WO2004003185A2 - Plant deoxyribonucleoside kinase enzymes and their use - Google Patents
Plant deoxyribonucleoside kinase enzymes and their use Download PDFInfo
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- WO2004003185A2 WO2004003185A2 PCT/DK2003/000429 DK0300429W WO2004003185A2 WO 2004003185 A2 WO2004003185 A2 WO 2004003185A2 DK 0300429 W DK0300429 W DK 0300429W WO 2004003185 A2 WO2004003185 A2 WO 2004003185A2
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
- C12N9/1211—Thymidine kinase (2.7.1.21)
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01076—Deoxyadenosine kinase (2.7.1.76)
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- A—HUMAN NECESSITIES
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- This invention relates to pharmaceutical compositions comprising plant deoxyribonucleoside kinase enzymes (dNK) capable of phosphorylating nucleoside analogs and to medical use of said dNKs. More specifically the invention relates to the medical use of deoxyribonucleoside kinase enzymes derived from (Arabidopsis thaliana), from loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa). The invention also relates to methods of sensitising cells to prodrugs, and to methods of inhibiting pathogenic agents in warm-blooded animals using said plant dNKs.
- dNK deoxyribonucleoside kinase enzymes
- the invention relates to plant derived deoxyribonucleoside kinase enzymes provided in isolated form from loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa).
- the invention provides polynucleotides encoding the plant dNKs, vector constructs comprising the polynucleotide, packaging cell lines capable of producing said vector, and genetically modified isolated host cells transduced/transfected/transformed with the vector.
- DNA is made of four deoxyribonucleoside triphosphates, provided by the de novo and the salvage pathway.
- the key enzyme of the de novo pathway is ribonucleotide reductase, which catalyses the reduction of the 2'-OH group of the nucleoside diphosphates
- the key salvage enzymes are the deoxyribonucleoside kinases, which phosphorylate deoxyribonucleosides to the corresponding deoxyribonucleoside monophosphates.
- 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.
- TK1 is cytosolic, and TK2 and dGK are localised in the mitochondria, although recent reports indicate a cytoplasmic localisation of TK2 as well.
- WO 01/88106 describes multi-substrate deoxyribonucleoside kinase variants derived from insects, in particular Drosophila melanogaster, and Bombyx mori, and amphibians Xenopus laevis.
- the invention in a first aspect relates to a pharmaceutical composition
- a pharmaceutical composition comprising at least one plant deoxyribonucleoside kinase enzyme, or a polynucleotide sequence encoding said at least one plant deoxyribonucleoside kinase enzyme, or an expression vector comprising said polynucleotide sequence, or a packaging cell line capable of producing an infective virion comprising said vector, or an isolated host cell transduced with the vector, and a pharmaceutically acceptable carrier or diluent.
- Plant deoxyribonucleoside kinases have never been contemplated for use in a pharmaceutical composition or for medical use at all.
- the present inventors have determined that plant deoxyribonucleoside kinases have properties different from those of deoxyribonucleoside kinases from other taxons, in particular virus and animals, which properties make plant deoxyribonucleoside kinases better for medical use than other deoxyribonucleoside kinases.
- the plant deoxyribonucleoside kinase enzyme is selected from the group consisting of:
- a plant deoxyribonucleoside kinase enzyme derived from thale cress (Arabidopsis thaliana), loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa), - a plant deoxyribonucleoside kinase enzyme comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, - a plant deoxyribonucleoside kinase enzyme comprising an amino acid sequence of at least 70% identity with any one of said SEQ ID Nos, when determined over its entire length,
- a plant deoxyribonucleoside kinase enzyme having an amino acid sequence which, when aligned with a sequence selected from those presented in Table 1 , comprises 60% or more of the residues identified in Table 1 as conserved residues, and
- the present inventors have determined that the plant deoxyribonucleoside kinases as defined above have unexpected properties in terms of substrate specificity. This means that the enzymes are essentially multi-substrate, plant dCK/dGK-like kinase enzymes. It also means that the enzymes are more efficient that the known dNKs of which the deoxyribonucleoside kinase derived from human Herpes simplex virus type 1 (HSV-1) is the most promising.
- HSV-1 Herpes simplex virus type 1
- the preferred enzymes of the pharmaceutical compositions decrease at least 3 fold the lethal dose (LD-ioo) of at least one nucleoside analogue when compared to the action of a deoxyribonucleoside kinase derived from human Herpes simplex preferably human Herpes simplex virus type 1 (HSV-1).
- a deoxyribonucleoside kinase derived from human Herpes simplex preferably human Herpes simplex virus type 1 (HSV-1).
- HSV-1 Herpes simplex virus type 1
- One preferred example of such nucleoside analogues used for reference is gemcitabine (dFdC).
- the invention relates to articles containing a nucleoside analogue and a plant deoxyribonucleoside kinase, or a gene coding for said plant deoxyribonucleoside kinase, or vector comprising said gene coding for said plant deoxyribonucleoside kinase, or a packaging cell line capable of producing an infective virion comprising said vector, as a combination for the simultaneous, separate or successive administration in cancer therapy.
- said plant deoxyribonucleoside kinase is enzyme is selected from the group consisting of: a plant deoxyribonucleoside kinase enzyme derived from thale cress
- a plant deoxyribonucleoside kinase enzyme comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, a plant deoxyribonucleoside kinase enzyme comprising an amino acid sequence of at least 70% identity with any one of said SEQ ID NOs, when determined over its entire length, - a plant deoxyribonucleoside kinase enzyme having an amino acid sequence which, when aligned with a sequence selected from those presented in Table 1, comprises 60% or more of the residues identified in Table 1 as conserved residues, and - a plant deoxyribonucleoside kinase as defined above having a C-
- nucleoside analogue containd in said articles is selected from the group consisting of cytidine, adenosine and guanosine analogs. These nucleoside analogues can all be converted by the kinases described in the present invention, and provide a high therapeutic efficiency. More preferably the nucleoside analog is Gemcitabine.
- the invention in a further aspect relates to a method of inhibiting a pathogenic agent in a warm-blooded animal, which method comprises administering to said animal a polynucleotide or a vector encoding a plant deoxyribonucleoside kinase enzyme, or a packaging cell line capable of producing an infective virion comprising said vector.
- the encoded plant deoxyribonucleoside kinase enzyme is selected from the group consisting of: - a plant deoxyribonucleoside kinase enzyme derived from thale cress (Arabidopsis thaliana), loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa),
- a plant deoxyribonucleoside kinase enzyme comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12,
- a plant deoxyribonucleoside kinase enzyme having an amino acid sequence which, when aligned with a sequence selected from those presented in Table 1, comprises 60% or more of the residues identified in Table 1 as conserved residues, and
- the plant deoxyribonucleoside kinase is derived from thale cress (Arabidopsis thaliana), and the enzyme shows at least 80%, preferably at least 85%, more preferred at least 90%, most preferred at least 95% identity with the amino acid sequence presented as SEQ ID NO: 2.
- the invention provides plant deoxyribonucleoside kinase enzymes derived from loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa), as well as the nucleotide sequences coding for said plant deoxyribonucleoside kinases, vector constructs comprising said polynucleotide sequences and a promoter operably linked to the polynucleotide, packaging cell lines capable of producing infective virions, which cell lines comprises said expression vector of the invention, isolated host cells transduced/transfected/transformed with the expression vector of the invention.
- the dCK/dGK like kinase enzymes from these four plant species constitute enzymes provided for the first time in purified form by the present invention. Due to the unexpected broad substrate specificity and the ability to phosphorylate a broad range of nucleoside analogues the kinases can be used for a wide range of applications, including medical use. In a further aspect the invention relates to the use of the plant deoxyribonucleoside kinase enzyme of the invention for phosphorylation of nucleosides or nucleoside analogs.
- the invention provides methods of phosphorylating nucleosides or nucleoside analogs, comprising the steps of subjecting the nucleosides or nucleoside analogs to the action of the plant deoxyribonucleoside kinase enzyme of the invention, and recovering the phosphorylated nucleosides or nucleoside analogs.
- the invention provides a method of non-invasive nuclear imaging of transgene expression of a plant deoxyribonucleoside kinase enzyme of the invention in a cell or subject.
- Radionuclide imaging techniques like single photon emission computed tomography (SPECT) and positron emission tomography (PET), which can non- invasively visualize and quantify metabolic processes in vivo, are being evaluated for repetitive monitoring of transgene expression in living animals and humans.
- Transgene expression can be monitored directly by imaging the expression of the therapeutic gene itself, or indirectly using a reporter gene that is coupled to the therapeutic gene.
- Various radiopharmaceuticals have been developed and are now being evaluated for imaging of transgene expression.
- the invention relates to a method of controlling or modifying growth of a plant, which plant comprises plant cells comprising a polynucleotide encoding a plant deoxyribonucleoside kinase enzyme of the invention which method comprises the step of exposing the plant or plant cells to a nucleoside analogue.
- a nucleoside analogue is gemcitabine.
- the invention relates to a pharmaceutical composition
- a pharmaceutical composition comprising a therapeutically effective amount of at least one plant deoxyribonucleoside kinase enzyme, or a polynucleotide sequence encoding said at least one plant deoxyribonucleoside kinase enzyme, or an expression vector comprising said polynucleotide sequence, or a packaging cell line capable of producing an infective virion comprising said vector, or an isolated host cell transduced with the vector, and a pharmaceutically acceptable carrier or diluent.
- the plant deoxyribonucleoside kinase enzyme is selected from the group consisting of:
- a plant deoxyribonucleoside kinase enzyme derived from thale cress (Arabidopsis thaliana), loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa), - a plant deoxyribonucleoside kinase enzyme comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12,
- - a plant deoxyribonucleoside kinase enzyme having an amino acid sequence which, when aligned with a sequence selected from those presented in Table 1 , comprises 60% or more of the residues identified in Table 1 as conserved residues, and - a plant deoxyribonucleoside kinase as . defined above having a C-terminal and/or N-terminal deletion in the order of 1-80 amino acid residues.
- the pharmaceutical composition comprises a gene coding for the deoxyribonucleoside kinase, an expression vector comprising said gene or a packaging cell line capable of producing an infective virion comprising said vector.
- the preferred polynucleotide sequence comprising the sequence coding for the deoxyribonucleoside kinase is selected from the group consisting of:
- polynucleotide sequence capable of hybridising under at least medium stringency conditions with a polynucleotide sequence presented as SEQ ID No 1 , 3, 5, 7, 9, or 11 ; and - a functional analog of any of SEQ ID No 1 , 3, 5, 7, 9, or 11.
- the preferred plant deoxyribonucleoside kinase enzymes are selected from the group consisting of:
- a plant deoxyribonucleoside kinase enzyme comprising an amino acid sequence of at least 80% identity with any one said SEQ ID Nos, when determined over its entire length, and
- These enzymes have improved kinetic properties and broader substrate specifictity compared to known dNKs, in particular compared to a deoxyribonucleoside kinase derived from human Herpes simplex, in particular human Herpes simplex virus type 1 (HSV-1).
- HSV-1 Herpes simplex virus type 1
- polynucleotide sequences encoding a plant deoxyribonucleoside kinase enzyme the corresponding preferred sequences are selected from the group consisiting of: - a polynucleotide sequence represented by SEQ ID No 1 or 5;
- polynucleotide sequence having at least 80% sequence identity with SEQ ID No 1 or 5 when determined over its entire length;
- the enzymes for the medical, pharmaceutical and therapeutic aspects are derived from thale cress (Arabidopsis thaliana), and which enzyme shows at least 80%, preferably at least 85%, more preferred at least 90%, most preferred at least 95% identity with the amino acid sequence presented as SEQ ID NO: 2.
- the dNK from thale cress was most efficient in phosphorylating both deoxyribonucleosides and analogs.
- polynucleotide sequences corresponds to the plant deoxyribonucleoside kinase encoding sequence derived from thale cress (Arabidopsis thaliana), which sequence shows at least 80%, preferably at least 85%, more preferred at least 90%, most preferred at least 95% identity with the polynucleotide sequence presented as SEQ ID No. 1.
- plant deoxyribonucleoside kinase enzyme of the invention may be administered in any convenient form.
- plant deoxyribonucleoside kinase enzyme of the invention is incorporated into a pharmaceutical composition together with one or more adjuvants, excipients, carriers and/or diluents, and the pharmaceutical composition prepared by the skilled person using conventional methods known in the art.
- the composition may be administered alone or in combination with one or more other agents, drugs or hormones.
- the pharmaceutical composition of this invention may be administered by any suitable route, including, but not limited to oral, intravenous, intramuscular, inter- arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, anteral, topical, sublingual or rectal application, buccal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intracistemal, intracapsular, intrapulmonary, transmucosal, or via inhalation.
- the active ingredient may be administered in one or several doses per day.
- appropriate dosages are between 0.5 ng to about 50 ⁇ g/kg deoxyribonucleoside kinase/kg body weight per administration, and from about 1.0 ng/kg to about 100 ⁇ g/kg daily.
- the dose administered must of course be carefully adjusted to the age, weight and condition of the individual being treated, as well as the route of administration, dosage form and regimen, and the result desired, and the exact dosage should of course be determined by the practitioner.
- the plant deoxyribonucleoside kinase of the invention may be administered by genetic delivery, using cell lines and vectors as described below under methods of treatment. Therefore, in another preferred embodiment, the invention provides pharmaceutical compositions comprising the polynucleotide of the invention, or a vector of the invention, or a packaging cell of the invention, or a host cell of the invention, and a pharmaceutically acceptable carrier or diluent. To generate such therapeutic cell lines, the polynucleotide of the invention may be inserted into an expression vector, e.g.
- 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.
- the present invention which relates to polynucleotides and proteins, polypeptides, or derivatives produced therefrom, may be used for treating or alleviating a disorder or disease of a living animal body, including a human, which disorder or disease is responsive to the activity of a cytotoxic agent.
- the disorder, disease or condition may in particular be a cancer or a viral infection.
- the polynucleotides of the present invention may in particular be used as a "suicide gene", i.e. a drug-susceptibility gene. Transfer of a suicide gene to a target cell renders the cell sensitive to compounds or compositions that are relatively non- toxic to normal cells.
- suicide gene i.e. a drug-susceptibility gene.
- the invention provides a method for sensitising target cells to prodrugs, which method comprises the steps of
- transfecting the target cell with a polynucleotide sequence encoding a plant deoxyribonucleoside kinase enzyme that promotes the conversion of said prodrug into a (cytotoxic) drug; and (ii) delivering said prodrug to said target cell; wherein said target cell is more sensitive to said (cytotoxic) drug than to said prodrug.
- a plant deoxyribonucleoside kinase enzyme may be used.
- the encoded plant deoxyribonucleoside kinase enzyme is selected from the group consisting of: - a plant deoxyribonucleoside kinase enzyme derived from thale cress Arabidopsis thaliana, loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa),
- a plant deoxyribonucleoside kinase enzyme comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ
- - a plant deoxyribonucleoside kinase enzyme comprising an amino acid sequence of at least 70% identity with any one of said SEQ ID Nos, when determined over its entire length, - a plant deoxyribonucleoside kinase enzyme having an amino acid sequence which, when aligned with a sequence selected from those presented in Table 1 , comprises 60% or more of the residues identified in Table 1 as conserved residues, and
- the preferred plant deoxyribonucleoside kinase enzymes are selected from the group consisting of:
- - a plant deoxyribonucleoside kinase enzyme comprising an amino acid sequence of at least 80% identity with any one said SEQ ID Nos, when determined over its entire length, and - a plant deoxyribonucleoside kinase as defined above having an N-terminal or C-terminal deletion in the order of 1-80 amino acid residues.
- These enzymes have improved kinetic properties and broader substrate specifictity compared to known dNKs, in particular compared to a deoxyribonucleoside kinase derived from human Herpes simplex, in particular human Herpes simplex virus type l (HSV-1).
- the enzymes for the medical, pharmaceutical and therapeutic aspects are derived from thale cress (Arabidopsis thaliana), and which enzyme shows at least 80%, preferably at least 85%, more preferred at least 90%, most preferred at least 95% identity with the amino acid sequence presented as SEQ ID NO: 2.
- the dNK from thale cress was most efficient in phosphorylating both deoxyribonucleosides and analogs.
- the deoxyribonucleoside kinase enzyme invention may be used directly via e.g., injected, implanted or ingested pharmaceutical compositions to treat a pathological process responsive to the deoxyribonucleoside kinase enzyme.
- the polynucleotide of the invention including the complementary sequences thereof, may be used for the expression of the deoxyribonucleoside kinase enzyme of the invention. This may be achieved by cell lines expressing such proteins, peptides or derivatives of the invention, or by virus vectors encoding such proteins, peptides or derivatives of the invention, or by host cells expressing such proteins, peptides or derivatives. These cells, vectors and compositions may be administered to treatment target areas to affect a disease process responsive to cytotoxic agents.
- Suitable expression vectors may be a viral vector derived from Herpes simplex, adenovira, lentivira, retrovira, or vaccinia vira, or from various bacterially produced plasmids, and may be used for in vivo delivery of nucleotide sequences to a whole organism or a target organ, tissue or cell population. Other methods include, but are not limited to, liposome transfection, electroporation, transfection with carrier peptides containing nuclear or other localising signals, and gene delivery via slow- release systems.
- "antisense" nucleotide sequences complementary to the nucleotide of the invention or portions thereof may be used to inhibit or enhance deoxyribonucleoside kinase enzyme expression.
- the invention provides methods for inhibiting pathogenic agents in warm-blooded animals, which methods comprises the step of administering to said animal a polynucleotide of the invention, or an expression vector of the invention.
- polynucleotide sequence or the expression vector is administered in vivo.
- the pathogenic agent is a virus, a bacteria or a parasite, or even a tumour cell. In another preferred embodiment the pathogenic agent is an autoreactive immune cell.
- the method further comprises the step of administering a nucleoside analogue to said warm-blooded animal.
- nucleoside analogs exist that can be converted into a toxic product by the dNKs described in the present invention.
- the prodrug is a nucleoside analogue.
- Nucleoside analogues that are suitable for use according to the invention include aciclovir (9-[2-hydroxy-ethoxy]-methyl-guanosine), buciclovir, famciclovir, ganciclovir (9-[2-hydroxy-1 -(hydroxymethyl)ethoxyl-methyl]-guanosine), penciclovir, valciclovir, trifluorothymidine, AZT (3'-azido-3'-thymidine), AIU (5'-iodo-5'-amino-2',5'- dideoxyuridine), ara-A (adenosine-arabinoside; Vivarabine), ara-C (cytidine- arabinoside), ara-G (9-beta-D-arabinofuranosylguanine), ara-T, 1-beta-D- arabinofuranosyl thymine, 5-e
- the nucleoside analog is cytidine analog, a guanosine analog or an adenosine analog.
- cytidine, guanosine and adenosine analogs include dFdC gemcitabine (2 ,2 ⁇ -difluorodeoxycytidine), 2-chloro- 2 -deoxyadenosine (2CdA), CaFdA (2-chloro-2-ara-fluoro-deoxyadenosine), fludarabine (2-Fluoroadenine 9-beta-D-Arabinofuranoside), 2 ⁇ 3 -dideoxycytidine (ddC), 2 ⁇ 3 x -dideoxyadenosine (ddA), 2 ⁇ 3 ' -dideoxyguanosine (ddG), ara-A (adenosine-arabinoside; Vivarabine), ara-C (cytidine-arabinoside),
- nucleoside analog is Gemcitabine which is shown in the examples to be converted effectively by the enzymes of the present invention into a toxic product.
- the invention relates to plant dCK/dGK-like deoxyribonucleoside kinase enzymes.
- a dCK/dGK-like deoxyribonucleoside kinase enzyme is understood an enzyme which can phosphorylate at least dAdo, dGuo, and dCyd as shown in Example 3. This does not exclude that the kinases have activity towards other deoxyribonucleosides as can be seen in the examples.
- the plant dCK/dGK-like deoxyribonucleoside kinase enzymes upon transduction into a cell, decreases at least 3 fold the lethal dose (LD 100 ) of at least one nucleoside analogue when compared to the action of a deoxyribonucleoside kinase derived from human Herpes simplex, in particular human Herpes simplex virus type 1 (HSV-1).
- a deoxyribonucleoside kinase derived from human Herpes simplex, in particular human Herpes simplex virus type 1 (HSV-1).
- HSV-1 Herpes simplex virus type 1
- said at least one nucleoside analogue is Gemcitabine.
- the plant dCK/dGK-like deoxyribonucleoside kinase of the invention is derived from thale cress Arabidopsis thaliana, from loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa).
- the most preferred plant deoxyribonucleoside kinase enzymes are selected from the group consisting of:
- a plant deoxyribonucleoside kinase enzyme comprising an amino acid sequence of at least 70% identity with any one said SEQ ID Nos, when determined over its entire length, and
- dNKs deoxyribonucleoside kinase derived from human Herpes simplex, in particular human Herpes simplex virus type 1 (HSV-1).
- HSV-1 human Herpes simplex virus type 1
- the enzymes for the medical, pharmaceutical and therapeutic aspects are derived from thale cress (Arabidopsis thaliana), and which enzyme shows at least 80%, preferably at least 85%, more preferred at least 90%, most preferred at least 95% identity with the amino acid sequence presented as SEQ ID NO: 2.
- the dNK from thale cress was most efficient in phosphorylating both deoxyribonucleosides and analogs.
- the plant deoxyribonucleoside kinases of the invention are multi-substrate kinases, capable of converting all four natural deoxyribonucleosides (dThd, dCyd, dAdo, dGuo). This can in particular be obtained by C- and/or N-terminal deletions.
- the invention provides isolated plant deoxyribonucleoside kinase enzymes (dNK) derived from loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) and from rice (Oryza sativa). More specifically the isolated plant deoxyribonucleoside kinase enzymes of the invention are deoxycytidine kinases (dCK) and/or deoxyguanosine kinases (dGK), herein designated as dCK/dGK-like kinases.
- dCK deoxycytidine kinases
- dGK deoxyguanosine kinases
- AT-dCK/dGK (SEQ ID NO: 2) Tomato-dCK/dGK (SEQ ID NO: 6) Pine-dCK/dGK (SEQ ID NO: 4) Maize-dCK/dGK ( SEQ ID NO: 8) Rice-dCK/dGKII (SEQ ID NO: 12) Rice-dCK/dGKI (SEQ ID NO: 10) + N-terminal extension * C-terminal extension Black: identical residues - threshold 50% Grey: similar residues - threshold 50% % conserved residues : semiconserved residues
- amino acid sequence of the plant deoxyribonucleoside kinase enzyme of the invention when aligned with a sequence selected from those presented in Table 1 , comprises 60% or more of the residues identified in Table 1 as conserved residues, preferably more than 70%, more preferred more than 80%, even more preferred more than 90%, still more preferred more than 9 ⁇ %, most preferred every one of the conserved residues identified in Table 1.
- amino acid sequence of the plant deoxyribonucleoside kinase enzyme of the invention when aligned with a sequence selected from those presented in Table 1 , in addition to comprising conserved residues, also comprises a semi-conserved residue at 60% or more of the positions identified in Table 1 as semi-conserved residues, preferably more than 70%, more preferred more than 80%, even more preferred more than 90%, still more preferred more than 9 ⁇ %, most preferred every one of the semi-conserved residues identified in Table 1.
- the plant deoxyribonucleoside kinase enzyme of the invention comprises the amino acid sequence presented as SEQ ID NO: 2, as SEQ ID NO: 4, as SEQ ID NO: 6, as SEQ ID NO: 8, as SEQ ID NO: 10, or as SEQ ID NO: 12, or an amino acid sequence that has at least 80%, preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 9 ⁇ % identity with any of said sequences, when determined over the entire length of the SEQ ID NO.
- 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 "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 174 247-2 ⁇ 0], which is available from the National Center for Biotechnology Information (NCBI) web site, and using the default settings suggested here (i.e.
- NCBI National Center for Biotechnology Information
- the BLAST algorithm determines the % sequence identity in a range of overlap between two aligned sequences.
- the percent sequence identity is preferably calculated in a range of overlap of at least ⁇ O amino acids, more preferably at least 7 ⁇ amino acids, more preferably at least 100 amino acids, the range being calculated by BLASTP under default settings.
- the plant deoxyribonucleoside kinase enzyme of the invention is derived from loblolly pine, and showing at least 76%, preferably at least 80%, more preferred at least 85%, even more preferred at least 90%, most preferred at least 95% identity with the amino acid residues presented in SEQ ID NO:
- the plant deoxyribonucleoside kinase enzyme of the invention is derived from tomato, and showing at least 80%, preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 9 ⁇ % identity with the amino acid residues presented in SEQ ID NO: 6, when determined over its entire length.
- the plant deoxyribonucleoside kinase enzyme of the invention is derived from maize, and showing at least 76%, preferably at least 80%, more preferred at least 8 ⁇ %, even more preferred at least 90%, most preferred at least 9 ⁇ % identity with the amino acid residues presented in SEQ ID NO:
- the plant deoxyribonucleoside kinase enzyme of the invention is derived from rice, and showing at least 80%, preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 9 ⁇ % identity with the amino acid residues presented in SEQ ID NO: 12, when determined over its entire length.
- the plant deoxyribonucleoside kinase enzyme of the invention is derived from rice, and showing at least 60%, more preferably at least 70% more preferably at least 80%, preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 9 ⁇ % identity with the amino acid residues presented in SEQ ID NO: 10, when determined over its entire length.
- the plant deoxyribonucleoside kinase enzyme used for the pharmaceutical compositons and for medical and therapeutic use of the invention is derived from thale cress (Arabidopsis thaliana), and showing at least 76%, preferably at least 80%, more preferred at least 8 ⁇ %, even more preferred at least 90%, most preferred at least 9 ⁇ % identity with the amino acid residues presented in SEQ ID NO: 2, when determined over its entire length.
- the plant deoxyribonucleoside kinase enzyme of the invention comprises the amino acid sequence presented as SEQ ID NO: 2, as SEQ ID NO: 4, as SEQ ID NO: 6, as SEQ ID NO: 8, as SEQ ID NO: 10, or as SEQ ID NO: 12, or a functional analogue thereof.
- the term "functional analog” means a polypeptide (or protein) having an amino acid sequence that differs from the sequence presented as SEQ ID NO: 2, as SEQ ID NO: 4, as SEQ ID NO: 6, as SEQ ID NO: 8, as SEQ ID NO: 10, or as SEQ ID NO: 12, at one or more amino acid positions and has dCK/dGK activity.
- Such analogous polypeptides include polypeptides comprising conservative substitutions, splice variants, isoforms, homologues from other species, and polymorphisms.
- conservative substitutions denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative substitutions include
- conservative substitution also include the use of a substituted amino acid residue in place of a parent amino acid residue, provided that antibodies raised to the substituted polypeptide also immunoreact with the un-substituted polypeptide. 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.
- plant 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 plant deoxyribonucleoside kinase enzymes of the invention may be converted into essentially multi-substrate enzymes, having ability to phosphorylate all four deoxyribonucleosides.
- dCK deoxycytidine kinase
- dGK deoxyguanosine kinase
- the plant kinase enzyme of the invention upon transduction into a cell, decreases at least 3 fold the lethal dose (LD-ioo) of at least one nucleoside analogue when compared to the action of a deoxyribonucleoside kinase derived from human Herpes simplex preferably human Herpes simplex virus type 1 (HSV-1).
- LD-ioo lethal dose
- the invention provides plant deoxyribonucleoside kinase enzymes having C- and/or N-terminal deletions when compared to the parent un-truncated (parent) enzyme.
- deletions may be obtained by conventional techniques, e.g. site-directed mutagenesis, or as described in the working examples.
- the kinase enyme upon transduction into a cell dereceases at least 3 fold the lethal dose (LD-ioo) of at least one nucleoside analogue when compared to the action of the untruncated deoxyribonucleoside enzyme.
- the kinase enzyme of the invention is plant deoxyribonucleoside kinase enzyme having a C-terminal and/or N-terminal deletion of up to about 100 amino acids.
- the kinase enzyme of the invention is plant deoxyribonucleoside kinase enzyme having a C-terminal deletion in the order of 1-80, preferably 1-60 amino acid residues, more preferred 1- ⁇ 0 amino acid residues, even more preferred 1-40 amino acid residues, still more preferred 1-30 amino acid residues, most preferred 1-20 amino acid residues.
- the kinase enzyme of the invention is plant deoxyribonucleoside kinase enzyme having a C-terminal deletion of 61 amino acid residues.
- said C-terminally deleted variant is derived from tomato.
- the kinase enzyme of the invention is plant deoxyribonucleoside kinase enzyme having a N-terminal deletion in the order of 1-80, preferably 1-60 amino acid residues, more preferred 1- ⁇ 0 amino acid residues, even more preferred 1-40 amino acid residues, still more preferred 1-30 amino acid residues, most preferred 1-20 amino acid residues.
- the N-terminally deleted variant dCK/dGK enzyme is derived from tomato.
- C-terminal deletions create enzymes of changed and often broader substrate specificities, when compared to the un-truncated (parent) enzyme.
- the invention relates to isolated polynucleotides encoding a plant dCK7dGK-like deoxyribonucleoside kinase, which upon transduction into a cell decreases at least 3 fold the lethal dose (LD-ioo) of at least one nucleoside analogue when compared to the action of a deoxyribonucleoside kinase derived from human
- LD-ioo lethal dose
- HSV-1 Herpes simplex virus type 1
- the invention relates to isolated polynucleotides encoding plant deoxyribonucleoside kinase enzymes derived from loblolly pine (Pinus taeda), from tomato (Lycopersicum esculentum), from maize (Zea mays) or from rice (Oryza sativa), preferably encoding the plant deoxyribonucleoside kinase enzyme of the invention. )
- the isolated polynucleotide of the invention is capable of hybridising with the polynucleotide sequence presented as SEQ ID NO: 1, as SEQ ID NO: 3, as SEQ ID NO: ⁇ , as SEQ ID NO: 7, as SEQ ID NO: 9, or as SEQ ID NO: 11 , or its complementary strand.
- Hybridization 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 ⁇ x SSC [Sodium chloride/Sodium citrate; cf. Sambrook et al.; Molecular Cloning: A Laboratory Manual. 2 nd Ed., Cold Spring Harbor Lab., Cold Spring Harbor, NY 1989] for 10 minutes, and prehybridization of the filter in a solution of ⁇ x SSC, ⁇ x Denhardt's solution [cf.
- the filter is then washed twice for 30 minutes in 2 x SSC, 0. ⁇ % SDS at a temperature of at least ⁇ °C (low stringency conditions), more preferred of at least 60°C (medium stringency conditions), still more preferred of at least 6 ⁇ °C (medium/high stringency conditions), even more preferred of at least 70°C (high stringency conditions), and yet more preferred of at least 7 ⁇ °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. 3 H, 125 l, 35 S, 14 C, or 32 P-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.
- the isolated polynucleotide of the invention has at least 81 %, preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 9 ⁇ % identity to the polynucleotide sequence presented as SEQ ID NO: 1 , as SEQ ID NO: 3, as SEQ ID NO: ⁇ , as SEQ ID NO: 7, as SEQ ID NO: 9, or as SEQ ID NO: 11 , when determined over the entire length of the SEQ ID NO:.
- the percent sequence identity is calculated when BLASTN provides a range of overlap of at least 100 nucleotides, the range being determined under default settings. More preferably the range of overlap is at least 1 ⁇ 0 nucleotides, more preferably at least 22 ⁇ nucleotides, more preferably at least 300 nucleotides.
- identity is a measure of the degree of homology of nucleotide 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 "percent identity" of two amino acid sequences or of two nucleic acids is determined using the BLASTN algorithm [Tatiana A. Tatusova, Thomas L. Madden: Blast 2 sequences - a new tool for comparing protein and nucleotide sequences; FEMS Microbiol. Lett. 1999 174 247-260], which is available from the National Center for Biotechnology Information (NCBI) web site, and using the default settings suggested here (i.e.
- NCBI National Center for Biotechnology Information
- the BLASTN algorithm determines the % sequence identity in a range of overlap between two aligned nucleotide sequences.
- the percent sequence identity is preferably calculated in a range of overlap of at least 100 nucleotides, the range being determined by BLASTN under default settings. More preferably the range of overlap is at least 300 nucleotides.
- the isolated polynucleotide for 5 medical/pharmaceutical use is derived from thale cress (Arabidopsis thaliana) and shows at least 70%, preferably at least 80%, more preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 9 ⁇ % identity to the polynucleotide sequence presented as SEQ ID NO: 1 , when determined over its entire length.
- the isolated polynucleotide of the .0 invention is derived from loblolly pine and shows at least 81 %, preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 9 ⁇ % identity to the polynucleotide sequence presented as SEQ ID NO: 3, when determined over its entire length.
- the isolated polynucleotide of the invention is derived from tomato and shows at least 79%, preferably at least 80%,
- the isolated polynucleotide of the invention is derived from maize and shows at least 79%, preferably at least 80%, more preferred at least 8 ⁇ %, even more preferred at least 90%, most preferred at least 9 ⁇ % identity to the polynucleotide sequence presented as SEQ ID NO: 7, when determined over its entire length.
- the isolated polynucleotide of the invention is derived from rice and shows at least 82%, preferably at least 8 ⁇ %, more preferred at least 90%, most preferred at least 95% identity to the polynucleotide sequence presented as SEQ ID NO: 11, when determined over its entire length.
- the isolated polynucleotide of the invention is derived from rice and shows at least 82%, preferably at least 85%, more preferred at least 90%, most preferred at least 95% identity to the polynucleotide sequence presented as SEQ ID NO: 9, when determined over its entire length.
- the isolated polynucleotide of the invention comprises the polynucleotide sequence presented as SEQ ID NO: 1 , as SEQ ID NO: 3, as SEQ ID NO: 5, as SEQ ID NO: 7, as SEQ ID NO: 9, or as SEQ ID NO: 11 , or a functional analog thereof.
- the term "functional analog” covers conservatively modified polynucleotides, and polynucleotides encoding functionally equivalent polypeptides.
- functionally equivalent polypeptides is meant a polypeptide having dCK/dGK enzyme activity as herein defined.
- nucleic acids refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences.
- nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein, which encodes a polypeptide, also describes every possible silent variation of the nucleic acid.
- the invention provides recombinant expression vectors comprising the isolated polynucleotide of the invention and a promoter operably linked to the polynucleotide.
- the isolated polynucleotides are selected from the group consisting of
- the expression vector of the invention preferably is one suitable for carrying out expression in a eukaryotic organism.
- the expression vector of the invention is a viral vector, in particular a Herpes simplex viral vector, an adenoviral vector, an adenovirus-associated viral vector, a lentivirus vector, a retroviral vector or a vacciniaviral vector.
- the invention provides packaging cell lines capable of producing an infective virion, which cell line comprises a vector of the invention.
- Packaging cells refers to cells, which contains those elements necessary for production of infectious recombinant vira, which are lacking in a recombinant virus vector. Methods for preparing packaging cells are known in the prior art.
- the invention provides a host cell comprising the polynucleotide sequence of the invention or the vector of the invention.
- the invention provides an isolated host cell transduced, transfected, or transformed with the expression vector of the invention.
- the host cell of the invention is a eukaryotic cell, in particular a mammalian cell, an oocyte, or a yeast cell.
- the host cell of the invention is a human cell, a dog cell, a monkey cell, a rat cell or a mouse cell.
- dCK/dGK enzyme encoding genes of the present invention is for suicide systems in cell and gene based therapy.
- all types of cell and gene therapy on mammals there is a need to have systems, which enable the irreversible killing of transplanted cells or cells which have been transduced by the gene therapy.
- the nucleoside analogue can be administered if the transplanted cells start to proliferate in an uncontrolled manner. One may also wish to terminate the treatment simply because there is no need for the replacement cells anymore or because further treatment is by some other route.
- the other type of cell-based therapy includes therapeutic ceils which are transplanted into the body to secrete e.g. a growth factor in a certain location. Often such therapeutic cells are encapsulated and can relatively easily be removed from the body again but the incorporation of a suicide system is preferred because the cells can be killed selectively without the use of surgery. In in vivo gene therapy the same considerations apply as with replacement cell therapy.
- a suicide gene can be achieved by constructing a viral vector comprising both the therapeutic gene and a dCK/dGK according to the present invention.
- the therapeutic gene and the dCK/dGK are inserted under the control of the same promoter, optionally by separating them with an IRES construct.
- a dCK/dGK enzyme is inserted into the vector construct under the control of the same promoter (or using an IRES construct). This ensures that whenever the oncogene is transcribed, the deoxyribonucleoside kinase is also transcribed and the transduced and tumorigenic cells can be selectively killed by administering a nucleoside analogue, such as Gemcitabine.
- a nucleoside analogue such as Gemcitabine.
- the deoxyribonucleoside kinase enzyme of the invention may find different utility, including both therapeutic and biotechnological applications.
- the invention relates to use of the plant deoxyribonucleoside kinase enzyme of the invention for phosphorylating nucleosides or a nucleoside analogs.
- the invention provides a method for phosphorylating a nucleoside or a nucleoside analog, comprising the steps of i) subjecting the nucleoside or nucleoside analog to the action of the plant deoxyribonucleoside kinase enzyme of the invention; and ii) recovering the phosphorylated nucleoside or nucleoside analog.
- Suicide gene therapy i.e. transfection of a so-called suicide gene that sensitizes target cells towards a prodrug, offers an attractive approach for treating malignant tumors.
- a non-invasive method to assay the extent, the kinetics and the spatial distribution of transgene expression is essential.
- imaging methods allow investigators and physicians to assess the efficiency of experimental and therapeutic gene transfection protocols and would enable early prognosis of therapy outcome.
- Radionuclide imaging techniques like single photon emission computed tomography (SPECT) and positron emission tomography (PET), which can non- invasively visualize and quantify metabolic processes in vivo, are being evaluated for repetitive monitoring of transgene expression in living animals and humans.
- Transgene expression can be monitored directly by imaging the expression of the therapeutic gene itself, or indirectly using a reporter gene that is coupled to the therapeutic gene.
- Various radiopharmaceuticals have been developed and are now being evaluated for imaging of transgene expression.
- the invention provides a method of noninvasive nuclear imaging of transgene expression of a plant deoxyribonucleoside kinase enzyme of the invention in a cell or subject, which method comprises the steps of
- step (iii) non-invasively monitoring the change to said prodrug in said cell or subject.
- the monitoring carried out in step (iii) is performed by Single Photon Emission Computed Tomography (SPECT), by Positron Emission Tomography (PET), by Magnetic Resonance Spectroscopy (MRS), by Magnetic Resonance Imaging (MRI), or by Computed Axial X-ray Tomography (CAT), or a combination thereof
- the substrate is a labelled nucleoside analogue selected from those listed above.
- the labelled nucleoside analogue preferably contains at least one radionuclide as a label. Positron emitting radionuclides are all candidates for usage.
- the radionuclide is preferably selected from 2 H (deuterium), 3 H (tritium), 11 C, 13 C, 14 C, 15 0, 13 N, 123 l, 125 l,
- labelling agents which can be used in the preparation of the labelled nucleoside analogue is [ 11 C]0 2 , 18 F, and Nal with different isotopes of Iodine.
- [ 11 C]0 2 may be converted to a [ 11 C]- methylating agent, such as [ 11 C]H 3 I or [ 11 C]-methyl triflate.
- the deoxyribonucleosides of the invention may also find utility in methods for modifying or controlling plant growth. Therefore, in a further aspect, the invention relates to a method of controlling or modifying growth of a plant, which plant comprises plant cells comprising a polynucleotide encoding a plant dCK/dGK enzyme of the invention, which method comprises the step of exposing the plant or plant cell to a nucleoside analog.
- nucleoside analogues can be used as herbicides for plants having these dCK/dGK enzymes either by nature or as a heterologous gene.
- the deoxyribonucleoside kinases of the present invention By inserting the deoxyribonucleoside kinases of the present invention into plants not having a deoxyribonucleoside kinase with these properties, the plant is rendered susceptible to nucleoside analogs.
- the polynucleotide encoding plant dCK/dGK enzyme of the invention preferably is a heterologous polynucleotide, and the plant subjected to the method of the invention preferably is a transgenic plant.
- the invention provides transgenic plants comprising an expressible heterologous nucleic acid encoding the plant deoxyribonucleoside enzyme of the invention, wherein the heterologous nucleic acid is introduced into the transgenic plant, or an ancestor of the transgenic plant.
- the transgenic plant may be obtained by known techniques for producing genetically modified plants, e.g. by introducing into a plant cell an expression vector of the invention.
- Any plant deoxyribonucleoside kinase may also be knocked out and/or functionally replaced by another plant dCK/dGK, said other kinase not being able to convert a certain nucleoside analog.
- a nucleoside analogue may then be used to kill all other plants around this genetically modified plant.
- Any transformed plant obtained according to the invention can be used in a conventional breeding scheme or in vitro plant propagation to produce more transformed plants with the same characteristics and/or can be used to introduce the same characteristics in other varieties of the same or related species. Such plants are also part of the invention. Seeds obtained from the transformed plants contain the same genetical characteristics and also form part of the invention.
- polynucleotide sequence encoding a plant dCK/dGK is selected from the group consisiting of:
- polynucleotide sequence having at least 80% sequence identity with SEQ ID No 1 or ⁇ when determined over its entire length;
- polynucleotide sequences encode plant dCK/dGK enzymes with preferred kinetic properties.
- polynucleotide sequence is derived from thale cress
- thale cress dCK/dGK is the most efficient in terms of conversion of nucleoside analogs.
- the plasmid containing ACCN AW223792 (clone cLEN13L23) was obtained from the Clemson University Genomics Institute and the insert sequenced.
- cLEN13L23 an ORF of 1098 bp encoding a protein of 36 ⁇ amino acids with a calculated molecular mass of 42.2 kDa was found.
- the ORF was submitted to GenBankTM with the ACCN AF614776 (SEQ ID NO ⁇ ).
- the plasmid containing ACCN AW036847 was obtained from V. Walbot, Department of Biological Sciences, Stanford University, 8 ⁇ California Ave, Palo Alto, CA 94304, USA.
- the plasmid containing ACCN C73813 was obtained from Takuji Sasaki, National Institute of Agrobiological Resources, Rice Genome Research Program, Kannondai 2-1-2, Tsukuba, Ibaraki, 306-8602, Japan.
- the inserts of the three plasmids were completely sequenced, and partial sequences for deoxyribonucleoside kinases (Loblolly pine, Maize and Rice I) were found (see SEQ ID NOS: 3, 7 and 9).
- Lycopersicum esculentum deoxyribonculeoside kinases to genomic sequences of rice, and by combining several DNA fragments from ACCN AP003922, the following full- length ORF for a deoxyribonucleoside kinase from rice presented as SEQ ID NO: 11 could be assembled, encoding the protein presented as SEQ ID NO: 12.
- SEQ ID NO: 10 is not completely identical to SEQ ID NO 12 implies the existence of isoforms of rice deoxyribonculeoside kinases (Rice dNK I and Rice dNK II).
- This example describes the preparation of bacterial expression plasmids for full-length and N- and/or C-terminal truncated deoxyribonucleoside kinases.
- the deoxyribonucleoside kinase from Lycopersicum esculentum and its truncated mutants were amplified and subcloned as follows: The ORF- of AF614776 (SEQ ID NO ⁇ ) was amplified by PCR using the primers
- TOMrevlA (SEQ ID NO: 14) were used, the resulting plasmid was named pGEX-2T-TOM- ⁇ NdCK/dGK.
- HSV1-TK Herpes simplex virus type 1 thymidine kinase
- pGEX-2T-HSV-TK using the plasmid pCMV-pacTK described by Karreman [Christiaan Karreman; Gene 1998 218 67-62] as template.
- the PCR fragment was subsequently cut by EcoRI/BamHI and ligated into pGEX-2T vector that was also cut by EcoRI/BamHI.
- the resulting plasmid was named pGEX-2T-HSV-TK.
- the E. coli strain KY896 (F " , tdk-1, ilv) [Knecht W, Munch-Petersen B and Piskur J: Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster; J. Mol. Biol. 2000 301 827-837] was transformed by various expression plasmids using standard techniques. Transformed KY896 strains were grown to an OD 6 oonm of O. ⁇ -0.6 In LB/Ampicillin (100 ⁇ g/ml) medium at 37°C, and protein expression was induced by addition of 100 ⁇ M IPTG.
- the cells were further grown for 4 hours at 25°C and subsequently harvested by centrifugation. Pellets were stored at -80°C until activity testing. Pellets were submitted to brief sonification in extraction buffer (60 mM Tris/HCI pH 7.6, 1 mM DTT, 10% (v/v) glycerol, 1% (v/v) Triton X-100, protease inhibitor cocktail (CompleteTM from Roche Diagnostics). Deoxyribonucleoside kinase activities were determined in the KY896 extracts by initial velocity measurements based on four time samples by the DE-81 filter paper assay using tritium-labelled nucleoside substrates. 4 to 20 ⁇ g extracts were used in the assays.
- the protein concentration was determined according to Bradford with BSA as standard protein [Bradford M M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding; Anal. Biochem. 1976 72 248-254].
- SDS-PAGE was done according to the procedure of Laemmli [Laemmli U K: Cleavage of structural proteins during the assembly of the head of bacteriophage T4; Nature 1970 227 680-685], and proteins were visualized by Coomassie staining to verify recombinant protein expression.
- the four natural deoxyribonucleosides were tested at a fixed concentration of 100 ⁇ M. The highest specific activity in each extract was set to 100%.
- pGEX-2T-AT-dCK/dGK is the vector containing the gene encoding an Arabidopsis thaliana dNK enzyme.
- deoxyribonucleoside kinases from Arabidopsis thaliana pGEX-2T-AT- dCK/dGK
- tomato pGEX-2T-TOM-dCK/dGK
- pGEX-2T-TOM- ⁇ NdCK/dGK The deoxyribonucleoside kinases from Arabidopsis thaliana
- the extended N- and C-terminus together play a vital role in substrate specificity and structural integrity of plant deoxyribonucleoside kinases.
- Deoxyribonucleoside kinases are of interest as suicide-genes to be used in gene-mediated therapy of cancer or viral infections.
- the potential of the plant kinases of the invention to convert different nucleoside analogs are compared to that of the human Herpes simplex virus type 1 thymidine kinase (HSV1-TK) in a bacterial test system.
- HSV1-TK Herpes simplex virus type 1 thymidine kinase
- AT-dCK/dGK was most efficient, as reflected by the lowest LD 10 o, in killing KY896 on dFdC plates.
- the LD-ioo was 10000-fold lower than that of HSV1-TK, that sensitised the cells to the same degree as the empty plasmid pGEX-2T.
- Cells transfected with the expression plasmid for TOM-dCK/dGK or TOM- ⁇ NdCK/dGK could be killed at 10-fold lower concentrations than cells transformed with pGEX-2T-HSV-TK or pGEX-2T.
- the cDNA of plant kinases were cloned into a retrovirus vector based on the Moloney murine leukemia (MLV) virus to generate a replication-deficient recombinant retrovirus containing the kinases.
- MLV Moloney murine leukemia
- DNA fragments were amplified with Pfu polymerase (Stratagene) using primers with designed flanking restriction enzyme sites and containing Kozak sequence at ⁇ ' end.
- Pfu polymerase (Stratagene) using primers with designed flanking restriction enzyme sites and containing Kozak sequence at ⁇ ' end.
- A. thaliana and tomato constructs based on PCR fragment were cut with
- AtdCGK PZG190
- TomdCGK PZG193
- LCXSN alone was used as a control.
- the plasmids were purified using the Qiagen plasmid kit (QIAGEN) and DNA sequences of the constructed plasmids were verified by DNA sequence determination.
- HE 293 T packaging cells (ATCC CRL-11268) were cultured at 37°C in OPTIMEM 1 medium (Life Technologies, Inc.)
- the constructed pLCXSN plasmid vector was transfected into the packaging cells using LipofectAMINE PLUS (Life Technologies, Inc.) according to the protocol provided by the supplier.
- the medium from the transfected cells was collected 48 hours after transfection, filtered through a 0.45 ⁇ m filter, pelleted by ultracentrifugation (50.000xg, 90 minutes at 4°C) and dissolved in DMEM (Cambrex, Bio Whittaker Cat. No. 12-741-F).
- the virus containing medium was subsequently used to transduce the cancer cell lines with a MOI of ⁇ .
- Human breast MCF-7 (ATCC HTB-22) and Glioblastoma U-118-MG (ATCC HTB-16) cancer cells were purchased from the American Type Culture Collection. Cells were cultured in RPMI, E-MEM or D-MEM (Cambrex, Bio Whittaker Cat. No. 12- 116-F, 12-611 and 12-741-F) with 10% (v/v) Australian originated fetal calf serum (Cambrex, Bio Whittaker Cat. No. 12-611) and 1 ml/l of Gentamicin (Cambrex, Bio Whittaker Cat. No. 17-618). Cells were grown at 37 °C in a humidified incubator with a gas phase of ⁇ % C0 2 .
- the cells were transduced with the retrovirus containing medium mixed with 5 ⁇ g/ml of Polybrene, incubated for 48 hours and then cultured continuously for 3 weeks in the presence of 300- 400 ⁇ g/ml Genetecin® (Life Technologies Inc.).
- Cells were plated at densities range of 1.600-3.600 cells/well in 96-well plates coated with Poly-L-lysine (Sigma Cat. No. P6282) Gemcitabine (obtained from Orifarm A/S - DK) was added after 24 hours of incubation at 37°C, 5% C0 2 , and the medium containing the nucleoside analog. Each experiment was performed in four replicates. Cell survival was assayed after 96-120 hours of drug exposure, by XTT cell proliferation kit (XTT kit II, Roche Cat. No. 1 46 ⁇ 016). The data was corrected for background media-only absorbance where after the 60% cell killing drug concentration - (IC 50 value) was calculated. The IC50 value of the investigated drug/compound was calculated as the mean of these experiments using SigmaPlot® (SPSS Science, Dyr- berg Traiding - DK).
- the sensitivity of the untransduced cells, and of the cells transduced with either the retroviral vector alone ore the vector containing plant kinases for Gemcitabine was determined.
- cytotoxicity (IC5 0 value) was determined after 96-120 hours of drug ex- posure. The results are presented in the table below.
- the concentrations which cause 60% lethality are shown for each construct and the parental cell line.
- the factor of sensitivity increase is compared to the parental cell line.
- the difference in sensitivity between the parental cell line and the cells transduced with the pLCXSN vector alone was less than 1 -fold.
- the cell lines expressing the plant kinases showed an increase in sensitivity to Gemcitabine. The highest increase was detected in the glioma cell line (U-1 18- MG) expressing Arabidopsis thaliana dCK (PZG190) with a 20-fold increase in IC 50 compared with the untransduced cells.
Abstract
Description
Claims
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JP2004516512A JP2005530511A (en) | 2002-06-26 | 2003-06-24 | Plant deoxyribonucleoside kinase enzymes and methods of use thereof |
AU2003239778A AU2003239778A1 (en) | 2002-06-26 | 2003-06-24 | Plant deoxyribonucleoside kinase enzymes and their use |
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EP2931739A4 (en) * | 2012-12-05 | 2016-08-03 | Exxonmobil Res & Eng Co | Nannochloropsis kozak consensus sequence |
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WO2003100045A1 (en) | 2002-05-23 | 2003-12-04 | Wolfgang Knecht | Plant thymidine kinases and their use |
JP5211303B2 (en) * | 2007-10-25 | 2013-06-12 | 独立行政法人産業技術総合研究所 | Method for producing protein |
WO2010000270A1 (en) * | 2008-07-04 | 2010-01-07 | Zgene A/S | Prodrug activation therapy for the treatment of malignant brain tumours |
AU2010206374B2 (en) | 2009-01-23 | 2013-01-17 | Gloriana Therapeutics Sarl | Improved cell lines and their use in encapsulated cell biodelivery |
US20120021039A1 (en) | 2009-01-23 | 2012-01-26 | Nsgene A/S | Expression of neuropeptides in mammalian cells |
CN117016549B (en) * | 2023-03-13 | 2024-02-27 | 山东蓬勃生物科技有限公司 | Application of nucleoside compound and composition thereof in promoting plant growth |
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EP2931739A4 (en) * | 2012-12-05 | 2016-08-03 | Exxonmobil Res & Eng Co | Nannochloropsis kozak consensus sequence |
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US20060230467A1 (en) | 2006-10-12 |
US7666639B2 (en) | 2010-02-23 |
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AU2003239778A8 (en) | 2004-01-19 |
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