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WO2009099959A2 - Tumor cell expression of neuropilin as a target for cancer therapy - Google Patents

Tumor cell expression of neuropilin as a target for cancer therapy Download PDF

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Publication number
WO2009099959A2
WO2009099959A2 PCT/US2009/032689 US2009032689W WO2009099959A2 WO 2009099959 A2 WO2009099959 A2 WO 2009099959A2 US 2009032689 W US2009032689 W US 2009032689W WO 2009099959 A2 WO2009099959 A2 WO 2009099959A2
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cancer
nrp
nucleic acid
phosphatidylcholine
composition
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WO2009099959A3 (en
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Lee M. Ellis
Michael J. Gray
Anil K. Sood
Gabriel Lopez-Berestein
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University of Texas System
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University of Texas System
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • the present invention relates generally to the fields of molecular biology, oncology, and neuropilins. More particularly, the invention generally concerns compositions comprising an inhibitory nucleic acid, wherein the inhibitory nucleic acid is targeted to a nucleic acid encoding a neuropilin, and methods of treating cancer that involve administration of such compositions.
  • Tumor angiogenesis is a complex process that requires interactions among endothelial cells, tumor cells, and other components of the microenvironment.
  • One of the most important secreted factors that promotes angiogenesis is vascular endothelial growth factor (VEGF) (Folkman and Shing, 1992; Ferrara, 2002).
  • VEGF vascular endothelial growth factor
  • the VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (Ferrara, 2002; Hicklin and Ellis, 2005).
  • VEGF tyrosine kinase receptors VEGFR-I, VEGFR-2, and VEGFR-3 (Ferrara et al, 2003). Initially, these receptors were believed to be expressed only on the surface of endothelial cells, but subsequent findings revealed that VEGF tyrosine kinase receptors are expressed on numerous human cancer cells as well (reviewed in Wey et al., 2004).
  • Neuropilins are multifunctional non-tyrosine kinase receptors that bind to class 3 semaphorins and vascular endothelial growth factor (VEGF) (reviewed in Ellis, 2006).
  • This family of receptors includes neuropilin 1 (NRP-I) and neuropilin-2 (NRP -2).
  • NRP-I and NRP -2 have also been recently implicated in VEGF-mediated vascularization and lymphangio genesis (Ellis, 2006; Bielenberg et al., 2006; Karpanen et al., 2006).
  • NRP-I and NRP -2 were originally identified as neuronal patterning receptors for the class 3 semaphorin ligands (Sema3A, Sema3C, and Sema3F) (He and Tessier-Lavigne, 1997; Chen et al, 1997). Unlike the previously identified VEGF receptors, NRP-I and NRP-2 lack a tyrosine kinase domain.
  • NRPs function as obligate coreceptors by cooperatively enhancing the activity of the VEGF kinase receptors in non-neuronal tissues (Ellis, 2006; Fuh et al, 2000; Whitaker et al, 2001; Lee et al, 2002; Wang et al, 2003; Pan et al, 2007). While NRP-I and NRP-2 are not normally expressed in adult tissues, their expression is detected on some human tumor cells (Ellis, 2006; Beilenberg, 2006).
  • Neuropilins serve as receptors or coreceptors for multiple ligands, including class 3 semaphorins, and VEGF families (Gluzman-Poltorak et al, 2000; Gluzman-Poltorak et al, 2001; Makinen et al, 1999; Nasarre et al, 2003).
  • NRP-I expression has been reported in some human colon cancer samples (Parikh et al, 2004). The expression and function of NRP-2 on tumor cells has yet to be elucidated (Ellis, 2006; Beilenberg, 2006).
  • the present invention is based in part on the finding that neuropilins are involved in cancer development and progression.
  • the inventors have found that decreased NRP -2 expression, such as by shRNA targeting, results in reduction of tumor growth and metastasis in an animal model of human colorectal carcinoma.
  • the inventors have also shown that reduction of NRP-2 expression in pancreatic ductal adenocarcinoma results in decreased migration, invasion, and growth of tumor cells.
  • the present invention is in part based on the finding that NRP-2 expression by a tumor in a subject can be reduced by administering to the subject a composition that includes an agent that reduces NRP-2 expression, which results in reduced tumor growth.
  • compositions that include (1) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes a neuropilin; and (2) a lipid component that includes one or more neutral phospholipids.
  • the neuropilin may be NRP-I or NRP-2. In specific embodiments, the neuropilin is NRP-2.
  • compositions that include a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes neuropilin-2 (NRP-2) and a pharmaceutically acceptable carrier.
  • a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes neuropilin-2 (NRP-2) and a pharmaceutically acceptable carrier.
  • the nucleic acid component may be a DNA or an RNA.
  • the nucleic acid component is a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes an a neuropilin.
  • the nucleic acid component is a shRNA or a nucleic acid encoding a shRNA, wherein the shRNA inhibits the expression of a gene that encodes an a neuropilin.
  • the composition includes a lipid component that forms a liposome. In embodiments that include a siRNA or shRNA, the siRNA or shRNA may be encapsulated in the lipid component.
  • the lipid component may include any lipid known to those of ordinary skill in the art.
  • the lipid component includes one or more phospholipids.
  • Any neutral phospholipid known to those of ordinary skill in the art is contemplated as a phospholipid for use in the compositions of the present invention that include one or more phospholipids.
  • the neutral phospholipid may be a phosphatidylcholine or phosphatidylethanolamine.
  • neutral phospholipids include 1 ,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine (“EPC”), dilauryloylphosphatidylcholine (“DLPC”), dimyristoylphosphatidylcholine (“DMPC”), dipalmitoylphosphatidylcholine (“DPPC”), distearoylphosphatidylcholine (“DSPC”), 1- myristoyl-2-palmitoyl phosphatidylcholine (“MPPC”), l-palmitoyl-2-myristoyl phosphatidylcholine (“PMPC”), l-palmitoyl-2-stearoyl phosphatidylcholine (“PSPC”), 1- stearoyl-2-palmitoyl phosphatidylcholine (“SPPC”), dimyristyl phosphatidylcholine (“DMPC”), l ⁇
  • the lipid component is DOPC.
  • the lipid component includes two or more neutral phospholipids.
  • the composition that includes a lipid component and a nucleic acid component further includes a pharmaceutically acceptable carrier. Any pharmaceutically acceptable carrier known to those of ordinary skill in the art is contemplated for inclusion in the compositions of the present invention. Examples of such carriers are discussed in greater detail in the specification below.
  • the lipid component may further include a positively charged lipid or a negatively charged lipid. Any charged lipid is contemplated for inclusion in the compositions of the present invention.
  • the negatively charged phospholipid may be a phosphatidylserine or phosphatidylglycerol.
  • negatively charged phospholipids include dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine (“DPPS”), brain phosphatidylserine (“BPS”), dilauryloylphosphatidylglycerol (“DLPG”), dimyristoylphosphatidylglycerol (“DMPG”), dipalmitoylphosphatidylglycerol (“DPPG”), distearoylphosphatidylglycerol (“DSPG”), or dioleoylphosphatidylglycerol (“DOPG”).
  • DMPS dimyristoyl phosphatidylserine
  • DPPS dipalmitoyl phosphatidylserine
  • BPS brain phosphatidylserine
  • DLPG dimyristoylphosphatidylglycerol
  • DMPG dimyristoylphosphatidylglycerol
  • compositions of the present invention include cholesterol or polyethyleneglycol (PEG).
  • PEG polyethyleneglycol
  • the nucleic acid component can be of any length.
  • the nucleic acid component may be 5 to 500 nucleobases in length, 10 to 300 nucleobases in length, 18 to 100 nucleobases in length, 18 to 30 nucleobases in length.
  • the nucleic acid is a siRNA that is a double stranded nucleic acid of 18 to 100 nucleobases in length.
  • the siRNA is 18 to 30 nucleobases in length.
  • the chemotherapeutic agent may be docetaxel, paclitaxel, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastin, methotrexate, oxaliplatin, or combinations thereof.
  • CDDP docetaxel
  • carboplatin carboplatin
  • procarbazine mechlorethamine
  • the chemotherapeutic agent is 5-fluorouracil or oxaliplatin.
  • the composition includes one or more anti-VEGF therapeutic agents, EGFR antagonists, IGFR antagonists, or other angiogenic inhibitors such as those angiogenic inhibitors that target the notch system.
  • the composition may further include a signaling inhibitor, an apoptosis inducer, or any other antineoplastic or antiangiogenic therapy.
  • the present invention also generally concerns methods of treating a subject with a disease that involve administering to the subject a pharmaceutically effective amount of any of the aforementioned compositions.
  • the disease can be any disease process associated with neuropilin expression, wherein reduced neuropilin expression is sought to achieve a therapeutic effect.
  • the disease may be a hyperproliferative disease or any disease process associated with angiogenesis.
  • the disease is cancer.
  • the cancer may be of any cancer type known to those of ordinary skill in the art.
  • the cancer may be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colorectal cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia.
  • the cancer is colorectal cancer.
  • the cancer is pancreatic ductal adenocarcinoma.
  • the subject may be any subject, but in particular embodiments the subject is a mammal. Non-limiting examples include human, primate, horse, cow, dog, cat, rat, mouse, and so forth. In specific embodiments, the subject is a human subject.
  • the method may further involve administering one or more additional therapies to the subject.
  • the subject has cancer
  • the additional therapy is an anticancer therapy that is chemotherapy, radiation therapy, surgical therapy, immunotherapy, gene therapy, or a combination thereof.
  • the additional anti-cancer therapy is chemotherapy.
  • the chemotherapy may include, for example, any of those agents discussed above and elsewhere in this specification.
  • the chemotherapy is 5-fluorouracil or oxaliplatin.
  • compositions can be administered to the subject using any method known to those of ordinary skill in the art.
  • Non-limiting examples include intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, topically, or by direct injection or perfusion.
  • the composition includes an siRNA that inhibits the expression of a gene that encodes NRP-I.
  • the composition includes an siRNA that inhibits the expression of a gene that encodes NRP-2.
  • the composition includes a shRNA that inhibits the expression of a gene that encodes NRP-2.
  • the subject has a tumor and the method is further defined as a method to reduce tumor volume in the subject.
  • the tumor may be of any type. Non-limiting examples are set forth above and elsewhere in this specification.
  • the tumor is colorectal cancer.
  • the tumor is pancreatic ductal adenocarcinoma.
  • the subject has a cancer and the method is further defined as a method to prevent metastasis of the cancer.
  • the cancer is colorectal cancer, and the metastasis is metastasis to the liver.
  • any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
  • any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
  • FIG. IA, IB Assessment of NRP-2 expression in human colon tissues and cell lines.
  • IA Representative tissue sections (2Ox magnification) of nonmalignant human colonic mucosa (Normal), adjacent colon adenocarcinoma (Normal/ Tumor Interface), colon adenocarcinoma (Tumor), and liver metastasis stained for expression of NRP-2 protein.
  • IB Immunoblot analysis of NRP-I and NRP-2 expression in six colon adenocarcinoma cell lines. Immunostaining for vinculin was used as an internal loading control.
  • FIG. 2A, 2B, 2C, 2D, 2E, 2F Effect of NRP-2 expression on proliferation and VEGFR-I receptor phosphorylation in HCT-116 cells.
  • 2A Creation of stable cell lines with reduced NRP-2 levels using shRNAs.
  • Upper panel immunoblot analysis of NRP-2 levels in parental HCT-116 cells carrying no shRNA, control shRNA (sh-Con) or shRNA to NRP-2 (sh-NP2-C8 and sh-NP2-C9).
  • Lower panel Immunoblot analysis following transient transfections with increasing concentrations of siRNAs to NRP-2 demonstrating specificity of this siRNA for reducing NRP-2 levels in human tumor cells (SW480) but not murine cells (murine melanoma cell line B16BL6).
  • 2B MTT analysis of growth rates of control cells (parental, sh-Con) or cells with reduced NRP-2 expression (sh-NP2-C8, sh-NP2-C9). Bars indicate standard mean ⁇ 95% CI.
  • 2C RT-PCR analysis of VEGFR-I, VEGFR-2, and VEGFR-3 expression in HCT-116 cells. Human umbilical vein endothelial cells (Hu) were used as a positive control. GAPDH was used as a positive control to ensure the integrity of RNA.
  • 2D Immunoblot analysis of VEGFR-I pathway activation.
  • Antibodies are used to compare VEGFR-I, Akt, and Erk-1/2 and BAD protein levels to phosphorylated protein levels in parental and control siRNA-containing cells and the two NRP-2 siRNA-expressing cell lines. Membranes were reprobed with an antibody against vinculin as a loading control.
  • 2E Immunoblot analysis showing phosphorylated VEGFR-I levels in control siRNA- carrying cells (sh-Con) and NRP2-shRNA -expressing cells (sh-NP2-C8) grown in serum- reduced media with or without stimulation with VEGF-A (10 ⁇ g/ml).
  • VEGF-A 10 ⁇ g/ml
  • FIG. 3A, 3B, 3C Effect of NRP-2 expression on survival or apoptosis of colorectal cancer cells in vitro.
  • 3 A Representative images of soft agar assay of a well from control cells and cells with reduced NRP-2 levels (magnification 0.75x). Growth of control HCT-116 cells (sh-Con) and HCT-116 cells with reduced NRP-2 expression (sh-NRP2-C8 and sh-NP2-C9) in soft agar.
  • FIG. 4A, 4B, 4C, 4D Effect of NRP-2 expression on migration and invasion by colorectal cancer cells.
  • 4A Representative images (2Ox) of migration assays of colon cancer cells with normal levels of NRP-2 expression (Parental and sh-Con) and cells with suppressed levels of NRP-2 expression (sh-NP2-C8 and Sh-NP2-C9).
  • 4B Mean number of cells from 5 independent assays that migrated in 4A. Asterisks indicate statistically significant differences in NRP-2 siRNA-containing cells vs. sh-Con control.
  • 4C Invasion assay of Parental HCT-116 cells, sh-Con cells, sh-NP2-C8- and sh-NP2-C9-cells in modified Matrigel membrane coated Boyden chambers, in which 30,000 cells of each clone were plated and after 12 hours, invasive cells which had digested and moved through the matrigel membrane were stained and counted under a microscope (2Ox magnification).
  • 4D Graphical representation of invasive cells calculated as mean value ⁇ 95% CI per five fields at 2Ox magnification. Asterisks indicate statistically significant differences in NRP-2 siRNA- containing cells vs. sh-Con.
  • FIG. 5 A, 5B, 5C, 5D, 5E Effect of NRP-2 expression on in vivo growth and metastasis of colorectal cancer cells.
  • 5 A Tumor incidence in 10 nude mice per group 30 days after subcutaneous injection with control (sh-Con) HCT-116 cells or with HCT-116 cells with reduced NRP-2 expression (sh-NP2-C8 and sh-NP2-C9).
  • 5B Final tumor volumes 30 days after subcutaneous injection of stable clones of HCT-116 cells expressing control shRNA or shRNAs to reduce NRP-2 expression. Data are means ⁇ 95% CI from 10 mice. Asterisks indicate significant differences between NRP-2 siRNA-containing cells vs. sh-Con cells.
  • FIG. 6A, 6B Effect of NRP-2 expression on apoptosis in tumor xenografts.
  • 6A Inverted images of (4Ox magnification) tumor sections from subcutaneously injected nude mice (above) that have been immunofluorescently stained to detect annexin V expression as a measure of the number of apoptotic cells.
  • 6B Graphical representation of annexin V staining, calculated with the mean value of the control set as equaling 1.0 ⁇ 95% CI. Asterisks indicate statistically significant differences.
  • FIG. 7A, 7B, 7C, 7D, 7E, 7F Effect of administration of liposomal-conjugated siRNA on colorectal tumor growth in vivo.
  • 7A - Top In vitro assay demonstrating reduced lucif erase activity in HCT-116 cells carrying the lenti-luc gene after transient trans fection with luciferase siRNA. Twenty- four hours after plating the cells in 12-well dishes, siRNA to luciferase or scrambled control siRNA was transiently transfected at the indicated concentrations.
  • 7B Top: Graphical plot of in vivo bio luminescent activity/proton emissions from mice at 3, 14, and 28 days post hepatic inoculation with 1.0 xlO 6 HCT-116 cells harboring the lenti-luc gene. Mice (10 per group) were treated intraperitoneally at day 4 and every 5 days thereafter, with Control-siRNA-DOPC complex or NRP-2 siRNA-DOPC complex. 7C - Final tumor volumes from mice in FIG. 7B after 32 days post inoculation of 1.0 xl O 6 HCT- 116 cells.
  • mice received 6 total intraperitoneal injections of liposomal control- or NRP-2- specif ⁇ c siRNA complexes (Control siRNA; Con-#1 and Con-#1, NRP-2 siRNA; NP2-#1 and NP2-#2, 5 ug total of siRNA per treatment).
  • Final tumor volumes were calculated as [(length/2) x (width )] and presented as means 1.0 ⁇ 95% CI.
  • Asterisk indicates a statistically significant difference in NRP-2- versus control siRNA-DOPC.
  • Top panel Section from control (Control SiRNA-DOPC treated tumors showing NRP-2 expression (green fluorescence) in both human tumor cells and murine endothelial cells.
  • Bottom panel Section from NRP-2 siRNAs- DOPC treated tumors showing NRP-2 expression is substantially decreased in human tumor cells compared to control, yet NRP-2 expression in murine vascular/endothelial cells (yellow arrows) remains unchanged. Hoechst staining (blue) was utilized to demarcate nuclei.
  • FIG. 8 Knockdown of NRP-2 expression.
  • FIG. 9 Effect of reduced NRP-2 expression on constitutive signaling.
  • FIG. 10 Effect of reduced NRP-2 expression on angiogenic mediator expression.
  • FIG. 11 Effect of decreased NRP-2 expression on migration.
  • FIG. 16 Effect of decreased NRP-2 expression on angiogenesis.
  • Neuropilins are 120 to 130 kDa non- tyrosine kinase receptors (Soker et ai, 1998).
  • Neuropilins include NRP-I and NRP-2. Multiple NRP-I and NRP-2 isoforms exist, including soluble forms (Rossignol et al, 2000; Gluzman-Poltorak et ah, 2000).
  • the basic structure of neuropilins includes five domains, including three extracellular domains, a transmembrane domain, and a short cytoplasmic domain (reviewed in Ellis, 2006).
  • Table 1 lists the GenBank Accession numbers of neuropilin protein sequences from homo sapiens.
  • EAW70371 10 EAW70370 11 EAW70369 12 EAW70368 13 EAW70367 14 EAW70366 15 EAW70365 16 EAW70364 17 EAW70363 18 EAW70362 19 AAC51789 20 AAC51788 21
  • siRNA-mediated gene silencing guidelines for selection of target sites on mRNA have been developed for optimal design of siRNA (Soutschek et al., 2004; Wadhwa et al, 2004). These strategies may allow for rational approaches for selecting siRNA sequences to achieve maximal gene knockdown.
  • plasmids and viral vectors such as adenovirus, lentivirus, and retrovirus have been used (Wadhwa et al., 2004). While many of these approaches are successful for in vitro studies, in vivo delivery poses additional challenges based on the complexity of the tumor microenvironment.
  • Liposomes are a form of nanoparticles that are attractive carriers for delivering a variety of drugs into the diseased tissue.
  • Optimal liposome size depends on the tumor target. In tumor tissue, the vasculature is discontinuous, and pore sizes vary from 100 to 780 nm (Siwak et al., 2002). By comparison, pore size in normal vascular endothelium is ⁇ 2 nm in most tissues, and 6 nm in post-capillary venules. Most liposomes are 65-125 nm in diameter.
  • Negatively charged liposomes were believed to be more rapidly removed from circulation than neutral or positively charged liposomes; however, recent studies have indicated that the type of negatively charged lipid affects the rate of liposome uptake by the reticulo-endothelial system (RES). For example, liposomes containing negatively charged lipids that are not sterically shielded (phosphatidylserine, phosphatide acid, and phosphatidylglycerol) are cleared more rapidly than neutral liposomes.
  • RES reticulo-endothelial system
  • siRNA appears to be more stable than antisense molecules, serum nucleases can degrade siRNAs (Leung and Whittaker, 2005).
  • modifications such as chemically stabilized siRNAs with partial phosphorothioate backbone and 2'-0-methyl sugar modifications or boranophosphate siRNAs (Leung and Whittaker, 2005).
  • Elmen and colleagues modified siRNAs with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA), which significantly enhanced the serum half-life of siRNA and stabilized the structure without affecting the gene-silencing capability (Elmen et al, 2005).
  • LNA Locked Nucleic Acid
  • the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991).
  • the present invention also encompasses compositions that have different structures in solution than the normal vesicular structure.
  • the lipids may assume a micellar structure or merely exist as non-uniform aggregates of lipid molecules.
  • lipofectamine- nucleic acid complexes are also contemplated.
  • Liposome-mediated polynucleotide delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al. (1980) demonstrated the feasibility of liposome- mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
  • Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.
  • the lipid may be associated with a hemaglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • HVJ hemaglutinating virus
  • the lipid may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al, 1991).
  • HMG-I nuclear non-histone chromosomal proteins
  • the lipid may be complexed or employed in conjunction with both HVJ and HMG-I. In that such expression vectors have been successfully employed in transfer of a polynucleotide in vitro and in vivo, then they are applicable for the present invention.
  • a neutral liposome may be used to deliver a siRNA.
  • the neutral liposome may contain a siRNA directed to the suppression of translation of a single gene, or the neutral liposome may contain multiple siRNA that are directed to the suppression of translation of multiple genes.
  • the neutral liposome may also contain a chemo therapeutic in addition to the siRNA; thus, in certain embodiments, chemotherapeutic and a siRNA may be delivered to a cell (e.g., a cancerous cell in a human subject) in the same or separate compositions.
  • An advantage to using neutral liposomes is that, in contrast to the toxicity that has been observed in response to cationic liposomes, little to no toxicity has yet been observed as a result of neutral liposomes. 2.
  • Lipid compositions of the present invention may comprise phospholipids.
  • a single kind or type of phospholipid may be used in the creation of lipid compositions such as liposomes (e.g., DOPC used to generate neutral liposomes).
  • more than one kind or type of phospholipid may be used.
  • Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols, and phosphatidylethanolamines; because phosphatidylethanolamines and phosphatidyl cholines are non-charged under physiological conditions (i.e., at about pH 7), these compounds may be particularly useful for generating neutral liposomes.
  • the phospholipid DOPC is used to produce non-charged liposomes or lipid compositions.
  • a lipid that is not a phospholipid e.g., a cholesterol
  • Phospholipids may be from natural or synthetic sources. However, phospholipids from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine are not used in certain embodiments as the primary phosphatide (i.e., constituting 50% or more of the total phosphatide composition) because this may result in instability and leakiness of the resulting liposomes. 3. Production of Liposomes Liposomes and lipid compositions of the present invention can be made by different methods.
  • a nucleotide e.g., siRNA
  • a nucleotide may be encapsulated in a neutral liposome using a method involving ethanol and calcium (Bailey and Sullivan, 2000).
  • the size of the liposomes varies depending on the method of synthesis.
  • a liposome suspended in an aqueous solution is generally in the shape of a spherical vesicle, and may have one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel array of molecules represented by the formula XY, wherein X is a hydrophilic moiety and Y is a hydrophobic moiety.
  • the concentric layers are arranged such that the hydrophilic moieties tend to remain in contact with an aqueous phase and the hydrophobic regions tend to self-associate.
  • the lipid molecules may form a bilayer, known as a lamella, of the arrangement XY-YX. Aggregates of lipids may form when the hydrophilic and hydrophobic parts of more than one lipid molecule become associated with each other. The size and shape of these aggregates will depend upon many different variables, such as the nature of the solvent and the presence of other compounds in the solution. Lipids suitable for use according to the present invention can be obtained from commercial sources.
  • Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen- free water by shaking until all the lipid film is resuspended.
  • the aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum.
  • Liposomes can also be prepared in accordance with other known laboratory procedures: the method of Bangham et ⁇ l.
  • Dried lipids or lyophilized liposomes may be dehydrated and reconstituted in a solution of inhibitory peptide and diluted to an appropriate concentration with a suitable solvent ⁇ e.g., DPBS). The mixture may then be vigorously shaken in a vortex mixer. Unencapsulated nucleic acid may be removed by centrifugation at 29,00Og and the liposomal pellets washed. The washed liposomes may be resuspended at an appropriate total phospholipid concentration ⁇ e.g., about 50-200 mM). The amount of nucleic acid encapsulated can be determined in accordance with standard methods. After determination of the amount of nucleic acid encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentrations and stored at 4°C until use.
  • a suitable solvent ⁇ e.g., DPBS
  • Unencapsulated nucleic acid may be removed by centrifugation at 29,00Og and the liposomal pellets
  • siRNA e.g., siRNA
  • siRNA and double- stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Applications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety.
  • the components of a nucleic acid need not be of the same type or homogenous throughout (e.g., a siNA may comprise a nucleotide and a nucleic acid or nucleotide analog).
  • the siNA may comprise 17 to 35 contiguous nucleobases, more preferably 18 to 30 contiguous nucleobases, more preferably 19 to 25 nucleobases, more preferably 20 to 23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure.
  • Agents of the present invention useful for practicing the methods of the present invention include, but are not limited to siRNAs.
  • RNA interference double-stranded RNA
  • siRNA small interfering RNA
  • RNAi there are several factors that need to be considered such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system.
  • the siRNA that is introduced into the organism will typically contain exonic sequences.
  • the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences.
  • the siRNA exhibits greater than 80, 85, 90, 95, 98,% or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences less than about 80% identical to the target gene are substantially less effective.
  • the greater homology between the siRNA and the STAT gene to be inhibited the less likely expression of unrelated genes will be affected.
  • the invention generally features an isolated siRNA molecule of at least 19 nucleotides, having at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of a nucleic acid that encodes a neuropilin (such as NRP-2), and that reduces the expression of the neuropilin.
  • the siRNA molecule has at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of the mRNA that encodes NRP-2.
  • the siRNA molecule is at least 75, 80, 85, or 90% homologous, preferably 95%, 99%, or 100% homologous, to at least 10 contiguous nucleotides of any of the nucleic acid sequences encoding a full-length neuropilin protein, such as those in Table 1.
  • the siRNA may also comprise an alteration of one or more nucleotides.
  • Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA).
  • the RNA molecule contains a 3'-hydroxyl group.
  • Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides.
  • RNAi is capable of decreasing the expression of a neuropilin, such NRP -2, by at least 10%, 20%, 30%, or 40%, more preferably by at least 50%, 60%, or 70%, and most preferably by at least 75%, 80%, 90%, 95% or more.
  • a neuropilin such NRP -2
  • Certain embodiments of the present invention pertain to methods of inhibiting expression of a gene encoding a neuropilin in a cell.
  • the neuropilin is NRP -2.
  • Introduction of siRNA into cells can be achieved by methods known in the art, including for example, microinjection, electroporation, or transfection of a vector comprising a nucleic acid from which the siRNA can be transcribed.
  • a siRNA can be directly introduced into a cell in a form that is capable of binding to target mRNA transcripts.
  • the siRNA may be combined or modified with liposomes, poly-L-lysine, lipids, cholesterol, Hpofectine or derivatives thereof.
  • cholesterol-conjugated siRNA can be used (see, Song et ah, 2003).
  • the present invention provides methods and compositions for the delivery of siNA via neutral liposomes. Because a siNA is composed of a nucleic acid, methods relating to nucleic acids ⁇ e.g., production of a nucleic acid, modification of a nucleic acid, etc.) may also be used with regard to a siNA.
  • nucleic acid is well known in the art.
  • a "nucleic acid” as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase.
  • Double stranded nucleic acids are formed by fully complementary binding, although in some embodiments a double stranded nucleic acid may formed by partial or substantial complementary binding.
  • a nucleic acid may encompass a double-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence, typically comprising a molecule.
  • a single stranded nucleic acid may be denoted by the prefix "ss" and a double stranded nucleic acid by the prefix "ds”.
  • nucleobase(s) encompass naturally occurring purine and/or pyrimidine nucleobases and also derivative(s) and analog(s) thereof, including but not limited to, those a purine or pyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol or alkylthiol moeity.
  • Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.) moeities comprise of from about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms.
  • a nucleobase may be comprised in a nucleside or nucleotide, using any chemical or natural synthesis method described herein or known to one of ordinary skill in the art. 2. Nucleosides
  • nucleoside comprising a purine (i.e., A or G) or a 7-deazapurine nucleobase typically covalently attaches the 9 position of a purine or a 7-deazapurine to the l'-position of a 5-carbon sugar.
  • a nucleoside comprising a pyrimidine nucleobase typically covalently attaches a 1 position of a pyrimidine to a l'-position of a 5-carbon sugar (Kornberg and Baker, 1992).
  • a nucleic acid may comprise, or be composed entirely of, a derivative or analog of a nucleobase, a nucleobase linker moiety and/or backbone moiety that may be present in a naturally occurring nucleic acid.
  • a "derivative” refers to a chemically modified or altered form of a naturally occurring molecule
  • the terms “mimic” or “analog” refer to a molecule that may or may not structurally resemble a naturally occurring molecule or moiety, but possesses similar functions.
  • a “moiety” generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure. Nucleobase, nucleoside and nucleotide analogs or derivatives are well known in the art, and have been described (see for example, Scheit, 1980, incorporated herein by reference).
  • nucleosides, nucleotides, or nucleic acids comprising 5-carbon sugar and/or backbone moiety derivatives or analogs include those in U.S. Patent 5,681,947 which describes oligonucleotides comprising purine derivatives that form triple helixes with and/or prevent expression of dsDNA; U.S. Patents 5,652,099 and 5,763,167 which describe nucleic acids incorporating fluorescent analogs of nucleosides found in DNA or RNA, particularly for use as flourescent nucleic acids probes; U.S. Patent 5,614,617 which describes oligonucleotide analogs with substitutions on pyrimidine rings that possess enhanced nuclease stability; U.S.
  • Patents 5,378,825, 5,777,092, 5,623,070, 5,610,289 and 5,602,240 which describe oligonucleotides with three or four atom linker moeity replacing phosphodiester backbone moeity used for improved nuclease resistance, cellular uptake and regulating RNA expression
  • U.S. Patent 5,858,988 which describes hydrophobic carrier agent attached to the 2'-0 position of oligonuceotides to enhanced their membrane permeability and stability
  • U.S. Patent 5,214,136 which describes olignucleotides conjugated to anthraquinone at the 5' terminus that possess enhanced hybridization to DNA or RNA; enhanced stability to nucleases;
  • a nucleic acid comprising a derivative or analog of a nucleoside or nucleotide may be used in the methods and compositions of the invention.
  • a non-limiting example is a "polyether nucleic acid", described in U.S. Patent 5,908,845, incorporated herein by reference.
  • polyether nucleic acid one or more nucleobases are linked to chiral carbon atoms in a polyether backbone.
  • PNA protein nucleic acid
  • PENAM peptide-based nucleic acid analog
  • Peptide nucleic acids generally have enhanced sequence specificity, binding properties, and resistance to enzymatic degradation in comparison to molecules such as DNA and RNA (Egholm et ah, 1993; PCT/EP/01219).
  • a nucleic acid may be made by any technique known to one of ordinary skill in the art, such as chemical synthesis, enzymatic production or biological production.
  • Non-limiting examples of a synthetic nucleic acid include a nucleic acid made by in vitro chemically synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al, 1986 and U.S. Patent 5,705,629, each incorporated herein by reference.
  • one or more oligonucleotide may be used.
  • Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
  • a non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCRTM (see for example, U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference.
  • a non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al 2001, incorporated herein by reference).
  • a nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al, 2001, incorporated herein by reference).
  • the present invention concerns a nucleic acid that is an isolated nucleic acid.
  • isolated nucleic acid refers to a nucleic acid molecule (e.g., an RNA or DNA molecule) that has been isolated free of, or is otherwise free of, the bulk of the total genomic and transcribed nucleic acids of one or more cells.
  • isolated nucleic acid refers to a nucleic acid that has been isolated free of, or is otherwise free of, bulk of cellular components or in vitro reaction components such as for example, macromolecules such as lipids or proteins, small biological molecules, and the like. 8.
  • low stringency or “low stringency conditions”
  • non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20 0 C to about 50 0 C.
  • hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 20 0 C to about 50 0 C.
  • Treatment refers to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • a treatment may include administration of a pharmaceutically effective amount of a nucleic acid that inhibits the expression of a gene that encodes a neuropilin and a neutral lipid for the purposes of minimizing the growth or invasion of a tumor, such as a colorectal cancer.
  • a "disease” or “health-related condition” can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, and/or environmental stress.
  • the cause may or may not be known.
  • the methods include identifying a patient in need of treatment.
  • a patient may be identified, for example, based on taking a patient history, based on findings on clinical examination, based on health screenings, or by self- referral.
  • the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer is colorectal cancer (i.e., cancer involving the colon or rectum).
  • composition containing a siNA where clinical application of a composition containing a siNA is undertaken, it will generally be beneficial to prepare a pharmaceutical composition appropriate for the intended application. This will typically entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One may also employ appropriate buffers to render the complex stable and allow for uptake by target cells.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • preservatives e.g., antibacterial agents, antifungal agents
  • isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • a pharmaceutically acceptable carrier is preferably formulated for administration to a human, although in certain embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but which would not be acceptable (e.g., due to governmental regulations) for administration to a human. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a gene expression inhibitor may be administered in a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more ⁇ g of nucleic acid per dose.
  • Each dose may be in a volume of 1, 10, 50, 100, 200, 500, 1000 or more ⁇ l or ml.
  • Solutions of therapeutic compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms .
  • compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified.
  • a typical composition for such purpose comprises a pharmaceutically acceptable carrier.
  • the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline.
  • Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.
  • compositions include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
  • the compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
  • the therapeutic compositions of the present invention may include classic pharmaceutical preparations. Administration of therapeutic compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for the treatment of skin cancers, to prevent chemotherapy- induced alopecia or other dermal hyperproliferative disorder.
  • administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients.
  • aerosol delivery can be used for treatment of conditions of the lungs, or respiratory tract. Volume of the aerosol is between about 0.01 ml and 0.5 ml.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered both according to number of treatments and unit dose, depends on the protection or effect desired.
  • Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.
  • the methods and compositions including combination therapies enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy.
  • Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both an inhibitor of gene expression and a second therapy.
  • one agent may be given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any combination thereof, and another agent is given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
  • the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as their prognosis, strength, health, etc.
  • an inhibitor of gene expression therapy is "A” and an anti-cancer therapy is "B”: A/B/A BIAJB B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B/B
  • any compound or therapy of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
  • chemotherapeutic agents may be used in accordance with the present invention.
  • the term “chemotherapy” refers to the use of drugs to treat cancer.
  • chemotherapeutic agent is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (
  • anti-hormonal agents that act to regulate or inhibit hormone action on tumors
  • SERMs selective estrogen receptor modulators
  • aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole
  • anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3- diox
  • DNA damaging factors include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. 3.
  • immunotherapeutics In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Trastuzumab (HerceptinTM) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • toxin chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • the combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers.
  • Another immunotherapy could also be used as part of a combined therapy with gen silencing therapy discussed above.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention.
  • Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL- 12, GM-CSF, gamma-IFN, chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand.
  • immune stimulating molecules either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al, 2000).
  • antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein.
  • immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S.
  • Patents 5,830,880 and 5,846,945) and monoclonal antibodies e.g., anti-ganglioside GM2, anti-HER- 2, anti-pl85 (Pietras et al, 1998; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the gene silencing therapies described herein.
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
  • cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments.
  • human colorectal carcinoma cell lines (Geo, HCT-116, HT-29, RKO, and SW- 480 cells) were obtained from the American Type Culture Collection (Manassas, VA).
  • the human colorectal carcinoma cell line KM 12 and the murine melanoma B16BL6 cell lines were obtained from Dr. I. J. Fidler (M. D. Anderson Cancer Center). Unless stated otherwise, all cells were maintained at 37°C with 5% CO 2 in complete minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and streptomycin (200 ⁇ g/ml) (Life Technologies, Grand Island, NY).
  • VEGF-A vascular endothelial growth factor-A
  • VEGF-A R & D Systems, Minneapolis, MN
  • shRNA Expression Plasmids, shRNA Cell Lines, and transient siRNA targeting were used at a concentration of 10 ⁇ g/mL for 10 minutes.
  • NRP -2 deficient cell lines were created by transfecting HCT-116 cells with 0.5 ng of both shRNA expression plasmids (sh-NP2Vec-#l and sh-NP2Vec-#2), while control shRNA cells were created by transfecting both scrambled sequence encoding shRNA vectors (sh-ConVec-#l and sh-ConVec-#2) at similar concentrations.
  • Stable clones were isolated by growing each trans fected cell type in medium containing 850 ⁇ g/mL hygromycin B (Roche Diagnostics, Mannheim, Germany). NRP-2 expression levels in isolated clones of shRNA generated HCT-116 cells were determined by immunoblot analysis.
  • HCT-116 cells 2.5 x 10 5 HCT-116 cells were plated per well in medium in 6-well plates, and incubated for 24 hours. The cells were then transfected with increasing concentrations of siRNA oligos (both NRP-2 target sequences described above without hairpins) using the transfection reagent SiPORT/ NeoFX (Ambion, TX) according to the manufactures' protocol.
  • the cells were incubated for an additional 72 hours after transfection and solubilized in 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% Triton X- 100, 1 mM Na 3 VO 4 , 2 mM EDTA, and one complete Mini Protease Inhibitor Cocktail Tablet (per 10 ml of lysis buffer) (Roche Diagnostics) and subjected to western/immunoblot analysis to determine NRP-2 levels.
  • Antibodies used were, NRP-I (C- 19, Mu mAb, Santa Cruz Biotechnology) and NRP-2 (C-9 and/or H- 300 from Santa Cruz Biotechnology); phospho-Akt Sei473 (DE-9, Rb mAb), Akt (C67E7, Rb mAb), phospho-Erk-l ⁇ 1111 2027T ⁇ 204 (D13.14.4E, Rb mAb), Erk-1/2 (137F5, Rb mAb), phospho-BADSer 136 (9295 Rb mAb) and phospho-BAD Ser 112 (9291, Rb mAb), and BAD (9292, Rb pAb) (all from Cell Signaling Technology, Danvers, MA); phospho-VEGFR- l Try1213 (PC459, Rb pAb, Calbiochem, Boston, MA) /(07-75K, Rb pAb, Upstate/Millipore, Billerica, MA); VEGFR-I (Oncogene Research Products, San
  • RT-PCR Assays The relative expression levels of VEGFR-I, VEGFR-2 and VEGFR-3 in human HCT-116 colon cancer cells and human umbilical vein endothelial cells (HUVEC) were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis.
  • the primers used were; VEGFRl, 5'-tgaaagccttcagtcccgtg-3' (sense; SEQ ID NO:27), and 5'-atccgtgttgagggtggtcagc-3' (antisense; SEQ ID NO:28); VEGFR-2,5'- catcacatccactggtattgg-3 ' (sense; SEQ ID NO:29), and 5'-gccaagcttgtaccatgtgag-3' (antisense; SEQ ID NO:30);and VEGFR-3, 5'-cccacgcagacatcaagacg (sense; SEQ ID NO:31), 5'- tgcagaactccacgatcacc-3 ' (antisense; SEQ ID NO: 32).
  • TRIzol reagent Invitrogen, Carlsbad, CA
  • Synthesized cDNAs were diluted in 500 ⁇ l of diethylpyrocarbonate-treated water, and 3 ⁇ l of each reaction was used in each 25 - ⁇ l RT-PCR reaction. Amplifications were performed using the following parameters: 95 0 C for 1 min, followed by 35 cycles of 95 0 C for 30 s, 6O 0 C for 30 s and 72 C for 1 minute.
  • VEGF receptor expression gene expression was normalized using reference primers toward glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5'- ccttcattgacctcaactac-3' (sense; SEQ ID NO33), and 5'-gatgatgttctggagtgcc-3' (antisense; SEQ ID NO:34).
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • SEQ ID NO33 sense
  • 5'-gatgatgttctggagtgcc-3' antisense; SEQ ID NO:34
  • VEGF Enzyme-Linked Immunosorbent Assay VEGF Enzyme-Linked Immunosorbent Assay. VEGF production in culture supernatants from control cells (Parental and sh-Con) and cells with reduced NRP-2 levels (sh-NP2-C8 and sh-NP2-C9) were examined using a human VEGF-specific ELISA according to the manufacture's instructions (Quantikine; R&D Systems, Minneapolis, MN). Cells were plated out at 80% cell density in a 100mm cell culture dish in minimal essential medium supplemented with 1% fetal bovine serum, 2 mM L-glutamine, streptomycin (200 ⁇ g/ml).
  • VEGF concentration was normalized to the total protein content of each culture dish, as measured by the Bradford assay.
  • MTT substrate 2.5mg/ml of PBS
  • DMSO dimethyl sulfoxide
  • Annexin V Staining To determine the role of NRP-2 in mediating survival of HCT- 116 cells under conditions of hypoxic stress, cells with normal levels of NRP-2 (Parental and sh-Con) and reduced levels of NRP-2 (sh-NP2-C8 and sh-NP2-C9) were subjected to hypoxic conditions (1% O 2 , 99% N 2 ) for 6 and 24 hours. The relative percentage of apoptotic cells was assessed at these time points using the Annexin V-FITC apoptosis Detection Kit-1, (BD Pharmingen, San Diego, CA) according to the manufacturer's protocol. Annexin V quantitation was performed using a Coulter EPICS XL-MCL fluorescent-activated cell (FAC) analyzer (Beckman Coulter, Miami, FL) equipped with System II software (Beckman Coulter).
  • FAC Coulter EPICS XL-MCL fluorescent-activated cell
  • Migration and Invasion Assays were conducted as described previously with minor modifications. Equal numbers (30,000) of control cells (sh-Con) or cells with reduced NRP-2 expression (sh-NP2-C8 and sh-NP2-C9) were suspended in 0.5 ml of medium and placed in the top compartment of a standard 8- ⁇ m pour Boyden chamber with 0.5 ml of medium added to the bottom compartment. Following 12 hr incubation under standard conditions (37 0 C / 5% CO 2 ), non-migrating cells were scraped from the top compartment and cells that had migrated to the bottom compartment were fixed and stained using the Protocol HEMA 3 stain set (Fisher Scientific). Membranes were excised and mounted on a standard microscope slide (Curtis Matheson Scientific, Houston, TX). The numbers of migrated cells were determined from 5 random fields visualized at 2Ox magnifications.
  • siRNA-Containing Liposomes For experiments to test the efficacy of in vivo therapeutic targeting of NRP -2 in tumor xenografts in mice, liposomes containing siRNA's were prepared as previously described (Landen et ah, 2005).
  • lyophilized preparations were hydrated in 0.9% saline at a concentration of 5 ⁇ g of si-RNA/ 200 ⁇ L, and were purified by separating free siRNA from liposomes with filter units with a size exclusion limit of 30,000 Daltons (Millipore Corp).
  • NRP-2 Expression of NRP-2 in Human Colon Tissues and Cell Lines.
  • HCT-116 cells stably transfected with shRNA to NRP-2 showed no changes in proliferation rate relative to that of Parental and sh-Con-transfected HCT-116 cells (FIG. 2B).
  • Doubling times were approximately 25 hours for both the Parental and the sh-Con control cells, and 24-26 hours for sh-NP2-C8 and sh-NP2-C9.
  • VEGF-A (10 ⁇ g/mL) treatment did not alter the doubling times of any of these cell lines, regardless of NRP-2 expression level, compared with doubling times in the absence of VEGF-A.
  • control cells and NRP-2 shRNA-expressing cells were grown in serum-reduced medium (1% fetal calf serum) and stimulated with VEGF-A (10 ⁇ g/ml) for 10 minutes, followed by protein extraction and immunoblot analysis.
  • serum-reduced medium 1% fetal calf serum
  • VEGF-A 10 ⁇ g/ml
  • VEGFR-I phosphorylation was increased upon VEGF-A stimulation in control cells to a greater extent than in HCT- 116 cells with reduced shRNA mediated reduction of NRP-2 levels. Effect of NRP-2 Expression on Endogenous Expression of VEGF-A and SEMA3F.
  • NRP-2 expression on tumor growth equal amounts (1.0 xlO / mouse) of HCT-116 control cells (sh-Con) or NRP-2 siRNA-expressing cells (sh-NP2-C8 and sh-NP2-C9) were injected subcutaneous Iy into ten nude mice and assessed tumor incidence and volume 30 days later. All mice were of approximately the same overall weight when sacrificed. Tumor incidence was 100 % in control (sh-Con) mice, 40 % in sh-NP2-C9 mice, and 60 % in sh-NP2-C9 mice (FIG. 5A). Subcutaneous tumors produced by cells with reduced NRP -2 expression were significantly smaller than those produced by control cells expressing normal levels of NRP-2 (Sh-Con) (FIG.
  • mice 7A, top were complexed with DOPC and injected intraperitoneally into mice at a dose of 5 ⁇ g/mouse 10 days after the inoculation of 1.0 x 10 6 HCT-116 colon cancer cells expressing the lenti-luc gene.
  • siRNA-DOPC administration Prior to siRNA-DOPC administration, the mice were subjected to bio luminescent imaging to establish baseline activity, and again 48 hours after siRNA treatment, to examine whether administration of siRNA to luciferase in liposomes led to a reduction in luciferase activity in the tumor cells. Hepatic luciferase activity, as reflected by photon emission, was reduced by approximately 50% in treated mice compared with that in the same mice measured before SiRNA-Luc- DOPC administration (FIG. 7A, bottom).
  • FIG. 8 demonstrates that specific knockdown of NRP-2 without any effect on NRP- 1.
  • FIG. 15 shows that decreased NRP-2 expression was associated with reduced in vivo proliferation. The effect of decreased NRP-2 expression on angiogenesis was also examined.
  • the stable control-transfected cell line, shCon was generated similarly using both scramble sequence-encoding shNRA expression plasmids. Selected clones were isolated and maintained in medium containing 50 ⁇ g/mL hygromycin B (Roche Diagnostics, Mannheim, Germany). NRP-2 expression levels in all resulting cell lines were determined by Western blot analysis.
  • ⁇ -actin or vinculin Sigma-Aldrich, St. Louis, MO.
  • densitometric analysis was used to quantitate differences in protein levels from blots using NIH ImageJ vl.34 software (http://rsb.info.nih.gov/ij).
  • Invasion assays were performed using a similar protocol with minor modifications.
  • the inserts used in the invasion assays were coated with Matrigel (BD Biosciences, San Jose, CA) and prehydrated with 1% FBS-supplemented medium for 30 min prior to the addition of the cell suspension.
  • Invasion chambers were incubated for 48 h, and numbers of invading cells were again quantified.
  • Anchorage-Independent Growth Assays Soft-agar assays were used to determine the effect of reduced NRP-2 expression on the ability to grow in anchorage-independent conditions. Each well of a six -well plate was coated with 1 mL of 10% FBS-supplemented medium with 1% agarose. After 20 min, cell suspensions containing control and shNRP2- transfected cells (500 cells each) were added in 1 mL of medium with 0.5% agarose. Cells were incubated for 14 days under standard conditions (37 0 C, 5% CO 2 ) with the addition of 300 ⁇ L of medium every 3 days to hydrate the exposed agarose. At the end of the incubation period, wells were examined under a light microscope at 2Ox magnification, and the number of colonies larger than 50 ⁇ m was counted per well.
  • mice Male athymic nude mice, 6-8 weeks old, were obtained from the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD) and acclimated for 2 weeks. All animal studies were conducted under approved guidelines of the Animal Care and Use Committee of M. D. Anderson. Equal numbers of cells (10 6 ) from the shCon, shNRP2-C21, and shNRP2-C23 cell lines were suspended in 100 ⁇ L of PBS and injected subcutaneously into the right rear flank of each mouse (10 mice per group). Tumor growth was observed and recorded over 10 weeks. When tumors in the control group exceeded 1.5 cm in longest diameter, mice were killed by CO 2 asphyxiation according to protocol, and tumors were excised.
  • Tumors were weighed and measured, and a portion of each was placed in either 10% formalin (for paraffin embedding) or optimal cutting temperature (OCT) compound or was snap-frozen in liquid nitrogen. Tumor volume was calculated as 0.5 x (width 2 ) x (length).
  • OCT optimal cutting temperature
  • mice In each of 10 mice, a suspension of 2 x 10 6 luciferase-labeled shCon or shNRP2 cells in 50 ⁇ L of PBS was injected into the tail of the pancreas through a left-flank incision under ketamine/xylazine (Sigma- Aldrich) anesthesia. Mice were killed at 50 days, when 2-3 mice in any group showed signs of lethargy. Tumors were weighed, measured, and processed as in the subcutaneous model.
  • ketamine/xylazine Sigma- Aldrich
  • Tumors preserved in formalin were placed in paraffin blocks and sectioned onto positively charged microscope slides. They were deparaffmized in xylene, hydrated in graded alcohol, and pretreated for antigen retrieval in citrate buffer for 20 min in a 98 0 C steamer. Tumor sections embedded in OCT compound were sectioned onto positively charged microscopy slides and serially immersed in acetone, a 1 :1 (vol:vol) acetone: chloroform mixture, then acetone.
  • CD31 staining was isolated and digitized into a binary image, with black indicating stained vessels and white indicating no staining. Vessels with lumens were digitally filled, and a composite digitized microvascular area was quantitated.
  • Bioluminescence imaging of luciferase- expressing cells in the orthotopic tumor model was performed using the IVIS 100 imaging system coupled to a data-acquisition personal computer equipped with Living Image software (Xenogen Corp, Hopkinton, MA).
  • Tumor cell-inoculated mice were anesthetized with a 1.5% isoflurane-oxygen mixture and injected intraperitoneally with luciferase potassium salt solution (Sigma- Aldrich) at a dose of 150 mg/kg body weight immediately prior to imaging.
  • In vivo images were obtained on days 3, 18, 39, and 50, and photon emission representative of luciferase activity was used assess relative tumor burden in the mice.
  • FIG. 18A Western blot analysis of six commonly used pancreatic cancer cell lines demonstrated that five of six expressed NRP-2 to varying degrees (FIG. 18A).
  • NRP-2 was detected in 7 (64%) of 11 adenocarcinomas but not in any of four specimens of adjacent nonmalignant tissue; representative images are shown in FIG. 18B.
  • FIG. 18B In specimens designated positive, all visualized tumor cells stained positively for NRP-2 expression.
  • Reduced NRP-2 Expression Alters Constitutive Signaling in Pancreatic Cancer Cells.
  • ShRNA-NRP-2 decreased NRP-2 without any effect on NRP-I. Because the NRPs are co-receptors for VEGF receptors in both tumor cells and endothelial cells, we determined the effect of reduced NRP-2 expression on constitutive activation of VEGFR-I in BxPC3 cells. This cell line expresses VEGFR-I but not VEGFR-2 or VEGFR-3 (by reverse transcription polymerase chain reaction analysis). The reduction of NRP2 expression was associated with a decrease in phosphorylation of VEGFR-I in these cells relative to contra 1- transfected cells (FIG. 19A).
  • FIG. 22J-L Vessels in shCon tumors were subjectively larger with more visible patent lumens. Vessels were enumerated by counting the number of discrete stained structures within each field without regard to vessel size or patency. There was no difference in absolute vessel number between groups. D-MVA was analyzed to incorporate vessel size and patency into the analysis of the tumor vasculature by providing an estimate of integrated lumen area and presumably blood flow orthogonal to the tumor section. ShNRP2 tumors had a 61-66% decrease in D-MVA than shCon tumors (FIG. 21B and FIG. 22M-O; p ⁇ 0.05). Reduced NRP-2 Expression Is Associated with Decreased Jagged-1 Levels.
  • D-MVA The reduction of D-MVA led to the hypothesis that the effects on development of the tumor vasculature may be due to altered angiogenic mediator expression in the tumor cells themselves.
  • Western blotting was used to identify differences in protein levels of several known angiogenic mediators. There were no differences in VEGF-A, VEGF-C, or delta-like ligand-4 (DLL-4) levels between shCon- and shNRP2- transfected cells; however, there was a significant reduction (46% in shNRP2-C21 and 53% in shNRP2-C23) in Jagged-1 levels in cells deficient in NRP-2 relative to that in control cells (FIG. 24A, B).

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Abstract

Disclosed are compositions that include a nucleic acid component that include a nucleic acid that inhibits the expression of a gene that encodes a neuropilin, and a lipid component that includes one or more neutral phospholipids. Also disclosed are compositions that include a nucleic acid component that includes a nucleic acid that inhibits the expression of a gene that encodes neuropilin-2 (NRP-2). Also disclosed are methods of treating a subject with cancer that involve administering to the subject a pharmaceutically effective amount of a composition of the present invention.

Description

DESCRIPTION
TUMOR CELL EXPRESSION OF NEUROPILIN AS A TARGET FOR CANCER THERAPY
The present application claims the benefit of priority to U.S. Provisional Patent
Application Serial No. 61/025,213, filed January 31, 2008, which is hereby incorporated by reference in its entirety.
1. Field of the Invention
The present invention relates generally to the fields of molecular biology, oncology, and neuropilins. More particularly, the invention generally concerns compositions comprising an inhibitory nucleic acid, wherein the inhibitory nucleic acid is targeted to a nucleic acid encoding a neuropilin, and methods of treating cancer that involve administration of such compositions.
2. Description of Related Art
Tumor angiogenesis is a complex process that requires interactions among endothelial cells, tumor cells, and other components of the microenvironment. One of the most important secreted factors that promotes angiogenesis is vascular endothelial growth factor (VEGF) (Folkman and Shing, 1992; Ferrara, 2002). The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placental growth factor (Ferrara, 2002; Hicklin and Ellis, 2005). The activity of the VEGF family is mediated by the VEGF tyrosine kinase receptors VEGFR-I, VEGFR-2, and VEGFR-3 (Ferrara et al, 2003). Initially, these receptors were believed to be expressed only on the surface of endothelial cells, but subsequent findings revealed that VEGF tyrosine kinase receptors are expressed on numerous human cancer cells as well (reviewed in Wey et al., 2004).
Neuropilins are multifunctional non-tyrosine kinase receptors that bind to class 3 semaphorins and vascular endothelial growth factor (VEGF) (reviewed in Ellis, 2006). This family of receptors includes neuropilin 1 (NRP-I) and neuropilin-2 (NRP -2). NRP-I and NRP -2 have also been recently implicated in VEGF-mediated vascularization and lymphangio genesis (Ellis, 2006; Bielenberg et al., 2006; Karpanen et al., 2006). NRP-I and NRP -2 were originally identified as neuronal patterning receptors for the class 3 semaphorin ligands (Sema3A, Sema3C, and Sema3F) (He and Tessier-Lavigne, 1997; Chen et al, 1997). Unlike the previously identified VEGF receptors, NRP-I and NRP-2 lack a tyrosine kinase domain. Although the precise mechanisms for their activities are still a matter of debate, most studies suggest that the NRPs function as obligate coreceptors by cooperatively enhancing the activity of the VEGF kinase receptors in non-neuronal tissues (Ellis, 2006; Fuh et al, 2000; Whitaker et al, 2001; Lee et al, 2002; Wang et al, 2003; Pan et al, 2007). While NRP-I and NRP-2 are not normally expressed in adult tissues, their expression is detected on some human tumor cells (Ellis, 2006; Beilenberg, 2006). Neuropilins serve as receptors or coreceptors for multiple ligands, including class 3 semaphorins, and VEGF families (Gluzman-Poltorak et al, 2000; Gluzman-Poltorak et al, 2001; Makinen et al, 1999; Nasarre et al, 2003).
Studies have shown that neuropilin is expressed in the vasculature of certain tumors (Broholm and Laursen, 2004; Stephenson et al, 2002; Fakhari et al, 2002; Straume and Akslen, 2003), is upregulated in certain tumor types (Hansel et al, 2004; Lantuejoul et al, 2003; Kawakami et al, 2002; Fukahi et al, 2004; Parikh et al, 2003), and that neuropilin expression correlates with tumor progression and patient prognosis in specific tumor types (Broholm and Laursen, 2004; Stephenson et al, 2002; Hansel et al, 2004; Lantuejoul et al, 2003; Parikh et al, 2004). NRP-I expression has been reported in some human colon cancer samples (Parikh et al, 2004). The expression and function of NRP-2 on tumor cells has yet to be elucidated (Ellis, 2006; Beilenberg, 2006).
To date, most anti-VEGF therapeutic targeting has focused on attenuation of VEGFR- 2 kinase activity. Although anti-VEGF-targeted therapies for patients with cancer have led to incremental improvements in efficacy, the overall outcomes (e.g. prolonged tumor dormancy) have not met expectations. Regarding targeting of a neuropilin, two studies have suggested that interruption of NRP-I function with a monoclonal antibody in tumor-associated endothelial cells has an additive effect when used in conjunction with anti-VEGF therapy (Pan et al, 2007; Hong et al, 2007). NRP-2 has not been identified as a cancer target in any human cancer where its expression occurs. Thus, the precise role of neuropilins in cancer development and progression remains to be fully eludicated.
SUMMARY OF THE INVENTION
The present invention is based in part on the finding that neuropilins are involved in cancer development and progression. For example, the inventors have found that decreased NRP -2 expression, such as by shRNA targeting, results in reduction of tumor growth and metastasis in an animal model of human colorectal carcinoma. The inventors have also shown that reduction of NRP-2 expression in pancreatic ductal adenocarcinoma results in decreased migration, invasion, and growth of tumor cells. Further, the present invention is in part based on the finding that NRP-2 expression by a tumor in a subject can be reduced by administering to the subject a composition that includes an agent that reduces NRP-2 expression, which results in reduced tumor growth.
Certain embodiments of the present invention generally pertains to compositions that include (1) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes a neuropilin; and (2) a lipid component that includes one or more neutral phospholipids. The neuropilin may be NRP-I or NRP-2. In specific embodiments, the neuropilin is NRP-2.
Further embodiments of the present invention pertain to compositions that include a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes neuropilin-2 (NRP-2) and a pharmaceutically acceptable carrier.
The nucleic acid component may be a DNA or an RNA. In specific embodiments, the nucleic acid component is a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes an a neuropilin. In other specific embodiments, the nucleic acid component is a shRNA or a nucleic acid encoding a shRNA, wherein the shRNA inhibits the expression of a gene that encodes an a neuropilin. In certain embodiments, the composition includes a lipid component that forms a liposome. In embodiments that include a siRNA or shRNA, the siRNA or shRNA may be encapsulated in the lipid component.
The lipid component may include any lipid known to those of ordinary skill in the art. In particular embodiments, the lipid component includes one or more phospholipids. Any neutral phospholipid known to those of ordinary skill in the art is contemplated as a phospholipid for use in the compositions of the present invention that include one or more phospholipids. For example, the neutral phospholipid may be a phosphatidylcholine or phosphatidylethanolamine. Specific examples of neutral phospholipids include 1 ,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1- myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), 1- stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dimyristyl phosphatidylcholine ("DMPC"), l^-distearoyl-sn-glycero-S-phosphocholine ("DAPC"), 1 ,2-diarachidoyl-sn- glycero-3-phosphocholine ("DBPC"), 1 ,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), palmitoyloeoyl phosphatidylcholine ("POPC"), ^phosphatidylcholine, dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine ("DSPE"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), or lysophosphatidylethanolamine. In particular embodiments, the lipid component is DOPC. In some embodiments, the lipid component includes two or more neutral phospholipids. In some embodiments, the composition that includes a lipid component and a nucleic acid component further includes a pharmaceutically acceptable carrier. Any pharmaceutically acceptable carrier known to those of ordinary skill in the art is contemplated for inclusion in the compositions of the present invention. Examples of such carriers are discussed in greater detail in the specification below. The lipid component may further include a positively charged lipid or a negatively charged lipid. Any charged lipid is contemplated for inclusion in the compositions of the present invention. For example, the negatively charged phospholipid may be a phosphatidylserine or phosphatidylglycerol. Specific non-limiting examples of negatively charged phospholipids include dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), or dioleoylphosphatidylglycerol ("DOPG").
In some embodiments, the compositions of the present invention include cholesterol or polyethyleneglycol (PEG).
The nucleic acid component can be of any length. For example, the nucleic acid component may be 5 to 500 nucleobases in length, 10 to 300 nucleobases in length, 18 to 100 nucleobases in length, 18 to 30 nucleobases in length. In some specific embodiments, the nucleic acid is a siRNA that is a double stranded nucleic acid of 18 to 100 nucleobases in length. In more specific embodiments, the siRNA is 18 to 30 nucleobases in length.
The compositions of the present invention may further include one or more therapeutic agents. For example, the therapeutic agent may be an anti-inflammatory agent, an antibiotic, or a chemotherapeutic agent. In specific embodiments, the therapeutic agent is a chemotherapeutic agent (i.e., anti-cancer agent). Any chemotherapeutic agent known to those of ordinary skill in the art is contemplated for inclusion in the compositions of the present invention. For example, the chemotherapeutic agent may be docetaxel, paclitaxel, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastin, methotrexate, oxaliplatin, or combinations thereof. In specific embodiments, the chemotherapeutic agent is 5-fluorouracil or oxaliplatin. In particular embodiments, the composition includes one or more anti-VEGF therapeutic agents, EGFR antagonists, IGFR antagonists, or other angiogenic inhibitors such as those angiogenic inhibitors that target the notch system. The composition may further include a signaling inhibitor, an apoptosis inducer, or any other antineoplastic or antiangiogenic therapy. The present invention also generally concerns methods of treating a subject with a disease that involve administering to the subject a pharmaceutically effective amount of any of the aforementioned compositions. The disease can be any disease process associated with neuropilin expression, wherein reduced neuropilin expression is sought to achieve a therapeutic effect. For example, the disease may be a hyperproliferative disease or any disease process associated with angiogenesis.
In specific embodiments, the disease is cancer. The cancer may be of any cancer type known to those of ordinary skill in the art. For example, the cancer may be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colorectal cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, testicular cancer, lymphoma, or leukemia. In specific embodiments, the cancer is colorectal cancer. In further specific embodiments, the cancer is pancreatic ductal adenocarcinoma.
The disease may be any disease associated with abnormal angiogenesis. The disease may be a hyperproliferative disease such as cancer, any other disease that is associated with neovascularization. Examples of disease other than cancer that are associated with neovascularization include an inflammatory disease, an autoimmune disease, an arthritic condition, or ocular neovasculization. Examples of causes of ocular neovascularization include age-related macular degeneration and diabetic retinopathy. In such embodiments, for example, it may be desirable to reduce expression of NRP-2, which in turn, as set forth below, has the effect of decreasing the expression of the angiogenic factor Jaggedl.
The subject may be any subject, but in particular embodiments the subject is a mammal. Non-limiting examples include human, primate, horse, cow, dog, cat, rat, mouse, and so forth. In specific embodiments, the subject is a human subject.
Some methods of the present invention are further defined as including the step of identifying a subject in need of treatment. Any method known to those of ordinary skill in the art can be used to identify a subject in need of treatment.
The method may further involve administering one or more additional therapies to the subject. For example, in particular embodiments, the subject has cancer, and the additional therapy is an anticancer therapy that is chemotherapy, radiation therapy, surgical therapy, immunotherapy, gene therapy, or a combination thereof. In specific embodiments, the additional anti-cancer therapy is chemotherapy. The chemotherapy may include, for example, any of those agents discussed above and elsewhere in this specification. In specific embodiments, the chemotherapy is 5-fluorouracil or oxaliplatin.
The composition can be administered to the subject using any method known to those of ordinary skill in the art. Non-limiting examples include intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, topically, or by direct injection or perfusion. In some embodiments of the present methods, the composition includes an siRNA that inhibits the expression of a gene that encodes NRP-I. In other embodiments, the composition includes an siRNA that inhibits the expression of a gene that encodes NRP-2. In further embodiments, the composition includes a shRNA that inhibits the expression of a gene that encodes NRP-2. In some embodiments, the subject has a tumor and the method is further defined as a method to reduce tumor volume in the subject. The tumor may be of any type. Non-limiting examples are set forth above and elsewhere in this specification. In particular embodiments, the tumor is colorectal cancer. In further embodiements, the tumor is pancreatic ductal adenocarcinoma. In other embodiments, the subject has a cancer and the method is further defined as a method to prevent metastasis of the cancer. In particular embodiments, the cancer is colorectal cancer, and the metastasis is metastasis to the liver.
It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternative are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."
Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device and/or method being employed to determine the value.
As used herein the specification, "a" or "an" may mean one or more, unless clearly indicated otherwise. As used herein in the claim(s), when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
It is specifically contemplated that any limitation discussed with respect to one embodiment of the invention may apply to any other embodiment of the invention.
Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention.
BRIEF DESCRIPTION OF THE FIGURES The following figures form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. IA, IB. Assessment of NRP-2 expression in human colon tissues and cell lines. IA - Representative tissue sections (2Ox magnification) of nonmalignant human colonic mucosa (Normal), adjacent colon adenocarcinoma (Normal/ Tumor Interface), colon adenocarcinoma (Tumor), and liver metastasis stained for expression of NRP-2 protein. IB - Immunoblot analysis of NRP-I and NRP-2 expression in six colon adenocarcinoma cell lines. Immunostaining for vinculin was used as an internal loading control.
FIG. 2A, 2B, 2C, 2D, 2E, 2F. Effect of NRP-2 expression on proliferation and VEGFR-I receptor phosphorylation in HCT-116 cells. 2A - Creation of stable cell lines with reduced NRP-2 levels using shRNAs. Upper panel: immunoblot analysis of NRP-2 levels in parental HCT-116 cells carrying no shRNA, control shRNA (sh-Con) or shRNA to NRP-2 (sh-NP2-C8 and sh-NP2-C9). Lower panel: Immunoblot analysis following transient transfections with increasing concentrations of siRNAs to NRP-2 demonstrating specificity of this siRNA for reducing NRP-2 levels in human tumor cells (SW480) but not murine cells (murine melanoma cell line B16BL6). 2B - MTT analysis of growth rates of control cells (parental, sh-Con) or cells with reduced NRP-2 expression (sh-NP2-C8, sh-NP2-C9). Bars indicate standard mean ± 95% CI. 2C - RT-PCR analysis of VEGFR-I, VEGFR-2, and VEGFR-3 expression in HCT-116 cells. Human umbilical vein endothelial cells (Hu) were used as a positive control. GAPDH was used as a positive control to ensure the integrity of RNA. 2D - Immunoblot analysis of VEGFR-I pathway activation. Antibodies are used to compare VEGFR-I, Akt, and Erk-1/2 and BAD protein levels to phosphorylated protein levels in parental and control siRNA-containing cells and the two NRP-2 siRNA-expressing cell lines. Membranes were reprobed with an antibody against vinculin as a loading control. 2E - Immunoblot analysis showing phosphorylated VEGFR-I levels in control siRNA- carrying cells (sh-Con) and NRP2-shRNA -expressing cells (sh-NP2-C8) grown in serum- reduced media with or without stimulation with VEGF-A (10 μg/ml). Membranes were reprobed with an antibody against vinculin as a loading control. 2F - Enzyme-linked immunosorbent assay of VEGF expression in culture supernatants from parental cells and NRP-2 shRNA-expressing cells. Data are means ± 95% CI. FIG. 3A, 3B, 3C. Effect of NRP-2 expression on survival or apoptosis of colorectal cancer cells in vitro. 3 A - Representative images of soft agar assay of a well from control cells and cells with reduced NRP-2 levels (magnification 0.75x). Growth of control HCT-116 cells (sh-Con) and HCT-116 cells with reduced NRP-2 expression (sh-NRP2-C8 and sh-NP2-C9) in soft agar. Cells were plated at low density in medium containing 0.5% agarose and colonies greater than 50 μm in diameter were counted fourteen days later. Large colonies appearing in representative wells containing control cells (sh-Con) or cells expressing NRP-2 shRNA (sh-NP2-C8, sh-NP2-C9) are shown. 3B - Graphical representation of soft-agar assay. Viable cells in three wells were counted and values are means ± 95% CI. All soft-agar assays were performed in duplicate. Asterisks indicate statistically significant differences in NRP -2 shRNA-containing cells compared with controls (mean sh-Con = 384 colonies, 95% CI 340-428 colonies mean sh-NP2-C8 =112 colonies, 95% CI = 69 to 155 colonies, P = 0.002 versus sh-Con control; and mean sh-NP2-C9 = 81 colonies, 95% CI = 56 to 106 colonies, P = 0.002 versus sh-Con control). 3C - Graphical representation of Annexin V apoptosis detection assay. HCT-116 cells were either untreated or subjected to hypoxia (1% O2) for 6 hours and 24 hours, and the percent of apoptotic cells was determined. Values are means ± 95% CI. Asterisks indicate statistically significant differences in NRP2 siRNA- containing cells vs. controls (sh-Con 6 h hypoxia mean = 1.42 %, 95% CI = 1.22 to 1.69 %, mean sh-NP2-C8 6 h hypoxia = 2.9 %, 95% CI = 2.75 to 3.12 %, P = 0.01 vs sh-Con 6h hypoxia, mean sh-Con 24 h hypoxia = 1.96, 95% CI = 1.68 to 2.24 %, mean sh-NP2-C8 24 h hypoxia mean = 3.52 95% CI = 3.13 to 3.91 %, P = 0.008 vs sh-Con 24 h hypoxia).
FIG. 4A, 4B, 4C, 4D. Effect of NRP-2 expression on migration and invasion by colorectal cancer cells. 4A - Representative images (2Ox) of migration assays of colon cancer cells with normal levels of NRP-2 expression (Parental and sh-Con) and cells with suppressed levels of NRP-2 expression (sh-NP2-C8 and Sh-NP2-C9). 4B - Mean number of cells from 5 independent assays that migrated in 4A. Asterisks indicate statistically significant differences in NRP-2 siRNA-containing cells vs. sh-Con control. Mean Parental = 187 cells, 95% CI = 157 to 217 cells; mean sh-Con = 167 cells, 95% CI = 152 to 182 cells; mean sh-NP2-C8 = 91, 95% CI = 67 to 115 cells, P = 0.008 vs sh-Con; and mean sh-NP2-C9 = 64 cells, 95% CI = 39 to 89 cells, P = 0.008 vs sh-Con. 4C - Invasion assay of Parental HCT-116 cells, sh-Con cells, sh-NP2-C8- and sh-NP2-C9-cells in modified Matrigel membrane coated Boyden chambers, in which 30,000 cells of each clone were plated and after 12 hours, invasive cells which had digested and moved through the matrigel membrane were stained and counted under a microscope (2Ox magnification). 4D - Graphical representation of invasive cells calculated as mean value ± 95% CI per five fields at 2Ox magnification. Asterisks indicate statistically significant differences in NRP-2 siRNA- containing cells vs. sh-Con. Mean Parental = 83 cells, 95% CI = 69 to 97 cells; mean sh-Con = 89 cells, 95% CI = 76 to 102 cells; mean sh-NP2-C8 = 44 cells, 95% CI = 36 to 52 cells, P = 0.008 vs sh-Con; mean sh-NP2-C9 = 29 + 5 cells, 95% CI =19 to 39 cells, P = 0.008 vs sh- Con.
FIG. 5 A, 5B, 5C, 5D, 5E. Effect of NRP-2 expression on in vivo growth and metastasis of colorectal cancer cells. 5 A - Tumor incidence in 10 nude mice per group 30 days after subcutaneous injection with control (sh-Con) HCT-116 cells or with HCT-116 cells with reduced NRP-2 expression (sh-NP2-C8 and sh-NP2-C9). 5B - Final tumor volumes 30 days after subcutaneous injection of stable clones of HCT-116 cells expressing control shRNA or shRNAs to reduce NRP-2 expression. Data are means ± 95% CI from 10 mice. Asterisks indicate significant differences between NRP-2 siRNA-containing cells vs. sh-Con cells. Mean volume sh-Con = 1.0 cm3, 95% CI = .8 to 1.2 cm3; mean volume sh-NP2-C8 = 0.1 cm3, 95% CI = 0 to 0.2 cm3, P = 0.01 vs. sh-Con; mean sh-NP2-C9 = 0.2 cm3, 95% CI = 0.0 to 0.4 cm3, P = 0.01 vs. sh-Con. 5C - Average numbers of liver metastases 30 days after intrasplenic injection of stable clones of HCT-116 cells expressing control siRNA or siRNAs to reduce NRP-2 expression. Data are means ± 95% CI from 10 mice. Asterisks indicate statistically significant differences for sh-NP-C8 and sh-NP-C9 vs. sh-Con. Mean sh-Con = 20 metastases, 95% CI = 6 to 33 metastases; mean sh-NP2-C8 = 1 metastases, 95% CI = 0 to 1 metastases, P = 0.0002 vs. control; mean sh-NP2-C9 = 6 metastases, 95% CI = 0 to 16 metastases, P = 0.008 vs. control. 5D - Final volumes of the combined hepatic metastases from each mouse 30 days after intrasplenic injection of control cells or cells with reduced NRP-2 expression. Data are means ± 95% CI from 10 mice. Asterisks indicate significant differences for sh-NP2-C8 and sh-NP2-C9 vs. sh-Con. Mean sh-Con = 79 mm3, 95% CI = 0 to 159 ; mean sh-NP2-C8 = 1 mm3, 95% CI = 0 to 1 mm3, P = 0.0008 vs. sh-Con; mean sh- NP2-C9 = 9 mm3, 95% CI = 0 to 25 mm3, P = 0.006 vs. sh-Con. 5E - Photographs of excised livers and spleens from mice injected with control cells or NRP-2 siRNA-expressing cells. Arrows indicate metastases.
FIG. 6A, 6B. Effect of NRP-2 expression on apoptosis in tumor xenografts. 6A - Inverted images of (4Ox magnification) tumor sections from subcutaneously injected nude mice (above) that have been immunofluorescently stained to detect annexin V expression as a measure of the number of apoptotic cells. 6B - Graphical representation of annexin V staining, calculated with the mean value of the control set as equaling 1.0 ± 95% CI. Asterisks indicate statistically significant differences. Mean sh-Con = 4 apoptotic cells, 95% CI = 3 to 6; mean sh-NP2-C8 = 24 apoptotic cells, 95% CI = 9 to 39 apoptotic cells, P = 0.01 vs. sh-Con; mean sh-NP2-C9 = 36 apoptotic cells, 95% CI = 23 to 49 apoptotic cells, P = 0.05 vs. sh-Con.
FIG. 7A, 7B, 7C, 7D, 7E, 7F. Effect of administration of liposomal-conjugated siRNA on colorectal tumor growth in vivo. 7A - Top: In vitro assay demonstrating reduced lucif erase activity in HCT-116 cells carrying the lenti-luc gene after transient trans fection with luciferase siRNA. Twenty- four hours after plating the cells in 12-well dishes, siRNA to luciferase or scrambled control siRNA was transiently transfected at the indicated concentrations. Bottom: In vivo luciferase activity in a mouse carrying hepatic tumors after intrahepatic inoculation with 1.0 x 106 HCT-116 cells harboring the lenti-luc gene. Mice were treated with anti-siRNA-luciferase / DOPC complex (5 μg/mouse at ten days after tumor cell implantation), and were imaged again at 48 hours after siRNA treatment. The images shown are of a representative single mouse before and after treatment with luciferase siRNA- DOPC. 7B - Top: Graphical plot of in vivo bio luminescent activity/proton emissions from mice at 3, 14, and 28 days post hepatic inoculation with 1.0 xlO6 HCT-116 cells harboring the lenti-luc gene. Mice (10 per group) were treated intraperitoneally at day 4 and every 5 days thereafter, with Control-siRNA-DOPC complex or NRP-2 siRNA-DOPC complex. 7C - Final tumor volumes from mice in FIG. 7B after 32 days post inoculation of 1.0 xl O6 HCT- 116 cells. Mice received 6 total intraperitoneal injections of liposomal control- or NRP-2- specifϊc siRNA complexes (Control siRNA; Con-#1 and Con-#1, NRP-2 siRNA; NP2-#1 and NP2-#2, 5 ug total of siRNA per treatment). Final tumor volumes were calculated as [(length/2) x (width )] and presented as means 1.0 ± 95% CI. Asterisk indicates a statistically significant difference in NRP-2- versus control siRNA-DOPC. Mean Control siRNA-DOPC = 420 mm3, 95% CI = 212 to 628 mm3; mean NRP-2 siRNA-DOPC = 36 mm3, 95% CI = 6 to 65 mm3, P = 0.005 vs. Control siRNA-DOPC. 7D - Final liver weights/ total body weights of mice in FIG. 7B from in vivo siRNA treatment groups. Values are means + 95% CI. Asterisk indicates a statistically significant difference in liver weights / total body weights from NRP2- vs. control-siRNA treated mice. Mean Control siRNA-DOPC = 0.124, 95% CI = .104 to .144; mean NRP-2 siRNA-DOPC = 0.67, 95% CI = .052 to .081, P = 0.0005 vs. Control siRNA-DOPC. 7E - Representative photographs of excised livers from mice in FIG. 7B treated with either nonspecific or NRP-2-targeted siRNA and killed at 32 days after inoculation. 7F - Representative photographs of immunohistochemical staining of tumors from the mice described above showing the specificity of NRP-2 siRNAs (NP2-#1 and NP2- #2) for reducing human, and not mouse, NRP-2 levels. Top panel, Section from control (Control SiRNA-DOPC treated tumors showing NRP-2 expression (green fluorescence) in both human tumor cells and murine endothelial cells. Bottom panel, Section from NRP-2 siRNAs- DOPC treated tumors showing NRP-2 expression is substantially decreased in human tumor cells compared to control, yet NRP-2 expression in murine vascular/endothelial cells (yellow arrows) remains unchanged. Hoechst staining (blue) was utilized to demarcate nuclei.
FIG. 8 - Knockdown of NRP-2 expression.
FIG. 9 - Effect of reduced NRP-2 expression on constitutive signaling. FIG. 10 - Effect of reduced NRP-2 expression on angiogenic mediator expression.
FIG. 11 - Effect of decreased NRP-2 expression on migration.
FIG. 12 - Effect of decreased NRP-2 expression on invasion.
FIG. 13 - Effect of decreased NRP-2 expression on anchorage-independent growth.
FIG. 14 - Effect of decreased NRP-2 expression on in vivo growth. FIG. 15 - Effect of decreased NRP-2 expression on in vivo proliferation.
FIG. 16 - Effect of decreased NRP-2 expression on angiogenesis.
FIG. 17 - Effect of NRP-2 expression on tumor MVD and perfusion.
FIG. 18A, B. Expression of NRP-2 in pancreatic adenocarcinoma. 18A - NRP-2 was expressed in five of the six pancreatic cell lines as determined by Western blotting. 18B - Immunohistochemical staining of specimens from a nonmalignant pancreas and from pancreatic adenocarcinoma. NRP-2 was expressed in pancreatic adenocarcinoma cells (arrows) but not in normal ductal structures (arrowheads). Photomicrographs were obtained at 2Ox magnification.
FIG. 19A, B. Effect of shRNA to NRP-2 on intracellular signaling in BxPC3 cells. 19A - Western blots demonstrating specific knockdown of NRP-2 in the BxPC3 human pancreatic cancer cell line by plasmid-mediated stable trans fection using shRNA to NRP-2.
ShCon cells were transfected with a scrambled sequence. VEGFR-I and Akt phosphorylation was reduced in cells with reduced NRP-2 expression. Vinculin and β-actin levels served as loading controls. 19B - Src phosphorylation was moderately reduced in shNRP2 clones relative to that in control transfected cells. Levels of phosphorylated and total ERK1/2 were unchanged.
FIG. 2OA, B, C, D. Effect of reduced NRP-2 on the in vitro phenotype of pancreatic cancer cells. 2OA - Control-transfected cells and shNRP2 cells had similar proliferation rates. 2OB, C - Cells with reduced NRP2 were less migratory in Boyden chamber assays (20B) and less invasive across a biologic barrier (20C) than scramble control-transfected cells. 2OD - Reduction of NRP-2 led to decreased growth in soft agar. All error bars represent standard error of the mean, and asterisks denote p<0.05. (HPF = high power microscopic field.)
FIG. 2 IA, B. Effect of reduced NRP-2 expression on tumor growth and in vivo proliferation. 21A - Tumors derived from shNRP2 cells were significantly smaller than shCon-derived tumors. 21B - Quantification of digitized microvascular areas (D-MVA) of shCon and shNRP2 tumor sections revealed that shNRP2-derived tumors had 61-66% smaller D-MVA than that in control tumors (p<0.05). All error bars represent standard error of the mean, and asterisks denote p<0.05. FIG. 22A, B, C, D, E, F, G, H, I, J, K, L, M, N, O. Effect of reduced NRP-2 expression on tumor growth, in vivo proliferation and tumor vasculature in a subcutaneous model. 22A, B, C - Tumors derived from shNRP2 cells were significantly smaller than shCon-derived tumors. 22D, E, F - H&E staining of shNRP2 tumors showed more necrotic foci throughout the tumor than there were in shCon tumors. 22G, H, I - PCNA staining demonstrated significantly fewer proliferative cells in shNRP2 tumors than in shControl tumors. 22J, K, L - CD31 staining revealed more patent vessels per field in the shControl specimens. 22M, N, O - Binary images of CD31 staining with lumens digitally filled demonstrate higher D-MVA in shCon tumor sections relative to those with reduced NRP-2 expression. FIG. 23 A, B, C. Effect of reduced NRP-2 expression on orthotopic xenograft growth. Two million pancreatic cancer BxPC3 cells transfected with either a scrambled (shCon) or targeting sequence against NRP-2 (shNRP2) and labeled with firefly luciferase were injected orthotopically into the tail of each mouse pancreas, and tumors were allowed to grow for 50 days. Mice were injected with luciferin substrate and imaged 4 times during tumor growth. 23A - Representative mice from each group at the first (Day 3) and final (Day 50) time points are depicted. 23B - Photon activity was plotted against time for mice in each group. 23C - At the time of their harvest, tumors derived from shNRP2 cells were significantly smaller than those from shControl cells. Error bars represent standard error of the mean, and asterisks represent statistical significance at p<0.05. FIG. 24A, B. Effect of reduced NRP-2 expression on Jagged-1 levels. 24A -
Western blotting demonstrated that stable reduction of NRP-2 by shRNA was associated with a decrease in Jagged-1 protein levels. 24B - Densitometric analysis of three independent experiments confirmed that Jagged-1 protein levels were reduced by 46-53% in cells deficient in NRP-2. Error bars represent standard error of the mean, and asterisks denote p<0.05 vs. shCon. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention is based on the finding that neuropilins are involved in cancer development, progression, and metastasis. For example, the inventors have found that decreased NRP-2 expression, such as by shRNA targeting, results in reduction of tumor growth of human colorectal carcinoma. Further, the present invention is in part based on the finding that NRP-2 is expressed in most human colon tumor samples but not in the adjacent mucosa. It has been found that reduction of NRP-2 levels results in a concomitant reduction in phosphorylation of VEGFR-I, Akt, resulting in a reduction in phosphorylation of the anti- apoptosis protein BAD. The present invention is also partly based on the finding that reduction of NRP-2 reduces in vitro migration and invasion of colorectal cancer cells, that reduction of NRP-2 reduces the ability of colorectal cancer cells to resist cell death under environmental stress conditions, and that in vivo targeting of NRP-2 by systemic administration of siRNA-DOPC complexes is a viable therapeutic target in the treatment of colorectal carcinoma metastasis.
A. Neuropilin Proteins
Neuropilins are 120 to 130 kDa non- tyrosine kinase receptors (Soker et ai, 1998).
Neuropilins include NRP-I and NRP-2. Multiple NRP-I and NRP-2 isoforms exist, including soluble forms (Rossignol et al, 2000; Gluzman-Poltorak et ah, 2000). The basic structure of neuropilins includes five domains, including three extracellular domains, a transmembrane domain, and a short cytoplasmic domain (reviewed in Ellis, 2006).
Table 1 lists the GenBank Accession numbers of neuropilin protein sequences from homo sapiens.
Table 1
Neuropilin GenBank Accession No. SEQ ID NO.
NRP-I NP 003864 1 NP 001019799 2 NP 001019800 3 AAG 41896 4 AAG 41895 5 AAG 41894 6 AAG41893 7
NRP-2 AAI17414 8
AAG41405 9 Neuropilin GenBank Accession No. SEQ ID NO.
EAW70371 10 EAW70370 11 EAW70369 12 EAW70368 13 EAW70367 14 EAW70366 15 EAW70365 16 EAW70364 17 EAW70363 18 EAW70362 19 AAC51789 20 AAC51788 21
B. Therapeutic Gene Silencing
Since the discovery of RNAi by Fire and colleagues in 1981, the biochemical mechanisms have been rapidly characterized. Long double stranded RNA (dsRNA) is cleaved by Dicer, which is an RNAaseIII family ribonuclease. This process yields siRNAs of ~21 nucleotides in length. These siRNAs are incorporated into a multiprotein RNA-induced silencing complex (RISC) that is guided to target mRNA. RISC cleaves the target mRNA in the middle of the complementary region. In mammalian cells, the related microRNAs (miRNAs) are found that are short RNA fragments (-22 nucleotides). MiRNAs are generated after Dicer-mediated cleavage of longer (-70 nucleotide) precursors with imperfect hairpin RNA structures. The miRNA is incorporated into a miRNA-protein complex (miRNP), which leads to translational repression of target mRNA.
To improve the effectiveness of siRNA-mediated gene silencing, guidelines for selection of target sites on mRNA have been developed for optimal design of siRNA (Soutschek et al., 2004; Wadhwa et al, 2004). These strategies may allow for rational approaches for selecting siRNA sequences to achieve maximal gene knockdown. To facilitate the entry of siRNA into cells and tissues, a variety of vectors including plasmids and viral vectors such as adenovirus, lentivirus, and retrovirus have been used (Wadhwa et al., 2004). While many of these approaches are successful for in vitro studies, in vivo delivery poses additional challenges based on the complexity of the tumor microenvironment.
Liposomes are a form of nanoparticles that are attractive carriers for delivering a variety of drugs into the diseased tissue. Optimal liposome size depends on the tumor target. In tumor tissue, the vasculature is discontinuous, and pore sizes vary from 100 to 780 nm (Siwak et al., 2002). By comparison, pore size in normal vascular endothelium is <2 nm in most tissues, and 6 nm in post-capillary venules. Most liposomes are 65-125 nm in diameter. Negatively charged liposomes were believed to be more rapidly removed from circulation than neutral or positively charged liposomes; however, recent studies have indicated that the type of negatively charged lipid affects the rate of liposome uptake by the reticulo-endothelial system (RES). For example, liposomes containing negatively charged lipids that are not sterically shielded (phosphatidylserine, phosphatide acid, and phosphatidylglycerol) are cleared more rapidly than neutral liposomes. Interestingly, cationic liposomes (1,2-dioleoyl- 3-trimethylammonium-propane [DOTAP]) and cationic-liposome-DNA complexes are more avidly bound and internalized by endothelial cells of angiogenic blood vessels via endocytosis than anionic, neutral, or sterically stabilized neutral liposomes (Thurston et al, 1998; Krasnici et al, 2003). Cationic liposomes may not be ideal delivery vehicles for tumor cells because surface interactions with the tumor cells create an electrostatically derived binding-site barrier effect, inhibiting further association of the delivery systems with tumor spheroids (Kostarelos et al, 2004). However, neutral liposomes appear to have better intratumoral penetration. Toxicity with specific liposomal preparations has also been a concern. Cationic liposomes elicit dose-dependent toxicity and pulmonary inflammation by promoting release of reactive oxygen intermediates, and this effect is more pronounced with multivalent cationic liposomes than monovalent cationic liposomes such as DOTAP (Dokka et al, 2000). Neutral and negative liposomes do not appear to exhibit lung toxicity (Gutierrez -Puente et al, 1999). Cationic liposomes, while efficiently taking up nucleic acids, have had limited success for in vivo gene downregulation, perhaps because of their stable intracellular nature and resultant failure to release siRNA contents. In vivo siRNA delivery using neutral liposomes in an orthotopic model of advanced ovarian cancer has been described (Landen et al, 2005, which is incorporated herein by reference in its entirety). For example, intravenous injection of the DOPC-siRNA complex allowed a significantly greater degree of siRNA deposition into the tumor parenchyma than either delivery with cationic (positively charged) liposomes (DOTAP) or unpackaged "naked" siRNA. While the DOPC formulation delivered siRNA to over 30% of cells in the tumor parenchyma, naked siRNA was delivered only to about 3% of cells, and DOTAP delivered siRNA only to tumor cells immediately adjacent to the vasculature.
Although siRNA appears to be more stable than antisense molecules, serum nucleases can degrade siRNAs (Leung and Whittaker, 2005). Thus, several research groups have developed modifications such as chemically stabilized siRNAs with partial phosphorothioate backbone and 2'-0-methyl sugar modifications or boranophosphate siRNAs (Leung and Whittaker, 2005). Elmen and colleagues modified siRNAs with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA), which significantly enhanced the serum half-life of siRNA and stabilized the structure without affecting the gene-silencing capability (Elmen et al, 2005). Alternative approaches including chemical modification (conjugation of cholesterol to the 3' end of the sense strand of siRNA by means of a pyrrolidine linker) may also allow systemic delivery without affecting function (Soutschek et al, 2004). Apsects of the present invention can use each of these modification strategies in combination with the compositions and methods described.
C. Lipid Preparations
The present invention provides methods and compositions for associating an inhibitory nucleic acid that inhibits the expression of a neuropilin, such as a siNA (e.g., a siRNA) with a lipid and/or liposome. The siNA may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polynucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. The liposome or liposome/siNA associated compositions of the present invention are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates which are not uniform in either size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which are well known to those of skill in the art which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. An example is the lipid dioleoylphosphatidyl choline (DOPC). "Liposome" is a generic term encompassing a variety of unilamellar, multilamellar, and multivesicular lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). However, the present invention also encompasses compositions that have different structures in solution than the normal vesicular structure. For example, the lipids may assume a micellar structure or merely exist as non-uniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.
Liposome-mediated polynucleotide delivery and expression of foreign DNA in vitro has been very successful. Wong et al. (1980) demonstrated the feasibility of liposome- mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al. (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.
In certain embodiments of the invention, the lipid may be associated with a hemaglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, the lipid may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al, 1991). In yet further embodiments, the lipid may be complexed or employed in conjunction with both HVJ and HMG-I. In that such expression vectors have been successfully employed in transfer of a polynucleotide in vitro and in vivo, then they are applicable for the present invention. 1. Neutral Liposomes
"Neutral liposomes or lipid composition" or "non-charged liposomes or lipid composition," as used herein, are defined as liposomes or lipid compositions having one or more lipids that yield an essentially-neutral, net charge (substantially non-charged). By "essentially neutral" or "essentially non-charged", it is meant that few, if any, lipids within a given population (e.g. , a population of liposomes) include a charge that is not canceled by an opposite charge of another component (e.g., fewer than 10% of components include a non- canceled charge, more preferably fewer than 5%, and most preferably fewer than 1%). In certain embodiments of the present invention, a composition may be prepared wherein the lipid component of the composition is essentially neutral but is not in the form of liposomes.
In certain embodiments, neutral liposomes or lipid compositions may include mostly lipids and/or phospholipids that are themselves neutral. In certain embodiments, amphipathic lipids may be incorporated into or used to generate neutral liposomes or lipid compositions. For example, a neutral liposome may be generated by combining positively and negatively charged lipids so that those charges substantially cancel one another. For such a liposome, few, if any, charged lipids are present whose charge is not canceled by an oppositely-charged lipid (e.g., fewer than 10% of charged lipids have a charge that is not canceled, more preferably fewer than 5%, and most preferably fewer than 1%). It is also recognized that the above approach may be used to generate a neutral lipid composition wherein the lipid component of the composition is not in the form of liposomes.
In certain embodiments, a neutral liposome may be used to deliver a siRNA. The neutral liposome may contain a siRNA directed to the suppression of translation of a single gene, or the neutral liposome may contain multiple siRNA that are directed to the suppression of translation of multiple genes. Further, the neutral liposome may also contain a chemo therapeutic in addition to the siRNA; thus, in certain embodiments, chemotherapeutic and a siRNA may be delivered to a cell (e.g., a cancerous cell in a human subject) in the same or separate compositions. An advantage to using neutral liposomes is that, in contrast to the toxicity that has been observed in response to cationic liposomes, little to no toxicity has yet been observed as a result of neutral liposomes. 2. Phospholipids
Lipid compositions of the present invention may comprise phospholipids. In certain embodiments, a single kind or type of phospholipid may be used in the creation of lipid compositions such as liposomes (e.g., DOPC used to generate neutral liposomes). In other embodiments, more than one kind or type of phospholipid may be used.
Phospholipids include glycerophospholipids and certain sphingolipids. Phospholipids include, but are not limited to, dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), l-myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), l-stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin ("DSSP"), distearoylphophatidylethanolamine ("DSPE"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine ("DMPC"), l^-distearoyl-sn-glycero-S-phosphocholine ("DAPC"), 1,2- diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"), 1 ,2-dieicosenoyl-sn-glycero-3- phosphocholine ("DEPC"), dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl phosphatidylcholine ("POPC"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), lysophosphatidylcholine, lysophosphatidylethanolamine, and dilinoleoylphosphatidylcholine.
Phospholipids include, for example, phosphatidylcholines, phosphatidylglycerols, and phosphatidylethanolamines; because phosphatidylethanolamines and phosphatidyl cholines are non-charged under physiological conditions (i.e., at about pH 7), these compounds may be particularly useful for generating neutral liposomes. In certain embodiments, the phospholipid DOPC is used to produce non-charged liposomes or lipid compositions. In certain embodiments, a lipid that is not a phospholipid (e.g., a cholesterol) can also be used
Phospholipids may be from natural or synthetic sources. However, phospholipids from natural sources, such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine are not used in certain embodiments as the primary phosphatide (i.e., constituting 50% or more of the total phosphatide composition) because this may result in instability and leakiness of the resulting liposomes. 3. Production of Liposomes Liposomes and lipid compositions of the present invention can be made by different methods. For example, a nucleotide (e.g., siRNA) may be encapsulated in a neutral liposome using a method involving ethanol and calcium (Bailey and Sullivan, 2000). The size of the liposomes varies depending on the method of synthesis. A liposome suspended in an aqueous solution is generally in the shape of a spherical vesicle, and may have one or more concentric layers of lipid bilayer molecules. Each layer consists of a parallel array of molecules represented by the formula XY, wherein X is a hydrophilic moiety and Y is a hydrophobic moiety. In aqueous suspension, the concentric layers are arranged such that the hydrophilic moieties tend to remain in contact with an aqueous phase and the hydrophobic regions tend to self-associate. For example, when aqueous phases are present both within and without the liposome, the lipid molecules may form a bilayer, known as a lamella, of the arrangement XY-YX. Aggregates of lipids may form when the hydrophilic and hydrophobic parts of more than one lipid molecule become associated with each other. The size and shape of these aggregates will depend upon many different variables, such as the nature of the solvent and the presence of other compounds in the solution. Lipids suitable for use according to the present invention can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma Chemical Co., dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem- Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -200C. Chloroform may be used as the only solvent since it is more readily evaporated than methanol.
Liposomes within the scope of the present invention can be prepared in accordance with known laboratory techniques. In certain embodiments, liposomes are prepared by mixing liposomal lipids, in a solvent in a container (e.g., a glass, pear-shaped flask). The container will typically have a volume ten-times greater than the volume of the expected suspension of liposomes. Using a rotary evaporator, the solvent may be removed at approximately 400C under negative pressure. The solvent may be removed within about 5 minutes to 2 hours, depending on the desired volume of the liposomes. The composition can be dried further in a desiccator under vacuum. Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen- free water by shaking until all the lipid film is resuspended. The aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum. Liposomes can also be prepared in accordance with other known laboratory procedures: the method of Bangham et αl. (1965), the contents of which are incorporated herein by reference; the method of Gregoriadis (1979), the contents of which are incorporated herein by reference; the method of Deamer and Uster (1983), the contents of which are incorporated by reference; and the reverse-phase evaporation method as described by Szoka and Papahadjopoulos (1978). The aforementioned methods differ in their respective abilities to entrap aqueous material and their respective aqueous space-to-lipid ratios.
Dried lipids or lyophilized liposomes may be dehydrated and reconstituted in a solution of inhibitory peptide and diluted to an appropriate concentration with a suitable solvent {e.g., DPBS). The mixture may then be vigorously shaken in a vortex mixer. Unencapsulated nucleic acid may be removed by centrifugation at 29,00Og and the liposomal pellets washed. The washed liposomes may be resuspended at an appropriate total phospholipid concentration {e.g., about 50-200 mM). The amount of nucleic acid encapsulated can be determined in accordance with standard methods. After determination of the amount of nucleic acid encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentrations and stored at 4°C until use.
D. Inhibition of Gene Expression siNA (e.g., siRNA) are well known in the art. For example, siRNA and double- stranded RNA have been described in U.S. Patents 6,506,559 and 6,573,099, as well as in U.S. Patent Applications 2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161, and 2004/0064842, all of which are herein incorporated by reference in their entirety. Within a siNA, the components of a nucleic acid need not be of the same type or homogenous throughout (e.g., a siNA may comprise a nucleotide and a nucleic acid or nucleotide analog). Typically, siNA form a double-stranded structure; the double-stranded structure may result from two separate nucleic acids that are partially or completely complementary. In certain embodiments of the present invention, the siNA may comprise only a single nucleic acid (polynucleotide) or nucleic acid analog and form a double-stranded structure by complementing with itself (e.g., forming a hairpin loop). The double-stranded structure of the siNA may comprise 16, 20, 25, 30, 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90 to 100, 150, 200, 250, 300, 350, 400, 450, 500 or more contiguous nucleobases, including all ranges therebetween. The siNA may comprise 17 to 35 contiguous nucleobases, more preferably 18 to 30 contiguous nucleobases, more preferably 19 to 25 nucleobases, more preferably 20 to 23 contiguous nucleobases, or 20 to 22 contiguous nucleobases, or 21 contiguous nucleobases that hybridize with a complementary nucleic acid (which may be another part of the same nucleic acid or a separate complementary nucleic acid) to form a double-stranded structure. Agents of the present invention useful for practicing the methods of the present invention include, but are not limited to siRNAs. Typically, introduction of double-stranded RNA (dsRNA), which may alternatively be referred to herein as small interfering RNA (siRNA), induces potent and specific gene silencing, a phenomena called RNA interference or RNAi. This phenomenon has been extensively documented in the nematode C. elegans (Fire et ah, 1998), but is widespread in other organisms, ranging from trypanosomes to mouse. Depending on the organism being discussed, RNA interference has been referred to as "cosuppression," "post-transcriptional gene silencing," "sense suppression," and "quelling." RNAi is an attractive biotechnological tool because it provides a means for knocking out the activity of specific genes. In designing RNAi there are several factors that need to be considered such as the nature of the siRNA, the durability of the silencing effect, and the choice of delivery system. To produce an RNAi effect, the siRNA that is introduced into the organism will typically contain exonic sequences. Furthermore, the RNAi process is homology dependent, so the sequences must be carefully selected so as to maximize gene specificity, while minimizing the possibility of cross-interference between homologous, but not gene-specific sequences. Preferably the siRNA exhibits greater than 80, 85, 90, 95, 98,% or even 100% identity between the sequence of the siRNA and the gene to be inhibited. Sequences less than about 80% identical to the target gene are substantially less effective. Thus, the greater homology between the siRNA and the STAT gene to be inhibited, the less likely expression of unrelated genes will be affected.
In addition, the size of the siRNA is an important consideration. In some embodiments, the present invention relates to siRNA molecules that include at least about 19-25 nucleotides, and are able to modulate neuropilin gene expression. In the context of the present invention, the siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides in length. More preferably, the siRNA is from about 19 nucleotides to about 25 nucleotides in length. siRNA can be obtained from commercial sources, natural sources, or can be synthesized using any of a number of techniques well-known to those of ordinary skill in the art. For example, one commercial source of predesigned siRNA is Ambion®, Austin, TX. Another is Qiagen® (Valencia, CA). An inhibitory nucleic acid that can be applied in the compositions and methods of the present invention may be any nucleic acid sequence that has been found by any source to be a validated downregulator of a neuropilin.
In one aspect, the invention generally features an isolated siRNA molecule of at least 19 nucleotides, having at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of a nucleic acid that encodes a neuropilin (such as NRP-2), and that reduces the expression of the neuropilin. In a particular embodiment of the present invention, the siRNA molecule has at least one strand that is substantially complementary to at least ten but no more than thirty consecutive nucleotides of the mRNA that encodes NRP-2.
In another particular embodiment, the siRNA molecule is at least 75, 80, 85, or 90% homologous, preferably 95%, 99%, or 100% homologous, to at least 10 contiguous nucleotides of any of the nucleic acid sequences encoding a full-length neuropilin protein, such as those in Table 1. Without undue experimentation and using the disclosure of this invention, it is understood that additional siRNAs can be designed and used to practice the methods of the invention.
The siRNA may also comprise an alteration of one or more nucleotides. Such alterations can include the addition of non-nucleotide material, such as to the end(s) of the 19 to 25 nucleotide RNA or internally (at one or more nucleotides of the RNA). In certain aspects, the RNA molecule contains a 3'-hydroxyl group. Nucleotides in the RNA molecules of the present invention can also comprise non-standard nucleotides, including non-naturally occurring nucleotides or deoxyribonucleotides. The double-stranded oligonucleotide may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages. Additional modifications of siRNAs (e.g., 2'-O-methyl ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides, 5-C-methyl nucleotides, one or more phosphorothioate internucleotide linkages, and inverted deoxyabasic residue incorporation) can be found in U.S. Application Publication 20040019001 and U.S. Patent 6,673,611 (each of which is incorporated by referencein its entirety). Collectively, all such altered nucleic acids or RNAs described above are referred to as modified siRNAs.
Preferably, RNAi is capable of decreasing the expression of a neuropilin, such NRP -2, by at least 10%, 20%, 30%, or 40%, more preferably by at least 50%, 60%, or 70%, and most preferably by at least 75%, 80%, 90%, 95% or more. Certain embodiments of the present invention pertain to methods of inhibiting expression of a gene encoding a neuropilin in a cell. In a specific embodient, the neuropilin is NRP -2. Introduction of siRNA into cells can be achieved by methods known in the art, including for example, microinjection, electroporation, or transfection of a vector comprising a nucleic acid from which the siRNA can be transcribed. Alternatively, a siRNA can be directly introduced into a cell in a form that is capable of binding to target mRNA transcripts. To increase durability and membrane -permeability the siRNA may be combined or modified with liposomes, poly-L-lysine, lipids, cholesterol, Hpofectine or derivatives thereof. In certain aspects cholesterol-conjugated siRNA can be used (see, Song et ah, 2003).
E. Nucleic Acids
The present invention provides methods and compositions for the delivery of siNA via neutral liposomes. Because a siNA is composed of a nucleic acid, methods relating to nucleic acids {e.g., production of a nucleic acid, modification of a nucleic acid, etc.) may also be used with regard to a siNA. The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to a molecule (i.e., a strand) of DNA, RNA or a derivative or analog thereof, comprising a nucleobase. A nucleobase includes, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C). The term "nucleic acid" encompass the terms "oligonucleotide" and "polynucleotide," each as a subgenus of the term "nucleic acid." The term "oligonucleotide" refers to a molecule of between 3 and about 100 nucleobases in length. The term "polynucleotide" refers to at least one molecule of greater than about 100 nucleobases in length. These definitions refer to a single-stranded or double-stranded nucleic acid molecule.
Double stranded nucleic acids are formed by fully complementary binding, although in some embodiments a double stranded nucleic acid may formed by partial or substantial complementary binding. Thus, a nucleic acid may encompass a double-stranded molecule that comprises one or more complementary strand(s) or "complement(s)" of a particular sequence, typically comprising a molecule. As used herein, a single stranded nucleic acid may be denoted by the prefix "ss" and a double stranded nucleic acid by the prefix "ds". 1. Nucleobases
As used herein a "nucleobase" refers to a heterocyclic base, such as for example a naturally occurring nucleobase (i.e., an A, T, G, C or U) found in at least one naturally occurring nucleic acid (i.e., DNA and RNA), and naturally or non-naturally occurring derivative(s) and analogs of such a nucleobase. A nucleobase generally can form one or more hydrogen bonds ("anneal" or "hybridize") with at least one naturally occurring nucleobase in manner that may substitute for naturally occurring nucleobase pairing (e.g., the hydrogen bonding between A and T, G and C, and A and U). "Purine" and/or "pyrimidine" nucleobase(s) encompass naturally occurring purine and/or pyrimidine nucleobases and also derivative(s) and analog(s) thereof, including but not limited to, those a purine or pyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino, hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol or alkylthiol moeity. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.) moeities comprise of from about 1, about 2, about 3, about 4, about 5, to about 6 carbon atoms. A nucleobase may be comprised in a nucleside or nucleotide, using any chemical or natural synthesis method described herein or known to one of ordinary skill in the art. 2. Nucleosides
As used herein, a "nucleoside" refers to an individual chemical unit comprising a nucleobase covalently attached to a nucleobase linker moiety. A non-limiting example of a "nucleobase linker moiety" is a sugar comprising 5-carbon atoms (i.e., a "5-carbon sugar"), including but not limited to a deoxyribose, a ribose, an arabinose, or a derivative or an analog of a 5-carbon sugar. Non-limiting examples of a derivative or an analog of a 5-carbon sugar include a 2'-fluoro-2'-deoxyribose or a carbocyclic sugar where a carbon is substituted for an oxygen atom in the sugar ring.
Different types of covalent attachment(s) of a nucleobase to a nucleobase linker moiety are known in the art. By way of non- limiting example, a nucleoside comprising a purine (i.e., A or G) or a 7-deazapurine nucleobase typically covalently attaches the 9 position of a purine or a 7-deazapurine to the l'-position of a 5-carbon sugar. In another non-limiting example, a nucleoside comprising a pyrimidine nucleobase (i.e., C, T or U) typically covalently attaches a 1 position of a pyrimidine to a l'-position of a 5-carbon sugar (Kornberg and Baker, 1992).
3. Nucleotides
As used herein, a "nucleotide" refers to a nucleoside further comprising a "backbone moiety". A backbone moiety generally covalently attaches a nucleotide to another molecule comprising a nucleotide, or to another nucleotide to form a nucleic acid. The "backbone moiety" in naturally occurring nucleotides typically comprises a phosphorus moiety, which is covalently attached to a 5-carbon sugar. The attachment of the backbone moiety typically occurs at either the 3'- or 5 '-position of the 5-carbon sugar. However, other types of attachments are known in the art, particularly when a nucleotide comprises derivatives or analogs of a naturally occurring 5-carbon sugar or phosphorus moiety. 4. Nucleic Acid Analogs
A nucleic acid may comprise, or be composed entirely of, a derivative or analog of a nucleobase, a nucleobase linker moiety and/or backbone moiety that may be present in a naturally occurring nucleic acid. As used herein a "derivative" refers to a chemically modified or altered form of a naturally occurring molecule, while the terms "mimic" or "analog" refer to a molecule that may or may not structurally resemble a naturally occurring molecule or moiety, but possesses similar functions. As used herein, a "moiety" generally refers to a smaller chemical or molecular component of a larger chemical or molecular structure. Nucleobase, nucleoside and nucleotide analogs or derivatives are well known in the art, and have been described (see for example, Scheit, 1980, incorporated herein by reference).
Additional non-limiting examples of nucleosides, nucleotides, or nucleic acids comprising 5-carbon sugar and/or backbone moiety derivatives or analogs, include those in U.S. Patent 5,681,947 which describes oligonucleotides comprising purine derivatives that form triple helixes with and/or prevent expression of dsDNA; U.S. Patents 5,652,099 and 5,763,167 which describe nucleic acids incorporating fluorescent analogs of nucleosides found in DNA or RNA, particularly for use as flourescent nucleic acids probes; U.S. Patent 5,614,617 which describes oligonucleotide analogs with substitutions on pyrimidine rings that possess enhanced nuclease stability; U.S. Patents 5,670,663, 5,872,232 and 5,859,221 which describe oligonucleotide analogs with modified 5-carbon sugars (i.e., modified T- deoxyfuranosyl moieties) used in nucleic acid detection; U.S. Patent 5,446,137 which describes oligonucleotides comprising at least one 5-carbon sugar moiety substituted at the 4' position with a substituent other than hydrogen that can be used in hybridization assays; U.S. Patent 5,886,165 which describes oligonucleotides with both deoxyribonucleotides with 3'-5' internucleotide linkages and ribonucleotides with 2'-5' internucleotide linkages; U.S. Patent 5,714,606 which describes a modified internucleotide linkage wherein a 3'-position oxygen of the internucleotide linkage is replaced by a carbon to enhance the nuclease resistance of nucleic acids; U.S. Patent 5,672,697 which describes oligonucleotides containing one or more 5' methylene phosphonate internucleotide linkages that enhance nuclease resistance; U.S. Patents 5,466,786 and 5,792,847 which describe the linkage of a substituent moeity which may comprise a drug or label to the 2' carbon of an oligonucleotide to provide enhanced nuclease stability and ability to deliver drugs or detection moieties; U.S. Patent 5,223,618 which describes oligonucleotide analogs with a 2 or 3 carbon backbone linkage attaching the 4' position and 3' position of adjacent 5-carbon sugar moiety to enhanced cellular uptake, resistance to nucleases and hybridization to target RNA; U.S. Patent 5,470,967 which describes oligonucleotides comprising at least one sulfamate or sulfamide internucleotide linkage that are useful as nucleic acid hybridization probe; U.S. Patents 5,378,825, 5,777,092, 5,623,070, 5,610,289 and 5,602,240 which describe oligonucleotides with three or four atom linker moeity replacing phosphodiester backbone moeity used for improved nuclease resistance, cellular uptake and regulating RNA expression; U.S. Patent 5,858,988 which describes hydrophobic carrier agent attached to the 2'-0 position of oligonuceotides to enhanced their membrane permeability and stability; U.S. Patent 5,214,136 which describes olignucleotides conjugated to anthraquinone at the 5' terminus that possess enhanced hybridization to DNA or RNA; enhanced stability to nucleases; U.S. Patent 5,700,922 which describes PNA-DNA-PNA chimeras wherein the DNA comprises 2'-deoxy-erythro- pentofuranosyl nucleotides for enhanced nuclease resistance, binding affinity, and ability to activate RNase H; and U.S. Patent 5,708,154 which describes RNA linked to a DNA to form a DNA-RNA hybrid.
5. Polyether and Peptide Nucleic Acids
In certain embodiments, it is contemplated that a nucleic acid comprising a derivative or analog of a nucleoside or nucleotide may be used in the methods and compositions of the invention. A non-limiting example is a "polyether nucleic acid", described in U.S. Patent 5,908,845, incorporated herein by reference. In a polyether nucleic acid, one or more nucleobases are linked to chiral carbon atoms in a polyether backbone.
Another non-limiting example is a "peptide nucleic acid", also known as a "PNA", "peptide-based nucleic acid analog" or "PENAM", described in U.S. Patent 5,786,461, 5,891,625, 5,773,571, 5,766,855, 5,736,336, 5,719,262, 5,714,331, 5,539,082, and WO 92/20702, each of which is incorporated herein by reference. Peptide nucleic acids generally have enhanced sequence specificity, binding properties, and resistance to enzymatic degradation in comparison to molecules such as DNA and RNA (Egholm et ah, 1993; PCT/EP/01219). A peptide nucleic acid generally comprises one or more nucleotides or nucleosides that comprise a nucleobase moiety, a nucleobase linker moeity that is not a 5- carbon sugar, and/or a backbone moiety that is not a phosphate backbone moiety. Examples of nucleobase linker moieties described for PNAs include aza nitrogen atoms, amido and/or ureido tethers (see for example, U.S. Patent 5,539,082). Examples of backbone moieties described for PNAs include an aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfmamide or polysulfonamide backbone moiety. In certain embodiments, a nucleic acid analogue such as a peptide nucleic acid may be used to inhibit nucleic acid amplification, such as in PCR™, to reduce false positives and discriminate between single base mutants, as described in U.S. Patent 5,891,625. Other modifications and uses of nucleic acid analogs are known in the art, and it is anticipated that these techniques and types of nucleic acid analogs may be used with the present invention. In a non-limiting example, U.S. Patent 5,786,461 describes PNAs with amino acid side chains attached to the PNA backbone to enhance solubility of the molecule. In another example, the cellular uptake property of PNAs is increased by attachment of a lipophilic group. U.S. Application Ser. No. 117,363 describes several alkylamino moeities used to enhance cellular uptake of a PNA. Another example is described in U.S. Patents 5,766,855, 5,719,262, 5,714,331 and 5,736,336, which describe PNAs comprising naturally and non-naturally occurring nucleobases and alkylamine side chains that provide improvements in sequence specificity, solubility and/or binding affinity relative to a naturally occurring nucleic acid.
6. Preparation of Nucleic Acids A nucleic acid may be made by any technique known to one of ordinary skill in the art, such as chemical synthesis, enzymatic production or biological production. Non-limiting examples of a synthetic nucleic acid (e.g., a synthetic oligonucleotide), include a nucleic acid made by in vitro chemically synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al, 1986 and U.S. Patent 5,705,629, each incorporated herein by reference. In the methods of the present invention, one or more oligonucleotide may be used. Various different mechanisms of oligonucleotide synthesis have been disclosed in for example, U.S. Patents 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which is incorporated herein by reference.
A non-limiting example of an enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCR™ (see for example, U.S. Patent 4,683,202 and U.S. Patent 4,682,195, each incorporated herein by reference), or the synthesis of an oligonucleotide described in U.S. Patent 5,645,897, incorporated herein by reference. A non-limiting example of a biologically produced nucleic acid includes a recombinant nucleic acid produced (i.e., replicated) in a living cell, such as a recombinant DNA vector replicated in bacteria (see for example, Sambrook et al 2001, incorporated herein by reference).
7. Purification of Nucleic Acids
A nucleic acid may be purified on polyacrylamide gels, cesium chloride centrifugation gradients, or by any other means known to one of ordinary skill in the art (see for example, Sambrook et al, 2001, incorporated herein by reference).
In certain embodiments, the present invention concerns a nucleic acid that is an isolated nucleic acid. As used herein, the term "isolated nucleic acid" refers to a nucleic acid molecule (e.g., an RNA or DNA molecule) that has been isolated free of, or is otherwise free of, the bulk of the total genomic and transcribed nucleic acids of one or more cells. In certain embodiments, "isolated nucleic acid" refers to a nucleic acid that has been isolated free of, or is otherwise free of, bulk of cellular components or in vitro reaction components such as for example, macromolecules such as lipids or proteins, small biological molecules, and the like. 8. Hybridization
As used herein, "hybridization", "hybridizes" or "capable of hybridizing" is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature. The term "anneal" as used herein is synonymous with "hybridize." The term "hybridization", "hybridize(s)" or "capable of hybridizing" encompasses the terms "stringent condition(s)" or "high stringency" and the terms "low stringency" or "low stringency condition(s)."
As used herein "stringent condition(s)" or "high stringency" are those conditions that allow hybridization between or within one or more nucleic acid strand(s) containing complementary sequence(s), but precludes hybridization of random sequences. Stringent conditions tolerate little, if any, mismatch between a nucleic acid and a target strand. Such conditions are well known to those of ordinary skill in the art, and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof, or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like.
Stringent conditions may comprise low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 500C to about 700C. It is understood that the temperature and ionic strength of a desired stringency are determined in part by the length of the particular nucleic acid(s), the length and nucleobase content of the target sequence(s), the charge composition of the nucleic acid(s), and to the presence or concentration of formamide, tetramethylammonium chloride or other solvent(s) in a hybridization mixture.
It is also understood that these ranges, compositions and conditions for hybridization are mentioned by way of non-limiting examples only, and that the desired stringency for a particular hybridization reaction is often determined empirically by comparison to one or more positive or negative controls. Depending on the application envisioned it is preferred to employ varying conditions of hybridization to achieve varying degrees of selectivity of a nucleic acid towards a target sequence. In a non-limiting example, identification or isolation of a related target nucleic acid that does not hybridize to a nucleic acid under stringent conditions may be achieved by hybridization at low temperature and/or high ionic strength. Such conditions are termed "low stringency" or "low stringency conditions", and non-limiting examples of low stringency include hybridization performed at about 0.15 M to about 0.9 M NaCl at a temperature range of about 200C to about 500C. Of course, it is within the skill of one in the art to further modify the low or high stringency conditions to suite a particular application.
F. Treatment of Disease 1. Definitions
"Treatment" and "treating" refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition. For example, a treatment may include administration of a pharmaceutically effective amount of a nucleic acid that inhibits the expression of a gene that encodes a neuropilin and a neutral lipid for the purposes of minimizing the growth or invasion of a tumor, such as a colorectal cancer.
A "subject" refers to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.
The term "therapeutic benefit" or "therapeutically effective" as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.
A "disease" or "health-related condition" can be any pathological condition of a body part, an organ, or a system resulting from any cause, such as infection, genetic defect, and/or environmental stress. The cause may or may not be known. . In some embodiments of the invention, the methods include identifying a patient in need of treatment. A patient may be identified, for example, based on taking a patient history, based on findings on clinical examination, based on health screenings, or by self- referral.
2. Diseases The present invention may be used to treat any disease associated with increased expression of a neuropilin. For example, the disease may be a hyperproliferative disease, such as cancer. For example, a siRNA that binds to a nucleic acid that encodes a neuropilin may be administered to treat a cancer. The cancer may be a solid tumor, metastatic cancer, or non- metastatic cancer. In certain embodiments, the cancer may originate in the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, colon, rectum, anus, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In certain embodiments, the cancer is colorectal cancer (i.e., cancer involving the colon or rectum).
The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadeno carcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia. Nonetheless, it is also recognized that the present invention may also be used to treat a non-cancerous disease (e.g., a fungal infection, a bacterial infection, a viral infection, and/or a neurodegenerative disease).
G. Pharmaceutical Preparations
Where clinical application of a composition containing a siNA is undertaken, it will generally be beneficial to prepare a pharmaceutical composition appropriate for the intended application. This will typically entail preparing a pharmaceutical composition that is essentially free of pyrogens, as well as any other impurities that could be harmful to humans or animals. One may also employ appropriate buffers to render the complex stable and allow for uptake by target cells.
The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as a human, as appropriate. The preparation of a pharmaceutical composition comprising a siNA or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington (2005), incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. A pharmaceutically acceptable carrier is preferably formulated for administration to a human, although in certain embodiments it may be desirable to use a pharmaceutically acceptable carrier that is formulated for administration to a non-human animal but which would not be acceptable (e.g., due to governmental regulations) for administration to a human. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
The actual dosage amount of a composition of the present invention administered to a patient or subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 μg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. , can be administered.
A gene expression inhibitor may be administered in a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more μg of nucleic acid per dose. Each dose may be in a volume of 1, 10, 50, 100, 200, 500, 1000 or more μl or ml.
Solutions of therapeutic compositions can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms .
The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. These preparations also may be emulsified. A typical composition for such purpose comprises a pharmaceutically acceptable carrier. For instance, the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like.
Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components the pharmaceutical composition are adjusted according to well known parameters.
Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. The therapeutic compositions of the present invention may include classic pharmaceutical preparations. Administration of therapeutic compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Topical administration may be particularly advantageous for the treatment of skin cancers, to prevent chemotherapy- induced alopecia or other dermal hyperproliferative disorder. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. For treatment of conditions of the lungs, or respiratory tract, aerosol delivery can be used. Volume of the aerosol is between about 0.01 ml and 0.5 ml.
An effective amount of the therapeutic composition is determined based on the intended goal. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each unit containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection or effect desired.
Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dose include the physical and clinical state of the patient, the route of administration, the intended goal of treatment (e.g., alleviation of symptoms versus cure) and the potency, stability and toxicity of the particular therapeutic substance.
H. Combination Treatments
In certain embodiments, the compositions and methods of the present invention involve an inhibitor of expression of a neuropilin, or construct capable of expressing an inhibitor of neuropilin expression, in combination with a second or additional therapy. Such therapy can be applied in the treatment of any disease that is associated with increased expression or activity of a neuropilin. For example, the disease may be a hyperproliferative disease, such as cancer.
The methods and compositions including combination therapies enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy. Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both an inhibitor of gene expression and a second therapy. A tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) including one or more of the agents (i.e., inhibitor of gene expression or an anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) an inhibitor of gene expression; 2) an anti-cancer agent, or 3) both an inhibitor of gene expression and an anticancer agent. Also, it is contemplated that such a combination therapy can be used in conjunction with a chemotherapy, radiotherapy, surgical therapy, or immunotherapy.
An inhibitor of gene expression may be administered before, during, after or in various combinations relative to an anti-cancer treatment. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the inhibitor of gene expression is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the inhibitor of gene expression therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more preferably, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between respective administrations.
In certain embodiments, a course of treatment will last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 days or more. It is contemplated that one agent may be given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, any combination thereof, and another agent is given on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and/or 90, or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered. This time period may last 1, 2, 3, 4, 5, 6, 7 days, and/or 1, 2, 3, 4, 5 weeks, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, depending on the condition of the patient, such as their prognosis, strength, health, etc.
Various combinations may be employed. For the example below an inhibitor of gene expression therapy is "A" and an anti-cancer therapy is "B": A/B/A BIAJB B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
BIBIBIh BIBIAJB A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A
Administration of any compound or therapy of the present invention to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, as well as surgical intervention, may be applied in combination with the described therapy.
In specific aspects, it is contemplated that a standard therapy will include chemotherapy, radiotherapy, immunotherapy, surgical therapy or gene therapy and may be employed in combination with the inhibitor of gene expression therapy, anticancer therapy, or both the inhibitor of gene expression therapy and the anti-cancer therapy, as described herein.
1. Chemotherapy
A wide variety of chemotherapeutic agents may be used in accordance with the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. A
"chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis. Most chemotherapeutic agents fall into the following categories: alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, and nitrosoureas.
Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC- 1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBl-TMl); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfϊromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex); razoxane; rhizoxin; sizofϊran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-I l); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; capecitabine; cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, paclitaxel, docetaxel, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate and pharmaceutically acceptable salts, acids or derivatives of any of the above.
Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestanie, fadrozole, vorozole, letrozole, and anastrozole; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3- dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, RaIf and H-Ras; ribozymes such as a VEGF expression inhibitor and a HER2 expression inhibitor; vaccines such as gene therapy vaccines and pharmaceutically acceptable salts, acids or derivatives of any of the above. 2. Radiotherapy
Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287) and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
The terms "contacted" and "exposed," when applied to a cell, are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell. To achieve cell killing, for example, both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing. 3. Immunotherapy
In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Trastuzumab (Herceptin™) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells. The combination of therapeutic modalities, i.e., direct cytotoxic activity and inhibition or reduction of ErbB2 would provide therapeutic benefit in the treatment of ErbB2 overexpressing cancers. Another immunotherapy could also be used as part of a combined therapy with gen silencing therapy discussed above. In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention. Common tumor markers include carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and pi 55. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines such as IL-2, IL-4, IL- 12, GM-CSF, gamma-IFN, chemokines such as MIP-I, MCP-I, IL-8 and growth factors such as FLT3 ligand. Combining immune stimulating molecules, either as proteins or using gene delivery in combination with a tumor suppressor has been shown to enhance anti-tumor effects (Ju et al, 2000). Moreover, antibodies against any of these compounds can be used to target the anti-cancer agents discussed herein. Examples of immunotherapies currently under investigation or in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al, 1998), cytokine therapy, e.g., interferons α, β and γ; IL-I, GM-CSF and TNF (Bukowski et al, 1998; Davidson et al, 1998; Hellstrand et al, 1998) gene therapy, e.g., TNF, IL-I, IL-2, p53 (Qin et al, 1998; Austin-Ward and Villaseca, 1998; U.S. Patents 5,830,880 and 5,846,945) and monoclonal antibodies, e.g., anti-ganglioside GM2, anti-HER- 2, anti-pl85 (Pietras et al, 1998; Hanibuchi et al, 1998; U.S. Patent 5,824,311). It is contemplated that one or more anti-cancer therapies may be employed with the gene silencing therapies described herein. In active immunotherapy, an antigenic peptide, polypeptide or protein, or an autologous or allogenic tumor cell composition or "vaccine" is administered, generally with a distinct bacterial adjuvant (Ravindranath and Morton, 1991; Morton et al, 1992; Mitchell et al, 1990; Mitchell et al, 1993).
In adoptive immunotherapy, the patient's circulating lymphocytes, or tumor infiltrated lymphocytes, are isolated in vitro, activated by lymphokines such as IL-2 or transduced with genes for tumor necrosis, and readministered (Rosenberg et al, 1988; 1989). 4. Surgery
Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue. Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
5. Other Agents
It is contemplated that other agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-I, MIP-lbeta, MCP-I, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas / Fas ligand, DR4 or DR5 / TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing abilities of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
There have been many advances in the therapy of cancer following the introduction of cytotoxic chemotherapeutic drugs. However, one of the consequences of chemotherapy is the development/acquisition of drug-resistant phenotypes and the development of multiple drug resistance. The development of drug resistance remains a major obstacle in the treatment of such tumors and therefore, there is an obvious need for alternative approaches such as gene therapy. Another form of therapy for use in conjunction with chemotherapy, radiation therapy or biological therapy includes hyperthermia, which is a procedure in which a patient's tissue is exposed to high temperatures (up to 1060F). External or internal heating devices may be involved in the application of local, regional, or whole-body hyperthermia. Local hyperthermia involves the application of heat to a small area, such as a tumor. Heat may be generated externally with high-frequency waves targeting a tumor from a device outside the body. Internal heat may involve a sterile probe , including thin, heated wires or hollow tubes filled with warm water, implanted microwave antennae, or radiofrequency electrodes.
A patient's organ or a limb is heated for regional therapy, which is accomplished using devices that produce high energy, such as magnets. Alternatively, some of the patient's blood may be removed and heated before being perfused into an area that will be internally heated. Whole-body heating may also be implemented in cases where cancer has spread throughout the body. Warm-water blankets, hot wax, inductive coils, and thermal chambers may be used for this purpose. Hormonal therapy may also be used in conjunction with the present invention or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.
I. Kits and Diagnostics
In various aspects of the invention, a kit is envisioned containing therapeutic agents and/or other therapeutic and delivery agents. In some embodiments, the present invention contemplates a kit for preparing and/or administering a therapy of the invention. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions of the present invention. In some embodiments, the lipid is in one vial, and the nucleic acid component is in a separate vial. The kit may include, for example, at least one inhibitor of neuropilin expression, one or more lipid component, as well as reagents to prepare, formulate, and/or administer the components of the invention or perform one or more steps of the inventive methods. In some embodiments, the kit may also comprise a suitable container means, which is a container that will not react with components of the kit, such as an eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container may be made from sterilizable materials such as plastic or glass.
The kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill. The instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent.
J. Examples
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1
Therapeutic Targeting of Neuropilin-2 on Colorectal Carcinoma Cells Implanted in the
Murine Liver
Materials and Methods Tissue Specimens and Cell Lines. Specimens of colon adenocarcinoma, adjacent non-malignant colonic mucosa, and colon cancer liver metastases were obtained from an established tissue bank in the Department of Surgical Oncology at The University of Texas M. D. Anderson Cancer Center (MDACC), following protocols approved by the institutional review board of MDACC. Specimens were fixed in formalin at the time of collection. Histopathologic confirmation was provided by the M. D. Anderson Cancer Center's Department of Pathology.
Five human colorectal carcinoma cell lines (Geo, HCT-116, HT-29, RKO, and SW- 480 cells) were obtained from the American Type Culture Collection (Manassas, VA). The human colorectal carcinoma cell line KM 12 and the murine melanoma B16BL6 cell lines were obtained from Dr. I. J. Fidler (M. D. Anderson Cancer Center). Unless stated otherwise, all cells were maintained at 37°C with 5% CO2 in complete minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and streptomycin (200 μg/ml) (Life Technologies, Grand Island, NY). For hypoxic studies, cells were plated at 80% density and were incubated at 37 0C, 1 % O2, in an MC0-5M hypoxic chamber (Sanyo Scientific, Bensenville, IL). For VEGF-A stimulation, studies cells were incubated in minimal essential medium supplemented with 1% fetal bovine serum, 2 mM L-glutamine, streptomycin (200 μg/ml). VEGF-A (R & D Systems, Minneapolis, MN) was used at a concentration of 10 μg/mL for 10 minutes. shRNA Expression Plasmids, shRNA Cell Lines, and transient siRNA targeting. shRNA expression vectors were created with the use of pSilencer 4.0 shRNA expression system (Ambion, Austin, TX), and followed the manufacturer's directions. Using the Ambion siRNA web design tool, two potential NRP-2-specifϊc target sequences (NP2-#1, 5'- CCCAACCAGAAGATTGTCC-3'; SEQ ID NO:22) and NP2-#2, 5'- GTCAGCACTAATGGAGAGG-3'; SEQ ID NO:23) were identified on the World Wide Web at Ambion.com. Two annealed oligonucleotides, each encoding one of the target sequences, followed by a with a 9-bp hairpin sequence (ttcaagaga; SEQ ID NO:24) and flanked by a 5' BamRl and a 3' HmdIII overhangs, were ligated into the pSilencer 4.0 expression plasmid at compatible sites. The two generated shRNA expression plasmids, sh- NP2Vec-#l and sh-NP2Vec-#2 were confirmed by sequencing. For negative controls, shRNA vectors were created using similar methods to generate the NRP -2 specific shRNA plasmids, but utilized a scrambled sequence of each of the NRP -2 target sequences identified (Con-#1, 5'-agatcggtggcctatagaacg-3' (SEQ ID NO:25) and Con-#2, 5'-gatcatcaccttggaccagac-3'; SEQ ID NO:26). Sequences were confirmed by NIH BLAST analysis to have no substantial homology to sequences in other vertebrate genes. Control shRNA control plasmids were designated sh-ConVec-#l and sh-ConVec-#2. NRP -2 deficient cell lines were created by transfecting HCT-116 cells with 0.5 ng of both shRNA expression plasmids (sh-NP2Vec-#l and sh-NP2Vec-#2), while control shRNA cells were created by transfecting both scrambled sequence encoding shRNA vectors (sh-ConVec-#l and sh-ConVec-#2) at similar concentrations. Stable clones were isolated by growing each trans fected cell type in medium containing 850 μg/mL hygromycin B (Roche Diagnostics, Mannheim, Germany). NRP-2 expression levels in isolated clones of shRNA generated HCT-116 cells were determined by immunoblot analysis. For transient transfections, 2.5 x 105 HCT-116 cells were plated per well in medium in 6-well plates, and incubated for 24 hours. The cells were then transfected with increasing concentrations of siRNA oligos (both NRP-2 target sequences described above without hairpins) using the transfection reagent SiPORT/ NeoFX (Ambion, TX) according to the manufactures' protocol. The cells were incubated for an additional 72 hours after transfection and solubilized in 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% Triton X- 100, 1 mM Na3VO4, 2 mM EDTA, and one complete Mini Protease Inhibitor Cocktail Tablet (per 10 ml of lysis buffer) (Roche Diagnostics) and subjected to western/immunoblot analysis to determine NRP-2 levels.
Immunoprecipitation and Immunoblot Analyses. Immunoprecipitation and immunoblot analyses were performed as previously described with minor modifications (Gray et ah, 2005). Briefly, human colon carcinoma and murine melonoma cells were grown to 85%-90% confluence in complete minimum essential medium (unless otherwise stated), and solubilized in 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 2 mM EDTA, and one complete Mini Protease Inhibitor Cocktail Tablet (per 10 ml of lysis buffer) (Roche Diagnostics). 50 μg of whole-cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). All Membranes were probed with antibodies at a concentration of 1 :1000 unless otherwise stated. Antibodies used were, NRP-I (C- 19, Mu mAb, Santa Cruz Biotechnology) and NRP-2 (C-9 and/or H- 300 from Santa Cruz Biotechnology); phospho-AktSei473 (DE-9, Rb mAb), Akt (C67E7, Rb mAb), phospho-Erk-l^1111 2027T^204 (D13.14.4E, Rb mAb), Erk-1/2 (137F5, Rb mAb), phospho-BADSer136 (9295 Rb mAb) and phospho-BAD Ser112 (9291, Rb mAb), and BAD (9292, Rb pAb) (all from Cell Signaling Technology, Danvers, MA); phospho-VEGFR- lTry1213 (PC459, Rb pAb, Calbiochem, Boston, MA) /(07-75K, Rb pAb, Upstate/Millipore, Billerica, MA); VEGFR-I (Oncogene Research Products, San Diego, CA). As a loading control, all membranes were stripped and reprobed for vinculin (V4505, Mu mAb, Sigma- Aldrich, St Louis, MO, 1 :10,000 dilution). AU antibodies were diluted in Tris-buffered saline and 0.1% (v/v) Tween 20 containing 5% dried milk. Membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies, and antibody-bound proteins were visualized by chemiluminescence (New England Nuclear, Boston, MA).
RT-PCR Assays. The relative expression levels of VEGFR-I, VEGFR-2 and VEGFR-3 in human HCT-116 colon cancer cells and human umbilical vein endothelial cells (HUVEC) were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The primers used were; VEGFRl, 5'-tgaaagccttcagtcccgtg-3' (sense; SEQ ID NO:27), and 5'-atccgtgttgagggtggtcagc-3' (antisense; SEQ ID NO:28); VEGFR-2,5'- catcacatccactggtattgg-3 ' (sense; SEQ ID NO:29), and 5'-gccaagcttgtaccatgtgag-3' (antisense; SEQ ID NO:30);and VEGFR-3, 5'-cccacgcagacatcaagacg (sense; SEQ ID NO:31), 5'- tgcagaactccacgatcacc-3 ' (antisense; SEQ ID NO: 32). RNA was extracted from HCT-116 and HUVEC cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. For cDNA synthesis, 4 μg of total RNA of each sample was used with oligo-dT and MMLV RT-polymerase (Epicenter, Madison, WI). Synthesized cDNAs were diluted in 500 μl of diethylpyrocarbonate-treated water, and 3 μl of each reaction was used in each 25 -μl RT-PCR reaction. Amplifications were performed using the following parameters: 950C for 1 min, followed by 35 cycles of 950C for 30 s, 6O0C for 30 s and 72 C for 1 minute. VEGF receptor expression gene expression was normalized using reference primers toward glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5'- ccttcattgacctcaactac-3' (sense; SEQ ID NO33), and 5'-gatgatgttctggagtgcc-3' (antisense; SEQ ID NO:34). PCR products were visualized in a 1.2 % agarose gel stained with ethidium bromide.
VEGF Enzyme-Linked Immunosorbent Assay (ELISA). VEGF production in culture supernatants from control cells (Parental and sh-Con) and cells with reduced NRP-2 levels (sh-NP2-C8 and sh-NP2-C9) were examined using a human VEGF-specific ELISA according to the manufacture's instructions (Quantikine; R&D Systems, Minneapolis, MN). Cells were plated out at 80% cell density in a 100mm cell culture dish in minimal essential medium supplemented with 1% fetal bovine serum, 2 mM L-glutamine, streptomycin (200 μg/ml). 24 h later media was removed for VEGF analysis, and cells solubilized in 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 2 mM EDTA, and one complete Mini Protease Inhibitor Cocktail Tablet (per 10 ml of lysis buffer). VEGF concentration was normalized to the total protein content of each culture dish, as measured by the Bradford assay.
MTT Analysis of Cell Proliferation. Cell proliferation in vitro was analyzed with the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described previously (Gray, 2005). The yellow dye MTT is reduced to a blue formazan product by respiratory enzymes active only in viable cells, making the amount of color change indicative of cell proliferation. Briefly, 2000 cells of each clone (Parental, sh-Con, sh- NP2-C8 and sh-NP2-C9) were plated per well in five 96-well microtiter plates in 200 μL of medium. For analysis, 20 μl of MTT substrate (2.5mg/ml of PBS) was added to each well, and the plates returned to standard tissue incubator conditions for an additional 2 hours. Medium was then removed and the cells solubilized in 200 μL of dimethyl sulfoxide (DMSO), and colorimetric analysis was performed (wavelength, 570 nm). One plate underwent was analyzed immediately after the cells adhered (approximately 4 hours after plating), and the remaining plates were assayed every 24 hours for the next 4 consecutive days.
Annexin V Staining. To determine the role of NRP-2 in mediating survival of HCT- 116 cells under conditions of hypoxic stress, cells with normal levels of NRP-2 (Parental and sh-Con) and reduced levels of NRP-2 (sh-NP2-C8 and sh-NP2-C9) were subjected to hypoxic conditions (1% O2, 99% N2) for 6 and 24 hours. The relative percentage of apoptotic cells was assessed at these time points using the Annexin V-FITC apoptosis Detection Kit-1, (BD Pharmingen, San Diego, CA) according to the manufacturer's protocol. Annexin V quantitation was performed using a Coulter EPICS XL-MCL fluorescent-activated cell (FAC) analyzer (Beckman Coulter, Miami, FL) equipped with System II software (Beckman Coulter).
Migration and Invasion Assays. Migration assays were conducted as described previously with minor modifications. Equal numbers (30,000) of control cells (sh-Con) or cells with reduced NRP-2 expression (sh-NP2-C8 and sh-NP2-C9) were suspended in 0.5 ml of medium and placed in the top compartment of a standard 8-μm pour Boyden chamber with 0.5 ml of medium added to the bottom compartment. Following 12 hr incubation under standard conditions (37 0C / 5% CO2), non-migrating cells were scraped from the top compartment and cells that had migrated to the bottom compartment were fixed and stained using the Protocol HEMA 3 stain set (Fisher Scientific). Membranes were excised and mounted on a standard microscope slide (Curtis Matheson Scientific, Houston, TX). The numbers of migrated cells were determined from 5 random fields visualized at 2Ox magnifications.
Invasion assays used identical methods, but the cells were placed in the top compartment of modified Boyden chamber with a Matrigel coated membrane. The numbers of invasive cells were determined from 5 random fields visualized at 2Ox magnifications.
Soft- Agar Colony-Forming Assay. To determine the effect of NRP -2 expression on anchorage-independent growth, 500 cells of each clone (sh-Con, sh-NP2-C8, and sh-NP2-C9) were plated per well onto six-well plates in 1 ml of medium containing 0.5% agarose, which previously were overlaid with 1 ml of medium with 0.8% agarose. Cells were incubated at standard conditions for 12 days (37°C, 5% CO2). Colonies >50 μm in diameter were counted under a light microscope at 2Ox magnifications. Five field of each clone were counted and expressed as mean ± 95% CI.
Preparation of siRNA-Containing Liposomes. For experiments to test the efficacy of in vivo therapeutic targeting of NRP -2 in tumor xenografts in mice, liposomes containing siRNA's were prepared as previously described (Landen et ah, 2005). Briefly, siRNA oligos (without hairpins) to NRP-2 target sequences (NP2-#1 and NP2-#2) or scrambled control sequences (CON-#1 and CON-#2) were mixed with l,2-dioleoyl-sn-glycero-3- phosphatidylcholine (DOPC) (Avanti Polar Lipids, Alabaster, Al) at a ratio of 10:1 (w/w) DOPC/siRNA and lyophilized. Immediately prior to in vivo administration, lyophilized preparations were hydrated in 0.9% saline at a concentration of 5 μg of si-RNA/ 200 μL, and were purified by separating free siRNA from liposomes with filter units with a size exclusion limit of 30,000 Daltons (Millipore Corp).
In vivo Modeling. Male athymic nude mice, 6-8 weeks old, were purchased from the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD) and maintained under specific pathogen-free conditions. All animal experiments met the requirements of the University of Texas M. D. Anderson Cancer Center Animal Care Facility and the National Institutes of Health guidelines on animal care and use. Each HCT-116 clone was inoculated into 10 mice per group as previously described (Gray et al., 2005). In brief, 1.0 x 106 viable control (sh-Con) or NRP-2 suppressed cell lines (sh-NP2-C8, or sh-NP2-C9) cells were injected in 0.1 rnL of Hanks balanced salt solution per mouse, either subcutaneously (right rear flank) or into the spleen of each nude mouse. Tumor growth was monitored, and when 3 mice in any group become moribund (lethargy, decreased grooming, wt loss) or had palpable tumors ~1 cm in diameter, mice were sacrificed by CO2 asphyxia and final tumor mass and volume were recorded. Volume was calculated as [(length/2) x (width2)].
For the in vivo siRNA delivery experiments, colon cancer cells that expresses the Lenti-luc reporter gene were created by infecting parental HCT-116 cells with a recombinant lentivirus as previously described (Arumugam et al, 2006). Luciferase expressing HCT-116 cells were suspended in 0.1 mL of Hanks balanced salt solution and inoculated directly into the liver of each mouse (1.0 x 106 cell per mouse, 20 mice total). Four days later, 10 mice were treated with either Control- or NRP-2-siRNA / liposomal complexes (5 μg siRNA per injection), which were administered intraperitoneally as a 200-μL bolus. Treatments continued every 5 days thereafter for a total of six treatments per group. At 32 days mice were sacrificed and total body weight, liver weight, and tumor volumes were calculated. Tumor specimens from each treatment group were snap-frozen and fixed in formalin or frozen in optimum cutting temperature solution (OCT). Histopathologic Analysis and Immunohistochemical Staining of Tissues. Patient tissue specimens were analyzed as previously described (Parikh et al, 2004) with an anti- NRP-2 antibody (C-9; Santa Cruz Biotechnology, Santa Cruz, CA, 1:500 dilution). Tumor xenografts from mice were fixed in 10% neutral formalin and paraffin or OCT solution (Miles, Elkhart, IN). Immunostaining was performed according to standard procedures (Camp et al, 2006; Yang et al, 2006). Immunofluorescent staining for NRP-2 in OCT- embedded xenografts was performed with a rabbit anti-NRP-2 antibody (H-300, Santa Cruz Biotechnology 1 :200 dilution), which recognizes human, rat, and mouse NRP-2, followed by anti-rabbit fluorescent- conjugated antibody (Alexa 488, dilution 1 :1000) and counterstained with the DNA-specifϊc dye Hoechst 33345 (both from Molecular Probes, Eugene, OR). Stained tissue sections were analyzed with NIH Image J 1.34 software. All analyses involved examining a minimum of five randomly selected fields or images. Fluorescent- labeled cells were examined using a Nikon Microphot-FXA fluorescent microscope and representative images recorded. Bioluminescence Imaging. In vitro and in vivo bio luminescence imaging of luciferase-expressing HCT-116 cells was conducted as previously described (Arumugam et al, 2006) using a cryogenically cooled IVIS 100 imaging system coupled to a data- acquisition personal computer equipped with Living Image software (all from Xenogen Corp, Hopkinton, MA). For in vitro imaging, 25 ul of luciferase potassium salt solution (15mg/ml PBS)(Sigma-Aldrich, St Louis, MO) was added to each well of a 12-well plate containing 25,000 HCT-116 cells per well. For in vivo studies, nude mice were anesthetized with 1.5% isofluorane-oxygen mixture, and injected intraperitoneally with of luciferase potassium salt solution (15mg/ml PBS), at a dose of 150 mg/kg body weight before each imaging session. Initial in vivo images at day 3 were obtained to establish baseline tumor volume as measured by luciferase/ photon activity. Additional images were obtained at 14 and 28 days after inoculation to monitor tumor growth kinetics.
Statistical Analyses. Data are presented as means, differences in means and 95% confidence intervals (CI) for the mean differences. For the in vitro and in vivo studies, statistical significance was determined by using Fisher exact tests (comparison of incidence) or Mann- Whitney tests (comparison of means) as indicated. For the in vivo experiments, 10 mice in each group were used, as directed by power analysis to detect a 50% reduction in tumor size or weight, with a beta error of 3%. All statistical tests were two-sided and p- values less than 0.05 were deemed statistically significant. Results
Expression of NRP-2 in Human Colon Tissues and Cell Lines. The expression of NRP -2 in paraffin-embedded samples of human primary colon tumors, adjacent normal colonic mucosa, and colon cancer liver metastases were first examined by immunoperoxidase staining (FIG. IA). NRP-2 expression was undetectable in nonmalignant mucosa (0 [0%] of 10 specimens) but was expressed in 10 (83%) of 12 adjacent adenocarcinomas (P = .001 vs. nonmalignant mucosa) and five (71%) of seven liver metastases (P = .003 vs. nonmalignant mucosa). NRP-2 protein expression was also detected in the Geo, HCT-116, HT-29, KM-12, and SW480 colorectal cancer cell lines. Geo, HCT-116, and SW-480 cells expressed the highest levels (FIG. IB). Effect of NRP-2 siRNA on NRP-2 Levels in Human and Murine Cell Lines. The effect of using targeted shRNAs to reduce NRP-2 levels in human HCT-116 colorectal cancer cells was next examined. Specificity of siRNA targeting for NRP-2 was first confirmed by western analysis of NRP-I and NRP-2 levels in control human HCT-116 cells and cells that had been transfected with control or NRP-2 siRNAs (FIG. 2A, top panel). To further examine specificity of the human NRP-2 siRNA sequence for human NRP-2, human SW480 colorectal cancer cells and B16BL6 melanoma cells were transiently transfected with increasing concentrations of human NRP-2 siRNA (NP#1 and NP#2) (FIG. 2 A, bottom panel). Transfection of 10 μg of siRNA strongly suppressed the expression of NRP-2 in human cells but its expression remained unchanged in the murine B16BL6 cell line.
Effect of NRP-2 Expression on Cell Proliferation Rates in vitro. An MTT assay was next used to determine the effect of reduced NRP-2 expression on colorectal cancer cell growth rates in vitro. HCT-116 cells stably transfected with shRNA to NRP-2 showed no changes in proliferation rate relative to that of Parental and sh-Con-transfected HCT-116 cells (FIG. 2B). Doubling times were approximately 25 hours for both the Parental and the sh-Con control cells, and 24-26 hours for sh-NP2-C8 and sh-NP2-C9. VEGF-A (10 μg/mL) treatment did not alter the doubling times of any of these cell lines, regardless of NRP-2 expression level, compared with doubling times in the absence of VEGF-A. Effect of NRP-2 Expression on VEGFR-I and Akt Signaling. NRP-2 can act as a coreceptor for VEGFR -1, VEGFR-2 and VEGFR-3 (Favier et al, 2006; Gluzman-Poltorak et al, 2001). HCT-116 cells, like other colon cancer cells (Fan et al, 2005), express only one of the three known VEGF tyrosine kinase receptors, VEGFR-I (FIG. 2C). NRP-2-mediated activation of any of the VEGF receptors would be expected to result in phosphorylation of the coexpressed VEGFR's, and activation of its downstream tyrosine kinase substrates. Immunoblot analysis was used to examine whether loss of NRP-2 expression was associated with changes in VEGFR-I receptor phosphorylation. NRP-2 siRNA-expressing cells showed no change in VEGFR-I levels but did show a discernable reduction in VEGFR-I phosphorylation compared with controls (sh-Con and parental cells) (FIG. 2D). Examination of the activation of downstream signaling intermediates using immunoblots probed with phospho-specifϊc antibodies showed that loss of NRP-2 also led to substantial reductions in the phosphorylation of Akt and the downstream anti-apoptosis protein BAD (Serl36), whereas the phosphorylation of Erk-1/2 remained unchanged (FIG. 2D). To confirm the effects of decreased NRP-2 expression on downstream pathways, signaling intermediates in a second colorectal cancer cell line, (SW480) were studied after transient transfection with NRP-2 siRNAs. Transient reduction of NRP-2 levels by siRNA in SW480 cells also led to decreased Akt phosphorylation (without altering the levels of Erk-1/2 phosphorylation), similar to what was observed in HCT-116 cells. To examine whether the loss of VEGFR-I phosphorylation accompanying reduced NRP -2 levels in HCT-116 cells could be overcome by the addition of exogenous VEGF-A, control cells and NRP-2 shRNA-expressing cells were grown in serum-reduced medium (1% fetal calf serum) and stimulated with VEGF-A (10 μg/ml) for 10 minutes, followed by protein extraction and immunoblot analysis. In agreement with the pattern shown in FIG. 2D, basal phosphorylation of VEGFR-I was mitigated in cells with reduced NRP-2 levels (FIG. 2E). VEGFR-I phosphorylation was increased upon VEGF-A stimulation in control cells to a greater extent than in HCT- 116 cells with reduced shRNA mediated reduction of NRP-2 levels. Effect of NRP-2 Expression on Endogenous Expression of VEGF-A and SEMA3F.
To determine whether the changes in VEGFR-I phosphorylation observed in the NRP-2 shRNA-expressing cells had resulted from potential changes in NRP-2 ligand expression, levels of VEGF-A and Sema3F in HCT-116 cells with varying levels of NRP-2 expression were next examined. VEGF-A levels, as determined by enzyme-linked immunosorbent assay (ELISA), were nearly the same in parental and sh-RNA control cells, and in both clones with suppressed NRP-2 expression (sh-NP2-C8 and sh-NP2-C9). Western analysis of control cells (Parental and sh-Con) and NRP-2 siRNA-expressing cells (sh-NP2-C8 and sh-NP2-C9) showed levels of Sema3F expression remained unchanged.
Effect of NRP-2 Expression on Anchorage Independent Survival of Colon Cancer Cells. On the basis of the findings that NRP-2 gene silencing was associated with reduced phosphorylation of anti-apoptosis proteins, studies were next conducted to assess the effect of NRP-2 expression on survival of colon cancer cells under anchorage-independent conditions. Control cells (sh-Con) formed abundant large (> 50 μm in diameter) colonies in soft agar, whereas cells with reduced levels of NRP-2 (sh-NP2-C8 and sh-NP2-C9) formed statistically significantly fewer colonies in soft agar, and the colonies that did form were generally smaller than those derived from control cells (FIG. 3A, 3B; mean number of colonies: sh-Con = 384, sh-NP2-C8 = 112, sh-NP2-C9 = 81; sh-NP2-C8 vs. control: difference = 272 colonies, 95% CI = 210 to 334 colonies, P =.002; sh-NP2-C9 vs. sh-Con control: difference = 303 colonies, 95% CI = 253 to 353 colonies, P =.002). Effect of NRP-2 Expression on Colon Cancer Cell Survival during Hypoxia. To further investigate the mechanism by which reduced expression of NRP-2 altered survival of CRC cells, HCT-116 cells with normal and reduced levels of NRP-2 were subjected to normoxic or hypoxic growth conditions (1% O2, 99% N2), and the relative percentages of Annexin V staining-cells were quantified as a measure of apoptosis. Under normoxic conditions, no discernable difference in constitutive apoptotic rates was detected between the two cell types. However, at 6 hours and 24 hours of hypoxic conditions, cells with decreased NRP -2 expression demonstrated a statistically significant increase in apoptosis (FIG. 3C; at 6 hours of hypoxia mean sh-Con apoptotic rate= 1.46 %, mean sh-NP2-C8 rate = 2.94 %, difference = 1.48%, 95% CI = 1.11 to 1.85 %, P =.01 ; at 24 hours of hypoxia mean sh-Con apoptotic rate = 1.96%, mean sh-NP2-C8 rate = 3.52 %, difference= 1.56%, 95% CI = 0.98 to 2.14 %, P = 0.008).
Effect of NRP-2 on Migration and Invasion. VEGFR-I activity has previously been shown to mediate tumor cell migration and invasion (Barleon et al., 1996; Wey et al., 2005; Lesslie et al, 2006). The observation that loss of NRP-2 expression reduced VEGFR-I phosphorylation and activation of the VEGFR-I pathway, led to studies to assess the effects of NRP-2 on the migration and invasion capabilities of colorectal cancer cells with the use of standard and Matrigel-coated Boyden chamber assays, respectively. NRP-2 shRNA- expressing cells displayed statistically significantly reduced migration compared with control cells (FIG. 4A, 4B; mean Parental migration = 187,cells, sh-Con = 167 cells, sh-NP2-C8 = 91 cells, sh-NP2-C9 = 64 cells; sh-NP2-C8 vs. sh-Con: difference = 76 cells, 95% CI = 48 to 104 cells, P =.008; sh-NP2-C9 vs; sh-Con: difference = 103 cells, 95% CI = 73 to 133 cells, P =.008). In Matrigel invasion assays, clones with reduced NRP-2 levels showed reduced invasiveness compared with controls (FIG. 4C, 4D; mean Parental invasion = 83 cells, mean sh-Con = 89 cells, mean sh-NP2-C8 = 44 cells; mean sh-NP2-C9 = 29 cells; sh-NP2-C8 vs. sh-Con: difference = 45 cells, 95% CI = 30 to 60 cells, P =.008; sh-NP2-C9 vs. sh-Con: difference = 60 cells, 95% CI = 44 to 76 cells, P =.008). To examine the effect of VEGF-A stimulation on cell migration in cells with decreased NRP-2 levels, control HCT-116 cells (sh-Con) and cells with reduced NRP-2 expression (sh-NP2-C8) were grown in serum- reduced medium (1% fetal calf serum), and stimulated with VEGF-A (10 μg/ml) for 24 hours. Although migration was higher in each VEGF-A-treated cell line compared with untreated cells, cells with reduced NRP-2 levels (sh-NP2-C8) migrated less in response to VEGF-A stimulation than cells with normal levels of NRP-2 (sh-Con). Effect of NRP-2 Expression on Tumor Growth in vivo. To examine the effect of
NRP-2 expression on tumor growth, equal amounts (1.0 xlO / mouse) of HCT-116 control cells (sh-Con) or NRP-2 siRNA-expressing cells (sh-NP2-C8 and sh-NP2-C9) were injected subcutaneous Iy into ten nude mice and assessed tumor incidence and volume 30 days later. All mice were of approximately the same overall weight when sacrificed. Tumor incidence was 100 % in control (sh-Con) mice, 40 % in sh-NP2-C9 mice, and 60 % in sh-NP2-C9 mice (FIG. 5A). Subcutaneous tumors produced by cells with reduced NRP -2 expression were significantly smaller than those produced by control cells expressing normal levels of NRP-2 (Sh-Con) (FIG. 5B: mean volume sh-Con = 1.0 cm3, sh-NP2-C8 = 0.1 cm3, sh-NP2-C9 = 0.2 cm3; sh-NP2-C8 vs. control: difference = 0.9 cm3, 95% CI = 0.7 to 1.2 cm3, P =.01; sh-NP2- C9 vs. control: difference = 0.8 cm3, 95% CI = 0.5 to 1.1 mm3, P =.01). To examine the effect of NRP-2 expression on hepatic metastasis of HCT-116 colorectal cancer cells, equal amounts of cells (1.0 xlO6 / mouse) of each clone were inoculated into the spleens often nude mice and the resulting liver metastases were counted and measured 30 days later. Incidence of hepatic metastases was lower in the mice inoculated with NRP-2 siRNA-expressing cells than in the mice injected with control cells (FIG. 5 A: mean sh-Con = 100%, mean sh-NP2-C8 = 20%, mean sh-NP2-C9 = 40%, sh-NP2-C8 vs. sh-Con control: difference = 80%, 95% CI = 44% to 97%, P =.002; sh-NP2-C9 vs. sh-Con control: difference = 60%, 95% CI = 26% to 88%, P =.01). Mice that developed hepatic metastases revealed that inoculation with cells with reduced NRP-2 expression produced fewer number of hepatic than did cells with normal levels of NRP-2 (FIG. 5C: mean sh-Con = 19 metastases, mean sh-NP2-C8 = 1 metastases, mean sh-NP2-C9 = 6 metastases; sh-NP2-C8 vs. sh-Con control: difference = 19 metastases, 95% CI = 6 to 32 metastases, P =.0001; sh-NP2-C9 vs. sh-Con control: difference = 13 metastases, 95% CI = -4 to 30 metastases, P = 0.008). In addition, the final volumes of the metastases that did form were smaller for NRP-2 shRNA-expressing cells (sh-NP2-C8 and sh-NP2-C9) than for shRNA control cells (FIG. 5D and 5E; mean sh-Con = 79 mm3, mean sh-NP2-C8 = 1 mm3, mean sh-NP2-C9 = 9 mm3; sh-NP2-C8 vs. control: difference = 78 , 95% CI = -1 to 158 mm3, P =.0008; sh-NP2-C9 vs. sh-Con control: difference = 70 mm3, 95% CI = -11 to 151 mm3, P =.006) NRP-2 Expression and Apoptosis in vivo. To examine the effect of NRP-2 expression on apoptosis in vivo, xenograft tissue samples derived from the sh-Con, sh-NP2- CS, and sh-NP2-C9 cell lines were stained as outlined above for Annexin V for immunohistochemical analysis. Tumors with reduced NRP-2 expression exhibited 5 to 10 times more apoptotic cells per 2Ox field than did the control tumors (FIG. 6: mean sh-Con control= 4 apoptotic cells, mean sh-NP2-C8 = 24 apoptotic cells, mean sh-NP2-C9 = 36 apoptotic cells, sh-NP2-C8 vs. sh-Con control: difference = 20 apoptotic cells, 95% CI = 5 to 35 apoptotic cells, P =.01 vs sh-Con; sh-NP2-C9 to sh-Con control: difference = 31 apoptotic cells, 95% CI = 18 to 45 apoptotic cells, P =.005). Effect of in vivo Targeting of NRP-2 on Colon Cancer Progression. To determine whether siRNAs to NRP-2 might be used to treat human tumors (derived from untransfected cells), studies were conducted to explore the use of liposomes carrying NRP-2 siRNA to target tumor xenografts in mice. Before targeting NRP-2 expression in vivo, a study was conducted to test whether siRNA incorporated into liposomes made of the neutral lipid DOPC could effectively reduce gene expression in tumors growing in the murine liver. For these experiments, luciferase-specifϊc siRNA, which was confirmed to be effective in attenuating luciferase activity in vitro (FIG. 7A, top), was complexed with DOPC and injected intraperitoneally into mice at a dose of 5 μg/mouse 10 days after the inoculation of 1.0 x 106 HCT-116 colon cancer cells expressing the lenti-luc gene. Prior to siRNA-DOPC administration, the mice were subjected to bio luminescent imaging to establish baseline activity, and again 48 hours after siRNA treatment, to examine whether administration of siRNA to luciferase in liposomes led to a reduction in luciferase activity in the tumor cells. Hepatic luciferase activity, as reflected by photon emission, was reduced by approximately 50% in treated mice compared with that in the same mice measured before SiRNA-Luc- DOPC administration (FIG. 7A, bottom).
Finally, studies were conducted to examine the effect of in vivo administration of siRNA to NRP-2 in liposomes on the growth of colorectal cancer xenografts in nude mice. For these experiments, control siRNA scrambled sequences (Con-#1 and Con-#2), and NRP-2 target sequences (NP2-#1 and NP2-#2) were complexed with DOPC and injected intraperitoneally into mice at a dose of 5 μg/mouse every 5 days, starting 4 days after the direct hepatic inoculation of 1.0 x 106 HCT-116 colorectal cancer cells per mouse. Bioluminescent imaging of tumor cell growth showed that administration of NRP2 siRNA- DOPC impaired the growth kinetics of colorectal cancer cells compared with those of control cells at 28 days after inoculation (FIG. 7B: mean control activity = 8.1 x 108 protons, mean NRP2 siRNA-DOPC = 0.5 x 108 protons). Excised livers had a much greater tumor burden in the control groups (FIG. 7E) than in mice treated with siRNA to NRP-2 (FIG. 7C: mean control siRNA-DOPC = 420 mm3, mean NRP-2 siRNA-DOPC = 36 mm3, NRP-2 vs. control: difference = 385 mm3, 95% CI = 174 to 595, P =.005). In addition, the ratios for liver to total body weight were statistically significantly higher in the control group than in the NRP-2- treated group (FIG. 7D: mean control siRNA-DOPC = 0.124; mean NRP-2 siRNA-DOPC = 0.067; NRP-2 vs. control: difference = 0.057, 95% CI = 0.032 to 0.082, P =.0005). Immunohistochemical examination of tumor samples revealed that only human NRP-2 expression (in tumor xenografts), and not murine NRP-2 expression (on murine endothelial cells), was decreased in mice treated with NRP -2 siRNA-DOPC compared to mice treated with control siRNA-DOPC (FIG. 7F).
EXAMPLE 2
Knockdown of NRP-2 Decreases the Expression of the Angiogenic Factor Jaggedl Studies were conducted to examine pancreatic cancer for expression of NRP-2, and to examine whether the expression of the angiogenic factor Jaggedl can be decreased in colon cancer and pancreatic cancer.
NRP-2 expression in human non-malignant pancreas and in pancreatic adenocarcinoma samples was examined. It was found that expression of NRP-2 was significantly greater in pancreatic adenocarcinoma that in uninvolved pancreas. A summary of results is shown in Table 2:
Table 2. Neuropilin-2 Expression in Pancreatic Cancer
Figure imgf000059_0001
The knockdown of NRP-2 expression was next examined. FIG. 8 demonstrates that specific knockdown of NRP-2 without any effect on NRP- 1.
The effect of reduced NRP-2 expression on constitutive signaling was next evaluated. Results in FIG. 9 demonstrate that NRP-2 knockdown is associated with a decrease in phosphorylated VEGFR-I and Akt. In addition, Src activation was also decreased by NRP-2 knockdown. Further, the effect of reduced NRP-2 expression on angiogenic mediator expression was examined. Results, shown in FIG. 10, demonstrate that NRP-2 knockdown decreases levels of Jaggedl with no effect on VEGF.
The effect of decreased NRP-2 expression on cellular migration (FIG. 11) and invasion (FIG. 12) was next examined. The results show that NRP-2 knockdown decreased tumor cell migration and invasion. The effect of decreased NRP-2 expression on anchorage independent growth, in vivo growth, and in vivo proliferation was also examined. FIG. 13 demonstrates that reduced NRP-2 expression was associated with reduced anchorage- independent growth. FIG. 14 shows that decreased NRP-2 expression was associated with reduced in vivo growth. Decreased NRP-2 in pancreatic cancer led to a decrease in tumor cell proliferation in vivo.
FIG. 15 shows that decreased NRP-2 expression was associated with reduced in vivo proliferation. The effect of decreased NRP-2 expression on angiogenesis was also examined. FIG.
16 demonstrates that NRP-2 knockdown led to a decrease in microvascular area. The effect of decreased NRP-2 expression on tumor mean vessel density (MVD) and perfusion was also examined. FIG. 17 shows that vessel perfusion was decreased even though VEGF levels and vessel counts were unchanged.
EXAMPLE 3
Neuropilin-2-Mediated Tumor Growth and Angiogenesis in Pancreatic
Adenocarcinoma
Materials and Methods
Human Tissue Specimens and Cell Lines. Formalin-fixed, paraffin-embedded primary pancreatic ductal adenocarcinoma (PDAC) specimens and adjacent nonmalignant pancreatic tissue were obtained from an established tumor bank at The University of Texas M. D. Anderson Cancer Center following protocols approved by the institutional review board. The human PDAC cell lines AsPC-I, BxPC3, MiaPaCa2, MPanc96, and Panc-1 were obtained from the American Type Culture Collection (Manassas, VA). All cells were cultured and maintained at 370C with 5% CO2 in minimal essential medium supplemented with 10% fetal bovine serum (FBS), 2 U/mL of a penicillin-streptomycin mixture (Flow Laboratories, Rockville, MD), vitamins (Life Technologies, Inc., Grand Island, NY), 1 mM sodium pyruvate, 2 mM L-glutamine, and nonessential amino acids. In vitro experiments were performed at 60-80% cell confluence and at early passages after receipt from their supplier.
Generation of NRP-2 shRNA Cell Lines. Short-hairpin RNA (shRNA) expression vectors targeting NRP-2 were created as described in Example 1. Briefly, two NRP-2 targeting sequences were identified, and oligonucleotides encoding each sequence followed by a 9-bp hairpin sequence were generated. The targeting sequences were as follow: for NP2#1, 5 '-CCCAACCAGAAGATTGTCC-S ' (SEQ ID NO:35), and for NP2#2, 5'- GTCAGC ACTAATGGAGAGG-3' (SEQ ID NO:36). The oligonucleotides were then ligated into the pSilencer 4.0 shRNA expression system (Ambion, Austin, TX) at compatible sites, generating the shRNA expression plasmids, sh-NP2Vec-#l and sh-NP2Vec-#2. Negative-control shRNA expression plasmids, sh-ConVec#l and sh-ConVec#2, were generated in a similar fashion by scrambling the NRP -2 targeting sequences and verifying through National Institutes of Health Basic Local Alignment Search Tool (BLAST) analysis that the scrambled sequences were not substantially homologous with any vertebrate genes.
The stable NRP-2 knockdown cell lines, shNRP2-C21 and shNRP2-C23, were created by transfecting BxPC3 cells with 0.5 ng of each sh-NP2Vec plasmid using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocols. The stable control-transfected cell line, shCon, was generated similarly using both scramble sequence-encoding shNRA expression plasmids. Selected clones were isolated and maintained in medium containing 50 μg/mL hygromycin B (Roche Diagnostics, Mannheim, Germany). NRP-2 expression levels in all resulting cell lines were determined by Western blot analysis.
Western Blotting. Cells were plated and grown to 70-80% confluence prior to protein extraction. Whole-cell lysates were obtained using radioimmunoprecipitation assay B protein lysis buffer as previously described Jung et al, 2001). In order to isolate secreted proteins, cells were plated in 1% FBS-supplemented medium for 48 h, and conditioned medium was harvested and concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore Corp., Billerica, MA). Isolated proteins were quantitated using a modified Bradford assay (Bio-Rad Laboratories, Hercules, CA). Protein samples for Western blotting were prepared, boiled, and separated using SDS-PAGE on an 8% or 15% gel and transferred to a polyvinylidene difluoride membrane (Millipore Corp.) by electroblotting. Antibodies were diluted in TBS and 0.1% (v/v) Tween with 5% nonfat dry milk after 1 h of protein blocking in the absence of antibody. Membranes were incubated at 40C overnight, then washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ) for 1 h at room temperature. Protein bands were visualized using a commercially available enhanced chemiluminescence kit (Amersham Biosciences). Antibodies used included NRP-I, NRP-2, Jagged-1 (Santa Cruz Biotechnology, Santa Cruz, CA); phos-AktSer473, Akt, phos-ERKl/2Ηir202/Tyr204, ERK 1/2, phos-SrcTyr416, Src (Cell Signaling Technology, Danvers, MA); phos-VEGFRlTyr1213 (Millipore Corp.); VEGFR-I (Oncogene Research Products, San Diego, CA); and VEGF-A (R&D Systems, Inc., Minneapolis, MN). For verification of equal protein loading, all membranes were stripped and reprobed for either β-actin or vinculin (Sigma-Aldrich, St. Louis, MO). When indicated, densitometric analysis was used to quantitate differences in protein levels from blots using NIH ImageJ vl.34 software (http://rsb.info.nih.gov/ij).
Cell Proliferation and Chemosensitivity. In vitro cellular proliferation was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as previously described in Example 1. Briefly, 2,500 cells of each cell line (parental, shCon, shNRP2-C21, and shNRP2-C23) were plated in each well. At each time point (0 h, 24 h, 48 h, 72 h), 40 μL of MTT solution was added to each well, and the plates were incubated for 1 h at 370C. Colorimetric analysis after the addition of dimethyl sulfoxide was performed using a standard microplate reader. Migration and Invasion Assays. Migration assays were performed as previously described (Wey et al, 2005). Briefly, 75,000 control or shNRP2-transfected cells were suspended in 500 μL of 1% FBS -supplemented medium and placed in an insert with 8-μm pores, which was lowered into 750 μL of 10% FBS-supplemented medium in a standard Boyden chamber assay. After 24 h of incubation, migrated cells on the underside of the membrane were fixed and stained using the Protocol HEMA 3 stain set (Fisher Scientific, Pittsburgh, PA). Membranes were excised, mounted, and examined under light microscopy at 2Ox magnification. Migrated cells were counted in five random fields.
Invasion assays were performed using a similar protocol with minor modifications. The inserts used in the invasion assays were coated with Matrigel (BD Biosciences, San Jose, CA) and prehydrated with 1% FBS-supplemented medium for 30 min prior to the addition of the cell suspension. Invasion chambers were incubated for 48 h, and numbers of invading cells were again quantified.
Anchorage-Independent Growth Assays. Soft-agar assays were used to determine the effect of reduced NRP-2 expression on the ability to grow in anchorage-independent conditions. Each well of a six -well plate was coated with 1 mL of 10% FBS-supplemented medium with 1% agarose. After 20 min, cell suspensions containing control and shNRP2- transfected cells (500 cells each) were added in 1 mL of medium with 0.5% agarose. Cells were incubated for 14 days under standard conditions (370C, 5% CO2) with the addition of 300 μL of medium every 3 days to hydrate the exposed agarose. At the end of the incubation period, wells were examined under a light microscope at 2Ox magnification, and the number of colonies larger than 50 μm was counted per well.
Xenograft Models. Male athymic nude mice, 6-8 weeks old, were obtained from the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD) and acclimated for 2 weeks. All animal studies were conducted under approved guidelines of the Animal Care and Use Committee of M. D. Anderson. Equal numbers of cells (106) from the shCon, shNRP2-C21, and shNRP2-C23 cell lines were suspended in 100 μL of PBS and injected subcutaneously into the right rear flank of each mouse (10 mice per group). Tumor growth was observed and recorded over 10 weeks. When tumors in the control group exceeded 1.5 cm in longest diameter, mice were killed by CO2 asphyxiation according to protocol, and tumors were excised. Tumors were weighed and measured, and a portion of each was placed in either 10% formalin (for paraffin embedding) or optimal cutting temperature (OCT) compound or was snap-frozen in liquid nitrogen. Tumor volume was calculated as 0.5 x (width2) x (length). The results of the subcutaneous xenograft study were validated with an orthotopic pancreatic tumor model. BxPC3-shCon and shNRP2 cells were infected with a luciferase- reporter gene using a recombinant lentivirus as previously described (Arumugam et al., 2006). In each of 10 mice, a suspension of 2 x 106 luciferase-labeled shCon or shNRP2 cells in 50 μL of PBS was injected into the tail of the pancreas through a left-flank incision under ketamine/xylazine (Sigma- Aldrich) anesthesia. Mice were killed at 50 days, when 2-3 mice in any group showed signs of lethargy. Tumors were weighed, measured, and processed as in the subcutaneous model.
Immunohistochemical and Immunofluorescence Analysis. Tumors preserved in formalin were placed in paraffin blocks and sectioned onto positively charged microscope slides. They were deparaffmized in xylene, hydrated in graded alcohol, and pretreated for antigen retrieval in citrate buffer for 20 min in a 980C steamer. Tumor sections embedded in OCT compound were sectioned onto positively charged microscopy slides and serially immersed in acetone, a 1 :1 (vol:vol) acetone: chloroform mixture, then acetone. Slides were then stained with hematoxylin and eosin (H&E) to assess morphology, with proliferating cell nuclear antigen (PCNA) to visualize proliferative nuclei, with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) to visualize apoptotic cells, or with CD31 to visualize blood vessels. AU immunohistochemical sections were counterstained with Gill No. 3 hematoxylin (Sigma-Aldrich).
Antibodies and materials used included rat anti-mouse CD31 (Pharmingen, San Diego, CA), mouse anti-human PCNA PC-10 (Dako, Carpinteria, CA, USA), and the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI). Immunofluorescence slides were examined using a Nikon Microphot FXA fluorescence microscope, and representative images were obtained. To determine the digitized microvascular area (D-MVA), CD31 fluorescently stained slides were analyzed using NIH ImageJ vl.34 software. The red channel, corresponding to
CD31 staining, was isolated and digitized into a binary image, with black indicating stained vessels and white indicating no staining. Vessels with lumens were digitally filled, and a composite digitized microvascular area was quantitated.
In vivo Bioluminescence Imaging. Bioluminescence imaging of luciferase- expressing cells in the orthotopic tumor model was performed using the IVIS 100 imaging system coupled to a data-acquisition personal computer equipped with Living Image software (Xenogen Corp, Hopkinton, MA). Tumor cell-inoculated mice were anesthetized with a 1.5% isoflurane-oxygen mixture and injected intraperitoneally with luciferase potassium salt solution (Sigma- Aldrich) at a dose of 150 mg/kg body weight immediately prior to imaging. In vivo images were obtained on days 3, 18, 39, and 50, and photon emission representative of luciferase activity was used assess relative tumor burden in the mice.
Results
Expression of Neuropilin-2 in Human Pancreatic Adenocarcinoma. Western blot analysis of six commonly used pancreatic cancer cell lines demonstrated that five of six expressed NRP-2 to varying degrees (FIG. 18A). When tissue from PDAC surgical specimens was analyzed for NRP-2 expression, NRP-2 was detected in 7 (64%) of 11 adenocarcinomas but not in any of four specimens of adjacent nonmalignant tissue; representative images are shown in FIG. 18B. In specimens designated positive, all visualized tumor cells stained positively for NRP-2 expression.
Reduced NRP-2 Expression Alters Constitutive Signaling in Pancreatic Cancer Cells. ShRNA-NRP-2 decreased NRP-2 without any effect on NRP-I. Because the NRPs are co-receptors for VEGF receptors in both tumor cells and endothelial cells, we determined the effect of reduced NRP-2 expression on constitutive activation of VEGFR-I in BxPC3 cells. This cell line expresses VEGFR-I but not VEGFR-2 or VEGFR-3 (by reverse transcription polymerase chain reaction analysis). The reduction of NRP2 expression was associated with a decrease in phosphorylation of VEGFR-I in these cells relative to contra 1- transfected cells (FIG. 19A). Furthermore, survival signaling, as measured by Western blotting for phosphorylated Akt, was reduced in cells transfected with shRNA to NRP-2. This finding was confirmed in transfected MPanc96 cells. Other intracellular signaling molecules were evaluated to investigate NRP-2's role in cellular signaling. Src phosphorylation was also found to be moderately reduced in shNRP-2 cells, but ERK1/2 levels were unchanged relative to those in control-transfected cells (FIG. 19B).
Reduced NRP-2 Expression Decreases Migration, Invasion, and Anchorage- Independent Growth. An MTT assay was used to determine the effect of reduced NRP2 expression on in vitro proliferation rates. The shCon and shNRP2 clones demonstrated similar in vitro proliferation rates up to 72 h after plating (FIG. 20A).
To evaluate the effect of reduced NRP-2 expression on in vitro migration and invasion, a standard Boyden chamber assay was used. Pancreatic cancer cells with shRNA to NRP-2 demonstrated a 40-70% decrease in ability to migrate (FIG. 2OB; p<0.05) and a 50- 70% decrease in ability to invade through a Matrigel-coated membrane (FIG. 2OC; p<0.05). The ability of cells to survive and replicate under anchorage-independent conditions was evaluated using a soft-agar growth assay. Cells with reduced NRP-2 produced significantly fewer clones in soft agar than did control-transfected cells (p<0.05).
Reduced NRP-2 Expression Leads to Decreased In Vivo Tumor Growth and Alters the Tumor Vasculature. Given the decrease in migratory and invasive behavior and the reduction in anchorage-independent growth of cells deficient in NRP-2, the role of NRP-2 in in vivo growth was next investigated. Growth characteristics were first evaluated using a murine subcutaneous xenograft model. After 10 weeks, mice inoculated with shNRP2- transfected cells grew tumors that were 63-95% smaller than those of mice inoculated with shCon-transfected cells (FIG. 2 IA, FIG. 22A-C; p<0.05). Furthermore, despite similar in vitro proliferation rates, analysis of tumors by PCNA staining demonstrated a significant decrease in in vivo proliferation in the shNRP2 groups. Specifically, tumors derived from shNRP2-transfected cells had -65% fewer proliferative nuclei per field relative to those in tumors from shCon-transfected cells (p<0.05). No differences were seen in the numbers of apoptotic cells in the tumors by TUNEL staining.
A follow-up study was designed to validate our findings in an orthotopic model using shCon- and shNRP2-transfected cells labeled with firefly luciferase. Prior to mouse implantation, 2 x 105 cells were plated in 12- well plates, and equal luciferase activity in each cell line was verified in vitro. Cells were then prepared and injected orthotopically into the tails of the mouse pancreases. As seen on bioluminescence imaging, tumors derived from shNRP2 cells grew more slowly than did the shCon-derived tumors (FIG. 23A, B; p<0.05). At the time of tumor harvest, shNRP2 tumors were significantly smaller than shCon tumors; average tumor masses were 0.50 g for shCon tumors and 0.08 g for shNRP2 tumors (FIG. 23C; p<0.05), corroborating the data from the subcutaneous model.
Reduced NRP-2 Expression on Tumor Cells Alters the Tumor Vasculature.
Tumor morphologic characteristics were evaluated by standard H&E staining, which demonstrated qualitative differences between groups (FIG. 22D-F). Although tumors derived from shCon-transfected cells formed contiguous tumor cell clusters with stromal elements, those from shNRP2-transfected cells contained patchy acellular areas suggestive of regions of restrained growth within the tumors. Given the decreased number of proliferative cells in the shNPR2-derived tumor sections (FIG. 22G-I), it was hypothesized that the inhibition of growth may be due to a secondary effect via limited tumor angiogenesis. Immunohistochemical staining for CD31 was then used to evaluate the number and morphologic characteristics of blood vessels within each tumor. Vessels in shCon tumors were subjectively larger with more visible patent lumens (FIG. 22J-L). Vessels were enumerated by counting the number of discrete stained structures within each field without regard to vessel size or patency. There was no difference in absolute vessel number between groups. D-MVA was analyzed to incorporate vessel size and patency into the analysis of the tumor vasculature by providing an estimate of integrated lumen area and presumably blood flow orthogonal to the tumor section. ShNRP2 tumors had a 61-66% decrease in D-MVA than shCon tumors (FIG. 21B and FIG. 22M-O; p<0.05). Reduced NRP-2 Expression Is Associated with Decreased Jagged-1 Levels. The reduction of D-MVA led to the hypothesis that the effects on development of the tumor vasculature may be due to altered angiogenic mediator expression in the tumor cells themselves. To investigate this hypothesis, Western blotting was used to identify differences in protein levels of several known angiogenic mediators. There were no differences in VEGF-A, VEGF-C, or delta-like ligand-4 (DLL-4) levels between shCon- and shNRP2- transfected cells; however, there was a significant reduction (46% in shNRP2-C21 and 53% in shNRP2-C23) in Jagged-1 levels in cells deficient in NRP-2 relative to that in control cells (FIG. 24A, B).
* * * * * All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. AU such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
U.S. Patent 4,659,774
U.S. Patent 4,682,195
U.S. Patent 4,683,202 U.S. Patent 4,816,571
U.S. Patent 4,870,287
U.S. Patent 4,959,463
U.S. Patent 5,141,813
U.S. Patent 5,214,136 U.S. Patent 5,223,618
U.S. Patent 5,264,566
U.S. Patent 5,378,825
U.S. Patent 5,428,148
U.S. Patent 5,446,137 U.S. Patent 5,466,786
U.S. Patent 5,470,967
U.S. Patent 5,539,082
U.S. Patent 5,539,082
U.S. Patent 5,554,744 U.S. Patent 5,574,146
U.S. Patent 5,602,240
U.S. Patent 5,602,244
U.S. Patent 5,610,289
U.S. Patent 5,614,617 U.S. Patent 5,623,070
U.S. Patent 5,645,897
U.S. Patent 5,652,099
U.S. Patent 5,670,663 U.S. Patent 5,672,697
U.S. Patent 5,681,947
U.S. Patent 5,700,922
U.S. Patent 5,705,629 U.S. Patent 5,708,154
U.S. Patent 5,714,331
U.S. Patent 5,714,331
U.S. Patent 5,714,606
U.S. Patent 5,719,262 U.S. Patent 5,719,262
U.S. Patent 5,736,336
U.S. Patent 5,736,336,
U.S. Patent 5,739,169
U.S. Patent 5,760,395 U.S. Patent 5,763,167
U.S. Patent 5,766,855
U.S. Patent 5,766,855
U.S. Patent 5,773,571
U.S. Patent 5,777,092 U.S. Patent 5,786,461
U.S. Patent 5,786,461
U.S. Patent 5,792,847
U.S. Patent 5,801,005
U.S. Patent 5,830,880 U.S. Patent 5,846,945
U.S. Patent 5,858,988
U.S. Patent 5,859,221
U.S. Patent 5,872,232
U.S. Patent 5,886,165 U.S. Patent 5,891,625
U.S. Patent 5,908,845
U.S. Patent 5,891,625
U.S. Patent 6,506,559
U.S. Patent 6,573,099 U.S. Patent 6,673,611
U.S. Patent Publn. 2002/0168707
U.S. Patent Publn. 2003/0051263
U.S. Patent Publn. 2003/0055020 U.S. Patent Publn. 2003/0159161
U.S. Patent Publn. 2004/0064842
U.S. Patent Publn. 2004/0265839
U.S. Patent Publn. 20040019001
U.S. Patent Serial 117,363
Arumugam et al., J. Natl. Cancer Inst., 98(24):1806-1818, 2006.
Austin- Ward and Villaseca, Revista Medica de Chile, 126(7):838-845, 1998.
Bailey and Sullivan, Biochimica. Biophys. Acts., 239-252, 2000.
Bangham et al, J. MoI. Biol, 13(l):238-252; 253-259, 1965. Barleon et al, Blood, 87(8):3336-3343, 1996.
Bielenberg et al, Exp. Cell Res., 312(5):584-593, 2006.
Broholm and Laursen, APMIS, 112(4-5):257-263, 2004.
Bukowski et al, Clinical Cancer Res., 4(10):2337 -2347, 1998.
Camp et al, Clin. Cancer Res., 12(8):2628-2633, 2006. Chen et al, Neuron., 19(3):547-559, 1997.
Christodoulides et al, Microbiology, 144(Pt l l):3027-3037, 1998.
Davidson et al, J. Immunother., 21(5):389-398, 1998.
Deamer and Uster, In: Liposome Preparation: Methods and Mechanisms, Ostro (Ed.),
Liposomes, 1983. Dokka et al, Pharm. Res., 17: 521-25, 2000.
Egholm et al, Nature, 365(6446):566-568, 1993.
Ellis et al, Clin. Adv. Hematol Oncol, 4(l):suppl 1-10:11-2, 2006.
Ellis, MoI Cancer Ther., 5(5):1099-107, 2006.
Elmen et al, Nucleic Acids Res., 33(l):439-447, 2005. EP 266,032,
Fakhari et al, Cancer, 94(l):258-263, 2002.
Fan et al, Oncogene, 24(16):2647-2653, 2005.
Favier et al, Blood, 108(4):1243-1250, 2006.
Ferrara et al, Nat. Med, 9(6):669-676, 2003. Ferrara, Nat. Rev. Cancer, 2(10):795-803, 2002.
Fire et al, Nature, 391(6669):806-811, 1998.
Folkman and Shing, J. Biol. Chem., 267(16): 10931-10934, 1992.
Froehler et al, Nucleic Acids Res., 14(13):5399-5407, 1986. Fuh et al, J. Biol. Chem., 275(35):26690-26695, 2000.
Fukahi et al, Clin. Cancer Res., 10(2):581-590, 2004.
Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors andLigands, Wu et al. (Eds.), Marcel Dekker, NY, 87-104, 1991.
Gluzman-Poltorak et al, Biol. Chem., 275(38):29922, 2000. Gluzman-Poltorak et al, J. Biol. Chem., 276(22): 18688-18694, 2001.
Gray et al, Cancer Res., 65(9):3664-36670, 2005.
Gray et al, Oncogene, 24(19):3110-3120, 2005.
Gregoriadis, In: Drug Carriers in Biology and Medicine, Gregoriadis (Ed.), 287-341, 1979.
Gutierrez-Puente et al, J. Pharmacol. Exp. Ther., 291(2):865-869, 1999. Hanibuchi et al, Int. J. Cancer, 78(4):480-485, 1998.
Hansel et al, Am. J. Surg. Pathol, 28(3):347-356, 2004.
He and Tessier-Lavigne, Cell, 90(4):739-751, 1997.
Hellstrand et al, Acta Oncol, 37(4):347-353, 1998.
Hicklin et al, J. Clin. Oncol, 23(5):1011-1027, 2005. Hong et al, Clin. Cancer Res., 13(16):4759-4768, 2007.
Hui and Hashimoto, Infection Immun., 66(l l):5329-5336, 1998.
Ju et al, Gene Ther., 7(19):1672-1679, 2000.
Jung et al, Angiogenesis, 4:155-62, 2001.
Kaneda et al, Science, 243:375-378, 1989.
Karpanen et al, Faseb J., 20(9): 1462-1472, 2006.
Kato et al, J. Biol. Chem., 266:3361-3364, 1991.
Kawakami et al, Cancer, 95(10):2196-21201, 2002.
Kornberg and Baker, In: DNA Replication, 2d Ed., Freeman, San Francisco, 1992. Kostarelos et al, Int. J. Cancer, 112: 713-21, 2004.
Krasnici et al, Int. J. Cancer, 105(4):561-567, 2003.
Landen et al, Cancer Res., 65(15):6910-6918, 2005.
Lantuejoul et al, J. Pathol, 200(3):336-347, 2003.
Lee et al, Proc. Natl. Acad. Sci. USA, 99(16):10470-10475, 2002. Lesslie et al, Br. J. Cancer, 94(11): 1710-1717, 2006.
Leung and Whittaker, Pharmacol. Ther., 107(2):222-239, 2005.
Makinen et al, J. Biol. Chem., 274(30):21217-21222, 1999.
Mitchell et al, Ann. NY Acad. ScL, 690:153-166, 1993. Mitchell et al, J. Clin. Oncol, 8(5):856-869, 1990.
Morton et al, Arch. Surg., 127:392-399, 1992.
Nasarre et al, Neoplasia., 5(l):83-92, 2003.
Nicolau et al, Methods Enzymol, 149:157-176, 1987.
Pan et al, Cancer Cell, 1 l(l):53-67, 2007. Pan et al, J. Biol. Chem., 282(33):24049-24056, 2007.
Parikh et al, Am. J. Pathol, 164(6):2139-2151, 2004.
Parikh et al, Cancer, 98(4):720-729, 2003.
PCT Appln. PCT/EP/01219
PCT Appln. WO 92/20702 Pietras et al, Oncogene, 17(17):2235-2249, 1998.
Qin et al, Proc. Natl. Acad. Sci. USA, 95(24): 14411-14416, 1998.
Ravindranath and Morton, Intern. Rev. Immunol, 7: 303-329, 1991.
Remington's Science and Practice of Pharmacy, 21st Ed., Mack Printing Company, 2005.
Rosenberg et al, Ann. Surg. 210(4):474-548, 1989. Rosenberg et al , N Engl. J. Med. , 319:1676, 1988.
Rossignol et α/,, 2000;
Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd Ed., Cold Spring Harbor Laboratory Press, 2001.
Siwak et al, Clin. Cancer Res., 8: 955-56, 2002. Soker et al, Cell, 92(6):735-745, 1998.
Song et al, Nature Med. 9:347-351, 2003.
Soutschek et al, Nature, 432:173-178, 2004.
Stephenson and Zambon, Occup. Med. (Lond), 52(5):241-247, 2002.
Straume and Akslen, Angiogenesis, 6(4):295-301, 2003. Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA, 75:4194 4198, 1978.
Thurston et al, J. Clin. Invest., 101(7): 1401-1413, 1998.
Wadhwa et al, Curr. Opin. MoI Ther., 6(4):367-372, 2004.
Wang et al, Biol. Chem., 278(49):48848-48860, 2003.
Wey et al, Cancer, 104(2):427-438, 2005. Wey et aL, Clin. Adv. Hematol. Oncol, 2(l):37-45, 2004. Whitaker et al, J. Biol. Chem., 276(27):25520-25531, 2001. Wong et al, Gene, 10:87-94, 1980. Yang et al, Clin. Cancer Res., 12(14 Pt 1):4147-4153, 2006.

Claims

1. A composition comprising a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes NRP-2, and a pharmaceutically acceptable carrier.
2. The composition of claim 1, wherein the nucleic acid component comprises a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes NRP-2.
3. The composition of claim 2, wherein the nucleic acid component comprises a siRNA.
4. The composition of claim 2, wherein the nucleic acid component comprises a nucleic acid encoding a siRNA.
5. The composition of claim 1, wherein the nucleic acid component comprises a shRNA or a nucleic acid encoding a shRNA, wherein the shRNA inhibits the expression of a gene that encodes NRP-2.
6. The composition of claim 1, wherein the composition further comprises a lipid component.
7. The composition of claim 6, wherein the lipid component comprises a neutral lipid.
8. The composition of claim 7, wherein the lipid component comprises two or more neutral lipids.
9. The composition of claim 6, wherein the lipid component forms a liposome.
10. The composition of claim 6, wherein the nucleic acid component is encapsulated in the lipid component.
11. The composition of claim 7, wherein the neutral lipid is a neutral phospholipid.
12. The composition of claim 11, wherein the lipid component comprises two or more neutral phospholipids.
13. The composition of claim 11, wherein the neutral phospholipid is a phosphatidylcholine or phosphatidylethanolamine.
14. The composition of claim 11, wherein the neutral phospholipid is 1 ,2-dioleoyl-sn- glycero-3 -phosphatidylcholine (DOPC), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1- myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), 1- stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dimyristyl phosphatidylcholine ("DMPC"), l,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1 ,2-diarachidoyl-sn- glycero-3-phosphocholine ("DBPC"), l,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), palmitoyloeoyl phosphatidylcholine ("POPC"), ^phosphatidylcholine, dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine ("DSPE"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), or lysophosphatidylethanolamine.
15. The composition of claim 14, wherein the neutral phospholipid is DOPC.
16. The composition of claim 6, wherein the lipid component further comprises a positively charged lipid or a negatively charged lipid.
17. The composition of claim 16, wherein the lipid component comprises a negatively charged phospholipid.
18. The composition of claim 17, wherein the negatively charged phospholipid is phosphatidylserine or phosphatidylglycerol.
19. The composition of claim 17, wherein the negatively charged phospholipid is dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain phosphatidylserine ("BPS"), dilauryloylphosphatidylglycerol ("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), or dioleoylphosphatidylglycerol ("DOPG").
20. The composition of claim 1, wherein the composition further comprises cholesterol or polyethyleneglycol (PEG).
21. The composition of claim 3, wherein the siRNA is a double stranded nucleic acid of 18 to 100 nucleobases.
22. The composition of claim 21, wherein the siRNA is 18 to 30 nucleobases.
23. The composition of claim 1, further comprising a chemotherapeutic agent.
24. The composition of claim 23, wherein the chemotherapeutic agent is docetaxel, paclitaxel, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastin, methotrexate, oxaliplatin, or combinations thereof.
25. The composition of claim 24, wherein the chemotherapeutic agent is 5-fluorouracil or oxaliplatin.
26. A composition comprising a nucleic acid component comprising: a) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes a neuropilin; and b) a lipid component comprising one or more neutral phospholipids.
27. The composition of claim 26, wherein the neuropilin is NRP- 1.
28. The composition of claim 26, wherein the neuropilin is NRP-2.
29. The composition of claim 26, wherein the nucleic acid component comprises a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes a neuropilin.
30. The composition of claim 26, wherein the nucleic acid component comprises a shRNA or a nucleic acid encoding a shRNA, wherein the shRNA inhibits the expression of a gene that encodes a neuropilin.
31. The composition of claim 26, wherein the neutral phospholipid is a phosphatidylcholine or phosphatidylethanolamine.
32. The composition of claim 26, wherein the neutral phospholipid is 1 ,2-dioleoyl-sn- glycero-3 -phosphatidylcholine (DOPC), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1- myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), 1- stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dimyristyl phosphatidylcholine ("DMPC"), l,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1 ,2-diarachidoyl-sn- glycero-3-phosphocholine ("DBPC"), l,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), palmitoyloeoyl phosphatidylcholine ("POPC"), ^phosphatidylcholine, dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine ("DSPE"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), or lysophosphatidylethanolamine.
33. The composition of claim 32, wherein the neutral phospholipid is DOPC.
34. A method of treating a subject with cancer comprising administering to the subject a pharmaceutically effective amount of a composition comprising a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes NRP-2, and a pharmaceutically acceptable carrier.
35. The method of claim 34, wherein the subject is a human subject.
36. The method of claim 34, wherein the nucleic acid component comprises a siRNA or a nucleic acid encoding a siRNA, wherein the siRNA inhibits the expression of a gene that encodes NRP-2.
37. The method of claim 36, wherein the nucleic acid component comprises a siRNA.
38. The method of claim 36, wherein the nucleic acid component comprises a nucleic acid encoding a siRNA.
39. The composition of claim 34, wherein the nucleic acid component comprises a shRNA or a nucleic acid encoding a shRNA, wherein the shRNA inhibits the expression of a gene that encodes a neuropilin.
40. The method of claim 34, wherein the composition further comprises a lipid component.
41. The method of claim 40, wherein the lipid component comprises one or more neutral phospholipids.
42. The method of claim 41, wherein the neutral phospholipid is a phosphatidylcholine or phosphatidylethanolamine.
43. The method of claim 42, wherein the neutral phospholipid is 1 ,2-dioleoyl-sn-glycero- 3-phosphatidylcholine (DOPC), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1- myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), 1- stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dimyristyl phosphatidylcholine ("DMPC"), l,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"), 1 ,2-diarachidoyl-sn- glycero-3-phosphocholine ("DBPC"), 1 ,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), palmitoyloeoyl phosphatidylcholine ("POPC"), ^phosphatidylcholine, dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine ("DSPE"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), or lysophosphatidylethanolamine.
44. The method of claim 43, wherein the neutral phospholipid is DOPC.
45. The method of claim 40, wherein the lipid component further comprises a positively charged lipid or a negatively charged lipid.
46. The method of claim 34, wherein the cancer is breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, colorectal cancer, renal cancer, skin cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, testicular cancer, lymphoma, or leukemia.
47. The method of claim 46, wherein the cancer is colorectal cancer.
48. The method of claim 46, wherein the cancer is pancreatic ductal adenocarcinoma.
49. The method of claim 34, further comprising administering an additional anticancer therapy to the subject.
50. The method of claim 49, wherein the additional anticancer therapy is chemotherapy, radiation therapy, surgical therapy, immunotherapy, gene therapy, or a combination thereof.
51. The method of claim 50, wherein the additional anticancer therapy is chemotherapy.
52. The method of claim 51, wherein the chemotherapy comprises administration of docetaxel, paclitaxel, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, oxaliplatin, or a combination thereof.
53. The method of claim 52, wherein the chemotherapy comprises 5-fluorouracil or oxaliplatin.
54. The method of claim 34, wherein the composition is administered to the patient intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion.
55. The method of claim 34, wherein the subject has a tumor and the method is further defined as a method to reduce tumor volume in the subject.
56. The method of claim 55, wherein the tumor is colorectal cancer.
57. A method of treating a subject with cancer comprising administering to the subject a pharmaceutically effective amount of a composition comprising:
(a) a nucleic acid component comprising a nucleic acid that inhibits the expression of a gene that encodes a neuropilin; (b) a lipid component comprising one or more neutral phospholipids; and
(c) a pharmaceutically acceptable carrier.
58. The method of claim 57, wherein the neuropilin is NRP-I .
59. The method of claim 57, wherein the neuropilin is NRP -2.
60. The method of claim 57, wherein the subject is a human subject.
61. The method of claim 57, wherein the nucleic acid component comprises a siRNA.
62. The method of claim 57, wherein the neutral phospholipid is a phosphatidylcholine or phosphatidylethanolamine.
63. The method of claim 57, wherein the neutral phospholipid is 1 ,2-dioleoyl-sn-glycero- 3-phosphatidylcholine (DOPC), egg phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), 1- myristoyl-2-palmitoyl phosphatidylcholine ("MPPC"), l-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"), l-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"), 1- stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"), dimyristyl phosphatidylcholine ("DMPC"), l^-distearoyl-sn-glycero-S-phosphocholine ("DAPC"), 1 ,2-diarachidoyl-sn- glycero-3-phosphocholine ("DBPC"), 1 ,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"), palmitoyloeoyl phosphatidylcholine ("POPC"), ^phosphatidylcholine, dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine ("DSPE"), dimyristoyl phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), palmitoyloeoyl phosphatidylethanolamine ("POPE"), or lysophosphatidylethanolamine.
64. The method of claim 57, further defined as a method of preventing metastasis of a cancer in a subject.
65. The method of claim 64, wherein the cancer is colorectal cancer, and the metastasis is metastasis to the liver.
66. The method of claim 57, wherein the cancer is pancreatic ductal adenocarcinoma
PCT/US2009/032689 2008-01-31 2009-01-30 Tumor cell expression of neuropilin as a target for cancer therapy Ceased WO2009099959A2 (en)

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WO2014058915A3 (en) * 2012-10-08 2014-05-30 St. Jude Children's Research Hospital Therapies based on control of regulatory t cell stability and function via a neuropilin-1:semaphorin axis
EP2823816A1 (en) 2013-07-09 2015-01-14 Tragex Pharma Inhibitors of Neuropilin and use thereof for the treatment of Neuropilin-related diseases
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EP2522341A1 (en) 2011-05-13 2012-11-14 Tragex Pharma Pharmaceutical compositions comprising Neuropilin inhibitors, and their use for the prevention and/or treatment of angiogenic disorders and cancers
WO2012156289A1 (en) 2011-05-13 2012-11-22 Tragex Pharma Pharmaceutical compositions comprising neuropilin inhibitors, and their use for the prevention and/or treatment of angiogenic disorders and cancers
WO2014058915A3 (en) * 2012-10-08 2014-05-30 St. Jude Children's Research Hospital Therapies based on control of regulatory t cell stability and function via a neuropilin-1:semaphorin axis
US9540439B2 (en) 2012-10-08 2017-01-10 St. Jude Children's Research Hospital Therapies based on control of regulatory T cell stability and function via a neuropilin-1:semaphorin axis
EP2823816A1 (en) 2013-07-09 2015-01-14 Tragex Pharma Inhibitors of Neuropilin and use thereof for the treatment of Neuropilin-related diseases
CN108196071A (en) * 2018-02-09 2018-06-22 上海市第十人民医院 liver cancer serum marker NRP1
CN119931944A (en) * 2025-04-09 2025-05-06 济南大学 Application of purple sweet potato pigment in the preparation of products for inducing neural differentiation of stem cells

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