Abstract:
Methods of making vaccinia viruses for gene-directed prodrug therapy are disclosed and their use in the treatment of disease is provided. In particular, the anti-tumor effects of vaccinia viruses that are modified to express a prodrug-activating enzyme are disclosed.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to vaccinia viruses for gene-directed prodrug therapy and their use in the treatment of disease, in particular the treatment of tumours. 
       BACKGROUND OF THE INVENTION 
       [0002]    Gene-directed enzyme prodrug therapy (GDEPT) and virally directed enzyme prodrug therapies (VDEPT (Huber et al., Proc. Natl. Acad. Sci. 91, 8302-8306, 1994)) are suicide-gene therapy approaches that aim to increase the delivery of toxic metabolites to solid tumours. This aims to overcome one of the major problems associated with current therapies for cancer, i.e., the lack of specificity, resulting in harmful side effects to normal tissues, such as the gut lining and bone marrow. The term ‘GDEPT’ is used to include both the viral and non-viral delivery systems. In the first step, a gene encoding a foreign, prodrug-activating enzyme is delivered to tumour cells in such a fashion as to ensure its tumour-restricted expression. Subsequent systemic administration of an appropriate prodrug results in generation of toxic metabolites only at the tumour site and consequently tumour selective killing. 
         [0003]    The GDEPT system developed by the present inventors uses expression of the enzyme carboxypeptidase G2 (CPG2) from  Pseudomonas  strain RS16 (WO 96/03151). CPG2 activates prodrugs, such as benzoic acid nitrogen mustard prodrugs and phenol nitrogen mustard prodrugs, to release L-glutamic acid and an active cytotoxic drug. 
         [0004]    Non-viral gene delivery methods have been used to express the prodrug-activating enzyme in tumour cells in vitro (Marais et al., Cancer Res. 56, 4735-4942, 1996; Chen et al., Cancer Res. 55, 581-589, 1995) and various studies have shown that viral gene therapy can be used to achieve tumour-targeted expression of the prodrug-activating enzyme in vivo (Schepelmann et al., Cancer Res. 67, 4949-4955, 2007; Hedley et. al., Nat. Rev. Can. 7, 870-879, 2007). Both non-replicating viral vectors and conditionally replicating oncolytic viruses with tumour selectivity have been used as vectors in GDEPT; non-replicating retrovirus and conditionally replicating adenovirus have been used as vectors in GDEPT clinical trials (Hedley et. al., Nat. Rev. Can. 7, 870-879, 2007). 
         [0005]    Most viruses have one or more genes which enhance viral replication, termed virulence genes. Wild-type adenoviruses express several proteins which disrupt cell cycle regulators in normal cells in order to promote cell division, thus creating an intracellular environment that supports the adenoviral lifecycle. For example, adenoviral E1A binds the retinoblastoma tumour suppressor protein, and E1B sequesters p53 to promote cell division and virus replication. Hence, adenoviral vectors in which the E1B gene is mutated are selective for cells where p53 is non-functional, e.g. many cancer cells. Termed replication-conditional oncolytic adenovirus, such vectors have been used to deliver a wide selection of prodrug-activating enzymes to cancer cells in vivo (Jounaidi et al., Cur. Cancer Drug Targets. 7(3): 285-301, 2007). 
         [0006]    Suicide gene therapy of human colon carcinoma xenografts using an oncolytic adenovirus expressing CPG2, denoted AdV.hTERT-CPG2, has been previously presented (Schepelmann et al., Cancer Res. 67, 4949-4955, 2007). Nude mice with human colon carcinoma xenografts were treated with a single systemic administration of AdV.hTERT-CPG2 followed by six doses of the prodrug ZD2767P administered by injection at weekly intervals was shown to achieve significant antitumour efficacy. AdV.hTERT-CPG2 expresses E1A under the control of the human telomerase promoter, allowing it to replicate and express CPG2 in telomerase-positive tumour cells. A direct correlation between viral cytotoxicity and CPG2 production was shown in each colorectal carcinoma cell line that was tested. Whilst other studies had found prodrug activation to abolish the replication of vaccinia virus vectors used for GDEPT-type systems (Puhlmann et al., Hum. Gene Therapy, 10, 649-57, 1999; McCart et al., Gene Therapy, 7, 1217-23, 2000), Schepelmann et al. found that the adenoviral vector AdV.hTERT-CPG2 was found to replicate more effectively in tumours in animals that had received the prodrug, compared with the tumours in animals that had not received the prodrug. 
         [0007]    Despite progress in the field, adenovirus-based cancer therapies have achieved only limited therapeutic potency in clinical trials. It remains a problem in the art to provide further effective anticancer therapies. 
       SUMMARY OF THE INVENTION 
       [0008]    Broadly, the present invention is based on the unexpected finding that vaccinia viruses that are modified to express a prodrug-activating enzyme are capable of eliciting a more potent anti-tumour effect than the oncolytic adenoviral vector AdV.hTERT-CPG2. Vaccinia virus, which is a member of the poxvirus family, is a highly immunogenic DNA virus that was used to eradicate smallpox. Like adenovirus, poxviruses express virulence genes which can disrupt cell cycle in normal cells in order to transform cells to promote cell division. Vaccinia virus and other poxviruses, such as myxoma, have a degree of natural affinity for tumour cells due to the dysregulation of the cell cycle in tumour cells, and this selectivity for tumour cells can be enhanced by deleting certain poxvirus virulence genes. For example, vaccinia tumour selectivity can be increased by the inactivation of virulence genes, e.g. the deletion of the viral thymidine kinase gene (Hedley et. al., Nat. Rev. Can. 7, 870-879, 2007). The present inventors have developed a modified vaccinia virus which expresses a prodrug-activating enzyme for use in a GDEPT system for treating tumours. 
         [0009]    Accordingly, in a first aspect, the present invention provides a vaccinia virus for use in a method of treatment of a tumour in a subject,
       wherein the vaccinia virus is capable of expressing a glutamate carboxypeptidase within a cell,   wherein the method includes the administration of a prodrug to the subject, wherein the prodrug is capable of being converted into an active form by the glutamate carboxypeptidase.       
 
         [0012]    The vaccinia virus may be genetically modified to have enhanced tumour selectivity. The genetic modification may take the form of an inactivation mutation of one or more vaccinia virulence genes e.g., a deletion, an insertion or a substitution mutation. The inactivated virulence gene may include inactivated vaccinia virus thymidine kinase (vTK). The vaccinia virus may have additional virulence genes inactivated, e.g., the deletion of vaccinia growth factor (VGF). 
         [0013]    vTK may be inactivated by virtue of the insertion of a CPG2 expression cassette, which both expresses CPG2 or a mutant, variant or homologue thereof, and also inactivates the vTK gene by insertion mutation. vTK may additionally comprise a luciferase expression cassette insert. When both are present, the CPG2 expression cassette and the luciferase expression cassette may be adjacent. Preferably, the CPG2 expression cassette and the luciferase expression cassette are adjacent to each other with their respective transcriptional promoters adjacent and priming in opposite directions. 
         [0014]    Alternatively, a CPG2 expression cassette may be inserted elsewhere in the vaccinia virus genome, e.g. in another virulence gene. 
         [0015]    The tumour to be treated may be a solid tumour. The tumour may be a head and neck cancer, colorectal cancer, lung cancer, melanoma, or breast cancer, cervix cancer, CNS cancer, ovarian cancer, kidney cancer, leukemia, brain tumour, or prostate cancer. Preferably, the tumour is selected from head and neck cancer, colorectal cancer, lung cancer, melanoma, or breast cancer. 
         [0016]    The volume of the tumour treated according to the invention will preferably decrease following administration of the prodrug. Preferably, the subject has a chance of survival which will increase following administration of the prodrug. 
         [0017]    The vaccinia virus may be administered to the subject by intravenous or intratumoural injection. The method of treatment may include the administration of radiotherapy to the subject. Further anticancer agents may also be administered to the subject, e.g. Chk1 inhibitors. 
         [0018]    The glutamate carboxypeptidase may comprise CPG2 or a mutant, variant or homologue thereof. The CPG2 may have an amino acid sequence which is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identical to SEQ ID NO:2. 
         [0019]    The prodrug may comprise a glutamic acid group or a glutamic acid analogue group, the glutamic acid group or the glutamic acid analogue being connected via N to a linker which is connected to a cytotoxic agent. The linker may comprise a functional group selected from: carbonyl, carbamate, urea, and equivalents. The cytotoxic agent may comprise a nitrogen mustard or a nitrogen mustard analogue. The prodrug may be N-(4-[bis(2-iodoethyl)amino]phenoxycarbonyl)-L-glutamic acid or a salt thereof. 
         [0020]    In a further aspect, the invention provides a vaccinia virus which is capable of expressing a glutamate carboxypeptidase within a cell. The vaccinia virus may comprise CPG2 or a mutant, variant or homologue thereof. The CPG2 may have an amino acid sequence which is at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, or 100% identical to SEQ ID NO:2. The CPG2 gene may be expressed by a CPG2 expression cassette inserted into a vaccinia virus virulence gene. The vaccinia virus virulence gene may be vaccinia thymidine kinase (vTK), thereby effecting inactivation of vTK. The vaccinia virus may have additional virulence genes inactivated, e.g., the deletion of vaccinia growth factor (VGF). 
         [0021]    In another aspect, the invention provides a two component system for gene directed enzyme prodrug therapy which comprises: (a) a vaccinia virus which is capable of expressing a glutamate carboxypeptidase, and (b) a prodrug which can be converted into an active drug by a glutamate carboxypeptidase. 
         [0022]    In yet another aspect, a method of killing a neoplastic cell, the method comprising treating the neoplastic cell with: a vaccinia virus which is capable of expressing a glutamate carboxypeptidase; and, a prodrug which is capable of being converted into an active form by a glutamate carboxypeptidase. 
         [0023]    In a further aspect, the invention provides a method of treating a tumour in a patient in need of treatment by administering to the patient: a vaccinia virus which is capable of expressing a glutamate carboxypeptidase in a cell; and, a prodrug which is capable of being converted into an active form by the glutamate carboxypeptidase. 
         [0024]    Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. 
         [0025]    “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. 
         [0026]    Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0027]      FIG. 1 . CPG2-catalysed conversion of N-(4-[bis(2-iodoethyl)amino]phenoxycarbonyl)-L-glutamic acid to N-(4-[bis(2-iodoethyl)amino]phenol. 
           [0028]      FIG. 2 . Scheme of tumour cell death triggered by gene-directed enzyme prodrug therapy (GDEPT) following viral expression of a prodrug-activating enzyme in the tumour cells and administration of prodrug. 
           [0029]      FIG. 3 . Molecular biology steps in the generation of VV-CPG2. 
           [0030]      FIG. 4 . CPG2 expression in head &amp; neck FaDu xenografts after intratumoural (IT) or intravenous (IV) administration of AdV-hTert or VV-CPG2 vectors. 
           [0031]      FIG. 5 . AdV-hTert in GDEPT of FaDu head &amp; neck cancer xenografts. AdV-hTert delivered intratumouraly (IT). Tumour volume (mm 3 ) vs time (A); Percentage survival vs time (B). The top plot of (A) represents the control group. 
           [0032]      FIG. 6 . VV-CPG2 in GDEPT of FaDu head &amp; neck cancer xenografts. VV-CPG2 delivered intratumouraly (IT). Tumour volume (mm 3 ) vs time (A); Percentage survival vs time (B). The lowest plot of (A) and the upper-right plot of (B) represent the GDEPT group. 
           [0033]      FIG. 7 . AdV-hTert in GDEPT of FaDu head &amp; neck cancer xenografts. AdV-hTert delivered intravenously (IV). Tumour volume (mm 3 ) vs time (A); Percentage survival vs time (B). The top plot of (A) and the lowest plot of (B) represent the control group. 
           [0034]      FIG. 8 . VV-CPG2 in GDEPT of FaDu head &amp; neck cancer xenografts. VV-CPG2 delivered intravenously (IV). Tumour volume vs time (mm 3 ) (A); Percentage survival vs time (B). The lowest plot of (A) and the upper-right plot of (B) represent the GDEPT group. 
           [0035]      FIG. 9 . VV-CPG2 in GDEPT of FaDu head &amp; neck cancer xenografts in combination with radiotherapy (RT). VV-CPG2 delivered intratumouraly (IT). Tumour volume vs time (mm 3 ) (A); Percentage survival vs time (B). The lower plot of (A) (extending to 160 days) and the upper-right plot of (B) represent the RT+GDEPT group. 
           [0036]      FIG. 10 . VV-CPG2 in GDEPT of FaDu head &amp; neck cancer xenografts in combination with radiotherapy (RT). VV-CPG2 delivered intravenously (IV), tumour volume vs time. The lowest plot represents the RT+GDEPT group. 
           [0037]      FIG. 11 . MTS viability assay of WM266.4 cells treated with ZD2767P prodrug, either untransduced (RHS y-axis) or transduced with VV-CPG2 at MOI 0.1 (LHS y-axis). 
           [0038]      FIG. 12 . MTS viability assay of FaDu cells treated with ZD2767P prodrug, either untransduced (RHS y-axis) or transduced with VV-CPG2 at MOI 0.1 (LHS y-axis). 
           [0039]      FIG. 13 . MTS viability assay of SW620 cells treated with ZD2767P prodrug, either untransduced (RHS y-axis) or transduced with VV-CPG2 at MOI 0.1 (LHS y-axis). 
           [0040]      FIG. 14 . MTS viability assay of A549 cells treated with ZD2767P prodrug, either untransduced (RHS y-axis) or transduced with VV-CPG2 at MOI 0.1 (LHS y-axis). 
           [0041]      FIG. 15 . CPG2 expression in Lung A549 xenografts after intravenous (IV) administration of VV-CPG2 vectors (A); and CPG2 expression in colorectal SW620 xenografts after intravenous (IV) administration of VV-CPG2 vectors (B). 
           [0042]      FIG. 16 . Tumour/tissue CPG2 expression after intravenous (IV) administration of VV-CPG2 vectors in mice with head &amp; neck FaDu xenografts (A); colorectal SW620 xenografts (B); and lung cancer A549 xenografts (C). Activity is expressed in unit activity per gram (for solid tissues) and unit activity per ml (for plasma). 
           [0043]      FIG. 17 . VV-CPG2 in GDEPT of lung cancer A549 xenografts. VV-CPG2 delivered intravenously (IV). Tumour volume vs time (mm 3 ) (A); Percentage survival vs time (B). The lowest plot of (A) and the upper plot of (B) represent the GDEPT group. 
       
    
    
     DETAILED DESCRIPTION 
     Gene-Directed Enzyme Prodrug Therapy (GDEPT) 
       [0044]    GDEPT is a two-step treatment for tumours. Foreign, prodrug-activating enzymes are delivered to, and expressed in, target cells where they can activate subsequently administered non-toxic prodrugs to form active drugs.  FIG. 2  shows a scheme of a GDEPT system in which, in a first step, a tumour-infecting virus is used to deliver the gene expressing the prodrug-activating enzyme. In the second step, a prodrug is administered that can be activated to form a toxic drug by the enzyme that has been expressed in the tumour. The prodrug-activating enzyme gene should be expressed exclusively, or with a relatively high ratio, in tumour cells compared with normal tissues and blood, and should achieve a sufficient concentration for clinical benefit. After gene delivery, prodrug administration should be delayed to permit protein expression in the targeted cells. The catalytic activity of the expressed enzyme should be sufficient for activation of the prodrug. Since expression of the prodrug-activating enzymes will not occur in all cells of a targeted tumour in vivo, a bystander cytotoxic effect is beneficial, whereby the prodrug is cleaved to an active drug that kills not only tumour cells but also neighbouring non-expressing tumour cells. This means that expression in less than 100% of tumour cells can still result in killing of all tumour cells. The prodrug-activating enzyme is usually expressed intracellularly, but it may be a membrane-tethered variant that is directed to the cell surface. 
       Enzyme Systems 
       [0045]    A number of different prodrug-activating enzymes have been used in GDEPT therapy. For example, cytosine deaminase, HSV thymidine kinase and cytochrome P450 have been used in GDEPT (Hedley et. al., Nat. Rev. Can. 7, 870-879, 2007). Any one of these enzymes can be used with vaccinia virus according to the present invention. 
         [0046]    The GDEPT system developed by the present inventors uses the enzyme carboxypeptidase G2 (CPG2) from  Pseudomonas  strain RS16. Peptidases are a class of enzymes which act upon a substrate to cleave a peptide (amide) bond; carboxypeptidases cleave peptide bonds at the carboxy-terminal of a protein or peptide. 
         [0047]    In some embodiments of the present invention, the prodrug-activating enzyme is a carboxypeptidase, which converts the prodrug into an active drug by removing a protecting group from the prodrug. The prodrug-activating enzyme may be a glutamate carboxypeptidase, e.g., CPG1 or CPG2, which preferentially cleave glutamate and glutamate analogues from the prodrug. 
         [0048]    The preferred enzyme is truncated carboxypeptidase CPG2 from  Pseudomonas  strain RS16 (disclosed in WO88/07378), having the sequence shown in SEQ ID NO:2, although full-length CPG2, membrane anchored CPG2, other mutants, orthologues, homologues or variants of CPG2 may also be used. In the context of this application, a CPG2 homologue is an enzyme that shares a common ancestry with CPG2, which is able to convert a prodrug to an active drug at substantially the same rate as the CPG2 of SEQ ID NO:2. In the context of this application, CPG2 mutants have an amino acid sequence consisting of at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% of SEQ ID NO:2 and also optionally comprise a further amino acid sequence derived from another protein. Variants may comprise an amino acid sequence which has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:2. Variants may be encoded by a nucleotide sequence which has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:1. Sequence comparison may be made over the full-length of the relevant sequence shown herein, or may more preferably be over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267, 300, 333, or more amino acids or nucleotide triplets, compared with the relevant amino acid sequence or nucleotide sequence as the case may be. Alterations to the sequence will be such that the enzyme retains its ability to convert a prodrug to an active drug at substantially the same rate as the native enzyme. In this context, “substantially the same rate” will desirably be within 1 order of magnitude, and preferably from about 50-fold e.g. about 2-fold less to 2, 5 or 10 fold more. 
         [0049]    CPG2 variants used according to the invention may be engineered to be directed to the cell membrane (i.e. membrane-tethered CPG2), as described in WO 01/085960, to enable prodrugs with poor intracellular availability to be activated in the extracellular space. 
         [0050]    Alternatively, other bacterial carboxypeptidase enzymes may be used, e.g., CPG2 enzymes from other  Pseudomonas  species such as  Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Pseudomonas savastanoi . Preferred carboxypeptidases will have one or more of the following properties: immunological cross-reactivity with an antibody reactive to the polypeptide for which the sequence given in SEQ ID NO:2; sharing an epitope with the polypeptide for which the amino acid sequence is shown in SEQ ID NO:2 (as determined for example by immunological cross-reactivity between the two polypeptides); a biological activity which is inhibited by an antibody raised against the polypeptide whose sequence is shown in SEQ ID NO: 2; ability to release L-glutamic acid from benzoic acid mustard prodrugs. Alteration of sequence may change the nature and/or level of activity and/or stability of the carboxypeptidase enzyme. 
         [0051]    A preferred substrate for CPG2 is an L-glutamic acid group, linked to an aromatic ring via an amidic, carbamic, or ureidic linkage. Glutamic acid analogs are also acceptable substrates. For example, L-glutamic acid modified at the γ-carbon (e.g., with an amide, —CONH 2 , instead of an acid, —COOH) also serves as a suitable substrate for CPG2. 
       Prodrugs 
       [0052]    A range of prodrugs have been used in GDEPT therapy. For example, 5-fluorocytosine has been used in cytosine deaminase GDEPT systems, ganciclovir has been used in HSV thymidine kinase GDEPT systems and cyclophosphamide has been used in Cytochrome P450 GDEPT systems (Hedley et. al., Nat. Rev. Can. 7, 870-879, 2007). Any one of these prodrug/enzyme systems can be used with vaccinia virus according to the present invention. 
         [0053]    The GDEPT system that the present inventors have developed uses the enzyme carboxypeptidase G2 (CPG2) to activate prodrugs such as benzoic acid nitrogen mustard prodrugs, aniline nitrogen mustard prodrugs and phenol nitrogen mustard prodrugs such as ZD2767P (N-(4-[bis(2-iodoethyl)amino]phenoxycarbonyl)-L-glutamic acid) and salts thereof. CPG2 cleaves the carbamate linkage of ZD2767P to release L-glutamic acid, a carbon dioxide molecule and the DNA alkylating nitrogen mustard N-(4-[bis(2-iodoethyl)amino]phenol, which is a potent cytotoxic agent.  FIG. 1  shows the CPG2-catalysed conversion of ZD2767P to produce N-(4-[bis(2-iodoethyl)amino]phenol. 
         [0054]    Nitrogen mustards are related to sulfur mustard, (ClCH 2 CH 2 ) 2 S, the “mustard gas” used during the First World War. Nitrogen mustards have the general formula (ClCH 2 CH 2 ) 2 NR. In vivo, each 2-chloroethyl side-chain undergoes an intramolecular cyclisation with the release of a chloride ion. The resulting highly reactive ethylene immonium derivative can interact with DNA and other molecules, for example, as an alkylating and/or crosslinking agent. Nitrogen mustards are useful, for example, in the treatment of proliferative conditions, such as cancer. 
         [0055]    Nitrogen mustard analogues, in which the chloro group is replaced by other groups, such as other halogens (e.g., bromo and iodo, as exemplified by ZD2767P) and other good leaving groups (e.g., sulfonates, such as mesyloxy, —OSO 2 Me) are also known, and are included in the class denoted “nitrogen mustards”. Nitrogen mustards may conveniently be grouped according to the group R. For example, two groups are phenolic nitrogen mustards and anilinic nitrogen mustards. 
         [0056]    In GDEPT systems, nitrogen mustards or other cytotoxic drugs are provided in the form of a prodrug, which has a protecting group linked to the cytotoxic moiety. In most cases, the protecting group will be cleaved as a whole from the prodrug. However, it is also possible for the enzyme to cleave or simply alter part of the protecting group, resulting in a partially cleaved or altered protecting group which is unstable, leading to spontaneous removal of the remainder of the group. 
         [0057]    A range of prodrugs which are suitable substrates for CPG2 have been described previously (WO 94/002450, WO 04/020400). A partial structure of typical substrates is; -aromatic-ring-X—CO—NH-Glu, where X is —NH—, —O— or —CH2, and Glu is glutamic acid or a glutamic acid analogue. 
         [0058]    Besides ZD2767P, other nitrogen mustard prodrugs which can be used according to the present invention in CPG2 GDEPT systems include:
   (S)-2-(4-[bis(2-chloroethyl)amino]phenoxycarbonylamino)-4-(1H-1,2,3,4-tetrazol-5-yl)butyric acid and salts thereof;   N-(4-[bis(2-chloroethyl)amino]-3-fluorophenylcarbamoyl)-L-glutamic acid and salts thereof;   N-(4-[bis(2-chloroethyl)amino]phenylcarbamoyl)-L-glutamic acid and salts thereof; and   N-(4-[bis(2-chloroethyl)amino]phenoxycarbonyl)-L-glutamic acid and salts thereof.   
 
         [0063]    WO 04/020400 describes “self-immolative” CPG2 substrates, which are defined as compounds that, following an activation process, generate an unstable intermediate that releases the active drug in a number of subsequent steps. Hence, CPG2 is able to activate these self-immolative prodrugs by cleaving or simply altering part of the prodrug protecting group, resulting in a partially cleaved or altered protecting group which is unstable, leading to spontaneous removal of the remainder of the group. 
         [0064]    The skilled person would understand that any of the prodrugs described herein, or any other compounds that can be used with the prodrug-activating enzymes considered herein, can be used according to the invention without undue burden. 
       Vaccinia Virus 
       [0065]    Vaccinia virus is a member of the poxviridae family of large DNA viruses, which typically have a genome of between 130-300 kbp in size, which is encoded on a single length of linear double-stranded DNA. To accommodate the relatively large quantity of genetic material, poxviruses have a large capsid having dimensions of around 200 nm by 300 nm. Their large DNA carrying capacity allows poxviruses to be engineered to deliver multiple transgenes and/or transgenes of a large size. 
         [0066]    Poxvirus virions take different forms at different stages of their lifecycle. The extracellular enveloped virion is the infectious particle which binds and enters the cell. The extracellular enveloped virion (EEV) has a viral core within a capsid particle, which is sheathed in an outer membrane. The outer membrane is removed upon cell entry, thus defining a first part of the two-part process of uncoating of the EEV. In the second part of the uncoating process, the viral core containing the viral DNA is released into the cytoplasm. 
         [0067]    Vaccinia genes are expressed in the cytoplasm, and this process takes place in several stages including early gene expression, intermediate gene expression and late gene expression. Genes which are necessary for genome replication, e.g. viral DNA-dependent RNA polymerase, are early phase genes. Genes which modulate the host anti-viral response and promote virus replication are also predominantly expressed during the early phase of infection. In contrast, viral structural components are mainly expressed during the late phase, with a relatively small group of genes thought to be important in activating late gene expression being expressed in the intermediate phase. Structural genes assemble in the cytosol to form the intracellular mature virion (IMV), which becomes coated with two membranes by the Golgi apparatus to form the intracellular enveloped virion (IEV). 
         [0068]    The IEV is translocated along the cytoskeletal microtubules to the cell periphery where it fuses with the cell membrane to form the cell-associated enveloped virion (CEV), which is released as EEV when the cell is lysed, causing cell death. Vaccinia virus according to the present invention will predominantly be in the form of EEV, however even the intracellular forms of the vaccinia virion are known to be infectious and are also considered to form a part of the invention. 
         [0069]    The vaccinia virus genome contains many virulence genes which function to modulate the host anti-viral response or disrupt the host cell cycle in order to promote intracellular conditions suitable for viral replication. For example, vaccinia viruses contain genes which express thymidine kinase, interferon-binding proteins, serine proteinase inhibitors and epidermal growth factor receptor (EGFR)-binding growth factors. Mutations to members of each of these classes of vaccinia virulence genes in order to enhance vaccinia selectivity for infecting tumour cells have been previously reported (Kirn et al., Nat. Rev. Can., 9, 64-71, 2009). Vaccinia virus having a deletion of the type I IFN-binding binding protein selectively replicates in tumours with a loss of interferon response. Deletion of the serine proteinase inhibitors SPI-1 and SPI-2, which interfere with host antiviral defence, results in vaccinia that preferentially replicate in transformed cells. Deletion of vaccinia thymidine kinase leads to dependence of the virus on cellular thymidine kinase expression, which is constitutively expressed in the majority of cancers, regardless of proliferation status (Kirn et al., Nat. Rev. Can., 9, 64-71, 2009). 
         [0070]    Vaccinia viruses have been shown to efficiently infect and kill a range of tumour types including lung cancer, cervix cancer and CNS cancer (A Ascierto et al., BMC Cancer, 11:451, 2011) and melanoma, ovarian cancer, kidney cancer, breast cancer, leukemia, non-small cell lung carcinoma, colon cancer, brain tumour, prostate cancer (Parato et al., Mol. Ther., 20(4), 749-758, 2012). 
         [0071]    The tumour selectivity of a virus can be measured or quantified by using standard techniques. For example, viral DNA copy number in tumour samples can be compared with viral DNA copy number in non-tumour samples by performing the polymerase chain reaction (PCR) on each sample, thereby allowing the skilled person to quantitatively assess the degree of tumour selectivity of a virus. The skilled person would understand that alternative techniques such as viral protein-specific Enzyme-Linked Immunosorbent Assay (ELISA) may also be used to determine and compare viral load in in tumour and non-tumour samples. 
       VV-CPG2 
       [0072]    The present inventors have found that vaccinia viruses which are modified to express CPG2 are capable of eliciting a potent anti-tumour response in vivo when used in GDEPT protocols together with a suitable prodrug. DNA expressing CPG2 under the control of the vaccinia p7.5 promoter was inserted into the vaccinia thymidine kinase (TK) gene, resulting in vaccinia virus which has inactivated thymidine kinase. The vaccinia growth factor (VGF) gene, which is homologous to epidermal growth factor, was also deleted. Furthermore, DNA expressing luciferase under the control of the vaccinia p syn  (E/L) promoter was also inserted to allow infected cells to be conveniently identified.  FIG. 3  shows the orientation of luciferase and CPG2 in the TK gene of VV-CPG2. 
       Examples 
     Materials and Methods 
     Cell Lines, Parental Virus and Plasmids 
       [0073]    Authenticated FaDu, SW620 and WM266.4 were obtained from American Type Culture Collection (LGC Promochem). FaDu cells are a telomerase-positive human carcinoma cell line. The cell lines used for virus production and characterization, CV1 cells (from American Type Culture Collection), 143B TK- (authenticated by STR profiling) and the parental virus, VSC20.VGF- and the shuttle vector, pSC65.lacZ.luc, based on pSC65 (Chakrabarti et al., 1997), were supplied by Jennerex Inc. Construction of the plasmid containing the CPG2 sequence and AdV-hTert have been previously described (Marais et al., 1996) (Schepelmann et al, 2005, 2007). 
       Construction of VV-CPG2 
       [0074]      FIG. 2  shows a schematic of the molecular biology steps in the cloning of the VV-CPG2 plasmid. 
         [0075]    pSC65.lacZ.luc was digested with XhoI and BamHI to remove the lacZ sequence and religated with the polylinker sequence TCGAGACGCGTGATATCATGCATACATGTCACGTGGAATTCACTAGTG (top) and GATCCACTAGTGAATTCCACGTGACATGTATGCATGATATCACGCGTC (bottom) to produce pSC65.polylinker.luc, which was digested with MluI and SpeI. pEF.CPG2 was modified to contain the adaptor-Kozak sequence ACGCGTGCCGCCACC (top) and GGTGGCGGCACGCGT (bottom) immediately upstream of the CPG2 ATG start codon, giving pEF.adaptor.CPG2, which was digested with MluI and XbaI. The linearized shuttle plasmid, pSC65.polylinker.luc, was religated with the excised CPG2 sequence to give pSC65.CPG2.luc, in which CPG2 and luc are downstream of the vaccinia promoters p7.5 and psyn (E/L), respectively. 
         [0076]    Recombination of pSC65.CPG2.luc with VSC20.VGF- and plaque purification of recombinants was performed according to standard protocols (Earl et al., 1998). Briefly, CV1 cells were infected with VSC20.VGF- and subsequently transfected with pSC65.CPG2.luc using Lipofectamine (Invitrogen). Bioluminescence indicated cellular colocalisation of vaccinia transcription factors and the luciferase gene. Recombinants were selected by three rounds of plaque purification in 143B TK- cells in the presence of bromodeoxyuridine, and validated by transgene region sequencing, bioluminescence and CPG2 activity assay (Stribbling et al., 1997). Recombinant virus was expanded to working stocks on CV1 cells, purified by sucrose gradient centrifugation and quantified by plaque titration. The engineered virus containing deletions of tk and vgf and insertions of cpg2 and luc was designated VV-CPG2. Insert fidelity was confirmed by dye-terminator cycle sequencing. 
         [0000]    Infection of Tumour Cell Lines with VV-CPG2 
         [0077]    The following cell lines were tested for susceptibility to transduction with VV-CPG2;
       SW620 and HT29: colorectal cancer cell lines   A549: lung cancer   FaDu: head and neck cancer   WM266.4: melanoma   MDA-MB-231: Breast cancer       
 
         [0083]    Each cell tumour cell line tested was found to express CPG2 following infection with VV-CPG2. For example, A549 cells, MDA-MB-231 cells and HT29 cells each transduced with VV-CPG2 at a multiplicity of infection (MOI) of 0.1 had CPG2 activity of 1 unit/mg (U/mg), 1.2 U/mg or 1.1 U/mg at 72 hours post-transduction, respectively. The reduction of cell viability in WM226.4 cells, FaDu cells, SW620 and A549 cells is shown in  FIGS. 11 to 14 . In each case, cell viability was measured by MTS assay. A reduction of the EC50 of ZD2767P in cells transduced with VV-CPG2 at MOI of 0.1 is: 114-fold (WM226.4 cells); 119-fold (SW620 cells); and 103-fold (FaDu cells). Hence, in each case, transduction with VV-CPG2 at MOI=0.1 results in over 100-fold increase in sensitivity to ZD2767P. 
       In Vivo Studies 
       [0084]    All animal work was compliant with Personal and Project UK Home Office License restrictions and UKCCCR guidelines (Workman, 1998). CD1 nu/nu athymic female mice were obtained from Charles River at 5-7 weeks of age, and allowed to acclimatize for one week prior to study. A specific pathogen-free environment was maintained. Animals had unlimited access to food and water and were housed at no more than five per cage. To establish tumour xenografts, cells in exponential growth phase were injected into the subcutaneous compartment of the right flank. FaDu cells were injected at 10 6  cells per animal in PBS. Interventions were initiated at 14 or 15 days after cell instillation in the presence of established tumours. A549 cells were injected at 10 7  cells per animal in PBS and Matrigel, in a 1:1 ratio. Interventions were initiated when tumours were approximately 100 mm 3 . SW620 cells were injected at 10 6  cells per animal in PBS. 
         [0085]    Animals were sacrificed by cervical dislocation when the mean tumour diameter exceeded 15 mm or any single diameter reached 17 mm, or in the presence of progressive tumour ulceration, as directed by project license conditions. 
         [0086]    For intratumoural studies AdV-hTert was administered at 4×10 8  plaque forming units/kg (PFU/kg) in 0.2 ml/20 g bodyweight, while VV-CPG2 was administered 4×10 7  PFU/kg by intratumoral injection in a total volume of 20 μl vehicle, using a systematic method adapted from published precedent (Bazan-Peregrino et al., 2008). For intravenous studies, AdV-hTert was administered at 4×10 9  PFU/kg in 0.2 ml/20 g bodyweight by tail vein injection. VV-CPG2 was also administered at 4×10 9  PFU/kg in 0.2 ml/20 g bodyweight by tail vein injection. ZD2767P was administered by intraperitoneal (IP) injection at 150-300 mg/kg in three divided doses at one hour intervals. Four to six prodrug administrations were performed over 4-8 weeks, guided by toxicity monitoring. In control groups, substance administrations were substituted by appropriate vehicles. Tumour caliper measurements were taken twice weekly and volumes calculated using the formula length×width×height×π/6. Tissue CPG2 activities were quantified by an indirect endpoint assay as previously described (Stribbling et al., 1997). Briefly, samples were homogenized and incubated with methotrexate for 30 minutes when the reaction was terminated. The concentration of the degradation product, DAMPA, was measured by high performance liquid chromatography. The end product concentration was converted to CPG2 activity by reference to standard curves, generated for each analysis using an alternative kinetic assay. Irradiation was performed using a Gammacell 40 Exactor (Best Theratronics, Ottawa, Canada), adapted to deliver unilateral localised tumor irradiation from a single caesium-137 source at 662 kV. Offline dosimetry was performed using Gafchromic EBT film (International Specialty Products, Vertec Scientific, UK), giving a current dose rate of 0.6 Gy per minute. In control groups, substance administrations were substituted by appropriate vehicles, and mock-irradiation was performed by sedation and placement of animals in the treatment chamber for similar duration. 
       Results 
       [0087]      FIG. 4  shows concentration of the CPG2 enzyme in tumours following intratumoural (IT) or intravenous (IV) injection of AdV-hTERT or VV-CPG2 in the FaDu human tumour xenograft model. CPG2 enzyme levels in the FaDu human tumour xenograft model, measured in units per gram of tumour tissue (CPG2 U/g), were determined via an activity endpoint assay, as described below.  FIG. 4  shows that similar levels of CPG2 activity are achieved following IV injection of VV-CPG2 in the FaDu human tumour xenograft model compared with AdV-hTERT administered at the same dose of 4×10 9  plaque forming units/kg. For IT administration in the FaDu human tumour xenograft model, a 4×10 7  pfu/kg VV-CPG2 is sufficient to achieve a similar level of CPG2 activity to that achieved by a dosage of 4×10 8  of AdV-hTERT in the FaDu human tumour xenograft model. 
         [0088]      FIGS. 5 and 6  show, in the FaDu human tumour xenograft model, the effect of GDEPT on tumour volume and animal survival following IT administration of the AdV-hTERT vector or the VV-CPG2 vector, respectively.  FIG. 5A  shows that AdV-hTERT-mediated GDEPT does not effect a reduction in tumour volume. The tumour volume of AdV-hTERT-GDEPT treated animals is not significantly different from the tumour volume of control animals or animals treated with the AdV-hTERT vector alone (without subsequent prodrug administration).  FIG. 5B  shows that animal survival following AdV-hTERT-mediated GDEPT is not extended; if anything, survival time is shortened. In contrast, in the FaDu human tumour xenograft model,  FIG. 6A  shows that VV-CPG2-mediated GDEPT results in a reduction in tumour volume which is significant and persistent over time, with tumour volume tending to zero in surviving animals.  FIG. 6B  shows that animal survival following VV-CPG2-mediated GDEPT is extended, with approximately 35% of VV-CPG2-GDEPT animals surviving for over 100 days. 
         [0089]      FIGS. 7 and 8  show, in the FaDu human tumour xenograft model, the effect of GDEPT on tumour volume and animal survival following IV administration of the AdV-hTERT vector and the VV-CPG2 vector, respectively.  FIG. 7A  shows that AdV-hTERT-mediated GDEPT does not effect a reduction in tumour volume. The tumour volume of AdV-hTERT-GDEPT treated animals is not significantly different from the tumour volume of control animals or animals treated with the AdV-hTERT vector alone (without subsequent prodrug administration).  FIG. 7B  shows that animal survival following AdV-hTERT-mediated GDEPT is only slightly extended.  FIG. 8A , in the FaDu human tumour xenograft model, shows that VV-CPG2-mediated GDEPT results in a reduction in tumour volume over time.  FIG. 8B  shows that animal survival following VV-CPG2-mediated GDEPT is extended, with approximately 50% VV-CPG2-GDEPT animals surviving for over 100 days. 
         [0090]      FIG. 9  shows the combination efficacy between VV-CPG2-mediated GDEPT and radiotherapy (RT) in the FaDu human tumour xenograft model. VV-CPG2 was delivered intratumouraly. All animals, except controls, received a 20 Gy radiation dose.  FIG. 9A  shows that RT in combination with the vector alone results in a stabilisation of tumour volume over time and that RT in combination with VV-CPG2-mediated GDEPT results in reduced tumour volumes, which tend to zero.  FIG. 9B  shows that over 75% of animals that received VV-CPG2-mediated GDEPT in combination with radiotherapy survived for over 150 days. 
         [0091]      FIG. 10  shows that VV-CPG2-mediated GDEPT in combination with radiotherapy (RT) results in stable tumour volume when VV-CPG2 is delivered intravenously. All animals, except controls, received a 10 Gy radiation dose. 
         [0092]    Other combinations of anticancer therapy may be envisaged as forming a part of the invention. For example, cell checkpoint inhibitors such as Chk1, known to sensitise tumours to cytotoxic agents, may be used alongside the GDEPT systems described herein for cancer therapies. 
         [0093]      FIG. 15  shows concentration of the CPG2 enzyme in (A) Lung A549 tumours and (B) colorectal SW620 tumours following intravenous (IV) injection of VV-CPG2. CPG2 enzyme levels, measured in units per gram of tumour tissue (CPG2 U/g), were determined via an activity endpoint assay.  FIG. 15  shows that similar levels of CPG2 activity are achieved in lung cancer models as with the head &amp; neck cancer model shown in  FIG. 4 . 
         [0094]      FIG. 16  illustrates the excellent tumour specificity of the VV-CPG2 vaccinia vector for head &amp; neck FaDu tumours (A), colorectal SW620 tumours (B), and lung cancer A549 tumours (C). 
         [0095]      FIG. 17A  shows the effect of GDEPT on tumour volume and animal survival following IV administration of the VV-CPG2 to animals with lung cancer A549 tumours. The tumour volume of control animals or animals treated with the VV-CPG2 vector alone (without subsequent prodrug administration) was only slightly reduced (central plot). This antitumoural effect is enhanced when the prodrug is also administered (lower plot). The ZD2767P prodrug was administered starting 4 days after VV-CPG2 injection by intraperitoneal (IP) injection at 150 mg/kg in three divided doses at one hour intervals. Six prodrug administrations were performed over 7 weeks. In control groups, substance administrations were substituted by appropriate vehicles. 
         [0096]      FIG. 17B  shows that animal survival following VV-CPG2-mediated GDEPT results in 100% survival (top line) compared with approximately 40% mortality of the control animals after 50 days. 
         [0000]    
       
         
               
             
               
             
           
               
                   
               
               
                 Sequence Listing 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 SEQ ID NO: 1 - CPG2* DNA sequence: 
               
               
                 atggccggatccgccctggcccagaagcgcgacaacgtgctgttccaggcagctaccgac 
               
               
                 gagcagccggccgtgatcaagacgctggagaagctggtcaacatcgagaccggcaccggt 
               
               
                 gacgccgagggcatcgccgctgcgggcaacttcctcgaggccgagctcaagaacctcggc 
               
               
                 ttcacggtcacgcgaagcaagtcggccggcctggtggtgggcgacaacatcgtgggcaag 
               
               
                 atcaagggccgcggcggcaagaacctgctgctgatgtcgcacatggacaccgtctacctc 
               
               
                 aagggcattctcgcgaaggccccgttccgcgtcgaaggcgacaaggcctacggcccgggc 
               
               
                 atcgccgacgacaagggcggcaacgcggtcatcctgcacacgctcaagctgctgaaggaa 
               
               
                 tacggcgtgcgcgactacggcaccatcaccgtgctgttcaacaccgacgaggaaaagggt 
               
               
                 tccttcggctcgcgcgacctgatccaggaagaagccaagctggccgactacgtgctctcc 
               
               
                 ttcgagcccaccagcgcaggcgacgaaaaactctcgctgggcacctcgggcatcgcctac 
               
               
                 gtgcaggtcaacatcaccggcaaggcctcgcatgccggcgccgcgcccgagctgggcgtg 
               
               
                 aacgcgctggtcgaggcttccgacctcgtgctgcgcacgatgaacatcgacgacaaggcg 
               
               
                 aagaacctgcgcttcaactggaccatcgccaaggccggcaacgtctcgaacatcatcccc 
               
               
                 gccagcgccacgctgaacgccgacgtgcgctacgcgcgcaacgaggacttcgacgccgcc 
               
               
                 atgaagacgctggaagagcgcgcgcagcagaagaagctgcccgaggccgacgtgaaggtg 
               
               
                 atcgtcacgcgcggccgcccggccttcaatgccggcgaaggcggcaagaagctggtcgac 
               
               
                 aaggcggtggcctactacaaggaagccggcggcacgctgggcgtggaagagcgcaccggc 
               
               
                 ggcggcaccgacgcggcctacgccgcgctctcaggcaagccagtgatcgagagcctgggc 
               
               
                 ctgccgggcttcggctaccacagcgacaaggccgagtacgtggacatcagcgcgattccg 
               
               
                 cgccgcctgtacatggctgcgcgcctgatcatggatctgggcgccggcaaggaattcctc 
               
               
                 gagatcgattagactagtctaga 
               
               
                   
               
               
                 SEQ ID NO: 2 - CPG2* aminoacidic sequence: 
               
               
                 MAGSALAQKRDNVLFQAATDEQPAVIKTLEKLVNIETGTGDAEGIAAAGNFLEAELKNLG 
               
               
                 FTVTRSKSAGLVVGDNIVGKIKGRGGKNLLLMSHMDTVYLKGILAKAPERVEGDKAYGPG 
               
               
                 IADDKGGNAVILHTLKLLKEYGVRDYGTITVLFNTDEEKGSFGSRDLIQEEAKLADYVLS 
               
               
                 FEPTSAGDEKLSLGTSGIAYVQVNITGKASHAGAAPELGVNALVEASDLVLRTMNIDDKA 
               
               
                 KNLRFNWTIAKAGNVSNIIPASATLNADVRYARNEDFDAAMKTLEERAQQKKLPEADVKV 
               
               
                 IVTRGRPAFNAGEGGKKLVDKAVAYYKEAGGTLGVEERTGGGTDAAYAALSGKPVIESLG 
               
               
                 LPGFGYHSDKAEYVDISAIPRRLYMAARLIMDLGAGKEFLEID*TSL 
               
               
                   
               
               
                 SEQ ID NO: 3 - pSC65.luc.lacZ: 
               
               
                 agcttttgcgatcaataaatggatcacaaccagtatctcttaacgatgttcttcgcagat 
               
               
                 gatgattcattttttaagtatttggctagtcaagatgatgaaatcttcattatctgatat 
               
               
                 attgcaaatcactcaatatctagactttctgttattattattgatccaatcaaaaaataa 
               
               
                 attagaagccgtgggtcattgttatgaatctctttcagaggaatacagacaattgacaaa 
               
               
                 attcacagactttcaagattttaaaaaactgtttaacaaggtccctattgttacagatgg 
               
               
                 aagggtcaaacttaataaaggatatttgttcgactttgtgattagtttgatgcgattcaa 
               
               
                 aaaagaatcctctctagctaccaccgcaatagatcctgttagatacatagatcctcgtcg 
               
               
                 caatatcgcattttctaacgtgatggatatattaaagtcgaataaagtgaacaataatta 
               
               
                 attctttattgtcatcatgaacggcggacatattcagttgataatcggccccatgttttc 
               
               
                 aggtaaaagtacagaattaattagacgagttagacgttatcaaatagctcaatataaatg 
               
               
                 cgtgactacaaaatattctaacgataatagatacggaacgggactatggacgcatgataa 
               
               
                 gaataattttgaagcattggaagcaactaaactatgtgatgtcttggaatcaattacaga 
               
               
                 tttctccgtgataggtatcgatgaaggacagttctttccagacattgttgaattagatcg 
               
               
                 ataaaaattaattaattacccgggtaccacatttgtagagagttttacttgctttaaaaa 
               
               
                 acctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaact 
               
               
                 tgtttattgcagcttacaatggttacaaataaagcaatagcatcacaaatttcacaaata 
               
               
                 aagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatc 
               
               
                 atgtctgctcgaagcggccggccgccccgactccagaattacacggcgatcttttccgcc 
               
               
                 cttcttgggcctttatgagagatctctctgatttttcttggcgtcgagttttccgggtaa 
               
               
                 gaccttttcggtactttcgtccacaaacacaactcctccgcgcaactttttcgcggttgt 
               
               
                 tacttgactggcgacgtaatccacgatctctttttccgtcatcgtctttccgtgctccaa 
               
               
                 aacaacaacggcggcgggaagttcaccggcgtcatcgtcgggaagacctgcgacacctgc 
               
               
                 gtcgaagatgttggggtgttggagcaagatggattccaattcagcgggagccacctgata 
               
               
                 gcctttgtacttaatcagagacttcaggcggtcaacgatgaagaagtgttcgtcttcgtc 
               
               
                 ccagtaagctatgtctccagaatgtagccatccatccttgtcaatcaaggcgttggtcgc 
               
               
                 ttccggattgtttacataaccggacataatcataggacctctcacacacagttcgcctct 
               
               
                 ttgattaacgcccagcgttttcccggtatccagatccacaaccttcgcttcaaaaaatgg 
               
               
                 aacaactttaccgaccgcgcccggtttatcatccccctcgggtgtaatcagaatagctga 
               
               
                 tgtagtctcagtgagcccatatccttgcctgatacctggcagatggaacctcttggcaac 
               
               
                 cgcttccccgacttccttagagaggggagcgccacccgtttgccatttcgtgtaaattag 
               
               
                 ataaatcgtatttgtcaatcagagtgcttttggcgaagaaggagaatagggttggcacca 
               
               
                 gcagcgcactttgaatcttgtaatcctgaaggctcctcagaaacagctcttcttcaatct 
               
               
                 atacattatgacgactcgaaatccacatatcaaatatccgagtgtagtaaacattccaaa 
               
               
                 accgtgatggaatggaacaacacttaaaatcgcagtatccggaatgatttgattgccaaa 
               
               
                 aataggatctctggcatgcgagaatctcacgcaggcagttctatgaggcagagcgacacc 
               
               
                 tttaggcagaccagtagatccagaggagttcatgatcagtgcaattgtcttgtccctatc 
               
               
                 gaaggactctggcacaaaatcgtattcattaaaaccgggaggtagatgagatgtgacgaa 
               
               
                 cgtgtacatcgactgaaatccctggtaatccgttttagaatccatgataataattttttg 
               
               
                 gatgattgggagctttttttgcacgttcaaaattttttgcaacccctttttggaaacgaa 
               
               
                 caccacggtaggctgcgaaatgcccatactgttgagcaattcacgttcattataaatgtc 
               
               
                 gttcgcgggcgcaactgcaactccgataaataacgcgcccaacaccggcataaagaattg 
               
               
                 aagagagttttcactgcatacgacgattctgtgatttgtattcagcccatatcgtttcat 
               
               
                 agcttctgccaaccgaacggacatttcgaagtactcagcgtaagtgatgtccacctcgat 
               
               
                 atgtgcatctgtaaaagcaattgctccaggaaccagggcgtatctcttcatagccttatg 
               
               
                 cagttgctctccagcggttccatcttccagcggatagaatggcgccgggcctttctttat 
               
               
                 gtttttggcgtcttccatggtaccaggcctagatctgtcgacttcgagcttatttatatt 
               
               
                 ccaaaaaaaaaaaataaaatttcaatttttaagctttcactaattccaaacccacccgct 
               
               
                 ttttatagtaagtttttcacccataaataataaatacaataattaatttctcgtaaaagt 
               
               
                 agaaaatatattctaatttattgcacggtaaggaagtagatcataactcgagcatgggag 
               
               
                 atcccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc 
               
               
                 ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcc 
               
               
                 cttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccag 
               
               
                 aagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcc 
               
               
                 cctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtaacctatccca 
               
               
                 ttacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacat 
               
               
                 ttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgtta 
               
               
                 actcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtt 
               
               
                 tgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtga 
               
               
                 tggtgctgcgttggagtgacggcagttatctggaagatcaggatatgtggcggatgagcg 
               
               
                 gcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatg 
               
               
                 ttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgt 
               
               
                 gcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcagg 
               
               
                 tcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccg 
               
               
                 atcgcgtcacactacgtctcaacgtcgaaaacccgaaactgtggagcgccgaaatcccga 
               
               
                 atctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaag 
               
               
                 cctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggca 
               
               
                 agccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtca 
               
               
                 tggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccg 
               
               
                 tgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcc 
               
               
                 tgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctga 
               
               
                 ccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcg 
               
               
                 atcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgcta 
               
               
                 atcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgccaggtgcagtatg 
               
               
                 aaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtgg 
               
               
                 atgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctac 
               
               
                 ctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttg 
               
               
                 gcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcg 
               
               
                 tctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcgg 
               
               
                 cttacggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctgg 
               
               
                 tctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttt 
               
               
                 tccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcata 
               
               
                 gcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtg 
               
               
                 aagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgc 
               
               
                 agccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccg 
               
               
                 catggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctca 
               
               
                 gtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatt 
               
               
                 tttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcac 
               
               
                 agatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtg 
               
               
                 caccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctggg 
               
               
                 tcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacgg 
               
               
                 cagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcagggga 
               
               
                 aaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgatta 
               
               
                 ccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgcc 
               
               
                 agctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatc 
               
               
                 ccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtata 
               
               
                 ccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatg 
               
               
                 gcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaac 
               
               
                 tgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcg 
               
               
                 acggtttccatatggggattggtggcgacgactcctggagcccgtcagtatcggcggaat 
               
               
                 tcagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaataataataacc 
               
               
                 gggcaggggggatccttctgtgagcgtatggcaaacgaaggaaaaatagttatagtagcc 
               
               
                 gcactcgatgggacatttcaacgtaaaccgtttaataatattttgaatcttattccatta 
               
               
                 tctgaaatggtggtaaaactaactgctgtgtgtatgaaatgctttaaggaggcttccttt 
               
               
                 tctaaacgattgggtgaggaaaccgagatagaaataataggaggtaatgatatgtatcaa 
               
               
                 tcggtgtgtagaaagtgttacatcgactcataatattatattttttatctaaaaaactaa 
               
               
                 aaataaacattgattaaattttaatataatacttaaaaatggatgttgtgtcgttagata 
               
               
                 aaccgtttatgtattttgaggaaattgataatgagttagattacgaaccagaaagtgcaa 
               
               
                 atgaggtcgcaaaaaaactgccgtatcaaggacagttaaaactattactaggagaattat 
               
               
                 tttttcttagtaagttacagcgacacggtatattagatggtgccaccgtagtgtatatag 
               
               
                 gatctgctcccggtacacatatacgttatttgagagatcatttctataatttaggagtga 
               
               
                 tcatcaaatggatgctaattgacggccgccatcatgatcctattttaaatggattgcgtg 
               
               
                 atgtgactctagtgactcggttcgttgatgaggaatatctacgatccatcaaaaaacaac 
               
               
                 tgcatccttctaagattattttaatttctgatgtgagatccaaacgaggaggaaatgaac 
               
               
                 ctagtacggcggatttactaagtaattacgctctacaaaatgtcatgattagtattttaa 
               
               
                 accccgtggcgtctagtcttaaatggagatgcccgtttccagatcaatggatcaaggact 
               
               
                 tttatatcccacacggtaataaaatgttacaaccttttgctccttcatattcagctgaaa 
               
               
                 tgagattattaagtatttataccggtgagaacatgagactgactcgggccgcgttgctgg 
               
               
                 cgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcaga 
               
               
                 ggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcg 
               
               
                 tgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgg 
               
               
                 gaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcggtgtaggtcgttc 
               
               
                 gctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccg 
               
               
                 gtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagcca 
               
               
                 ctggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggt 
               
               
                 ggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccag 
               
               
                 ttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcg 
               
               
                 gtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatc 
               
               
                 ctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt 
               
               
                 tggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtt 
               
               
                 ttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatca 
               
               
                 gtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccg 
               
               
                 tcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgatac 
               
               
                 cgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaaggg 
               
               
                 ccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgcc 
               
               
                 gggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctg 
               
               
                 caggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaac 
               
               
                 gatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtc 
               
               
                 ctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcac 
               
               
                 tgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtact 
               
               
                 caaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaa 
               
               
                 cacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgtt 
               
               
                 cttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaaccca 
               
               
                 ctcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaa 
               
               
                 aaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatac 
               
               
                 tcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcg 
               
               
                 gatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttcccc 
               
               
                 gaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaata 
               
               
                 ggcgtatcacgaggccctttcgtcttcgaataaatacctgtgacggaagatcacttcgca 
               
               
                 gaataaataaatcctggtgtccctgttgataccgggaagccctgggccaacttttggcga 
               
               
                 aaatgagacgttgatcggcacgtaagaggttccaactttcaccataatgaaataagatca 
               
               
                 ctaccgggcgtattttttgagttatcgagattttcaggagctaaggaagctaaaatggag 
               
               
                 aaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacatttt 
               
               
                 gaggcatttcagtcagttgctcaatgtacctataaccagaccgttcag 
               
               
                   
               
             
          
         
       
     
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