WO2001025407A2 - Tyrosine-dna phosphodiesterases (tdp) and related polypeptides, nucleic acids, vectors, tdp-producing host cells, antibodies and methods of use - Google Patents

Abstract

The present invention provides a nucleic acid molecule encoding a tyrosine-DNA phosphodiesterase (TDP), and a related vector, host cell, polypeptide, antibody, antisense nucleic acid molecule, and ribozyme. Also provided are a method of altering the level of TDP in a cell, tissue, organ or organism, as well as the resulting cell, tissue, organ or non-human organism, as well as a method of identifying a TDP-resistant compound, a method of assessing TDP1 activity in an animal, and a method of assessing the efficacy of a topoisomerase I inhibitor.

Description

TYROSINE-DNA PHOSPHODIESTERASES (TDP) AND RELATED

POLYPEPTIDES, NUCLEIC ACIDS, VECTORS, TDP-PRODUCING HOST

CELLS, ANTIBODIES AND METHODS OF USE TECHNICAL FIELD OF THE INVENTION

The present invention relates to tyrosine-DNA phosphodiesterases and related polypeptides, nucleic acids, vectors, TDP-producing host cells, antibodies and methods of use in the identification of a TDP-resistant compound, in the assessment of TDP 1 activity in an animal, and in the assessment of efficacy of a topoisomerase I inhibitor.

BACKGROUND OF THE INVENTION Topoisomerases are cellular enzymes that are crucial for replication and transcription of the cellular genome. Topoisomerases cleave the DNA backbone, thereby allowing topological change for replication and transcription of the cellular genome to occur, after which topoisomerases reseal the DNA backbone (Wang, Ann. Rev. Biochem. 65: 635 (1996)). Topoisomerases are efficient because DNA breakage is accompanied by covalent bonding between the enzyme and the DNA to create an intermediate that is resolved during the resealing step. This mechanism, while elegant, makes topoisomerases potentially dangerous. If the resealing step fails, a normally transient break in DNA becomes a long-lived disruption, one with a topoisomerase covalently joined to it. Unless a way is found to restore the continuity of the DNA, the cell will die.

In virtually all topoisomerases, the heart of the covalent complex is a phosphodiester bond between a specific tyrosine residue of the enzyme and one end of the break (the 3' end for eukaryotic topoisomerase I and the 5' end for topoisomerases II and IH). The high-energy nature of this bond normally ensures the resealing step.

Failure of resealing is dramatically increased by several drugs, such as camptothecin, a promising anti-cancer agent that specifically targets eukaryotic topoisomerase I (Chen et al., Ann. Rev. Pharmacol. Toxicol. 34: 191 (1994)).

Protein-linked breaks also accumulate when topoisomerases act on DNA containing structural lesions like thymine dimers, abasic sites and mismatched base pairs (Pommier et al, Biochim. Biophys. Acta 1400: 83 (1998)). To the extent that such lesions arise during the normal life of a cell, topoisomerase-associated damage may be unavoidable.

Repair of topoisomerase-DNA covalent complexes is of obvious value to the cell but, until the present invention, very little was known about the mechanisms involved in such repair. Hydrolysis of the bond joining the topoisomerase to DNA had been proposed as a way to effect release of the topoisomerase such that the cleaved DNA could undergo conventional modes of break repair (Friedberg et al.,

DNA Repair and Mutagenesis (ASM Press, Washington, D.C. (1995)); Kanaar et al.,

Trends Cell. Biol. 8: 483 (1998)). Although no such hydrolysis has been reported for 0 covalent complexes between DNA and topoisomerase II or III, such hydrolysis has been described for covalent complexes between DNA and topoisomerase I (Yang et al., PNAS USA 93: 11534 (1996)).

The present invention seeks to provide the enzyme responsible for hydrolysis of the covalent complexes between DNA and topoisomerase I, specifically tyrosine- 5 DNA phosphodiesterase, which acts on a tyrosine linked through the side-chain oxygen to the 3' phosphate of DNA. This and other objects and advantages, as well as additional inventive features, will become apparent to one of ordinary skill in the art from the detailed description provided herein.

o BRIEF SUMMARY OF THE INVENTION

The present invention provides an isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a mammalian, in particular a human, tyrosine-DNA phosphodiesterase (TDP1) and a continuous fragment thereof of at least about 36 nucleotides. Also provided is an isolated or purified nucleic acid 5 molecule encoding a modified mammalian TDP1, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof of at least about 36 nucleotides. The modified mammalian TDP1 does not differ functionally from the corresponding unmodified mammalian TDP1.

An isolated and purified nucleic acid molecule consisting essentially of a o nucleotide sequence encoding a yeast, in particular a Saccharomyces cerevisiae,

TDP1 and a continuous fragment thereof comprising at least about 36 nucleotides are also provided by the present invention. In this regard, an isolated or purified nucleic acid molecule encoding a modified yeast TDP1, which comprises one or more insertions, deletions and/or substitutions, and a continuous fragment thereof of at least about 36 nucleotides, are also provided by the present invention. The modified yeast TDPl does not differ functionally from the corresponding unmodified yeast TDPl.

The present invention further provides a vector comprising an above-described nucleic acid molecule and a vector comprising or encoding an antisense molecule of at least about 20 nucleotides that hybridizes to or a ribozyme that cleaves an RNA molecule encoding an above-described TDPl, as well as the antisense molecule and the ribozyme.

Also provided by the present invention is a host cell comprising an above- described vector, a polypeptide produced by such a host cell, and a polyclonal or monoclonal antibody that binds to an above-described TDPl.

Further provided by the present invention is a method of altering the level of TDPl in a cell, a tissue, an organ or an organism. The method comprises contacting a cell, a tissue, an organ or an organism with a vector comprising a (i) a nucleic acid molecule encoding a TDPl, (ii) a nucleic acid molecule comprising or encoding an antisense molecule of at least about 20 nucleotides to an RNA molecule transcribed from (i), or (iii) a nucleic acid molecule comprising or encoding a ribozyme to an RNA molecule transcribed from (i). The vector comprising (i) increases or decreases the level of TDPl in the cell, the tissue, the organ or the organism, whereas the vector comprising (ii) or (iii) decreases the level of TDPl in the cell, the tissue, the organ or the organism. In this regard, the present invention also provides a cell, a tissue, an organ or a nonhuman organism in which the level of TDPl has been altered in accordance with such a method.

Another method provided by the present invention is a method of identifying a compound that stabilizes a covalent bond complex that forms between DNA and topoisomerase I, wherein the covalent bond cannot be cleaved by or is resistant to cleavage by a TDPl . The method comprises (a) contacting a compound, which stabilizes a covalent bond complex that forms between DNA and topoisomerase I such that the covalent bond cannot be cleaved by or is resistant to cleavage by a TDPl, with DNA and topoisomerase I under conditions suitable for a covalent bond complex to form between the DNA and the topoisomerase I and for the compound to stabilize the covalent bond complex, (b) contacting the covalent bond complex with a TDPl under conditions suitable for the cleavage of the covalent bond between the DNA and topoisomerase I by TDPl , and (c) detecting cleavage of the covalent bond by the TDPl . The amount of cleavage detected is indicative of whether or not the compound stabilizes a covalent bond complex that forms between DNA and topoisomerase I such that the covalent bond cannot be cleaved or is resistant to cleavage by the TDP 1.

Yet another method provided by the present invention is a method of assessing TDPl activity in an animal. The method comprises (a) obtaining a sample of a cellular extract from an animal, wherein the cellular extract comprises TDPl, and (b) measuring the level of TDPl activity in the sample. Still yet another method provided by the present invention is a method of assessing the efficacy of a topoisomerase I inhibitor. The method comprises (a) obtaining a sample of DNA to which is covalently bound topoisomerase I after contact with a topoisomerase I inhibitor, (b) contacting the sample with a TDPl under conditions suitable for cleavage of the covalent bond between the DNA and the topoisomerase I by TDPl, and (c) measuring the amount of topoisomerase I that is cleaved from the DNA. The amount of topoisomerase I that is cleaved from the DNA is indicative of the efficacy of the topoisomerase I inhibitor.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is the cDNA sequence of a human TDPl [SEQ ID NO: 1].

Fig. 2 is the deduced amino acid sequence [SEQ ED NO: 2] of the cDNA of Fig. 1. The start codon (M) is circled.

Fig. 3 is the genomic DNA sequence of a yeast TDPl [SEQ ID NO: 3]. Fig. 4 is the deduced amino acid sequence [SEQ ID NO: 4] of the genomic DNA of Fig 3.

Fig. 5 is an alignment of TDPl homologs from various organisms. DETAILED DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides isolated or purified nucleic acid molecules. By "isolated" is meant the removal of a nucleic acid from its natural environment. By "purified" is meant that a given nucleic acid, whether one that has been removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified under laboratory conditions, has been increased in purity, wherein "purity" is a relative term, not "absolute purity." "Nucleic acid molecules" is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides.

One isolated or purified nucleic acid molecule consists essentially of a nucleotide sequence encoding a mammalian TDPl or a continuous fragment thereof of at least about 36 nucleotides. Preferably, the mammalian TDPl is a human TDPl. Also, preferably, the mammalian TDPl is (i) DNA and consists essentially of SEQ ID NO: 1 or a sequence that encodes SEQ ID NO: 2, (ii) RNA and consists essentially of a sequence encoded by SEQ ID NO: 1 or a sequence that encodes SEQ ID NO: 2, or (iii) a nucleic acid molecule consisting essentially of a nucleotide sequence that encodes a mammalian TDPl or a continuous fragment of at least about 12 amino acids thereof and that hybridizes to either one of the foregoing under low stringency conditions.

Also provided is an isolated or purified nucleic acid molecule encoding a modified mammalian TDPl, which comprises one or more insertions, deletions and/or substitutions, wherein the modified mammalian TDPl encoded by the isolated or purified nucleic acid molecule does not differ functionally from the corresponding unmodified mammalian TDPl, or a continuous fragment thereof of at least about 36 nucleotides. Desirably, the modified mammalian TDPl does not differ functionally from the corresponding unmodified mammalian TDPl, such as that comprising SEQ ID NO: 2. Preferably, the modified mammalian TDPl cleaves a covalent bond complex between DNA and topoisomerase I at least about 90% as well as the corresponding unmodified mammalian TDPl, such as that comprising SEQ ID NO: 2, as determined by in vitro assay using labeled topoisomerase I or an oligonucleotide comprising a 3' phosphotyrosine (see, e.g., Yang et al. (1996), supra). Use of the word "labeled" herein is intended to mean any means of detection, such as a radioactive isotope.

Another isolated or purified nucleic acid molecule consists essentially of a nucleotide sequence encoding a yeast TDPl or a continuous fragment thereof comprising at least about 36 nucleotides. Preferably, the yeast TDPl is a

Saccharomyces cerevisiae TDPl. Also, preferably, the yeast TDPl is (i) DNA and consists essentially of SEQ ID NO: 3 or a sequence that encodes SEQ ID NO: 4, (ii) RNA and consists essentially of a sequence encoded by SEQ ED NO: 3 or a sequence that encodes SEQ ID NO: 4, or (iii) a nucleic acid molecule consisting essentially of a nucleotide sequence that encodes a yeast TDPl or a continuous fragment of at least about 12 amino acids thereof and that hybridizes to either one of the foregoing under low stringency conditions. Yeast TDPl will act on a tyrosine linked through the side- chain oxygen to the 3' phosphate of single-stranded and double-stranded DNA. Also provided is an isolated or purified nucleic acid molecule encoding a modified yeast TDPl, which comprises one or more insertions, deletions and/or substitutions, wherein the modified yeast TDPl encoded by the isolated or purified nucleic acid molecule does not differ functionally from the corresponding unmodified yeast TDPl, or a continuous fragment thereof of at least about 36 nucleotides. Desirably, the modified yeast TDPl does not differ functionally from the corresponding unmodified yeast TDPl, such as that comprising SEQ ED NO: 4.

Preferably, the modified yeast TDPl cleaves a covalent bond complex between DNA and topoisomerase I at least about 90% as well as the corresponding unmodified yeast TDPl, such as that comprising SEQ ED NO: 4, as determined by in vitro assay using labeled topoisomerase I or an oligonucleotide comprising a 3' phosphotyrosine. With respect to the above, one of ordinary skill in the art knows how to generate insertions, deletions and/or substitutions in a given nucleic acid molecule. Also with respect to the above, "does not differ functionally from" is intended to mean that the modified enzyme has enzymatic activity characteristic of the unmodified enzyme. In other words, it acts upon the same substrate and generates the same product. The modified enzyme, however, can be more or less active than the unmodified enzyme as described in accordance with the present invention.

Nucleic acid molecules encoding TDPl can be isolated from numerous eukaryotic sources. In this regard, TDPl is highly conserved among eukaryotes. With respect to the above isolated or purified nucleic acid molecules, it is preferred that the one or more substitution(s) do(es) not result in a change in an amino acid of the enzyme. Alternatively, and also preferred, is that the one or more subsitution(s) result(s) in the substitution of an amino acid of the encoded yeast TDPl with another amino acid of approximately equivalent size, shape and charge.

Also with respect to the above isolated or purified nucleic acid molecules, a "continuous fragment of at least about 36 nucleotides of the isolated or purified nucleic acid molecule," preferably encodes a polypeptide that can carry out the same function as the corresponding complete polypeptide or protein. For example, a fragment of an isolated or purified nucleic acid molecule encoding a mammalian

TDPl can be a continuous fragment of the TDPl -encoding nucleic acid molecule that encodes a polypeptide that can cleave a covalent bond complex between DNA and topoisomerase I, but not necessarily as well as the corresponding complete polypeptide or protein. The above isolated or purified nucleic acid molecules also can be characterized in terms of "percentage of sequence identity." In this regard, a given nucleic acid molecule as described above can be compared to a nucleic acid molecule encoding a corresponding gene (i.e., the reference sequence) by optimally aligning the nucleic acid sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage of sequence identity is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences, i.e., the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison can be conducted by computerized implementations of known algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, or BlastN and BlastX available from the National Center for Biotechnology Information, Bethesda, MD), or by inspection. Sequences are typically compared using BESTFIT or BlastN with default parameters. "Substantial sequence identity" means that at least about 75%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95% of the sequence of a given nucleic acid molecule is identical to a given reference sequence or that at least about 40%, preferably at least about 60%, more preferably at least about 90%, and most preferably at least about 95% of the amino acids of which a given polypeptide is comprised are identical to or represent conservative substitutions of the amino acids of a given reference sequence.

Another indication that polynucleotide sequences are substantially identical is if two molecules selectively hybridize to each other under stringent conditions. The phrase "selectively hybridizing to" refers to the selective binding of a single-stranded nucleic acid probe to a single-stranded target DNA or RNA sequence of complementary sequence when the target sequence is present in a preparation of heterogeneous DNA and/or RNA. Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. "Low stringency conditions," as that term is used herein, means those conditions that allow for as much as about 80% mismatch. The above-described nucleic acid molecules can be used, in whole or in part

(i.e., as fragments), to identify and isolate corresponding genes from other eukaryotes for use in the context of the present inventive method using conventional means as known in the art. For example, such molecules or fragments thereof can be used in chromosome walking, genomic subtraction, which requires the availability of strains having deletions of the target gene (Strauss and Ausubel, PNAS USA 87: 1889-1893 (1990); and Sun et al., Plant Cell 4: 119-128 (1992)), transposon (Chuck et al., Plant Cell 5: 371-378 (1993); Dean et al., Plant J. 2: 69-81 (1992); Grevelding et al., PNAS USA 899: 6085-6089 (1992); Swinburne et al, Plant Cell 4: 583-595 (1992); Fedoroff and Smith, Plant J. 3: 273-289 (1993); and Tsay et al, Science 260: 342-344 (1993)) and T-DNA tagging (Feldmann, Plant J. 1 : 71-82 (1991); Feldmann et al., Science 243: 1351-1354 (1989); Herman et al, Plant Cell 11 : 1051-1055 (1989); Konz et al., EMBO J. 9: 1337-1346 (1989); and Kieber et al., Cell 72: 427-441 (1993)), and heterologous probe selection techniques in accordance with methods well-known in the art. Although T-DNA tagging, chromosome walking or heterologous probe selection can identify a DNA fragment that putatively contains the gene of interest, the DNA fragment must be confirmed by genetic complementation or some other means. In another embodiment, the present invention also provides a vector comprising a nucleic acid molecule as described above. A nucleic acid molecule as described above can be cloned into any suitable vector and can be used to transform or transfect any suitable host cell, whether a single cell or a collection of cells, such as in the context of a tissue, an organ or an organism. The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (see, in general, "Recombinant DNA Part D," Methods in Enzγmology, Vol. 153, Wu and Grossman, eds., Academic Press (1987)). Desirably, the vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA or RNA.

Preferably, the vector comprises regulatory sequences that are specific to the genus of the host. Most preferably, the vector comprises regulatory sequences that are specific to the species of the host.

Constructs of vectors, which are circular or linear, can be prepared to contain an entire nucleic acid sequence as described above or a portion thereof ligated to a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived from ColEl, 2 mμ plasmid, λ, SV40, bovine papilloma virus, and the like. Yeast centromeric plasmid (YCp50) constructs can be used to express TDPl in yeast. In addition to the replication system and the inserted nucleic acid, the construct can include one or more marker genes, which allow for selection of transformed or transfected hosts, if so desired. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc.. complementation in an auxotrophic host to provide pro to trophy, and the like. Suitable vectors include those designed for propagation and expansion or for expression or both. A preferred cloning vector is selected from the group consisting of the pUC series, the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clonetech, Palo Alto, CA). Bacteriophage vectors, such as λ GT10, λGTl 1, λZapII (Stratagene), λ EMBL4, and λ NM1149, also can be used. Examples of plant expression vectors include pBHOl, pBI101.2, pBI101.3, pBI121 and pBEN19 (Clonetech, Palo Alto, CA). Examples of animal expression vectors include pEUK-C 1 , pMAM and pMAMneo (Clonetech, Palo Alto, CA). Other examples of suitable vectors include the yeast centromeric plasmid (YCp50), 2u and integrative vectors, such as the pRS series (Bachmann et al., Yeast 14: 115 (1998)) or PYES2 (Invitrogen).

An expression vector can comprise a native or normative promoter operably linked to a nucleic acid molecule encoding a TDPl as described above. The selection of promoters, e.g., strong, weak, inducible, repressible, cell-specific, tissue-specific, organ-specific and developmental-specific, is within the skill in the art. Similarly, the combining of a nucleic acid molecule as described above with a promoter is also within the skill in the art. The present invention not only provides a vector comprising a nucleic acid molecule as described above but also provides a vector comprising or encoding an antisense molecule that hybridizes to or a ribozyme that cleaves an RNA molecule encoding a TDPl as described above. The present invention also provides the antisense molecules and ribozymes, themselves. Antisense nucleic acids can be generated in accordance with methods known in the art. The nucleic acid molecule introduced in antisense inhibition generally is substantially identical to at least a portion, preferably at least about 20 continuous nucleotides, of the nucleic acid to be inhibited, but need not be identical. The complex can, thus, be designed such that the inhibitory effect applies to other proteins within a family of genes exhibiting homology or substantial homology to the nucleic acid. The introduced sequence also need not be full-length relative to either the primary transcription product or fully processed mRNA. Generally, higher homology can be used to compensate for the use of a shorter sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and homology of non-coding segments will be equally effective.

The inclusion of ribozyme sequences within antisense RNAs confers RNA- cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334: 585-591 (1988). Preferably, the ribozyme comprises at least about 20 continuous nucleotides complementary to the target sequence on each side of the active site of the ribozyme.

In view of the above, the present invention provides a host cell comprising a vector as described above. In addition, the present invention provides a polypeptide produced by a host cell comprising a vector as described above.

Suitable hosts of cells include mammalian, such as human, and insect cells, yeast (e.g., S288c strain (wild-type) S. cerevisiae), and bacteria (e.g., E. coli strain BL21 (DΕ3)). Also provided by the present invention is a purified and isolated mammalian, in particular human, TDPl . A mammalian, in particular a human, TDPl can be purified and isolated using methods known to those of ordinary skill in the art. For example, a human, TDPl can be isolated or purified by forming a human TDPl fusion protein containing a polyhistidine tail and purifying via nickel-chelation chromatography.

In addition to the above, the present invention provides polyclonal and monoclonal antibodies to TDPl . Preferably, the antibody binds to a mammalian TDPl but does not bind to a nonmammalian TDPl or the antibody binds to a yeast TDPl but does not bind to a non-yeast TDPl. Methods of polyclonal and monoclonal antibody production are known in the art. See, for example, Harlow and Lane, in Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988, pp. 1-725). For example, a yeast TDP fusion protein containing a polyhistidine tail was expressed in a bacterial expression vector and purified via nickel-chelation chromatography. Pure protein was isolated from bacterial cultures and injected into rabbits using standard methods to generate polyclonal antibodies.

Antibodies were harvested ten weeks later and were shown to interact effectively with and to precipitate yeast TDPl .

In another embodiment, the present invention provides a method of altering the level of TDPl in a cell, tissue, organ or organism. By "altering" is meant that the TDPl level in a given cell, tissue, organ or organism is different as a result of the practice of the present inventive method as compared to a like cell, tissue, organ or organism in which the level of TDPl has not been altered as a result of the practice of the present inventive method. The method comprises contacting the cell, the tissue, the organ or the organism with a vector comprising a nucleic acid molecule selected from the group consisting of (i) a nucleic acid molecule encoding a TDPl, (ii) a nucleic acid molecule comprising or encoding an antisense sequence of at least about 20 nucleotides to an RNA molecule transcribed from (i), and (iii) a nucleic acid molecule comprising or encoding a ribozyme to an RNA molecule transcribed from (i). The vector comprising a nucleic acid molecule of (i) increases or decreases the level of TDPl in the cell, the tissue, the organ or the organism, whereas the vector comprising a nucleic acid molecule of (ii) or (iii) decreases the level of TDPl in the cell, the tissue, the organ or the organism. Accordingly, the present invention further provides a cell, a tissue, an organ or a nonhuman organism in which the level of TDPl has been altered in accordance with the method.

By "contacting" is meant bringing the cell, tissue, organ or organism into sufficiently close proximity with the vector such that the vector is taken up by the cell or by cells in the tissue, organ or organism, wherein it can be expressed. The method is not dependent on any particular means of contact and is not to be so construed. Means of contact are well-known to those skilled in the art, and also are exemplified herein.

Accordingly, contact can be effected, for instance, either in vitro (e.g., in an ex vivo type method of gene therapy) or in vivo, which includes the use of electroporation, transformation, transduction, conjugation or triparental mating, transfection, infection, membrane fusion with cationic lipids, high-velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate- DNA precipitate, direct micro injection into single cells, and the like. Other methods also are available and are known to those skilled in the art.

Preferably, however, the vectors (including antisense molecules and ribozymes) are introduced by means of cationic lipids, e.g., liposomes. Such liposomes are commercially available (e.g., Lipofectin^®, Lipofectamine™, and the like, supplied by Life Technologies, Gibco BRL, Gaithersburg, MD). Moreover, liposomes having increased transfer capacity and/or reduced toxicity in vivo (e.g., as reviewed in PCT patent application no. WO 95/21259) can be employed in the present invention. For liposome administration, the recommendations identified in the PCT patent application no. WO 93/23569 can be followed. Generally, with such administration the formulation is taken up by the majority of lymphocytes within 8 hr at 37°C, with more than 50% of the injected dose being detected in the spleen an hour after intravenous administration. Similarly, other delivery vehicles include hydrogels and controlled-release polymers. The form of the vector introduced into a host cell can vary, depending in part on whether the vector is being introduced in vitro or in vivo. For instance, the nucleic acid can be closed circular, nicked, or linearized, depending on whether the vector is to be maintained extragenomically (i.e., as an autonomously replicating vector), integrated as a provirus or prophage, transiently transfected, transiently infected as with use of a replication-deficient or conditionally replicating virus, or stably introduced into the host genome through double or single crossover recombination events.

Preferably, the nucleic acid molecule used in the above-described method is one of those described above. In this regard, nucleic acid molecules that correspond to the above-described nucleic acid molecules but which have been isolated from other eukaryotic sources, in particular other mammalian or yeast sources, can be used in the context of the present inventive method to increase the level of TDPl in a cell, tissue, organ or organism, provided that a cDNA sequence is used in those instances where the genomic sequence contains introns that may not be properly processed in a given cell, tissue, organ or organism. In addition, it may be necessary to alter the cDNA sequence so that it contains codon sequences that are preferred in a given species. However, to the extent that antisense or ribozyme sequences are employed in the present inventive method, it would be advantageous to use a nucleic acid molecule isolated from a eukaryotic source that is of the same origin as the cell, the tissue, the organ or the organism in which the level of TDPl is to be altered.

If it is desired to increase the expression of TDPl, it is preferred to do so by introducing a gene encoding TDPl . Preferably, a vector comprising a nucleotide sequence encoding TDPl operably linked to a promoter that is functional in the cell, the tissue, the organ or the organism with which it is brought into contact is used. It is preferred that either multiple extra copies of the gene are introduced into the cell, the tissue, the organ or the organism or that a vector comprising a strong promoter is introduced into the cell, the tissue, the organ or the organism such that the coding sequence is expressed at a higher rate, thereby generating more mRNA, which, in turn, is translated into more of the encoded enzyme.

In this regard, if expression is desired in a given cell, tissue or organ, a cell-, tissue- or organ specific promoter can be used in the vector. Developmentally specific promoters and regulatable, i.e., inducible or repressible, e.g., metallothionem promoter and radiation-responsive promoter, also can be used. Examples of such promoters, as well as enhancer elements and suppressor elements, are known in the art. Promoters can be found on the Internet in the eukaryotic promoter database, for example, at http://www.genomes.ad.ip/dbgetbin/www bFind? epdtable and other such databases. In addition, a nucleic acid can be directly or indirectly linked to a targeting moiety. A "targeting moiety," such as that term is used herein is any molecule that can be linked with an above-described nucleic acid directly or indirectly, such as through a suitable delivery vehicle, such that the targeting moiety preferentially binds to a target cell as compared to a non-target cell. The targeting moiety can bind to a target cell through a receptor, a substrate, an antigenic determinant or another binding site on the target cell. Examples of a targeting moiety include an antibody (i.e., a polyclonal or a monoclonal antibody), an immunologically reactive fragment of an antibody, an engineered immunoprotein and the like, a protein (target is receptor, as substrate, or regulatory site on DNA or RNA), a polypeptide (target is receptor), a peptide (target is receptor), a nucleic acid, which is DNA or RNA (i.e., single-stranded or double-stranded, synthetic or isolated and purified from nature; target is complementary nucleic acid), a steroid (target is steroid receptor), and the like. In general, there are a number of databases for targeting moieties (see, e.g., ftp://kegg.genome.ad.jp, http://broweb.pasteur.fr/docs/versions, http://ampere.doe- mbi.ucla.edu:8801/dat/dip.dat, or http:/ bones.biochem.ualberta.ca/pedro/rt-l .html 1). Analogs of targeting moieties that retain the ability to bind to a defined target also can be used. In addition, synthetic targeting moieties can be designed, such as to fit a particular epitope. Alternatively, the therapeutic nucleic acid can be encapsulated in a liposome comprising on its surface the targeting moiety. The targeting moiety can include a linking group that can be used to join a targeting moiety to, in the context of the present invention, an above-described nucleic acid. It will be evident to one skilled in the art that a variety of linking groups, including bifunctional reagents, can be used. The targeting moiety can be linked to the nucleic acid by covalent or non-covalent bonding. If bonding is non-covalent, the conjugation can be through hydrogen bonding, ionic bonding, hydrophobic or van der Waals interactions, or any other appropriate type of binding.

If it is desired to decrease the expression of TDPl, it is preferred to do so by 5 introducing either a nucleic acid molecule comprising (i.e., in the case of an RNA vector) or encoding (i.e., in the case of a DNA vector) an antisense nucleic acid molecule to an RNA molecule transcribed from an aforementioned gene or a nucleic acid molecule comprising a ribozyme to an RNA molecule transcribed from such a gene. In antisense technology, a nucleic acid segment from the desired gene can be 0 cloned and operably linked to the promoter sequence such that the anti-sense strand of RNA is transcribed. Another alternative method to decrease TDPl is to use a compound that inhibits the transcription, translation or activity of TDPl.

In addition to the above, gene replacement technology can be used to increase or decrease the expression of TDPl. Gene replacement technology is based on 5 homologous recombination. The nucleic acid of TDPl can be manipulated by mutagenesis (e.g., insertions, deletions, duplications or replacements) to either increase or decrease its function. The altered sequence can be introduced into the genome to replace the existing, e.g., wild-type, gene via homologous recombination. The activity of TDPl can be measured using labeled substrates in vitro. TDPl o activity can be assay quickly and conveniently in vitro by mixing a radioactively 5'- labeled 18-mer oligonucleotide with a 3'phosphotyrosyl group and TDPl. The material is then run on a polyacrylamide sequencing gel and exposed to autoradiographic film. Product bands then can be quantitated (see Yang et al. (1996), supra). TDPl will also act on thymidine 3'-nitrophenyl phosphate in vitro. 5 In addition to being useful in the study of TDPl, the above-described method is useful in the context of prophylactic and therapeutic treatment, such as chemotherapy, in particular where the chemotherapeutic agent is an inhibitor of topoisomerase I and causes the formation of DNA-topoisomerase I complexes that cannot be cleaved or are resistant to cleavage by TDPl, such as campothecin, o topotecan and irinotecan (CPT- 11 ) and analogs thereof (see, e.g., Slichenmyer et al., J.

Natl. Cancer Inst. 85(4): 271-291 (1993); Slichenmyer et al., Cancer Chemother. Pharmacol. 34(Suppl.): S53-57 (1994); Burns & Fields, Hemtol. Onol. Clin. North Am. 8(2): 333-355 (1994); Hawkins, Oncology 6(12): 17-23 (1992); Emerson et al, Cancer Res. 55(3): 603-609 (1995); Sugimori et al., J. Med. Chem. 37(19): 3033-3039 (1994); Wall et al, J. Med. Chem. 36(18): 2689-2700 (1993); Kingsbury et al., J. Med. Chem. 34(1): 98-107 (1991); Wani et al.,J. Med. Chem. 30(10): 1774-1779 (1987); Wani et al., J. Med. Chem. 23: 554-560 (1980); and Wani et al. (1986)). In this regard, a patient with low levels of TDPl might be overly sensitive to compounds that inhibit topoisomerase I and might benefit from treatment with lower doses of . topoisomerase I inhibitors or increased expression of TDPl . In contrast, a patient with high levels of TDPl might be resistant to compounds that inhibit topoisomerase I and might benefit from treatment with higher doses of topoisomerase I inhibitors or reduced expression of TDPl.

In the context of chemotherapy and other methods of treatment, a topoisomerase I inhibitor and an above-described nucleic acid molecule can be administered simultaneously or sequentially in either order, by the same route of administration or by different routes of administration. In this regard, the topoisomerase I inhibitor and the above-described nucleic acid molecule can be present in a single biologically or pharmaceutically acceptable composition or in separate biologically or pharmaceutically acceptable compositions. Pharmaceutically acceptable compositions comprise pharmaceutically acceptable carriers and diluents as appropriate, for example, for human or veterinary applications, as are known in the 0 art.

Thus, a composition for use in the method of the present invention can comprise an above-described nucleic acid molecule, e.g., vector, preferably in combination with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well-known to those skilled in the art, as are suitable methods of 5 administration. The choice of carrier will be determined, in part, by the particular nucleic acid molecule, as well as by the particular method used to administer the composition. One skilled in the art will also appreciate that various routes of administering a composition are available, and, although more than one route can be used for administration, a particular route can provide a more immediate and more o effective reaction than another route. Accordingly, there are a wide variety of suitable formulations of the composition of the present invention.

A composition comprised of a nucleic acid molecule of the present invention, alone or in combination with another active agent, such as a chemotherapeutic agent that inhibits topoisomerase I by causing formation of complexes between DNA and topoisomerase I that cannot be cleaved or are resistant to cleavage by TDPl, can be made into a formulation suitable for parenteral administration, preferably intraperitoneal administration. Such a formulation can include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit dose or multidose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophihzed) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneously injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets, as described herein. A formulation suitable for oral administration can consist of liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or fruit juice; capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solid or granules; solutions or suspensions in an aqueous liquid; and oil-in-water emulsions or water-in-oil emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. An aerosol formulation suitable for administration via inhalation also can be made. The aerosol formulation can be placed into a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Similarly, a formulation suitable for oral administration can include lozenge forms that can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier; as well as creams, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art. A formulation suitable for topical application can be in the form of creams, ointments, or lotions.

A formulation for rectal administration can be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate. A formulation suitable for vaginal administration can be presented as a pessary, tampon, cream, gel, paste, foam, or spray formula containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the infected individual over a reasonable time frame. The dose will be determined by the potency of the particular vector employed for treatment, the severity of the disease state, as well as the body weight and age of the infected individual. The size of the dose also will be determined by the existence of any adverse side effects that can accompany the use of the particular vector employed. It is always desirable, whenever possible, to keep adverse side effects to a minimum.

The dosage can be in unit dosage form, such as a tablet or capsule. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a vector, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound or compounds employed and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host. The dose administered should be an "effective amount" or an amount necessary to achieve an "effective level" in the individual patient. Since the "effective level" is used as the preferred endpoint for dosing, the actual dose and schedule can vary, depending on interindividual differences in pharmacokinetics, drug distribution, and metabolism.

One skilled in the art can easily determine the appropriate dose, schedule, and method of administration for the exact formulation of the composition being used, in order to achieve the desired "effective level" in the individual patient. One skilled in the art also can readily determine and use an appropriate indicator of the "effective level" of the compounds of the present invention by a direct (e.g., analytical chemical analysis) or indirect analysis of appropriate patient samples (e.g., blood and/or tissues).

Generally, an amount of vector sufficient to achieve a tissue concentration of the administered ribozyme (or vector) of from about 50 to about 300 mg/kg of body weight per day is preferred, especially of from about 100 to about 200 mg/kg of body weight per day. In certain applications, e.g., topical, multiple daily doses are prefeπed. Moreover, the number of doses will vary depending on the means of delivery and the particular vector administered. In the treatment of some individuals, it can be desirable to utilize a "mega-dosing" regimen. In yet another embodiment, the present invention provides a method of identifying a compound that stabilizes a covalent bond complex that forms between DNA and topoisomerase I such that the covalent bond cannot be cleaved by or is resistant to cleavage by a TDPl . The method comprises (a) contacting a compound, which stabilizes a covalent bond complex that forms between DNA and topoisomerase I such that the convalent bond cannot be cleaved by or is resistant to cleavage by a TDPl, with DNA and topoisomerase I under conditions suitable for a covalent bond complex to form between the DNA and the topoisomerase I and for the compound to stabilize the covalent bond complex, (b) contacting the covalent bond complex with a TDPl under conditions suitable for cleavage of the covalent bond between the DNA and topoisomerase I by TDP 1 , and (c) detecting cleavage of the covalent bond by the TDPl . The amount of cleavage detected is indicative of whether or not the compound stabilizes a covalent bond complex that forms between DNA and topoisomerase I such that the covalent bond cannot be cleaved or is resistant to cleavage by the TDPl . For example, the more cleavage detected, the less the compound stabilizes a covalent bond complex that forms between DNA and topoisomerase I, and the less cleavage detected, the more the compound stabilizes a covalent bond complex that forms between DNA and topoisomerase I. Preferably, the compound is an analog of camptothecin, topotecan, or irinotecan (CPT-11).

In still yet another embodiment, the present invention provides a method of assessing TDPl activity in an animal. The method comprises (a) obtaimng a sample of a cellular extract from an animal, wherein the cellular extract comprises TDPl, and (b) measuring the level of TDPl activity in the sample. Assessing the level of TDPl activity in a patient may be useful in predicting the patient's sensitivity to a topoisomerase I inhibitor, such as camptothecin, topotecan, irinotecan (CPT-11), an analog of any of the foregoing, and the like. For example, the more TDPl activity a patient has, the less sensitive the patient will be to a topoisomerase I inhibitor, and the less TDPl activity a patient has, the more sensitive the patient will be to a topoisomerase I inhibitor.

A still further embodiment of the present invention is a method of assessing the efficacy of a topoisomerase I inhibitor. The method comprises (a) obtaining a sample of DNA to which is covalently bound topoisomerase I after contact with a topoisomerase I inhibitor, (b) contacting the sample with a TDPl under conditions suitable for cleavage of the covalent bond between the DNA and the topoisomerase I by TDPl, and (c) measuring the amount of topoisomerase I that is cleaved from the DNA. The amount of topoisomerase I that is cleaved from the DNA is indicative of the efficacy of the topoisomerase I inhibitor. Preferably, the sample of DNA is obtained from a patient undergoing treatment with the topoisomerase I inhibitor and the patient's dosage or frequency of administration of topoisomerase I is adjusted down or up based on the high or low efficacy, respectively, of the topoisomerase I inhibitor. Also, preferably, the sample is obtained from peripheral blood cells of the patient. This method can be adapted for screening potential environmental mutagens for DNA-topoisomerase I complexes that are noncleavable by or resistant to cleavage by TDPl.

With respect to the above three methods, methods of contacting a compound with DNA and an enzyme, the determination of conditions suitable for formation, stabilization and cleavage of a DNA - topoisomerase I covalent bond complex, such as physiological conditions, and the detection and measurement of enzyme activity, as well as the preparation of cellular extracts are within the skill in the art (see also, the paragraph bridging pages 5-6, the fourth full paragraph on page 11, and the Examples herein and Yang et al. (1996), supra).

EXAMPLES The present invention is described further in the context of the following examples. These examples serve to illustrate further the present invention and are not intended to limits its scope in any way. In the following examples using yeast, standard protocols of yeast growth, mutagenesis, mating and sporulation were used (see, e.g., Sherman, Methods Enzymol. 194:3 (1991); Treco and Lundblad, in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley, NY (1991)), vol. 2. pp. 13.1.1-13.1.7.).

Example 1

This example describes how the genomic DNA sequence of the yeast TDPl gene was obtained.

Extracts from colonies of chemically mutagenized Saccharomyces cervisiae were assayed for TDP activity. A single strain, KYY337, had very low TDP activity. In backcrosses to the parental line, the enzyme defect appeared to reflect a single mutation (designated enz). That is, when a diploid between the parental line and a defective line was sporulated and haploid colonies were assayed at random, approximately equal numbers were found with normal and with low enzyme activity. The strains were compared for sensitivity to killing by camptothecin. Despite the dramatic difference in TDP activity, the parental line and the backcrossed enz mutant were insensitive to camptothecin. When combined with a disruption of the RAD9 gene, the camptothecin sensitivity of the low activity mutant (strain HNY244) was increased by a factor of 12 relative to the rad9 derivative of the parental strain HNY243. The same difference was observed after the mutant had undergone two additional rounds of backcrossing. In order to confirm that camptothecin-induced damage was due to topoisomerase trapping, the TOPI gene of HNY244 was disrupted and survival increased nearly 1 ,000-fold.

While the mutant line was sensitized to killing by camptothecin, the mutant line was not sensitized to all sources of DNA damage. For example, the mutant line was not sensitized to killing by methyl methane sulfonate, a DNA-alkylating agent. In addition, independent overexpression of two mutant yeast topoisomerase I genes that depress resealing of DNA, thereby leading to an accumulation of covalent complexes, were more toxic in a strain with low TDP activity than in a corresponding control strain.

In view of the above, a library of yeast genomic fragments was screened for the ability to improve the camptothecin resistance of HNY244 and restore its TDP activity. The cloning scheme was based on the assessment that (i) the signa noise ratio of the TDP assay would permit detection of one positive colony in a group of 5- 10 mutants and (ii) one cycle of camptothecin killing would enrich a positive colony by approximately ten-fold.

Strain HNY244 was transformed by electroporation with a genomic library that had been made in a low-copy number vector (Rose et al., Gene 60: 237 (1987)). Transformants were picked from uracil-selective plates and pooled in groups of about 50.

Each pool was separately grown and treated with camptothecin for 24 hours. Cells were grown to near-saturation in medium with glycerol in place of dextrose (YPG) to ensure a starting population with few or no petite derivatives in accordance with standard methods. These cells were resuspended at OD₆₅o=0.4 in YPD (bacto- yeast extract, peptone and dextrose standard yeast growth medium), grown for 2 hrs and diluted again in YPD to OD₆₅o-0.4. Drug was then added and samples were withdrawn immediately and after 24 hrs at 30°C. After dilution and plating on YPD, surviving colonies were counted after 3-4 days of growth. When plasmid-containing strains were assessed for camptothecin sensitivity, YPD was replaced throughout by uracil-deficient minimal medium to ensure plasmid retention. The survivors were amplified by growth in YPD.

An extract of an aliquot of the resulting cells was assayed for TDP activity. 0 From 30 such pools, one was identified that had increased activity. Growth and assay of 15 colonies from this pool identified a single clone, LI 0-13, with nearly wild-type levels of activity. DNA sequence from the insert of the plasmid in LI 0-13 placed its centromere-distal end at coordinate 673926 of chromosome 11.

Several subclones of the approximately 8 kb insert in this plasmid were 5 generated. Plasmid pNS2 was made from pL10-13 by elimination of a Notl/Sall fragment. Elimination of an AatEE-Xbal fragment from pNS2 yielded pAXb, which has a 3.2 kb insert. Several subclones retained full activity.

Transformation of HNY244 with pNS2 or pAXb restored TDP activity and improved camptothecin resistance. A control plasmid, pXl, that failed to restore TDP o activity was made by removal of the central Xbal fragment from pL 10- 13.

The smallest subclone contained a single open reading frame (ORF), namely YBR223c, which encodes a protein of 544 amino acids with a molecular weight of about 62,000. A disruption that removed all but the first 32 amino acids of the ORF was generated by PCR (Baudin, Nucleic Acids Res. 21 : 3329 (1993); Ozier- Kalogeropoulos et al., Nucleic Acids Res. 21 : 3329 (1993); Brachman et al., Yeast 14: 115 (1998)) in strain HNY243. The resulting strain had an enzymatic defect and camptothecin sensitivity was very similar to that of HNY244, indicating that YBR223c is involved in TDP activity.

In order to distinguish whether YBR223c encodes or controls TDP activity, a histidine-tagged version of YBR223c was introduced into E. coli, which, by itself, has no detectable TDP activity. A PCR fragment containing the entire ORF YBR223c was cloned into the BamHI site of pΕT15b. The resulting plasmid, pHN1856, was transformed into strain BL21(DE3) (Novagen, Madison, WI). Bacterial pellets from 3 liters of a culture that had been induced for 2 hrs were resuspended in 100 ml of disruption buffer (Yang et al., PNAS USA 93: 11534 (1996)), sonicated (7 x 3 min), clarified by centrifugation at 20,000 g, and assayed as described above. Induction of bacteria bearing this construct (but not a control construct) resulted in massive overproduction of TDP, since crude extracts of such cells had a specific activity greater than 10,000-fold higher than that of extracts from a standard yeast strain. Moreover, most of the induced activity was bound to a tag-specific column. Specific elution released more than 75% of the bound activity, resulting in a fraction with a single Coomasie-stainable band of the expected molecular size. Based on the above, it was concluded that YBR223c encodes TDPl.

Database searches failed to reveal homology between TDPl and any genes of known function. Even individualized comparisons to motifs identified in various phosphodiesterases and phosphatases were, at best, marginal. Thus, TDPl encodes a novel enzyme. Eukaryotic databases contain several unannotated sequences that match TDPl . The complete genome sequence of the nematode Caenorhabditis elegans contains a single ORF with significant similarity to TDPl. Probing EST databases with the yeast and nematode proteins revealed many significant matches (see Figure 4, which is an alignment of TDP homologs from various organisms, in which "hs" is the human deduced amino acid sequence, "mm" is the mouse amino acid sequence (Mus muscularis; assembly of mouse ESTs GenBank AA940134, W89267 and W13117), "dm" is the fruit fly deduced amino acid sequence (Drosophila melanogaster; GenBank Al 517253), "ce" is the nematode deduced amino acid sequence (Caenorhabditis elegans (ce; gene F52C12.1; GenBank AF100657.2)), "sp" is the Schitosaccharomyces pombe (Sanger Centre Sequencing Group: ftp://ftp.sanger.ac.uk/pub/) deduced amino acid sequence, and "sc" is the yeast deduced amino acid sequence (Saccharomyces cerevisiae; gene YBR223c; GenBank Z36092.1). Black boxes indicate identities, whereas shaded boxes indicate similarities and "x" indicates uncertainty in the GenBank entry AA48921. X's were confirmed by sequence analysis of the product of a 3' RACE of a human EST that showed that the sequence in the region of ambiguity is identical to that shown for the mouse).

Example 2

This example describes how the cDNA sequence of the human TDPl gene was obtained.

A human database and a mouse EST database were searched with the yeast sequence and several EST's were identified that could be aligned to make up a single ORF with substantial similarity to the carboxy-terminal half of TDP 1. In order to determine if the homology extends further, PCR was performed on a collection of human cDNAs (Marathon-Ready; Clontech Laboratories, Palo Alto, CA) with a primer complementary to an EST sequence identified in the human EST database and a primer complementary to the tag affixed to the 5' end of the cDNAs. The resulting

5'-RACE products were cloned. The sequence of one of the longest clones aligned well to most of the 5' half of the yeast and nematode ORFs. Based on the above, it was concluded that the TDPl gene is highly conserved in eukaryotes.

Partial 5' and 3' sequences of the human TDPl have been deposited as GenBank AF 182002 and AF 182003, respectively. The complete cDNA sequence is shown in Figure 1 as SEQ ID NO: 1. The deduced amino acid sequence is shown in Figure 2 as SEQ ID NO: 2.

All of the references cited herein, including patents, patent applications, and publications, are hereby incorporated in their entireties by reference.

While this invention has been described with an emphasis upon preferred embodiments, it will be apparent to those of ordinary skill in the art that variations in the preferred embodiments can be prepared and used and that the invention can be practiced otherwise than as specifically described herein. The present invention is intended to include such variations and alternative practices. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

WHAT IS CLAEMED IS

1. An isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a mammalian tyrosine-DNA phosphodiesterase (TDPl) or a continuous fragment thereof of at least about 36 nucleotides.

2. The isolated and purified nucleic acid molecule of claim 1, wherein said mammalian TDPl is a human TDPl.

3. The isolated and purified nucleic acid molecule of claim 1, wherein said nucleic acid molecule is (i) DNA and consists essentially of SEQ ED NO: 1 or a sequence that encodes SEQ ED NO: 2, (ii) RNA and consists essentially of a sequence encoded by SEQ ED NO: 1 or a sequence that encodes SEQ ED NO: 2, or (iii) a nucleic acid molecule consisting essentially of a nucleotide sequence that encodes a mammalian TDPl or a continuous fragment of at least about 12 amino acids thereof and that hybridizes to either one of the foregoing under low stringency conditions.

4. An isolated or purified nucleic acid molecule encoding a modified mammalian TDPl, which comprises one or more insertions, deletions and/or substitutions, wherein the modified mammalian TDPl encoded by said isolated or purified nucleic acid molecule does not differ functionally from the coπesponding unmodified mammalian TDPl, or a continuous fragment thereof of at least about 36 nucleotides.

5. The isolated or purified nucleic acid molecule of claim 4, wherein the corresponding unmodified mammalian TDPl has the amino acid sequence of SEQ ID NO: 2.

6. The isolated or purified nucleic acid molecule of claim 4, wherein the modified mammalian TDPl encoded by said isolated or purified nucleic acid molecule cleaves a covalent bond complex between DNA and topoisomerase I at least about 90% as well as the unmodified mammalian TDPl comprising SEQ ED NO: 2 as determined by in vitro assay using labeled topoisomerase I or an oligonucleotide comprising a 3' phosphotyrosine.

7. The isolated or purified nucleic acid molecule of claim 4, wherein said one or more substitution(s) do(es) not result in a change in an amino acid of the encoded mammalian TDPl or results in the substitution of an amino acid of the encoded mammalian TDPl with another amino acid of approximately equivalent size, shape and charge.

8. A vector comprising a nucleic acid molecule of any of claims 1-7.

9. A vector comprising or encoding an antisense molecule of at least about 20 nucleotides that hybridizes to or a ribozyme that cleaves an RNA molecule encoding a mammalian TDPl .

10. A host cell comprising a vector of claim 8 or claim 9.

11. A polypeptide produced by a host cell comprising the vector of claim 8.

12. A purified and isolated human TDPl .

13. A polyclonal or monoclonal antibody that binds to a mammalian TDPl but does not bind to a nonmammalian TDPl .

14. An antisense molecule of at least about 20 nucleotides that can hybridize to an RNA molecule encoding a mammalian TDP 1.

15. A ribozyme that can cleave an RNA molecule encoding a mammalian TDPl.

16. An isolated and purified nucleic acid molecule consisting essentially of a nucleotide sequence encoding a yeast tyrosine-DNA phosphodiesterase (TDPl) or a continuous fragment thereof comprising at least about 36 nucleotides.

17. The isolated and purified nucleic acid molecule of claim 16, wherein said yeast TDPl is a Saccharomyces cerevisiae TDPl.

18. The isolated and purified nucleic acid molecule of claim 16, wherein said nucleic acid molecule is (i) DNA and consists essentially of SEQ ED NO: 3 or a sequence that encodes SEQ ED NO: 4, (ii) RNA and consists essentially of a sequence encoded by SEQ ED NO: 3 or a sequence that encodes SEQ ED NO: 4, or (iii) a nucleic acid molecule consisting essentially of a nucleotide sequence that encodes a yeast TDPl or a continuous fragment of at least about 12 amino acids thereof and that hybridizes to either one of the foregoing under low stringency conditions.

19. An isolated or purified nucleic acid molecule encoding a modified yeast TDPl, which comprises one or more insertions, deletions and/or substitutions, wherein the modified yeast TDPl encoded by said isolated or purified nucleic acid molecule does not differ functionally from the corresponding unmodified yeast TDPl, or a continuous fragment thereof of at least about 36 nucleotides.

20. The isolated or purified nucleic acid molecule of claim 19, wherein the corresponding unmodified yeast TDPl has the amino acid sequence of SEQ ED NO: 4.

21. The isolated or purified nucleic acid molecule of claim 20, wherein the modified yeast TDPl encoded by said isolated or purified nucleic acid molecule cleaves a covalent bond complex between DNA and topoisomerase I at least about 90% as well as the unmodified yeast TDPl of SEQ ED NO: 4 as determined by in vitro assay using labeled topoisomerase I or an oligonucleotide comprising a 3'- phosphotyrosine.

22. The isolated or purified nucleic acid molecule of claim 21, wherein said one or more substitution(s) do(es) not result in a change in an amino acid of the encoded yeast TDPl or results in the substitution of an amino acid of the encoded yeast TDPl with another amino acid of approximately equivalent size, shape and charge.

23. A vector comprising a nucleic acid molecule of any of claims 16-22.

24. A vector comprising or encoding an antisense molecule of at least about 20 nucleotides that hybridizes to or a ribozyme that cleaves an RNA molecule encoding a yeast TDPl.

25. A host cell comprising a vector of claim 23 or claim 24.

26. A polypeptide produced by a host cell comprising the vector of claim 23.

27. A polyclonal or monoclonal antibody that binds to a yeast TDPl but does not bind to a nonyeast TDPl.

28. An antisense molecule of at least about 20 nucleotides that can hybridize to an RNA molecule encoding a yeast TDPl .

29. A ribozyme that can cleave an RNA molecule encoding a yeast TDPl .

30. A method of altering the level of TDPl in a cell, tissue, organ or organism, which method comprises contacting said cell, tissue, organ or organism with a vector comprising a nucleic acid molecule selected from the group consisting of (i) a nucleic acid molecule encoding a TDPl, (ii) a nucleic acid molecule comprising or encoding an antisense molecule of at least about 20 nucleotides to an RNA molecule transcribed from (i), and (iii) a nucleic acid molecule comprising or encoding a ribozyme to an RNA molecule transcribed from (i), wherein said vector comprising a nucleic acid molecule of (i) increases or decreases the level of TDPl in said cell, tissue, organ or organism, and wherein said vector comprising a nucleic acid molecule of (ii) or (iii) decreases the level of TDPl in said cell, tissue, organ or organism.

31. A cell, tissue, organ or nonhuman organism in which the level of TDPl has been altered in accordance with the method of claim 30.

32. A method of identifying a compound that stabilizes a covalent bond complex that forms between DNA and topoisomerase I, wherein said covalent bond cannot be cleaved by or is resistant to cleavage by a TDPl, which method comprises: (a) contacting a compound, which stabilizes a covalent bond complex that forms between DNA and topoisomerase I such that the covalent bond cannot be cleaved by or is resistant to cleavage by a TDPl , with DNA and topoisomerase I under conditions suitable for a covalent bond complex to form between the DNA and the topoisomerase I and for the compound to stabilize the covalent bond complex, 0 (b) contacting the covalent bond complex with a TDPl under conditions suitable for the cleavage of the covalent bond between the DNA and topoisomerase I by TDPl, and

(c) detecting cleavage of said covalent bond by said TDPl, wherein the amount of cleavage detected is indicative of whether or not 5 said compound stabilizes a covalent bond complex that forms between DNA and topoisomerase I such that the covalent bond cannot be cleaved or is resistant to cleavage by said TDPl .

33. The method of claim 32, wherein said compound is an analog of o camptothecin, topotecan, or irinotecan (CPT- 11).

34. A method of assessing TDPl activity in an animal, which method comprises:

(a) obtaining a sample of a cellular extract from the animal, wherein 5 said cellular extract comprises TDP 1 , and

(b) measuring the level of TDPl activity in said sample.

35. A method of assessing the efficacy of a topoisomerase I inhibitor, which method comprises: 0 (a) obtaining a sample of DNA to which is covalently bound topoisomerase I after contact with a topoisomerase I inhibitor, (b) contacting said sample with a TDPl under conditions suitable for cleavage of the covalent bond between the DNA and the topoisomerase I by TDPl, and

(c) measuring the amount of topoisomerase I that is cleaved from the DNA, wherein the amount of topoisomerase I that is cleaved from the DNA is indicative of the efficacy of the topoisomerase I inhibitor.

36. The method of claim 35, wherein said sample of DNA is obtained from a patient undergoing treatment with said topoisomerase I inhibitor.

37. The method of claim 36, wherein said sample is obtained from peripheral blood cells of said patient.