                   
                 9 
                 caaagtgcctatcaagtggat 
                 62 
               
               
                   
                   
                   
                   
                 10 
                 ctggatcccagaaggtgagaa 
                 63 
               
               
                   
                   
                   
                 23-mer 
                 1 
                 gacaaccctgactaccagcagga 
                 64 
               
               
                   
                   
                   
                   
                 2 
                 caaccctgactaccagcaggact 
                 65 
               
               
                   
                   
                   
                   
                 3 
                 ccctgactaccagcaggacttct 
                 66 
               
               
                   
                   
                   
                   
                 4 
                 cagggggatgaaagaatgcattt 
                 67 
               
               
                   
                   
                   
                   
                 5  
                 ggatgaaagaatgcatttgccaa 
                 68 
               
               
                   
                   
                   
                   
                 6 
                 gaattctccaaaatggcccgaga 
                 69 
               
               
                   
                   
                   
                   
                 7 
                 cgatgtctacatgatcatggtca 
                 70 
               
               
                   
                   
                   
                   
                 8 
                 ctacatgatcatggtcaagtgct 
                 71 
               
               
                   
                   
                   
                   
                 9 
                 ggcaaagtgcctatcaagtggat 
                 72 
               
               
                   
                   
                   
                   
                 10 
                 ctctggatcccagaaggtgagaa 
                 73 
               
               
                   
                   
                   
                 25-mer 
                 1 
                 gacaaccctgactaccagcaggact 
                 74 
               
               
                   
                   
                   
                   
                 2 
                 ggggatgaaagaatgcatttgccaa 
                 75 
               
               
                   
                   
                   
                   
                 3 
                 ccatcgatgtctacatgatcatggt 
                 76 
               
               
                   
                   
                   
                   
                 4 
                 gatgtctacatgatcatggtcaagt 
                 77 
               
               
                   
                   
                   
                   
                 5 
                 gtctacatgatcatggtcaagtgct 
                 78 
               
               
                   
                   
                   
                   
                 6 
                 gatcatggtcaagtgctggatgata 
                 79 
               
               
                   
                   
                   
                   
                 7 
                 gatcacagattttgggctggccaaa 
                 80 
               
               
                   
                   
                   
                   
                 8 
                 cagattttgggctggccaaactgct 
                 81 
               
               
                   
                   
                   
                   
                 9 
                 cacagattttgggctggccaaactg 
                 82 
               
               
                   
                   
                   
                   
                 10 
                 ctctggatcccagaaggtgagaaag 
                 83 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 The targeted sequences of MGMT gene of both human and mouse 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Organism 
                 Gene 
                 homology 
                 Length 
                 No. 
                 Sense Sequences 
                 SEQ ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Human 
                 MGMT 
                 Human 
                 21-mer 
                 1 
                 caccagacaggtgttatggaa 
                 84 
               
               
                 Mouse 
                   
                 Mouse 
                   
                 2 
                 cagacaggtgttatggaagct 
                 85 
               
               
                   
                   
                   
                   
                 3 
                 ggtgttatggaagctgctgaa 
                 86 
               
               
                   
                   
                   
                   
                 4 
                 ggaagctgctgaaggttgtga 
                 87 
               
               
                   
                   
                   
                   
                 5 
                 gctgctgaaggttgtgaaatt 
                 88 
               
               
                   
                   
                   
                   
                 6 
                 gaaggttgtgaaattcggaga 
                 89 
               
               
                   
                   
                   
                   
                 7 
                 cagcaattagcagccctggca 
                 90 
               
               
                   
                   
                   
                   
                 8 
                 cagcaattagcagccctggca 
                 90 
               
               
                   
                   
                   
                   
                 9 
                 cagccctggcaggcaacccca 
                 91 
               
               
                   
                   
                   
                   
                 10 
                 gccctggcaggcaaccccaaa 
                 92 
               
               
                   
                   
                   
                 23-mer 
                 1 
                 ccagacaggtgttatggaagct 
                 93 
               
               
                   
                   
                   
                   
                 2 
                 gacaggtgttatggaagctgct 
                 94 
               
               
                   
                   
                   
                   
                 3 
                 caggtgttatggaagctgctga 
                 95 
               
               
                   
                   
                   
                   
                 4 
                 gttatggaagctgctgaaggtt 
                 96 
               
               
                   
                   
                   
                   
                 5  
                 ggaagctgctgaaggttgtgaa 
                 97 
               
               
                   
                   
                   
                   
                 6  
                 gctgaaggttgtgaaattcgga 
                 98 
               
               
                   
                   
                   
                   
                 7 
                 gaaggttgtgaaattcggagaa 
                 99 
               
               
                   
                   
                   
                   
                 8 
                 cttaccagcaattagcagccct 
                 100 
               
               
                   
                   
                   
                   
                 9 
                 ccagcaattagcagccctggca 
                 101 
               
               
                   
                   
                   
                   
                 10 
                 gcagccctggcaggcaacccca 
                 102 
               
               
                   
                   
                   
                 25-mer 
                 1 
                 ccagacagguguuauggaagcugcu 
                 103 
               
               
                   
                   
                   
                   
                 2 
                 gacagguguuauggaagcugcugaa 
                 104 
               
               
                   
                   
                   
                   
                 3 
                 gguguuauggaagcugcugaagguu 
                 105 
               
               
                   
                   
                   
                   
                 4 
                 ggaagcugcugaagguugugaaauu 
                 106 
               
               
                   
                   
                   
                   
                 5 
                 gcugcugaagguugugaaauucgga 
                 20 
               
               
                   
                   
                   
                   
                 6 
                 gcugaagguugugaaauucggagaa 
                 18 
               
               
                   
                   
                   
                   
                 7 
                 cagcaauuagcagcccuggcaggca 
                 107 
               
               
                   
                   
                   
                   
                 8 
                 cuuaccagcaauuagcagcccuggc 
                 108 
               
               
                   
                   
                   
                   
                 9 
                 ccagcaauuagcagcccuggcaggc 
                 109 
               
               
                   
                   
                   
                   
                 10 
                 gcaauuagcagcccuggcaggcaac 
                 110 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 The targeted sequences of MMP-2 gene of both human and mouse 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 SEQ ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Human 
                 MMP-2 
                 Human 
                 21-mer 
                 1 
                 cccttgtttccgctgcatcca 
                 111 
               
               
                 Mouse 
                   
                 Mouse 
                   
                 2 
                 catcatcaagttccccggcga 
                 112 
               
               
                   
                   
                   
                   
                 3 
                 gacaaagagttggcagtgcaa 
                 113 
               
               
                   
                   
                   
                   
                 4 
                 gcaacccagatgtggccaact 
                 114 
               
               
                   
                   
                   
                   
                 5 
                 caagcccaagtgggacaagaa 
                 115 
               
               
                   
                   
                   
                   
                 6 
                 gcccaagtgggacaagaacca 
                 116 
               
               
                   
                   
                   
                   
                 7 
                 caactttgagaaggatggcaa 
                 117 
               
               
                   
                   
                   
                   
                 8 
                 gatggcatcgctcagatccgt 
                 118 
               
               
                   
                   
                   
                   
                 9 
                 cctggatgccgtcgtggacct 
                 119 
               
               
                   
                   
                   
                   
                   
                 gccagggatctcttcaatgct 
                 120 
               
               
                   
                   
                   
                 23-mer 
                 1 
                 cccttgtttccgctgcatccaga 
                 121 
               
               
                   
                   
                   
                   
                 2  
                 ccatcatcaagttccccggcgat 
                 122 
               
               
                   
                   
                   
                   
                 3 
                 gacaaagagttggcagtgcaata 
                 123 
               
               
                   
                   
                   
                   
                 4 
                 ggcaacccagatgtggccaacta 
                 124 
               
               
                   
                   
                   
                   
                 5 
                 cgcaagcccaagtgggacaagaa 
                 125 
               
               
                   
                   
                   
                   
                 6 
                 gcccaagtgggacaagaaccaga 
                 126 
               
               
                   
                   
                   
                   
                 7 
                 ggacaagaaccagatcacataca 
                 127 
               
               
                   
                   
                   
                   
                 8 
                 caactttgagaaggatggcaagt 
                 128 
               
               
                   
                   
                   
                   
                 9 
                 ggcatcgctcagatccgtggtga 
                 129 
               
               
                   
                   
                   
                   
                 10 
                 ctggatgccgtcgtggacctgca 
                 130 
               
               
                   
                   
                   
                 25-mer 
                 1 
                 cccttgtttccgctgcatccagact 
                 131 
               
               
                   
                   
                   
                   
                 2 
                 ccatcatcaagttccccggcgatgt 
                 132 
               
               
                   
                   
                   
                   
                 3 
                 gagttggcagtgcaatacctgaaca 
                 133 
               
               
                   
                   
                   
                   
                 4 
                 gcaacccagatgtggccaactacaa 
                 134 
               
               
                   
                   
                   
                   
                 5 
                 gcaagcccaagtgggacaagaacca 
                 135 
               
               
                   
                   
                   
                   
                 6 
                 cccaagtgggacaagaaccagatca 
                 136 
               
               
                   
                   
                   
                   
                 7 
                 gacaagaaccagatcacatacagga 
                 137 
               
               
                   
                   
                   
                   
                 8 
                 ggcatcgctcagatccgtggtgaga 
                 138 
               
               
                   
                   
                   
                   
                 9 
                 gagcgtgaagtttggaagcatcaaa 
                 139 
               
               
                   
                   
                   
                   
                 10 
                 gagatcttcttcttcaaggaccggt 
                 140 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 The targeted sequences of MMP-9 gene of both human and mouse 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Organism 
                 Gene 
                 homology 
                 Length 
                 No. 
                 Sense Sequences 
                 SEQ ID NO: 
               
               
                   
               
               
                 Human 
                 MMP-9 
                 Human 
                 21-mer 
                 1 
                 catccagtttggtgtcgcgga 
                 141 
               
               
                 Mouse 
                   
                 Mouse 
                   
                 2 
                 ccagtttggtgtcgcggagca 
                 142 
               
               
                   
                   
                   
                   
                 3 
                 gcggagcacggagacgggtat 
                 143 
               
               
                   
                   
                   
                   
                 4 
                 cggagacgggtatcccttcga 
                 144 
               
               
                   
                   
                   
                   
                 5 
                 gagctgtgcgtcttccccttc 
                 145 
               
               
                   
                   
                   
                 23-mer 
                 1 
                 gtcatccagtttggtgtcgcgga 
                 146 
               
               
                   
                   
                   
                   
                 2  
                 gcgcggagcacggagacgggtat 
                 147 
               
               
                   
                   
                   
                   
                 3 
                 ggagcacggagacgggtatccct 
                 148 
               
               
                   
                   
                   
                 25-mer 
                 1 
                 ccagtttggtgtcgcggagcacgga 
                 149 
               
               
                   
                   
                   
                   
                 2 
                 cgcgcgcggagcacggagacgggta 
                 150 
               
               
                   
                   
                   
                   
                 3 
                 cggagcacggagacgggtatccctt 
                 151 
               
               
                   
                   
                 Human only 
                 21-mer 
                 1s 
                 CCACCACAACAUCACCUAUTT 
                 152 
               
               
                   
                   
                 s: sense 
                   
                 1a 
                 AUAGGUGAUGUUGUGGUGGTT 
                 153 
               
               
                   
                   
                 a: antisense 
                   
                 2s 
                 GCCAGUUUCCAUUCAUCUUTT 
                 154 
               
               
                   
                   
                   
                   
                 2a 
                 AAGAUGAAUGGAAACUGGCTT 
                 155 
               
               
                   
                   
                   
                   
                 3s 
                 GCGCUGGGCUUAGAUCAUUTT 
                 156 
               
               
                   
                   
                   
                   
                 3a 
                 AAUGAUCUAAGCCCAGCGCTT 
                 157 
               
               
                   
                   
                   
                   
                 4s 
                 GCAUAAGGACGACGUGAAUTT 
                 158 
               
               
                   
                   
                   
                   
                 4a 
                 AUUCACGUCGUCCUUAUGCTT 
                 159 
               
               
                   
                   
                   
                   
                 5s 
                 CCUGCAACGUGAACAUCUUTT 
                 160 
               
               
                   
                   
                   
                   
                 5a 
                 AAGAUGUUCACGUUGCAGGTT 
                 161 
               
               
                   
                   
                   
                   
                 6s 
                 GGAACCAGCUGUAUUUGUUTT 
                 162 
               
               
                   
                   
                   
                   
                 6a 
                 AACAAAUACAGCUGGUUCCTT 
                 163 
               
               
                   
                   
                   
                   
                 7s 
                 GCCAGUUUGCCGGAUACAATT 
                 164 
               
               
                   
                   
                   
                   
                 7a 
                 UUGUAUCCGGCAAACUGGCTT 
                 165 
               
               
                   
                   
                   
                   
                 8s 
                 CCAGUUUGCCGGAUACAAATT 
                 166 
               
               
                   
                   
                   
                   
                 8a 
                 UUUGUAUCCGGCAAACUGGTT 
                 167 
               
               
                   
                   
                   
                   
                 9s 
                 GCCGGAUACAAACUGGUAUTT 
                 168 
               
               
                   
                   
                   
                   
                 9a 
                 AUACCAGUUUGUAUCCGGCTT 
                 169 
               
               
                   
                   
                   
                   
                 10s  
                 CCGGAUACAAACUGGUAUUTT 
                 170 
               
               
                   
                   
                   
                   
                 10a  
                 AAUACCAGUUUGUAUCCGGTT 
                 171 
               
               
                   
                   
                 Human only 
                 25-mer 
                 1s 
                 UGGCACCACCACAACAUCACCUAUU 
                 172 
               
               
                   
                   
                 s: sense 
                   
                 1a 
                 AAUAGGUGAUGUUGUGGUGGUGCCA 
                 173 
               
               
                   
                   
                 a: antisense 
                   
                 2s 
                 CACAACAUCACCUAUUGGAUCCAAA 
                 174 
               
               
                   
                   
                   
                   
                 2a 
                 UUUGGAUCCAAUAGGUGAUGUUGUG 
                 175 
               
               
                   
                   
                   
                   
                 3s 
                 GACGCAGACAUCGUCAUCCAGUUUG 
                 176 
               
               
                   
                   
                   
                   
                 3a 
                 CAAACUGGAUGACGAUGUCUGCGUC 
                 177 
               
               
                   
                   
                   
                   
                 4s 
                 GGAAACCCUGCCAGUUUCCAUUCAU 
                 178 
               
               
                   
                   
                   
                   
                 4a 
                 AUGAAUGGAAACUGGCAGGGUUUCC 
                 179 
               
               
                   
                   
                   
                   
                 5s 
                 CAUUCAUCUUCCAAGGCCAAUCCUA 
                 180 
               
               
                   
                   
                   
                   
                 5a 
                 UAGGAUUGGCCUUGGAAGAUGAAUG 
                 181 
               
               
                   
                   
                   
                   
                 6s 
                 ACGAUGCCUGCAACGUGAACAUCUU 
                 182 
               
               
                   
                   
                   
                   
                 6a 
                 AAGAUGUUCACGUUGCAGGCAUCGU 
                 183 
               
               
                   
                   
                   
                   
                 7s 
                 GCGGAGAUUGGGAACCAGCUGUAUU 
                 184 
               
               
                   
                   
                   
                   
                 7a 
                 AAUACAGCUGGUUCCCAAUCUCCGC 
                 185 
               
               
                   
                   
                   
                   
                 8s 
                 CGGAGAUUGGGAACCAGCUGUAUUU 
                 186 
               
               
                   
                   
                   
                   
                 8a 
                 AAAUACAGCUGGUUCCCAAUCUCCG 
                 187 
               
               
                   
                   
                   
                   
                 9s 
                 CAGUACCGAGAGAAAGCCUAUUUCU 
                 188 
               
               
                   
                   
                   
                   
                 9a 
                 AGAAAUAGGCUUUCUCUCGGUACUG 
                 189 
               
               
                   
                   
                   
                   
                 10s  
                 AAGCCUAUUUCUGCCAGGACCGCUU 
                 190 
               
               
                   
                   
                   
                   
                 10a  
                 AAGCGGUCCUGGCAGAAAUAGGCUU 
                 191 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 The targeted sequences of PDGFa gene of both human and mouse 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Organism 
                 Gene 
                 homology 
                 Length 
                 No. 
                 Sense Sequences 
                 SEQ ID NO: 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Human 
                 PDGF a 
                 Human 
                 21-mer 
                 1 
                 caccctcctccgggccgcgct 
                 192 
               
               
                 Mouse 
                   
                 Mouse 
                   
                 2 
                 ctcctccgggccgcgctccct 
                 193 
               
               
                   
                   
                   
                   
                 3 
                 gtactgaatttcgccgccaca 
                 194 
               
               
                   
                   
                   
                   
                 4 
                 ctgaatttcgccgccacagga 
                 195 
               
               
                   
                   
                   
                   
                 5 
                 ggagcgcccgccccgcggcct 
                 196 
               
               
                   
                   
                   
                   
                 6 
                 ctgctgctcctcggctgcgga 
                 197 
               
               
                   
                   
                   
                   
                 7 
                 gctgctcctcggctgcggata 
                 198 
               
               
                   
                   
                   
                   
                 8 
                 gatccacagcatccgggacct 
                 199 
               
               
                   
                   
                   
                   
                 9 
                 ccacagcatccgggacctcca 
                 200 
               
               
                   
                   
                   
                   
                 10 
                 catccgggacctccagcgact 
                 201 
               
               
                   
                   
                   
                 23-mer 
                 1 
                 gccaccctcctccgggccgcgct 
                 202 
               
               
                   
                   
                   
                   
                 2 
                 ccctcctccgggccgcgctccct 
                 203 
               
               
                   
                   
                   
                   
                 3 
                 gatggtactgaatttcgccgcca 
                 204 
               
               
                   
                   
                   
                   
                 4 
                 ctggagcgcccgccccgcggcct 
                 205 
               
               
                   
                   
                   
                   
                 5 
                 gcgcccgccccgcggcctcgcct 
                 206 
               
               
                   
                   
                   
                   
                 6 
                 gcctcgggacgcgatgaggacct 
                 207 
               
               
                   
                   
                   
                   
                 7 
                 ggcttgcctgctgctcctcggct 
                 208 
               
               
                   
                   
                   
                   
                 8 
                 gcctgctgctcctcggctgcgga 
                 209 
               
               
                   
                   
                   
                   
                 9 
                 cagatccacagcatccgggacct 
                 210 
               
               
                   
                   
                   
                   
                 10 
                 gaccaggacggtcatttacgaga 
                 211 
               
               
                   
                   
                   
                 25-mer 
                 1 
                 gcgccaccctcctccgggccgcgct 
                 212 
               
               
                   
                   
                   
                   
                 2 
                 caccctcctccgggccgcgctccct 
                 213 
               
               
                   
                   
                   
                   
                 3 
                 gggatggtactgaatttcgccgcca 
                 214 
               
               
                   
                   
                   
                   
                 4  
                 gatggtactgaatttcgccgccaca 
                 215 
               
               
                   
                   
                   
                   
                 5 
                 ggtactgaatttcgccgccacagga 
                 216 
               
               
                   
                   
                   
                   
                 6  
                 ggctggagcgcccgccccgcggcct 
                 217 
               
               
                   
                   
                   
                   
                 7  
                 gagcgcccgccccgcggcctcgcct 
                 218 
               
               
                   
                   
                   
                   
                 8 
                 ccagcgcctcgggacgcgatgagga 
                 219 
               
               
                   
                   
                   
                   
                 9 
                 gcgcctcgggacgcgatgaggacct 
                 220 
               
               
                   
                   
                   
                   
                 10 
                 gcctgctgctcctcggctgcggata 
                 221 
               
               
                   
               
            
           
         
       
     
     The following siRNA duplexes were designed to target human/mouse EGFR gene (BCER series, eight siRNAs), human/mouse VEGF gene (BCVF series, eight siRNAs) and human/mouse MGMT gene (MGMT series, eight siRNAs): 
     
       
         
           
               
             
               
                   
               
             
            
               
                 BCER siRNA, siRNA duplexes targeting EGFR genes (both mouse and human), 25 mer 
               
               
                 blunt ended: 
               
            
           
           
               
               
               
            
               
                 Sense: 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 (SEQ ID NO: 14) 
               
               
                 Antisense: 
                 5′-ACCAUGAUCAUGUAGACAUCGAUGG-3′ 
                 (SEQ ID NO: 15) 
               
               
                 BCER-b 
                   
                   
               
               
                 Sense: 
                 5′-GAUGUCUACAUGAUCAUGGUCAAGU-3′ 
                 (SEQ ID NO: 222) 
               
               
                 Antisense: 
                 5′-ACUUGACCAUGAUCAUGUAGACAUC-3′ 
                 (SEQ ID NO: 223) 
               
               
                 BCER-c: 
                   
                   
               
               
                 Sense: 
                 5′-GUCUACAUGAUCAUGGUCAAGUGCU-3′  
                 (SEQ ID NO: 224) 
               
               
                 Antisense: 
                 5′-AGCACUUGACCAUGAUCAUGUAGAC-3′ 
                 (SEQ ID NO: 225) 
               
               
                 BCER-d: 
                   
                   
               
               
                 Sense: 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 (SEQ ID NO: 16) 
               
               
                 Antisense: 
                 5′-UAUCAUCCAGCACUUGACCAUGAUC-3′ 
                 (SEQ ID NO: 17) 
               
               
                 BCER-e: 
                   
                   
               
               
                 Sense: 
                 5′-GAUCACAGAUUUUGGGCUGGCCAAA-3′ 
                 (SEQ ID NO: 226) 
               
               
                 Antisense: 
                 5′-UUUGGCCAGCCCAAAAUCUGUGAUC-3′ 
                 (SEQ ID NO: 227) 
               
               
                 BCER-f: 
                   
                   
               
               
                 Sense: 
                 5′-CAGAUUUUGGGCUGGCCAAACUGCU-3′  
                 (SEQ ID NO: 228) 
               
               
                 Antisense: 
                 5′-AGCAGUUUGGCCAGCCCAAAAUCUG-3′ 
                 (SEQ ID NO: 229) 
               
               
                 BCER-g: 
                   
                   
               
               
                 Sense: 
                 5′-CACAGAUUUUGGGCUGGCCAAACUG-3′  
                 (SEQ ID NO: 230) 
               
               
                 Antisense: 
                 5′-CAGUUUGGCCAGCCCAAAAUCUGUG-3′  
                 (SEQ ID NO: 231) 
               
               
                 BCER-h: 
                   
                   
               
               
                 Sense: 
                 5′-CUCUGGAUCCCAGAAGGUGAGAAAG-3′  
                 (SEQ ID NO: 232) 
               
               
                 Antisense: 
                 5′-CUUUCUCACCUUCUGGGAUCCAGAG-3′  
                 (SEQ ID NO: 233) 
               
            
           
           
               
            
               
                 BCVF siRNA, siRNA duplexes targeting VEGF gene (both mouse and human) 25 mer  
               
               
                 blunt ended: 
               
            
           
           
               
               
               
            
               
                 BCVF-a 
                   
                   
               
               
                 Sense: 
                 5′-CCAUGCCAAGUGGUCCCAGGCUGCA-3′ 
                 (SEQ ID NO: 234) 
               
               
                 Antisense: 
                 5′-UGCAGCCUGGGACCACUUGGCAUGG-3′  
                 (SEQ ID NO: 235) 
               
               
                 BCVF-b 
                   
                   
               
               
                 Sense: 
                 5′-CCAACAUCACCAUGCAGAUUAUGCG-3′ 
                 (SEQ ID NO: 236) 
               
               
                 Antisense: 
                 5′-CGCAUAAUCUGCAUGGUGAUGUUGG-3′ 
                 (SEQ ID NO: 237) 
               
               
                 BCVF-c 
                   
                   
               
               
                 Sense: 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 (SEQ ID NO: 10) 
               
               
                 Antisense: 
                 5′-UGUAUGUGGGUGGGUGUGUCUACAG-3′  
                 (SEQ ID NO: 11) 
               
               
                 BCVF-d 
                   
                   
               
               
                 Sense: 
                 5′-CACUUUGGGUCCGGAGGGCGAGACU-3′  
                 (SEQ ID NO: 238) 
               
               
                 Antisense: 
                 5′-AGUCUCGCCCUCCGGACCCAAAGUG-3′  
                 (SEQ ID NO: 239) 
               
               
                 BCVF-e 
                   
                   
               
               
                 Sense: 
                 5′-CCAUGCCAAGUGGUCCCAGGCUGCA-3′  
                 (SEQ ID NO: 234) 
               
               
                 Antisense: 
                 5′-UGCAGCCUGGGACCACUUGGCAUGG-3′  
                 (SEQ ID NO: 235) 
               
               
                 BCVF-f 
                   
                   
               
               
                 Sense: 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′  
                 (SEQ ID NO: 12) 
               
               
                 Antisense: 
                 5′-UACUCCUGGAAGAUGUCCACCAGGG-3′  
                 (SEQ ID NO: 13) 
               
               
                 BCVF-g 
                   
                   
               
               
                 Sense: 
                 5′-CGCAGACGUGUAAAUGUUCCUGCAA-3′ 
                 (SEQ ID NO: 240) 
               
               
                 Antisense: 
                 5′-UUGCAGGAACAUUUACACGUCUGCG-3′ 
                 (SEQ ID NO: 241) 
               
               
                 BCVF-h 
                   
                   
               
               
                 Sense: 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 (SEQ ID NO: 12) 
               
               
                 Antisense: 
                 5′-UACUCCUGGAAGAUGUCCACCAGGG-3′ 
                 (SEQ ID NO: 13) 
               
            
           
           
               
            
               
                 BCMM siRNA, siRNA duplexes targeting MGMT gene (both human and mouse) 25 mer  
               
               
                 blunt ended: 
               
            
           
           
               
               
               
            
               
                 BCMM-a: 
                   
                   
               
               
                 Sense: 
                 5′-GGUGUUAUGGAAGCUGCUGAAGGUU-3′ 
                 (SEQ ID NO: 105) 
               
               
                 Antisense: 
                 5′-AACCUUCAGCAGCUUCCAUAACACC-3′ 
                 (SEQ ID NO: 242) 
               
               
                 BCMM-b: 
                   
                   
               
               
                 Sense: 
                 5′-GGAAGCUGCUGAAGGUUGUGAAAUU-3′  
                 (SEQ ID NO: 106) 
               
               
                 Antisense: 
                 5′-AAUUUCACAACCUUCAGCAGCUUCC-3′ 
                 (SEQ ID NO: 243) 
               
               
                 BCMM-c: 
                   
                   
               
               
                 Sense: 
                 5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′ 
                 (SEQ ID NO: 20) 
               
               
                 Antisense: 
                 5′-UCCGAAUUUCACAACCUUCAGCAGC-3′  
                 (SEQ ID NO: 21) 
               
               
                 BCMM-d: 
                   
                   
               
               
                 Sense: 
                 5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′ 
                 (SEQ ID NO: 18) 
               
               
                 Antisense: 
                 5′-UUCUCCGAAUUUCACAACCUUCAGC-3′ 
                 (SEQ ID NO: 19) 
               
               
                 BCMM-e: 
                   
                   
               
               
                 Sense: 
                 5′-CAGCAAUUAGCAGCCCUGGCAGGCA-3′  
                 (SEQ ID NO: 107) 
               
               
                 Antisense: 
                 5′-UGCCUGCCAGGGCUGCUAAUUGCUG-3′ 
                 (SEQ ID NO: 244) 
               
               
                 BCMM-f: 
                   
                   
               
               
                 Sense: 
                 5′-CUUACCAGCAAUUAGCAGCCCUGGC-3′ 
                 (SEQ ID NO: 108) 
               
               
                 Antisense: 
                 5′-GCCAGGGCUGCUAAUUGCUGGUAAG-3′ 
                 (SEQ ID NO: 245) 
               
               
                 BCMM-g: 
                   
                   
               
               
                 Sense: 
                 5′-CCAGCAAUUAGCAGCCCUGGCAGGC-3′ 
                 (SEQ ID NO: 109) 
               
               
                 Antisense: 
                 5′-GCCUGCCAGGGCUGCUAAUUGCUGG-3′ 
                 (SEQ ID NO: 246) 
               
               
                 BCMM-h: 
                   
                   
               
               
                 Sense: 
                 5′-GCAAUUAGCAGCCCUGGCAGGCAAC-3′ 
                 (SEQ ID NO: 110) 
               
               
                 Antisense: 
                 5′-GUUGCCUGCCAGGGCUGCUAAUUGC-3′  
                 (SEQ ID NO: 247) 
               
               
                   
               
            
           
         
       
     
     Example I 
     Identification of the Most Potent siRNA Duplex Targeting the Above Three Genes to Demonstrate/Validate the Potency of siRNA Against Specific Target Genes 
     To accomplish this objective, we generated EGFR, VEGF, and MGMT siRNAs and quantitatively evaluated the effect of siRNA-mediated gene knockdown in vitro using cell-culture-based assays. Gene knockdown was confirmed and evaluated by immunoblotting. We selected the most potent siRNA for each target gene for subsequent experiments. Using RT-PCR and Western blot analyses, we measured the gene expression silencing by each of the siRNA duplexes listed above, with optimized transfection followed by RNA and protein isolation.  FIGS. 1-9  demonstrate the identified potent siRNA duplexes targeting each of the genes discussed. 
     Selection of Potent siRNA Sequences 
                                A. Two siRNA duplexes were selected for MGMT knockdown:                         MGMT-D               Sense   5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′   (SEQ ID NO: 18)       Antisense   5′-UUCUCCGAAUUUCACAACCUUCAGC-3′   (SEQ ID NO: 19)       MGMT-C               Sense   5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′   (SEQ ID NO: 20)       Antisense   5′-UCCGAAUUUCACAACCUUCAGCAGC-3′   (SEQ ID NO: 21)                 B. Two siRNA duplexes were selected for EGFR knockdown:                         EGFR-A               Sense   5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′   (SEQ ID NO: 14)       Antisense   5′-ACCAUGAUCAUGUAGACAUCGAUGG-3′   (SEQ ID NO: 15)       EGFR-D               Sense   5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′   (SEQ ID NO: 16)       Antisense   5′-UAUCAUCCAGCACUUGACCAUGAUC-3′   (SEQ ID NO: 17)                 C. Two siRNA duplexes were selected for human and mouse VEGF knockdown:                         VEGF-C               Sense   5&#39;-CUGUAGACACACCCACCCACAUACA-3&#39;   (SEQ ID NO: 10)       Antisense   5&#39;-UGUAUGUGGGUGGGUGUGUCUACAG-3&#39;   (SEQ ID NO: 11)       VEGF-H               Sense   5&#39;-CCCUGGUGGACAUCUUCCAGGAGUA-3&#39;   (SEQ ID NO: 12)       Antisense   5&#39;-UACUCCUGGAAGAUGUCCACCAGGG-3&#39;   (SEQ ID NO: 13)                 D. One siRNA duplex was selected for human and mouse PDGF knockdown:                         hmPD-25-3:               sense:   5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′   (SEQ ID NO: 5)       antisense:   5′-UAUCCGCAGCCGAGGAGCAGCAGGC-3′   (SEQ ID NO: 248)                 E. One siRNA duplex was selected for human and mouse MMP-2 knockdown:                         hmM2-25-3:               sense   5&#39;-CCCAAGUGGGACAAGAACCAGAUCA-3&#39;   (SEQ ID NO: 249)       antisense   5&#39;-UGAUCUGGUUCUUGUCCCACUUGGG-3&#39;   (SEQ ID NO: 250)                 F. One siRNA duplex was selected for human and mouse MMP-9 knockdown:                         hmM9-25-1:               sense   5′-CCAGUUUGGUGUCGCGGAGCACGGA-3′   (SEQ ID NO: 251)       antisense   5′-UCCGUGCUCCGCGACACCAAACUGG-3′   (SEQ ID NO: 252)                    
SiRNA Cocktail Therapeutics for GBM
 
     We designed a therapeutic siRNA cocktail targeting multiple-disease controlling genes for treating several types of brain cancer, including GBM, acting on multiple aspects of the diseases and reducing potential toxicity. 
     The siRNA cocktail has the following characteristics:
         (1) The siRNA cocktail contains at least three siRNA duplexes targeting at least three genes at a ratio required by the therapy.   (2) The cocktail design for each combination follows the understanding of the role of each gene in a background of the system biology network, such as whether these genes are functioning in the same pathway or in different ones.   (3) The chemical property of each siRNA molecule in the cocktail is the same in terms of source of supply, manufacturing process, chemical modification, storage conditions, and formulation procedures.   (4) The individual siRNA molecules in the cocktail can be of different lengths, with either blunt or sticky end, as long as their potencies have been defined.   (5) Since an siRNA cocktail is targeting multiple genes and a single cell type usually does not express all those factors, the efficacy of the siRNA cocktail is tested in a relevant disease model, either a multiple cell model, a tissue model, or an animal model, after confirmation of the potency of each individual siRNA duplex in the cell culture.   (6) Each validated siRNA cocktail can be used for addressing one or more pathological conditions or for treating one or multiple types of diseases, such as an siRNA cocktail for suppressing inflammation, siRNA cocktail for anti-angiogenesis activity and siRNA cocktail for autoimmune conditions.   (7) The siRNA cocktail is 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 requirements.   (8) 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 drugs.       

     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 VEGF-EGFR-MGMT siRNA cocktails 
               
               
                 siRNA Cocktail Combinations (siRNA sense sequences) 
               
            
           
           
               
               
               
               
            
               
                 VEGF 
                   
                   
                   
               
               
                 EGFR 
                   
                   
                 SEQ ID  
               
               
                 MGMT 
                   
                 Human and Mouse homologues 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 1 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MGMT 
                 5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′ 
                 18 
               
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 2 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MGMT 
                 5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′ 
                 20 
               
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 3 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MGMT 
                 5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′ 
                 20 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 4 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MGMT 
                 5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′ 
                 20 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 5 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MGMT 
                 5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′ 
                 18 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 VEGF-EGFR-PDGF siRNA cocktails 
               
               
                 siRNA Cocktail Combinations (siRNA sense sequences) 
               
            
           
           
               
               
               
               
            
               
                 VEGF 
                   
                   
                   
               
               
                 EGFR 
                   
                   
                 SEQ ID  
               
               
                 PDGF 
                   
                 Human and Mouse homologues 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 1 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 2 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 3 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 4 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 VEGF-EGFR-MMP-9 siRNA cocktails 
               
               
                 siRNA Cocktail Combinations (siRNA sense sequences) 
               
            
           
           
               
               
               
               
            
               
                 VEGF 
                   
                   
                   
               
               
                 EGFR 
                   
                   
                 SEQ ID  
               
               
                 MMP-9 
                   
                 Human and Mouse homologues 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 1 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MMP-9 
                 5′-CCAGUUUGGUGUCGCGGAGCACGGA-3′ 
                 251 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 2 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MMP-9 
                 5′-CCAGUUUGGUGUCGCGGAGCACGGA-3′ 
                 251 
               
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 3 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MMP-9 
                 5′-CCAGUUUGGUGUCGCGGAGCACGGA-3′ 
                 251 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 4 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MMP-9 
                 5′-CCAGUUUGGUGUCGCGGAGCACGGA-3′ 
                 251 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 VEGF-EGFR-MMP-2 siRNA cocktails 
               
               
                 siRNA Cocktail Combinations (siRNA sense sequences) 
               
            
           
           
               
               
               
               
            
               
                 VEGF 
                   
                   
                   
               
               
                 EGFR 
                   
                   
                 SEQ ID  
               
               
                 MMP-2 
                   
                 Human and Mouse homologues 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 1 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MMP-2 
                 5′-CCCAAGUGGGACAAGAACCAGAUCA-3′ 
                 249 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 2 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MMP-2 
                 5′-CCCAAGUGGGACAAGAACCAGAUCA-3′ 
                 249 
               
               
                 Cock- 
                 VEGF 
                 5′-CUGUAGACACACCCACCCACAUACA-3′ 
                 10 
               
               
                 tail 3 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MMP-2 
                 5′-CCCAAGUGGGACAAGAACCAGAUCA-3′ 
                 249 
               
               
                 Cock- 
                 VEGF 
                 5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′ 
                 12 
               
               
                 tail 4 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MMP-2 
                 5′-CCCAAGUGGGACAAGAACCAGAUCA-3′ 
                 249 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 PDGF-EGFR-MGMT siRNA cocktails 
               
               
                 siRNA Cocktail Combinations (siRNA sense sequences) 
               
            
           
           
               
               
               
               
            
               
                 PDGF 
                   
                   
                   
               
               
                 EGFR 
                   
                   
                 SEQ ID  
               
               
                 MGMT 
                   
                 Human and Mouse homologues 
                 NO: 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Cock- 
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                 tail 1 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MGMT 
                 5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′ 
                 18 
               
               
                 Cock- 
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                 tail 2 
                 EGFR 
                 5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′ 
                 14 
               
               
                   
                 MGMT 
                 5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′ 
                 20 
               
               
                 Cock- 
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                 tail 3 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MGMT 
                 5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′ 
                 20 
               
               
                 Cock- 
                 PDGF 
                 5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′ 
                 5 
               
               
                 tail 4 
                 EGFR 
                 5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′ 
                 16 
               
               
                   
                 MGMT 
                 5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′ 
                 18 
               
               
                   
               
            
           
         
       
     
                     TABLE 12                  VEGF-EGFR-MGMT-other siRNA cocktails       siRNA Cocktail Combinations (siRNA sense sequences)                             VEGF                   EGFR           SEQ ID        MGMT   other   Human and Mouse homologues   NO:                                     Cock-   VEGF   5′-CUGUAGACACACCCACCCACAUACA-3′   10       tail 1   EGFR   5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′   14           MGMT   5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′   18           PDGF   5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′   5       Cock-   VEGF   5′-CUGUAGACACACCCACCCACAUACA-3′   10       tail 2   EGFR   5′-CCAUCGAUGUCUACAUGAUCAUGGU-3′   14           MGMT   5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′   20           MMP-2   5′-CCCAAGUGGGACAAGAACCAGAUCA-3′   249       Cock-   VEGF   5′-CUGUAGACACACCCACCCACAUACA-3′   10       tail 3   EGFR   5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′   16           MGMT   5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′   20           MMP-9   5′-CCAGUUUGGUGUCGCGGAGCACGGA-3′   251       Cock-   VEGF   5′-CCCUGGUGGACAUCUUCCAGGAGUA-3′   12       tail 4   MMP-2   5′-CCCAAGUGGGACAAGAACCAGAUCA-3′   249           MGMT   5′-GCUGCUGAAGGUUGUGAAAUUCGGA-3′   20           MMP-9   5′-CCAGUUUGGUGUCGCGGAGCACGGA-3′   251       Cock-   MMP-2   5′-CCCAAGUGGGACAAGAACCAGAUCA-3′   249       tail 5   EGFR   5′-GAUCAUGGUCAAGUGCUGGAUGAUA-3′   16           MGMT   5′-GCUGAAGGUUGUGAAAUUCGGAGAA-3′   18           PDGF   5′-GCCUGCUGCUCCUCGGCUGCGGAUA-3′   5                    
Experimental Design:
 
     The design is summarized for three experiments that may be performed to evaluate the antitumorigenic effects of this method. 
     Experiment A. 
     To evaluate the antitumorigenic effects of the selected siRNAs individually and collectively in vitro as well as in vivo. The most potent siRNA inhibitors for each gene are transfected into established cell lines. Measuring cellular proliferation, apoptosis, and signaling pathways downstream of these genes serves as parameters for drug-target validation and potential siRNA-mediated antitumorigenesis in vivo. Then, all three siRNAs are transfected simultaneously into these cell lines to validate the biological potential of a combinatorial approach. This method is applied in vivo to GBM xenograft nude mice models. Evaluation of angiogenesis, proliferation, and apoptosis in these models is used to assess the efficacy of siRNA-mediated antitumorigenesis in vivo.
 
Experiment B.
 
To evaluate, both in vitro and in vivo, the antitumorigenic effects of using individual siRNA and a combination of the three siRNAs in conjunction with clinically available agents, including Tarceva (anti-EGFR), Avastin (anti-VEGF), and Temozolomide (TMZ). Each candidate siRNA, coupled with its clinically available agent, is transfected into established cell lines in vitro to assess the antitumorigenic potential of combined administration. For example, the most potent siRNA for EGFR can be delivered, along with Tarceva, to evaluate the combined effect of these two agents. Comparing proliferation, apoptosis, and downstream signaling activity in these trials to those conducted with siRNA only provides a means to evaluate the effects of combined treatment, as well as the potential for enhanced therapeutic vulnerability. After each siRNA-therapeutic agent pair has been evaluated individually in vitro, all three siRNAs, along with their existing therapeutic agents, are transfected into cell lines simultaneously. These trials are used as a proof of concept for achieving synergistic antitumorigenesis. Corresponding in vivo experiments are conducted using nude mouse models.
 
Experiment C.
 
To validate the efficacy of siRNA-mediated antitumorigenesis in vivo using a nanotechnology-based delivery system. Nanoparticle delivery of the siRNA and siRNA-therapeutic agent cocktails via local and systemic injection to the GBM mouse models is a means to validate multiple drug targets, as well as to indicate the potential therapeutic applications of siRNA. Moreover, the success of a systemic, nanoparticle delivery method in GBM animal models to effect antitumorigenesis serves to validate this approach for clinical trials.
 
Polymers Enhance siRNA Cocktail Delivery
 
     As shown in a study using siRNA cocktails to inhibit ocular neovascularization induced by a herpes simplex viral sequence, the histidine-lysine (HK) polymer-siRNA nanoparticle-mediated local delivery has achieved potent anti-angiogenesis activity. In a separate study using HK polymer to enhance siRNA delivery intratumorally, the tumor growth curves showed significant anti-tumor efficacy with clear downregulation of the target gene expression. At 10 days after the injection of MDA-MB-435 cells into the mammary fat pad, mice with visible tumors were separated into treatment groups. Each group had four mice with eight tumors, and tumor size was assessed in two dimensions and calculated. Mice received 4 μg/tumor of siRNA with each intratumoral injection every 5 days. To confirm the antitumor efficacy of siRNA Raf-1 with the optimal polymer in greater detail, mice with tumors were divided into these groups: untreated, b-galactosidase siRNA, and Raf-1 siRNA. Clearly, HK polymer has been validated as an effective local siRNA delivery carrier. 
     Over the past few decades, biodegradable polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), have been extensively studied for a wide variety of pharmaceutical and biomedical applications. The biodegradable polyester family has been regarded as one of the few synthetic biodegradable polymers with controllable biodegradability, excellent biocompatibility, and high safety. The need for a variety of drug formulations for different drugs and delivery pathways resulted in development of various types of block copolymers (e.g., diblock, triblock, multiblock, and star-shaped block) consisting of the biodegradable polyesters and PEG. 
     PAMAM dendrimers represent an exciting new class of macromolecular architecture called “dense star” polymers. Unlike classical polymers, dendrimers have a high degree of molecular uniformity, narrow molecular weight distribution, specific size and shape characteristics, and a highly functionalized terminal surface. The manufacturing process is a series of repetitive steps starting with a central initiator core. Each subsequent growth step represents a new “generation” of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately double the molecular weight of the preceding generation. PAMAM dendrimers are the most common class of dendrimers suitable for many materials science and biotechnology applications. PAMAM dendrimers consist of alkyl-diamine core and tertiary amine branches. 
     Cationic polymers have gained prominence in nonviral DNA delivery, although their use in siRNA delivery is much more recent. Of these polymers, PEI, which contains primary, secondary, and tertiary amines, has the unique abilities to complex nucleic acid and serve as a low pH (4-5) buffer. Owing to this buffering capacity or “proton sponge” effect, PEI has shown higher transfection efficiency than other cationic polymers. Recently, several groups have reported the use of PEI-siRNA complexes for the treatment of influenza in mice and systemic delivery for treatment of breast cancer in a mouse model, etc. However, in vivo toxicity has been noted for PEI upon intravenous administration. To reduce PEI toxicity, PEG was grafted onto PEI, greatly reducing toxicity for the resulting gene delivery complexes. The PEI component of the copolymer allows for complexation with polynucleotides, and it increases endosomal release of the nanocomplexes into the cytoplasm. The PEG component not only reduces toxicity of the PEI component, but also stabilizes the resulting nanocomplex. This modified vector was recently used to deliver siRNA targeting VEGF for treatment of angiogenic tumors. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2 k 10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and it had the smallest nanocomplex size (115±13 nm diameter). When we tested this PEG-PEI-siRNA nanoparticle in mouse for pulmonary delivery, the anti-influenza A activity was very much enhanced, which allows us to achieve potent therapeutic effect. Therefore, this PEG-PEI could be used in our multi-targeted siRNA regimen for treatment of glioblastoma. 
     DOTAP Chloride has been used as DNA transfection reagent. Racemic DOTAP Chloride is a cationic lipid which forms liposomes in aqueous media, alone or in combination with other lipids. These liposomes can carry polar and non-polar molecules (APIs or diagnostics). They can enter cells carrying their load through the cell membrane. We will use either R DOTAP Chloride or S DOTAP Chloride for siRNA cocktail delivery into the tumor tissue. 
     To test HK polymer, PLGA, and PAMAM for their role in enhancement of siRNA delivery in vivo, we need to define the formulations for each of these carrier-siRNA nanoparticles. 
     Ligands Useful for Targeted siRNA Delivery 
     The targeting ligands may also improve intracerebral or intratumoral siRNA delivery. The EGF receptor ligands, IL13 ligand, hepatocyte growth factor ligand, and other single chain monoclonal antibodies may be used for this type of targeting design. 
     RGD (arginine-glycine-aspartic acid) peptide ligands, such as the ‘cyclic’ 10mer RGD peptide with the sequence H-ACRGDMFGCA-OH (SEQ ID NO: 22), and -(D)CR(D)WKTCT-(ol) (SEQ ID NO: 253) have been used for neovasculature targeted nucleic acid delivery. 
     A short peptide derived from rabies virus glycoprotein (RVG) recently has been identified for its ability to enable the transvascular delivery of small interfering RNA (siRNA) (27). This 29-amino-acid peptide specifically binds to the acetylcholine receptor expressed by neuronal cells. To enable siRNA binding, a chimaeric peptide was synthesized by adding nonamer arginine residues at the carboxy terminus of RVG. This RVG-9R peptide was able to bind and transduce siRNA to neuronal cells in vitro and in vivo, resulting in efficient gene silencing. Peptides: RVG (YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 23)), RVG-9R (YTIWMPENPRPGTPCDIFTNS-RGKRASNGGGGRRRRRRRRR (SEQ ID NO: 254)). 
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     All publications, including issued patents and published applications, and all database entries identified by url addresses or accession numbers are incorporated herein by reference in their entirety. 
     Although this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.