Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Transferase other than ribonuclease
Reexamination Certificate
2001-06-19
2003-06-24
Patterson, Jr., Charles L. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Transferase other than ribonuclease
C435S235100, C435S320100, C536S023200, C424S093200, C514S04400A
Reexamination Certificate
active
06582947
ABSTRACT:
This is a national stage application under 35 U.S.C. 371 of PCT/SE99/01434, filed on Dec. 10, 1999, now abandoned.
The present invention relates to a gene encoding a multisubstrate deoxyribonucleoside kinase of
Drosophila melanogaster
, and vectors and recombinant viruses containing said gene, as well as pharmaceutical compositions comprising such a vector and/or a virus. It also relates to production of said multisubstrate deoxyribonucleoside kinase and a process for phosphorylating nucleosides and nucleoside analogs.
TECHNICAL BACKGROUND
Nucleoside analogs are commonly used in treatment of virus infections and cancer. The therapeutic nucleoside analogs are inactive prodrugs that are dependent on intracellular phosphorylation for pharmacological activity. The majority of nucleoside analogs in clinical use are phosphorylated by deoxyribonucleoside kinases (Arnér et al., (1995)
Pharmac. Ther
. 67, 155-186). These enzymes are intensively studied since they catalyze the rate limiting step in the pharmacological activation of the nucleoside analogs. There are four major deoxyribonucleoside kinases in human cells: deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) (1995)
Pharmac. Ther
. 67, 155-186). DCK, dGK and TK2 are closely sequence-related enzymes whereas TK1 has low similarity with the other deoxyribonucleoside kinases (Johansson et al., (1996)
Proc. Natl. Acad. Sci. USA
. 93, 7258-7262; Johansson et al., (1997)
J. Biol. Chem
. 272, 8454-8458; Chottiner et al., (1991)
Proc. Natl. Acad. Sci. USA
. 88, 1531-1535). The human deoxyribonucleoside kinases have distinct substrate specificities in regard to phosphorylation of both deoxyribonucleosides as well as nucleoside analogs.
WO95/14102 discloses recombinant adenoviruses comprising a DNA sequence coding for herpes simplex thymidine kinase under the control of a heterologous expression signal that can be associated with certain form of cancer. These recombinant viruses are then used to infect tumours of such a cancer. As a result, thymidine kinase is expressed in the cancer tumour. Subsequently, a therapeutic nucleoside analog prodrug, such as acyclovir (ACV, 9-(hydroxy ethoxymethyl)-guanine) and gancyclovir (GCV), is administred. Due to the enhanced expression of thymidine kinase in the tumor, the prodrug is only converted to the active form in the tumour, resulting in death of the tumour. However, the ability of thymidine kinase to phosphorylate potentially useful nucleoside analog prodrugs is limited.
WO97/29196 also relates to recombinant adenoviruses comprising a DNA sequence encoding herpes simplex thymidine kinase (HSV-TK). The kinase is mutated in order to increase the phosphorylation rate and to broaden the substrate specificity.
WO96/21724 discloses recombinant virus particles, such as recombinant retroviruses, contaning RNA encoding human deoxycytidine kinase 2. These virus particles are used for the same purposes as the virus particles described in WO97/29196 and WO95/14102, but the enzyme has another substrate specificity.
Accordingly, it is known to insert a “suicide” nucleic acid sequence, such as a nucleic acid sequence encoding a nucleoside kinase, into the genome of a virus or some other kind of vector capable of transferring nucleic acid sequences into tumour cells of a human or animal patient, and subsequently administer a therapeutic nucleoside analog prodrug. Known nucleoside kinases have a limited substrate specificity. HSV-TK, which is described in the above cited WO97/29196 and WO95/14102, cannot phosphorylate 2′,2′-difluorodeoxycytidine, 2-chloro-2′-deoxyadenosine, 1-&bgr;-D-arabinofuranosylcytosine, 2′,3′-dideoxycytidine and 2′-deoxy-3-thiacytidine. Also deoxyguanosine kinase disclosed in the above cited WO96/2 1724 has been shown to have a limited substrate specificity. This enzyme does not phosphorylate), (E)-5-(2-bromovinyl)-2′-deoxyuridine, (E)-5-(2-bromovinyl)-1-&bgr;-D-arabinofuranosyl-uracil, 2′,2′-difluorodeoxycytidine, 1-&bgr;-D-arabinofuranosylcytosine, 2′,3′-dideoxycytidine or 3TC.
Consequently, there is a need for a DNA sequence encoding a nucleotide kinase having a broad specificity and a high catalytic rate of phosphozylation, in order to obtain flexibility regarding the use possible nucleoside analog prodrugs, and in order to reduce the dose amount required to obtain a sufficient therapeutic effect, resulting in a minimized risk for undesired side effects in the patient.
When comercially producing phosphoiylated nucleoside analogs., there is also a need for a nucleotide kinase having a broad specificity and a high catalytic rate of phosphorylation.
SUMMARY OF THE INVENTION
It has now turned out that by inserting a DNA or RNA sequence comprising a sub-sequence showing a homology of at least 60%, preferably at least 80%, and most preferably at least 90% of the DNA sequence of SEQ.ID.NO.1 into a cell, that cell will obtain a broad specificity for changing nucleoside analog prodrugs to active drugs by phosphorylation. Likewise this changement will occur at a high catalytic rate. Preferably the DNA sequence is inserted into the cell by transformation with a suitable virus or another suitable vector. Such viruses and vectors also constitute a part of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
A recent report (Munch-Petersen et al., (1998)
J. Biol. Chem
. 273, 3926-3931) shows that cell lines from the fruit fly
Drosophila melanogaster
contains only a single deoxyribonucleoside kinase. The report does neither reveal anything about the aminoacid sequence of the enzyme, nor about any DNA sequence encoding it. This enzyme, namned DM-dNK, is in contrast to the human deoxyribonucleoside kinases a multisubstrate enzyme. Although pyrimidine nucleosides are the preferred substrates of this enzyme, it catalyzes phosphorylation of both pyrimidine and purine deoxyribonucleosides. The enzyme also efficiently phosphorylates several anti-viral and anti-cancer nucleoside analogs. The catalytic rates of deoxyribonucleoside and nucleoside analog phosphorylation are, depending on the substrate, 10- to 100-folds higher than the maximal catalytic rates reported for the mammalian enzymes. The broad substrate specificity and high catalytic rate in phosphorylation of deoxyribonucleosides render DM-dNK unique among the family members of deoxyribonucleoside kinases.
Accordingly, an object of the present invention is to provide a nucleic acid sequence encoding a multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2. Depending on the vector into which the nucleic acid sequence is intended to be inserted, the nucleic acid sequence can be a DNA sequence or a RNA sequence. The DNA may be cDNA, genomic DNA and synthetic DNA. It may also be double-stranded or single-stranded, and if single-stranded it may be the coding strand or the anti-sense strand. SEQ.ID.NO.1 discloses a cDNA sequence encoding the multisubstrate deoxyribonucleoside kinase. However, because of the fact that the genetic code is degenerated, other nucleic acid sequences encoding the same enzyme can be used in connection to the present invention.
Another object of the present invention is to provide a nucleic acid sequence comprising a disease-associated promoter and/or signal sequence operatively linked to a nucleic acid subsequence encoding a multisubstrate deoxyribonucleoside kinase showing at least 70% homology, preferably at least 90% homology with the amino acid sequence of SEQ.ID.NO.2. Depending on the vector into which the nucleic acid sequence is intended to be inserted, the nucleic acid sequence can be a DNA sequence or a RNA sequence. It is also possible to inject an expression cassette comprising such a DNA sequence directly into cells that are to be killed.
Yet another object of the present invention is to provide a vector, such as a plasmid, cosmid or a bacteriophage, which vector contains a DNA sequ
Johansson Magnus
Karlsson Anna
BIO VICI
Nixon & Vanderhye P.C.
Patterson Jr. Charles L.
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