ONCOLYTIC VIRUS

Abstract

Malignant tumors that are intrinsically resistant to conventional therapies are significant therapeutic challenges. An embodiment of the present invention provides an oncolytic virus capable of killing target cells, such as a tumor cells. In various embodiments presented herein, the oncolytic virus is armed or encodes a therapeutic polypeptide. In at least one embodiment, a recombinant oncolytic virus has been generated that can specifically replicate in cancer cells leading to their destruction and at the same time secrete robust amounts of a therapeutic polypeptide. Compositions and methods disclosed herein have broad therapeutic applicability.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This non-provisional application is a divisional of, and claims a priority benefit to, U.S. patent application Ser. No. 12/697,891, filed Feb. 1, 2010 and issued as U.S. Pat. No. 8,450,106 on May 28, 2013, which application in turn claims the priority benefit of U.S. Provisional Patent Application No. 61/256,644, filed Oct. 30, 2009, and U.S. Provisional Patent Application No. 61/148,870, filed Jan. 30, 2009. U.S. patent application Ser. No. 12/697,891 is also a continuation of International Patent Application No. PCT/US2008/080367, filed Oct. 17, 2008, which claims the priority benefit of U.S. Provisional Patent Application No. 60/980,664, filed Oct. 17, 2007. All of the above referenced applications are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The present invention is directed to the fields of virology, cancer biology, and medicine. More particularly, it concerns compositions and methods of treating cancer of the brain in a patient using oncolytic herpes simplex virus 1(HSV-1) armed with therapeutic transgenes.

BACKGROUND OF THE ART

[0003] Malignant tumors that are intrinsically resistant to conventional therapies are significant therapeutic challenges. Such malignant tumors include, but are not limited to malignant gliomas and recurrent systemic solid tumors such as lung cancer. Malignant gliomas are the most abundant primary brain tumors having an annual incidence of 6.4 cases per 100,000 (CBTRUS, 2002-2003). These neurologically devastating tumors are the most common subtype of primary brain tumors and are one of the deadliest human cancers. In the most aggressive cancer, manifestation glioblastoma multiforme (GBM), median survival duration for patients is 14 months, despite maximum treatment efforts. A prototypic disease, malignant glioma is inherently resistant to current treatment regimens. In fact, in approximately 1/3 of patients with GBM the tumor will continue to grow despite treatment with radiation and chemotherapy. Median survival even with aggressive treatment including surgery, radiation, and chemotherapy is less than 1 year (Schiffer, 1998). Because few good treatment options are available for many of these refractory tumors, the exploration of novel and innovative therapeutic approaches is essential.

[0004] Gene therapy is a promising treatment for tumors including gliomas because conventional therapies typically fail and are toxic. In addition, the identification of genetic abnormalities contributing to malignancies is providing crucial molecular genetic information to aid in the design of gene therapies. Genetic abnormalities indicated in the progression of tumors include the inactivation of tumor suppressor genes and the overexpression of numerous growth factors and oncogenes. Tumor treatment may be accomplished by supplying a polynucleotide encoding a therapeutic polypeptide or other therapeutic that target the mutations and resultant aberrant physiologies of tumors. It is these mutations and aberrant physiology that distinguishes tumor cells from normal cells. A tumor-selective virus would be a promising tool for gene therapy. Recent advances in the knowledge of how viruses replicate have been used to design tumor-selective oncolytic viruses.

[0005] In gliomas, several kinds of conditionally replication competent viruses have been shown to be useful in animal models for example: reoviruses that can replicate selectively in tumors with an activated ras pathway (Coffey et al., 1998); genetically altered herpes simplex viruses (Martuza et al., 1991; Mineta et al., 1995; Andreanski et al., 1997), including those that can be activated by the different expression of proteins in normal and cancer cells (Chase et al., 1998); and mutant adenoviruses that are unable to express the E1B55 kDa protein and are used to treat p53-mutant tumors (Bischof et al., 1996; Heise et al., 1997; Freytag et al., 1998; Kim et al., 1998). Taken together, these reports confirm the relevance of oncolytic viruses as anti-cancer agents. In all three systems, the goal is the intratumoral spread of the virus and the ability to selectively kill cancer cells. Along with directly killing the cancers cells, agents that can also influence the microenvironment surrounding the tumor may enhance the therapeutic effect of the OV.

[0006] Replication selective oncolytic viruses have shown great promise as anti-tumor agents for solid tumors. The viruses have been constructed genetically so that they are able to preferentially replicate within tumor cells, while being restricted in their ability to replicate in normal cells. The principal anti-tumor mechanism of oncolytic viruses is through a direct cytopathic effect as they propagate and spread from initially infected tumor cells to surrounding tumor cells, achieving a larger volume of distribution and anticancer effects. Oncolytic herpes simplex virus (HSV) were initially designed and constructed for the treatment of brain tumors. Subsequently, they have been found to be effective in a variety of other human solid tumors, including breast, prostate, lung, ovarian, colon and liver cancers. The safety of oncolytic HSVs has also been extensively tested in mice and primates (Aotus), which are extremely sensitive to HSV.

[0007] HSV-1 based oncolytic viruses are particularly exciting because of: 1) their ability to infect a wide variety of tumors; 2) their inherent cytolytic nature; 3) their well characterized large genome (152 Kb) that provides ample opportunity for genetic manipulations wherein many of the non-essential genes (up to 30 kb) can be replaced by therapeutic genes; 4) their ability to remain as episomes that avoid insertional mutagenesis in infected cells; and 5) the availability of anti-herpetic drugs to keep in check possible undesirable replication.

[0008] Despite these encouraging preclinical studies, results from early clinical trials have suggested that the current versions of oncolytic viruses, although safe, may only have limited anti-tumor activity on their own. One of the main reasons for the sub-optimal oncolytic efficacy is probably because viral gene deletions that confer tumor selectivity also result in reduced potency of the virus in tumors. For example, the complete elimination of endogenous γ34.5 function from HSV, one of the common approaches for the construction of oncolytic HSV, significantly reduces viral replication potential and therefore may compromise the ability of the virus to spread within the targeted tumors (Kramm et al., 1997). Therefore, strategies designed to further enhance the potency of oncolytic viruses will likely increase their chance of clinical success.

[0009] The tumor microenvironment is also recognized as an important determinant for tumor progression. Vasculature is a major component of the microenvironment of solid tumors such as malignant gliomas. Solid tumors depend on the development of a vasculature to provide them with nutrients. While tumor oncolysis is thought to set the stage for activating a systemic adaptive immune surveillance, innate defense mechanisms elicited upon OV infection are thought to be responsible for rapid viral clearance from tumor. Thus, OV-induced inflammation and its attendant increased vasculature may be counterproductive to the goal of killing cancer cells.

[0010] Therefore, there is an unmet need for an OV therapy that is tumor selective yet robust enough to kill tumor cells over an organism's natural defenses.

SUMMARY

[0011] Embodiments address a long-felt need in the art by providing a potent oncolytic virus for therapy of undesirable cells, such as malignant cells.

[0012] A preferred embodiment provides an oncolytic virus capable of killing target cells, such as a tumor cells. In preferred embodiments, the conditionally replicating HSV comprises at least two mechanisms to rid a culture, tissue or organism of at least some undesirable cells, to inhibit proliferation of at least some undesirable cells, to prevent proliferation of at least some desirable cells, or a combination thereof. A preferred embodiment may not only directly act on the cancer itself, but also may enhance tumor cell killing by influencing the microenvironment around the physical tumor.

[0013] In various embodiments presented herein, the oncolytic virus is armed or encodes a therapeutic polypeptide. "Armed" is a term that indicates that the virus contains a heterologous nucleic acid sequence encoding a polypepitde of interest or a nucleic acid comprising a polynucleotide of interest. In certain embodiments, the nucleic acid encoding a therapeutic polypeptide may encode an angiostatic factor. In various embodiments, the nucleic acid encoding a therapeutic polypeptide encodes the polypeptide Vasculostatin. In an alternative embodiment, the transgene encodes an enzyme that can depolymerize at least a portion of the extracellular matrix scaffold. Preferably, the transgene encodes a choindroitinase ABC 1 (Chase ABC), a bacterial enzyme. In yet another embodiment, the oncolytic virus may be armed with multiple heterologous nucleic acid sequences, for example, the oncoyltic virus may contain transgenes encoding both Vasculostatin and Chase ABC.

[0014] In various embodiments, the OV comprises a transcriptionally targeted OV wherein at least one of the deleted ICP34.5 genes may be reinserted into the HSVQ backbone, under the transcriptional control of a glioma specific nestin enhancer element. In at least one such exemplary embodiment, the OV is a Vasculostatin expressing virus within the rQnestin34.5 virus backbone. In another such exemplary embodiment, the OV is a Chase ABC expressing virus within the rQnestin34.5 virus backbone.

[0015] Oncolytic viruses expressing angiostatic factors using a CMV promoter have demonstrated limited efficacy in rodent models of glioma (for example, US Pub. No. 2006/0147420 and US Pub. No. 2004/0009604, incorporated herein by reference). Given the rapid lytic cycle of HSV, the challenge has been to ensure robust expression of the therapeutic protein before lysis of the infected cell.

[0016] Disclosed herein, a preferred embodiment overcomes this challenge by utilizing the immediate early HSV promoter IE4/5 operably linked to a therapeutic transgene. In at least one embodiment, the transgene is angiostatic. In a preferred embodiment, the transgene is a novel, brain specific angiostatic polypeptide, Vasculostatin. In another embodiment, the transgene is Chase ABC, a bacterial enzyme. In a preferred embodiment, robust expression of a therapeutic gene can be seen at least as early as 4 hours.

[0017] In various embodiments, the transgene encodes an enzyme that can depolymerize at least a portion of the extracellular matrix scaffold. Preferrably, the transgene encodes a choindroitinase ABC 1 (Chase ABC), a bacterial enzyme. The ECM forms an inhibitory scaffold organized by CSPGs that bind the HA scaffold, other key matrix molecules (tenascins and link proteins) and cellular receptors (CD44, NCAM, integrins, etc). This scaffold inhibits the spread of infectious OV particles from one infected cell (bottom) to the next (top). A preferred embodiment overcomes this obstacle by utilizing the immediate early HSV promoter IE4/5 operably linked to a nucleic acid that encodes a choindroitinase ABC 1 (Chase ABC). An exemplary embodiment gains increased therapeutic efficacy by delivering Choindroitinase ABC in tumors to increase dispersal of a large therapeutic molecule(s). Furthermore, an exemplary embodiment combines the use of an oncolytic virus (OV) with an HSV immediate early IE 4/5 viral promoter that drives expression of a secreted glycosidase as a means of increasing viral dispersal in tumors. The oncolytic virus of an exemplary embodiment can be delivered by a number routes including, but not limited to intracranial (into the skull cavity) intra-tumoral or intravenous administration. The tumor may be a primary tumor or it may be a tumor resulting from a metastasis to the skull or brain.

[0018] In an exemplary embodiment, a recombinant oncolytic virus has been generated that can specifically replicate in cancer cells leading to their destruction and at the same time secrete robust amounts of an angiostatic factor to inhibit the regrowth of residual disease. Such a dually armed OV destroys cancer cells through its tumor specific replication potential and also targets tumor vasculature to enhance therapeutic efficacy.

[0019] In a preferred embodiment, a dually armed OV has been generated and shown that it does express and secrete the therapeutic anti-angiogenic factor (Vasculostatin), a novel brain specific anti-angiogenic factor, even at early time points after infection. One embodiment, an embodiment referred to herein as RAMBO (Rapid Anti-angiogenesis Mediated By Oncolytic virus) is a Vasculostatin expressing virus within a HSVQ backbone. In another embodiment, an embodiment referred to herein as Nested-RAMBO, is a Vasculostatin expressing virus within the rQnestin34.5 virus backbone (see Kambara, H.; Okano, H.; Chiocca, E. A. Saeki, Y. An oncolytic HSV-1 mutant expressing ICP34.5 under control of a nestin enhancer element increases survival of animals even when symptomatic from a brain tumor. Cancer Res 2005, 65, 2832-9, expressly incorporated by reference in its entirety.) In various embodiments, Vasculostatin is operably linked to and expressed under the control of an HSV immediate early IE4/5 promoter.

[0020] In order to maximize the expression of Vasculostatin, at least one embodiment exploits the robust transgene expression from an early viral promoter to maximize the expression of Vasculostatin. The expression profile of a preferred embodiment allows for maximal expression of therapeutic transgenes before the lytic phase. At least one embodiment has shown efficacy in mice with established brain tumors. Compositions and methods disclosed herein have broad therapeutic applicability to most solid cancers.

[0021] Accordingly, embodiments include a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a therapeutic polypeptide operably linked to an immediate early HSV promoter IE4/5. In some embodiments the vector is a modified herpes simplex virus. In various embodiments, the modified herpes simplex virus is a mutant herpes simplex virus deficient for both copies of its native γ₁₃₄.5 gene. In exemplary embodiments, the therapeutic polypeptide may comprise Vasculostatin, a proteolytic polypeptide fragment of BAI1. In alternative embodiments, the therapeutic polypeptide comprises a Chase ABC polypeptide.

[0022] Various embodiments comprise a recombinant Herpes Simplex Virus, comprising: a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 6, or of a degenerate variant of SEQ ID NO: 1 or SEQ ID NO: 6, operably linked to an expression control sequence. The expression control sequence may be an immediate early HSV promoter IE4/5. In various embodiments, the expression control sequence comprises the nucleotide sequence of SEQ ID NO: 5.

[0023] In some embodiments, the recombinant Herpes Simplex Virus is deficient for both copies of its native γ₁₃₄.5 gene. However, various embodiments include a nucleic acid comprising a nucleotide sequence encoding a replacement γ₁₃₄.5 gene to conditionally restore this deficiency. In preferred embodiments, the replacement γ₁₃₄.5 gene is operably linked to a tumor specific promoter.

[0024] Exemplary embodiments include a method of killing intracranial tumor cells in a mammal comprising introducing into the vicinity of the tumor cells an expression vector, the vector comprises a modified herpes virus deficient for both copies of its native γ₁₃₄.5 gene; a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 1, or of a degenerate variant of SEQ ID NO: 1, operably linked to an expression control sequence; and a nucleic acid encoding a replacement γ₁₃₄.5 gene, the replacement γ₁₃₄.5 gene is operably linked to a nestin enhancer element. In some embodiments, the expression control sequence comprises an immediate early HSV promoter IE4/5. In various embodiments, the method further comprises the step of mixing a pharmacologically acceptable carrier with the expression vector prior to the introducing step.

[0025] Embodiments include compositions comprising a vector comprising: a nucleic acid encoding the polypeptide Vasculostatin operably linked to an immediate early HSV IE4/5 promoter. In alternative embodiments, the composition comprises a vector comprising: a nucleic acid encoding the polypeptide Chase ABC operably linked to an immediate early HSV IE4/5 promoter. In various embodiments, the vector is a mutant Herpes Simplex Virus comprising a nucleic acid encoding a replacement γ₁₃₄.5 gene inserted into an otherwise γ₁₃₄.5-deleted viral genome, the replacement γ₁₃₄.5 gene is operably linked to a tumor specific promoter. Preferrably, the tumor specific promoter comprises a nestin enhancer element.

[0026] Embodiments comprises a recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a Chase ABC polypeptide operably linked to an immediate early HSV promoter IE4/5. In various embodiments the vector is a modified herpes simplex virus. In some embodiments, the modified herpes simplex virus is deficient for both copies of its native γ₁₃₄.5 gene. In exemplary embodiments, the nucleic acid sequence encoding a Chase ABC polypeptide comprises the nucleotide sequence of SEQ ID NO: 6 or of a degenerate variant of SEQ ID NO: 6.

[0027] In exemplary embodiments, expression of the therapeutic transgene (e.g., Vasculostatin and/or Chase ABC) is functional and does not interfere with the virus's cytotoxicity toward tumor cells (e.g., glioma cells). Additionally, the generated OV has therapeutic advantage over the control virus for the treatment of mice with established brain tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of a preferred embodiment. 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:

[0029] FIG. 1 shows the survival of rats implanted with U87 human glioma cells stably expressing Vasculostatin (clones U14 and U18) with parental untransfected glioma cells (U87). Note that survival of rats implanted with cells expressing Vasculostatin was significantly greater than that of rats implanted with control parental U87MG cells (P<0.05). (Kaur et al., unpublished results).

[0030] FIG. 2 provides a schematic illustration of the steps utilized to first clone the cDNA encoding for Vasculostatin under IE4/5 promoter into a shuttle plasmid (ptransferIE4/5) in order to generate pVasculo-transfer (SEQ ID NO: 10)

[0031] FIG. 3 provides a schematic illustration of the steps utilized to generate the OV, rHSVQvasculo (also called "RAMBO")

[0032] FIG. 4 shows a western blot analysis confirming the expression of Vasculostatin by the recombinant viral isolates.

[0033] FIG. 5 shows a time course western analysis of Vasculostatin production by both OV isolates in LN229 cells

[0034] FIG. 6 shows a cytotoxicity assay for both viral isolates with U87ΔEGFR cells. Note that there is no significant difference in cytotoxicity between the control rHSVQ virus and the isolated rHSVQvasculo1, and rHSVQvasculo2 expressing Vasculostatin.

[0035] FIG. 7 demonstrates the anti-angiogenic capabilities of rHSVQvasculo. The experiment was performed using the Trevigen Direct In Vivo Angiogenesis Assay (DIVAA®) Inhibition Kit.

[0036] FIG. 8 shows a Kaplan-Meier survival analysis of mice treated with rHSVQ control virus or rHSVQvasculo (the virus generated to express Vasculostatin). In this experiment mice were treated by direct intratumoral injection on day 5 after tumor implantation.

[0037] FIG. 9 shows the results of an experiment to compare the cytotoxicity of rHSVQvasculo to rHSVQ (an OV equivalent to the G207 being tested in clinical trials) toward normal human astrocytes. Normal human astrocytes (NHA, CellSciences Canton, Mass.) were infected at different multiplicities of infection (MOI) with rHSVQ and rHSVQvasculo to evaluate potential cytotoxicity of OV produced Vasculostatin towards NHA.

[0038] FIGS. 10A-10L show a comparison of the effect of Vasculostatin expression on ability of OV to be cytotoxic to glioma cells in the glioma cells U87ΔEGFR (FIGS. 10A-10D), LN229 (FIGS. 10E-10H), and U343 (FIGS. 10I-10L), using a standard colorimetric assay. Note that there are no significant differences in the cytotoxicity for the two viruses.

[0039] FIG. 11 is a Western blot demonstrating that cyclophosphamide (CPA) pretreatment enhances the anti-tumor ability of Vasculostatin.

[0040] FIG. 12 is a schematic representation of the maps of various oncolytic virus embodiments, included Nested-RAMBO. Nested-RAMBO is a Vasculostatin expressing virus within the rQnestin34.5 virus backbone.

[0041] FIG. 13 is a Western blot of U251 glioma cells treated with PBS (lane 1), HSVQ (Lane 2), rQnestin34.5 (lane 3), RAMBO (lane 4), or 2 different isolates of Nested-Rambo (lanes 5 and 6) showing expression of Vstat120, elF2α, and phospho-elF2α in cell lysate 14 hrs after OV infection.

[0042] FIG. 14 is a graph of the results from an experiment comparing the cytolytic ability of Nested-RAMBO, to rQnestin34.5 in glioma cells with high and low nestin expression.

[0043] FIG. 15 is a graph detailing the results from an experiment comparing the antitumor efficacy of rQnestin34.5, RAMBO, and Nested-RAMBO against subcutaneous glioma in athymic nude mice.

[0044] FIG. 16 shows representative fluoresecent images of glioma spheres infected with HSVQ (right panel) or OV-Chase (left panel). Human glioma spheres infected with HSVQ or OV-Chase (green cells are GFP positive infected cells) were stained for the appearance of immunoreactive sugar stubs (red staining, white arrows) obtained by Chase ABC mediated digestion of cell secreted CSPG.

[0045] FIG. 17 shows fluorescent images of glioma spheroids infected with HSVQ or OV-Chase (n=6/group). Human glioma spheres grown on mouse brain slices, were infected with HSVQ (A) or OV-Chase (B) (n=6/group), and followed over a period of time (24 hrs: top row, 48 hrs: middle row, 60 hrs: bottom row). Only the rim of the spheres was infected with HSVQ treated spheres (bottom panel is bright field image showing the intact sphere at 60 hrs post infection). Note the spread of infectious virus particles into the sphere in all 6 spheroids treated with OV-Chase.

DETAILED DESCRIPTION

[0046] Gliomas are the most common primary tumors of the central nervous system (CNS). Glioblastoma multiforme (GBM), the most aggressive form (WHO grade IV) of malignant astrocytoma, is highly invasive and vascularized [40] and characterized by 1) rapidly proliferating endothelial cells that form tufted aggregates referred to as glomeruloid bodies and 2) multiple hypoxic-necrotic areas within the tumor that drive hypoxia-mediated activation of hypoxia inducible factor (HIF), which thereby leads to increased transcription of factors, such as vascular endothelial growth factor (VEGF), that heralds a phase of more malignant tumor growth [41]. Their very aggressive growth and highly vascular nature makes malignant gliomas an attractive target for testing the effects of anti-angiogenic gene therapy. The increased vascularization essential for malignant progression is triggered by disruption of the normal homeostasis between angiogenic and angiostatic factors within the tumor microenvironment. It has been shown that expression of angiogenesis inhibitors is reduced in GBMs but not in normal brain and benign gliomas. Although not to be limited by theory, physiologically occurring factors that inhibit angiogenesis which are lost during tumor progression should represent molecules of choice for restoration by gene therapy.

[0047] Vasculostatin, a fragment of Brain Angiogenesis inhibitor 1 (BAI1) (GenBank Accession No. AB005297), can inhibit angiogenesis, permeability, and subcutaneous as well as intracerebral tumor growth. Vasculostatin 1) is expressed at high levels primarily in normal brain but not in most GBMs and 2) has potent anti-angiogenic, anti-tumorigenic, and anti-permeability properties and 3) the ability to target multiple receptors on endothelial cells (αvβ3, αvβ5, and CD36), and 4) its over expression is well tolerated in brain tissue. Hence, Vasculostatin may be a better candidate than other more popular and not so novel anti-angiogenic factors for therapy of GBMs.

[0048] HSV-1-derived OVs that express endostatin and, more recently, oncolytic viruses that express platelet factor IV and dominant negative FGF receptor under the control of a CMV promoter have been described. However, at least one embodiment disclosed herein is better for GBM therapy because of its combination with an IE4/5 promoter (SEQ ID NO: 5) that drives the expression of the transgene to levels unseen with a CMV promoter. More particularly, in certain embodiments disclosed herein Vasculostatin, a novel angiostatic factor, has been successfully expressed as part of an oncolytic viral stategy and shown to successfully counter anti-therapeutic changes in residual disease after oncolysis.

[0049] Specific replication within tumor cells can be achieved by OVs genetically engineered for that purpose or by naturally occurring strains of some viruses that have such propensity [25]. Specific embodiments utilize one such mutant, designated G207, which comprises an F-strain derived HSV-1 with deletions in both copies of the γ₁₃₄.5 gene (encoding for the viral ICP34.5 protein) and an inactivating insertion of Escherichia coli (E. coli) lacZ into the viral ICP6/RR gene (encoding for the large subunit of ribonucleotide reductase). The available human evidence shows that the injection of HSV-1 with γ₁₃₄.5 deletion (and intact vhs) does not lead to the reactivation of wild-type HSV-1, produce toxicity from infection of neurons surrounding the glioma cavity, or lead to encephalitis or meningitis.

[0050] HSV-1 infection of cells activates, a cellular "stress" or "defense" response consisting of activation of the PKR (double stranded RNA protein kinase) enzyme, ultimately leading to blockage of protein synthesis. HSV1 gene product ICP34.5 (γ₁₃₄.5) activates a cellular phosphatase (PP1α) that dephosphorylates eiF2α, thus allowing for viral protein translation and replication to occur. Complete deletion of ICP34.5 severely attenuates the replication potential of oncolytic HSV, and this has been deleted in clinical viruses tested to date. Various embodiments overcome this replication deficiency using tumor specific promoters to drive viral potency. More specifically, various embodiments include a transcriptionally targeted OV, wherein at least one copy of the γ₁₃₄.5 gene may be conditionally expressed under the governance of a nestin enhancer element when this expression cassette is reinserted into a γ₁₃₄.5 null OV backbone.

[0051] Angiogenesis is critical for the development and maintenance of glioblastomas, the most malignant and common form of primary brain tumors. Combining oncolysis with anti-angiogenesis may produce a synergistic effect since the anti-cancer mechanisms are different but complementary. A preferred embodiment allows an anti-angiogenic nucleic acid or polypeptide, such as, but not limited to a Vasculostatin protein, to be produced, ultimately favoring delivery to the extracellular compartment. For that reason, the oncolytic HSV-1 is used as an improved HSV vector to deliver high and continuous levels of Vasculostatin to the tumor.

[0052] Angiogenesis refers to vessel formation by remodeling the primary vascular network or by sprouting from existing vessels (reviewed in Yancopoulos et al., 2000). The "angiogenesis switch" is "off" when the effect of pro-angiogenic molecules is balanced by the activity of anti-angiogenic molecules, and is "on" when the net balance between the molecules is tipped in favor of angiogenesis (reviewed in Carmeliet and Jain, 2000). Angiogenesis has an essential role in the development and maintenance of solid tumors, including malignant gliomas.

[0053] One of the major barriers for effective drug delivery within the tumor parenchyma is the ubiquitous extracellular matrix (ECM) secreted by glioma cells. This matrix forms a complex scaffold that modulates tumor cell proliferation, cell adhesion, and motility. Glioma ECM is based on a scaffold of hyaluronic acid (HA) with associated glycoproteins and proteoglycans, which resemble the composition of the normal brain ECM. However, the ECM of gliomas also includes mesenchymal proteins that are absent in normal brain and that make the matrix of these tumors distinct from the ECM of normal neural tissue and of other solid tumors. Increased expression and extracellular accumulation of ECM reduces the interstitial spaces and increases the internal pressure in the tumor. This leads to an increase in the fractional volume and tortuosity of the extracellular space, which are the major biophysical factors that limit passive molecular diffusion in the tumor tissue and are limiting for spread of therapeutics.

[0054] Inefficient viral dispersal through the tumor interstitium can lead to poor viral spread hence not permitting efficient tumor cell infection and oncolysis. Efforts to increase viral spread within the tumor should lead to improved efficacy. Glioma extracellular matrix (ECM) poses a significant barrier for efficient viral spread through the tumor, and hence limits its efficacy. Chondroitin sulfate proteoglycans (CSPG) are one of the major inhibitory components of glioma ECM. A specific bacterial glycosidase, such as Choindroitinase ABC 1 (Chase ABC) can depolymerize this ECM scaffold to provide a long-lasiting "loosening" effect on the inhibitory scaffold. Accordingly, in various exemplary embodiments, Chase ABC mediated digestion of glioma CSPG may enhance OV dissemination and efficacy.

[0055] Embodiments of this invention may include multiple other heterologous genes. For example, they may include therapeutic genes, pro-drug converting enzymes, cytosine deaminase (to convert 5-FC to 5-FU), a yeast cytosine deaminase, a humanized yeast cytosine deaminase, an image enhancing polypeptides, a sodium-iodide symporter, anti-sense or inhibitory VEGF, Bcl-2, Ang-2, or interferons alpha, beta or gamma.

[0056] In describing the exemplary embodiments, the following terms will be employed, and are intended to be defined as indicated below.

[0057] The term "recombinant HSV-1 vector" as used herein defines a recombinant HSV-1 vector comprising: (a) the DNA of, or corresponding to, at least a portion of the genome of an HSV-1 which portion is capable of transducing into a target cell at least one selected gene and is capable of promoting replication and packaging; and (b) at least one selected gene (or transgene) operatively linked to regulatory sequences directing its expression, the gene flanked by the DNA of (a) and capable of expression in the target cell in vivo or in vitro. Thus, when referring to a "recombinant HSV" (rHSV) it is meant the HSV that has been genetically altered, e.g., by the addition or insertion of a selected gene.

[0058] A "gene," or a "sequence which encodes" a particular protein, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the gene are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence. Typically, polyadenylation signal is provided to terminate transcription of genes inserted into a recombinant virus.

[0059] As is known to those of skill in the art, the term "polypeptide" or "protein" means a linear polymer of amino acids joined in a specific sequence by peptide bonds. As used herein, the term "amino acid" refers to either the D or L stereoisomer form of the amino acid, unless otherwise specifically designated.

[0060] The term "transgene" refers to a particular nucleic acid sequence encoding a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence is inserted. The term "transgene" is meant to include (1) a nucleic acid sequence that is not naturally found in the cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been inserted; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been inserted; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been inserted. By "mutant form" is meant a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and/or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that the transgene product may be secreted from the cell.

[0061] In a preferred embodiment, a proteolytic fragment of BAI1, Vasculostatin (also referred to herein as Vstat120), is utilized as the anti-angiogenic transgene. A preferred embodiment comprises a polypeptide having the angiostatic activity of Vasculostatin, including, but not limited to the polypeptide of SEQ ID NO:2 that is encoded by the nucleic acid sequence of SEQ ID NO:1. Additionally, the transgene may optionally include nucleotides encoding a his and/or myc tag as in SEQ ID NO:3.

[0062] In various embodiments, the OV may contain a heterologous transgene encoding an enzyme that can depolymerize at least a portion of the extracellular matrix scaffold. Preferrably, the OV comprises a heterologous nucleic acid encoding a choindroitinase ABC 1 (Chase ABC), a bacterial enzyme (SEQ ID NOS: 6 or 8), or a degenerate variant. In yet another embodiment, the oncolytic virus may be armed with multiple heterologous nucleic acid sequences, for example, the oncoyltic virus may contain transgenes encoding both Vasculostatin and Chase ABC. The nucleic acid and amino acid sequence of Chase ABC enzyme provided herein is provided as SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Additionally, also provided are polypeptides that comprise the amino acid sequence of SEQ ID NO: 7, and fragments thereof. The fragment can be of any size greater than 15 amino acids in length. In some embodiments the fragment is at least 20, 30, 50, 75, 125, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 950, 975, 990 or more amino acids in length.

[0063] The Chase ABC enzymes provided also include modified Chase ABC enzymes. Such enzymes include those that can contain amino acid substitutions (e.g., at one or more of the important amino acid residues) of native Chase ABC as provided herein. The Chase ABC enzymes provided can have altered enzymatic activity and/or substrate specificity as compared to a native Chase ABC. In some embodiments, the Chase ABC enzymes have increased enzymatic activity. In others, the Chase ABC enzymes have diminished enzymatic activity. "Modified Chase ABC enzymes", are Chase ABC enzymes that are not as they would be found in nature and are somehow altered or modified. As used herein the "sequence of a modified Chase ABC" is intended to include the sequences of the modified enzymes provided with conservative substitutions therein and functional equivalents thereof, including, but not limited to fragments of the enzymes. The nucleic acid and amino acid sequence of an exemplary modified Chase ABC is provided herein is provided as SEQ ID NO: 8 and SEQ ID NO: 9, respectively. These modified sequences in some embodiment include a heterologous signal sequence, such as a secretion signal (SEQ ID NO: 8). In other embodiments, a signal sequence is not included. Modified Chase ABC enzymes can be produced using conservative substitutions, nonconservative substitutions, deletions, additions or a multiple mutant combination. In various exemplary embodiments, nucleic acids encoding modified Chase ABC enzymes may include mutations to prevent cryptic signals in the bacterial sequence from causing inappropriate modifications in animal cells. Such mutations may be made to prevent eukaryotic N-glycosylation systems from interfering with folding and secretion of active Chase ABC, a protein whose sequence has not naturally been adapted for glycosylation in structurally appropriate locations. (See Elizabeth M. Muir, et al, Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells, Journal of Biotechnology. 2009 (article in press)); see also, U.S. Pat. No. 7,553,950, incorporated by reference herein.

[0064] The promoter operably linked to heterologous transgenes in exemplary embodiments is preferably the immediate early promoter IE4/5 (SEQ ID NO:5). However, the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.

[0065] A preferred embodiment provides a method for treating a neoplastic disease in a subject, the subject being an animal or human, comprising administering to the subject a therapeutically effective amount of a recombinant tumor-specific conditional replication oncolytic activity, the vector comprising a DNA sequence encoding an anti-angiogenic agent, the DNA is operably linked to a promoter. Preferably, the anti-angiogenic agent is vasculostatin (which is a fragment of brain angiogenesis inhibitor 1 (BAI1)) or a biologically active variant thereof.

[0066] The term "operably linked" refers to the arrangement of various nucleic acid molecule elements relative to each other such that the elements are functionally connected and are able to interact with each other. Such elements may include, without limitation, a promoter, an enhancer, a polyadenylation sequence, one or more introns and/or exons, and a coding sequence of a gene of interest to be expressed (i.e., the transgene). The nucleic acid sequence elements, when operably linked, act together to modulate the activity of one another, and ultimately may affect the level of expression of the transgene. By modulate is meant increasing, decreasing, or maintaining the level of activity of a particular element. Typically, transduction of the transgene of the invention increases the expression of the transgene, preferably that of the angiostatic polypeptide Vasculostatin. The position of each element relative to other elements may be expressed in terms of the 5' terminus and the 3' terminus of each element.

[0067] The term "transfection" is used to refer to the uptake of foreign DNA by a mammalian cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are known in the art. See, Graham et al. (1973) Virology, 52:456; and Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Such techniques can be used to introduce one or more exogenous DNA moieties, such as a viral vector and other nucleic acid molecules, into suitable host cells. The term refers to both stable and transient uptake of the genetic material.

[0068] The vectors of the preferred embodiments may be useful for the introduction of additional genes in gene therapy. Thus, for example, the HSV vector of this invention can contain an additional exogenous gene for the expression of a protein effective in regulating the cell cycle, such as p53, Rb, or mitosin, or a biologically active variant thereof, or in inducing cell death, such as the conditional suicide gene thymidine kinase, the latter must be used in conjunction with a thymidine kinase metabolite in order to be effective, or any other anti-tumor gene, such as for example a toxin.

[0069] As used hereafter, the terms "neoplasm" and "neoplastic" refer to a tumor and/or to an abnormal tissue, including metastatic disease, that grows by cellular proliferation more rapidly than normal, continues to grow after the stimuli that initiated the new growth cease, shows partial or complete lack of structural organization and functional coordination with normal tissue, and usually forms a distinct mass of tissue which may be either benign or malignant.

[0070] A wide variety of neoplastic diseases can be treated by the same therapeutic strategy of exemplary embodiments. Neoplastic diseases include, but are not limited to, benign solid tumors, malignant solid tumors, benign proliferative diseases of the blood, and malignant proliferative diseases of the blood. Representative examples include colon carcinoma, prostate cancer, breast cancer, lung cancer, skin cancer, liver cancer, bone cancer, ovary cancer, pancreas cancer, brain cancer, head and neck cancer, and lymphoma.

[0071] As used throughout this application, the term animal is intended to be synonymous with mammal and is to include, but not be limited to, bovine, porcine, feline, simian, canine, equine, murine, rat or human. Host cells include, but are not limited to, any neoplastic or tumor cell, such as osteosarcoma, ovarian carcinoma, breast carcinoma, melanoma, hepatocarcinoma, lung cancer, brain cancer, colorectal cancer, hematopoietic cell, prostate cancer, cervical carcinoma, retinoblastoma, esophageal carcinoma, bladder cancer, neuroblastoma, or renal cancer.

[0072] When used pharmaceutically, OVs embodiments discussed herein can be combined with various pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers are well known to those of skill in the art. The compositions can then be administered therapeutically or prophylactically, in effective amounts, described in more detail below.

[0073] As used herein, the term "therapeutically effective amount" is intended to mean the amount of vector or of transformed cells, which exerts oncolytic activity, causing attenuation or inhibition of tumor cell proliferation leading to tumor regression. An effective amount will vary on the pathology or condition to be treated, by the patient and his status, and other factors well known to those of skill in the art. Effective amounts are easily determined by those of skill in the art.

[0074] The term "oncolytic activity" as used herein refers to cytotoxic effects in vitro and/or in vivo exerted on tumor cells without any appreciable or significant deleterious effects to normal cells under the same conditions. The cytotoxic effects under in vitro conditions are detected by various means as known in prior art, for example, by staining with a selective stain for dead cells, by inhibition of DNA synthesis, or by apoptosis. Detection of the cytotoxic effects under in vivo conditions is performed by methods known in the art.

[0075] Methods of treating a neoplastic disease may include administration of the compounds of exemplary embodiments as a single active agent, or in combination with additional methods of treatment including, but not limited to, irradiation therapy, therapy with immunosuppressive agents, chemotherapeutic or anti-proliferative agents, including cytokines. The methods of treatment of the invention may be in parallel to, prior to, or following additional methods of treatment.

[0076] Any of the vectors described herein are useful for the treatment of a neoplastic disease. When used pharmaceutically, the vectors of the invention can be combined with one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, vegetable oils (e.g., olive oil) or injectable organic esters. A pharmaceutically acceptable carrier can be used to administer the compositions of the invention to a cell in vitro or to a subject in vivo.

[0077] A pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the composition or to increase the absorption of the agent. A physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the polypeptide. For example, a physiologically acceptable compound such as aluminum monosterate or gelatin is particularly useful as a delaying agent, which prolongs the rate of absorption of a pharmaceutical composition administered to a subject. Further examples of carriers, stabilizers or adjutants can be found in Martin, Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton, 1975), incorporated herein by reference.

[0078] As used herein, "pharmaceutical composition" or "composition" refers to any of the compositions of matter described herein. The compositions can then be administered therapeutically or prophylactically. They can be contacted with the host cell in vivo, ex vivo, or in vitro, in a therapeutically effective amount. In vitro and in vivo means of transfecting the vectors of the invention are provided below.

[0079] According to the invention, any suitable route of administration of the vectors may be adapted, including but not limited to, intravenous, oral, buccal, intranasal, inhalation, topical application to a mucosal membrane or injection, including intratumoral, intradermal, intrathecal, intracisternal, intralesional or any other type of injection. Administration can be effected continuously or intermittently and will vary with the subject and the condition to be treated.

[0080] An exemplary embodiment includes an oncolytic HSV, such as created by methods described herein, for example random mutagenesis, and further comprises a nucleic acid encoding an angiostatic polypeptide, such as Vasculostatin.

[0081] Nucleic Acid-Based Expression Systems

[0082] An exemplary embodiment is directed to an HSV vector. In specific embodiments, the vector comprises some or all of the following components.

[0083] The term "vector" is used to refers to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0084] The term "expression vector" refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

[0085] Promoters and Enhancers

[0086] In exemplary embodiments, an HSV immediate early viral promoter is operably linked to the transgene in order to drive the expression of the heterologous transgene. More preferrably, the early viral promoter utilized is the HSV immediate early viral promoter IE4/5 (SEQ ID NO:5).

[0087] Various embodiments employ a tumor specific promoter element to achieve tumor specific replication of the mutant oncolytic virus. In various embodiments, the tumor specific promoter comprises a nestin enhancer element (6454 bp-7082 bp of SEQ ID NO: 12). In an exemplary embodiment, the nestin enhancer element may be operably linked to a suitable promoter, for example, a heat shock protein 68 (hsp68) promoter (5576 bp-6412 bp of SEQ ID NO: 12). Various embodiments comprise a mutant HSV-1 vector in which a nestin enhancer element (6454 bp-7082 bp of SEQ ID NO: 12) drives expression of a replacement γ₁₃₄.5 gene, in an otherwise γ₁₃₄.5-deleted viral genome. In this manner, exemplary embodiments achieve increased tumor selectivity, particularly with respect to glioma cells. This promoter/enhancer arrangement was previously described by Kambara, et al. (See reference 15, below, incorporated by reference in its entirety).

[0088] The term "promoter" refers to a nucleic acid sequence that regulates, either directly or indirectly, the transcription of a corresponding nucleic acid coding sequence to which it is operably linked. The promoter may function alone to regulate transcription, or, in some cases, may act in concert with one or more other regulatory sequences such as an enhancer or silencer to regulate transcription of the transgene. The promoter comprises a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene, which is capable of binding RNA polymerase and initiating transcription of a downstream (3'-direction) coding sequence.

[0089] A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence "under the control of" a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame "downstream" of (i.e., 3' of) the chosen promoter. The "upstream" promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.

[0090] The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0091] A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not "naturally occurring," i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the β-lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0092] The nucleic acid molecules of the invention are not limited strictly to molecules including the sequences set forth as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 8. Rather, specific embodiments encompasses nucleic acid molecules carrying modifications such as substitutions, small deletions, insertions, or inversions, which nevertheless encode proteins having substantially the biochemical activity of the polypeptide according to the specific embodiments, and/or which can serve as hybridization probes for identifying a nucleic acid with one of the disclosed sequences. Included in the invention are nucleic acid molecules, the nucleotide sequence of which is at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical) to the nucleotide sequence shown as SEQ ID NOS: 1, 3, 5, 6, 8, and 10-12 in the Sequence Listing.

[0093] The determination of percent identity or homology between two sequences is accomplished using the algorithm of Karlin and Altschul (1990) Proc. Nat'l Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

[0094] The term "stringent hybridization conditions" is known in the art from standard protocols (e.g., Current Protocols in Molecular Biology, editors F. Ausubel et al., John Wiley and Sons, Inc. 1994) and is to be understood as conditions as stringent as those defined by the following: hybridization to filter-bound DNA in 0.5 M NaHPO₄ (pH 7.2), 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65° C., and washing in 0.1×SSC/0.1% SDS at +68° C.

[0095] Also included in the invention is a nucleic acid molecule that has a nucleotide sequence which is a degenerate variant of a nucleic acid disclosed herein, e.g., SEQ ID NOS: 1, 3, 6, and 8. A sequential grouping of three nucleotides, a "codon," encodes one amino acid. Since there are 64 possible codons, but only 20 natural amino acids, most amino acids are encoded by more than one codon. This natural "degeneracy" or "redundancy" of the genetic code is well known in the art. It will thus be appreciated that the nucleic acid sequences shown in the Sequence Listing provide only an example within a large but definite group of nucleic acid sequences that will encode the polypeptides as described above.

[0096] The invention also includes an isolated polypeptide encoded by a nucleic acid of the invention. An "isolated" polypeptide is a polypeptide that is substantially free from the proteins and other naturally occurring organic molecules with which it is naturally associated. Purity can be measured by any art-known method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC. An isolated polypeptide may be obtained, for example, by extraction from a natural source (e.g., a human cell); by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical synthesis of the polypeptide. In the context of a polypeptide obtained by extraction from a natural source, "substantially free" means that the polypeptide constitutes at least 60% (e.g., at least 75%, 90%, or 99%) of the dry weight of the preparation. A protein that is chemically synthesized, or produced from a source different from the source from which the protein naturally originates, is by definition substantially free from its naturally associated components. Thus, an isolated polypeptide includes recombinant polypeptides synthesized, for example, in vivo, e.g., in the milk of transgenic animals, or in vitro, e.g., in a mammalian cell line, in E. coli or another single-celled microorganism, or in insect cells.

[0097] In various embodiments, the polypeptide of the invention include an amino acid sequence as set forth in SEQ ID NOS: 2, 4, 7 and 9. However, polypeptides of the exemplary embodiments are not to limited to those having an amino acid sequence identical to one of SEQ ID NOS: 2, 4, 7 and 9 in the Sequence Listing. Rather, the invention also encompasses conservative variants of the disclosed sequences. "Conservative variants" include substitutions within the following groups: glycine and alanine; valine, alanine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine, and threonine; lysine, arginine, and histidine; and phenylalanine and tyrosine.

[0098] Also included in the invention are polypeptides carrying modifications such as substitutions, small deletions, insertions, or inversions, which polypeptides nevertheless have substantially the biological activities of the Vasculostatin polypeptide. Consequently, included in the invention is a polypeptide, the amino acid sequence of which is at least 95% identical (e.g., at least 96%, 97%, 98%, or 99% identical) to an amino acid sequence set forth as SEQ ID NOS: 2, 4, 7 and 9 in the Sequence Listing. "Percent identity" is defined in accordance with the algorithm described above.

[0099] Also included in the invention are polypeptides of the invention that have been post-translationally modified, e.g., by cleavage of an N-terminal signal sequence, which can be, e.g., 1 to 25 amino acids long.

Examples

[0100] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors 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 concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0101] Oncolytic HSV

[0102] Recent developments in the use of gene therapy vectors have utilized viral and nonviral vectors to transduce cancer or stem cells [18-20]. Oncolytic viral treatment exploits tumor-specific conditional replication of viruses to lyse tumor cells [21-23]. Genetic modifications of viral proteins to infect tumor cells specifically has been exploited to enhance tumor-specific viral tropism [24]. Specific replication within tumor cells can be achieved by OVs genetically engineered for that purpose or by naturally occurring strains of some viruses that have such propensity [25]. In a preferred embodiment, an HSV-1 derived OV deleted for both copies of the γ34.5 gene and additionally disrupted for the ICP6/RR genes that expresses Vasculostatin under the control of an early viral promoter, IE4/5, was constructed.

Example 1

[0103] Vasculostatin is Anti-Tumorigenic:

[0104] We have recently discovered Vasculostatin, a fragment of brain angiogenesis inhibitor 1 (BAI1), which inhibits angiogenesis, tumor growth, and vascular permeability. We have found that BAI1 is differentially expressed in normal and neoplastic brain. This is consistent with its reduced expression in pulmonary adenocarcinoma and pancreatic and gastric cancers, we and others have found it to be absent from brain tumors, but present in normal brain and benign gliomas. The protein's brain specific expression along with its absence in a majority of human GBM specimens implies that loss of BAI1 during tumor progression may give the tumors a growth advantage.

[0105] Previous work has demonstrated that BAI1 is processed at a conserved GPS site through proteolysis, and this processing leads to the secretion of the extracellular domain of the protein. The cleaved extracellular domain yields a 120-kDa secreted fragment called Vasculostatin.

[0106] To investigate if Vasculostatin could inhibit the growth of intracranial tumors, we compared the survival of rats implanted with U87 human glioma cells stably expressing Vasculostatin (clones U14 and U18) with parental untransfected glioma cells. Referring to FIG. 1, 1×10⁶ cells, derived from parental U87MG and Vasculo-expressing clones U14 and U18 were implanted stereotactically in the brains of athymic nude rats as described. Animals were carefully monitored for signs of necropsy and were sacrified according to our IACUC guidelines. Survival of rats implanted with cells expressing Vasulostatin was significantly greater than that of rats implanted with control parental U87MG cells (P<0.05).

[0107] Therefore, Vasculostatin is a novel and potent inhibitor of angiogenesis tumor growth and vascular permeability. However, because of the many variables involved with oncolytic viral expression, it was unknown whether or not it would make an effective therapeutic factor in the context of an oncolytic virus. Furthermore, it was unknown whether or not the presence of vasculostatin would interfere with the cytoxic ability of the virus.

Example 2

[0108] Creation of rHSVQvasculo:

[0109] In order to produce an oncolytic virus expressing vasculostatin HSVQuick methodology was employed[1]. The HSVQuik methodology is a novel BAC-based method that utilizes two different site-specific recombination systems to introduce a transgene of interest into the deleted UL39 locus. The fHsvQuik-1 is the BAC DNA with the incorporation of the entire HSV-1 genome lacking a functional ICP6 gene and deleted in both copies of the γ34.5 gene incorporated in it. ICP34.5 allows the virus to replicate in non-dividing cells and dephosphorylates the cellular translation initiation factor (elF-2α) that is phosphorylated in response to activation of double-stranded RNA activated protein kinase (PKR). These modifications allow the virus to replicate selectively in cancer cells. Additionally, fHsvQuik-1 has an insertion of a red fluorescent protein (RFP) in the middle of the BAC (bacterial artificial chromosome) backbone and thereby allows for efficient monitoring of the presence of BAC sequences in the vector genome.

[0110] Using this methodology, one OV embodiment was created and named rHSVQvasculo (also named "RAMBO," Rapid Anti-angiogenesis Mediated By Oncolysis virus). In that OV, a Vasculostatin transgene is driven by the viral immediate early IE4/5 promoter.

[0111] Referring to FIG. 2, to construct rHSVQvasculo, we used standard molecular biology approaches to first clone the cDNA encoding for Vasculostatin under IE4/5 promoter into a shuttle plasmid (ptransferIE4/5 which is published in Yamamoto et al, Gene Therapy 13, 1731-1736 (2006), incorporated herein by reference) to generate the pVasculo-transfer plasmid (SEQ ID NO: 10). The generated pVasculo-transfer plasmid is a replication-conditional plasmid (cannot replicate at 43° C.) in which the Vasculostatin gene is flanked by one loxP site and an FRT site. The early viral promoter IE4/5 was selected to drive Vasculostatin expression because it has an early and robust expression profile in the context of an oncolytic herpes simplex virus [1]. The generated plasmid was verified by restriction digest analysis and confirmed by sequencing (not shown).

[0112] Referring to FIG. 3, the Vasculostatin cassette along with the entire shuttle plasmid is inserted by Flp-mediated recombination into the disrupted ICP6 locus of the mutant HSV (Deleted for both copies of the γ34.5 gene) genome in the fHSVQuik-1 BAC DNA. To accomplish this pVasculo-transfer (ampicillin [Amp] resistant), along with fHSVQuik-1 (Chloramphenicol [Cm] resistant) and an Flp-expressing plasmid (pFTP-T) is electroporated into bacteria carrying fHSVQuik-1 DNA and grown at 43° C. The pCMVvasculo-transfer and pFTP-T cannot replicate at this temperature, and 80% of the Cm- and Amp-resistant recombinants have the correct recombination to generate fHSVQ1-Vasculo. The harvested BACs are analyzed by PCR and restriction analysis for integration of pVasculo.

[0113] Again referring to FIG. 3, the selected recombinant fHSVQ1-Vasculo BAC is then transfected into Vero cells with a Cre-expressing helper plasmid. The Cre-mediated recombination results in the excision of the bacterial plasmid sequences flanked by LoxP sites. HSV recombinants generated by this process are easily identified because they express GFP, but not the RFP (excised by Cre-mediated recombination) in infected Vero cells. The isolated recombinants are purified through subsequent plaque purifications or serial dilutions, and confirmed by further southern blot analysis. The generated rHSVQvasculo from at least 2 isolates may be confirmed for correct insertion of IE4/5-Vasculostatin by Southern blot analysis. Briefly, viral DNA isolated from infected Vero cells was digested with XhoI, resolved by agarose gel electrophoresis, and transferred to nylon membranes. Probes specific for IE4/5-Vasculostatin were used to confirm its correct size and insertion [15]. The selected virus was confirmed for correct insertion and recombination events by sequencing both of the sites of recombination.

Example 3

[0114] Confirmation of Expression of Vasculostatin by the Recombinant Viral Isolates:

[0115] Referring to FIG. 4, three viral isolates from each recombinant BAC ((fHSVQ1vasulo1, and fHSVQ1 vasulo2) were selected for further analysis. The resulting six viral isolates were used to infect two different glioma cell lines (LN229, and U87AEGFR) to evaluate Vasculostatin and viral ICP4 expression (FIG. 1). The indicated glioma cells were infected with the six viruses, 3 isolated from fHSVQ1vasculo1 (lanes 1-3) and with 3 isolated from fHSVQ1vasulo2 (lanes 4-6).

[0116] The infected cells and conditioned media were harvested 10 hours post infection and the cell lysate and TCA precipitated conditioned media was analyzed for Vasculostatin protein expression by western blot analysis. Note the presence of Vasculostatin in infected cell lysate and harvested conditioned media from infected cells but not in the control rHSVQ infected cells (bottom panel).

Example 4

[0117] Initial Characterization of Two Selected Viral Isolates:

[0118] Referring to FIG. 5, from this initial screen we selected two viruses rHSVQvasculo 1 (lane 3), and rHSVQvasculo 2 (lane 5). We have purified both of these viruses. Since Vasculostatin expression in this recombinant virus is under the control of ICP4 promoter we checked the temporal pattern of expression of Vasculostatin and ICP4 in LN229 cells transfected with these viruses (FIG. 5). Briefly, LN229 glioma cells were transfected with the indicated viral isolate at an MOI of 0.1. Cells were harvested at the indicated times after infection and analyzed for expression of Vasculostatin and ICP4 by western blot analysis. Note the expression of both Vasculostatin and ICP4 come up as early as 4 hours after infection.

[0119] These results confirm that a recombinant oncolytic HSV which also expresses vasculostatin was generated.

Example 5

[0120] Vasculostatin does not Affect the Cytotoxicity of the Recombinant Oncolytic Virus:

[0121] Referring to FIG. 6, to compare the effect of Vasculostatin expression on oncolytic virus replication we compared the cytotoxixcity of the control rHSVQ virus with the 2 selected viral isolates rHSVQvasculo1, and rHSVQvasculo2. Six thousand U87AEGFR glioma cells were infected with the indicated virus at MOI of 1, 0.5, 0.1, 0.01, and 0.05, on day zero. The number of viable cells was measured by a standard crystal violet assay on day 1, day, 2, day, 3 and day 5. Briefly, the cells at the indicated time point were fixed with 1% glutaraldehyde for 15 minutes and then stained with 5% crystal violet (dissolved in 4.75% ethanol) for 15 minutes. The plates were washed to remove unbound stain and the crystal violet crystals were dissolved in Sorensen's buffer prior to reading absorbance read at 590 nm. Note that there is no significant difference in cytotoxicity between the control rHSVQ virus and the isolated rHSVQvasculo1, and rHSVQvasculo2 expressing Vasculostatin.

Example 6

[0122] DIVAA Assay Confirms the In Vivo Anti-Angiogenic Capability of an Embodiment:

[0123] Referring to FIG. 7, the anti-angiogenic capabilities of rHSVQvasculo was tested using the Trevigen Direct In Vivo Angiogenesis Assay (DIVAA®) Inhibition Kit (Cat #: 3450-048-IK). Briefly, 2.5×10⁵ U87ΔEGFR cells treated with PBS or infected with rHSVQ, rHSVQvasculo was mixed with basement membrane. The samples were then pipette into angioreactors and allowed to polymerize for 1 hour at 37° C. The tubes were then implanted into the rear flanks of nu/nu mice and 12 days later the mice were sacrifice and angioreactors removed. The amount of angiogenesis that occurred in the angioreactors was quantified using the Wako Hemoglobin B kit. Angiogenesis is initiated into the tubes from the one open end in the tubes. Note both visually and graphically the significant reduction in angiogenesis for samples infected with RAMBO compared to rHSVQ (n=10/group, and p=0.07) and PBS control (FIG. 6).

Example 7

[0124] The Oncolytic Virus rHSVQvasculo is Therapeutically Effective:

[0125] We have tested the therapeutic potential of rHSVQvasculo in human glioma cells grown in a mouse brain tumor model. U87ΔEGFR human glioma cells were implanted intracranially in mice. Later, Mice were treated with direct intratumoral injection of rHSVQ or rHSVQvasculo. Animals were carefully monitored for any signs of morbidity and were sacrificed in accordance with our IACUC guidelines.

[0126] FIG. 8 shows a Kaplan-Meier survival analysis of mice treated with rHSVQ control virus or rHSVQvasculo (the virus generated to express Vasculostatin). Briefly, Mice with intracranial tumors (U87ΔEGFR) were treated with a single dosage (1×10⁵ pfu) of the control rHSVQ or the rHSVQvasculo at day 5 after tumor implantation. As can be observed from the figure, all of the rHSVQ mice died of tumor burden by day 55. However, there were 20% survivors in rHSVQvasculo treated animals. Survival of mice treated with rHSVQvasculo was significantly greater than that of mice treated with the rHSVQ virus (P=0.0246). Hence, rHSVQvasculo has potent anti-tumor efficacy compared to the parent control oncolytic virus.

Example 8

[0127] Comparison of rHSVQvasculo and rHSVQ Mediated Cytotoxicity to Normal Human Astrocytes (NHA):

[0128] Referring to FIG. 9, to evaluate the cytotoxicity of rHSVQvasculo produced by the recombinant OV, we infected normal human astrocytes (NHA, CellSciences Canton, Mass.) at different multiplicities of infection (MOI) with rHSVQ and rHSVQvasculo to evaluate potential cytotoxicity of OV produced Vasculostatin towards NHA. Briefly NHA: cells were plated into 96 well plates (10,000 cells/well). The cells were infected with the indicated virus at MOI of 1, 0.5, 0.1, 0.01, and 0.05. Forty-eight hours post infection the number of viable cells measured by a standard Colorimetric crystal violet assay. Note no significant difference in the cytotoxicity to NHA at any of the indicated multiplicity of infection between rHSVQVasculo and rHSVQ. This indicated that rHSVQvasculo was as cytotoxic to NHA cells as rHSVQ.

Example 9

[0129] Viral Replication of rHSVQvasculo is Similar to rHSVQ Control Virus:

[0130] Referring to Table 1 below, glioma cell lines: LN229, and U87ΔEGFR were infected with rHSVQ and rHSVQvasculo at an MOI 0.05. Seventy-two hours post infection the cells and supernatants were harvested and the number of infectious viral particles (pfu) in each cell line was assessed by a standard viral titration assay. Table 1 below shows the results of viral titration in each indicated cell line. Note: The results indicate no significant difference in the replication ability of rHSVQvasculo compared to rHSVQ.

TABLE-US-00001 TABLE 1 LN229 U87ΔEGFR rHSVQ rHSVQvasculo rHSVQ rHSVQvasculo 7500 pfu/mL 8125 pfu/mL 56250 pfu/mL 31250 pfu/mL

Example 10

[0131] Cellular Cytotoxicity Results in Multiple Glioma Cell Lines Demonstrating the Ability of the rHSVQvasculo to be Cytotoxic to Multiple Glioma Cells In Vitro:

[0132] Referring to FIGS. 10A-10L, the effect of Vasculostatin expression on ability of OV to be cytotoxic to glioma cells was compared in the glioma cells LN229, U87ΔEGFR, and U343, using a standard colorimetric assay. All cell lines were infected with control rHSVQ virus or rHSVQvasculo at indicated MOIs (1, 0.5, 0.1, 0.01, or 0.05). The number of viable cells was measured by a standard Colorimetric crystal violet assay on day 1, day, 2, day, 3 and day 5. Note that there are no significant differences in the cytotoxicity for the two viruses.

Example 11

[0133] Cyclophosphamide (CPA) Pretreatment Further Enhances the Anti-Tumor Ability of Vasculostatin:

[0134] Referring to FIG. 11, Athymic nude mice were injected with 2.5×10⁶ U87ΔEGFR cells. Seventeen days later, when the tumors were of sufficient size (>750 mm³), the mice were treated with PBS or CPA (200 mg/kg) by intraperitoneal injection. Two days after CPA/PBS treatment the animals were anesthetized and tumors were injected with 1×10⁶ pfu rHSVQvasculo, or control rHSVQ. Animals were sacrificed 48 hrs after OV treatment and the tumors were explanted sectioned into small pieces, and snap frozen. The tumors were lysed and equal amounts of lysate was then assayed for the presence of Vasculostatin by western blot analysis. Western blot analysis for expression of Vasculostatin in subcutaneous tumors (U87ΔEGFR glioma) injected with rHSVQvasculo or control rHSVQ OV. Positive control is cell lysate from LN229 cells infected with rHSVQvasculo, (MOI 0.05) for 48 hours. Note the presence of vasculostatin in the rHSVQvasculo treated tumors indicating the ability of rHSVQvasculo to express Vasculostatin in vivo. Note also the increase in Vasculostatin expression in the CPA treated animals.

Example 12

[0135] Creation of "Nested" RAMBO Vector

[0136] The HSVQuik methodology was employed to engineer a Nested-RAMBO Oncolytic virus. See Edyta Tyminski, et al; Brain Tumor Oncolysis with Replication-Conditional Herpes Simplex Virus Type 1 Expressing the Prodrug-Activating Genes, CYP2B1 and Secreted Human Intestinal Carboxylesterase, in Combination with Cyclophosphamide and Irinotecan; Cancer Res 2005 Aug. 1; 65(15):6850-6857, (this reference incorporated by reference in its entirety). HSVQuik methodology is a BAC-based method which utilizes two different site specific recombination systems to introduce a transgene of interest into the deleted UL39 locus. The fHsvQuik-1 is lacking a functional ICP6 gene and is deleted in both the copies of the γ34.5 gene. Additionally fHsvQuik-1 has an insertion of RFP in frame and downstream of a truncated ICP6 coding sequence resulting in the loss of ICP6 (large subunit of viral ribonucleotide reductase) viral protein and an RFP (red fluorescent protein) in the middle of the BAC backbone to monitor the presence of BAC sequences in the vector genome.

[0137] The pTnestin34.5 plasmid (SEQ ID NO: 11) containing the nestin enhancer driven ICP34.5 (RL1 gene) in it was used as a starting material. Initially, a fragment (BstBI and XbaI) corresponding to Vasculostatin under the control of HSV-1 IE4/5 derived from pVasculo transfer plasmid (2276 bp-6641 bp of SEQ ID NO: 10) was inserted into the BstB1 and XbaI site of pT nestin34.5 plasmid (SEQ ID NO: 11). This step allowed for most of the Vasculostatin gene and HSV-1 IE4/5 promotor sequence to be inserted into the pTnestin 34.5 plasmid. Subsequently, a PCR fragment comprising of C-terminal region of Vasculostatin tagged with myc-His and a polyA site was inserted into XbaI site, resulting in complete Vasculostatin cDNA formed in the plasmid PNested-RAMBO-BGH. The primers were designed to recreate the disrupted FRT site. The Vasculostain and ICP34.5 expressing cassette along with the entire shuttle plasmid is inserted by Flp mediated recombination, into the fHSVQuik-1 BAC plasmid into the disrupted ICP6 locus of the mutant HSV (deleted for both copies of γ34.5 gene) genome in the BAC. pNested-RAMBO-BGH (ampicillin (Amp) resistant), along with fHSVQuik-1 (Chloramphenicol (Cm) resistant) and a Flp-expressing plasmid (pFTP-T) is electroporated into bacteria carrying fHSVQuik-1 DNA and grown at 43° C. The pNested-RAMBO-BGH, and pFTP-T can not replicate at this temperature and 80% of the Cm and Amp resistant recombinants have the correct recombination to generate fHSVQ1-vasculo. The harvested BACs were analyzed by PCR, and restriction analysis for integration of PNested-RAMBO-BGH.

[0138] The selected recombinant fHSVQ1-Nested-RAMBO was transfected into Vero cells with a Cre expressing helper plasmid. The Cre mediated recombination results in the excision of the bacterial plasmid sequences flanked by LoxP sites. HSV Recombinants generated by this process are easily identified as they express EGFP but not the RFP (excised by Cre-mediated recombination) in infected Vero cells. The isolated recombinants are purified through subsequent plaque purifications or serial dilutions.

[0139] The final insertion in the virus corresponds to the sequences 9278-7344 of the plasmid p-Nested RAMBO BGH (9278-7344 of SEQ ID No. 12). A map of the Nested RAMBO virus is provided in FIG. 12.

Example 13

[0140] Expression of Vstat120 and Suppression of elF2α Phosphorylation

[0141] Having engineered Nested-RAMBO, an OV expressing Vstat120 in the backbone of rQnestin34.5 (FIG. 12), the correct insertion of Vstat120 was confirmed by restriction digest and PCR (not shown). The ability of this virus to express Vstat120 and to suppress elF2α phosphorylation was tested in U251 glioma cells following infection (FIG. 13). U251 glioma cells express high levels of nestin and so permit efficient expression of ICP34.5 from its nestin promotor from rQnestin34.5 and Nested-RAMBO. Note the production of Vstat120 in cells infected with RAMBO (lane 4) and two different isolates of Nested-RAMBO (lane 5 and 6). Note also increased phosphorylation of elF2α in HSVQ and RAMBO infected cells (Lanes 2 and 4) but not in rQnestin34.5 and Nested-RAMBO cells (Lanes 3, 5, and 6). This indicates that Nested RAMBO has both ICP34.5 and Vstat120 expression in glioma cells expressing high levels of nestin. The expression of Vstat120 (ICP4 driven) in Qnestin negative cells was also confirmed (data not shown).

Example 14

[0142] Specificity of Nested-RAMBO

[0143] With reference to FIG. 14, to test the specificity of nestin enhancer element driven ICP34.5 in Nested-RAMBO, the cytolytic ability of Nested-RAMBO, to rQnestin34.5 in glioma cells with high and low nestin expression was compared. U251 and U87ΔEGFR cells express high levels of nestin, and T98G cells express low levels of nestin (8). Percent of cell survival relative to HSVQ cells was measured in these cells by a standard crystal violet assay.

[0144] The indicated cells were infected with the indicated virus (MOI=0.1) and the relative cell killing was measured. Briefly, the indicated glioma cells were seeded on 96-well plates. The following day, cells were infected with HSVQ, rQnestin 34.5, Rambo, or Nested-Rambo. After 2 days of incubation, cells were stained with crystal violet and the percentage of viable cells relative to HSVQ infected cells was measured at A₅₆₀ nm. The level of endogenous nestin expression in each cell is indicated in brackets. FIG. 14 shows the % cell survival relative to HSVQ infected cells. Both rQnestin34.5 and Nested-RAMBO showed increased levels of cell killing relative to HSVQ infected cells in both U251 and U87ΔEGFR cells (74%-86%). In contrast, in T98G cells which express much lower levels of nestin (8) the difference between HSVQ and rQnestin34.5/Nested-RAMBO mediated cell killing was reduced to about 36-38%. This indicated that transcriptional control of ICP34.5 by the nestin enhancer element in Nested-RAMBO was similar to that in rQnestin34.5 in vitro (FIG. 14). There was no significant difference between HSVQ and RAMBO mediated glioma cell killing in vitro in any of the cell lines.

Example 15

[0145] Dramatically Improved Antitumor Efficacy of Nested RAMBO, Compared to rQnestin34.5 and RAMBO Against Subcutaneous Glioma In Vivo.

[0146] Referring to FIG. 15, U251T3 tumor bearing mice were treated with the indicated virus or PBS (not shown) once the tumors reached an average size of 200 mm³. The mice were treated by direct intratumoral injection of 1×10⁵ pfu of virus on days 0, 2, 4, 6, 8, and 10. Tumor volume was measured over a period of time. The tumor volume of individual mice in each group on day 21 after initiation of treatment is shown. PBS treated mice had to be all sacrificed by day 21 due to tumor burden in accordance with our IACUC protocol (not shown). Not the significant reduction in tumor volume in mice treated with Nested-RAMBO compared to rQnestin34.5 and RAMBO treated mice.

Example 16

[0147] Creation of OV Expressing Chase ABC

[0148] The HSVQuik methodology was also used to engineer OV-Chase oncolytic virus (Tyminski, et al. 2005).

[0149] Step 1: Generation of Chase transfer plasmid: The cDNA encoding for Chase ABC was PCR amplified from DNA prepared from P. Vulgaris cells (ATCC). The resulting DNA was cloned into psecTAG/FRT/V5 plasmid (Invitrogen), which incorporated cDNA encoding for a secretion signal at the 5' terminus of Chase ABC cDNA. This was then subcloned into a shuttle plasmid under the control of viral IE4/5 promoter to generate pChase-transfer. The generated pChase-transfer plasmid is a replication-conditional plasmid (cannot replicate at 43° C.) in which the Chase ABC gene (from P vulgaris) under the control of IE4/5 promotor is flanked by one loxP site and an FRT site. The generated plasmid was verified by restriction digest analysis and confirmed by sequencing (not shown).

[0150] Step 2: Generation of the recombinant BAC fChase: Next, the Chase ABC expression cassette along with the entire shuttle plasmid is inserted by Flp-mediated recombination into the disrupted ICP6 locus of the mutant HSVQ (Deleted for both copies of the γ34.5 gene) genome in the fHSVQuik-1 BAC DNA. To accomplish this pChase-transfer (ampicillin [Amp] resistant), along with fHSVQuik-1 (Chloramphenicol [Cm] resistant) and an Flp-expressing plasmid (pFTP-T) is electroporated into bacteria carrying fHSVQuik-1 DNA and grown at 43° C. The Chasetransfer plasmid and pFTP-T cannot replicate at this temperature, and 80% of the Cm- and Amp-resistant recombinants have the correct recombination to generate Bac fChase. The harvested BACs are analyzed by PCR and restriction analysis for integration of Chase-transfer plasmid. The resulting Bac DNA was confirmed by restriction digest and PCR to confirm correct insertion of the transgene plasmid.

[0151] Step 3: Generation of OV-Chase: The selected recombinant Bac fChase is then transfected into Vero cells with a Cre-expressing helper plasmid. The Cre-mediated recombination results in the excision of the bacterial plasmid sequences flanked by LoxP sites. HSV recombinants generated by this process are easily identified because they express GFP, but not the RFP (excised by Cre-mediated recombination) in infected Vero cells. The isolated recombinants are purified through subsequent plaque purifications or serial dilutions, and confirmed by further southern blot analysis. The generated ChaseQ from at least 2 isolates has been confirmed for correct insertion of Chase ABC by PCR analysis.

Example 17

[0152] Confirmation of Active Chase ABC Provided by OV-Chase

[0153] To confirm functionality of the secreted Chase ABC produced by OV-Chase we tested for the appearance of immunoreactive stubs in cells treated with OV-Chase compared to cells infected with HSVQ. FIG. 16 shows representative fluorescent images of glioma spheres infected with HSVQ (control OV) or Chase HSVQ. Human glioma spheres, were infected with HSVQ or OV-Chase and were stained for the appearance of immunoreactive sugar stubs (red staining, white arrows) obtained by Chase mediated digestion of CSPG. Infected cells are made obvious by the presence of virally encoded GFP. Note the presence of red staining sugar stubs (arrows) in Chase-HSVQ infected glioma cells around infected green cells, indicating functionality of ChaseABC produced OV-Chase.

Example 18

[0154] Enhanced OV spread in ex vivo cultures of glioma infected with OV-chase compared to glioma spheres infected with HSVQ.

[0155] Referring to FIG. 17, U87ΔEGFR human glioma spheroids (hanging drop method) were placed on organotypic brain slice cultures (from 5-7-day-old mice). The spheroids were infected with 1×10⁴ pfu of HSVQ or OV-Chase (as described above). We have previously worked and published with this ex vivo model Spread of OV in the sphere was visualized by fluorescent imaging of OV encoded GFP in infected cells over a period of time (24 hrs: top row, 48 hrs: middle row, 60 hrs: bottom row). Infection was apparent only in the rim of the spheres infected with HSVQ, compared to the increased spread to the core of spheres infected with OV-Chase (FIG. 17).

[0156] Publications

[0157] The following references and others cited herein but not listed here, to the extent that they provide exemplary procedural and other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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[0244] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the exemplary embodiments, suitable methods and materials are described above. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other Embodiments

[0245] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

1212796DNAHomo sapiensCDS(1)..(2796) 1atg agg ggc cag gcc gcc gcc ccg ggc ccc gtc tgg atc ctc gcc ccg 48Met Arg Gly Gln Ala Ala Ala Pro Gly Pro Val Trp Ile Leu Ala Pro 1 5 10 15 ctg cta ctg ctg ctg ctg ctg ctg gga cgc cgc gcg cgg gcg gcc gcc 96Leu Leu Leu Leu Leu Leu Leu Leu Gly Arg Arg Ala Arg Ala Ala Ala 20 25 30 gga gca gac gcg ggg ccc ggg ccc gag ccg tgc gcc acg ctg gtg cag 144Gly Ala Asp Ala Gly Pro Gly Pro Glu Pro Cys Ala Thr Leu Val Gln 35 40 45 gga aag ttc ttc ggc tac ttc tcc gcg gcc gcc gtg ttc ccg gcc aac 192Gly Lys Phe Phe Gly Tyr Phe Ser Ala Ala Ala Val Phe Pro Ala Asn 50 55 60 gcc tcg cgc tgc tcc tgg acg cta cgc aac ccg gac ccg cgg cgc tac 240Ala Ser Arg Cys Ser Trp Thr Leu Arg Asn Pro Asp Pro Arg Arg Tyr 65 70 75 80 act ctc tac atg aag gtg gcc aag gcg ccc gtg ccc tgc agc ggc ccc 288Thr Leu Tyr Met Lys Val Ala Lys Ala Pro Val Pro Cys Ser Gly Pro 85 90 95 ggc cgc gtg cgc acc tac cag ttc gac tcc ttc ctc gag tcc acg cgc 336Gly Arg Val Arg Thr Tyr Gln Phe Asp Ser Phe Leu Glu Ser Thr Arg 100 105 110 acc tac ctg ggc gtg gag agc ttc gac gag gtg ctg cgg ctc tgc gac 384Thr Tyr Leu Gly Val Glu Ser Phe Asp Glu Val Leu Arg Leu Cys Asp 115 120 125 ccc tcc gca ccc ctg gcc ttc ctg cag gcc agc aag cag ttc ctg cag 432Pro Ser Ala Pro Leu Ala Phe Leu Gln Ala Ser Lys Gln Phe Leu Gln 130 135 140 atg cgg cgc cag cag ccg ccc cag cac gac ggg ctc cgg ccc cgg gcc 480Met Arg Arg Gln Gln Pro Pro Gln His Asp Gly Leu Arg Pro Arg Ala 145 150 155 160 ggg ccg ccg ggc ccc acc gac gac ttc tcc gtg gag tac ctg gtg gtg 528Gly Pro Pro Gly Pro Thr Asp Asp Phe Ser Val Glu Tyr Leu Val Val 165 170 175 ggg aac cgc aac ccc agc cgt gcc gcc tgc cag atg ctg tgc cgc tgg 576Gly Asn Arg Asn Pro Ser Arg Ala Ala Cys Gln Met Leu Cys Arg Trp 180 185 190 ctg gac gcg tgt ctg gcc ggt agt cgc agc tcg cac ccc tgc ggg atc 624Leu Asp Ala Cys Leu Ala Gly Ser Arg Ser Ser His Pro Cys Gly Ile 195 200 205 atg cag acc ccc tgc gcc tgc ctg ggc ggc gag gcg ggc ggc cct gcc 672Met Gln Thr Pro Cys Ala Cys Leu Gly Gly Glu Ala Gly Gly Pro Ala 210 215 220 gcg gga ccc ctg gcc ccc cgc ggg gat gtc tgc ttg aga gat gcg gtg 720Ala Gly Pro Leu Ala Pro Arg Gly Asp Val Cys Leu Arg Asp Ala Val 225 230 235 240 gct ggt ggc cct gaa aac tgc ctc acc agc ctg acc cag gac cgg ggc 768Ala Gly Gly Pro Glu Asn Cys Leu Thr Ser Leu Thr Gln Asp Arg Gly 245 250 255 ggg cac ggc gcc aca ggc ggc tgg aag ctg tgg tcc ctg tgg ggc gaa 816Gly His Gly Ala Thr Gly Gly Trp Lys Leu Trp Ser Leu Trp Gly Glu 260 265 270 tgc acg cgg gac tgc ggg gga ggc ctc cag acg cgg acg cgc acc tgc 864Cys Thr Arg Asp Cys Gly Gly Gly Leu Gln Thr Arg Thr Arg Thr Cys 275 280 285 ctg ccc gcg ccg ggc gtg gag ggc ggc ggc tgc gag ggg gtg ctg gag 912Leu Pro Ala Pro Gly Val Glu Gly Gly Gly Cys Glu Gly Val Leu Glu 290 295 300 gag ggt cgc cag tgc aac cgc gag gcc tgc ggc ccc gct ggg cgc acc 960Glu Gly Arg Gln Cys Asn Arg Glu Ala Cys Gly Pro Ala Gly Arg Thr 305 310 315 320 agc tcc cgg agc cag tcc ctg cgg tcc aca gat gcc cgg cgg cgc gag 1008Ser Ser Arg Ser Gln Ser Leu Arg Ser Thr Asp Ala Arg Arg Arg Glu 325 330 335 gag ctg ggg gac gag ctg cag cag ttt ggg ttc cca gcc ccc cag acc 1056Glu Leu Gly Asp Glu Leu Gln Gln Phe Gly Phe Pro Ala Pro Gln Thr 340 345 350 ggt gac cca gca gcc gag gag tgg tcc ccg tgg agc gtg tgc tcc agc 1104Gly Asp Pro Ala Ala Glu Glu Trp Ser Pro Trp Ser Val Cys Ser Ser 355 360 365 acc tgc ggc gag ggc tgg cag acc cgc acg cgc ttc tgc gtg tcc tcc 1152Thr Cys Gly Glu Gly Trp Gln Thr Arg Thr Arg Phe Cys Val Ser Ser 370 375 380 tcc tac agc acg cag tgc agc gga ccc ctg cgc gag cag cgg ctg tgc 1200Ser Tyr Ser Thr Gln Cys Ser Gly Pro Leu Arg Glu Gln Arg Leu Cys 385 390 395 400 aac aac tct gcc gtg tgc cca gtg cat ggt gcc tgg gat gag tgg tcg 1248Asn Asn Ser Ala Val Cys Pro Val His Gly Ala Trp Asp Glu Trp Ser 405 410 415 ccc tgg agc ctc tgc tcc agc acc tgt ggc cgt ggc ttt cgg gat cgc 1296Pro Trp Ser Leu Cys Ser Ser Thr Cys Gly Arg Gly Phe Arg Asp Arg 420 425 430 acg cgc acc tgc agg ccc ccc cag ttt ggg ggc aac ccc tgt gag ggc 1344Thr Arg Thr Cys Arg Pro Pro Gln Phe Gly Gly Asn Pro Cys Glu Gly 435 440 445 cct gag aag caa acc aag ttc tgc aac att gcc ctg tgc cct ggc cgg 1392Pro Glu Lys Gln Thr Lys Phe Cys Asn Ile Ala Leu Cys Pro Gly Arg 450 455 460 gca gtg gat gga aac tgg aat gag tgg tcg agc tgg agc gcc tgc tcc 1440Ala Val Asp Gly Asn Trp Asn Glu Trp Ser Ser Trp Ser Ala Cys Ser 465 470 475 480 gcc agc tgc tcc cag ggc cga cag cag cgc acg cgt gaa tgc aac ggg 1488Ala Ser Cys Ser Gln Gly Arg Gln Gln Arg Thr Arg Glu Cys Asn Gly 485 490 495 cct tcc tac ggg ggt gcg gag tgc cag ggc cac tgg gtg gag acc cga 1536Pro Ser Tyr Gly Gly Ala Glu Cys Gln Gly His Trp Val Glu Thr Arg 500 505 510 gac tgc ttc ctg cag cag tgc cca gtg gat ggc aag tgg cag gcc tgg 1584Asp Cys Phe Leu Gln Gln Cys Pro Val Asp Gly Lys Trp Gln Ala Trp 515 520 525 gcg tca tgg ggc agt tgc agc gtc acg tgt ggg gct ggc agc cag cga 1632Ala Ser Trp Gly Ser Cys Ser Val Thr Cys Gly Ala Gly Ser Gln Arg 530 535 540 cgg gag cgt gtc tgc tct ggg ccc ttc ttc ggg gga gca gcc tgc cag 1680Arg Glu Arg Val Cys Ser Gly Pro Phe Phe Gly Gly Ala Ala Cys Gln 545 550 555 560 ggc ccc cag gat gag tac cgg cag tgc ggc acc cag cgg tgt ccc gag 1728Gly Pro Gln Asp Glu Tyr Arg Gln Cys Gly Thr Gln Arg Cys Pro Glu 565 570 575 ccc cat gag atc tgt gat gag gac aac ttt ggt gct gtg atc tgg aag 1776Pro His Glu Ile Cys Asp Glu Asp Asn Phe Gly Ala Val Ile Trp Lys 580 585 590 gag acc cca gcg gga gag gtg gct gct gtc cgg tgt ccc cgc aac gcc 1824Glu Thr Pro Ala Gly Glu Val Ala Ala Val Arg Cys Pro Arg Asn Ala 595 600 605 aca gga ctc atc ctg cga cgg tgt gag ctg gac gag gaa ggc atc gcc 1872Thr Gly Leu Ile Leu Arg Arg Cys Glu Leu Asp Glu Glu Gly Ile Ala 610 615 620 tac tgg gag ccc ccc acc tac atc cgc tgt gtt tcc att gac tac aga 1920Tyr Trp Glu Pro Pro Thr Tyr Ile Arg Cys Val Ser Ile Asp Tyr Arg 625 630 635 640 aac atc cag atg atg acc cgg gag cac ctg gcc aag gct cag cga ggg 1968Asn Ile Gln Met Met Thr Arg Glu His Leu Ala Lys Ala Gln Arg Gly 645 650 655 ctg cct ggg gag ggg gtc tcg gag gtc atc cag aca ctg gtg gag atc 2016Leu Pro Gly Glu Gly Val Ser Glu Val Ile Gln Thr Leu Val Glu Ile 660 665 670 tct cag gac ggg acc agc tac agt ggg gac ctg ctg tcc acc atc gat 2064Ser Gln Asp Gly Thr Ser Tyr Ser Gly Asp Leu Leu Ser Thr Ile Asp 675 680 685 gtc ctg agg aac atg aca gag att ttc cgg aga gcg tac tac agc ccc 2112Val Leu Arg Asn Met Thr Glu Ile Phe Arg Arg Ala Tyr Tyr Ser Pro 690 695 700 acc cct ggg gac gta cag aac ttt gtc cag atc ctt agc aac ctg ttg 2160Thr Pro Gly Asp Val Gln Asn Phe Val Gln Ile Leu Ser Asn Leu Leu 705 710 715 720 gca gag gag aat cgg gac aag tgg gag gag gcc cag ctg gcg ggc ccc 2208Ala Glu Glu Asn Arg Asp Lys Trp Glu Glu Ala Gln Leu Ala Gly Pro 725 730 735 aac gcc aag gag ctg ttc cgg ctg gtg gag gac ttt gtg gac gtc atc 2256Asn Ala Lys Glu Leu Phe Arg Leu Val Glu Asp Phe Val Asp Val Ile 740 745 750 ggc ttc cgc atg aag gac ctg agg gat gca tac cag gtg aca gac aac 2304Gly Phe Arg Met Lys Asp Leu Arg Asp Ala Tyr Gln Val Thr Asp Asn 755 760 765 ctg gtt ctc agc atc cat aag ctc cca gcc agc gga gcc act gac atc 2352Leu Val Leu Ser Ile His Lys Leu Pro Ala Ser Gly Ala Thr Asp Ile 770 775 780 agc ttc ccc atg aag ggc tgg cgg gcc acg ggt gac tgg gcc aag gtg 2400Ser Phe Pro Met Lys Gly Trp Arg Ala Thr Gly Asp Trp Ala Lys Val 785 790 795 800 cca gag gac agg gtc act gtg tcc aag agt gtc ttc tcc acg ggg ctg 2448Pro Glu Asp Arg Val Thr Val Ser Lys Ser Val Phe Ser Thr Gly Leu 805 810 815 aca gag gcc gat gaa gca tcc gtg ttt gtg gtg ggc acc gtg ctc tac 2496Thr Glu Ala Asp Glu Ala Ser Val Phe Val Val Gly Thr Val Leu Tyr 820 825 830 agg aac ctg ggc agc ttc ctg gcc ctg cag agg aac acg acc gtc ctg 2544Arg Asn Leu Gly Ser Phe Leu Ala Leu Gln Arg Asn Thr Thr Val Leu 835 840 845 aat tct aag gtg atc tcc gtg act gtg aaa ccc ccg cct cgc tcc ctg 2592Asn Ser Lys Val Ile Ser Val Thr Val Lys Pro Pro Pro Arg Ser Leu 850 855 860 cgc aca ccc ttg gag atc gag ttt gcc cac atg tat aat ggc acc acc 2640Arg Thr Pro Leu Glu Ile Glu Phe Ala His Met Tyr Asn Gly Thr Thr 865 870 875 880 aac cag acc tgt atc ctg tgg gat gag acg gat gta ccc tcc tcc tcc 2688Asn Gln Thr Cys Ile Leu Trp Asp Glu Thr Asp Val Pro Ser Ser Ser 885 890 895 gcc ccc ccg cag ctc ggg ccc tgg tcg tgg cgc ggc tgc cgc acg gtg 2736Ala Pro Pro Gln Leu Gly Pro Trp Ser Trp Arg Gly Cys Arg Thr Val 900 905 910 ccc ctc gac gcc ctc cgg acg cgc tgc ctc tgt gac cgg ctc tcc acc 2784Pro Leu Asp Ala Leu Arg Thr Arg Cys Leu Cys Asp Arg Leu Ser Thr 915 920 925 ttc gat atc tta 2796Phe Asp Ile Leu 930 2932PRTHomo sapiens 2Met Arg Gly Gln Ala Ala Ala Pro Gly Pro Val Trp Ile Leu Ala Pro 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Leu Gly Arg Arg Ala Arg Ala Ala Ala 20 25 30 Gly Ala Asp Ala Gly Pro Gly Pro Glu Pro Cys Ala Thr Leu Val Gln 35 40 45 Gly Lys Phe Phe Gly Tyr Phe Ser Ala Ala Ala Val Phe Pro Ala Asn 50 55 60 Ala Ser Arg Cys Ser Trp Thr Leu Arg Asn Pro Asp Pro Arg Arg Tyr 65 70 75 80 Thr Leu Tyr Met Lys Val Ala Lys Ala Pro Val Pro Cys Ser Gly Pro 85 90 95 Gly Arg Val Arg Thr Tyr Gln Phe Asp Ser Phe Leu Glu Ser Thr Arg 100 105 110 Thr Tyr Leu Gly Val Glu Ser Phe Asp Glu Val Leu Arg Leu Cys Asp 115 120 125 Pro Ser Ala Pro Leu Ala Phe Leu Gln Ala Ser Lys Gln Phe Leu Gln 130 135 140 Met Arg Arg Gln Gln Pro Pro Gln His Asp Gly Leu Arg Pro Arg Ala 145 150 155 160 Gly Pro Pro Gly Pro Thr Asp Asp Phe Ser Val Glu Tyr Leu Val Val 165 170 175 Gly Asn Arg Asn Pro Ser Arg Ala Ala Cys Gln Met Leu Cys Arg Trp 180 185 190 Leu Asp Ala Cys Leu Ala Gly Ser Arg Ser Ser His Pro Cys Gly Ile 195 200 205 Met Gln Thr Pro Cys Ala Cys Leu Gly Gly Glu Ala Gly Gly Pro Ala 210 215 220 Ala Gly Pro Leu Ala Pro Arg Gly Asp Val Cys Leu Arg Asp Ala Val 225 230 235 240 Ala Gly Gly Pro Glu Asn Cys Leu Thr Ser Leu Thr Gln Asp Arg Gly 245 250 255 Gly His Gly Ala Thr Gly Gly Trp Lys Leu Trp Ser Leu Trp Gly Glu 260 265 270 Cys Thr Arg Asp Cys Gly Gly Gly Leu Gln Thr Arg Thr Arg Thr Cys 275 280 285 Leu Pro Ala Pro Gly Val Glu Gly Gly Gly Cys Glu Gly Val Leu Glu 290 295 300 Glu Gly Arg Gln Cys Asn Arg Glu Ala Cys Gly Pro Ala Gly Arg Thr 305 310 315 320 Ser Ser Arg Ser Gln Ser Leu Arg Ser Thr Asp Ala Arg Arg Arg Glu 325 330 335 Glu Leu Gly Asp Glu Leu Gln Gln Phe Gly Phe Pro Ala Pro Gln Thr 340 345 350 Gly Asp Pro Ala Ala Glu Glu Trp Ser Pro Trp Ser Val Cys Ser Ser 355 360 365 Thr Cys Gly Glu Gly Trp Gln Thr Arg Thr Arg Phe Cys Val Ser Ser 370 375 380 Ser Tyr Ser Thr Gln Cys Ser Gly Pro Leu Arg Glu Gln Arg Leu Cys 385 390 395 400 Asn Asn Ser Ala Val Cys Pro Val His Gly Ala Trp Asp Glu Trp Ser 405 410 415 Pro Trp Ser Leu Cys Ser Ser Thr Cys Gly Arg Gly Phe Arg Asp Arg 420 425 430 Thr Arg Thr Cys Arg Pro Pro Gln Phe Gly Gly Asn Pro Cys Glu Gly 435 440 445 Pro Glu Lys Gln Thr Lys Phe Cys Asn Ile Ala Leu Cys Pro Gly Arg 450 455 460 Ala Val Asp Gly Asn Trp Asn Glu Trp Ser Ser Trp Ser Ala Cys Ser 465 470 475 480 Ala Ser Cys Ser Gln Gly Arg Gln Gln Arg Thr Arg Glu Cys Asn Gly 485 490 495 Pro Ser Tyr Gly Gly Ala Glu Cys Gln Gly His Trp Val Glu Thr Arg 500 505 510 Asp Cys Phe Leu Gln Gln Cys Pro Val Asp Gly Lys Trp Gln Ala Trp 515 520 525 Ala Ser Trp Gly Ser Cys Ser Val Thr Cys Gly Ala Gly Ser Gln Arg 530 535 540 Arg Glu Arg Val Cys Ser Gly Pro Phe Phe Gly Gly Ala Ala Cys Gln 545 550 555 560 Gly Pro Gln Asp Glu Tyr Arg Gln Cys Gly Thr Gln Arg Cys Pro Glu 565 570 575 Pro His Glu Ile Cys Asp Glu Asp Asn Phe Gly Ala Val Ile Trp Lys 580 585 590 Glu Thr Pro Ala Gly Glu Val Ala Ala Val Arg Cys Pro Arg Asn Ala 595 600 605 Thr Gly Leu Ile Leu Arg Arg Cys Glu Leu Asp Glu Glu Gly Ile Ala 610 615 620 Tyr Trp Glu Pro Pro Thr Tyr Ile Arg Cys Val Ser Ile Asp Tyr Arg 625 630 635 640 Asn Ile Gln Met Met Thr Arg Glu His Leu Ala Lys Ala Gln Arg Gly 645 650 655 Leu Pro Gly Glu Gly Val Ser Glu Val Ile Gln Thr Leu Val Glu Ile 660 665 670 Ser Gln Asp Gly Thr Ser Tyr Ser Gly Asp Leu Leu Ser

Thr Ile Asp 675 680 685 Val Leu Arg Asn Met Thr Glu Ile Phe Arg Arg Ala Tyr Tyr Ser Pro 690 695 700 Thr Pro Gly Asp Val Gln Asn Phe Val Gln Ile Leu Ser Asn Leu Leu 705 710 715 720 Ala Glu Glu Asn Arg Asp Lys Trp Glu Glu Ala Gln Leu Ala Gly Pro 725 730 735 Asn Ala Lys Glu Leu Phe Arg Leu Val Glu Asp Phe Val Asp Val Ile 740 745 750 Gly Phe Arg Met Lys Asp Leu Arg Asp Ala Tyr Gln Val Thr Asp Asn 755 760 765 Leu Val Leu Ser Ile His Lys Leu Pro Ala Ser Gly Ala Thr Asp Ile 770 775 780 Ser Phe Pro Met Lys Gly Trp Arg Ala Thr Gly Asp Trp Ala Lys Val 785 790 795 800 Pro Glu Asp Arg Val Thr Val Ser Lys Ser Val Phe Ser Thr Gly Leu 805 810 815 Thr Glu Ala Asp Glu Ala Ser Val Phe Val Val Gly Thr Val Leu Tyr 820 825 830 Arg Asn Leu Gly Ser Phe Leu Ala Leu Gln Arg Asn Thr Thr Val Leu 835 840 845 Asn Ser Lys Val Ile Ser Val Thr Val Lys Pro Pro Pro Arg Ser Leu 850 855 860 Arg Thr Pro Leu Glu Ile Glu Phe Ala His Met Tyr Asn Gly Thr Thr 865 870 875 880 Asn Gln Thr Cys Ile Leu Trp Asp Glu Thr Asp Val Pro Ser Ser Ser 885 890 895 Ala Pro Pro Gln Leu Gly Pro Trp Ser Trp Arg Gly Cys Arg Thr Val 900 905 910 Pro Leu Asp Ala Leu Arg Thr Arg Cys Leu Cys Asp Arg Leu Ser Thr 915 920 925 Phe Asp Ile Leu 930 32962DNAHomo sapiensCDS(1)..(2961) 3atg agg ggc cag gcc gcc gcc ccg ggc ccc gtc tgg atc ctc gcc ccg 48Met Arg Gly Gln Ala Ala Ala Pro Gly Pro Val Trp Ile Leu Ala Pro 1 5 10 15 ctg cta ctg ctg ctg ctg ctg ctg gga cgc cgc gcg cgg gcg gcc gcc 96Leu Leu Leu Leu Leu Leu Leu Leu Gly Arg Arg Ala Arg Ala Ala Ala 20 25 30 gga gca gac gcg ggg ccc ggg ccc gag ccg tgc gcc acg ctg gtg cag 144Gly Ala Asp Ala Gly Pro Gly Pro Glu Pro Cys Ala Thr Leu Val Gln 35 40 45 gga aag ttc ttc ggc tac ttc tcc gcg gcc gcc gtg ttc ccg gcc aac 192Gly Lys Phe Phe Gly Tyr Phe Ser Ala Ala Ala Val Phe Pro Ala Asn 50 55 60 gcc tcg cgc tgc tcc tgg acg cta cgc aac ccg gac ccg cgg cgc tac 240Ala Ser Arg Cys Ser Trp Thr Leu Arg Asn Pro Asp Pro Arg Arg Tyr 65 70 75 80 act ctc tac atg aag gtg gcc aag gcg ccc gtg ccc tgc agc ggc ccc 288Thr Leu Tyr Met Lys Val Ala Lys Ala Pro Val Pro Cys Ser Gly Pro 85 90 95 ggc cgc gtg cgc acc tac cag ttc gac tcc ttc ctc gag tcc acg cgc 336Gly Arg Val Arg Thr Tyr Gln Phe Asp Ser Phe Leu Glu Ser Thr Arg 100 105 110 acc tac ctg ggc gtg gag agc ttc gac gag gtg ctg cgg ctc tgc gac 384Thr Tyr Leu Gly Val Glu Ser Phe Asp Glu Val Leu Arg Leu Cys Asp 115 120 125 ccc tcc gca ccc ctg gcc ttc ctg cag gcc agc aag cag ttc ctg cag 432Pro Ser Ala Pro Leu Ala Phe Leu Gln Ala Ser Lys Gln Phe Leu Gln 130 135 140 atg cgg cgc cag cag ccg ccc cag cac gac ggg ctc cgg ccc cgg gcc 480Met Arg Arg Gln Gln Pro Pro Gln His Asp Gly Leu Arg Pro Arg Ala 145 150 155 160 ggg ccg ccg ggc ccc acc gac gac ttc tcc gtg gag tac ctg gtg gtg 528Gly Pro Pro Gly Pro Thr Asp Asp Phe Ser Val Glu Tyr Leu Val Val 165 170 175 ggg aac cgc aac ccc agc cgt gcc gcc tgc cag atg ctg tgc cgc tgg 576Gly Asn Arg Asn Pro Ser Arg Ala Ala Cys Gln Met Leu Cys Arg Trp 180 185 190 ctg gac gcg tgt ctg gcc ggt agt cgc agc tcg cac ccc tgc ggg atc 624Leu Asp Ala Cys Leu Ala Gly Ser Arg Ser Ser His Pro Cys Gly Ile 195 200 205 atg cag acc ccc tgc gcc tgc ctg ggc ggc gag gcg ggc ggc cct gcc 672Met Gln Thr Pro Cys Ala Cys Leu Gly Gly Glu Ala Gly Gly Pro Ala 210 215 220 gcg gga ccc ctg gcc ccc cgc ggg gat gtc tgc ttg aga gat gcg gtg 720Ala Gly Pro Leu Ala Pro Arg Gly Asp Val Cys Leu Arg Asp Ala Val 225 230 235 240 gct ggt ggc cct gaa aac tgc ctc acc agc ctg acc cag gac cgg ggc 768Ala Gly Gly Pro Glu Asn Cys Leu Thr Ser Leu Thr Gln Asp Arg Gly 245 250 255 ggg cac ggc gcc aca ggc ggc tgg aag ctg tgg tcc ctg tgg ggc gaa 816Gly His Gly Ala Thr Gly Gly Trp Lys Leu Trp Ser Leu Trp Gly Glu 260 265 270 tgc acg cgg gac tgc ggg gga ggc ctc cag acg cgg acg cgc acc tgc 864Cys Thr Arg Asp Cys Gly Gly Gly Leu Gln Thr Arg Thr Arg Thr Cys 275 280 285 ctg ccc gcg ccg ggc gtg gag ggc ggc ggc tgc gag ggg gtg ctg gag 912Leu Pro Ala Pro Gly Val Glu Gly Gly Gly Cys Glu Gly Val Leu Glu 290 295 300 gag ggt cgc cag tgc aac cgc gag gcc tgc ggc ccc gct ggg cgc acc 960Glu Gly Arg Gln Cys Asn Arg Glu Ala Cys Gly Pro Ala Gly Arg Thr 305 310 315 320 agc tcc cgg agc cag tcc ctg cgg tcc aca gat gcc cgg cgg cgc gag 1008Ser Ser Arg Ser Gln Ser Leu Arg Ser Thr Asp Ala Arg Arg Arg Glu 325 330 335 gag ctg ggg gac gag ctg cag cag ttt ggg ttc cca gcc ccc cag acc 1056Glu Leu Gly Asp Glu Leu Gln Gln Phe Gly Phe Pro Ala Pro Gln Thr 340 345 350 ggt gac cca gca gcc gag gag tgg tcc ccg tgg agc gtg tgc tcc agc 1104Gly Asp Pro Ala Ala Glu Glu Trp Ser Pro Trp Ser Val Cys Ser Ser 355 360 365 acc tgc ggc gag ggc tgg cag acc cgc acg cgc ttc tgc gtg tcc tcc 1152Thr Cys Gly Glu Gly Trp Gln Thr Arg Thr Arg Phe Cys Val Ser Ser 370 375 380 tcc tac agc acg cag tgc agc gga ccc ctg cgc gag cag cgg ctg tgc 1200Ser Tyr Ser Thr Gln Cys Ser Gly Pro Leu Arg Glu Gln Arg Leu Cys 385 390 395 400 aac aac tct gcc gtg tgc cca gtg cat ggt gcc tgg gat gag tgg tcg 1248Asn Asn Ser Ala Val Cys Pro Val His Gly Ala Trp Asp Glu Trp Ser 405 410 415 ccc tgg agc ctc tgc tcc agc acc tgt ggc cgt ggc ttt cgg gat cgc 1296Pro Trp Ser Leu Cys Ser Ser Thr Cys Gly Arg Gly Phe Arg Asp Arg 420 425 430 acg cgc acc tgc agg ccc ccc cag ttt ggg ggc aac ccc tgt gag ggc 1344Thr Arg Thr Cys Arg Pro Pro Gln Phe Gly Gly Asn Pro Cys Glu Gly 435 440 445 cct gag aag caa acc aag ttc tgc aac att gcc ctg tgc cct ggc cgg 1392Pro Glu Lys Gln Thr Lys Phe Cys Asn Ile Ala Leu Cys Pro Gly Arg 450 455 460 gca gtg gat gga aac tgg aat gag tgg tcg agc tgg agc gcc tgc tcc 1440Ala Val Asp Gly Asn Trp Asn Glu Trp Ser Ser Trp Ser Ala Cys Ser 465 470 475 480 gcc agc tgc tcc cag ggc cga cag cag cgc acg cgt gaa tgc aac ggg 1488Ala Ser Cys Ser Gln Gly Arg Gln Gln Arg Thr Arg Glu Cys Asn Gly 485 490 495 cct tcc tac ggg ggt gcg gag tgc cag ggc cac tgg gtg gag acc cga 1536Pro Ser Tyr Gly Gly Ala Glu Cys Gln Gly His Trp Val Glu Thr Arg 500 505 510 gac tgc ttc ctg cag cag tgc cca gtg gat ggc aag tgg cag gcc tgg 1584Asp Cys Phe Leu Gln Gln Cys Pro Val Asp Gly Lys Trp Gln Ala Trp 515 520 525 gcg tca tgg ggc agt tgc agc gtc acg tgt ggg gct ggc agc cag cga 1632Ala Ser Trp Gly Ser Cys Ser Val Thr Cys Gly Ala Gly Ser Gln Arg 530 535 540 cgg gag cgt gtc tgc tct ggg ccc ttc ttc ggg gga gca gcc tgc cag 1680Arg Glu Arg Val Cys Ser Gly Pro Phe Phe Gly Gly Ala Ala Cys Gln 545 550 555 560 ggc ccc cag gat gag tac cgg cag tgc ggc acc cag cgg tgt ccc gag 1728Gly Pro Gln Asp Glu Tyr Arg Gln Cys Gly Thr Gln Arg Cys Pro Glu 565 570 575 ccc cat gag atc tgt gat gag gac aac ttt ggt gct gtg atc tgg aag 1776Pro His Glu Ile Cys Asp Glu Asp Asn Phe Gly Ala Val Ile Trp Lys 580 585 590 gag acc cca gcg gga gag gtg gct gct gtc cgg tgt ccc cgc aac gcc 1824Glu Thr Pro Ala Gly Glu Val Ala Ala Val Arg Cys Pro Arg Asn Ala 595 600 605 aca gga ctc atc ctg cga cgg tgt gag ctg gac gag gaa ggc atc gcc 1872Thr Gly Leu Ile Leu Arg Arg Cys Glu Leu Asp Glu Glu Gly Ile Ala 610 615 620 tac tgg gag ccc ccc acc tac atc cgc tgt gtt tcc att gac tac aga 1920Tyr Trp Glu Pro Pro Thr Tyr Ile Arg Cys Val Ser Ile Asp Tyr Arg 625 630 635 640 aac atc cag atg atg acc cgg gag cac ctg gcc aag gct cag cga ggg 1968Asn Ile Gln Met Met Thr Arg Glu His Leu Ala Lys Ala Gln Arg Gly 645 650 655 ctg cct ggg gag ggg gtc tcg gag gtc atc cag aca ctg gtg gag atc 2016Leu Pro Gly Glu Gly Val Ser Glu Val Ile Gln Thr Leu Val Glu Ile 660 665 670 tct cag gac ggg acc agc tac agt ggg gac ctg ctg tcc acc atc gat 2064Ser Gln Asp Gly Thr Ser Tyr Ser Gly Asp Leu Leu Ser Thr Ile Asp 675 680 685 gtc ctg agg aac atg aca gag att ttc cgg aga gcg tac tac agc ccc 2112Val Leu Arg Asn Met Thr Glu Ile Phe Arg Arg Ala Tyr Tyr Ser Pro 690 695 700 acc cct ggg gac gta cag aac ttt gtc cag atc ctt agc aac ctg ttg 2160Thr Pro Gly Asp Val Gln Asn Phe Val Gln Ile Leu Ser Asn Leu Leu 705 710 715 720 gca gag gag aat cgg gac aag tgg gag gag gcc cag ctg gcg ggc ccc 2208Ala Glu Glu Asn Arg Asp Lys Trp Glu Glu Ala Gln Leu Ala Gly Pro 725 730 735 aac gcc aag gag ctg ttc cgg ctg gtg gag gac ttt gtg gac gtc atc 2256Asn Ala Lys Glu Leu Phe Arg Leu Val Glu Asp Phe Val Asp Val Ile 740 745 750 ggc ttc cgc atg aag gac ctg agg gat gca tac cag gtg aca gac aac 2304Gly Phe Arg Met Lys Asp Leu Arg Asp Ala Tyr Gln Val Thr Asp Asn 755 760 765 ctg gtt ctc agc atc cat aag ctc cca gcc agc gga gcc act gac atc 2352Leu Val Leu Ser Ile His Lys Leu Pro Ala Ser Gly Ala Thr Asp Ile 770 775 780 agc ttc ccc atg aag ggc tgg cgg gcc acg ggt gac tgg gcc aag gtg 2400Ser Phe Pro Met Lys Gly Trp Arg Ala Thr Gly Asp Trp Ala Lys Val 785 790 795 800 cca gag gac agg gtc act gtg tcc aag agt gtc ttc tcc acg ggg ctg 2448Pro Glu Asp Arg Val Thr Val Ser Lys Ser Val Phe Ser Thr Gly Leu 805 810 815 aca gag gcc gat gaa gca tcc gtg ttt gtg gtg ggc acc gtg ctc tac 2496Thr Glu Ala Asp Glu Ala Ser Val Phe Val Val Gly Thr Val Leu Tyr 820 825 830 agg aac ctg ggc agc ttc ctg gcc ctg cag agg aac acg acc gtc ctg 2544Arg Asn Leu Gly Ser Phe Leu Ala Leu Gln Arg Asn Thr Thr Val Leu 835 840 845 aat tct aag gtg atc tcc gtg act gtg aaa ccc ccg cct cgc tcc ctg 2592Asn Ser Lys Val Ile Ser Val Thr Val Lys Pro Pro Pro Arg Ser Leu 850 855 860 cgc aca ccc ttg gag atc gag ttt gcc cac atg tat aat ggc acc acc 2640Arg Thr Pro Leu Glu Ile Glu Phe Ala His Met Tyr Asn Gly Thr Thr 865 870 875 880 aac cag acc tgt atc ctg tgg gat gag acg gat gta ccc tcc tcc tcc 2688Asn Gln Thr Cys Ile Leu Trp Asp Glu Thr Asp Val Pro Ser Ser Ser 885 890 895 gcc ccc ccg cag ctc ggg ccc tgg tcg tgg cgc ggc tgc cgc acg gtg 2736Ala Pro Pro Gln Leu Gly Pro Trp Ser Trp Arg Gly Cys Arg Thr Val 900 905 910 ccc ctc gac gcc ctc cgg acg cgc tgc ctc tgt gac cgg ctc tcc acc 2784Pro Leu Asp Ala Leu Arg Thr Arg Cys Leu Cys Asp Arg Leu Ser Thr 915 920 925 ttc gat atc tta atc aag ctt ggt acc gag ctc gga tcc act agt cca 2832Phe Asp Ile Leu Ile Lys Leu Gly Thr Glu Leu Gly Ser Thr Ser Pro 930 935 940 gtg tgg tgg aat tct gca gat atc cag cac agt ggc ggc cgc tcg agt 2880Val Trp Trp Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg Ser Ser 945 950 955 960 cta gag ggc ccg cgg ttc gaa caa aaa ctc atc tca gaa gag gat ctg 2928Leu Glu Gly Pro Arg Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 965 970 975 aat atg cat acc ggt cat cat cac cat cac cat t 2962Asn Met His Thr Gly His His His His His His 980 985 4987PRTHomo sapiens 4Met Arg Gly Gln Ala Ala Ala Pro Gly Pro Val Trp Ile Leu Ala Pro 1 5 10 15 Leu Leu Leu Leu Leu Leu Leu Leu Gly Arg Arg Ala Arg Ala Ala Ala 20 25 30 Gly Ala Asp Ala Gly Pro Gly Pro Glu Pro Cys Ala Thr Leu Val Gln 35 40 45 Gly Lys Phe Phe Gly Tyr Phe Ser Ala Ala Ala Val Phe Pro Ala Asn 50 55 60 Ala Ser Arg Cys Ser Trp Thr Leu Arg Asn Pro Asp Pro Arg Arg Tyr 65 70 75 80 Thr Leu Tyr Met Lys Val Ala Lys Ala Pro Val Pro Cys Ser Gly Pro 85 90 95 Gly Arg Val Arg Thr Tyr Gln Phe Asp Ser Phe Leu Glu Ser Thr Arg 100 105 110 Thr Tyr Leu Gly Val Glu Ser Phe Asp Glu Val Leu Arg Leu Cys Asp 115 120 125 Pro Ser Ala Pro Leu Ala Phe Leu Gln Ala Ser Lys Gln Phe Leu Gln 130 135 140 Met Arg Arg Gln Gln Pro Pro Gln His Asp Gly Leu Arg Pro Arg Ala 145 150 155 160 Gly Pro Pro Gly Pro Thr Asp Asp Phe Ser Val Glu Tyr Leu Val Val 165 170 175 Gly Asn Arg Asn Pro Ser Arg Ala Ala Cys Gln Met Leu Cys Arg Trp 180 185 190 Leu Asp Ala Cys Leu Ala Gly Ser Arg Ser Ser His Pro Cys Gly Ile 195 200 205 Met Gln Thr Pro Cys Ala Cys Leu Gly Gly Glu Ala Gly Gly Pro Ala 210 215 220 Ala Gly Pro Leu Ala Pro Arg Gly Asp Val Cys Leu Arg Asp Ala Val 225 230 235 240 Ala Gly Gly Pro Glu Asn Cys Leu Thr Ser Leu Thr Gln Asp Arg Gly 245 250 255 Gly His Gly Ala Thr Gly Gly Trp Lys Leu Trp Ser Leu Trp Gly Glu 260 265 270 Cys Thr Arg Asp Cys Gly Gly Gly Leu Gln Thr Arg Thr Arg Thr Cys 275 280 285 Leu Pro Ala Pro Gly Val Glu Gly Gly Gly Cys Glu Gly Val Leu Glu 290 295 300 Glu Gly Arg Gln Cys Asn Arg Glu Ala Cys Gly Pro Ala Gly Arg Thr 305 310 315 320 Ser Ser Arg Ser Gln Ser Leu Arg Ser Thr Asp Ala Arg Arg Arg Glu 325 330 335 Glu Leu Gly

Asp Glu Leu Gln Gln Phe Gly Phe Pro Ala Pro Gln Thr 340 345 350 Gly Asp Pro Ala Ala Glu Glu Trp Ser Pro Trp Ser Val Cys Ser Ser 355 360 365 Thr Cys Gly Glu Gly Trp Gln Thr Arg Thr Arg Phe Cys Val Ser Ser 370 375 380 Ser Tyr Ser Thr Gln Cys Ser Gly Pro Leu Arg Glu Gln Arg Leu Cys 385 390 395 400 Asn Asn Ser Ala Val Cys Pro Val His Gly Ala Trp Asp Glu Trp Ser 405 410 415 Pro Trp Ser Leu Cys Ser Ser Thr Cys Gly Arg Gly Phe Arg Asp Arg 420 425 430 Thr Arg Thr Cys Arg Pro Pro Gln Phe Gly Gly Asn Pro Cys Glu Gly 435 440 445 Pro Glu Lys Gln Thr Lys Phe Cys Asn Ile Ala Leu Cys Pro Gly Arg 450 455 460 Ala Val Asp Gly Asn Trp Asn Glu Trp Ser Ser Trp Ser Ala Cys Ser 465 470 475 480 Ala Ser Cys Ser Gln Gly Arg Gln Gln Arg Thr Arg Glu Cys Asn Gly 485 490 495 Pro Ser Tyr Gly Gly Ala Glu Cys Gln Gly His Trp Val Glu Thr Arg 500 505 510 Asp Cys Phe Leu Gln Gln Cys Pro Val Asp Gly Lys Trp Gln Ala Trp 515 520 525 Ala Ser Trp Gly Ser Cys Ser Val Thr Cys Gly Ala Gly Ser Gln Arg 530 535 540 Arg Glu Arg Val Cys Ser Gly Pro Phe Phe Gly Gly Ala Ala Cys Gln 545 550 555 560 Gly Pro Gln Asp Glu Tyr Arg Gln Cys Gly Thr Gln Arg Cys Pro Glu 565 570 575 Pro His Glu Ile Cys Asp Glu Asp Asn Phe Gly Ala Val Ile Trp Lys 580 585 590 Glu Thr Pro Ala Gly Glu Val Ala Ala Val Arg Cys Pro Arg Asn Ala 595 600 605 Thr Gly Leu Ile Leu Arg Arg Cys Glu Leu Asp Glu Glu Gly Ile Ala 610 615 620 Tyr Trp Glu Pro Pro Thr Tyr Ile Arg Cys Val Ser Ile Asp Tyr Arg 625 630 635 640 Asn Ile Gln Met Met Thr Arg Glu His Leu Ala Lys Ala Gln Arg Gly 645 650 655 Leu Pro Gly Glu Gly Val Ser Glu Val Ile Gln Thr Leu Val Glu Ile 660 665 670 Ser Gln Asp Gly Thr Ser Tyr Ser Gly Asp Leu Leu Ser Thr Ile Asp 675 680 685 Val Leu Arg Asn Met Thr Glu Ile Phe Arg Arg Ala Tyr Tyr Ser Pro 690 695 700 Thr Pro Gly Asp Val Gln Asn Phe Val Gln Ile Leu Ser Asn Leu Leu 705 710 715 720 Ala Glu Glu Asn Arg Asp Lys Trp Glu Glu Ala Gln Leu Ala Gly Pro 725 730 735 Asn Ala Lys Glu Leu Phe Arg Leu Val Glu Asp Phe Val Asp Val Ile 740 745 750 Gly Phe Arg Met Lys Asp Leu Arg Asp Ala Tyr Gln Val Thr Asp Asn 755 760 765 Leu Val Leu Ser Ile His Lys Leu Pro Ala Ser Gly Ala Thr Asp Ile 770 775 780 Ser Phe Pro Met Lys Gly Trp Arg Ala Thr Gly Asp Trp Ala Lys Val 785 790 795 800 Pro Glu Asp Arg Val Thr Val Ser Lys Ser Val Phe Ser Thr Gly Leu 805 810 815 Thr Glu Ala Asp Glu Ala Ser Val Phe Val Val Gly Thr Val Leu Tyr 820 825 830 Arg Asn Leu Gly Ser Phe Leu Ala Leu Gln Arg Asn Thr Thr Val Leu 835 840 845 Asn Ser Lys Val Ile Ser Val Thr Val Lys Pro Pro Pro Arg Ser Leu 850 855 860 Arg Thr Pro Leu Glu Ile Glu Phe Ala His Met Tyr Asn Gly Thr Thr 865 870 875 880 Asn Gln Thr Cys Ile Leu Trp Asp Glu Thr Asp Val Pro Ser Ser Ser 885 890 895 Ala Pro Pro Gln Leu Gly Pro Trp Ser Trp Arg Gly Cys Arg Thr Val 900 905 910 Pro Leu Asp Ala Leu Arg Thr Arg Cys Leu Cys Asp Arg Leu Ser Thr 915 920 925 Phe Asp Ile Leu Ile Lys Leu Gly Thr Glu Leu Gly Ser Thr Ser Pro 930 935 940 Val Trp Trp Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg Ser Ser 945 950 955 960 Leu Glu Gly Pro Arg Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 965 970 975 Asn Met His Thr Gly His His His His His His 980 985 5144DNAHerpes simplex viruspromoter(1)..(144) 5ttcgcacttc gtcccaatat atatatatta ttagggcgaa gtgcgagcac tggcgccgtg 60cccgactccg cgccggcccc gggggcgggc ccgggcggcg gggggcgggt ctctccggcg 120cacataaagg cccggcgcga ccga 14462997DNAProteus vulgarisCDS(1)..(2997) 6atg gcc acc agc aat cct gca ttt gat cct aaa aat ctg atg cag tca 48Met Ala Thr Ser Asn Pro Ala Phe Asp Pro Lys Asn Leu Met Gln Ser 1 5 10 15 gaa att tac cat ttt gca caa aat aac cca tta gca gac ttc tca tca 96Glu Ile Tyr His Phe Ala Gln Asn Asn Pro Leu Ala Asp Phe Ser Ser 20 25 30 gat aaa aac tca ata cta acg tta tct gat aaa cgt agc att atg gga 144Asp Lys Asn Ser Ile Leu Thr Leu Ser Asp Lys Arg Ser Ile Met Gly 35 40 45 aac caa tct ctt tta tgg aaa tgg aaa ggt ggt agt agc ttt act tta 192Asn Gln Ser Leu Leu Trp Lys Trp Lys Gly Gly Ser Ser Phe Thr Leu 50 55 60 cat aaa aaa ctg att gtc ccc acc gat aaa gaa gca tct aaa gca tgg 240His Lys Lys Leu Ile Val Pro Thr Asp Lys Glu Ala Ser Lys Ala Trp 65 70 75 80 gga cgc tca tct acc ccc gtt ttc tca ttt tgg ctt tac aat gaa aaa 288Gly Arg Ser Ser Thr Pro Val Phe Ser Phe Trp Leu Tyr Asn Glu Lys 85 90 95 ccg att gat ggt tat ctt act atc gat ttc gga gaa aaa ctc att tca 336Pro Ile Asp Gly Tyr Leu Thr Ile Asp Phe Gly Glu Lys Leu Ile Ser 100 105 110 acc agt gag gct cag gca ggc ttt aaa gta aaa tta gat ttc act ggc 384Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asp Phe Thr Gly 115 120 125 tgg cgt act gtg gga gtc tct tta aat aac gat ctt gaa aat cga gag 432Trp Arg Thr Val Gly Val Ser Leu Asn Asn Asp Leu Glu Asn Arg Glu 130 135 140 atg acc tta aat gca acc aat acc tcc tct gat ggt act caa gac agc 480Met Thr Leu Asn Ala Thr Asn Thr Ser Ser Asp Gly Thr Gln Asp Ser 145 150 155 160 att ggg cgt tct tta ggt gct aaa gtc gat agt att cgt ttt aaa gcg 528Ile Gly Arg Ser Leu Gly Ala Lys Val Asp Ser Ile Arg Phe Lys Ala 165 170 175 cct tct aat gtg agt cag ggt gaa atc tat atc gac cgt att atg ttt 576Pro Ser Asn Val Ser Gln Gly Glu Ile Tyr Ile Asp Arg Ile Met Phe 180 185 190 tct gtc gat gat gct cgc tac caa tgg tct gat tat caa gta aaa act 624Ser Val Asp Asp Ala Arg Tyr Gln Trp Ser Asp Tyr Gln Val Lys Thr 195 200 205 cgc tta tca gaa cct gaa att caa ttt cac aac gta aag cca caa cta 672Arg Leu Ser Glu Pro Glu Ile Gln Phe His Asn Val Lys Pro Gln Leu 210 215 220 cct gta aca cct gaa aat tta gcg gcc att gat ctt atc cgc caa cgt 720Pro Val Thr Pro Glu Asn Leu Ala Ala Ile Asp Leu Ile Arg Gln Arg 225 230 235 240 cta att aat gaa ttt gtc gga ggt gaa aaa gag aca aac ctc gca tta 768Leu Ile Asn Glu Phe Val Gly Gly Glu Lys Glu Thr Asn Leu Ala Leu 245 250 255 gaa gag aat atc agc aaa tta aaa agt gat ttc gat gct ctt aat att 816Glu Glu Asn Ile Ser Lys Leu Lys Ser Asp Phe Asp Ala Leu Asn Ile 260 265 270 cac act tta gca aat ggt gga acg caa ggc aga cat ctg gtc act gat 864His Thr Leu Ala Asn Gly Gly Thr Gln Gly Arg His Leu Val Thr Asp 275 280 285 aaa caa atc att att tat caa cca gag aat cct aac tct caa gat aaa 912Lys Gln Ile Ile Ile Tyr Gln Pro Glu Asn Pro Asn Ser Gln Asp Lys 290 295 300 caa cta ttt gat aat tat gtt att tta ggt aat tac acg aca tta atg 960Gln Leu Phe Asp Asn Tyr Val Ile Leu Gly Asn Tyr Thr Thr Leu Met 305 310 315 320 ttt aat att agc cgt gct tat gtg ctg gaa aaa gat ccc aca caa aag 1008Phe Asn Ile Ser Arg Ala Tyr Val Leu Glu Lys Asp Pro Thr Gln Lys 325 330 335 gcg caa cta aag cag atg tac tta tta atg aca aag cat tta tta gat 1056Ala Gln Leu Lys Gln Met Tyr Leu Leu Met Thr Lys His Leu Leu Asp 340 345 350 caa ggc ttt gtt aaa ggg agt gct tta gtg aca acc cat cac tgg gga 1104Gln Gly Phe Val Lys Gly Ser Ala Leu Val Thr Thr His His Trp Gly 355 360 365 tac agt tct cgt tgg tgg tat att tcc acg tta tta atg tct gat gca 1152Tyr Ser Ser Arg Trp Trp Tyr Ile Ser Thr Leu Leu Met Ser Asp Ala 370 375 380 cta aaa gaa gcg aac cta caa act caa gtt tat gat tca tta ctg tgg 1200Leu Lys Glu Ala Asn Leu Gln Thr Gln Val Tyr Asp Ser Leu Leu Trp 385 390 395 400 tat tca cgt gag ttt aaa agt agt ttt gat atg aaa gta agt gct gat 1248Tyr Ser Arg Glu Phe Lys Ser Ser Phe Asp Met Lys Val Ser Ala Asp 405 410 415 agc tct gat cta gat tat ttc aat acc tta tct cgc caa cat tta gcc 1296Ser Ser Asp Leu Asp Tyr Phe Asn Thr Leu Ser Arg Gln His Leu Ala 420 425 430 tta tta cta cta gag cct gat gat caa aag cgt atc aac tta gtt aat 1344Leu Leu Leu Leu Glu Pro Asp Asp Gln Lys Arg Ile Asn Leu Val Asn 435 440 445 act ttc agc cat tat atc act ggc gca tta acg caa gtg cca ccg ggt 1392Thr Phe Ser His Tyr Ile Thr Gly Ala Leu Thr Gln Val Pro Pro Gly 450 455 460 ggt aaa gat ggt tta cgc cct gat ggt aca gca tgg cga cat gaa ggc 1440Gly Lys Asp Gly Leu Arg Pro Asp Gly Thr Ala Trp Arg His Glu Gly 465 470 475 480 aac tat ccg ggc tac tct ttc cca gcc ttt aaa aat gcc tct cag ctt 1488Asn Tyr Pro Gly Tyr Ser Phe Pro Ala Phe Lys Asn Ala Ser Gln Leu 485 490 495 att tat tta tta cgc gat aca cca ttt tca gtg ggt gaa agt ggt tgg 1536Ile Tyr Leu Leu Arg Asp Thr Pro Phe Ser Val Gly Glu Ser Gly Trp 500 505 510 aat aac ctg aaa aaa gcg atg gtt tca gcg tgg atc tac agt aat cca 1584Asn Asn Leu Lys Lys Ala Met Val Ser Ala Trp Ile Tyr Ser Asn Pro 515 520 525 gaa gtt gga tta ccg ctt gca gga aga cac cct ttt aac tca cct tcg 1632Glu Val Gly Leu Pro Leu Ala Gly Arg His Pro Phe Asn Ser Pro Ser 530 535 540 tta aaa tca gtc gct caa ggc tat tac tgg ctt gcc atg tct gca aaa 1680Leu Lys Ser Val Ala Gln Gly Tyr Tyr Trp Leu Ala Met Ser Ala Lys 545 550 555 560 tca tcg cct gat aaa aca ctt gca tct att tat ctt gcg att agt gat 1728Ser Ser Pro Asp Lys Thr Leu Ala Ser Ile Tyr Leu Ala Ile Ser Asp 565 570 575 aaa aca caa aat gaa tca act gct att ttt gga gaa act att aca cca 1776Lys Thr Gln Asn Glu Ser Thr Ala Ile Phe Gly Glu Thr Ile Thr Pro 580 585 590 gcg tct tta cct caa ggt ttc tat gcc ttt aat ggc ggt gct ttt ggt 1824Ala Ser Leu Pro Gln Gly Phe Tyr Ala Phe Asn Gly Gly Ala Phe Gly 595 600 605 att cat cgt tgg caa gat aaa atg gtg aca ctg aaa gct tat aac acc 1872Ile His Arg Trp Gln Asp Lys Met Val Thr Leu Lys Ala Tyr Asn Thr 610 615 620 aat gtt tgg tca tct gaa att tat aac aaa gat aac cgt tat ggc cgt 1920Asn Val Trp Ser Ser Glu Ile Tyr Asn Lys Asp Asn Arg Tyr Gly Arg 625 630 635 640 tac caa agt cat ggt gtc gct caa ata gtg agt aat ggc tcg cag ctt 1968Tyr Gln Ser His Gly Val Ala Gln Ile Val Ser Asn Gly Ser Gln Leu 645 650 655 tca cag ggc tat cag caa gaa ggt tgg gat tgg aat aga atg cca ggg 2016Ser Gln Gly Tyr Gln Gln Glu Gly Trp Asp Trp Asn Arg Met Pro Gly 660 665 670 gca acc act att cac ctt cct ctt aaa gac tta gac agt cct aaa cct 2064Ala Thr Thr Ile His Leu Pro Leu Lys Asp Leu Asp Ser Pro Lys Pro 675 680 685 cat acc tta atg caa cgt gga gag cgt gga ttt agc gga aca tca tcc 2112His Thr Leu Met Gln Arg Gly Glu Arg Gly Phe Ser Gly Thr Ser Ser 690 695 700 ctt gaa ggt caa tat ggc atg atg gca ttc gat ctt att tat ccc gcc 2160Leu Glu Gly Gln Tyr Gly Met Met Ala Phe Asp Leu Ile Tyr Pro Ala 705 710 715 720 aat ctt gag cgt ttt gat cct aat ttc act gcg aaa aag agt gta tta 2208Asn Leu Glu Arg Phe Asp Pro Asn Phe Thr Ala Lys Lys Ser Val Leu 725 730 735 gcc gct gat aat cac tta att ttt att ggt agc aat ata aat agt agt 2256Ala Ala Asp Asn His Leu Ile Phe Ile Gly Ser Asn Ile Asn Ser Ser 740 745 750 gat aaa aat aaa aat gtt gaa acg acc tta ttc caa cat gcc att act 2304Asp Lys Asn Lys Asn Val Glu Thr Thr Leu Phe Gln His Ala Ile Thr 755 760 765 cca aca tta aat acc ctt tgg att aat gga caa aag ata gaa aac atg 2352Pro Thr Leu Asn Thr Leu Trp Ile Asn Gly Gln Lys Ile Glu Asn Met 770 775 780 cct tat caa aca aca ctt caa caa ggt gat tgg tta att gat agc aat 2400Pro Tyr Gln Thr Thr Leu Gln Gln Gly Asp Trp Leu Ile Asp Ser Asn 785 790 795 800 ggc aat ggt tac tta att act caa gca gaa aaa gta aat gta agt cgc 2448Gly Asn Gly Tyr Leu Ile Thr Gln Ala Glu Lys Val Asn Val Ser Arg 805 810 815 caa cat cag gtt tca gcg gaa aat aaa aat cgc caa ccg aca gaa gga 2496Gln His Gln Val Ser Ala Glu Asn Lys Asn Arg Gln Pro Thr Glu Gly 820 825 830 aac ttt agc tcg gca tgg atc gat cac agc act cgc ccc aaa gat gcc 2544Asn Phe Ser Ser Ala Trp Ile Asp His Ser Thr Arg Pro Lys Asp Ala 835 840 845 agt tat gag tat atg gtc ttt tta gat gcg aca cct gaa aaa atg gga 2592Ser Tyr Glu Tyr Met Val Phe Leu Asp Ala Thr Pro Glu Lys Met Gly 850 855 860 gag atg gca caa aaa ttc cgt gaa aat aat ggg tta tat cag gtt ctt 2640Glu Met Ala Gln Lys Phe Arg Glu Asn Asn Gly Leu Tyr Gln Val Leu 865 870 875 880 cgt aag gat aaa gac gtt cat att att ctc gat aaa ctc agc aat gta 2688Arg Lys Asp Lys Asp Val His Ile Ile Leu Asp Lys Leu Ser Asn Val 885 890 895 acg gga tat gcc ttt tat cag cca gca tca att gaa gac aaa tgg atc 2736Thr Gly Tyr Ala Phe Tyr Gln Pro Ala Ser Ile Glu Asp Lys Trp Ile 900 905 910 aaa aag gtt aat aaa cct gca att gtg atg act cat cga caa aaa gac 2784Lys Lys Val Asn Lys Pro Ala Ile Val Met Thr His Arg Gln Lys Asp 915 920 925 act ctt att gtc agt gca gtt aca cct gat tta aat atg act cgc caa 2832Thr Leu Ile Val Ser Ala Val Thr Pro Asp Leu Asn Met Thr Arg Gln 930 935 940

aaa gca gca act cct gtc acc atc aat gtc acg att aat ggc aaa tgg 2880Lys Ala Ala Thr Pro Val Thr Ile Asn Val Thr Ile Asn Gly Lys Trp 945 950 955 960 caa tct gct gat aaa aat agt gaa gtg aaa tat cag gtt tct ggt gat 2928Gln Ser Ala Asp Lys Asn Ser Glu Val Lys Tyr Gln Val Ser Gly Asp 965 970 975 aac act gaa ctg acg ttt acg agt tac ttt ggt att cca caa gaa atc 2976Asn Thr Glu Leu Thr Phe Thr Ser Tyr Phe Gly Ile Pro Gln Glu Ile 980 985 990 aaa ctc tcg cca ctc cct tga 2997Lys Leu Ser Pro Leu Pro 995 7998PRTProteus vulgaris 7Met Ala Thr Ser Asn Pro Ala Phe Asp Pro Lys Asn Leu Met Gln Ser 1 5 10 15 Glu Ile Tyr His Phe Ala Gln Asn Asn Pro Leu Ala Asp Phe Ser Ser 20 25 30 Asp Lys Asn Ser Ile Leu Thr Leu Ser Asp Lys Arg Ser Ile Met Gly 35 40 45 Asn Gln Ser Leu Leu Trp Lys Trp Lys Gly Gly Ser Ser Phe Thr Leu 50 55 60 His Lys Lys Leu Ile Val Pro Thr Asp Lys Glu Ala Ser Lys Ala Trp 65 70 75 80 Gly Arg Ser Ser Thr Pro Val Phe Ser Phe Trp Leu Tyr Asn Glu Lys 85 90 95 Pro Ile Asp Gly Tyr Leu Thr Ile Asp Phe Gly Glu Lys Leu Ile Ser 100 105 110 Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asp Phe Thr Gly 115 120 125 Trp Arg Thr Val Gly Val Ser Leu Asn Asn Asp Leu Glu Asn Arg Glu 130 135 140 Met Thr Leu Asn Ala Thr Asn Thr Ser Ser Asp Gly Thr Gln Asp Ser 145 150 155 160 Ile Gly Arg Ser Leu Gly Ala Lys Val Asp Ser Ile Arg Phe Lys Ala 165 170 175 Pro Ser Asn Val Ser Gln Gly Glu Ile Tyr Ile Asp Arg Ile Met Phe 180 185 190 Ser Val Asp Asp Ala Arg Tyr Gln Trp Ser Asp Tyr Gln Val Lys Thr 195 200 205 Arg Leu Ser Glu Pro Glu Ile Gln Phe His Asn Val Lys Pro Gln Leu 210 215 220 Pro Val Thr Pro Glu Asn Leu Ala Ala Ile Asp Leu Ile Arg Gln Arg 225 230 235 240 Leu Ile Asn Glu Phe Val Gly Gly Glu Lys Glu Thr Asn Leu Ala Leu 245 250 255 Glu Glu Asn Ile Ser Lys Leu Lys Ser Asp Phe Asp Ala Leu Asn Ile 260 265 270 His Thr Leu Ala Asn Gly Gly Thr Gln Gly Arg His Leu Val Thr Asp 275 280 285 Lys Gln Ile Ile Ile Tyr Gln Pro Glu Asn Pro Asn Ser Gln Asp Lys 290 295 300 Gln Leu Phe Asp Asn Tyr Val Ile Leu Gly Asn Tyr Thr Thr Leu Met 305 310 315 320 Phe Asn Ile Ser Arg Ala Tyr Val Leu Glu Lys Asp Pro Thr Gln Lys 325 330 335 Ala Gln Leu Lys Gln Met Tyr Leu Leu Met Thr Lys His Leu Leu Asp 340 345 350 Gln Gly Phe Val Lys Gly Ser Ala Leu Val Thr Thr His His Trp Gly 355 360 365 Tyr Ser Ser Arg Trp Trp Tyr Ile Ser Thr Leu Leu Met Ser Asp Ala 370 375 380 Leu Lys Glu Ala Asn Leu Gln Thr Gln Val Tyr Asp Ser Leu Leu Trp 385 390 395 400 Tyr Ser Arg Glu Phe Lys Ser Ser Phe Asp Met Lys Val Ser Ala Asp 405 410 415 Ser Ser Asp Leu Asp Tyr Phe Asn Thr Leu Ser Arg Gln His Leu Ala 420 425 430 Leu Leu Leu Leu Glu Pro Asp Asp Gln Lys Arg Ile Asn Leu Val Asn 435 440 445 Thr Phe Ser His Tyr Ile Thr Gly Ala Leu Thr Gln Val Pro Pro Gly 450 455 460 Gly Lys Asp Gly Leu Arg Pro Asp Gly Thr Ala Trp Arg His Glu Gly 465 470 475 480 Asn Tyr Pro Gly Tyr Ser Phe Pro Ala Phe Lys Asn Ala Ser Gln Leu 485 490 495 Ile Tyr Leu Leu Arg Asp Thr Pro Phe Ser Val Gly Glu Ser Gly Trp 500 505 510 Asn Asn Leu Lys Lys Ala Met Val Ser Ala Trp Ile Tyr Ser Asn Pro 515 520 525 Glu Val Gly Leu Pro Leu Ala Gly Arg His Pro Phe Asn Ser Pro Ser 530 535 540 Leu Lys Ser Val Ala Gln Gly Tyr Tyr Trp Leu Ala Met Ser Ala Lys 545 550 555 560 Ser Ser Pro Asp Lys Thr Leu Ala Ser Ile Tyr Leu Ala Ile Ser Asp 565 570 575 Lys Thr Gln Asn Glu Ser Thr Ala Ile Phe Gly Glu Thr Ile Thr Pro 580 585 590 Ala Ser Leu Pro Gln Gly Phe Tyr Ala Phe Asn Gly Gly Ala Phe Gly 595 600 605 Ile His Arg Trp Gln Asp Lys Met Val Thr Leu Lys Ala Tyr Asn Thr 610 615 620 Asn Val Trp Ser Ser Glu Ile Tyr Asn Lys Asp Asn Arg Tyr Gly Arg 625 630 635 640 Tyr Gln Ser His Gly Val Ala Gln Ile Val Ser Asn Gly Ser Gln Leu 645 650 655 Ser Gln Gly Tyr Gln Gln Glu Gly Trp Asp Trp Asn Arg Met Pro Gly 660 665 670 Ala Thr Thr Ile His Leu Pro Leu Lys Asp Leu Asp Ser Pro Lys Pro 675 680 685 His Thr Leu Met Gln Arg Gly Glu Arg Gly Phe Ser Gly Thr Ser Ser 690 695 700 Leu Glu Gly Gln Tyr Gly Met Met Ala Phe Asp Leu Ile Tyr Pro Ala 705 710 715 720 Asn Leu Glu Arg Phe Asp Pro Asn Phe Thr Ala Lys Lys Ser Val Leu 725 730 735 Ala Ala Asp Asn His Leu Ile Phe Ile Gly Ser Asn Ile Asn Ser Ser 740 745 750 Asp Lys Asn Lys Asn Val Glu Thr Thr Leu Phe Gln His Ala Ile Thr 755 760 765 Pro Thr Leu Asn Thr Leu Trp Ile Asn Gly Gln Lys Ile Glu Asn Met 770 775 780 Pro Tyr Gln Thr Thr Leu Gln Gln Gly Asp Trp Leu Ile Asp Ser Asn 785 790 795 800 Gly Asn Gly Tyr Leu Ile Thr Gln Ala Glu Lys Val Asn Val Ser Arg 805 810 815 Gln His Gln Val Ser Ala Glu Asn Lys Asn Arg Gln Pro Thr Glu Gly 820 825 830 Asn Phe Ser Ser Ala Trp Ile Asp His Ser Thr Arg Pro Lys Asp Ala 835 840 845 Ser Tyr Glu Tyr Met Val Phe Leu Asp Ala Thr Pro Glu Lys Met Gly 850 855 860 Glu Met Ala Gln Lys Phe Arg Glu Asn Asn Gly Leu Tyr Gln Val Leu 865 870 875 880 Arg Lys Asp Lys Asp Val His Ile Ile Leu Asp Lys Leu Ser Asn Val 885 890 895 Thr Gly Tyr Ala Phe Tyr Gln Pro Ala Ser Ile Glu Asp Lys Trp Ile 900 905 910 Lys Lys Val Asn Lys Pro Ala Ile Val Met Thr His Arg Gln Lys Asp 915 920 925 Thr Leu Ile Val Ser Ala Val Thr Pro Asp Leu Asn Met Thr Arg Gln 930 935 940 Lys Ala Ala Thr Pro Val Thr Ile Asn Val Thr Ile Asn Gly Lys Trp 945 950 955 960 Gln Ser Ala Asp Lys Asn Ser Glu Val Lys Tyr Gln Val Ser Gly Asp 965 970 975 Asn Thr Glu Leu Thr Phe Thr Ser Tyr Phe Gly Ile Pro Gln Glu Ile 980 985 990 Lys Leu Ser Pro Leu Pro 995 83099DNAArtificial SequenceHuman Igk attached to N terminal to permit secretion of bacterial protein. N terminal 72 bp of bacterial Chase ABC are omitted. 8atg gag aca gac aca ctc ctg cta tgg gta ctg ctg ctc tgg gtt cca 48Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 ggt tcc act ggt gac gcg gcc cag ccg gcc agg cgc gcg cgc cgt acg 96Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr 20 25 30 aag ctc gcc ctt acc agc aat cct gca ttt gat cct aaa aat ctg atg 144Lys Leu Ala Leu Thr Ser Asn Pro Ala Phe Asp Pro Lys Asn Leu Met 35 40 45 cag tca gaa atc tat cat ttt gcg caa agt aac cca tta gag gac ttt 192Gln Ser Glu Ile Tyr His Phe Ala Gln Ser Asn Pro Leu Glu Asp Phe 50 55 60 tca tca gat aaa aac tca gca ctg acg tta tct gat aaa cgt agc att 240Ser Ser Asp Lys Asn Ser Ala Leu Thr Leu Ser Asp Lys Arg Ser Ile 65 70 75 80 atg gga aac caa tcc ctt ttg tgg aaa tgg aaa ggt ggc agt agt ttt 288Met Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys Gly Gly Ser Ser Phe 85 90 95 act ttg cat aaa aaa ctt att gtc cca aca gat aaa gaa gcg tct aaa 336Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp Lys Glu Ala Ser Lys 100 105 110 gca tgg gga cga gca tca aca cct gtt tta tca ttt tgg ctt tat aat 384Ala Trp Gly Arg Ala Ser Thr Pro Val Leu Ser Phe Trp Leu Tyr Asn 115 120 125 gaa aaa ccc att gat ggc tat ctt act atc gat ttt gga gag aag ctg 432Glu Lys Pro Ile Asp Gly Tyr Leu Thr Ile Asp Phe Gly Glu Lys Leu 130 135 140 aat tca acg agc gaa gcg caa gcg ggt ttt aaa gta aaa cta aat ttc 480Asn Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asn Phe 145 150 155 160 act ggc tgg cgt gct gtt ggg atc tct tta aat aac gat ctt gaa aat 528Thr Gly Trp Arg Ala Val Gly Ile Ser Leu Asn Asn Asp Leu Glu Asn 165 170 175 cga gag atg acc tta aat gca atg aat acc tct tct gat ggt acg caa 576Arg Glu Met Thr Leu Asn Ala Met Asn Thr Ser Ser Asp Gly Thr Gln 180 185 190 gac agc att ggt cgc tct tta ggt gct aat gtc gat agt att cga ttt 624Asp Ser Ile Gly Arg Ser Leu Gly Ala Asn Val Asp Ser Ile Arg Phe 195 200 205 aaa gcc cca tca aat ata ggt cag ggt gaa atc tat atc gat cgt att 672Lys Ala Pro Ser Asn Ile Gly Gln Gly Glu Ile Tyr Ile Asp Arg Ile 210 215 220 atg ttt tct atc gat gat gct cgc tat caa tgg tct gat tat caa gtt 720Met Phe Ser Ile Asp Asp Ala Arg Tyr Gln Trp Ser Asp Tyr Gln Val 225 230 235 240 aaa aca cgc tta tca gag cct gag atc caa ttt cat aac gta caa cct 768Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe His Asn Val Gln Pro 245 250 255 cag tta ccg gtg aca cct gaa aat cta gct gca att gat ctt att cgc 816Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala Ile Asp Leu Ile Arg 260 265 270 caa cgt ttg ata aat gag ttt gtt ggt ggc gaa aaa gag aca aat ctc 864Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu Lys Glu Thr Asn Leu 275 280 285 gca cta gaa gaa aat att ggt aag tta aaa agt gat ttt gat gct ctt 912Ala Leu Glu Glu Asn Ile Gly Lys Leu Lys Ser Asp Phe Asp Ala Leu 290 295 300 aat att cac gct tta gag aat ggc aca ata caa ggg cga cac ctg atc 960Asn Ile His Ala Leu Glu Asn Gly Thr Ile Gln Gly Arg His Leu Ile 305 310 315 320 aca gac aaa caa act att att tac caa cct gaa aat ctc aac cct caa 1008Thr Asp Lys Gln Thr Ile Ile Tyr Gln Pro Glu Asn Leu Asn Pro Gln 325 330 335 gat aaa caa cta ttt gat aat tat gtc att tta ggt aat tac aca aca 1056Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile Leu Gly Asn Tyr Thr Thr 340 345 350 ttg atg ttt aat att agt cgg gct tat gtg ctg gaa aaa gat ccc tca 1104Leu Met Phe Asn Ile Ser Arg Ala Tyr Val Leu Glu Lys Asp Pro Ser 355 360 365 caa aaa gct caa cta aag cag atg tac tta tta atg aca aaa cac tta 1152Gln Lys Ala Gln Leu Lys Gln Met Tyr Leu Leu Met Thr Lys His Leu 370 375 380 tta gat caa ggc ttt gtt aaa gga agt gca tta gtc aca acc cat cac 1200Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu Val Thr Thr His His 385 390 395 400 tgg ggg tat agt tct cgt tgg tgg tat att tcc aca tta cta atg tct 1248Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser Thr Leu Leu Met Ser 405 410 415 gat gca cta aaa gag gca aac tta caa act caa gtt tat gat tca ttg 1296Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln Val Tyr Asp Ser Leu 420 425 430 ttg tgg tat tca cga gag ttt aaa agt agt ttt gat atg aaa gtg ggt 1344Leu Trp Tyr Ser Arg Glu Phe Lys Ser Ser Phe Asp Met Lys Val Gly 435 440 445 gca gat agc tcc gac tta gac tat ttc aat acc cta tct cgt caa cac 1392Ala Asp Ser Ser Asp Leu Asp Tyr Phe Asn Thr Leu Ser Arg Gln His 450 455 460 tta gcc tta tta cta cta gag cct gat gat caa aaa cgt atc aac tta 1440Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln Lys Arg Ile Asn Leu 465 470 475 480 gtt aat acc ttc agc cat tac atc act ggc gca tta act caa gta ccg 1488Val Asn Thr Phe Ser His Tyr Ile Thr Gly Ala Leu Thr Gln Val Pro 485 490 495 ccg ggt ggt aaa ggt ggt tta cgc cct gac ggt act gcg tgg cga cat 1536Pro Gly Gly Lys Gly Gly Leu Arg Pro Asp Gly Thr Ala Trp Arg His 500 505 510 gaa ggc aac tat cca ggc tac tct ttc cca gct ttt aaa aat gcc tct 1584Glu Gly Asn Tyr Pro Gly Tyr Ser Phe Pro Ala Phe Lys Asn Ala Ser 515 520 525 caa ctt att tat tta cta cgt ggt acc cca ttt tca gta ggt gaa agc 1632Gln Leu Ile Tyr Leu Leu Arg Gly Thr Pro Phe Ser Val Gly Glu Ser 530 535 540 ggt tgg aat aac cta aaa aaa gcg atg att tca gcg tgg atc tac agt 1680Gly Trp Asn Asn Leu Lys Lys Ala Met Ile Ser Ala Trp Ile Tyr Ser 545 550 555 560 aat cca gaa gtt gga tta cct ctt gct gga agg cac cct ttt aac tca 1728Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg His Pro Phe Asn Ser 565 570 575 cct tcg tta aaa tca gtc gct caa ggt tac tac tgg ctt gcc atg tct 1776Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr Trp Leu Ala Met Ser 580 585 590 gca aaa ccg tct cca gat aaa act ctc gcg tct att tat ctt gca ata 1824Ala Lys Pro Ser Pro Asp Lys Thr Leu Ala Ser Ile Tyr Leu Ala Ile 595 600 605 agt gat aaa aca caa aat gaa tcg aag gct att ttt gga gaa act att 1872Ser Asp Lys Thr Gln Asn Glu Ser Lys Ala Ile Phe Gly Glu Thr Ile 610 615 620 gca cca gca gcg ttg cct caa ggt ttc tat gcc ttt aat ggc ggg gct 1920Ala Pro Ala Ala Leu Pro Gln Gly Phe Tyr Ala Phe Asn Gly Gly Ala 625 630 635 640 ttt ggt att cat cgt tgg caa gat aaa atg gtg aca ctg aaa gct tat 1968Phe Gly Ile His Arg Trp Gln Asp Lys Met Val Thr Leu Lys Ala Tyr 645 650 655 aat acc aat gtt tgg tca tct gaa att tat aat aaa gat aac cgt tat 2016Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn Lys Asp Asn Arg Tyr 660 665 670 ggt cgt tat caa agt cat ggt gtc gct caa ata gtg agt aat ggt tcg 2064Gly Arg Tyr Gln Ser His Gly Val Ala Gln Ile Val Ser Asn Gly Ser

675 680 685 cag ctc tca cag ggc tat cag caa gaa ggt tgg gat tgg aat agg atg 2112Gln Leu Ser Gln Gly Tyr Gln Gln Glu Gly Trp Asp Trp Asn Arg Met 690 695 700 cca gga gca acc act att cat ctt tct ctt aaa gag tta gat agc cct 2160Pro Gly Ala Thr Thr Ile His Leu Ser Leu Lys Glu Leu Asp Ser Pro 705 710 715 720 aaa cct cat aca tta atg caa cgt ggc gag cgt gga ttt agc gga aca 2208Lys Pro His Thr Leu Met Gln Arg Gly Glu Arg Gly Phe Ser Gly Thr 725 730 735 tct gcc ctt gat ggt aaa tat ggg atg atg gca ttc gat ctt att tat 2256Ser Ala Leu Asp Gly Lys Tyr Gly Met Met Ala Phe Asp Leu Ile Tyr 740 745 750 cct acc aac ctt gaa cgt ttt gat cct aat ttc act gcg aaa aag agt 2304Pro Thr Asn Leu Glu Arg Phe Asp Pro Asn Phe Thr Ala Lys Lys Ser 755 760 765 gta tta gcc gtt gat aat cac tta att ttt att ggt agc aat ata aat 2352Val Leu Ala Val Asp Asn His Leu Ile Phe Ile Gly Ser Asn Ile Asn 770 775 780 agt agt gat aaa aat aaa aat gtt gaa acg act tta ttt caa cat gct 2400Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr Leu Phe Gln His Ala 785 790 795 800 ata aca cca aca tta aat acc att tgg gtt aat gga caa aaa gtt gag 2448Ile Thr Pro Thr Leu Asn Thr Ile Trp Val Asn Gly Gln Lys Val Glu 805 810 815 acc ttt cct tat caa gca aca ctt aaa caa ggt gat tgg tta att gat 2496Thr Phe Pro Tyr Gln Ala Thr Leu Lys Gln Gly Asp Trp Leu Ile Asp 820 825 830 agc aac ggt aat ggc tat tta att acg caa gct gaa aaa gta aat att 2544Ser Asn Gly Asn Gly Tyr Leu Ile Thr Gln Ala Glu Lys Val Asn Ile 835 840 845 agc cgt caa cac cag act tcg gct gaa aat aaa aat cgc caa cca aca 2592Ser Arg Gln His Gln Thr Ser Ala Glu Asn Lys Asn Arg Gln Pro Thr 850 855 860 gaa gga aac ttt agc tcg gca tgg att gat cac agt gtt caa cct aaa 2640Glu Gly Asn Phe Ser Ser Ala Trp Ile Asp His Ser Val Gln Pro Lys 865 870 875 880 gat tcc aac tat gag tat atg gtc ttt tta gat gct act cct gaa aga 2688Asp Ser Asn Tyr Glu Tyr Met Val Phe Leu Asp Ala Thr Pro Glu Arg 885 890 895 atg gga gaa ata gca caa aaa ttt cgt gaa aat aat ggg tta tat cag 2736Met Gly Glu Ile Ala Gln Lys Phe Arg Glu Asn Asn Gly Leu Tyr Gln 900 905 910 gtt ctt cgt aag gat aaa gac gtt cat att att ctc gat aaa ctc agt 2784Val Leu Arg Lys Asp Lys Asp Val His Ile Ile Leu Asp Lys Leu Ser 915 920 925 aat gta acg gga tat gcc ttt tat cag tct gct tca att gaa gat aaa 2832Asn Val Thr Gly Tyr Ala Phe Tyr Gln Ser Ala Ser Ile Glu Asp Lys 930 935 940 tgg atc aaa aaa gtt gat aaa ccc gcg att gtg atg act cat cga caa 2880Trp Ile Lys Lys Val Asp Lys Pro Ala Ile Val Met Thr His Arg Gln 945 950 955 960 aaa gat act ctt att gtc agt gct gtt aca cct gat tta aat atg act 2928Lys Asp Thr Leu Ile Val Ser Ala Val Thr Pro Asp Leu Asn Met Thr 965 970 975 cgc caa aaa gca gca act cct gtc atc atc aat gtc acg att aat ggt 2976Arg Gln Lys Ala Ala Thr Pro Val Ile Ile Asn Val Thr Ile Asn Gly 980 985 990 aaa tgg caa tct gct gat aaa aat agc aaa gta aaa tat agt gtt tca 3024Lys Trp Gln Ser Ala Asp Lys Asn Ser Lys Val Lys Tyr Ser Val Ser 995 1000 1005 ggt gat aac act gaa ctg act ttt acc agc tac ttt ggt att cca 3069Gly Asp Asn Thr Glu Leu Thr Phe Thr Ser Tyr Phe Gly Ile Pro 1010 1015 1020 caa gaa atc aaa ctc tcg cca ctc cct tga 3099Gln Glu Ile Lys Leu Ser Pro Leu Pro 1025 1030 91032PRTArtificial SequenceSynthetic Construct 9Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr 20 25 30 Lys Leu Ala Leu Thr Ser Asn Pro Ala Phe Asp Pro Lys Asn Leu Met 35 40 45 Gln Ser Glu Ile Tyr His Phe Ala Gln Ser Asn Pro Leu Glu Asp Phe 50 55 60 Ser Ser Asp Lys Asn Ser Ala Leu Thr Leu Ser Asp Lys Arg Ser Ile 65 70 75 80 Met Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys Gly Gly Ser Ser Phe 85 90 95 Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp Lys Glu Ala Ser Lys 100 105 110 Ala Trp Gly Arg Ala Ser Thr Pro Val Leu Ser Phe Trp Leu Tyr Asn 115 120 125 Glu Lys Pro Ile Asp Gly Tyr Leu Thr Ile Asp Phe Gly Glu Lys Leu 130 135 140 Asn Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys Val Lys Leu Asn Phe 145 150 155 160 Thr Gly Trp Arg Ala Val Gly Ile Ser Leu Asn Asn Asp Leu Glu Asn 165 170 175 Arg Glu Met Thr Leu Asn Ala Met Asn Thr Ser Ser Asp Gly Thr Gln 180 185 190 Asp Ser Ile Gly Arg Ser Leu Gly Ala Asn Val Asp Ser Ile Arg Phe 195 200 205 Lys Ala Pro Ser Asn Ile Gly Gln Gly Glu Ile Tyr Ile Asp Arg Ile 210 215 220 Met Phe Ser Ile Asp Asp Ala Arg Tyr Gln Trp Ser Asp Tyr Gln Val 225 230 235 240 Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe His Asn Val Gln Pro 245 250 255 Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala Ile Asp Leu Ile Arg 260 265 270 Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu Lys Glu Thr Asn Leu 275 280 285 Ala Leu Glu Glu Asn Ile Gly Lys Leu Lys Ser Asp Phe Asp Ala Leu 290 295 300 Asn Ile His Ala Leu Glu Asn Gly Thr Ile Gln Gly Arg His Leu Ile 305 310 315 320 Thr Asp Lys Gln Thr Ile Ile Tyr Gln Pro Glu Asn Leu Asn Pro Gln 325 330 335 Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile Leu Gly Asn Tyr Thr Thr 340 345 350 Leu Met Phe Asn Ile Ser Arg Ala Tyr Val Leu Glu Lys Asp Pro Ser 355 360 365 Gln Lys Ala Gln Leu Lys Gln Met Tyr Leu Leu Met Thr Lys His Leu 370 375 380 Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu Val Thr Thr His His 385 390 395 400 Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser Thr Leu Leu Met Ser 405 410 415 Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln Val Tyr Asp Ser Leu 420 425 430 Leu Trp Tyr Ser Arg Glu Phe Lys Ser Ser Phe Asp Met Lys Val Gly 435 440 445 Ala Asp Ser Ser Asp Leu Asp Tyr Phe Asn Thr Leu Ser Arg Gln His 450 455 460 Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln Lys Arg Ile Asn Leu 465 470 475 480 Val Asn Thr Phe Ser His Tyr Ile Thr Gly Ala Leu Thr Gln Val Pro 485 490 495 Pro Gly Gly Lys Gly Gly Leu Arg Pro Asp Gly Thr Ala Trp Arg His 500 505 510 Glu Gly Asn Tyr Pro Gly Tyr Ser Phe Pro Ala Phe Lys Asn Ala Ser 515 520 525 Gln Leu Ile Tyr Leu Leu Arg Gly Thr Pro Phe Ser Val Gly Glu Ser 530 535 540 Gly Trp Asn Asn Leu Lys Lys Ala Met Ile Ser Ala Trp Ile Tyr Ser 545 550 555 560 Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg His Pro Phe Asn Ser 565 570 575 Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr Trp Leu Ala Met Ser 580 585 590 Ala Lys Pro Ser Pro Asp Lys Thr Leu Ala Ser Ile Tyr Leu Ala Ile 595 600 605 Ser Asp Lys Thr Gln Asn Glu Ser Lys Ala Ile Phe Gly Glu Thr Ile 610 615 620 Ala Pro Ala Ala Leu Pro Gln Gly Phe Tyr Ala Phe Asn Gly Gly Ala 625 630 635 640 Phe Gly Ile His Arg Trp Gln Asp Lys Met Val Thr Leu Lys Ala Tyr 645 650 655 Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn Lys Asp Asn Arg Tyr 660 665 670 Gly Arg Tyr Gln Ser His Gly Val Ala Gln Ile Val Ser Asn Gly Ser 675 680 685 Gln Leu Ser Gln Gly Tyr Gln Gln Glu Gly Trp Asp Trp Asn Arg Met 690 695 700 Pro Gly Ala Thr Thr Ile His Leu Ser Leu Lys Glu Leu Asp Ser Pro 705 710 715 720 Lys Pro His Thr Leu Met Gln Arg Gly Glu Arg Gly Phe Ser Gly Thr 725 730 735 Ser Ala Leu Asp Gly Lys Tyr Gly Met Met Ala Phe Asp Leu Ile Tyr 740 745 750 Pro Thr Asn Leu Glu Arg Phe Asp Pro Asn Phe Thr Ala Lys Lys Ser 755 760 765 Val Leu Ala Val Asp Asn His Leu Ile Phe Ile Gly Ser Asn Ile Asn 770 775 780 Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr Leu Phe Gln His Ala 785 790 795 800 Ile Thr Pro Thr Leu Asn Thr Ile Trp Val Asn Gly Gln Lys Val Glu 805 810 815 Thr Phe Pro Tyr Gln Ala Thr Leu Lys Gln Gly Asp Trp Leu Ile Asp 820 825 830 Ser Asn Gly Asn Gly Tyr Leu Ile Thr Gln Ala Glu Lys Val Asn Ile 835 840 845 Ser Arg Gln His Gln Thr Ser Ala Glu Asn Lys Asn Arg Gln Pro Thr 850 855 860 Glu Gly Asn Phe Ser Ser Ala Trp Ile Asp His Ser Val Gln Pro Lys 865 870 875 880 Asp Ser Asn Tyr Glu Tyr Met Val Phe Leu Asp Ala Thr Pro Glu Arg 885 890 895 Met Gly Glu Ile Ala Gln Lys Phe Arg Glu Asn Asn Gly Leu Tyr Gln 900 905 910 Val Leu Arg Lys Asp Lys Asp Val His Ile Ile Leu Asp Lys Leu Ser 915 920 925 Asn Val Thr Gly Tyr Ala Phe Tyr Gln Ser Ala Ser Ile Glu Asp Lys 930 935 940 Trp Ile Lys Lys Val Asp Lys Pro Ala Ile Val Met Thr His Arg Gln 945 950 955 960 Lys Asp Thr Leu Ile Val Ser Ala Val Thr Pro Asp Leu Asn Met Thr 965 970 975 Arg Gln Lys Ala Ala Thr Pro Val Ile Ile Asn Val Thr Ile Asn Gly 980 985 990 Lys Trp Gln Ser Ala Asp Lys Asn Ser Lys Val Lys Tyr Ser Val Ser 995 1000 1005 Gly Asp Asn Thr Glu Leu Thr Phe Thr Ser Tyr Phe Gly Ile Pro 1010 1015 1020 Gln Glu Ile Lys Leu Ser Pro Leu Pro 1025 1030 106915DNAArtificial SequenceShuttle plasmid containing BstBI to XbaI fragment with IE 4/5 operably linked to partial Vasculostatin sequence. 10atccagcaca gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct 60cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 120ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 180gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 240attgggaaga caatagcgat ccggaaccct taatataact tcgtataatg tatgctatac 300gaagttatta ggtccctcga cctgcaggca tggctaattc tgtcagccgt taagtgttcc 360tgtgtcactg aaaattgctt tgagaggctc taagggcttc tcagtgcgtt acatccctgg 420cttgttgtcc acaaccgtta aaccttaaaa gctttaaaag ccttatatat tctttttttt 480cttataaaac ttaaaacctt agaggctatt taagttgctg atttatatta attttattgt 540tcaaacatga gagcttagta cgtgaaacat gagagcttag tacgttagcc atgagagctt 600agtacgttag ccatgagggt ttagttcgtt aaacatgaga gcttagtacg ttaaacatga 660gagcttagta cgtgaaacat gagagcttag tacgtactat caacaggttg aactgcggat 720cttgcggccg gccgcaatcg ggcaaatcgc tgaatattcc ttttgtctcc gaccatcagg 780cacctgagtc gctgtctttt tcgtgacatt cagttcgctg cgctcacggc tctggcagtg 840aatgggggta aatggcacta caggcgcctt ttatggattc atgcaaggaa actcaaaata 900atacaagaaa agcccgtcac gggcttctca gggcgtttta tggcgggtct gctatgtggt 960gctatctgac tttttgctgt tcagcagttc ctgccctctg attttccagt ctgaccactt 1020cggattatcc cgtgacaggt cattcagact ggctaatgca cccagtaagg cagcggtatc 1080atcaacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 1140attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 1200ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 1260tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 1320aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 1380acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 1440aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 1500agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 1560ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 1620agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 1680tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 1740tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 1800attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 1860taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 1920aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 1980caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 2040gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 2100cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 2160tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 2220acttgcatcg atgaattgat ccgaagttcc tattctctag aaagtatagg aacttcgaat 2280tgtcgaggcc gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt 2340gaatcgatag tactaacata cgctctccat caaaacaaaa cgaaacaaaa caaactagca 2400aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctctatcg ataggtaccg 2460tcgacctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata 2520cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 2580tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 2640ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagcc 2700ggccattatg cacgaccccg ccccgacgcc ggcacgccgg gggcccgtgg ccgcggcccg 2760ttggtcgaac ccccggcccc gcccatccgc gccatctgcc atgggcgggg cgcgagggcg 2820ggtgggtccg cgccccgccc cgcatggcat ctcattaccg cccgatccgg cggtttccgc 2880ttccgttccg catgctaacg aggaacgggc agggggcggg gcccgggccc cgacttcccg 2940gttcggcggt aatgagatac gagccccgcg cgcccgttgg ccgtccccgg gccccccggt 3000cccgcccgcc ggacgccggg accaacggga cggcgggcgg cccaagggcc gcccgccttg 3060ccgccccccc attggccggc gggcgggacc gccccaaggg ggcggggccg ccgggtaaaa 3120gaagtgagaa cgcgaagcgt tcgcacttcg tcccaatata tatatattat tagggcgaag 3180tgcgagcact ggcgccgtgc ccgactccgc gccggccccg ggggcgggcc cgggcggcgg 3240ggggcgggtc tctccggcgc acataaaggc ccggcgcgac cgacgcccgc agacggcgcc 3300ggccacgaac gacgggagcg gctgcggagc acgcggaccg ggagcgggag tcgcagaggg 3360ccgtcggagc ggacggcgtc ggcatcgcga cgccccggct cgggatcggg atcgcatcgg 3420aaagggacac gcggacgcgg gggggaaaga cccgcccacc ccacccacga aacacagggg 3480acgcaccccg ggggcctccg acgacagaaa cccaccggtc cgcctttttt gcacgggtaa 3540gcaccttggg tgggcggagg agggggggac gcgggggtgg aggagggggg acgcgggggc 3600ggaggagggg ggacgcgggg gcggaggagg ggggacgcgg gggcggagga ggggggacgc 3660gggggcggag gagggggctc acccgcgttc gtgccttccc gcaggaggaa cgtcctcgtc 3720gataagctag cgcggccgca tcgataagct tcgagctagg atgaggggcc aggccgccgc 3780cccgggcccc gtctggatcc tcgccccgct gctactgctg ctgctgctgc tgggacgccg 3840cgcgcgggcg gccgccggag cagacgcggg gcccgggccc gagccgtgcg ccacgctggt 3900gcagggaaag ttcttcggct acttctccgc ggccgccgtg ttcccggcca acgcctcgcg 3960ctgctcctgg acgctacgca acccggaccc gcggcgctac actctctaca tgaaggtggc 4020caaggcgccc gtgccctgca gcggccccgg ccgcgtgcgc acctaccagt tcgactcctt 4080cctcgagtcc acgcgcacct acctgggcgt ggagagcttc gacgaggtgc

tgcggctctg 4140cgacccctcc gcacccctgg ccttcctgca ggccagcaag cagttcctgc agatgcggcg 4200ccagcagccg ccccagcacg acgggctccg gccccgggcc gggccgccgg gccccaccga 4260cgacttctcc gtggagtacc tggtggtggg gaaccgcaac cccagccgtg ccgcctgcca 4320gatgctgtgc cgctggctgg acgcgtgtct ggccggtagt cgcagctcgc acccctgcgg 4380gatcatgcag accccctgcg cctgcctggg cggcgaggcg ggcggccctg ccgcgggacc 4440cctggccccc cgcggggatg tctgcttgag agatgcggtg gctggtggcc ctgaaaactg 4500cctcaccagc ctgacccagg accggggcgg gcacggcgcc acaggcggct ggaagctgtg 4560gtccctgtgg ggcgaatgca cgcgggactg cgggggaggc ctccagacgc ggacgcgcac 4620ctgcctgccc gcgccgggcg tggagggcgg cggctgcgag ggggtgctgg aggagggtcg 4680ccagtgcaac cgcgaggcct gcggccccgc tgggcgcacc agctcccgga gccagtccct 4740gcggtccaca gatgcccggc ggcgcgagga gctgggggac gagctgcagc agtttgggtt 4800cccagccccc cagaccggtg acccagcagc cgaggagtgg tccccgtgga gcgtgtgctc 4860cagcacctgc ggcgagggct ggcagacccg cacgcgcttc tgcgtgtcct cctcctacag 4920cacgcagtgc agcggacccc tgcgcgagca gcggctgtgc aacaactctg ccgtgtgccc 4980agtgcatggt gcctgggatg agtggtcgcc ctggagcctc tgctccagca cctgtggccg 5040tggctttcgg gatcgcacgc gcacctgcag gcccccccag tttgggggca acccctgtga 5100gggccctgag aagcaaacca agttctgcaa cattgccctg tgccctggcc gggcagtgga 5160tggaaactgg aatgagtggt cgagctggag cgcctgctcc gccagctgct cccagggccg 5220acagcagcgc acgcgtgaat gcaacgggcc ttcctacggg ggtgcggagt gccagggcca 5280ctgggtggag acccgagact gcttcctgca gcagtgccca gtggatggca agtggcaggc 5340ctgggcgtca tggggcagtt gcagcgtcac gtgtggggct ggcagccagc gacgggagcg 5400tgtctgctct gggcccttct tcgggggagc agcctgccag ggcccccagg atgagtaccg 5460gcagtgcggc acccagcggt gtcccgagcc ccatgagatc tgtgatgagg acaactttgg 5520tgctgtgatc tggaaggaga ccccagcggg agaggtggct gctgtccggt gtccccgcaa 5580cgccacagga ctcatcctgc gacggtgtga gctggacgag gaaggcatcg cctactggga 5640gccccccacc tacatccgct gtgtttccat tgactacaga aacatccaga tgatgacccg 5700ggagcacctg gccaaggctc agcgagggct gcctggggag ggggtctcgg aggtcatcca 5760gacactggtg gagatctctc aggacgggac cagctacagt ggggacctgc tgtccaccat 5820cgatgtcctg aggaacatga cagagatttt ccggagagcg tactacagcc ccacccctgg 5880ggacgtacag aactttgtcc agatccttag caacctgttg gcagaggaga atcgggacaa 5940gtgggaggag gcccagctgg cgggccccaa cgccaaggag ctgttccggc tggtggagga 6000ctttgtggac gtcatcggct tccgcatgaa ggacctgagg gatgcatacc aggtgacaga 6060caacctggtt ctcagcatcc ataagctccc agccagcgga gccactgaca tcagcttccc 6120catgaagggc tggcgggcca cgggtgactg ggccaaggtg ccagaggaca gggtcactgt 6180gtccaagagt gtcttctcca cggggctgac agaggccgat gaagcatccg tgtttgtggt 6240gggcaccgtg ctctacagga acctgggcag cttcctggcc ctgcagagga acacgaccgt 6300cctgaattct aaggtgatct ccgtgactgt gaaacccccg cctcgctccc tgcgcacacc 6360cttggagatc gagtttgccc acatgtataa tggcaccacc aaccagacct gtatcctgtg 6420ggatgagacg gatgtaccct cctcctccgc ccccccgcag ctcgggccct ggtcgtggcg 6480cggctgccgc acggtgcccc tcgacgccct ccggacgcgc tgcctctgtg accggctctc 6540caccttcgat atcttaatca agcttggtac cgagctcgga tccactagtc cagtgtggtg 6600gaattctgca gatatccagc acagtggcgg ccgctcgagt ctagagggcc cgcggttcga 6660acaaaaactc atctcagaag aggatctgaa tatgcatacc ggtcatcatc accatcacca 6720ttgagtttat ctctagagct gagaacttca gggtgagttt ggggaccctt gattgttctt 6780tctttttcgc tattgtaaaa ttcatgttat atggaggggg caaagttttc agggtgttgt 6840ttagaatggg aagatgtccc ttgtatcacc atggaccctc atgataattt tgtttctttc 6900actttctact ctgtt 6915114594DNAArtificial Sequenceplasmid containing Nestin enhancer driven ICP34.5 11tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 60gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 120caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 180ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 240gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 300tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 360tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 420cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 480cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 540gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 600atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 660agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 720ccccgaaaag tgccacttgc atcgatgaat tgatccgaag ttcctattct ctagaaagta 780taggaacttc gaattgtcga cggatccnnn nnccgcggaa gacccaggcc gcctcgggtg 840taacgttaga ccgagttcgc cgggccggct ccgcgggcca gggcccgggc acgggcctcg 900ggccccaggc acggcccgat gaccgcctcg gcctccgcca cccggcgccg gaaccgagcc 960cggtcggccc gctcgcgggc ccacgagccg cggcgcgcca ggcgggcggc cgaggcccag 1020accaccaggt ggcgcacccg gacgtggggc gagaagcgca cccgcgcggg ggtcgcgggg 1080gtcgcggggg tcgcgggggt cgcgggggtc gcggggggct ccggcgcccc ctccccgccc 1140gcgcgtcgca ggcgcaggcg cgccaggtgc tccgcggtga cgcgcaggcg gagggcgagg 1200cgcggcggaa ggcggaaggg gcgcgagggg gggtgggagg ggtcagcccc gccccccggg 1260cccacgccgg gcggtggggg ccggggccgg ggggcggcgg cggtgggccg ggcctctggc 1320gccggctcgg gcggggggct gtccggccag tcgtcgtcat cgtcgtcgtc ggacgcggac 1380tcgggaacgt ggagccactg gcgcagcagc agcgaacaag aaggcggggg cccaccggcg 1440gggggcggcg gcggggcggc cgcgggcgcg ctcctgaccg cgggttccga gttgggcgtg 1500gaggttacct gggactgtgc ggttgggacg gcgcccgtgg gcccgggcgg ccgggggcgg 1560cgggggccgc gatggcggcg gcggcgggcc atgatcaagc tcatggcgcc gcgctctgct 1620tctggaaggc tgcgctccgc ggcgtggatg ctccggggaa agttgcgctc cgcggcaggg 1680atgctcctgg gaaggttgcg ctccgcggca gggatgctct ggggaaggct ggtcctggcc 1740gaggatcggg aacgcgccgc tcgctctgct tctcttgtct tcgcttgtct ctggatggaa 1800ccagatttgg ttctgagtag ctgtcagcgt ctggtgacct gctcgccgcc ctgcgccttt 1860aaggagtctt caccggcccc gcccactctc cgctgggcca atcagcgagc cggaggaggc 1920cttggggcca ggaatcttcc agcagtttcg cgtctggtgg agcttccccg cctcccttga 1980gtaatcggag ttgtgggttc cgcccttgtc cagaactctc cagaggtttc tggggttcac 2040tggagagtac ggattcctga gggggagggt gtggggaagt gctggtgcta ctagtgacac 2100tgttgctatg gcgacgcatt actaaggcct gtgtggaatg gacaagaaag atcacctcta 2160gctcggtgtt gtgtacagtt tgttgtgatt tgtggggttt cgccaactcg cacagttctg 2220aatatggggg ttaaaggcta aaacttaagg gctaaaactt ctccccgcca agtttaggag 2280acccagggag atgcctgggg gcgtgtccgg tgacgtgatc ctctccaatc gcgttacaat 2340ggcagtgctg cctctgacct catggactaa tttaggaact agaggctctg tcccagcaca 2400ggctcaaagt tgccgggagg ggcggggtgg ggggtggggg ggaccccggc tgctcagttt 2460ggatgttcct ggagctcggt acccgcgatc gcccctagag gatctactag tcatatgata 2520agcctgaacc tcgtccaggt gtctgcaacc gagagttctc agcctccagc agagtcctgg 2580tggggagtgg ggagataggg tcagctccag ctgaggtagc atgtcctgcc actgcaggat 2640caatctctat tgtgaccatt gtcatataaa agccacacag tcatataccc acagatatat 2700acttagccaa cccatatttg agacacaggg agaccccaca tgcagattcc cacagtcgga 2760ggcagggcca aatgaattgc taacacttat atcagactcc tcagatcagt ctccgcctcc 2820ccacccaagg ccaaggccga tgacctcatc ctctgggagg gaggccgatt ctcatgctaa 2880ttattgcctt ttgtccacac taccatctgg agggcctaag aagggagggc tcctcagggg 2940aagtgggaat tctcaggctg ttcccagggg atggctctct ctctgccccc agagctggta 3000acagacaaaa gcaaatgaat tcagctcccc ttctccaaat ccttttcaga cctcaaacgc 3060cagtggttac attcctcaga gctgcctgga cccttcccct cagaggactg actggggcta 3120aagccctcat ctcaggatca caaactcttc agggnnngga tctcgagccc gggctagcac 3180gcgtaagagc tcggtaccta tcgatagaga aatgttctgg cacctgcact tgcactgggg 3240acagcctatt ttgctagttt gttttgtttc gttttgtttt gatggagagc gtatgttagt 3300actatcgatt cacacaaaaa accaacacac agatgtaatg aaaataaaga tattttattg 3360cggccgatcc ggaaccctta atataacttc gtataatgta tgctatacga agttattagg 3420tccctcgacc tgcaggcatg gctaattctg tcagccgtta agtgttcctg tgtcactgaa 3480aattgctttg agaggctcta agggcttctc agtgcgttac atccctggct tgttgtccac 3540aaccgttaaa ccttaaaagc tttaaaagcc ttatatattc ttttttttct tataaaactt 3600aaaaccttag aggctattta agttgctgat ttatattaat tttattgttc aaacatgaga 3660gcttagtacg tgaaacatga gagcttagta cgttagccat gagagcttag tacgttagcc 3720atgagggttt agttcgttaa acatgagagc ttagtacgtt aaacatgaga gcttagtacg 3780tgaaacatga gagcttagta cgtactatca acaggttgaa ctgcggatct tgcggccggc 3840cgcaatcggg caaatcgctg aatattcctt ttgtctccga ccatcaggca cctgagtcgc 3900tgtctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa 3960tggcactaca ggcgcctttt atggattcat gcaaggaaac tcaaaataat acaagaaaag 4020cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc tatctgactt 4080tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg gattatcccg 4140tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat caacggggtc 4200tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 4260gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 4320tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 4380ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 4440ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 4500tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 4560aactttatcc gcctccatcc agtctattaa ttgt 4594129292DNAArtificial SequencePlasmid with IE 4/5 - Vaculostatin with Nestin enhancer driven ICP34.5 cassette 12ctagaaagta taggaacttc ccatagagcc caccgcatcc ccagcatgcc tgctattgtc 60ttcccaatcc tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct 120actcagacaa tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc 180ttccagggtc aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg 240cacagtcgag gctgatcagc gggtttaaac tcaatggtga tggtgatgat gaccggtatg 300catattcaga tcctcttctg agatgagttt ttgttcgaac cgcgggccct ctagactcga 360gcggccgcca ctgtgctgga tatctgcaga attccaccac actggactag tggatccgag 420ctcggtacca agcttgatta agatatcgaa ggtggagagc cggtcacaga ggcagcgcgt 480ccggagggcg tcgaggggca ccgtgcggca gccgcgccac gaccagggcc cgagctgcgg 540gggggcggag gaggagggta catccgtctc atcccacagg atacaggtct ggttggtggt 600gccattatac atgtgggcaa actcgatctc caagggtgtg cgcagggagc gaggcggggg 660tttcacagtc acggagatca ccttagaatt caggacggtc gtgttcctct gcagggccag 720gaagctgccc aggttcctgt agagcacggt gcccaccaca aacacggatg cttcatcggc 780ctctgtcagc cccgtggaga agacactctt ggacacagtg accctgtcct ctggcacctt 840ggcccagtca cccgtggccc gccagccctt catggggaag ctgatgtcag tggctccgct 900ggctgggagc ttatggatgc tgagaaccag gttgtctgtc acctggtatg catccctcag 960gtccttcatg cggaagccga tgacgtccac aaagtcctcc accagccgga acagctcctt 1020ggcgttgggg cccgccagct gggcctcctc ccacttgtcc cgattctcct ctgccaacag 1080gttgctaagg atctggacaa agttctgtac gtccccaggg gtggggctgt agtacgctct 1140ccggaaaatc tctgtcatgt tcctcaggac atcgatggtg gacagcaggt ccccactgta 1200gctggtcccg tcctgagaga tctccaccag tgtctggatg acctccgaga ccccctcccc 1260aggcagccct cgctgagcct tggccaggtg ctcccgggtc atcatctgga tgtttctgta 1320gtcaatggaa acacagcgga tgtaggtggg gggctcccag taggcgatgc cttcctcgtc 1380cagctcacac cgtcgcagga tgagtcctgt ggcgttgcgg ggacaccgga cagcagccac 1440ctctcccgct ggggtctcct tccagatcac agcaccaaag ttgtcctcat cacagatctc 1500atggggctcg ggacaccgct gggtgccgca ctgccggtac tcatcctggg ggccctggca 1560ggctgctccc ccgaagaagg gcccagagca gacacgctcc cgtcgctggc tgccagcccc 1620acacgtgacg ctgcaactgc cccatgacgc ccaggcctgc cacttgccat ccactgggca 1680ctgctgcagg aagcagtctc gggtctccac ccagtggccc tggcactccg cacccccgta 1740ggaaggcccg ttgcattcac gcgtgcgctg ctgtcggccc tgggagcagc tggcggagca 1800ggcgctccag ctcgaccact cattccagtt tccatccact gcccggccag ggcacagggc 1860aatgttgcag aacttggttt gcttctcagg gccctcacag gggttgcccc caaactgggg 1920gggcctgcag gtgcgcgtgc gatcccgaaa gccacggcca caggtgctgg agcagaggct 1980ccagggcgac cactcatccc aggcaccatg cactgggcac acggcagagt tgttgcacag 2040ccgctgctcg cgcaggggtc cgctgcactg cgtgctgtag gaggaggaca cgcagaagcg 2100cgtgcgggtc tgccagccct cgccgcaggt gctggagcac acgctccacg gggaccactc 2160ctcggctgct gggtcaccgg tctggggggc tgggaaccca aactgctgca gctcgtcccc 2220cagctcctcg cgccgccggg catctgtgga ccgcagggac tggctccggg agctggtgcg 2280cccagcgggg ccgcaggcct cgcggttgca ctggcgaccc tcctccagca ccccctcgca 2340gccgccgccc tccacgcccg gcgcgggcag gcaggtgcgc gtccgcgtct ggaggcctcc 2400cccgcagtcc cgcgtgcatt cgccccacag ggaccacagc ttccagccgc ctgtggcgcc 2460gtgcccgccc cggtcctggg tcaggctggt gaggcagttt tcagggccac cagccaccgc 2520atctctcaag cagacatccc cgcggggggc caggggtccc gcggcagggc cgcccgcctc 2580gccgcccagg caggcgcagg gggtctgcat gatcccgcag gggtgcgagc tgcgactacc 2640ggccagacac gcgtccagcc agcggcacag catctggcag gcggcacggc tggggttgcg 2700gttccccacc accaggtact ccacggagaa gtcgtcggtg gggcccggcg gcccggcccg 2760gggccggagc ccgtcgtgct ggggcggctg ctggcgccgc atctgcagga actgcttgct 2820ggcctgcagg aaggccaggg gtgcggaggg gtcgcagagc cgcagcacct cgtcgaagct 2880ctccacgccc aggtaggtgc gcgtggactc gaggaaggag tcgaactggt aggtgcgcac 2940gcggccgggg ccgctgcagg gcacgggcgc cttggccacc ttcatgtaga gagtgtagcg 3000ccgcgggtcc gggttgcgta gcgtccagga gcagcgcgag gcgttggccg ggaacacggc 3060ggccgcggag aagtagccga agaactttcc ctgcaccagc gtggcgcacg gctcgggccc 3120gggccccgcg tctgctccgg cggccgcccg cgcgcggcgt cccagcagca gcagcagcag 3180tagcagcggg gcgaggatcc agacggggcc cggggcggcg gcctggcccc tcatcctagc 3240tcgaagctta tcgatgcggc cgcgctagct tatcgacgag gacgttcctc ctgcgggaag 3300gcacgaacgc gggtgagccc cctcctccgc ccccgcgtcc cccctcctcc gcccccgcgt 3360cccccctcct ccgcccccgc gtcccccctc ctccgccccc gcgtcccccc tcctccaccc 3420ccgcgtcccc ccctcctccg cccacccaag gtgcttaccc gtgcaaaaaa ggcggaccgg 3480tgggtttctg tcgtcggagg cccccggggt gcgtcccctg tgtttcgtgg gtggggtggg 3540cgggtctttc ccccccgcgt ccgcgtgtcc ctttccgatg cgatcccgat cccgagccgg 3600ggcgtcgcga tgccgacgcc gtccgctccg acggccctct gcgactcccg ctcccggtcc 3660gcgtgctccg cagccgctcc cgtcgttcgt ggccggcgcc gtctgcgggc gtcggtcgcg 3720ccgggccttt atgtgcgccg gagagacccg ccccccgccg cccgggcccg cccccggggc 3780cggcgcggag tcgggcacgg cgccagtgct cgcacttcgc cctaataata tatatatatt 3840gggacgaagt gcgaacgctt cgcgttctca cttcttttac ccggcggccc cgcccccttg 3900gggcggtccc gcccgccggc caatgggggg gcggcaaggc gggcggccct tgggccgccc 3960gccgtcccgt tggtcccggc gtccggcggg cgggaccggg gggcccgggg acggccaacg 4020ggcgcgcggg gctcgtatct cattaccgcc gaaccgggaa gtcggggccc gggccccgcc 4080ccctgcccgt tcctcgttag catgcggaac ggaagcggaa accgccggat cgggcggtaa 4140tgagatgcca tgcggggcgg ggcgcggacc cacccgccct cgcgccccgc ccatggcaga 4200tggcgcggat gggcggggcc gggggttcga ccaacgggcc gcggccacgg gcccccggcg 4260tgccggcgtc ggggcggggt cgtgcataat ggccggctgc ctcgcgcgtt tcggtgatga 4320cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 4380tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc 4440agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca 4500gagcagattg tactgagagt gcaccatagg tcgacggtac ctatcgatag agaaatgttc 4560tggcacctgc acttgcactg gggacagcct attttgctag tttgttttgt ttcgttttgt 4620tttgatggag agcgtatgtt agtactatcg attcacacaa aaaaccaaca cacagatgta 4680atgaaaataa agatatttta ttgcggcctc gacaattcga attgtcgacg gatccnnnnn 4740ccgcggaaga cccaggccgc ctcgggtgta acgttagacc gagttcgccg ggccggctcc 4800gcgggccagg gcccgggcac gggcctcggg ccccaggcac ggcccgatga ccgcctcggc 4860ctccgccacc cggcgccgga accgagcccg gtcggcccgc tcgcgggccc acgagccgcg 4920gcgcgccagg cgggcggccg aggcccagac caccaggtgg cgcacccgga cgtggggcga 4980gaagcgcacc cgcgcggggg tcgcgggggt cgcgggggtc gcgggggtcg cgggggtcgc 5040ggggggctcc ggcgccccct ccccgcccgc gcgtcgcagg cgcaggcgcg ccaggtgctc 5100cgcggtgacg cgcaggcgga gggcgaggcg cggcggaagg cggaaggggc gcgagggggg 5160gtgggagggg tcagccccgc cccccgggcc cacgccgggc ggtgggggcc ggggccgggg 5220ggcggcggcg gtgggccggg cctctggcgc cggctcgggc ggggggctgt ccggccagtc 5280gtcgtcatcg tcgtcgtcgg acgcggactc gggaacgtgg agccactggc gcagcagcag 5340cgaacaagaa ggcgggggcc caccggcggg gggcggcggc ggggcggccg cgggcgcgct 5400cctgaccgcg ggttccgagt tgggcgtgga ggttacctgg gactgtgcgg ttgggacggc 5460gcccgtgggc ccgggcggcc gggggcggcg ggggccgcga tggcggcggc ggcgggccat 5520gatcaagctc atggcgccgc gctctgcttc tggaaggctg cgctccgcgg cgtggatgct 5580ccggggaaag ttgcgctccg cggcagggat gctcctggga aggttgcgct ccgcggcagg 5640gatgctctgg ggaaggctgg tcctggccga ggatcgggaa cgcgccgctc gctctgcttc 5700tcttgtcttc gcttgtctct ggatggaacc agatttggtt ctgagtagct gtcagcgtct 5760ggtgacctgc tcgccgccct gcgcctttaa ggagtcttca ccggccccgc ccactctccg 5820ctgggccaat cagcgagccg gaggaggcct tggggccagg aatcttccag cagtttcgcg 5880tctggtggag cttccccgcc tcccttgagt aatcggagtt gtgggttccg cccttgtcca 5940gaactctcca gaggtttctg gggttcactg gagagtacgg attcctgagg gggagggtgt 6000ggggaagtgc tggtgctact agtgacactg ttgctatggc gacgcattac taaggcctgt 6060gtggaatgga caagaaagat cacctctagc tcggtgttgt gtacagtttg ttgtgatttg 6120tggggtttcg ccaactcgca cagttctgaa tatgggggtt aaaggctaaa acttaagggc 6180taaaacttct ccccgccaag tttaggagac ccagggagat gcctgggggc gtgtccggtg 6240acgtgatcct ctccaatcgc gttacaatgg cagtgctgcc tctgacctca tggactaatt 6300taggaactag aggctctgtc ccagcacagg ctcaaagttg ccgggagggg cggggtgggg 6360ggtggggggg accccggctg ctcagtttgg atgttcctgg agctcggtac ccgcgatcgc 6420ccctagagga tctactagtc atatgataag cctgaacctc gtccaggtgt ctgcaaccga 6480gagttctcag cctccagcag agtcctggtg gggagtgggg agatagggtc agctccagct 6540gaggtagcat gtcctgccac tgcaggatca atctctattg tgaccattgt catataaaag 6600ccacacagtc atatacccac agatatatac ttagccaacc catatttgag acacagggag 6660accccacatg cagattccca cagtcggagg cagggccaaa tgaattgcta acacttatat 6720cagactcctc agatcagtct ccgcctcccc acccaaggcc aaggccgatg acctcatcct 6780ctgggaggga ggccgattct catgctaatt attgcctttt gtccacacta ccatctggag 6840ggcctaagaa gggagggctc ctcaggggaa gtgggaattc tcaggctgtt cccaggggat 6900ggctctctct ctgcccccag agctggtaac agacaaaagc aaatgaattc agctcccctt 6960ctccaaatcc ttttcagacc tcaaacgcca gtggttacat tcctcagagc tgcctggacc 7020cttcccctca gaggactgac tggggctaaa gccctcatct caggatcaca aactcttcag 7080ggnnnggatc tcgagcccgg gctagcacgc gtaagagctc ggtacctatc gatagagaaa 7140tgttctggca cctgcacttg cactggggac agcctatttt gctagtttgt tttgtttcgt 7200tttgttttga tggagagcgt atgttagtac tatcgattca cacaaaaaac caacacacag 7260atgtaatgaa aataaagata ttttattgcg gccgatccgg aacccttaat ataacttcgt 7320ataatgtatg ctatacgaag ttattaggtc cctcgacctg caggcatggc taattctgtc 7380agccgttaag tgttcctgtg tcactgaaaa ttgctttgag

aggctctaag ggcttctcag 7440tgcgttacat ccctggcttg ttgtccacaa ccgttaaacc ttaaaagctt taaaagcctt 7500atatattctt ttttttctta taaaacttaa aaccttagag gctatttaag ttgctgattt 7560atattaattt tattgttcaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg 7620ttagccatga gagcttagta cgttagccat gagggtttag ttcgttaaac atgagagctt 7680agtacgttaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg tactatcaac 7740aggttgaact gcggatcttg cggccggccg caatcgggca aatcgctgaa tattcctttt 7800gtctccgacc atcaggcacc tgagtcgctg tctttttcgt gacattcagt tcgctgcgct 7860cacggctctg gcagtgaatg ggggtaaatg gcactacagg cgccttttat ggattcatgc 7920aaggaaactc aaaataatac aagaaaagcc cgtcacgggc ttctcagggc gttttatggc 7980gggtctgcta tgtggtgcta tctgactttt tgctgttcag cagttcctgc cctctgattt 8040tccagtctga ccacttcgga ttatcccgtg acaggtcatt cagactggct aatgcaccca 8100gtaaggcagc ggtatcatca acggggtctg acgctcagtg gaacgaaaac tcacgttaag 8160ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 8220gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 8280taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 8340tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 8400tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 8460gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 8520gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 8580ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 8640cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 8700tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 8760cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 8820agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 8880cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 8940aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 9000aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 9060gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 9120gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 9180tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 9240ttccccgaaa agtgccactt gcatcgatga attgatccga agttcctatt ct 9292

Claims

1. A recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding an angiostatic polypeptide operably linked to an immediate early HSV promoter IE4/5.

2. The recombinant expression vector of claim 1, wherein: the vector is a modified herpes simplex virus.

3. The recombinant expression vector of claim 2, wherein: the modified herpes simplex virus is a mutant herpes simplex virus deficient for both copies of its native γ.sub.134.5 gene.

4. The recombinant viral expression vector of claim 1, wherein: the angiostatic polypeptide comprises a proteolytic polypeptide fragment of BAI1.

5. The recombinant expression vector of claim 1, wherein: the nucleic acid encoding the angiostatic polypeptide comprises the nucleotide sequence of SEQ ID NO: 1 or of a degenerate variant of SEQ ID NO: 1.

6-16. (canceled)

17. A composition, comprising: a vector comprising: a nucleic acid encoding the polypeptide Chase ABC operably linked to an immediate early HSV IE4/5 promoter.

18. (canceled)

19. The composition of claim 17, wherein said vector is a mutant Herpes Simplex Virus comprising a nucleic acid encoding a replacement γ.sub.134.5 gene inserted into an otherwise γ.sub.134.5-deleted viral genome, the replacement γ.sub.134.5 gene is operably linked to a tumor specific promoter.

20. The composition of claim 19, wherein, the tumor specific promoter comprises a nestin enhancer element.

21. A recombinant expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a Chase ABC polypeptide operably linked to an immediate early HSV promoter IE4/5.

22. The recombinant expression vector of claim 21, wherein: the vector is a modified herpes simplex virus.

23. The recombinant expression vector of claim 22, wherein: the modified herpes simplex virus is deficient for both copies of its native γ.sub.134.5 gene.

24. The recombinant expression vector of claim 21, wherein: the nucleic acid sequence encoding a Chase ABC polypeptide comprises the nucleotide sequence of SEQ ID NO: 6 or of a degenerate variant of SEQ ID NO: 6.

25. The recombinant expression vector of claim 23, wherein: the nucleic acid sequence encoding a Chase ABC polypeptide comprises the nucleotide sequence of SEQ ID NO: 6 or of a degenerate variant of SEQ ID NO: 6.

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