WO2010000270A1 - Prodrug activation therapy for the treatment of malignant brain tumours - Google Patents

Abstract

The present invention relates to a prodrug activation therapy comprised of a neural progenitor cell line comprising a deoxyribonucleoside kinase gene, such as Thymidine Kinase (TK) gene, and a nucleoside analog for the use as a medicament, in particular for the treatment of malignant glioma, such as glioblastoma multiforme (GBM).

Description

Prodrug activation therapy for the treatment of malignant brain tumours

The present invention relates to a prodrug activation therapy comprised of a neural progenitor cell line comprising a deoxyribonucleoside kinase gene, such as Thymidine Kinase (TK) gene, and a nucleoside analog for the use as a medicament, in particular for the treatment of malignant glioma, such as glioblastoma multiforme (GBM).

The present application claims priority from Danish patent application no. PA 2008 00947, filed 4 July 2008. All references cited in those applications and in the present application are hereby incorporated by reference in their entirety.

Background

Glioma is a type of central nervous system tumour that arises from glial cells. The most common site of involvement of glioma is the brain, and it only rarely spreads further. The current standard therapy involves surgically removing the solid tumor mass and initiating radiotherapy and/or chemotherapy. Even when the solid tumor mass has been removed, pre-cancerous or isolated cancerous cells can exist in the brain. In the majority of these patients, a new tumor grows and a repeated operation is frequently required. Malignant glioma tumours progress rapidly and are largely unresponsive to surgery, radio- and chemotherapy because of their exceptional migratory nature and ability to insinuate themselves seamlessly and extensively into normal brain tissue. Currently most available anti-cancer drugs are generally very toxic and many do not readily reach the brain tumor. Malignant brain tumours are an appealing target for suicide gene delivery.

Tumour cells are modified to express a deoxyribonucleoside kinase gene, such as a Thymidine Kinase (TK) gene, thereby acquiring the ability to convert a non-toxic nucleoside analog, e.g. Zidovudine (AZT), to its cytotoxic metabolite. Cells genetically engineered to express this "suicide" gene are eliminated if exposed to e.g. Zidovudine (AZT). This way tumour cells engineered to express the suicide gene as well as unmodified "native" tumour cells regress following nucleoside analog treatment without harm to adjacent normal tissue. This phenomenon, where a minority of TK-expressing cells lead to the death and elimination of adjacent native tumour cells not expressing TK, has been termed the "bystander effect". Malignant brain tumours are an appealing target for suicide gene delivery, since the entire malignancy is confined to the brain and amenable to eradication by the bystander effect. Key components for the success of this strategy are the genetic vector from which the suicide gene is expressed, its delivery vehicle and an effective nucleoside analog. As it is impossible to target all individual tumours in e.g. glioblastoma multiforme with separate injections of a gene therapy vector another delivery strategy is needed. Migrating cells that are capable of tracking down glioma cells and that have been engineered to deliver a therapeutic molecule represent an ideal solution to the problem of glioma cells invading normal brain tissue. It has been demonstrated that the migratory capacity of neural stem cells (NSCs) is ideally suited to therapy in neurodegenerative disease models that require brain-wide cell replacement and gene expression. Studies have also yielded the intriguing observation that transplanted NSCs are able to home into a primary tumour mass when injected at a distance from the tumour itself; furthermore, NSCs were observed to distribute themselves throughout the tumour bed, even migrating in juxtaposition to advancing single tumour cells (Dunn & Black, Neurosurgery 2003, 52:141 1-1424; Aboody et al, PNAS, 2000, 97:12846-12851 ).

The use of the neural progenitor cell (NPC) line NGC-407 in combination with tomato TK and the nucleoside analogue AZT for use in treatment of glioma multiforme in humans has been indicated in WO 2006/102902.

Summary of the invention

It is generally assumed that the suicide enzymes should activate their prodrugs to achieve at least a 100-fold increase in cytotoxic effect compared to the preactivated prodrug form ('Connors, T. A. The choice of prodrugs for gene directed enzyme prodrug therapy of cancer. Gene Ther., 2, 702-709, 1995/

Therefore it is expected that high expression of a thymidine kinase is required for the migrating cells to be effective in the treatment of e.g. cancer. The inventors of the present invention have surprisingly found that a human neural precursor cell line with a high expression (3.0 units or higher, where one unit (u) of thymidine kinase activity is defined as 1 nmol of the AZT monophosphate formed per minute, see Example 3) of a thymidine kinase is unstable regardless of the presence or absence of a nucleoside analog and thus not as useful in treatment of malignant glioma as expected, whereas a neural progenitor cell line with a relatively lower (0.12 - 0.017 units, app. 10 times above thymidine expression in parental cells) expression of a o

deoxyribonucleoside kinase, such as a thymidine kinase, remains stable while at the same time being able of reducing the size of malignant glioma tumours. The present invention relates to a neural progenitor or stem cell line expressing a deoxyribonucleoside kinase resulting in increase in sensitivity, when applying the assay disclosed in Example 2 of the present invention, in a level of not less than 5 fold and not more than 50 fold in combination with a nucleoside analog for the use as a medicament, such as for the treatment of cancer. In a particular embodiment the sensitivity is increased in a level of not less than 5 fold and not more than 40 fold, such as not less than 5 fold and not more than 30 fold, such as not less than 5 fold and not more than 20 fold, in combination with a nucleoside analog for the use as a medicament. In one embodiment the sensitivity is increased in a level of not less than 10 fold and not more than 50 fold in combination with a nucleoside analog for the use as a medicament, such as for the treatment of cancer. In a particular embodiment the sensitivity is increased in a level of not less than 10 fold and not more than 40 fold, such as not less than 10 fold and not more than 30 fold, such as not less than 10 fold and not more than 20 fold, in combination with a nucleoside analog for the use as a medicament. In another particular embodiment the sensitivity is increased in a level of not less than 20 fold and not more than 50 fold, such as not less than 20 fold and not more than 40 fold, such as not less than 20 fold and not more than 30 fold, in combination with a nucleoside analog for the use as a medicament. In yet another particular embodiment the sensitivity is increased in a level of not less than 30 fold and not more than 50, such as not less than 40 fold and not more than 50 fold, in combination with a nucleoside analog for the use as a medicament.

In a particular embodiment the cancer is glioma, such as ependymomas, astrocytomas, oligodendrogliomas or mixed gliomas. In an even more particular embodiment the astrocytomas is pilocytic astrocytoma, diffuse astrocytoma, malignant astrocytoma or glioblastoma multiforme.

In a particular embodiment the neural progenitor cell line is obtainable from or derived from or constituted by NGC-407 cells (WO 2006/102902). The cell line has been deposited by the applicant of WO 2006/102902 under the Budapest Treaty with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124 Braunschweig, Germany on the 31^st of March, 2005 under accession number DSM ACC2718. In a more particular embodiment the cell line is a polyclonal cell line or a monoclonal cell line, in particular a cell line being capable of differentiating into astrocytes or glia. In a preferred embodiment the deoxyribonucleoside kinase (dNK) expressed by the cell line is a thymidine kinase (TK) having an amino acid sequence which differs by 60 amino acids or less when compared to SEQ ID NO: 3 or SEQ ID NO: 18. In particular the said TK has an amino acid sequence which differs by 50 amino acids or less, such as by 40, 30, 20 or 15 amino acids or less when compared to SEQ ID NO: 3 or S EQ ID NO: 18. In a preferred embodiment the said TK has an amino acid sequence which differs by 10 amino acids or less, such as by 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acids or less when compared to SEQ ID NO: 3 or SEQ ID NO: 18. In a most preferred embodiment the said TK has an amino acid sequence comprising the amino acids of SEQ ID NO: 3 or SEQ ID NO: 18. In an even more preferred embodiment the said TK has an amino acid sequence consisting of or comprising the amino acids of SEQ ID NO: 3 or SEQ ID NO: 18.

In another preferred embodiment the dNK expressed by the cell line is a TK which has at least 70% identity to SEQ ID NO: 3 SEQ ID NO: 18. In a preferred embodiment the plant thymidine kinase has at least 75%, such as 80% or 85% identity to SEQ ID NO: 3 or SEQ ID NO: 18. In a more preferred embodiment the plant thymidine kinase has at least 90%, such as 95% or 96% or 97% or 98% or even 99% identity to SEQ ID NO: 3 or SEQ ID NO: 18. In a most preferred embodiment the plant thymidine kinase is identical to SEQ ID NO: 3 or SEQ ID NO: 18. The NGC-407 cell line has several advantages. It is a stable, immortalised cell line which has been expanded and has remained stable during more than 130 population doublings. The cell line is a neural progenitor cell line, which can differentiate into neurons, astrocytes and dopaminergic neurons depending on the differentiation conditions. The NGC-407 cell line can be used for transplantation. It has been shown that the cell line can survive transplantation for at least 3 weeks in rats. It is therefore expected that the NGC-407 cell line can survive for an even longer time in human brains. During the transplantation period, the cell line can stably express a heterologous gene.

The cell line has been transduced to express a heterologous thymidine kinase. These cell lines can be used as vehicles for delivery of thymidine kinase to tumour cells in the nervous system. It has also been shown that the NGC-407 cell line can migrate towards cancer cells in the central nervous system, and that the NGC-407 cell line can form gap junctions with cancer cells and transfer low molecular weight compounds from the cell line to the cancer cells. The NGC-407 cell line can therefore be used as a delivery vehicle to activate prodrugs (e.g. AZT, ganciclovir) after the cell line has migrated to cancer cells and formed gap junctions with these. The activated prodrugs will then be transferred to the cancer cells and kill both these and the delivery cell line. This is a feasible and promising way of treating glioblastoma multiforme.

The nucleoside analog used in the present invention is selected from the group consisting of Zidovudine (AZT); Ganciclovir (GCV); Acyclovir (ACV); Penciclovir;

Buciclovir; Gemcitabine; (dFdC); cladribine (CdA); Fludarabine (FaraA); Clofarabine;

Cytarabine (Arabinocytidine, (araC), Dideoxycytidine (ddC), Stavudine (D4T), 2', 3'- dideoxythymidine (ddT), fluorouridine (F-Urd).

Accordingly, in a most preferred embodiment the cell line NGC-407 has been transduced or transfected to express a heterologous thymidine kinase comprising the amino acids of SEQ ID NO: 3 or SEQ ID NO: 18, and is applied for the treatment of cancer in combination with a nucleoside analog selected from the group consisting of Zidovudine (AZT), Stavudine (D4T), 2'-3'-dideoxythimidine and fluorouridine.

In one embodiment of the present invention, "treatment", "therapy", and "medical use" is intended to cover prophylaxis. "Treatment", "therapy" and "medical use" may also cover inhibition of a disease or disorder, protection against a disease or disorder, and/or prevention (not absolute) of a disease or disorder. "Treatment", "therapy" and "medical use" may also comprise curative, ameliorative, and/or symptomatic treatment, therapy and medical use.

Brief description of the drawings

Figure 1 : In vivo migration and distribution of NGC-407 cells. The cartoon shows how the cells are injected, whereas the immunohistochemistry (marked b) shows positive (marked with GFP) NGC-407 cells that are infiltrating the tumor bed.

Figure 2 : TTK1 expressing neural stem cells of the invention improve survival in animals with human glioblastoma xenografts. Kaplan-Meier survival plots of nude rats upon exposure to azidothymidine (AZT) or vehicle for 21 days after intracranial implantation of U87MG glioblastoma cells and TTK1 expressing NGC-407 stem cells. NGC-407 and U87MG cells at the ratio of 1 :1 or 1 :3 or 1 :30 or U87MG cells alone were injected. Each group (n = 6) was then subgroupped to assign AZT or vehicle treatment. Comparison was made between these two treatment groups. ^* indicates P < 0.05 (α = 0.05, n = 3) in both comparisons (Log-rank (Mantel-Cox) Test). Animals with the clinical signs of decreased locomotion, decreased alertness, loss of weight and reduced food- intake, were sacrifized. Two animals died overnight. The clinical signs were observed by a person blinded to the groups and treatment paradigm. Each group contained three animals. Mean survival time is indicated on the figure.

Figure 3: MRI showing reduced size of glioblastoma xenograft after treatment with TTK1 expressing NGC-407 progenitor/stem cells and azidothymidine (AZT). Sagittal, coronal and axial images to simulate 3D imaging of brains from one animal of U87MG alone, 1 :30, 1 :3 and 1 : 1 (NGC-407: U87MG) groups are shown. The human glioblastoma cell line U87MG was used to create the intracranial xenografts in nude rats. TTK1 expressing NGC-407 cells were co-injected with U87MG cells at indicated ratios. Another group received only U87MG cells. Animals were treated with AZT or vehicle for 21 days starting 24 hours after implantation and the MRI experiments were performed on day 22 or 23 post-implantation. Scale bar is 1 cm.

Figure 4: Reduced tumor volume when stem cell-delivery of TTK1 into intracranial glioblastoma xenografts was present. Bar graphs show the volume of xenografts measured from multislice brain images of MRI experiments in sagittal, coronal and axial planes on day 22 or 23 post-implantation. The neural progenitor progenitor/stem cell line NGC-407 expressing TTK1 was co-implanted with the human glioblastoma cell line U87MG at 1 :30, 1 :3 (n = 6) or 1 :1 (n = 6) ratios. Each group of animals were then subgrouped and treated with azidothymidine (AZT) or vehicle for 21 days starting 24 hours after implantation. U87MG cells alone were injected in another group (n = 6) which was also treated similarly to examine the AZT effect in absence of TTK1. Comparisons were made between AZT and vehicle treatment. ^** indicates P < 0.01 (two-tailed, α = 0.05, n = 3) (Student's t-test for unpaired values). Values are the means ± SEM. Two animals in each of U87MG-alone and vehicle-treated 1 :3 groups reached the end point before the scheduled MRI experiments. Therefore, error bars in these groups are not presented. If those animals survived, mean tumor volume in each of these groups might be larger than the bar graph indicates. This suggests that the difference between AZT and vehicle treatment in the 1 :3 group is representative, if not even underestimated. It also indicates that there is likely no TTK1/AZT effect in U87MG-alone group.

Detailed description

Transfection or transduction of NGC-407 cell line In a preferred embodiment, cells derived from the NGC-407 cell line comprise, integrated into the genome and replicated together with the chromosome(s) into which it has been integrated, the heterologous DNA elements, in operable combination, of a eukaryotic promoter, a heterologous therapeutic gene, a polyadenylation signal (pA). The heterologous DNA elements may be of any suitable origin, but preferably selected among those described herein.

In a preferred embodiment, the heterologous therapeutic gene may be expressed under the transcriptional control of the human ubiquitin (UbC) promoter.

A possible down-regulation of expression may be circumvented by procedures that direct a site specific integration of the transgene and its accompanying promoter.

According to one embodiment of the invention, the promoter is a constitutive promoter selected from the group consisting of: ubiquitin promoter, CMV promoter, JeT promoter (US 6,555,674), SV40 promoter, Elongation Factor 1 alpha promoter (EF1- alpha), RSV, and Mo-MLV-LTR. Examples of inducible/repressible promoters include: Tet-On, Tet-Off,

Rapamycin-inducible promoter, Mx1.

Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, splicing signals for introns, and stop codons, all maintained in the correct reading frame of the polynucleotide of the invention so as to permit proper translation of mRNA. Expression control sequences may also include additional components such as leader sequences and fusion partner sequences.

Suitable expression vectors may be a viral vector derived from Herpes simplex, alphavirus, adenovirus, adeno associated virus, baculovirus, HSV, coronavirus, Bovine papilloma virus, Mo-MLV, preferably adeno associated virus, or from various bacterially produced plasmids.

Other transfection methods include, but are not limited to, liposome transfection, electroporation, and transfection with carrier peptides containing nuclear or other localising signals.

Other suitable expression vectors include general purpose mammalian vectors which are also obtained from commercial sources (Invitrogen Inc., Clontech, Promega,

BD Biosecences, etc) and contain selection for Geneticin/neomycin (G41 8), hygromycin B, puromycin, Zeocin/bleomycin, blasticidin Sl, mycophenolic acid or histidinol.

The vectors include the following classes of vectors: general eukaryotic expression vectors, vectors for stable and transient expression and epitag vectors as o

well as their TOPO derivatives for fast cloning of desired inserts (see list below for non- limiting examples of vectors).

Ecdysone-lnducible Expression: plND(SP1 ) Vector; plND/V5-His Tag Vector Set; plND(SP1 )A/5-His Tag Vector Set; EcR Cell Lines; Muristerone A. Stable Expression: pcDNA3.1/Hygro; PCI; PSI; pSecTag A, B & C; pcDNA3.1 (-

)/Myc H i s A, B & C; pcDNA3.1 +/-; pcDNA3.1/Zeo (+) and pcDNA3.1/Zeo (-); pcDNA3.1/His A, B, & C; pRc/CMV2; pZeoSV2 (+) and pZeoSV2 (-); pRc/RSV; pTracer™-CMV; pTracer™-SV40.

Transient Expression: pCDM8; pcDNA1.1 ; pcDNA1.1/Amp. Epitag Vectors: pcDNA3.1/MycHis A, B & C; pcDNA3.1/V5-His A, B, & C.

Neural stem and/or progenitor cells

The invention can be put to practise using any kind of neural stem and/or progenitor cells. Neural stem and progenitor cells are known in the art. The following is a non- limiting disclosure of suitable sources and methods for establishment of neural stem and/or progenitor cells and cell lines. Preferably the neural stem cells and/or progenitor cells are human.

A "neural stem cell" is a stem cell in the neural cell lineage. A stem cell is a cell which is capable of reproducing itself. In other words, daughter cells which result from stem cell divisions include stem cells. The neural stem cells are capable of ultimately differentiating into all the cell types in the neural cell lineage, including neurons, astrocytes and oligodendrocytes (astrocytes and oligodendrocytes are collectively called glia or glial cells). Thus, the neural stem cells referred to herein are multipotent neural stem cells.

For the purposes of this disclosure, the terms "neural progenitor cell" or "neural precursor cell" mean a cell that can generate progeny that are either neuronal cells (such as neuronal precursors or mature neurons) or glial cells (such as glial precursors, mature astrocytes, or mature oligodendrocytes). Typically, the cells express some of the phenotypic markers that are characteristic of the neural lineage. Typically, they do not produce progeny of other embryonic germ layers when cultured by themselves in vitro, unless dedifferentiated or reprogrammed in some fashion. A "neuronal progenitor cell" or "neuronal precursor cell" is a cell that can generate progeny that are mature neurons. These cells may or may not also have the capability to generate glial cells.

A "neurosphere" is a group of cells derived from a single neural stem cell as the result of clonal expansion. A "primary neurosphere" refers to the neurospheres generated by plating as primary cultures brain tissue which contains neural stem cells. The method for culturing neural stem cells to form neurospheres has been described in, for example, U.S. Pat. No. 5,750,376. A "secondary neurosphere" refers to the neurospheres generated by dissociating primary neurospheres and allowing the individual dissociated cells to form neurospheres again.

In embodiments of the invention, the neural stem cell or neural progenitor cell is derived from fetal brain, adult brain, neural cell culture, a neurosphere, tissue enclosed by dura mater, peripheral nerves, ganglia, pancreas, skin, muscle, adult bone marrow, umbilical cord tissue and umbilical cord blood. Preferably the cell line is a human cell line.

Recent developments in stem cell technology have enabled the isolation of neural stem cells from embryonic stem (ES) cell cultures. Methods of isolating neural stem from murine (Okabe et al. Mech Dev 59, 89-102. 1996) and human ES cells (Zhang et al. Nat Biotechnol 19, 1 129-1 133. 2001 and Reubinoff et al. Nat Biotechnol 19, 1 134- 1 140. 2001 ) have been previously described. These and other similar methods are based on culture conditions, which induce the formation of ES cell aggregates, which develop into so-called embryoid bodies (Martin et al. Dev. Biol. 61 . 230. 1977). In serum free defined media (Rizzino & Crowley Proc. Natl. Acad. Sci. USA 77. 457. 1980; Okabe et al. Mech. Dev. 59. 89. 1996) most non-neural elements of the embryoid body die and round cell aggregates, which are termed "neurospheres" appear. Neurospheres contain neural stem cells, which can be induced to differentiate into neurons and glia cells either in vitro or in vivo.

Multipotent neural stem cells, capable of producing progeny that differentiate into neurons and glia, exist in adult mammalian neural tissue. (Reynolds and Weiss, 1992). Methods have been provided for the proliferation of these stem cells to provide large numbers of neural cells that can differentiate into neurons and glia (See, e.g., US 5,750,376, and WO 93/01275). Various factors can be added to neural cell cultures to influence the make-up of the differentiated progeny of multipotent neural stem cell, as disclosed in WO 94/10292. Additional methods for directing the differentiation of stem cell progeny were disclosed in US 6,165,783 utilizing erythropoietin and various growth factors.

A neural stem cell is a stem cell found in adult neural tissue and may give rise to neurons, astrocytes, and oligodendrocytes. A review of neural stem cells is given in GaIIi et al., Circulation Research 92 (6):598; Gage F H. Science. 2000; 287: 1433- 1438.

Neural stem cells can be isolated from both the subventricular zone (SVZ), a thin layer of dividing cells that lies along the length of the lateral wall of the lateral ventricles, and the hippocampus, a cortical structure in the medial portion of the temporal lobe. Indeed, in the adult mammalian brain, the genesis of new neurons has been consistently documented in the subgranular layer of the dentate gyrus of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles (GaIIi et al., Circulation Research 92 (6):598; Gage F H. Science. 2000; 287: 1433-1438; Luskin M B. Neuron. 1993; 1 1 ; 173-189; Lois C, Alvarez-Buylla A. Science. 1994; 264: 1 145-1 148).

The SVZ contains four main cell types: newly generated neurons, astrocytes, rapidly dividing precursors, and ependymal cells. The rapidly dividing immature precursors are closely associated with the chains of newly generated neurons that migrate through the glial tubes formed by the processes of SVZ astrocytes. They are scattered in focal clusters along the network of chains. The multiciliated ependymal cells line the ventricular cavity. A series of observations indicate that a specific subtype of SVZ astroglial cells is the actual neural stem cell (Alvarez-Buylla A., J Neurosci. 2002; 22: 629-34).

It has been shown that NSCs can be isolated and grown in vitro from non-canonical neurogenic periventricular regions, in which the mature parenchyma is directly in contact with the ependymal monolayer, such as the fourth ventricle or the spinal cord (Johansson et al., Cell. 1999; 96: 25-34). The use of specific systems has permitted the isolation and expansion of NSCs ex vivo, and has allowed the establishment of NSC lines from various species, including humans (Gage, Science. 2000; 287: 1433-1438; McKay, Science. 1997; 276; 66-71 ; Gritti et al., Cultures of stem cells of the central nervous system. Chapter 14. In: Fedoroff S, Richardson A, eds. Protocols for Neural Cell Culture. 3rd ed. Totowa, N. J.: Humana Press; 2001 ).

Under appropriate conditions and in the presence of mitogens (epidermal growth factor (EGF) and/or fibroblast growth factor [FGF]), it is possible to induce the proliferation of rapidly dividing precursors from certain neurogenic areas of the adult mammalian brain, for example, the stratum, SVZ, hippocampus and olfactory bulb (Gritti et al., J. Neurosci 19, 3287-97 (1999); Gritti et al., J Neurosci 16, 1091-100 (1996)). At least in vitro, these precursors fulfill most of the criteria of bona fide stem cells (Gritti et al., J Physiol Paris. 2002; 96: 81-90; Vescovi et al., Brain Pathol. 1999; 9: 569-598; Weiss et al., Trends Neurosci. 1996; 19: 387-393). On removal of the mitogens, the progeny of NSCs promptly differentiate into the three main cell types of the CNS (astrocytes, oligodendrocytes, and neurons) (McKay R. Science (1997) 276: 66-71 ).

As in the primary culture, differentiating/differentiated cells rapidly die while the NSCs continue to proliferate, giving rise to many secondary spheres and exponential growth in vitro. In this way, stable though heterogeneous NSCs cell lines can be obtained (GaIIi et al., Circ. Res 92, 598-608 (2003)) Due to these properties, it has been possible to establish continuous mouse transgenic/genetically mod ified (GaIIi et al . , Development. 2002; 129: 1633-1644) or human NSC lines (Carpenter et al., Exp Neurol. 1999; 158: 265-278; Vescovi et al., Exp Neurol. 1999; 156: 71-83).

The cells of the present invention are cultured according to methods known in the art. Neural stem cells are typically derived from the CNS, often from the hippocampal or subventricular region, but can be derived from any suitable region of the brain or other source. The neural stem cells of the invention are isolated and cultured according to methods known in the art (see, e. g., Gage, et al., Proc Natl Acad Sc USA. 92 : 11879- 83 (1995); Palmer, et al., MoI Cell Neurosci. 8: 389-404 (1997); Ray, et al., Proc Natl Acad Sci USA. 90: 3602-6 (1993) ). Other methods of isolating neural stem cells are described, e. g., in US Patent publication 20030095956; US Patent publication 20030082160 ; US Patent publication 20020039724 ; US 6,767,738; US 6,498,018; US 6,071 ,889; US 5,980,885; US 5,968,829; US 5,851 ,832; and US 5,750,376. Established neural stem cell lines can be used as well as primary cultures.

Suitable cell culture methods and conditions can be determined by those of skill in the art using known methodology (see, e. g., Freshney et al., CULTURE OF ANIMAL CELLS (3rd ed. 1994)). In general, the cell culture environment includes consideration of such factors as the substrate for cell growth, cell density and cell contract, the gas phase, the medium, and temperature.

Typically plastic dishes or flasks are used. Other artificial substrates can be used such as glass and metals. The substrate is often treated by etching, or by coating with substances such as collagen, chondronectin, fibronectin, and laminin. The type of culture vessel depends on the culture conditions, e. g., multi-well plates, petri dishes, tissue culture tubes, flasks, and the like. Cells are grown at optimal densities that are determined empirically based on the cell type. For example, before adherence, a typical cell density for mononuclear cell cultures varies from about 1 x 10⁶ to about 1 x 10⁸ per ml of medium, and after adherence-the typical cell density is about 1 x 10⁴ to about 1 x 10⁶ cells per ml.

Important constituents of the gas phase are oxygen and carbon dioxide. Typically, atmospheric oxygen tensions are used for the cultures. Culture vessels are usually vented into the incubator atmosphere to allow gas exchange by using gas permeable caps or by preventing sealing of the culture vessels. Carbon dioxide plays a role in pH stabilization , along with buffer in the cell media and is typically present at a concentration of 1-10% in the incubator. The preferred CO₂ concentration is 5%.

Cultured cells are normally grown in an incubator that provides a suitable temperature, e. g., the body temperature of the animal from which is the cells were obtained, accounting for regional variations in temperature. Generally, 37 C. is the preferred temperature for cell culture. Most incubators are humidified to approximately atmospheric conditions.

Defined cell media are available as packaged, premixed powders or presterilized solutions. Examples of commonly used media include Iscove's media, AIM-V, RPMI O

1640, DMEM, and McCoy's Medium (see, e.g., GibcoBRL/Life Technologies Catalogue and Reference Guide; Sigma Catalogue). Defined cell culture media are often supplemented with 5-20% serum, e.g., human horse, calf, and fetal bovine serum. Preferably the serum is 10% non-heat inactivated human serum (Sigma). The culture medium is usually buffered to maintain the cells at a pH preferably from 7.2-7. 4. Other supplements to the media include, e.g., antibiotics, amino acids, sugars, and growth factors.

Differentiation The neural stem or progenitor cell lines may be subjected to differentiation treatments generally known in the art. For example, the NGC-407 cell line may be subjected to known differentiation treatments in vitro such as those described in Example 1 in WO 2006/102902.

Suicide gene therapy

Neural stem or progenitor cells can be used as a delivery vehicle to deliver the product of a suicide gene to cancer cells. As evidenced by the examples herein NGC- 407 is indeed capable of migrating to gliobastoma tumours while maintaining expression of a marker gene (Green Fluorescent Protein, GFP). The cell line may therefore be used as a vehicle to deliver a heterologous suicide gene to tumours. It has been observed that administration of 4-PB (phenyl butyrate) increases the number of GFP positive cells around the implanted tumours. Thus in a preferred embodiment, 4- PB is administered to a patient to whom suicide gene expressing NGC-407 cells or other suicide gene expressing neural stem or precursor cells, have been implanted. Methods and dosages for administration of 4-PB and analogs in connection with suicide gene therapy are described in WO 2005/079849.

Treatment strategy

Following the standard surgery to remove the solid tumour mass, the cell line expressing a deoxyribonucleoside kinase may be injected into the cavity left behind by the surgical removal of the solid tumour. Furthermore, patients with inoperable tumours may be treated by deposition the cell line expressing a deoxyribonucleoside kinase into the tumour after biopsies. Due to the ability of the cell line to migrate, the cells will make close connections to the remaining cancer cells, while expressing deoxyribonucleoside ki n ase . A n u cleosi d e i s g iven to th e pati ent pri or to , simultaneously with or after the injection. In a preferred embodiment the nucleoside analog is given to the patient after the injected cells have had time to migrate, such as one, two, three or four days after the injection, in a more preferred embodiment five, six, seven, eight or even nine days after the injection. Neither the deoxyribonucleoside kinase, nor the nucleoside analog are individually active, but together they produce a toxic compound which destroys dividing cell. Cell division is a key characteristic of cancer cells while the normal brain neuronal cells are not actively dividing. Tumour cells that try to form a new tumour around the site of the removal of the original tumour are targeted for destruction due to the above outlined treatment. Furthermore, cancer cells that have migrated away from the tumor cavity will be tracked down by the NPCs which will connect also to these distant tumour cells thereby inhibiting the infiltration of the cancer cells into the healthy brain tissue.

Deoxyribonucleoside kinases In a preferred embodiment, the cell line of the invention has been genetically engi neered to overexpress a heterologou s deoxyri bon ucleoside kin ase. Deoxyribonucleoside kinases (dNK) from various organisms differ in their substrate specificity, regulation of gene expression and cellular localisation. In mammalian cells there are four enzymes with overlapping specificities, the thymidine kinases 1 (TK1 ) and 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) phosphorylate purine and pyrimidine deoxyribonucleosides. TK1 and TK2 are pyrimidine specific and phosphorylate deoxyuridine (dUrd) and thymidine (dThd), and TK2 also phosphorylates deoxycytidine (dCyd). dCK phosphorylates dCyd , deoxyadenosine (dAdo) and deoxyguanosine (dGuo), but not dThd. dGK phosphorylates dGuo and dAdo. In mammals, TK1 is cytosolic, and TK2 and dGK are localised in the mitochondria, although recent reports indicate a cytoplasmic localisation of TK2 as well.

The best known and most studied example of suicide gene therapy is Herpes simplex virus (HSV) thymidine kinase {tk) gene (Karreman, 1998, A new set of positive/negative selectable markers for mammalian cells. Gene. 218: 57-61 ). The HSV tk gene leads to cell death when growing cells are exposed to antiherpetic nucleoside analogs such as ganciclovir (GCV), as this and other prodrugs are metabolised by HSV TK to toxic metabolites.

A Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) phosphorylates all four natural deoxyribonucleosides as well as several nucleoside analogs (Munch-Petersen et al., 1998, Four deoxynucleoside kinase activities from Drosophila melanogaster are contained within a single monomeric enzyme, a new multifunctional deoxynucleoside kinase. J Biol Chem. 273: 3926-31 ; Munch-Petersen et al 2000, Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants. J Biol Chem. 275: 6673- 9; WO 00/36099 "New medical use of gene and vector encoding a multisubstrate deoxyribonucleoside kinase (dNK)"). The broad substrate specificity of this enzyme together with a high catalytic rate makes it unique among the nucleoside kinases for use as a suicide gene in combined gene/chemotherapy of cancer. Mutant forms of the Drosophila melanogaster Dm dNK have been developed, which have broad substrate specificities (WO 01/88106 "Multi-substrate insect deoxynucleoside kinase variants"). A particularly preferred variant is the variant B5 (SEQ ID NO: 5) because its degree of activation is approximately 50 times better than wild type Dm dNK for gemcitabine. The degree of activation is defined as the ratio of the IC₅₀ of the prodrug in the nontransfected cell line to the IC₅₀ of the nucleoside analogue in the transfected cell line.

Tomato (Lycopersicon esculentum) TK1 is not able to phosphorylate deoxyadenosine, deoxycytidine or deoxyguanosine but could efficiently phosphorylate thymidine (Thd) and AZT. The Vmax and Km parameters for Thd and AZT were very similar suggesting that this kinase could be called a thymidine/azidothymidine kinase. Even more importantly, TTK1 surprisingly also accepts mono phosphate substrates, such as thymidine monophosphate (TMP) and azidothymidine monophosphate (AZT- MP). AZT exhibits better biopharmaceutical properties than other nucleoside analogs e.g. GCV, with better solubility and blood-brain-barrier penetration. The cerebrospinal fluid-plasma ratio of AZT increases in a l inear fash ion with time after d rug administration, while GCV is rapidly eliminated from the brain.

These and other recombinant kinases in a gene therapy approach can be overexpressed in NGC-407 cells by placing them under the control of a strong constitutive promoter, such as the CMV promoter, human UbiC promoter, JeT promoter (US 6,555,674), SV40 promoter, and Elongation Factor 1 alpha promoter (EF1 -alpha).

Non-limiting examples of specific known sequences of deoxyribonucleoside kinases comprise for example the following:

HSV-tk wild type ACCESSION V00470 (SEQ ID NO 1) MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQKMPTLLRVYIDG PHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQHRLDQGEISA GDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDRHPIAALLCYP AARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAML AAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTL FTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFILDYDQSPAGCRDALLQLTSG MVQTHVTTPGSIPTICDLARTFAREMGEAN

Drosophila melanogaster wildtype kinase GenBanK ACCN Y18048 (SEQ ID NO 2)

MAEAASCARKGTKYAEGTQPFTVLIEGNIGSGKTTYLNHFEKYKNDICLLTEPVEKWR NVNGVNLLELMYKDPKKWAMPFQSYVTLTMLQSHTAPTNKKLKIMERSIFSARYCFVE NMRRNGSLEQGMYNTLEEWYKFIEESIHVQADLIIYLRTSPEVAYERIRQRARSEESC VPLKYLQELHELHEDWLIHQRRPQSCKVLVLDADLNLENIGTEYQRSESSIFDAISSNQ QPSPVLVSPSKRQRVAR

Tomato TK (SEQ ID NO 3) MAFSSSARNPVDLRNGSKNSFCPVGEIHVIVGPMFAGKTTALLRRVNLESNDGRNVV

LIKSSKDARYAVDAWTHDGTRFPCWSLPDLSSFKQRFGKDAYEKVDVIGIDEAQFFG DLYEFCCNAADFDGKIIVVAGLDGDYLRKSFGSVLDIIPLADTVTKLTARCELCNRRAFF TFRKTNETETELIGGADIYM PVCRQHYVNGQSVNESAKMVLESHKVSNELILESPLVD P

Arabidopsis thaliana cINK (SEQ ID NO 4)

MVDYLRSSVGIIHRNHAESITTFIKESVDDELKDSGPEPNLNVKKRLTFCVEGNISVGK STFLQRIANETVELQDLVEIVPEPVDKWQDVGPDHFNILDAFYSEPQRYAYTFQNYVF VTRLMQEKESASGVKPLRLMERSVFSDRMVFVRAVHEAKWMNEMEISIYDSWFDPV VSSLPGLVPDGFIYLRASPDTCHKRMMLRKRAEEGGVSLKYLQDLHEKHESWLLPFE SGNHGVLSVSRPSLHMDNSLHPDIKDRVFYLEGNHMHSSIQKVPALVLDCEPNIDFSR DIEAKTQYARQVAEFFEFVKKKQETSTEKSNSQSPVLLPHQNGGLWMGPAGNHVPG LDLPPLDLKSLLTRPSA Drosophila melanogaster, mutant B5 (SEQ ID NO 5)

MAEAASCARKGTKYAEGTQPFTVLIEGNIGSGKTTYLNHFEKYKNDICLLTEPVEKWR NVNGVNLLELMYKDPKKWAMPFQSYATLTMLQSHTAPTNKKLKIMERSIFSARYCFVE NMRRNGSLEQGMYNTLEEWYKFIEESIHVQADLIIYLRTSPEVAYERIRQRARSEESC VPLKYLQELHELHEDWLIHQRRPQSCKVLVLDADLDLENIGTEYQRSESSIFDAISSNQ QPSPVPVSPSKRQRVAR

Arabidopsis thaliana dCGK NP_565032 (SEQ ID NO 6)

MQKILCKSTTSSTPVLSTPVNSLAAGFISLGFKTPVKNLPPCSTTKPLSTCFFSTSAMP TTTASVSSGGVGFSAYLQRTVHKPAPASVRFSTAGYRTCRCSIDGTN RAWVRTGSW RALFCSDSTGGLTPVNATAGAVVESEEESDGEDEDEEKDEKPVRMNRRNRSSSGSG EFVGNPDLLKIPGVGLRNQRKLVDNGIGDVAELKKLYKDKFWKASQKMVDYLRSSVGI IHRN HAESITTFIKESVDDELKDSGPEPNLNVKKRLTFCVEGNISVGKSTFLQRIANETV ELQDLVEIVPEPVDKWQDVGPDHFNILDAFYSEPQRYAYTFQNYVFVTRLMQEKESA SGVKPLRLMERSVFSDRMVFVRAVHEAKWMNEMEISIYDSWFDPWSSLPGLVPDG FIYLRASPDTCHKRMMLRKRAEEGGVSLKYLQDLHEKHESWLLPFESGNHGVLSVSR PSLHMDNSLHPDIKDRVFYLEGNHMHSSIQKVPALVLDCEPNIDFSRDIEAKTQYARQ VAEFFEFVKKKQETSTEKSNSQSPVLLPHQNGGLWMGPAGNHVPGLDLPPLDLKSLL

TRPSA Oryza sativa dCGK BAB86213 (SEQ ID NO 7) MVEFLQSSVGIIHKN HAESITLFIKESVDEELKGTDSPNVSKNKRLTFCVEGNISVGKTT FLQRIANETIELRDLVEIVPEPIAKWQDVGPDHFNILDAFYAEPQRYAYTFQNYVFVTR VMQEKESSSGIKPLRLMERSVFSDRMWKFLKVFVRAVHEANWMNEMEISIYDSWFD PWSSLPGLIPDGFIYLRASPDTCHKRMMVRKRSEEGGVTLDYLRGLHEKHESWLLP SKGQGPGVLSVSQVPVHMEGSLPPDIRERVFYLEGDHMHSSIQKVPALVLDCEHDID FNKDIEAKRQ

H. sapiens dCK XP_003471 (SEQ ID NO 8)

MATPPKRSCPSFSASSEGTRIKKISIEGNIAAGKSTFVNILKQLCEDWEWPEPVARWC NVQSTQDEFEELTMSQKNGGNVLQMMYEKPERWSFTFQTYACLSRIRAQLASLNGK

LKDAEKPVLFFERSVYSDRYIFASNLYESECMNETEWTIYQDWHDWMNNQFGQSLEL DGIIYLQATPETCLHRIYLRGRNEEQGIPLEYLEKLHYKHESWLLHRTLKTNFDYLQEV PILTLDVNEDFKDKYESLVEKVKEFLSTL H. sapiens dGK XP_002341 (SEQ ID NO 9)

MAAGRLFLSRLRAPFSSMAKSPLEGVSSSRGLHAGRGPRRLSIEGNIAVGKSTFVKLL TKTYPEWHVATEPVATWQNIQAAGNQKACTAQSLGNLLDMMYREPARWSYTFQTFS FLSRLKVQLEPFPEKLLQARKPVQIFERSVYSDRYIFAKNLFENGSLSDIEWHIYQDWH SFLLWEFASRITLHGFIYLQASPQVCLKRLYQRAREEEKGIELAYLEQLHGQHEAWLIH KTTKLHFEALMNIPVLVLDVNDDFSEEVTKQEDLMREVNTFVKNL

H. sapiens TK2 NP_004605 (SEQ ID NO 10)

MGAFCQRPSSDKEQEKEKKSVICVEGNIAGGKTTCLEFFSNATDVEVLTEPVSKWRN VRGHNPLGLMYHDASRWGLTLQTYVQLTMLDRHTRPQVSSVRLMERSIHSARYIFVE NLYRSGKMPEVDYWLSEWFDWILRNMDVSVDLIVYLRTNPETCYQRLKKRCREEEK

VIPLEYLEAIHHLHEEWLIKGSLFPMAAPVLVIEADHHMERMLELFEQNRDRILTPENRK HCP

H. sapiens TK1 XP_037195 (SEQ ID NO 11) MSCINLPTVLPGSPSKTRGQIQVILGPMFSGKSTELMRRVRRFQIAQYKCLVIKYAKDT

RYSSSFCTHDRNTMEALPACLLRDVAQEALGVAVIGIDEGQFFPDIMEFCEAMANAGK TVIVAALDGTFQRKPFGAILNLVPLAESWKLTAVCMECFREAAYTKRLGTEKEVEVIG GADKYHSVCRLCYFKKASGQPAGPDNKENCPVPGKPGEAVAARKLFAPQQILQCSP AN

Bombyx mori clNK AAK28318 (SEQ ID NO 12)

MSAN NVKPFTVFVEGNIGSGKTTFLEHFRQFEDITLLTEPVEMWRDLKGCNLLELMYK DPEKWAMTFQSYVSLTMLDMHRRPAPTPVKLMERSLFSARYCFVEHIMRNNTLHPA QFAVLDEWFRFIQHNIPIDADLIVYLKTSPSIVYQRIKKRARSEEQCVPLSYIEELHRLHE DWLINRIHAECPAPVLVLDADLDLSQITDEYKRSEHQILRKAVNWMSSPNKHSPKKPI

STTPIKITPHMRIL

Anopheles clNK AAO49462 (SEQ ID NO 13)

MPPIASEKLGASGKKPFTVFVEGNIGSGKTTFLNHFQKFNDICLLTEPVEKWRNCGGV NLLDLMYKESHRWAMPFQTYVTLTMLDMHTCQTDKSVKLMERSLFSARNCFVESML

ASGSLHQGMYNVLQEWYDFICCNIHIQADLIVYLQTSPEWYERMKQRARSEESCVPL EYLKELHELHENWLIHGASPRPAPVLVLNADLDLNTIGAEYERSETSILKPILIENTNQH AILTSPAKRAKTDF Rice TK1 (SEQ ID NO 14) o

MSSICAMRSLLAASTFLRSGASPLLRPLSRPLPSRLNLSRFGPVRPVSAAAAAADKSR GGGGSAMEAQPSYPGEIHVIVGPMFAGKTTALLRRVQVEAGTGRNVALIKSDKDNRY GLDSWTHDGTKMPCWALPELSSFQDKLGTEAYDKVDVIGIDEAQFFDDLHDFCCKA ADRDGKIVWAGLDGDYKRNKFGSVLDIIPLADSVTKLTARCELCGRRAFFTLRKTRET KTELIGGADVYMPVCRQHYLDGQIVIEATRIVLDLEKSKVIHAFK

A. thaliana TK1 AAF13097 (SEQ ID NO 15)

MATLKASFLIKTLDSDVTGDFLSDLERRGSGAVHVIMGPMFSGKSTSLLRRIKSEISDG RSVAMLKSSKDTRYAKDSVVTHDGIGFPCWALPDLMSFPEKFGLDAYNKLDVIGIDEA QFFGDLYEFCCKVADDDGKIVIVAGLDGDYLRRSFGAVLDIIPIADSVTKLTARCEVCG

HKAFFTLRKNCDTRTELIGGADVYMPVCRKHYITNHIVIKASKKVLEDSDKARAESCVA ATI

A. thaliana TK1 b (SEQ ID NO 16) MRTLISPSLAPFSLHLHKPSLFSTALRFSFSINNITPTNSPPSTISTRKLQTKATRVTSSS SSQPLSSSSPGEIHVWGPMFSGKTTTLLRRILAERETGKRIAIIKSNKDTRYCTESIVT HDGEKYPCWSLPDLSSFKERFGFDDYENRLDVIGIDEAQFFGDLYEFCREAADKEGK TVIVAGLDGDFMRRRFGSVLDLIPIADTVTKLTSRCEVCGKRALFTMRKTEEKETELIG GAEVYMPVCRSHYVCGQNVLETARAVLDSSNNHSVVASSL

Tomato dCGK (SEQ ID NO 17)

MVEFLQSSIGIIHRN HAESITTYIRKSVDEELKENNSDSNVKSTQKKRLTFCVEGNISVG KTTFLQRIANETLELQDLVEIVPEPIAKWQDIGPDHFNILDAFYAEPQRYAYTFQNYVFV TRVMQERESSGGIRPLRLMERSVFSDRMVFVRAVHEANWMNEMEISIYDSWFDPW STLPGLIPDGFIYLRASPDTCHKRMMLRKRTEEGGVSLEYLRGLHEKHESWLFPFESG NHGVLSVSELPLNFDKFCVPPEIRDRVFYLEGNHMHPSIQKVPALVLDCEPNIDFNRDI EAKRQYARQVADFFEFVKKKQEVMPGAGEEQPKGNQAPVMLPQNGGLWVPGGKFS ESTLNLDFRRNMSFMSH

Tomato TK, delta 26 (SEQ ID NO 18)

MAFSSSARNPVDLRNGSKNSFCPVGEIHVIVGPMFAGKTTALLRRVNLESNDGRNW LIKSSKDARYAVDAWTHDGTRFPCWSLPDLSSFKQRFGKDAYEKVDVIGIDEAQFFG DLYEFCCNAADFDGKIIWAGLDGDYLRKSFGSVLDIIPLADTVTKLTARCELCNRRAFF TFRKTNETETELIGGADIYMPVCRQHYVNGQSV

The corresponding nucleotide sequences can be found in Genbank using the accession numbers given above, in the references given above and for the plant kinases in WO 03/100045 (thymidine kinases), and WO 2004/003185 (dCK/dGK).

In a preferred embodiment, the deoxyribonucleoside kinase is selected from the group consisting of a) a deoxyribonucleoside kinase having the amino acid sequence of any of SEQ ID No 1 to 18; b) a deoxyribonucleoside kinase variant comprising an amino acid sequence having at least 70% sequence identity to any of SEQ ID No 1 to 18; c) a deoxyribonucleoside kinase variant having an amino acid sequence which has not more than 60 amino acid substitutions when compared to any of SEQ ID No 1 to 18; d) a deoxyribonucleoside kinase encoded by a nucleotide sequence capable of hybridising under conditions of high stringency to a nucleotide sequence encoding any of SEQ ID No 1 to 18.

In the context of this invention, the term deoxyribonucleoside kinase variant is a polypeptide (or protein) having an amino acid sequence that differs from the sequence presented as SEQ ID NO: 1 , as SEQ ID NO:2, as SEQ ID NO: 3, as SEQ ID NO: 4, as

SEQ ID NO: 5, as SEQ ID NO: 6, as SEQ ID NO: 7, as SEQ ID NO: 8, as SEQ ID NO:

9, as SEQ ID NO: 10, as SEQ ID NO: 1 1 , as SEQ ID NO: 12, as SEQ ID NO: 13, as SEQ ID NO: 14, as SEQ ID NO: 15, as SEQ ID NO: 16, as SEQ ID NO: 17, as SEQ ID NO: 18, at one or more amino acid positions and has dNK activity. In a particular embodiment the kinase variant has an amino acid sequence which differs by 60 amino acids or less when compared to SEQ ID NO: 1 , or SEQ ID NO:2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 11 , or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18. In particular the said kinase variant differs by 50 amino acids or less, such as by 40, 30, 20, 19, 18, 17, 16 or 15 amino acids or less when compared to SEQ ID NO: 1 , or SEQ ID NO:2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO:

10, or SEQ ID NO: 1 1 , or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ I D NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18. In a preferred embodiment the said kinase variant differs by 10 amino acids or less, such as by 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acids or less when compared to SEQ ID NO: 1 , or SEQ ID NO:2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 1 1 , or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or

SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, SEQ ID NO: 18. In a most preferred embodiment the kinase expressed by the neural progenitor cell line has an amino acid sequence comprising the amino acids of SEQ ID NO: 1 , or SEQ ID NO:2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6, or SEQ ID NO: 7, or SEQ ID NO: 8, or SEQ ID NO: 9, or SEQ ID NO: 10, or SEQ ID NO: 1 1 , or SEQ ID NO: 12, or SEQ ID NO: 13, or SEQ ID NO: 14, or SEQ ID NO: 15, or SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18.

In another aspect variant deoxyribonucleoside kinase can be defined with reference to the amino acid sequence of a known deoxyribonucleoside kinase, such as any of the kinases disclosed above. In a preferred embodiment, the variant kinase has at least 50% sequence identity to a reference sequence, more preferably at least 60% sequence identity, more preferably at least 70% sequence identity, more preferably at least 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95%, such as 96%, 97%, 98% or 99% sequence identity. The individual reference sequence may be either of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In the context of this invention sequence identity (or identity) is a measure of the degree of identical amino acid residues among sequences. In order to characterize the identity, subject sequences are aligned so that the highest order homology (match) is obtained. Based on these general principles the identity (or sequence identity or percent identity) of two amino acid sequences is determined using the BLASTP algorithm [Tatiana A. Tatusova, Thomas L. Madden: Blast 2 sequences - a new tool for comparing protein and nucleotide sequences; FEMS Microbiol. Lett. 1999 174 247- 250], which is available from the National Center for Biotechnology Information (NCBI) web site, and using the default settings suggested here (i.e. Matrix = Blosum62; Open gap = 1 1 ; Extension gap = 1 ; Penalties gab x_dropoff = 50; Expect = 10; Word size = 3; Filter on). The BLAST algorithm determines the % sequence identity in a range of overlap between two aligned sequences. For the purposes of the present invention, the percent sequence identity is preferably calculated in a range of overlap of at least 50 amino acids, more preferably at least 75 amino acids, more preferably at least 100 amino acids, the range being calculated by BLASTP under default settings.

In the case of plant thymidine kinases, a variant plant thymidine kinase, such as for example a variant tomato thymidine kinase comprises one or more of the following three motifs as defined in Table 1 of WO 03/100045: VaI lie GIy lie Asp GIu Ala GIn Phe Phe (Motif I) VaI Ala GIy Leu Asp GIy (Motif II) Tyr Met Pro VaI Cys Arg (Motif III)

In another preferred embodiment, the plant thymidine kinase enzyme comprises the Lid region:

VaI A1 Lys Leu A2 A3 Arg Cys GIu A4 (Lid region), wherein A1 is selected from Thr and VaI, A2 is selected from Thr and Lys, A3 is selected from Ala and Ser, and A4 is selected from Leu and VaI.

I n a more preferred embodiment the plant thymidine kinase enzyme of the invention comprises all of the conserved residues identified in Table 1 of WO 03/100045. Such analogous polypeptides include polypeptides comprising conservative substitutions, splice variants, isoforms, homologues from other species, and polymorphisms.

As defined herein, the term "conservative substitutions" denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include

(i) the substitution of one non-polar or hydrophobic residue such as alanine, leucine, isoleucine, valine, proline, methionine, phenylalanine or tryptophan for another, in particular the substitution of alanine, leucine, isoleucine, valine or proline for another; or (ii) the substitution of one neutral (uncharged) polar residue such as serine, threonine, tyrosine, asparagine, glutamine, or cysteine for another, in particular the substitution of arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine; or

(iii) the substitution of a positively charged residue such as lysine, arginine or histidine for another; or

(iv) the substitution of a negatively charged residue such as aspartic acid or glutamic acid for another.

Modifications of this primary amino acid sequence may result in proteins which have substantially equivalent activity as compared to the unmodified counterpart polypeptide, and thus may be considered functional analogous of the parent proteins. Such modifications may be deliberate, e.g. as by site-directed mutagenesis, or they may occur spontaneous, and include splice variants, isoforms, homologues from other species, and polymorphisms. Such functional analogous are also contemplated according to the invention. It has been found that deoxyribonucleoside kinase enzymes that are C- and/or

N-terminally altered significantly change their properties in particular in respect of kinetic properties such as turnover and substrate specificity. So from having a more restricted specificity, usually deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) activity, the deoxyribonucleoside kinase enzymes of the invention may be converted into essentially multi-substrate enzymes, having ability to phosphorylate all four deoxyribonucleosides.

It is also possible to administer two or more deoxyribonucleoside kinases to the same individual. Without being limiting the following combinations of kinase and nucleoside analogues are preferred:

HSV-tk - ganciclovir, acyclovir, penciclovir

Drosophila melanogaster dNK or B5- gemcitabine, cladribine, fludarabine, cytarabine, zalcitabine Plant TKs including Tomato TK- zidovudine (AZT), D4T, ddT, fluorouridine

Plant dNKs including Arabidopsis thaliana dNK- gemcitabine, cladribine, fludarabine, cytarabine, zalcitabine

Hybridisation Hybridisation should be accomplished under at least low stringency conditions, but preferably at medium or high stringency conditions.

Suitable experimental conditions for determining hybridisation at low, medium, or high stringency conditions, respectively, between a nucleotide probe and a homologous DNA or RNA sequence, involves pre-soaking of the filter containing the DNA fragments or RNA to hybridise in 5 x SSC [Sodium chloride/Sodium citrate; cf. Sambrook et al.; Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Lab., Cold Spring Harbor, NY 1989] for 10 minutes, and prehybridisation of the filter in a solution of 5 x SSC, 5 x Denhardt's solution [cf. Sambrook et al.; Op cit], 0.5% SDS and 100 μg/ml of denatured sonicated salmon sperm DNA [cf. Sambrook et al.; Op cit.], followed by hybridisation in the same solution containing a concentration of 10 ng/ml of a random-primed [Feinberg A P & Vogelstein B; Anal. Biochem. 1983 132 6-13], 32P- dCTP-labeled (specific activity > 1 x 109 cpm/μg) probe for 12 hours at approximately 45°C.

The filter is then washed twice for 30 minutes in 2 x SSC, 0.5% SDS at a temperature of at least 55°C (low stringency conditions), more preferred of at least 60°C (medium stringency conditions), still more preferred of at least 65°C (medium/high stringency conditions), even more preferred of at least 70°C (high stringency conditions), and yet more preferred of at least 75°C (very high stringency conditions). Molecules to which the oligonucleotide probe hybridises under these conditions may be labelled to detect hybridisation. The complementary nucleic acids or signal nucleic acids may be labelled by conventional methods known in the art to detect the presence of hybridised oligonucleotides. The most common method of detection is the use of autoradiography with e.g. 3H, 1251, 35S, 14C, or 32P-labelled probes, which may then be detected using an X-ray film. Other labels include ligands, which bind to labelled antibodies, fluorophores, chemoluminescent agents, enzymes, or antibodies, which can then serve as specific binding pair members for a labelled ligand.

In a preferred embodiment the dNK of the present invention is encoded by a nucleic acid sequence that hybridises to the complementary nucleic acid sequence of the nucleic acid sequence encoding any of SEQ ID NO: 1 to 18 under at least medium stringency conditions, such as medium/high or high stringency conditions or even very high stringency conditions. Preferably the SEQ ID NO is 3 or 18.

Examples

Materials ³H labelled Thymidine (dThd) was obtained from Amersham Corp., and ³H labeled AZT [methyl-³H] (AZT), thymidine monophosphate (TMP), thymidine diphopshate (TDP), AZT-MP and AZT-DP were from Moravek Biochemicals Inc., Brea, CA. Unlabelled nucleosides and nucleotides were from Sigma. AZT for the treatment of animals was purchased from Sigma (A2169) and was dissolved at 40 mg/ml of 15 % Methyl-beta-cyclodextrin (Kleptose CRYSMEB, Roquette).

Magnetic resonance imaging (MRI) and image processing. On day 12-13 and day 22- 23 post-implantation, MRI experiments were performed using a 4.7 T horizontal bore animal MR system operating on a Paravision (version 3.0.2) software platform (Bruker Biospec, Germany). Animals were anaesthetised with and maintained on isofluorane and body temperature was maintained with a heated air stream. Respiratory activity was monitored continuously throughout the experiments. Multislice T2-weighted images of rat brains were acquired in the coronal, sagittal and axial planes (TR 2500 ms, TE 95.6 ms, slice thickness 1.5 mm or 0.5 mm, FOV 3.5 cm, 5-16 slices depending on tumor size). Tumor volumes from stack images of each anatomical plane in each animal were measured by manual outlining tumors using the public domain program ImageJ (National Institutes of Health, Bethesda, Maryland, USA, hiΪΩlllF3hsΪDlo_:D}h.-.QoyJJΪL 1997-2007). Tumor volume in an animal was then averaged out from individual volumes in all 3 planes.

Statistical analysis. Kinetic data were evaluated by nonlinear regression analysis using the Michaelis-Menten equation v = V_max x [S]/(K_m + [S]). The equations were fitted to all available data in a global fit. Statistical significance between means of the tumor volumes in different experimental groups was assessed by Student's t-test for unpaired values. Kaplan-Meier survival analysis with Log-rank (Mantel-Cox) Test (including test for trend) was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA (jΛΛΛΛΛΛgraghgad_JLconi).

Example 1 : Cloning of TTK1 delta26 into the plasmid expression vector pCI-Hyg

Construction ofpCI-Hyg pZG752 (pCI-HygR) is a derivative of pCI-neo Mammalian Expression vector (Promega, catalog number E 1841 , Lot. number 15036808). The Xbal site in the MCS of pCI-neo was removed by Xbal digest followed by completely filled in of 5^'overhangs by Klenow resulting in the vector pZG733. Following, two Xbal sites flanking the neoR coding region were introduced using site-directed mutagenesis (Quick Change XL Site- Directed mutagenesis Kit from Stratagene, Catalog # 200516). The first Xbal site upstream neoR start codon was introduced using the primers CAGTCTCGAACTTAAGTCTAGAGCCACCATGATTG (SEQ ID NO: 19), and CAATCATGGTGGCTCTAGACTTAAGTTCGAGACG (SEQ ID NO: 20) resulting in the vector pZG744.

The second Xbal site downstream the neoR stop codon was introduced using the primers: δ-CTTCTGAGCGGGACTCTAGAGTTCGAAATGACCGAC-S (SEQ ID NO: 21 ), and δ-GTCGGTCATTTCGAACTCTAGAGTCCCGCTCAGAAG-S (SEQ ID NO: 22) resulting in the vecor pZG746.

The neoR gene was removed from pZG746 by Xbal digestion. The HygR gene with flanking Xbal sites was constructed in the following way: The HygR gene from pLHCX vector (LRCX retroviral vector set, Clonetech, cat.# K1061-1 , lot. # 2060034,) was PCR amplified using the forward primer δ-GCTCTAGAGCCACCATGGATAGATCCGGAAAGCCTG-S (SEQ ID NO: 23) containing Kozak sequence and Xbal site and the reverse primer 5-CGTCTAGACTCTATTCCTTT GCCCTCGGACGAG-3 (SEQ ID NO: 24) containing stop codon and Xbal site using Accuzyme DNA polymerase. The amplified PCR product was digested with Xbal and cloned into the Xbal cut pZG746 vector. The correct construction of the resulting vector, pZG752, was confirmed by sequencing.

Construction of tomato TK1delta26 synthetic gene:

Tomato TK1 delta 26 (pZG59) wt gene was codon optimized for expression in human cells (GENEART GmbH).

The 627 bp long synthetic gene (0427723, Geneart) corresponding to pZG59delta 26 was cloned into pPCR-Script using Kpnl and Sacl restriction sites.

The synthetic gene has the following sequence:

Tomato TK1 (CpG+) synthetic gene (SEQ ID NO: 25):

1 ATGGCCTTCAGCAGCAGCGCCCGGAACCCCGTCGACCTGCGGAACGGCAGCAAGAACAGC

61 TTCTGCCCTGTCGGCGAGATCCACGTGATTGTCGGCCCCATGTTCGCCGGCAAGACCACC

121 GCCCTGCTGCGGCGCGTGAACCTCGAAAGCAACGACGGCCGGAACGTCGTGCTGATCAAG

181 AGCAGCAAGGACGCCCGCTACGCCGTGGACGCCGTCGTGACCCACGACGGCACCCGGTTC 241 CCCTGCTGGAGCCTGCCCGACCTGAGCAGCTTCAAGCAGCGCTTCGGGAAGGACGCCTAC

301 GAGAAAGTCGACGTGATCGGCATCGACGAGGCCCAGTTCTTCGGCGACCTGTACGAGTTC

361 TGCTGCAACGCCGCCGACTTCGACGGCAAGATCATCGTCGTCGCCGGCCTCGACGGCGAC

421 TACCTGCGGAAGAGCTTCGGCAGCGTGCTCGACATCATCCCTCTCGCCGACACCGTGACC 481 AAGCTGACCGCCCGCTGCGAGCTGTGCAATCGGCGCGCTTTCTTCACCTTCCGCAAGACC 541 AACGAGACCGAGACCGAGCTGATCGGCGGAGCCGATATCTACATGCCCGTGTGTCGGCAG 601 CACTACGTGAACGGCCAGAGCGTGTAATGA

Construction of pCI-Hyg-TTK1 (CpG+) vector. pZG752 was digested with Xhol and Smal. The synthetic Tomato TK1 (CpG+) gene was isolated from pZG698. First, pZG698 was digested with BgIII and the 5^'-ends were completely filled in by Klenow. Following the synthetic Tomato TK1 (CpG+) gene was isolated by Xhol digestion. The isolated Xhol/Bglll (completely filled in) fragment containing the KOZAK sequence in front of the synthetic Tomato TK1 (CpG+) gene followed by two stop codons were cloned into pZG752 digested with Xhol/Smal resulting in the vector pZG757 (pCI-Hyg-tomato TK1 (CpG+). pZG757 was sent to Plamid Factory GmbH (Bielefeld, Germany) for preparation of a ccc Pilot Grade Quality Plasmid DNA.

The resulting plasmid from plasmid Factory ccc Plasmid pZG757 (Lot. No. PF584- 061009) was sequenced (both strands) as publication quality (PQ) sequencing grade by MWG Biotech (Ebersberg, Germany).

Example 2: Sensitivity test

The sensitivity test is based on increase in cytotoxicity (by application of the Cell Proliferation Assay) where viability of cells with different dosages of nucleoside analog is measured and the IC₅₀ value determined. Sensitivity increase is ratio between IC50 of parental cells and IC50 of the cells transduced with kinase gene.

Cell Proliferation Assays

3'-Azido 3'-deoxythymidine (AZT) was obtained from Sigma. The cells were plated at 1500 -2500 cells/well in 96-well plates coated with poly-L-lysine. AZT was added after 24 h, and the medium containing the nucleoside analogs was not changed. Cell survival was assayed by a XTT assay (Roche) after 5 days of drug exposure. Each experiment was performed in four replicates. The IC₅O value of the investigated compounds was calculated as the mean value of these experiments using SigmaPlot® and formula d = Vm/(1 +(1/IC)).

Example 3: Enzyme assay.

Deoxyribonucleoside kinase activities were determined by initial velocity measurements based on four time samples by the DE-81 filter paper assay using tritium-labeled nucleoside substrates and liquid scintillation as described previously

(Munch-Petersen et al, 1998). The standard assay conditions were: 50 mM Tris-HCI, pH 8.0, 2 mM MgCI2, 10 mM dithiothreiotol, 0.5 mM CHAPS, 3 mg/ml bovine serum albumine, 2.5 mM ATP.

One unit (U) of kinase activity is defined as 1 μmol of the corresponding monophosphate or diphosphate product formed per minute, and one small unit (mil) is

1.000 lower.

Example 4: Cell cultures and Establishment of TTK1 delta26 (SEQ ID NO: 18) expressing cells. U87MG human GBM cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Transduction of U87MG cells with TTK1 and HSV-TK was performed using retroviral vectors NGC-407 progenitor/stem cell line was established, maintained and cultured as previously described (Khan et al, ECR). Briefly, the culture medium DMEM-F12 (1 :1 ) with Glutamax I (Gibco, 31331-028) was supplemented with 40 ng/ml basic fibroblast growth factor (R&D Systems, 233-FB), 20 ng/ml epidermal growth factor (Invitrogen, 13247-051 ), 0.5% human serum albumin (Sigma, A1653) and other ingredients including N2 and B27 supplements, glucose and nonessential amino acids. NGC-407 cells were transfected with TTK1 plasmid DNA using FuGENE 6 Transfection Reagent (Roche) according to the manufacturer's instructions. Positive clones were then selected with hygromycin. The recombinant cells were also tested for the sensitivity to AZT.

Cells were seeded in equal number and in triplicates and viability was assayed using either MTT or XTT tetrazolium salt. MTT was dissolved in serum-free culture medium and isopropanol was used to dissolve the formazan dye formed by viable cells. Where XTT assay was used, the defrosted XTT solution was directly added to the plates. After incubation plates were read at ELISA Reader (570 nm with 650 nm reference for MTT and 450 nm with 690 nm reference for XTT). The table shows the IC50 for parental cells and for an exemplary NGC-407 cell line expressing TTK1-delta26 (SEQ ID NO 18) (NGC-407-TTK1 ) as well as the measured sensitivity increase.

Example 5: Stem cell-delivery of TTK1 delta 26 (SEQ ID NO: 18) to treat experimental glioblastomas

Method: All animal experiments were conducted in accordance with the ethical guidelines from Stockholm local ethics committee (permission no. N46/05). Male nude rats (rnu/rnu; Harlan, Germany), aged 8-9 weeks with an average weight of 150-20Og were housed in groups of 3 in standardized big cages at 50-70% relative humidity, o

20°C-24°C temperature and 12/12 day/night variation. Standard sterility was maintained for the materials including foods (Ad libitum) and water used for them. They were anesthesitized by isoflurane inhalation and on a stereo tactic platform a burr hole of <1 mm diameter was made on the skull over the right hemisphere under microscopic guidance. 3x10⁵ U87MG and NGC-407 TTK1 cells mixed in different ratios in a 4 μl volume were slowly injected through the burr hole using a 5 μl volume Hamilton syringe, 2 mm right lateral, 3 mm posterior to the bregma and at a depth of 5 mm from the surface. 24 hours after implantation, the rats were treated intraperitoneally with AZT 400 mg/kg of body weight per day in 2 divided doses for 14 days. The control groups received equal volume of vehicle (15 % Methyl-β-cyclodextrin). All animals were observed twice daily for significant weight loss (>10%), abnormal behaviour including food intake, mobility and other neurological symptoms which were set as the end point of experiment. After sacrificing the animal, brains were collected quickly and were immediately frozen in dry ice cooled 2-methyl butane (Sigma). The brains were then transferred into -75°C freezer until they were sectioned by cryostat onto SuperFrost Plus glass slides (Menzel-Glaser) at a 14μm thickness.

Human embryonic neural progenitor/stem cell line NGC-407 was used as the delivery vehicles of TTK1 to investigate its ablative effect on glioblastoma xenografts in the presence of AZT. When implanted into nude rat brains, they survived and migrated to glioblastoma cells (GBM) and did not form tumor themselves (Figure 1 ).

NGC-407-TTK1 cells stably expressing TTK1 were established using a TTK1 plasmid. Briefly, the tomato thymidine kinase gene (ZG59) was cloned into the pCI_hyg vector (Promega) using appropriate restriction enzymes. Plasmid DNA was introduced into NGC-407 cells by lipofection using FuGENE 6 Transfection Reagent (Roche) according to the manufacturer's instructions. Positive clones were selected using hygromycin and tested for TK activity using enzyme kinetics and cytotoxicity assays to verify expression. The enzymatic activity of TTK1 in the implanted NGC-407-TTK1 cells was 10 to 14-fold higher compared to parental NGC-407 cells. NGC-407-TTK1 cells were co-implanted with U87MG cells at different proportions (1 :1 , 1 :3, 1 :30) into nude rat brains and the rats were then treated intraperitoneally with AZT for up to 21 days. Survival of AZT-treated rats was significantly increased compared to control vehicle treated rats. Upon exposure to AZT, the animals which received NGC- 407 TTK1 and tumor cells at 1 :1 and 1 :3 ratios survived on an average 6-7 days longer than the controls (Fig. 2). In vehicle treated animals NGC-407-TTK1 cells were found intermingling with U87MG cells (Figure 1 ). AZT did not influence the survival where U87MG cells alone were implanted (Fig. 2). Tumor growth was followed by experimental MRI. Images were acquired at 3 anatomical projections (sagittal, coronal and axial) to simulate 3D imaging (Figure 3). Upon exposure to AZT for 3 weeks, the observed tumor volumes were reduced to one-fourth, and to half of the controls, at 1 :1 and 1 :3 ratios (NGC-407-TTK1 : U87MG), respectively. However, there was no difference in tumor size where only U87MG cells were injected (Figure 4). MRI follow- ups on day 12-13 and day 22-23 post-implantation revealed that after initial establishment, the U87MG xenografts grew very rapidly without AZT treatment. In the clinic, surgically debulked tumor will presumably require several injections of TTK1 expressing stem cells, a procedure which is technically not realistic in animal studies. Although, this experimental set-up does not closely mimic the clinical therapeutic approach where a glioblastoma patient gets treatment for an existing tumor, it does provide evidence in favour of the effectiveness of TTK1 in vivo.

Figure 3 shows 8 individual rats that have received U87 and NGC-407 as indicated on the Figure. Tumour growth was followed by experimental MRI. Images were acquired at 3 anatomical projections (sagittal, coronal and axial) to simulate 3D imaging (Fig. 3). Upon exposure to AZT for 3 weeks, the observed tumor volumes were reduced to one- fourth, and to half of the controls, at 1 :1 and 1 :3 ratios (NGC-407-ZG59: U87MG), respectively. At the 1 :30 ratio, exposure to AZT resulted in an apparent reduced tumor size and 3 days increased survival, although this was statistically insignificant (Fig. 2). MRI follow-ups on day 12-13 and day 22-23 post-implantation revealed that after initial establishment, the U87MG xenografts grew very rapidly without AZT treatment.

Figure 4 shows the average tumor volume for the different implantation groups. In the 1 :30 group, AZT treated animals had an average tumor-volume of 0.2 cm³ (n=3) versus 0.27 cm³ in the vehicle group, where only one animal survived until scanning. In the 1 :3 group, the average tumor-size of AZT-treated animals was 0.16 cm³ versus 0.29 cm³ in the vehicle group (just 1 animal survived until scanning). In the 1 :1 group, all animals survived until scanning. AZT-treated animals had an average tumor-size of 0.07±0.02 cm³ that was significant (p < 0.01 ) smaller than vehicle -treated animals (tumor-size of 0.29±0.07 cm³). There is a clear dose-dependent effect of the NGC-407 cells on the tumor-volumes. o

The study was not designed to include to survival, but when the first animal got the clinical signs of decreased locomotion, decreased alertness, loss of weight and reduced food-intake, these signs were used as clinical endpoints. Animals with these clinical signs were sacrificed. Two animals died overnight. The clinical signs were observed by a person blinded to the groups and treatment paradigm. Figure 2 shows the survival curves for the treatment groups. Survival of AZT-treated rats was significantly increased compared to control vehicle treated rats. Upon exposure to AZT, the animals which received NGC-407-ZG59 and tumour cells at 1 :1 and 1 :3 ratios survived on an average 6-7 days longer than the controls (Fig. 2). In vehicle treated animals NGC-407-ZG59 cells was found intermingling with U87MG cells. AZT did not influence the survival where U87MG cells alone were implanted (Fig. 2), i.e. animals implanted with U87 alone showed no difference in survival rate, whereas AZT-treated animals in the 1 :3 (mean survival 26.7 days) and the 1 :1 (mean survival 30.2 days) lived significantly longer than the vehicle-treated animals (Figure 2).

In the 1 :30 group, there was no significant difference with and without AZT (at the 5% level). The lack of difference is mot probably due to the limited number of animals in each group. The data for the 30:1 ratio (tumor volume, mean survival) with and without treatment indicates a small, if any, effect of NGC-407-ZG59 on the tumor-size. Hence, the minimal effective dose may be higher than 1 :30 as estimated from this experiment.

Claims

Claims

1. A neural progenitor or stem cell line expressing a deoxyribonucleoside kinase resulting in an increase in sensitivity, when applying the sensitivity test, in a level of not less than 5 fold and not more than 50 fold.

2. A neural progenitor or stem cell line expressing a deoxyribonucleoside kinase resulting in an increase in sensitivity, when applying the sensitivity test, in a level of not less than 5 fold and not more than 50 fold in combination with a nucleoside analog for the use as a medicament.

3. The neural progenitor or stem cell line according to claim 2 in combination with a nucleoside analog for the use in the treatment of cancer.

4. The neural progenitor or stem cell line according to claim 3 in combination with a nucleoside analog for the treatment of cancer, wherein the cancer is glioma

5. Use of a neural progenitor or stem cell line expressing a deoxyribonucleoside kinase resulting in increase in sensitivity in a level of not less than 5 fold and not more than 50 fold in combination with a nucleoside analog for the manufacture of a medicament for the treatment of cancer.

6. Use of a neural progenitor or stem cell line according to claim 4 for the manufacture of a medicament for the treatment of glioma

7. The neural progenitor or stem cell line according to claim 4 or 6 for the use in the treatment of a type of glioma selected from the group consisting of a. Ependymomas b. Astrocytomas c. Oligodendrogliomas d. Mixed gliomas

8. The neural progenitor or stem cell line according to claim 7 for the use in the treatment of a type of astrocytomas selected from the group consisting of a. Pilocytic astrocytoma b. Diffuse astrocytoma c. Malignant astrocytoma d. Gioblastoma multiforme

9. The cell line of any of the claims 1-8 wherein the expressed deoxyribonucleoside kinase is selected from the group consisting of a. a deoxyribonucleoside kinase having the amino acid sequence of any of SEQ ID NO: 1 to 18; b. a deoxyribonucleoside variant which differs by 60 amino acids or less when compared to any of SEQ ID NO: 1 to 18. c. a thymidine kinase variant comprising an amino acid sequence having at least 70 % identity to any of SEQ ID NO 1 to 18 and having thymidine kinase activity; and d. a thymidine kinase encoded by a nucleotide sequence capable of hybridising under conditions of high stringency to a nucleotide sequence encoding any of SEQ ID NO 1 to 18

10. The cell line of any of the claims 1-8 wherein the expressed deoxyribonucleoside kinase has at least 70% identity to SEQ ID NO: 3 or SEQ ID NO: 18

1 1. The cell line of any of the claims 1-8 wherein the expressed deoxyribonucleoside kinase is encoded by a nucleotide sequence capable of hybridising under conditions of high stringency to the complement of a nucleotide sequence encoding SEQ ID NO: 3 or SEQ ID NO: 18.

12. The cell line of any of the claims 1-11 obtainable from or derived from or constituted by NGC-407 cells deposited under the Budapest Treaty with Deutsche Sammlung von Mikroorganismen und Zellkulturen, on 31^st March, 2005, under accession number DSM ACC2718.

13. The cell line of claim 1-12, being a polyclonal cell line.

14. The cell line of claim 1-12, being a monoclonal cell line. oo

15. The cell line of claim 1-14, being capable of differentiating into astrocytes.

16. The cell line of claim 1 -14, being capable of differentiating into glia.

17. The cell line of any of the preceding claims, wherein the increase in sensitivity is not less than 5 fold and not more than 40 fold.

18. The cell line of any of the preceding claims, wherein the increase in sensitivity is not less than 5 fold and not more than 30 fold.

19. The cell line of any of the preceding claims, wherein the increase in sensitivity is not less than 5 fold and not more than 20 fold.

20. The cell line of any of the preceding claims, wherein the increase in sensitivity is not less than 10 fold and not more than 40 fold.

21. The cell line of any of the preceding claims, wherein the increase in sensitivity is not less than 10 fold and not more than 30 fold.

22. The cell line of any of the preceding claims, wherein the increase in sensitivity is not less than 10 fold and not more than 20 fold.

23. The cell line of any of the preceding claims, being a human cell line.

24. The cell line of any of the claims 2 to 16 selected from the group consisting of

Zidovudine (AZT), Ganciclovir (GCV), Acyclovir (ACV) Penciclovir, Gemcitabine, cladrbine (CdA), Fludarabine (FaraA) Cytarabine,

Arabinocytidine (araC), Dideoxycytidine (ddC), Stavudine (D4T), 2', 3'- dideoxythymidine (ddT), fluorouridine (F-Urd), Gemcitabine (dFdC).

25. The cell line of claim 24 in the form of salt or a prodrug.

26. A pharmaceutical composition comprising the cell line of any of the preceding claims. \

27. A process for the manufacture of the pharmaceutical composition of claim 26.